Binding of 2,4,6-Trinitrotoluene, Aniline, and Nitrobenzene to

The extensive use of 2,4,6-trinitrotoluene (TNT) in explosives and the toxicity and mutagenic effects of TNT and its degradation products pose an envi...
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Environ. Sci. Technol. 2004, 38, 3074-3080

Binding of 2,4,6-Trinitrotoluene, Aniline, and Nitrobenzene to Dissolved and Particulate Soil Organic Matter J O H A N E R I K S S O N , †,‡ S O F I A F R A N K K I , † ANDREI SHCHUKAREV,§ AND U L F S K Y L L B E R G * ,† Department of Forest Ecology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden, NBC-Defence, Swedish Defence Research Agency (FOI) SE-901 82 Umeå, Sweden, and Department of Inorganic Chemistry, Umeå University, SE-901 87 Umeå, Sweden

The distribution of TNT* (the sum of TNT and its degradation products), aniline, and nitrobenzene between particulate organic matter (POM), dissolved soil organic matter (DOM), and free compound was studied in controlled kinetic (with and without irradiation) and equilibrium experiments with mixtures of POM and DOM reflecting natural situations in organic rich soils. The binding of TNT* to POM was fast, independent of irradiation, and adsorption isotherms had a great linear contribution (as determined by a mixed model), indicative of a hydrophobic partitioning mechanism. The binding of TNT* to DOM was slower, strongly enhanced under nonirradiated conditions, and adsorption isotherms were highly nonlinear, indicative of a specific interaction between TNT derivatives and functional groups of DOM. Nitrobenzene was associated to both POM and DOM via hydrophobic partitioning, whereas aniline binding was dominated by specific binding to POM and DOM functional groups. On the basis of nitrobenzene and TNT* adsorption parameters determined by a mixed Langmuir + linear model, POM had 2-3 times greater density of hydrophobic moieties as compared to DOM. This difference was reflected by a greater (O + N)/C atomic ratio for DOM. The sum of C-C and C-H moieties, as determined by X-ray photoelectron spectroscopy (XPS), and the sum of aryl-C and alkyl-C, as determined by solidstate cross-polarization magic-angle spinning (CP-MAS) 13C NMR, could only qualitatively account for differences in adsorption parameters. Aliphatic C was found to be more important for the hydrophobic partitioning than aromatic C. On the basis of nonlinear adsorption parameters, the density of functional groups reactive with aniline and TNT derivatives was 1.3-1.4 times greater in DOM than in POM, which was in fair agreement with 13C NMR and XPS data for the sum of carboxyl and carbonyl groups as potential sites for electrostatic and covalent bonding. We conclude that in contaminated soils characterized by continuous leaching of DOM, formation of TNT derivatives (via biotic and abiotic reductive degradation) and their * Correspondingauthorphone: +46-907866865;fax: +46-907867750; e-mail: [email protected]. † Swedish University of Agricultural Sciences. ‡ Swedish Defense Research Agency. § Umeå University. 3074

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preference for specific functional groups in DOM may contribute to a significant transportation of potentially toxic TNT compounds into surface waters and groundwaters.

Introduction The extensive use of 2,4,6-trinitrotoluene (TNT) in explosives and the toxicity and mutagenic effects of TNT and its degradation products pose an environmental threat in soils and surface waters, especially in connection to shooting ranges, military bases and former munitions manufacturing plants. Severe local problems have put pressure on remediation measures. It is well-known that TNT is extensively adsorbed by negatively charged 2:1 clay mineral surfaces saturated by low hydrated cations (1). On the other hand, in most soils, TNT rapidly undergoes reductive degradation (2), and composting experiments have shown that biological degradation of TNT promotes an irreversible binding to SOM with time (3-5). Under aerobic and partly anaerobic conditions, one or more nitro groups may be reduced to amino groups, via nitroso- and hydroxylamino intermediates, forming aminodinitrotoluene (2- or 4-ADNT), diaminonitrotoluene (2,4- or 2,6-DANT), and under strictly anaerobic conditions, triaminotoluene (TAT). The nitroso- and hydroxylamino derivatives may also react to form azoxy compounds. Recent 15N NMR studies have revealed that amino groups of ADNT and DANT form covalent bonds with carbonyl groups in SOM through condensation reactions (6), similar to the reaction proposed to occur between aniline and humic substances (7). Aniline has been shown to form an electrostatic interaction with humic substances within hours, followed by a slower (days) formation of covalent bonds (8). Using 15N NMR, hydroxylamino and azoxy intermediates of TNT have been proposed to be involved in covalent bond formation with SOM during composting (9, 10). The reactivity of hydroxylamines have been partly questioned (5, 11), and in the latter study, a reaction between nitroso derivatives and thiol groups (and other nucleophiles such as amines) in DOM was suggested to be important for the association to humic substances. Even though 15N NMR studies and studies of 14C labeled TNT have produced important information on the incorporation of TNT and its degradation products (the sum of which are henceforth designated TNT*) into SOM, and different extracted fractions such as humic acids, fulvic acids, and humin (4, 9), we still know little about the potential mobility of TNT* in soils. The mobility and possible transportation of TNT* into surface waters and groundwaters is dependent on the ability of solid phase particulate OM (POM), and other soil surface components, to retain TNT* and the ability of dissolved organic matter (DOM) to bind and transport TNT*. In a 24 h equilibrium experiment, TNT* was found to bind more strongly to DOM than to POM, but the binding capacity was greater for POM (12). A nonlinear isotherm and a strong pH dependence of the association between TNT* and DOM was interpreted as an effect of electrostatic interactions, whereas a more linear and pHindependent reaction with POM was interpreted as caused by mainly hydrophobic partitioning. For aniline and nitrobenzene, studies on possible differences in their binding to DOM and POM today are lacking. In the present study, we conducted kinetic and equilibrium experiments to determine the distribution of TNT*, 10.1021/es035015m CCC: $27.50

 2004 American Chemical Society Published on Web 04/28/2004

aniline, and nitrobenzene between POM and DOM as a function of total concentration of chemical, under irradiated and nonirradiated conditions and at varying composition of adsorbed major metal cations. The fact that aniline and nitrobenzene do not undergo degradation under the experimental conditions used in this study makes them fair model compounds for the different characters of TNT and its major degradation products; benzene ring + nitro group (nitrobenzene) and benzene ring + amino group (aniline).

Experimental Procedures Sampling, Preparation, and Characterization of Soil Organic Matter Fractions. A 30 cm thick organic horizon from a Gleysol (13) was sampled at Svartberget Research Station, Vindeln, Sweden. A SOM (soil organic matter) sample was homogenized and separated from plant roots and large, undecomposed debris using a 4 mm cutting sieve. Total organic carbon was 46% and total nitrogen 2.2% of dry mass, as determined by dry combustion (2400 CHN elemental analyzer, Perkin-Elmer, CT). Soil pH was 3.16 determined at a 1:6 soil to solution mass ratio in 0.01 M CaCl2. Adsorbed metal ions were sequentially extracted with 0.5 M CuCl2 using the method of ref 14 and were determined on ICP AES (PerkinElmer). The concentration of adsorbed Na, Mg, Ca, and Al was 1, 3.5, 10, and 115 mmol kg-1, respectively. The total cation-exchange capacity (CECt), calculated as the sum of total acidity determined at pH 8.2 (15) and the sum of charges pertaining to adsorbed Na, Mg, and Ca was 2110 mmolc kg-1 soil. A stock solution of dissolved organic matter (DOM) was prepared by adding 7.0 g of the metal chelating resin Chelex 20 (Bio-Rad, Hercules, CA) to 60.0 g of SOM and 150 mL of deionized water in a 250 mL polycarbonate centrifugation tube. After 24 h of gentle end-over-end shaking, DOM was separated from the solid phase by centrifugation at 14600g for 15 min. The DOM stock solution had a pH of 6.0 and was stored at 4 °C in darkness. Hydrogen ion saturated particulate organic matter (POM) was prepared from SOM by a sequential treatment with 0.5, 0.1, and 0.05 M HCl solutions. After each HCl addition, the suspension was equilibrated for 2 h during end-over-end mixing, and then the solid phase (POM) was separated from the supernatant by centrifugation at 14600g for 15 min. Finally, the H+-saturated POM (H-POM) sample was washed with several portions of Milli-Q water until no precipitation of AgCl(s) was detected after addition of a droplet of washing solution to a saturated AgNO3 solution. Freeze-dried samples of SOM, H-POM, and DOM were characterized by XPS and CPMAS 13C NMR. XPS spectra were recorded with a Kratos Axis Ultra electron spectrometer using monochromated Al Ka source operated at 180 W. To compensate for surface charging, a low-energy electron gun was used. The binding energy (BE) scale was referenced to the C 1s line of aliphatic carbon, set at 285.0 eV. Processing of the spectra was accomplished with Kratos software. A Bruker DSX 200 was used for CP-MAS 13C NMR determination at a resonance frequency of 50.3 MHz, spinning rate 6.7 kHz, 1 ms contact time, a 90 °C 1H-pulse width of 5.75 µs and a time delay of 400 ms. The spectra were integrated into six chemical shift regions; -10 to 45 ppm as a measure of alkyl C, 45-110 ppm representing O- and N-alkyl C, 110-140 ppm representing aryl C, 140-160 representing O/N-aryl C, 160185 ppm representing carboxyl + amide C, and 185-220 ppm, as an estimate of carbonyl C. Chemicals. TNT (C7H5N3O6, mw 227.13, log KOW ) 1.86 (16), solubility in water 0.13 g L-1 (17)), nitrobenzene (C6H5NO2, mw 123.11, log KOW ) 1.84 (18), solubility in water 2.00 g L-1 (19)), and aniline (C6H7N, mw 93.13, pKa 4.6, log KOW ) 0.90 (18), solubility in water 34.97 g L-1 (19)) were the chemicals used in this study. Stock solutions of labeled and nonlabeled compounds were prepared in Milli-Q water

without cosolvent. TNT was synthesized from 14C-labeled toluene at the Swedish Defence Research Agency (FOI), Weapons and Protection Division, with a specific activity of 1.7 mCi mmol-1 and a purity of >95%. 14C-labeled aniline was purchased from Sigma-Aldrich (Steinheim, Germany) and had a specific activity in the prepared stock solutions of 0.05-0.4 mCi mmol-1. Nitrobenzene was purchased from American Radio-labeled Chemicals (St. Louis, MO) and had an activity of 0.1 mCi mmol-1 in the final stock solution. Kinetic Adsorption Experiments: Sterile and Nonsterile Conditions. The untreated SOM sample was used in timedependent adsorption experiments. A small volume of DOM stock solution was preequilibrated with 10 g of SOM in 500 mL of Milli-Q water added NaOH and NaCl to yield a DOC concentration of 300 mg C L-1, an ionic strength of 50 mM NaCl, and a pH of 5.1. After preequilibration during a week in darkness at 4 °C, the aqueous phase with DOM was separated from POM by centrifugation at 14600g during 15 min. The DOM solution was filtered through a sterile 0.22 µm filter to remove microorganisms. The POM sample was divided in two parts, one of which was sterilized with γ-radiation by a Scanditronix Gammarad 900 (Uppsala, Sweden) with a 137Cs-source delivering 20 TBq for 18 days. Replicates of filtered DOM solution were mixed with POM (irradiated and nonirradiated) in a 50:1 (DOM stock solution mass: POM dry mass) ratio in 4 or 7 mL glass vials with Teflon lined screw lids. After 24 h of end-over-end mixing, the vials were added TNT (labeled), nitrobenzene (nonlabeled), or aniline (nonlabeled). Duplicates of vials for a single compound were sampled at 0.03, 0.13, 0.32, 1, 2, 4, 8, and 28 (only for TNT) days. After centrifugation for 10 min at 14600g, the pH, DOM, and free and labeled organic compounds were determined as described next. Blanks without SOM or DOM were prepared and sampled after 0.03 and 8 days to monitor possible side reactions with, for example, glass walls. For irradiated series, all labware was autoclaved, heated at 160 °C for 2 h, or washed in methanol. The biological activity in irradiated and nonirradiated samples was determined by respiration according to a method described in ref 20, and the microbial activity in each vial was tested by a plate-count method described in ref 21. Absorbance at 254 nm revealed no direct effect of irradiation on the concentration of DOM. Equilibrium Adsorption Experiments. Manipulated DOM-POM systems were created with a varied composition of adsorbed Na and Al ions at constant ionic strength, a constant pH of 4.9-5.2, and DOM concentrations in the range of 500-590 mg C L-1. This was achieved by an addition of different concentrations of NaOH + NaCl (Na system) and 3NaOH + AlCl3 (Al system), to H+-saturated POM, as described by ref 22. The DOM concentration was adjusted by an addition of DOM stock solution. The DOM-POM system was prepared and equilibrated by gentle shaking for a week in darkness and 4 °C. After a separation of DOM solution from the solid phase (POM), by centrifugation at 14600g for 15 min, final DOM-POM systems were prepared by mixing in a 50:1 (DOM solution mass: POM dry mass) ratio in 4 or 7 mL glass vials with Teflon lined screw lids. To duplicates of these vials different concentrations of 14Clabeled TNT, nitrobenzene, or aniline were added. The DOM-POM systems were equilibrated in darkness at 22 °C by end-over-end shaking (30 rpm) during 15 min every second hour. The equilibrium time was chosen based on the kinetic experiments to reach as close as possible to chemical equilibrium and to avoid extensive biodegradation (relevant only for TNT). After 22 h (TNT and NB) or 72 h (aniline), the DOM solution and POM were separated by centrifugation at 14600g for 10 min. The concentration of DOM (as determined by absorbance at 254 nm) did not differ significantly after centrifugation at 14600g, as compared to filtration through VOL. 38, NO. 11, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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a 0.45 µm filter. For all series, blanks without DOM + POM, but otherwise treated the same way as other samples, were monitored for possible side reactions with , for example, glass walls. Chemical Analyses. Waters (Milford, MA) reversed-phase high-performance liquid chromatography (HPLC) system with a Lichrocart 125-4 column packed with 5 µm spheres of Purospher C-18e (Merck, Darmstadt, Germany) with a photodiode array detector was used to separate DOM and the analytes TNT, NB, or aniline. The composition of the mobile phase was adjusted to elute the free analyte at a retention time of 5-6 min. For TNT the mobile phase was 50:50 of methanol and PO4 buffer (5 mM, pH 7.0), for aniline 15:85 of acetonitrile (AcN) and PO4 buffer, and for nitrobenzene 45:55 AcN and PO4 buffer. The flow was constant at 1.1 mL min-1. The chromatograms were used for quantification of free analyte at wavelengths of 228 nm for TNT, 230 nm for aniline (280 nm at high concentrations), and 263 nm for NB (226 nm at high concentrations). Calibration standards of all substances were prepared in acetonitrile + PO4 buffer, and all substances were of analytical purity. The DOM concentration was calculated from adsorption at 254 nm using a relationship between absorbance and DOC concentration (measured by TOC-5000 Shimadzu, Japan) for similar types of DOM extracted from organic soil. In the kinetic studies of nitrobenzene and aniline, free concentrations of the analytes in aqueous phase were determined directly from the chromatogram and the concentration of SOM associated analyte was calculated as the difference between the total and the free concentration. In kinetic experiments of TNT, and in all equilibrium experiments, the concentration of the DOM-associated analyte was determined as the 14C activity in the DOM chromatogram peak cf. ref 12. The analyte associated with POM was calculated as total 14C activity subtracted by DOM 14C activity and free aqueous concentration. In the equilibrium experiments, the high DOM concentration gave rise to a tailing of the DOM peak in the chromatogram that increased with content of acetonitrile in the mobile phase. Since DOM retarded by the column may interact with the free analyte in other ways than the major portion of DOM passing more easily through the column, and because of uncertainties in quantification due to the DOM tailing, the concentration of free analyte was calculated as the difference between total 14C activity in the supernatant (after separation of POM by centrifugation) and 14C activity associated to DOM passing through the column. In the TNT kinetic experiments, direct HPLC determination of free TNT was shown to give very similar results as a subtraction of 14C activity in solution from 14C activity in DOM. DOM sorbed on the column was removed between runs by sequentially washing the column with Milli-Q water, followed by methanol, chloroform, 0.005 M H2SO4, and finally Milli-Q water. At least 20 column volumes of this sequence of solvents were used. The retardation/sorption of DOM in the Lichrocart 125-4 column at different pH values and composition of adsorbed major metal cations was quantified by use of a size-exclusion chromatography (SEC) column (a prepacked Superdex 75 HR with inner diameter of 10 mm and a length of 300 mm, with an approximately exclusion limit of 100 kDa, Amersham Pharmacia Biotech AB, Uppsala, Sweden) that was found not to sorb DOM. At pH 5 and with Na+ as the major adsorbed metal ion, the DOM recovery in the first three HPLC fractions (see next) was 0.86, 0.76, and 0.87 for the mobile phase used for TNT, NB, and aniline systems, respectively. At pH 5 and Al3+ as the major adsorbed metal ion, the corresponding DOM recovery was 0.73, 0.56, and 0.77, respectively. These DOM recoveries were used to linearly correct the 14C activity in DOM for retardation/sorption in the column at a certain pH and cation composition by 3076

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corrected 14C activity (DOM) ) determined 14C activity (DOM) recovery

(1)

14C Determination. The 14C activity of the supernatant (DOM + aqueous solution) was determined after POM had been separated by centrifugation. The HPLC analysis effluent was fractionated and subsequently analyzed for 14C activity using a modified version of the method of ref 12 to determine the 14C activity associated to DOM. The elute collected the first 15 s of each run was discarded to rinse the fraction collector. The elute from the following 3 × 1.5 min was collected in three fractions. The 14C activity determined in these three fractions, after blank subtraction, represented 14C associated with DOM. The 14C activity was determined on a liquid scintillation system, LS 5000CE (Beckman, Fullerton, CA). Liquid scintillation cocktails were prepared using ReadySafe (Beckman, Fullerton, CA). All activities were corrected for quenching and are henceforth expressed as disintegrations per minute (DPM). Isotherm Calculations. Three different models were used to analyze the data; the linear isotherm (eq 2), the Langmuir isotherm (eq 3), and a combined linear and Langmuir isotherm (eq 4), using a nonlinear least-squares fitting procedure. For data with nonuniform variance, the dependent variable was weighted by 1/abs (value). For these calculations the SigmaPlot computer software was used (23).

linear:

Cs ) KOCCw

Langmuir:

Cs )

(2)

qmaxKLCw 1 + K LC w

Langmuir + linear:

Cs )

qmaxKLCw + KOCCw 1 + KLCw

(3)

(4)

The term Cs is the sorbed concentration of the compound expressed in relation to the mass of organic carbon (mol kg-1 C), and Cw is the equilibrium concentration of free compound (mol L-1) in solution. The KOC is the partitioning coefficient for the linear equation (L kg-1 C). In the Langmuir equation, qmax is the maximum sorption capacity, assuming that the sorbate is arranged in a monolayer (µmol g-1 C) at the adsorbing surface, and KL (µM-1) is the Langmuir constant.

Results and Discussion Chemical Characterization of POM and DOM. The atomic ratio (O + N)/C was significantly greater in DOM, indicating more oxidized, polar moieties than in SOM and H-POM (Table 1). Both XPS and 13C NMR (Table 1 and Figure 1) data indicate that the density of carboxyl functionalities (NMR shift 160-185 ppm, XPS BE 289-290 eV) was greater in DOM, as compared to SOM and H-POM. Carbonyl functionalities determined by NMR (185-220 ppm) was indicated to be slightly more abundant in DOM, but small differences were seen in XPS if also amides were included (BE 287-288 eV). On the basis of both XPS and NMR data, the sum of CdO, N-CdO, and O-CdO groups accounted for 9-10% of total C in SOM and H-POM, whereas in DOM these groups accounted for 21% of total C, as determined by XPS, and 13%, as determined by NMR. This discrepancy between methods for DOM is not easily explained, but it should be remembered that especially NMR data are semiquantitative and affected by factors such as the contact time used and paramagnetic compounds such as, for example, Fe(III) (24). The effect of Fe(III) could be assumed small since in the original soil the content of Fe(III) was only 0.05 mass %, and after H+ saturation (H-POM) and chelate resin extraction

TABLE 1. Binding Energy (BE) and Chemical Shift for Carbon Bonds in Organic Soil (SOM), H+-Saturated POM (H-POM), and DOM at pH 5.1a BE, eV XPS

285.0 Cs(C, H)

286.5-286.7 CsOH, CsN, CsOsC

289-290 OsCdO

287-288 CdO, NsCdO

(O + N)/Cd

SOM H-POM DOM

65c (51)b 65c (49)b 53c (33)b

24c (19)b 24c (18)b 26c (16)b

5c (4)b 4c (3)b 16c (10)b

5c (4)b 6c (5)b 5c (3)b

0.26 0.27 0.33

shift, ppm 13C NMR H-POM DOM a

0-45 alkyl C

110-140 Ar-C

45-110 O-alkyl C

140-160 O/N-Ar-C

160-185 OsCdO, NsCdO

185-220 CdO

Caromatic/ Caliphatice

40c 34c

10c 12c

36c 34c

4c 5c

7c 10c

2c 3c

0.18 0.26

Errors are on the order of ( 1-2% C.

b

Atomic percent of all elements. c Percent of total C.

d

Atomic ratio. e (110-160 ppm)/(0-110 ppm).

FIGURE 1. 13C NMR spectra for dissolved organic matter (DOM) at pH 5.1 and hydrogen ion saturated particulate organic matter (HPOM). (DOM), insignificant amounts of Fe (III) could be expected. According to NMR data, the ratio of aromatic to aliphatic C was larger for DOM than for H-POM, whereas the sum of aliphatic and aromatic C (0-45 + 110-140 ppm) was quite similar, contributing to 50% of H-POM C and 46% of DOM C. The XPS data point at a greater percentage of C-C and C-H moieties in SOM and H-POM (65%) as compared to DOM (53%). In summary, NMR and XPS analyses showed that H-POM and SOM were less oxidized and had more hydrophobic C-C, C-H moieties than DOM, which in turn contained a higher density of hydrophilic O/N functional groups. Kinetic Experiments: Irradiated and Nonirradiated Systems. In the irradiated system, which can be assumed to be almost free from biological activity (see next), the extent of TNT binding to POM and DOM was quite similar on a mol g-1 [C] basis (Figure 2a). Equilibrium between TNT in solution and TNT bound to POM and DOM seemed to be obtained within approximately 1 day. In the nonirradiated system (Figure 2b), the binding of TNT* to POM was quite similar to the irradiated system, but the extent of reaction with DOM was much greater. In the nonirradiated system, TNT* bound to DOM reached 5 µmol g-1 [C] after 1 day, to be compared with ∼2 µmol g-1 [C] in the irradiated system, and the binding to DOM continued to increase with time until day 8 (Figure 2b). This increase is interpreted as an effect of biodegradation of TNT and that its derivatives have a greater reactivity toward DOM than nondegraded TNT. The fact that the extent of TNT* binding to POM was only marginally affected by irradiation suggests that TNT and its derivatives produced

FIGURE 2. Kinetics of TNT* association to DOM and POM. [DOM] ) 280 mg (C) L-1, [POM] ) 9.9 g (C) L-1, [TNT]0 ) 34 µM, and pH ) 5.1. (a) Irradiated and (b) nonirradiated. C × C0-1 is the ratio between measured and initial concentration of free compound in solution. by biodegradation bind to POM in a similar way. The reaction between TNT* and POM in addition reached equilibrium much faster than the reaction with DOM, in the nonirradiated system. In previous 15N NMR spectroscopy studies, hydroxylamines and azoxy derivatives (3, 25), nitrosarenes (11), or the TNT amines ADNT, DANT, and triamino-toluene (TAT) (5, 6) have been shown to form electrostatic and covalent bonds with mainly carbonyl groups (and in the case of nitrosoarenes, thiol groups) of SOM. Hydroxylamines are only stable under anaerobic conditions, whereas the other derivatives are formed also under aerobic conditions. In the study of ref 12, the association between TNT* and POM was found to be dominated by a hydrophobic partitioning mechanism, with equilibrium obtained within 24 h. Thus, based on the difference in binding to DOM and POM revealed VOL. 38, NO. 11, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Adsorption isotherms for the association of TNT* to POM and DOM. Na system: [DOM] ) 560 mg (C) L-1, [POM] ) 10.7 g (C) L-1, Naads ) 490 mmolc kg-1 (POM), Alads < 10 mmolc kg-1 (POM), and pH ) 5.0. Al system: [DOM] ) 540 mg (C) L-1, [POM] ) 9.5 g (C) L-1, Naads) 340 mmolc kg-1 (POM), Alads) 210 mmolc kg-1 (POM), and pH ) 4.9. Solid lines represent the mixed model (eq 4, parameters in Table 2).

FIGURE 3. (a) Kinetics of nitrobenzene association to SOM (POM + DOM). [DOM] ) 290 mg (C) L-1, [POM] ) 9.3 g (C) L-1, [NB]0 ) 124 µM, and pH ) 5.1. (b) Kinetics of aniline association to SOM. [DOM] ) 342 mg (C) L-1, [POM] ) 9.2 g (C) L-1, [aniline]0 ) 225 µM, and pH ) 5.3. C × C0-1 is the ratio between measured and initial concentration of free compound in solution. in the kinetic studies, we suggest that the initial, fast mechanism of TNT* association to DOM and POM is through hydrophobic partitioning and that TNT derivatives formed by biodegradation show a preference for DOM functional groups. All irradiated samples were tested for biological activity by the plate-count method. If biological activity was encountered by this method, the sample was omitted. Typically, less than 10% of the microbial community is detected by this method (26); therefore, some biological activity might have been present also in the irradiated systems tested by the plate-count method. During the first 80 h, a CO2 production of 0.8 and 12 µg h-1 g-1 (dry wt) was determined by the method of ref 20 in irradiated and nonirradiated systems, respectively. The fact that extent of TNT reaction with DOM increased again after 5 days in the irradiated system (Figure 2a), and interestingly enough, reached an concentration of TNT* bound to POM and DOM similar to the nonirradiated system after 28 days, demonstrates that the irradiated systems were not sterile and that the biodegradation of TNT was quite active after 5 days. The sorption of nitrobenzene to SOM (the binding to POM and DOM was not separated in the kinetic study) approached a chemical equilibrium already after 1 h (Figure 3a). As opposed to TNT, the insignificant effect of irradiation indicates that biotic degradation did not play any significant role for the adsorption of nitrobenzene to SOM in our kinetic experiment. Borisover and Graber (27) demonstrated that nitrobenzene showed no hydration-assisted sorption to NOM, strongly indicative of hydrophobic partitioning. The fast reaction kinetics between nitrobenzene and SOM gives additional support for the interpretation that the fast reaction 3078

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between TNT* and POM mainly is due to hydrophobic partitioning. The kinetics of the reaction between aniline and SOM was quite slow, and even after 3 days there was a small, but steady, increase in aniline adsorption to SOM with time (Figure 3b). Weber and co-workers (28) described a similar slow time-dependent reaction between aniline (5 µM) and aquatic humic substances (250 mg C L-1) by a simple secondorder rate expression. Similar to our results, approximately 75% of aniline was associated with humic substances after 7 days. The authors explained the slow kinetics by a transformation from an electrostatic interaction to successively more of covalent bonding, further demonstrated using 15N NMR spectroscopy (7). Functionalities in SOM likely involved in covalent bonds with amino groups are carbonyl moieties such as quinones and ketones (7). Equilibrium Experiments: Distribution of TNT, Nitrobenzene, and Aniline between POM and DOM. The mixed model (eq 4) gave a better fit to TNT* binding data than separate linear (eq 2) and Langmuir isotherms (eq 3) (Figure 4). Data from the Na system showed a linear contribution (KOC) (i.e., hydrophobic partitioning) of TNT* that was approximately three times greater in POM (KPOC, Table 2) than in DOM (KDOC), whereas the binding strength (KL) and the specific binding capacity (qmax) (i.e., the density of functional groups reactive with TNT derivatives) were approximately 1.5 times greater in DOM than in POM (Table 2). Thus, in agreement with the interpretations from the kinetic studies, specific (electrostatic and covalent) interactions with TNT derivatives seems to be the predominate mechanism for the binding to DOM, and hydrophobic partitioning of TNT* is indicated to be the major mechanism for the association to POM. In comparison with the Na system, KL modeled by eq 4 was greater, and qmax was similar in size or smaller, for both DOM and POM, when Al was the main adsorbed metal cation (Al system). This difference is quite interesting and may be explained by a mechanism where Al cations are strongly bound to (blocking) oxygen (carboxyl) functional groups, minimizing weak electrostatic interactions between negatively charged groups and positively charged TNT* (amino) derivatives. Thus, TNT* in the Al system may be associated to a small number of stronger sites, likely via stronger electrostatic and covalent bonds.

TABLE 2. Isotherm Parameters for the Association of Organic Compounds to DOM and POM DOM substance TNT* NB

Aniline

ion/pH/ DOC Na/5.0/560 Na/5.0/560a Al/4.9/540 Na/5.1/500 Na/5.1/500 Al/5.1/590 Al/5.1/590 Al/4.9/570 Al/4.9/570

KL (nM-1) 99d

qmax (µmol g-1 C) 3.3d

110d 330d 2 × 10-3 d

3.3d 2.4d 4 × 10-7 d

3 × 10-3 d

1 × 10-7 d

22d 19c

143d 160c

POM

KDOC (L kg-1 C)

r2

KL (nM-1)

41d

0.980d

52d

2.8d

148d

44d 68d 34d 34b 38d 38b 29d

0.983d 0.965d 0.969d 0.969b 0.962d 0.962b 0.965d 0.965c

80d 150d 4.8d

2.1d 2.2d 7.9d

19d

2.0d

146d 187d 62d 73b 64d 69b 31d

23d 19c

qmax (µmol g -1 C)

104d 124c

KPOC (L kg-1 C)

r2 0.998d 0.998d 0.995d 0.999d 0.989b 0.998d 0.993b 0.964d 0.964c

a TNT* bound to DOM was determined by difference of 14C in supernatant and free TNT determined by HPLC. b Linear model (eq 2). c Langmuir model (eq 3). d Langmuir + linear model (eq 4).

146-187 L kg C-1) and DOM (KDOC ) 41-68 L kg C-1) was approximately 3. This suggests a difference in the density of hydrophobic moieties between POM and DOM that is qualitatively reflected by the (O + N)/C ratio but not fully accounted for by XPS (C-C + C-H) or NMR (aryl-C + alkylC) data (Table 1). Aromatic C, being more abundant in DOM than in POM (Table 1), apparently was less important than aliphatic moieties for the hydrophobic partitioning of TNT* and nitrobenzene. Our results may in addition be explained by the fact that the volume of hydrophobic moieties in general increases with size of NOM macromolecules (29), thus most likely being greater in POM than in DOM. It was recently reported that humic acid molecules larger than 100 000 Da have a more aliphatic character as compared to molecules smaller than 30 000 Da (30).

FIGURE 5. Adsorption isotherms for DOM and POM. (a) Nitrobenzene Na system: [DOM] ) 500 mg (C) L -1, [POM] ) 8.5 g (C) L-1, Naads) 600 mmolc kg-1 (POM), Alads< 10 mmolc kg-1 (POM), and pH ) 5.1. Al system: [DOM] ) 590 mg (C) L-1, [POM] ) 9.6 g (C) L-1, Naads) 80 mmolc kg-1 (POM), Alads) 240 mmolc kg-1 (POM), and pH ) 5.1. Solid lines represent the fit of eq 2 (Table 2). (b) Aniline Al system: [DOM] ) 570 mg (C) L-1, [POM] ) 9.5 g (C) L-1, Naads ) 340 mmolc kg-1 (POM), Alads ) 210 mmolc kg-1 (POM), and pH ) 4.9. Solid lines represent the fit of eq 3 (Table 2). Nitrobenzene, with its less reactive nitro group, interacts with POM and DOM almost exclusively through a hydrophobic partitioning mechanism, best described by the linear isotherm (eq 2 and Figure 5a). The mixed model (eq 4) did not improve the fit. The KOC values reported in Table 2 suggest that the hydrophobic partitioning of nitrobenzene is approximately twice as great per organic C atom in POM (KPOC ) 69-73 L kg C-1), as compared to DOM (KDOC ) 34-38 L kg C-1). For TNT*, the ratio of KOC between POM (KPOC )

In contrast to nitrobenzene, aniline with its highly reactive amino group is well-known to form specific bonds with carbonyl functionalities. These interactions were best described by nonlinear equation such as the Langmuir (eq 3 and Figure 5b). Unfortunately, some data for the Na system were subjected to errors and are therefore not shown. On the basis of the Langmuir constant for the Al system (eq 4 and Table 2), the binding strength of aniline to POM and DOM was quite similar. The mixed model (eq 4) gave a qmax of 104 and 143 µmol g C-1 for sites involved in the specific bonding of aniline to POM and DOM, respectively. For TNT*, the corresponding values were 2.1 and 3.3 µmol g C-1 (Na + Al systems). Thus, the quotient qmax(DOM)/qmax(POM) was 1.4 for aniline and 1.3 for TNT*, suggesting 1.3-1.4 times higher density of sites reactive toward aniline and TNT derivatives in DOM, as compared to POM. This can be compared with XPS and NMR data showing insignificant (within analysis errors) differences in the density of carbonyl groups (NMR) and carbonyl + amides (XPS) between DOM and POM (Table 1). However, if carboxyl (and amide) groups are included as potential (electrostatic) binding sites, the 1.3-1.4 times higher density of reactive sites in DOM is in fair agreement with NMR data (quotient 1.4, shift 160-220 ppm, Table 1), whereas XPS data suggests an even greater density in favor of DOM (quotient 2.1, BE 287-290 eV). If it is assumed that the reaction of TNT derivatives with POM and DOM functional groups can be approximated by a reaction similar to the one between aniline amino groups and carbonyls, the concentration of reactive TNT derivatives can be calculated by applying a model combining KOC for TNT* and aniline KL and qmax parameters (from eq 4 and Table 2) to TNT* adsorption data. Recent 15N NMR spectroscopy studies showing that TNT amines and their isomers (ADNT, DANT, and TAT) undergo nucleophilic addition with ketone and quinone groups similar to aniline (5, 6) may be taken as support for such an approach. With such a model, VOL. 38, NO. 11, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the adsorption of TNT* to POM could be satisfactorily fitted with 7.1% of TNT* represented by amino derivatives, whereas the marked nonlinear shape of the TNT*-DOM isotherm (Figure 4), suggesting a small concentration of very strong sites, was not possible to fit with this model. Apparently, additional reactions involving even higher energy sites than the aniline-carbonyl reaction need to be considered to explain the adsorption of TNT* to DOM. The very complex composition of TNT derivatives occurring in solution already after 22 h cf. ref 25 likely give rise to an array of different specific interactions with POM and DOM functional groups (e.g., ref 11), each of which need to be described by a specific (Langmuir) equation. At this point, lack of definite information on which types of functional groups TNT derivatives interact with, such a complex model, cannot be justified. Our results, showing the ability of newly formed TNT derivatives to form specific bonds with DOM functional groups, suggest that soils with continuous production and leaching of DOM may increase the mobility of TNT*. This may be most relevant in organic rich top soils and discharge areas. Most field studies of TNT mobility have, however, been conducted in mineral soils with maximum 3% SOM and often with clay contents >10%. (e.g., ref 31). If, in these soils, TNT is extensively bound to 2.1 clays, and conditions promote biotic or abiotic reductive degradation, even small concentrations of mobile DOM in the long-term may result in a significant transport of TNT derivatives to ground and surface waters. Currently, there is a lack of studies in which the role of DOM-POM in soils with 2:1 clays have been investigated under naturally relevant conditions.

Acknowledgments We are grateful to Torbjo¨rn Karlsson for his technical assistance and to Heikki Knicker, Technische Universita¨t Mu ¨nchen, for help with the 13C NMR analyses. Swedish Armed Forces financially supported this project, and we are grateful for the help and support supplied by the personnel at the Swedish Defence Research Agency (FOI). The salary of S.F. was funded by North Sweden Soil Remediation Centre (MCN), EC Project 113-12534-00.

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Received for review September 15, 2003. Revised manuscript received December 23, 2003. Accepted March 24, 2004. ES035015M