Environ. Sci. Technol. 2009, 43, 6632–6638
Cation Binding of Antimicrobial Sulfathiazole to Leonardite Humic Acid M E R L E K . R I C H T E R , †,‡ M I C H A E L S A N D E R , * ,‡ M A R T I N K R A U S S , * ,† ISO CHRISTL,‡ MANUEL G. DAHINDEN,† MANUEL K. SCHNEIDER,§ AND ´ P. SCHWARZENBACH‡ RENE Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Duebendorf, Switzerland, Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, 8092 Zurich, Switzerland, and Agroscope Reckenholz-Ta¨nikon Research Station ART, 8046 Zurich, Switzerland
Received March 30, 2009. Revised manuscript received June 11, 2009. Accepted July 2, 2009.
Sorption of sulfathiazole (STA) and three structural analogs to Leonardite humic acid (LHA) was investigated in single- and binary-solute systems to elucidate the sorption mechanism of sulfonamides to soil organic matter (SOM). Cation binding of STA+ to anionic sites A- in LHA governed sorption up to circumneutral pH, based on the following findings: (i) From pH 7.7 to 3.3, the increase in extent and nonlinearity (i.e., concentration dependence) of STA sorption paralleled the increase in STA+. (ii) From pH 3.3 to 1.7, sorption decreased and nonlinearity increased, consistent with strong competition of STA+ and H+ for A-. (iii) Replacement of the protonable aniline group in STA by an apolar methylbenzene group resulted in much weaker, linear, and pH-independent sorption. (iv) Only analogs with aniline moieties displaced STA from LHA in binary-solute systems. Displacement occurred up to pH 5.4, at which 4. Each system consisted of two 4 mL half-cells separated by a dialysis membrane (Spectra/Por Biotech, Cellulose ester, 500 MWCO, Socochim, Lausanne, Switzerland), which was impermeable to LHA but permeable to the sorbates. Dialysis systems were loaded by pipetting 3.5 mL aliquots of the LHA stocks to one-half-cell and solutions containing one or two sorbates to the other half-cell. Cells were shaken horizontally (175 rpm) for 20 days at 19 °C in the dark, during which equilibrium was attained (see control experiments in SI, Section E). The LHA-free half-cells were subsequently analyzed for total solute aqueous concentrations C∑species (mol L-1), which was the sum of aqueous concentration of all species. Single solute STA experiments at pH 1.7 and 2.5, and STA-APBS competition experiments (see below) were carried out in 20 mL brown glass vials. At these pH values, the major fraction of added LHA remained particulate, such that C∑species could be determined in the supernatant after high-speed centrifugation (13 000 rpm, 30 min). Total sorbed concentrations of all sorbate species, Q ∑species (mol kg-1), were determined via mass balance on the initial and equilibrium C∑species and corrected for minor sorbate losses (SI, Section E). Separate control experiments were run to establish accuracy in the transfer of LHA stocks, impermeability of the membrane for LHA, attainment of equilibration in the dialysis systems in 20 days, sorbate losses to the membrane, and pH stability (results in SI, Section E). Furthermore, consistent results were obtained from selected sorption experiments that were run in both dialysis systems and glass vials. Complete mass recoveries of STA and MTBS were determined in separate controls by pressurized liquid extraction of LHA after equilibration according to ref 24 (SI, Section E). Analytical Procedure. Sorbates were quantified by liquid chromatography-tandem mass spectrometry using an autosampler (HTC PAL, CTC Analytics, Zwingen, Switzerland), Rheos 2000 LC pumps (Flux Instruments, Basel, Switzerland), a column oven at 30 °C (Jones, Omnilab, Mettmenstetten, Switzerland), and a triple quadrupole mass spectrometer with an ESI probe (TSQ Quantum, Thermo Finnigan, San Jose, VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. a. Sorption isotherms of sulfathiazole (STA), and its analogs 4-ethoxyaniline (EXA), 4-amino-N-(phenyl)-benzenesulfonamide (APBS), and 4-methyl-N-(2-thiazolyl)-benzene-sulfonamide (MTBS) to Leonardite humic acid at all experimental pH. Q ∑species (mol kg-1) and C∑species (mol L-1) are the total molar sorbed and aqueous concentrations including all sorbate species, respectively. Dashed lines on EXA, APBS, and MTBS data represent Freundlich model fits (parameters in Supporting Information Table S3, Section G). Solid lines on STA data at pH g 3.3 represent fits of the NICA-Donnan model (eq 2). b. Concentration and pH dependence of the apparent distribution coefficient KD ) Q ∑species · (C∑species)-1 (L kg-1) of STA. c. and d. STA sorption data at pH g 3.3 replotted from panel a as Q ∑species versus the calculated aqueous concentrations of the cationic and zwitterionic STA species, CSTA+and CSTA(, respectively. CA). All samples were adjusted to pH 4 with acetate buffer prior to analysis. Samples with analyte concentrations C∑species> 50 µg L-1 were directly injected into the column via a 20 µL sample loop. Samples with C∑species< 50 µg L-1 were preconcentrated by online enrichment (Oasis HLB extraction cartridges; Waters, Rupperswil, Switzerland) according to ref 25. Separation was achieved on a Nucleodur C18 Gravity column (Macherey-Nagel, Oensingen, Switzerland). The mass spectrometer was run in selected reaction monitoring (SRM) mode. Peak areas were integrated using Xcalibur software 2.0.7 (Thermo Finnigan, San Jose, CA). Further details on the analytical procedure are provided in the SI, Section F.
Results and Discussion Qualitative Analysis of STA Sorption. The single solute sorption isotherms of STA and its structural analogs MTBS, APBS, and EXA are shown in Figure 2a. All single-solute isotherms were fitted by the linearized Freundlich model log Q∑species ) log KF + N · log C∑species
(1)
where KF (mol1-N LN kg-1) is the Freundlich affinity constant and N the Freundlich exponent. The fitted Freundlich parameters, summarized in SI (Table S3, Section G), allow comparing the sorption of STA and its analogs. It is important to bear in mind that the fitted 6634
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Freundlich parameters do not carry any physical meaning with regard to the underlying sorption mechanism. The extent of STA sorption to LHA was strongly pHdependent (Figure 2a and b). Total sorption of STA increased as the pH decreased from pH 7.7 to 3.3, as shown by the shift of the STA sorption isotherms toward higher Q ∑species (Figure 2a) and the corresponding increase in the apparent distribution coefficients KD ) Q ∑species · (C∑species)-1 of STA at a given C∑species (Figure 2b; see also plot of KD versus pH in Figure S9 and Freundlich parameters in Table S3, both in SI, Section G). Conversely, decreasing the pH from 3.3 to 1.7 resulted in slightly decreasing total sorption at low STA concentrations of C∑species < ∼10-7 mol L-1, but in a significant decrease in total sorption at high STA concentrations C∑species> ∼10-7 mol L-1 (Figure 2b). At all experimental pHs, STA sorption isotherms were nonlinear (Figure 2a) with KD decreasing with increasing C∑species. The degree of sorption nonlinearity increased with decreasing pH: an increase in C∑species from ∼7.1 × 10-8 mol L-1 to ∼1.4 × 10-5 mol L-1 resulted in an 8-fold decrease in KD at pH 1.7 but only a 2.4-fold decrease at pH 7.7 (Figure 2b). Increasing nonlinearity with pH is also reflected by the gradual decrease in Freundlich exponents N from 0.85 ( 0.02 at pH 7.7 to 0.56 ( 0.02 at pH 1.7 (SI Table S3, Section G). The previously reported data for STA sorbing to LHA (11), which was collected over a narrower pH range
and at only one STA concentration, agree well with the data presented here. At pH 3.2, the sorption of APBS, which carries an apolar phenyl group as the N1 substituent instead of the H-bond accepting thiazolyl ring in STA, was indistinguishable from that of STA at pH 3.3 both in sorption affinity and nonlinearity (Figure 2a and Freundlich parameters in SI Table S3, Section G). This similarity implies that differences in the polarities of the N1 substituent in STA and APBS had no discernible effect on sorption. Furthermore, overlapping isotherms indicate that, at this pH, STA and APBS sorbed by the same mechanism and to identical sorption sites on LHA. In contrast, replacement of the aniline group in STA by the apolar methyl-benzene group in MTBS (see structure in Figure 1) had a pronounced effect on sorption. First, sorption of MTBS was much weaker than sorption of STA, even for pH 7.7 (Figure 2a). Second, MTBS exhibited close to linear sorption across the experimental pH range from pH 3.2 to 5.4 (Figure 2a and N ) 0.94 ( 0.02 in SI Table S3, Section G). Third, in contrast to STA, sorption of MTBS was pHindependent (overlapping isotherms of MTBS at pH 3.2, 5.0, and 5.4; Figure 2a). These differences imply that the sorption characteristics of STA (and APBS) were linked to its aniline moiety. Total sorption of EXA to LHA was much larger than that of STA at pH 5.4 (Figure 2), with 10-30-fold higher KD of EXA than STA depending on C∑species (SI Table S3, Section G). Sorption of EXA at pH 5.4 was highly nonlinear with N ) 0.55 ( 0.04 (SI Table S3, Section G). Notably, at pH 5.4, approximately 50% of the EXA molecules were cationic while less than 1% of the STA molecules in solution were cationic (Figure 1). The comparison of single solute sorption of STA and its analogs strongly suggests that sorption of STA to LHA was governed by an interaction mechanism that involved the aniline group in STA and sorption sites in LHA whose abundance decreased with decreasing pH. The following arguments show that this interaction mechanism must have been cation binding of STA+ to anionic sites A- on LHA. First, STA0 and MTBS0, the dominant species in solution between pH 2.5 and 7 (SI, Figure S10, Section G), have comparable molecular sizes. Consequently, sorption of these two species has similar free energy contributions from the formation of cavities in water and LHA phases to accommodate the sorbates and from van-der-Waals interactions between the sorbates and the phase molecules in LHA and water. In contrast, the free energy contributions from electron donor-acceptor interactions differ for STA0 and MTBS0. Whereas water molecules strongly H-bond with the aniline moiety in STA0, only weak H-bonds form between water and the methylbenzene group (weak H-bond acceptor) in MTBS0. Consequently, the sorption affinity of STA0 from water to an organic sorbent, such as LHA, is expected to be weaker or at most equal to that of MTBS0. However, STA exhibited higher overall sorption than MTBS for LHA at circumneutral pH. Therefore, sorption must have been dominated by STA+ (and potentially also STA(). Second, the decrease in sorption of STA from pH 3.3 to 7.7 paralleled the decrease in CSTA+ (SI Figure S10, Section G). In contrast, CSTA0 and CSTA( changed only slightly, whereas CSTA- increased from pH 3.3 to 7.7 (SI, Figure S10, Section G). The congruent trends in STA sorption and CSTA+ at pH g 3.3 strongly advocate that STA+ dominated sorption up to circumneutral pH. Species-specific sorption affinities therefore had to decrease in the order of STA+ . STA0 (and STA() . STA-. The STA sorption data from pH 7.7 to 3.3 in Figure 2a is replotted in Figure 2c as Q ∑STA species versus CSTA+, which was calculated from experimental C∑STA species. For a given Q ∑STA species, CSTA+ increased with increasing proton concentrations (i.e., decreasing pH) (Figure 2c). This demonstrates
that STA+ and H+ competed for the same sorption sites, A-, in LHA. The same pH-dependent spacing of isotherms was previously reported for sorption of inorganic cations to humic substances and was taken as evidence for cation binding to anionic sites A- (26). Conversely, plotting Q ∑STA species versus CSTA( resulted in closely spaced STA sorption isotherms that did not show a clear pH pattern (Figure 2d). This indicates that STA( did not sorb to LHA by cation binding. Strong competition between STA+ and H+ for A- also provides an explanation for the apex in KD of STA at approximately pH 3.3 (Figure 2b). From pH 3.3 to 1.7, successive protonation of A- reduced its availability for the binding of STA+ to a degree that total STA sorption decreased, particularly at high CSTA+ (i.e., highC∑species in Figure 2b). Third, the observed pH-dependent nonlinearity in STA sorption cannot be explained if partitioning of uncharged STA0 into LHA was assumed as the dominant sorption mechanism. The latter interaction mechanism typically results in linear and pH-independent sorption isotherms, as observed for the sorption of MTBS to LHA. Instead, the pHdependent sorption nonlinearity of STA to LHA resulted from the opposing pH dependence in the speciation of STA+ and LHA. Both competition of STA+ with H+ and self-competition of STA+ for sorption sites A- increased with decreasing pH, which resulted in the concomitant increase in isotherm nonlinearity. Competition was particularly strong at pH 2.5 and 1.7, resulting in very low Freundlich exponents N < 0.66 (SI, Table S3, Section G). Covalent bonding of STA to LHA played at most a minor role in sorption in the present study for the following reasons. First, the aniline group in STA is a relatively weak nucleophile due to the strong electron withdrawing effect of the sulfonamide moiety (high Hammett constant σp ) 0.60 for -SO2NH2 (27)). The kinetics of nucleophilic addition, therefore, should be slow, particularly when compared to more reactive aromatic amines that were previously investigated (17, 18). Second, while covalent bond formation between SA and humic acids has recently been demonstrated (20), these reactions only occurred to a detectable degree if sites in humic acid susceptible to nucleophilic attack were generated via oxidation by MnO2 or oxidase enzymes. No such oxidants were added in this study. Third, 97.0% ( 1.4% of added STA was recovered in control experiments at pH 5.8 by pressurized liquid extraction of LHA. Therefore, at most, 3% of the added STA molecules irreversibly coupled to LHA, which is much less than was sorbed at equilibrium (about 50%). A series of binary solute experiments were conducted to confirm the proposed cation binding mechanism. Competition between STA and APBS at pH 3.2 was investigated first to validate that these compounds sorbed by the same mechanism and to the same sites in LHA, as suggested by the overlapping single solute isotherms (Figure 2a). Figure 3a shows that the KD of STA to LHA successively decreased from its single-solute value (absence of APBS; open triangles) with increasing sorbed concentrations of APBS (final sorbed concentrations ratios Q ∑APBS species · (Q ∑STA species)-1 of 1, 3, 5, and 10. However, when considering sorption of both solutes STA and APBS to LHA by plotting KD ) (Q ∑APBS species + Q ∑STA species) · (C∑APBS species + C∑STA species)-1 versus C∑APBS species + C∑STA species, all binary data points (filled symbols, Figure 3a) fell onto the single-solute KD-C∑STA species relationship of STA. Furthermore, the single solute sorption isotherms of STA at pH 3.3 and of APBS at 3.2 overlapped when the total sorbed concentrations of STA and of APBS from Figure 2a were replotted versus CSTA+ and CAPBS+, respectively (SI, Figure S11, Section G). Therefore, STA and APBS sorbed by the same mechanism and affinities to the same sites in LHA. This suggests that SA with different N1 substituents, yet similar pKa values of their aniline moieties, show highly similar sorption characteristics to SOM. This finding is highly relevant VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Binary solute sorption experiments to Leonardite humic acid a. Apparent distribution coefficient KD (ratio of sorbed to solution concentration, Q ∑species · (C∑species)-1) of sulfathiazole (STA) at pH 3.2 in the absence (single solute; open triangles up) and in the presence of increasing sorbed concentrations of the cosolute 4-amino-N-(phenyl)-benzenesulfonamide (ABPS) (binary, open symbols). Full symbols are obtained when accounting for sorption of both sorbates. b. KD of STA sorption at pH 5.4 in the absence of cosolutes (open triangle down) and in the presence of cosolutes 4-methyl-N-(2-thiazolyl)-benzene-sulfonamide (MTBS) (open circle) and 4-ethoxyaniline (EXA) (open square). Small graph: KD of MTBS at pH 5.4 in absence of a competitor (open triangle up) and in presence of EXA (open diamond). The numbers in the figure legend correspond to the molar equilibrium sorbed concentrations of the target analyte to that of the respective cosolutes. to the fate modeling and risk assessment of SA in soils. This is discussed further in the Environmental Implications section. A second set of competition experiments using the cosolutes MTBS and EXA served to independently verify the predominance of cation binding of STA+ even at pH 5.4. Less than
