Silver Binding by Humic Acid as Determined by Equilibrium Ion

Article pubs.acs.org/JPCA

Silver Binding by Humic Acid as Determined by Equilibrium Ion-Exchange and Dialysis Zhongzhi Chen, Peter G. C. Campbell, and Claude Fortin* Institut national de la recherche scientifique, Centre Eau Terre Environnement, 490 de la Couronne, Québec, Canada, G1K 9A9 S Supporting Information *

ABSTRACT: One of the major challenges in environmental analytical chemistry is to develop methods for determining metal speciation in natural waters that contain low metal concentrations and dissolved organic matter (DOM). Because of its complex heterogeneous nature, metal binding to DOM cannot be predicted accurately using equilibrium models. Two independent speciation methods, the equilibrium ion-exchange technique (IET) and equilibrium dialysis (EqD), were used to determine silver binding by standard Suwannee River humic acid. Both approaches gave very similar results, although for a given silver loading, the concentration of free silver obtained by IET was somewhat higher than that determined by EqD. Our results suggest that any high-affinity binding sites present within the humic acid are likely saturated at [AgT] > 10−9 M. This comparison of free metal ion concentrations with two independent methods provides useful speciation information in the absence of reliable complexation constants for the reaction of silver with humic acid.

1. INTRODUCTION Silver has been used extensively in the 20th century for a plethora of purposes (coins, jewelry, dental alloys, electronics, photography, etc).1 More recently, the growing use of silver nanoparticles in consumer products has contributed to raising concerns about the ecological risk that silver poses in aquatic ecosystems.2,3 Moreover, silver nanoparticles are known to be unstable in aquatic environments, and their oxidative dissolution leads to the release of Ag+.4 It is generally accepted that to assess the bioavailability and toxicity of a trace metal in the aquatic environment, it is essential to consider the metal’s speciation.5 However, until recently, the chemical speciation of silver in natural waters has been largely overlooked, and few data are available in the literature. In natural waters, silver speciation can be strongly affected by simple inorganic ligands, such as chloride, thiosulfate, and reduced sulfur species, as well as by organic ligands such as simple thiols (e.g., cysteine) and natural dissolved organic matter (DOM). In principle, metal binding can be estimated by thermodynamic calculations, but the polymeric and heterogeneous nature of DOM results in metal-binding properties that cannot be described by a set of discrete binding constants. Ideally, metal speciation in natural waters should be determined © 2012 American Chemical Society

analytically, but the development of such methods remains a major challenge. Many existing analytical techniques are not specific for the free metal ion (e.g., diffusive gradients in thin films), suffer interference from the adsorption of surface-active organic matter (e.g., anodic stripping voltammetry), or are not sensitive enough (e.g., ion-selective electrodes).6,7 Here, we propose the use of a technique based on a column equilibration with an ion-exchange resin. This method involves passing a sufficient volume of test solution through a weakly binding cation exchange resin until equilibrium is reached between the solution and the resin. We previously demonstrated that this ion exchange technique (IET) could be used to determine free Cd, Cu, and Zn concentrations in lake waters.8 The technique has also been used to measure free Ni in complex solutions containing natural organic matter.9−11 In addition to the experimental measurements with the IET, free metal ion concentrations were compared with those determined by equilibrium dialysis (EqD) experiments. The Special Issue: Herman P. van Leeuwen Festschrift Received: December 22, 2011 Revised: February 29, 2012 Published: February 29, 2012 6532

dx.doi.org/10.1021/jp212403r | J. Phys. Chem. A 2012, 116, 6532−6539

The Journal of Physical Chemistry A

Article

come into contact with the resin, metal ions will bind to the resin until equilibrium is achieved. This reaction can be c described by the equilibrium constant KIE :

EqD technique was chosen to isolate silver bound to humic acid (HA) from the rest of the solution. This approach is convenient and inexpensive, it does not require the addition of any potentially binding reagents, and no specific equipment is required. This method has been successfully used in the past to determine the speciation of several metals in solutions containing humic acids.12,13 The purpose of this study was thus to determine and validate, by combining the results of two speciation methods applied to lab-defined solutions, the binding affinity of Suwannee River humic acid (SRHA) for Ag. Both speciation methods were optimized for determination of free silver. Recent comparisons among different speciation techniques have revealed large discrepancies in free metal ion concentrations for a common set of natural water solutions.14,15 Such discrepancies have highlighted the necessity to validate metal speciation techniques. Comparison of two or more independent methodologies is one approach to such validation. If the results obtained by the different methods agree, we can be increasingly confident that, whichever one we choose, it will give consistent and correct results.

c KIE

Mz + + z RNa ←→ ⎯ R zM + z Na+ c KIE =

(1)

[R zM][Na+]z [Mz +][RNa]z

(2)

where R = resin, RNa = resin binding sites occupied by Na+, RzM = resin binding sites occupied by metal Mz+, and z = the charge of metal M. A key characteristic of this approach is the choice of resin. Sulfonate functional groups have a very low affinity for metals such that, in the presence of a sufficiently high amount of strong electrolyte (e.g., NaNO3 or other background electrolyte), it is possible to minimize interactions between the resin and the Mz+ ions. Under such conditions, the concentrations of Na+ in solution and on the resin will not be significantly affected by the exchange of the trace metal ion (Mz+). Trace conditions are fulfilled when the metal Mz+ ions occupy a very small fraction of the total resin sites ([RNa+] ≫ [RZM]). Thus, at a fixed pH and ionic strength, eq 2 can be rearranged to yield a distribution coefficient (λo,i,pH expressed in L·g−1) describing the ratio of metal bound to the resin and the metal in its free form in solution:

2. EXPERIMENTAL METHODS 2.1. General: Reagents, Sample Preparation and Measurement. All plasticware was soaked in 15% HNO3 v/v for at least 24 h, thoroughly rinsed seven times with ultrapure water (18 MΩ·cm, Milli-Q3RO/Milli-Q2 system), and dried, when necessary, under a class 100 laminar flow hood under a positive pressure of filtered air prior to use. All exposure solutions were prefiltered (0.2-μm polycarbonate) to eliminate any fine particulate material from solution, to which silver could potentially adsorb. In addition, unless otherwise indicated, all experiments were performed at neutral pH (7.0 ± 0.1) and ambient temperature (22 °C) with a minimum of three replicates. A MOPS buffer (N-morpholino-3-propanesulfonic acid, Sigma-Aldrich; pKa = 7.20) was used at a concentration of 10 mM to maintain a constant pH throughout the experiments. Polycarbonate containers were used for silver solutions because preliminary tests showed that adsorptive losses of silver over time were minimized in these types of bottles. Salts used for experiments were of analytical grade or better. Radioactive silver (110mAg; 45 mCi·mmol−1) was purchased from Eckert & Ziegler Isotope Products (Atlanta, GA, USA). Acidic stock and intermediate solutions (0.2% HNO3 v/v) of nonradioactive and radioactive silver were kept at pH < 2 at 4 °C in the dark, to minimize adsorptive losses and potential photoreduction. All data presented were measured rather than nominal concentrations. A standard humic acid (Catalog no. 2S101H) isolated from the Suwannee River (Suwannee River Humic Acid Standard II, GA/FL, USA) was obtained from the International Humic Substances Society. Humic acid was wrapped in aluminum foil to prevent photo-oxidation and stored in a desiccator until use. Stock solutions of SRHA (∼250 mg·L−1) were prepared in a NaOH solution (0.01 M), left to agitate in the dark at room temperature for 24 h to ensure dissolution, and then filtered through 0.4-μm polycarbonate filter membranes to obtain the dissolved fraction. The filtered stock solution was used to prepare the SRHA experimental solution (final pH = 7.0). 2.2. IET Method. 2.2.1. Theory. The IET methodological details are given elsewhere.16 Briefly, this technique is based on the principles of chemical equilibrium and exchange between a metal, Mz+, present in solution and a resin. When the free ions (Mz+)

c λo, i,pH = KIE

[RNa]z

[R zM]

[Na ]

[Mz +]

+z =

(3)

The distribution coefficient for a given metal can be measured by passing solutions of a constant ionic composition and with known concentrations of the free metal of interest through the resin column. Note that eq 3 remains valid if Na is replaced by another counterion or a mixture of ions. It follows that a distribution coefficient can be obtained for any given combination of metal and solution matrix. The concentration of metal bound to the resin ([RzM]) can be measured by eluting the resin with a known volume (V) of eluant solution to dislodge the Mz+ ions bound to the functional groups of the resin. By measuring the metal concentration in the eluate (Meluate) and the exact quantity of the resin (mr), [RzM] can be calculated using the following equation: [R zM] =

[Meluate] × V mr

(4)

Finally, combining and rearranging the last two equations, the relationship between the concentration of metal bound to the resin and the free metal ion concentration in solution is obtained: [Mz +] =

[Meluate] × V λo,i,pH × mr

(5)

Once a distribution coefficient specific to the metal of interest has been determined, for the relevant concentration and nature of the electrolyte solution and a given pH, [Mz+] can be determined in samples of identical matrixes. 2.2.2. Experimental Setup. The experimental setup [see Fortin and Campbell16 for a more detailed description] consists of a peristaltic pump (Minipuls3, Gilson) and a six-position rotary valve carrying the required solutions to the resin column 6533



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chloride and thiosulfate are well-defined, and consensus values for the complexation constants are available.20 We tested various concentrations of chloride (0.5, 1, 2, and 3 mM) added to the medium using stock solutions made from ultrapure KCl salts (EM Science). With the increase in chloride ions in the medium, the concentration of the free Ag+ ion decreased, and that of the chloro complex AgCl0 increased. The ionic strength of the solution was kept constant by decreasing the concentration of NO 3 − ion gradually as the chloride concentration was increased. Thiosulfate concentrations tested were 50, 100, 150, and 200 nM. Thiosulfate stock solutions were prepared monthly from Na2S2O3·5H2O salts (EM Science) using autoclaved ultrapure water, kept in the dark at 4 °C. 2.2.5. Titration of DOM. To determine the percentage of free silver present in solution after the addition of 10 mg·L−1 humic acid (yielding values of ∼5 mg C·L−1 dissolved organic carbon; determined by total organic carbon analysis; Shimadzu TOC-5000A), a titration was performed (1 to 300 nM AgT). The solutions were allowed to equilibrate for 24 h in the dark with constant stirring at ∼30 rpm at room temperature (22 °C) before their passage through the column. The pH was adjusted to 7.0 after addition of silver and verified before measuring free Ag+ ion. In our earlier work with the IET, we found no evidence of DOM fouling of the ion-exchange resin.16 2.2.6. Influence of Experimental Conditions on Ag Binding by DOM. The evolution of the free silver concentration was monitored over 96 h at constant total nominal silver concentration ([AgT] = 200 nM). In addition, an experiment was also carried out to examine the potential effects of MOPS (10 mM), light, and temperature on the complexation kinetics. Three different sets of three flasks each containing 300 mL of MHSM medium in the presence of DOM and silver were prepared. Each series was kept under different conditions: one at 20 °C exposed to light (100 μmol·m−2·s−1), one at 20 °C in the dark, and the other at 4 °C in the dark. The concentration of free metal was measured in all cases at time 0 and 96 h. To test the effect of pH buffer, another set of four solutions was prepared containing Ag, Ag + DOM, Ag + MOPS, and Ag + DOM + MOPS, again at a nominal Ag concentration of 200 nM. Finally, another set of experiments was designed to verify if the Ag-binding properties of DOM changed with time (72 h) after preparation of the 5 mg C·L−1 DOM solution; solutions were kept at 20 °C in the dark. By comparing the results obtained, we were able to determine if light, temperature, pH buffer, or DOM incubation time would have an effect on the Ag−DOM complex formation. 2.3. Equilibrium Dialysis Method. Dialysis bags with a nominal molecular mass cutoff of 500 Da (Spectra/Por CE, Spectrum) were used to evaluate the extent of silver binding to high molecular weight humic acid. The dialysis bags were stored at 4 °C in a 0.05% sodium azide solution and were prepared by soaking in deionized water for 15 min at room temperature followed by rinsing thoroughly with deionized water. Powder-free gloves were worn when handling the dialysis tubing. Each bag was initially filled with matrix solution (∼1.5 mL) containing SRHA (5 mg C·L−1) with or without Ag (200 nM). For the first experiment, no Ag was added to the dialysis bag (only in the outer solution). For the following experiments, Ag was added both inside and outside the bag. The dialysis bags were introduced into a polypropylene flask containing 950 mL of a stirred solution with the same matrix solution composition, but without any humic acid. On one hand, free Ag+ and other

(Dowex 50W-X8, 50−100 mesh, sulfonic acid type, Sigma). Tubes and connections were made of polytetrafluoroethylene, with the exception of the pump tubing (Tygon, Technicon green−green). Solutions were kept in a custom-built acrylic box to protect them from contamination by ambient dust. 2.2.3. Methodology of the IET. The volume needed to reach equilibrium between the metal ion in solution and the resin was determined using several solutions (n = 3) with known concentrations of Ag (300 nM) at pH 7.0 (ionic strength was fixed at 20 meq·L−1 by adding different volumes of 1 M NaNO3; see below), and 5 mL subsamples of the outflow were taken after each 20 mL interval, up to a total effluent volume of 320 mL. The following sequence was systematically performed in each of the IET experiments using a flow rate of 5 mL·min−1 unless otherwise mentioned: (1) 2-min rinse of the resin using 1.5 M HNO3 (suprapure, J. T. Baker); (2) rinse with ultrapure water for 4 min; (3) conversion of the resin to sodium (Na) by passing a solution of NaOH (1 M) for 4 min; (4) another 2 min rinse in ultrapure water; (5) pre-equilibration with an electrolyte solution of the same pH and composition as the sample to analyze, without Ag and SRHA (15 min); (6) equilibration of the sample with the resin; (7) ultrapure water was then passed rapidly (0.5 min) to dislodge any sample solution entrapped in the interstitial spaces that could contribute to the Ag content of the eluate; and (8) an air pulse was applied to flush the solution from the column. After that, the thiosulfate eluant solution (200 μM; NaS2O3·5H2O, EM Science; prepared monthly and kept in the dark at 4 °C) was passed for 16 min at 0.5 mL·min−1 to strip the Ag from the column. All samples were collected in polystyrene containers before being placed in scintillation vials for γ counting (Wizard2, PerkinElmer) or analysis by ICP-AES (AtomScan 25 spectrophotometer, Thermo Jarrell Ash) in a 0.2% HNO3 matrix. For radioactivity measurement, counts per minute (cpm) were converted into Ag molar concentrations, taking into account the detector efficiency, radioactive decay, and the 110m Ag specific activity. The eluate vials were weighed to allow for the precise determination of the elution volumes and then stored at 4 °C until analysis of the metal concentrations. For each assay performed with the IET, a reference solution of known Ag concentration and containing no ligands was passed through the column before or after the sample solution to ensure that the distribution coefficient, λ0, was constant. The free ion concentration present in each sample was then calculated using eq 5. The distribution coefficients were determined using several solutions (n ≥ 3) of known Ag concentration (30 to 300 nM) at pH 7.0. The solution ionic composition used for all the Ag-binding experiments was based on the modified high salt medium (MHSM; [Na+] = 1.46 × 10−2 M; [K+] = 4.00 × 10−3 M; [NH4+] = 9.37 × 10−4 M; [Mg2+] = 8.12 × 10−5 M; [Ca2+] = 6.80 × 10−5 M; [NO3−] = 1.96 × 10−2 M; [SO42−] = 8.12 × 10−5 M; ionic strength = 20 meq·L−1). This simplified algal growth medium has been used for Ag bioavailability and toxicity tests in the past.5,17,18 2.2.4. Validation of the IET with Chloride and Thiosulfate as Ligands. To assess the selectivity of the IET for free silver, the calculated concentrations of free Ag+ using the chemical equilibrium program MINEQL+19 were compared with those measured in the laboratory with different concentrations of chloride or thiosulfate at a single nominal silver concentration of 300 nM. The inorganic complexation reactions of silver with 6534



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matrix (200 meq·L−1) showed that only 20 mL was required for the same quantity of resin.16 At lower ionic strength, more metal is being bound to the resin, thus increasing the volume needed to reach equilibrium. More recent work at lower ionic strength (10 meq·L−1) showed that ∼150 mL was required to reach equilibrium.8 Our current results are thus coherent with previous ones. The solution matrix used here was selected to mimic both an algal growth medium and natural freshwaters. Indeed, the Ca and Mg concentrations used are within the range of concentrations expected in natural waters. 3.1.2. Distribution Coefficient. Before we could measure the concentration of free silver in solution, we first had to determine the distribution coefficient (λ0; eq 3) for silver in the MHSM solution matrix. Thus, several different concentrations of total silver (tested in triplicate) were passed through the ion-exchange resin column to determine this coefficient for Ag and to verify whether the value of λ0 varied as a function of [Ag+]. Standard solutions tested were adjusted to pH 7.0, buffered with 10 mM MOPS. Results showed that the coefficient decreased slightly with increasing concentrations of Ag+, up to ∼100 nM; above this concentration, λ0 was stable (Table 1). As a precaution, from then on for each assay, a

ions as well as molecules with a molecular weight lower than 500 Da equilibrate across the dialysis tubing membrane such that the outer solution is identical to the inner solution with respect to these solution components. On the other hand, molecules with a molecular weight greater than 500 Da are retained inside the dialysis tubing. At equilibrium, the activities of all ions and complexes of molecular weight less than 500 Da are identical in the inner and outer solutions. The contribution of low-molecular-weight organic ligands (100 nM; Figure 2), presumably when all adsorption sites were saturated. We obtained similar results when we replaced the Dowex 50W-X8 resin by Dowex optipore (L494, polymeric adsorbent, Dow Chemicals), a resin sharing the same divinylbenzene backbone as the 50W-X8 resin but

Figure 1. Variation in the effluent silver concentration as a function of the volume passed through the ion-exchange column. The horizontal line corresponds to the influent Ag+ concentration. The vertical dotted line corresponds to the volume needed to achieve equilibrium between Ag+ in the MHSM solution and on the resin. 6535



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using MINEQL+. Second, thiosulfate was used as the model binding ligand with concentrations of 50, 100, 150, and 200 nM in the presence of 300 nM AgT (Figure 3B). Measured and calculated values using thiosulfate as a ligand were closer to each other than what was observed for chloride. This time, measured values were slightly lower than predicted ones except at the highest thiosulfate concentration. Predicted values of percent Ag+ were within experimental error of the measured values for three of the four thiosulfate concentrations tested. The slight difference between the measured and calculated free Ag+ concentrations in the presence of chloride could possibly be caused by the formation of a ternary complex on the resin, that is, resin−Ag−Cl. Note that the negatively charged AgCl2− complex is not likely to interact with the anionic resin because of electrostatic repulsion. Assuming that the thermodynamic calculations are correct, our observations suggest that chloro complexes may slightly interfere with the Ag+ measurement. The chloride concentration is, however, very low in our humic acid solutions (≤6 μM), resulting in a proportion of chloro complexes that can be considered negligible (∼1% of all species present). It follows that this potential interference will not influence the measurement of Ag+ in our study of Ag-SRHA interactions. Overall, the IET responds in the predicted manner to changes in the speciation of silver in the presence of complexing ligands. 3.1.4. Evaluation of Ag+ Concentration in the Presence of DOM. Using the IET, we measured the concentration of free Ag+ in solutions containing 5 mg C·L−1 HA and compared these results with those obtained in the absence of ligands. Different concentrations of total silver (1−300 nM) were used, resulting in a silver titration of HA at pH 7.0 (Figure 4). Under 7 nM AgT, no free Ag+ was detected, whereas above this concentration, the Ag+ concentration in solution increased linearly, with an average free silver ion proportion of 61 ± 12%. A small nonzero y-intercept was determined ([Ag+] = 0.71 × [AgT] − 4.5; P = 0.05), which was significantly different from zero. Humic acid from the Suwannee River, when present at a concentration of 5 mg C·L−1, complexes almost all the silver in solution until the total concentration of Ag reaches 6.3 ± 3.1 nM. This result indicates the presence of two types of complexation sites:21 the sites with the highest affinity for Ag would be filled first, and then the weaker sites would be occupied subsequently as the total Ag concentration increases. On the basis of the titration curve, the concentration of strong binding sites was 1.3 ± 0.6 nmol Ag·mg C−1 (determined with

Figure 2. Relationship between the concentration of free Ag+ and the amount of Ag in the eluate without resin (●) or with Dowex optipore resin (○). Silver in the eluate was expressed as a mass (μg) rather than as a concentration to avoid the variability caused by differences in the volume of the eluate from one experiment to another. Error bars represent standard deviations of the average of three measurements.

without the sulfonate functional groups, albeit with a higher apparent number of binding sites (Figure 2). These results indicate that the increase in the distribution coefficient observed when Ag+ concentration decreases is likely due to adsorption of silver ions to the tubing. At relatively high Ag+ concentrations, this adsorption is insignificant compared with the silver bound to the resin, but it becomes important at concentrations below ∼100 nM Ag+. This problem could be avoided by transferring the ion-exchange resin column to a “clean” system for the elution step. 3.1.3. Selectivity of the Resin. To test the selectivity of the IET method to the free Ag+ ions, we used different concentrations of chloride and thiosulfate to decrease the proportion of free Ag+ in solution. Measured free Ag+ concentrations were then compared with those predicted using the chemical equilibrium program MINEQL+. First, chloride levels of 0.5, 1, 2, and 3 mM were tested with a silver concentration of 276 ± 17 nM (Figure 3A). The results showed, as expected, a gradual decrease in Ag+ concentration as the chloride concentration increased, forming chloro complexes such as AgCl0 (aq) and AgCl2−. However, the measured values for Ag+ consistently exceeded the Ag+ concentrations calculated

Figure 3. Comparison of the proportion of free silver measured by the IET (black) with the value predicted by MINEQL+ (blank) with different concentrations of chloride (A) and thiosulfate (B). Error bars represent standard deviations of the average of three measurements at [Ag]nominal = 300 nM. 6536



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Figure 4. Titration curve of Suwannee River humic acid (5 mg C·L−1) with silver at pH 7.0 (n = 3).

Figure 5. Measured free Ag+ over time under different conditions at [Ag]nominal = 200 nM with 5 mg C·L−1 (●); for Ag added after 72 h preincubation of DOM solution (○); for Ag added at 4 °C (▼), with light (Δ), and without MOPS (■).

the linear regression equation when y = 0 and normalized for SRHA concentration). Beyond this initial complexation capacity, the concentration of free Ag+ increased more or less linearly as the dissolved total Ag concentration increased up to the highest concentration tested (300 nM). No sign of saturation of these weaker affinity sites was thus detected, and a conditional binding constant of 104.15 (spanning the range of 3.79−4.35 when including the standard deviation) was determined by assuming a binding site density of 10 μeq·mg C−1.22,23 3.1.5. Influence of Experimental Conditions on Ag Binding by DOM. Silver ions are known for their inherent fast rate of water exchange reactions (k−w = 1012 s−1).24 Solution equilibrium would thus be expected to be achieved rapidly. To confirm this assumption, we followed the speciation of silver over time in HA solutions ([AgT] = 200 nM; 5 mg C·L−1) at different temperatures as well as in the presence or absence of a pH buffer. We also tested whether the binding of silver changed after 72 h incubation with 5 mg C·L−1. We hypothesized here that the conformation of humic acid molecules may change with pH and ionic strength.25 Since our stock solution matrix of SRHA is drastically different from that in our experimental solutions (250 mg C·L−1 SRHA stock solutions are prepared in 0.01 M NaOH), we tested whether a change in silver binding occurred over time after dilution of SRHA from the stock solution. Our results showed that the concentration of free metal measured did not vary appreciably over time or with temperature (t test, P > 0.1; Figure 5), suggesting that equilibrium is reached in less than 1 day. Only exposure to light appreciably changed the speciation of Ag with an apparent decrease in the proportion of free Ag+ after 96 h to a final value of 50 ± 3%. For the same conditions, but in the dark, the final proportion of free Ag+ was ∼72%. This effect of light was not anticipated, and we can only speculate that perhaps photoreduction of Ag+ occurred, as suggested by Adams and Kramer,26 leading to an apparent increase in silver complexation, since the IET measures only free Ag+ (bound Ag−HA fraction is calculated by difference). The presence or absence of MOPS did not have an effect on silver speciation in the presence of SRHA (Figure 5). The hydrogen ion buffer MOPS was chosen, since it was reported to be noncomplexing for metals.27−30 Moreover, in the absence of

SRHA and MOPS, the recovery of Ag+ in solution was 113 ± 10%. We thus conclude that the addition of MOPS under our conditions did not alter silver speciation. 3.2. Equilibrium Dialysis. 3.2.1. Determination of Equilibrium Time. Similar to other studies, we used equilibrium dialysis to differentiate between (small) inorganic metal species and metal bound to humic acid.13,31 This distinction was obtained by difference between the outer solution (inorganic Ag species) and the inner solution (both inorganic and organic Ag species). When using dialysis, the time required to reach equilibrium between the two solutions depends on the surfaceto-volume ratio of the dialysis tubing as well as the stirring rate and the temperature.12 All these parameters were kept constant during and between the experiments. Preliminary experiments indicated that silver approached equilibrium in less than 72 h. A decrease in total Ag concentrations outside the tubing with time was observed, presumably due to adsorption to the container walls; total losses outside the tubing never exceeded 20%. The time required to reach equilibrium did not vary significantly in the presence or absence of DOM in the tubing. After 3 days, no significant further decrease in concentration of Ag in the outer solution was observed (the recovery of Ag in outer solutions between days 3 and 5 was 96 ± 6%), and this equilibration time was chosen for subsequent measurements. 3.2.2. Evaluation of Ag-DOM complex. Equilibrium dialysis results were obtained from two independent experiments (Figure 6). The EqD experiments showed that 48 ± 10% of the Ag was present in complexed form when the concentration of Ag outside the tubing was between 20 and 200 nM. The percentage of free Ag+ at a given time (as plotted in Figure 6) was calculated from the concentration inside and outside the tubing at the sampling time; this approach compensated for any losses through adsorption. A linear relationship between the concentration of total Ag and free Ag+ was observed ([Ag+] = 0.72 × [AgT] − 22.7, R2 = 0.94). The corresponding Ag binding capacity of high affinity sites was 6.3 ± 2.4 nmol Ag·mg C−1. 3.3. Comparison of IET and EqD. The analytical determination of Ag+ in aqueous solution is still challenging, although a few approaches have been used.26,32−38 The two speciation methods used in this study yielded consistent results 6537



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donor groups (log KAg−N = 3−6; log KAg−O < 2, respectively).39 Xia et al.40 reported that aquatic humic substances may contain an appreciable sulfur content, with 55% of the total S in SRHA present in reduced form (e.g., thiol groups). Even though these reduced sulfur functional groups are present at relatively low concentrations compared with O and C, they may play a disproportionate role in complexing soft trace metals present at trace concentrations.21 Several recent studies have suggested that reduced sulfur groups are involved in the binding of Ag to natural DOM. Schafer et al.41 studied the removal of Ag in water treatment plants and its fate in the receiving waters downstream. Their results showed a strong linear relationship between effluent DOC and the fraction of “dissolved” Ag with a large proportion of the Ag being associated with colloidal particles (

Silver Binding by Humic Acid as Determined by Equilibrium Ion

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