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Avi Aggarwal

silver concentration10. In this work we used GSH, a low molecular weight thiol-containing peptide produced by organisms in response to oxidative stress, to model the fate and transport of AgNP in thiol rich environments through the central processes of dissolution, aggregation, and li-gand surface sorption. Examples of thiol-rich environ-ments include the anaerobic pore water of sediments11, contaminated marine waters containing phytochelatins (product of plants response to increased metal concentra-tions12), inside of cells (where concentrations can reach up to hundreds of millimolars13), and blood14.

Nanoparticle suspensions are typically manufactured with a stabilizing coating to prevent flocculation of par-ticles. This coating can be steric, which involves polymers adsorbed to the surface to prevent contact, or electrostat-ic, such as citrate, which creates a barrier of counter ions around the particle surface and causes electrostatic repul-sion between particles. However, in natural environments, particles come in contact with organic ligands that can adsorb to particle surfaces and replace the coating. This sorption, along with changes in ionic strength in natural waters, can alter colloidal stability and lead to more ag-gregation, which in turn could decrease exposed NP sur-face area and slow dissolution. Conversely, because of their high affinities, the ligands may attach to and remove ions at the NP surface, inducing surface-modified dissolution (See Figure 1.) These inter-related processes occur simul-taneously. The goal of this research was to determine the mechanisms of the processes taking place and how they are related.

IntroductionSilver nanoparticles (AgNPs) have unique antimicro-

bial properties and are widely used in a variety of applica-tions including food storage, wound dressings, and fabrics. Applications for the NPs are increasing, but little is known about their environmental implications and risks associ-ated with their use. The silver ion has cytotoxic effects on organisms through oxidative damage1. While much of the recent work assessing risks of AgNPs has focused on path-ways of toxicity1, 15, it does not consider environmentally relevant means of exposure. An important first step in re-searching the risks posed to ecological systems and organ-isms is to determine means of exposure to AgNPs, which will influence transport, transformation, bioavailability, and toxicity of the nanomaterials2.

Silver nanoparticles are likely to reach aquatic environ-ments through runoff and consumer waste, and their fate is affected by dissolved organic matter (DOM) and ligands found within. Organic ligands can modify particle surfaces and alter the stability of the nanoparticles in water, caus-ing aggregation into nanoparticle conglomerates and dis-solution into other potentially bioavailable forms of silver. This potential for multiple transformations is what makes nanomaterials unique contaminants in the environment, unlike other emerging organic pollutants.

The thiol group (SH) has been shown to stabilize HgS and ZnS nanoparticles by adsorbing on their surface 3, 4, 5, and it is likely to play a key role in the fate of AgNPs because of its high affinity for silver. Silver nanoparticles are known to form highly insoluble sulfides or chlorides in freshwaters 6, 7, wastewater treatment plants 8, and sea-water 9, but when thiol containing organic compounds are present in significant concentrations, they may compete with sulfide for binding to silver and increase dissolved

Effect of Thiols on the Environmental Fate of Silver Nanoparticles

Abstract: Silver nanoparticles (AgNPs) are used in consumer goods for their antimicrobial properties, yet little is known about their environmental impact. When they are released into aquatic systems, their fate is influenced by organic ligands that may adsorb to particle surfaces and modify reactivity, causing particles to aggregate or dissolve into potentially bio-available forms of silver. The thiol (S-H) ligand is likely to play a key role because of its affinity for silver. We assessed the effect of thiols on citrate-coated AgNPs using glutathione (GSH), a low molecular weight peptide produced by organisms during oxidative stress and exposure to toxic metals. Aggregation of AgNPs was measured through time-resolved dynamic light scattering (DLS). Silver dissolution over time was determined by filtering batch suspensions to separate dissolved from particulate silver and measured with inductively coupled plasma mass spectroscopy (ICP-MS). Also, GSH concentrations measured with high pressure liquid chromatography (HPLC) and zeta potential measured with DLS were used to monitor surface modifications on the AgNPs. Results show that glutathione sorbs on the surface of AgNPs, reduces growth rate, and improves AgNP stability in solution, and moreover, that these processes occur simultaneously. This has implications for persistence of silver in aquatic systems and less bioavailability to organisms.

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of solutions containing 1 ppm AgNP-CIT (9.26 μM), 7 mM NaHCO3 buffer solution, 10 mM NaNO3, and varying GSH concentrations of 1 μM, 10 μM, and 100 μM and filtering at individual time points over the time range of zero to forty eight hours. Solutions were filtered with 0.025 μm membranes (VSWP Millipore) to sepa-rate particles from suspension. Membranes were rinsed with 7 mM NaHCO3 buffer solution before used for the suspensions. The duration of filtering ranged from eight to ten minutes. Silver concentration in the filtrate was measured after digesting samples with 2% HNO3 and 1% HCl by inductively coupled plasma mass spectroscopy (Agilent Technologies).The silver concentration of the acidified samples was kept below 50 ppb to avoid pre-cipitation of silver chloride. The ICP-MS measurements were taken by a member of the research group presiding over my work.

Glutathione measurements were also taken from filtrates; GSH measurements were quantified by reverse phase high performance liquid chromatography (Varian ProStar)16, 17. Samples were diluted in 0.5 M sodium acetate buffer dissolved in water and adjusted to pH 6 and 2, 2’-dithiobis(5-nitropyridine) (DTNP) dissolved in in acetonitrile to derivatize the thiol. HPLC-grade solvents were utilized for all reagents. HPLC involved a C18 column and UV-Vis detector. These measurements were also done by the same presiding member of the research group.

DLS (Zetasizer Nano Series, Malvern Instruments) was used to monitor particle growth by measuring the intensity-weighted average hydrodynamic diameter of particles precipitating in test solutions over a range of 96 hours using incident light (λ = 633 nm) scattered at 173°. Individual measurements were taken at 0, 3, 24, and 48 hours. Immediately after addition of AgNP to the test solution, a 1 mL aliquot was dispensed into a polycarbon-ate disposable 1 cm cuvette cleansed with ultrapure water and placed in the instrument. The remaining solution was then preserved for the next measurements. The aver-age hydrodynamic diameter of particles was estimated approximately every 3 min by averaging 16 individual measurements runs. All particle growth measurements were conducted at 25° C. Zeta (ζ) potential of Ag NPs, which is a measure of surface potential and stability, was calculated from electrophoretic mobility measurements of the particle suspensions (Zetasizer Nano Series, Malvern Instruments). Mobility was calculated by applying electric potential through a capillary cell with electrodes at either end of the sample holder. The velocity of the moving charged particles was measured and used to infer the magnitude of their surface charge and zeta potential. Zeta potential was measured 4 times for each time point after the initiation of AgNP aggregation at 25 °C. Addition-ally, a pH replicate for each test solution was measured at each of the time points.

Results and DiscussionPrevious data on silver dissolution, size, zeta potential,

and pH collected from control solution with no added organic ligands (1 ppm AgNP-CIT (9.26 μM), 7 mM NaHCO3, 10 mM NaNO3) by collaborating researcher

Methods and Materials

MaterialsAll chemicals used in this work were ACS reagent

grade and purchased from Sigma-Aldrich unless other-wise stated. Stock solutions were prepared using filtered (<0.2 μm) ultrapure water (Barnstead Nanopure, >17.8 MΩ-cm). Trace-metal grade acids were used to adjust the pH of solutions. Ultrahigh purity nitrogen was utilized for purging oxygen from aqueous samples. All borosilicate glass containers were acid-washed through soaking overnight in 1 N HCl followed by three rinses with ultrapure water. Stock solution of 3.25 mM L-glutathione was prepared with degassed water, stored at 4 °C, and utilized within 2 weeks of preparation. Synthesis of silver nanoparticles with citrate coating (Ag-CIT) fol-lowed previously published methods15. Particle mono-mer size and shape were characterized with Transmission Electron Microscopy (TEM). Synthesis and character-ization of NPs was done by research group associates. Ag-CIT consisted of mostly spherical with some oval shaped particles and an average geometric diameter of 19.1 ± 12.7 nm (average of 188 particles measured from TEM images). From the number-based size distribution of the two stock suspensions, surface area per mass was calculated: 16.1 m2/g. Stock solution of Ag-CIT was stored at 4 °C.

Experimental solutions were prepared of 1 ppm Ag-CIT (9.26 μM) in a pH 7.5 buffer solution with 7 mM NaHCO3, 10 mM NaNO3, and varying glutathione concentrations of 1 μM, 10 μM, and 100 μM GSH. Bicarbonate buffer solution was prepared fresh daily.

MethodsDissolved silver was measured by preparing replicates

Figure 1. Surface adsorption by ligands will interfere with NP solubility and bioavailability by influencing aggregation, dissolution kinetics, and metal speciation. (Diagram created by and used with persmission from mentor.)

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was added to measurements. All data presented are either individual measurements or averaged measurements with standard deviation error bars.

Silver Dissolution – (Figure 2) Solutions containing GSH displayed elevated dissolved Ag concentrations compared to solutions with no ligand. 10 μM and 100 μM GSH solutions generally showed greater dissolution than 1 μM GSH solution. Dissolution increased rapidly within the initial measurements (0, 3, 6hrs) and contin-ued to dissolve over the remaining period. Solution of 10 μM GSH appears to have the highest dissolved Ag but shows highly variable concentrations within replicate measurements, so a trend between dissolution and GSH concentration cannot be characterized.

Glutathione in filtrate - (Figure 3) GSH concentra-tions were measured from the same filtrates to quantify ad-sorption of GSH to the particle surface, disregarding the potential confounding factor of oxidation of GSH. GSH measured for each solution was relatively stable across all time points measured, indicating that ligand-NP adsorp-tion occurs soon after NPs are exposed to ligands and does not fluctuate much after the initial exchange. This suggests that GSH stabilizes AgNP for at least 48 hours. Right: Concentrations from A were converted to percentage of original GSH in solutions. Solution with 1 μM GSH so-lution had significantly lower percentage than solutions with 10 μM and 100 μM GSH, which could be attributed to limiting behavior of low GSH or inconsistency in mea-suring low concentrations.

FIGURE 3

Figure 3. GSH detected after filtering 1 ppm Ag-CIT in 7 mM NaHCO3, 10 mM NaNO3 and GSH concentrations of 1 μM GSH, 10 μM GSH, and 100 μM GSH. GSH was found to be stable over time in concentrations correspond-ing to original values. Bottom- Percent of GSH measured in filtrate of original GSH in solutions.

Zeta Potential – (Figure 4) Zeta potential, which is an indicator of particle surface charge and stability, was mea-sured over time. All solutions containing GSH displayed increasingly negative zeta potential values over time, sug-gesting increased repulsion between particles and more stability. Both 10 μM and 100 μM GSH solutions, which contained GSH in excess of Ag-CIT, stabilized at similar values; 1 μM GSH solution showed less negative values. This data shows that increased GSH causes more surface modification among AgNPs.

Figure 2. Dissolution of 9.3 μM Ag-CIT in the presence of 1 μM GSH, 10 μM GSH, 100 μM GSH, and no ligand. Solu-tion pH was 7.5-8.5 buffered with 7 mM NaHCO3. Ionic strength was controlled with 10 mM NaNO3. Measured to-tal silver. GSH significantly increases dissolution compared to solution with no ligand; however a definite trend with re-spect to concentration cannot be determined.

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on the growth, dissolution, and surface modification of citrate-coated silver nanoparticles can be summarized as follows:

Figure 4. (Previous page) Zeta potential of 9.3 μM Ag-CIT in the presence of 1 μM GSH, 10 μM GSH, 100 μM GSH, and no ligand. Solution pH was 7.5-8.5 buff-ered with 7 mM NaHCO3. Ionic strength was controlled with 10 mM NaNO3. The presence of GSH appears to decrease zeta potential, implying stability.

Aggregation – (Figure 5) Aggregation leads to large clus-ters that settle out of solution faster than small particles and is an important process in the behavior of nanopar-ticles. It also influences surface reactions by reducing the available surface area and slowing diffusion of chemicals. The presence of GSH at low concentration was found to induce NP aggregation compared to a control solu-tion with no ligand, but increasing GSH concentrations resulted in less aggregation. This suggests that GSH sig-nificantly alters NP stability even at low concentrations, but when present at high concentrations in excess of the AgNP, such as in the 10 μM and 100 μM solutions, it can alternately promote stability and prevent interaction of particles.

Figure 5. (above) Aggregation of 9.3 μM Ag-CIT in the presence of 1 μM GSH, 10 mM GSH, 100 μM GSH, and no ligand in 7 mM NaHCO3 solution pH buffered at 7.5-8.5. Ionic strength was controlled with 10 mM NaNO3. GSH promotes aggregation at low concentrations, but at high concentrations it reduces growth.

Figure 6. (below) The pH of all solutions was also measured over time with replicates to measure changes in solution be-havior and give context for zeta potential values. All solu-tions displayed an increase in pH value over the time range.

From the above data, the effects of GSH concentration

Figure 7. (above) Initial Ag-CIT growth rates were calculated by fitting a linear trend to the first 0-3 hours of hydrodynamic diameter measurements (see Fig. 5) for each solution and are plotted on a logarithmic scale. Additional measurements in this time range are necessary to characterize growth; values are imprecise and displayed with standard error. GSH was found to reduce growth rate.

Figure 8. Rates of dissolution over time range calculated from fitting a linear trend to the Ag dissolution measure-ments (see Fig. 2) normalized to initial total surface area (μmol/m2•h) and plotted over a logarithmic scale for each replicate tested. Definite trend cannot be determined, but shows that intermediate GSH concentrations increase dis-solution rates.

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Figure 9. Zeta potential for each GSH concentration (see Fig. 4) tested at two selected time points, 3 hours and 48 hours, plotted over a logarithmic scale. Shows that over mul-tiple time ranges, GSH decreases zeta potential of AgNPs; this implies increased charge, surface modification, and sta-bility.

Although other research confirms that the fate of nanomaterials is determined by several processes includ-ing dissolution, aggregation, and surface reactions18, few studies investigate these processes together. This research is unique in that it studies several inter-linked mechanisms simultaneously affecting the environmental fate and bioavailability of AgNPs in natural settings (See Figure 10.)

Figure 10. Processes affecting the fate of AgNP in the en-vironment. (Created by and used with permission from col-laborating member of research group.)

Measurements of zeta potential and of glutathione con-centrations in filtrates quantify the surface modification of the nanoparticles through measuring surface charge and GSH adsorption. Size measurements of the hydro-dynamic radius of nanoparticles over time quantify ag-gregation and speciation. Dissolved silver measurements quantify the dissolution of Ag+ from the NPs and specia-tion. Together, these results elucidate the role of the thiol ligand in the fate of AgNPs under various conditions and confirm that when present in significant concentrations, thiols can alter the behavior of silver nanomaterials in the environment and should be considered in assessing their fate along with other key inorganic ligands such as sulfides and chlorides.

Environmental ImplicationsEnvironmental implications of this work include the

persistence of AgNPs in aquatic systems. Glutathione appeared to stabilize the AgNPs at high concentrations through reduced aggregation, reduced Ag+ dissolu-tion, and greater surface modification, which could prevent phase separation or sedimentation of the NPs out of water. In thiol-rich environments, this may cause particles to persist in solution and remain subject to modification by other inorganic ligands, natural organic matter, and uptake by organisms.

However, the stabilization of Ag NPs through thiol-containing molecules such as glutathione can be environmentally beneficial in wastewater treatment plants. Concerns have arisen about the negative effects of Ag NP on bacteria in activated sludge9, but these concerns may not be valid if Ag NPs encounter bacte-rially secreted thiol-containing compounds before they reach the bacteria.Biological Implications

The behavior of Ag NP with GSH also has biologi-cal implications for the means of heavy metal toxicity in organisms. Glutathione and other thiol-containing antioxidants and enzymes are produced in response to oxidative stress and metal toxicity, but Ag NPs may sorb to these molecules and deplete their availability.Additionally, this research will affect models of Ag NP drug delivery. Ag NPs are being considered for use in drug delivery applications19, but in the body they are likely to come into contact with high concentrations of GSH and other thiols, which will alter their availability and solubility.

Conclusions and Future WorkThe environmental fate and bioavailability of silver

nanoparticles is likely to be affected by the thiol ligand, which, due to its high affinity for silver metal, can adsorb to particle surfaces and alter their stability in waters. We studied the nature of thiol-Ag NP interac-tions through measuring the aggregation, dissolution, and zeta potential of citrate-coated silver nanoparticles in the presence of varying concentrations of glutathi-one. Results show that GSH reduces aggregation rates, increases dissolution of Ag+ from nanoparticles, and decreases zeta potential, implying increased nanopar-ticle stability as a result of thiol sorption. Thiols play a critical role in the behavior and uptake of Ag NPs and should be included with other key ligands in assess-ing the risks of silver nanomaterials. Moreover, results stress the need for a multiple-focus holistic approach looking at all of the interacting processes that are oc-curring together and affecting one another.Further experimentation is necessary to determine if GSH is adsorbing to NP surfaces as inferred; measure-ments of GSH could be due to GSH oxidation as well as AgNP sorption; reliable methods to measure radical oxidative species or silver oxides are required. Future work should also include studies of additional biogenic thiols besides glutathione to confirm the role of the thiol ligand in the interactions.

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Fahey, R. C.; Brown, W. C.; Adams, W. B.; Worsham, M. B., Occurrence of glutathione in bacteria. J Bacteriol 1978, 133 (3), 1126-9.Meyer, J. N.; Lord, C. A.; Yang, X. Y. Y.; Turner, E. A.; Badireddy, A. R.; Marinakos, S. M.; Chilkoti, A.; Wiesner, M. R.; Auffan, M., Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans. Aquatic Toxicology 2010, 100 (2), 140-150.Hsu-Kim, H., Stability of metal - glutathione complexes during oxidation by hydrogen peroxide and Cu(II) catalysis. Environmen-tal Science & Technology 2007, 41, 2338-2342.Vairavamurthy, A.; Mopper, K., Field Method for Determination of Traces of Thiols in Natural-Waters. Analytica Chimica Acta 1990, 236 (2), 363-370Wiesner, M.R., Lowry, G.V., Alvarez, P., Dionysiou, D., Biswas, P., 2006. Assessing the risks of manufactured nanomaterials. Environ. Sci. Technol. 40, 4336-4345.Langer, R.; LaVan, D. A.; McGuire, T., Small-scale systems for in vivo drug delivery. Nat Biotechnol 2003, 21 (10), 1184-1191.

Also, the role of thiols is limited by their presence relative to competing ligands such as sulfide and chloride6, 7, 8, 9, and the behavior of AgNP in environments with multiple influential ligands should be examined. Furthermore, we only conducted experiments at a single ionic strength, controlled at 10mM NaNO3, but ionic strength varies between freshwater and saline environments, and a gradi-ent of ionic strengths should be considered. Likewise, changes in GSH content significantly affected Ag NP behavior in experiments, and additional GSH contents should be tested to characterize silver nanoparticle be-havior for a full spectrum of possible thiol concentrations in various environments.

References

Ercal, N.; Gurer-Orhan, H; Aykin-Burns, N. Toxic metals and oxidative stress part I: Mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 2001, 1, 529-539.Aiken, G., et. al. Influence of Dissolved Organic Matter on the Environmental Fate of Metals, Nanoparticles, and Colloids. Envi-ron. Sci. Technol., 2011, 45(8), 3196-3201.Gondikas, A. P.; Jang, E. K.; Hsu-Kim, H., Influence of amino ac-ids cysteine and serine on aggregation kinetics of zinc and mercury sulfide colloids. Journal of Colloid and Interface Science 2010, 347 (2), 167-171.Lau, B. L. T.; Hsu-Kim, H., Precipitation and growth of zinc sulfide nanoparticles in the presence of thiol-containing natural organic ligands. Environmental Science & Technology 2008, 42 (19), 7236-7241.Deonarine, A.; Hsu-Kim, H., Precipitation of Mercuric Sulfide Nanoparticles in NOM-Containing Water: Implications for the Natural Environment. Environmental Science & Technology 2009, 43 (7), 2368-2373.Adams, N. W. H.; Kramer, J. R., Silver speciation in wastewater effluent, surface waters, and pore waters. Environ Toxicol Chem 1999, 18 (12), 2667-2673.Kramer, J. R.; Adams, N. W. H.; Manolopoulos, H.; Collins, P. V., Silver at an old mining camp, Cobalt, Ontario, Canada. Environ Toxicol Chem 1999, 18 (1), 23-29.Kim, B.; Park, C. S.; Murayama, M.; Hochella, M. F., Discovery and Characterization of Silver Sulfide Nanoparticles in Final Sew-age Sludge Products. Environmental Science & Technology 2010, 44 (19), 7509-7514.Miller, L. A.; Bruland, K. W., Organic Speciation of Silver in Marine Waters. Environmental Science & Technology 1995, 29 (10), 2616-2621.Adams, N. W. H.; Kramer, J. R., Reactivity of Ag+ ion with thiol ligands in the presence of iron sulfide. Environ Toxicol Chem 1998, 17 (4), 625-629.Shea, D.; Maccrehan, W. A., Determination of Hydrophilic Thiols in Sediment Porewater Using Ion-Pair Liquid-Chromatography Coupled to Electrochemical Detection. Analytical Chemistry 1988, 60 (14), 1449-1454. Ahner, B. A.; Morel, F. M. M., Phytochelatin Production in Marine-Algae .2. Induction by Various Metals. Limnology and Oceanography 1995, 40 (4), 658-665.