Formation of Silver Nanoparticles in Visible Light-Illuminated Waters:Mechanism and Possible Impacts on the Persistence

of AgNPs and Bacterial Lysis

Appala Raju Badireddy,1,2 Jeffrey Farner Budarz,1,2 Stella M. Marinakos,2

Shankararaman Chellam,3,4 and Mark R. Wiesner1,2,*

1Department of Civil and Environmental Engineering, Duke University, Durham, North Carolina.2Center for the Environmental Implications of Nanotechnology, Duke University, Durham, North Carolina.

3Department of Civil and Environmental Engineering, University of Houston, Houston, Texas.4Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas.

Received: October 25, 2013 Accepted in revised form: January 29, 2014

Abstract

A confluence of water and silver-containing products or compounds results in unintentional or intentional dis-charge of silver species (including silver nanoparticles [AgNPs], free or organic matter-bound Ag+, Ag-halides, orsoluble Ag-complexes) into visible light-illuminated waters. To date, little is known about the transformations ofAg species and mechanisms of AgNP formation in light-illuminated waters consisting of components such asbacteria, extracellular polymeric substances (EPS), natural organic matter (NOM), chloride, and/or nitrate. In thiswork, we demonstrate that in the presence of combinations of water components the transformation of ionic Ag to‘‘new’’ AgNPs occurs via heterogeneous nucleation followed by (1) light-induced growth mediated by surfaceplasmon resonance activity of AgNPs, and (2) Ostwald ripening in the absence of light and chloride anions.Further, oxidative dissolution of AgNPs at near-neutral pH (6.8) releases Ag ions, which either complex with thecomponents or form ‘‘new’’ AgNPs depending on solution chemistry. AgNP nucleation and growth on bacterialcells (e.g., Pseudomonas aeruginosa) may cause cell lysis. This study highlights that light-illuminated waterscomprising common aquatic components are highly favorable for the AgNP formation over dissolution underneutral pH conditions, in which the presence of chloride accelerates AgNP formation and the reducing capacity ofEPS/chloride medium may be greater than NOM (humics and fulvics). Findings of this study suggest that AgNPformation and dissolution may be a continual process and AgNPs thus formed may be stabilized by organicmatter, which possibly leads to persistence of the nanoparticles in sunlit waters.

Key words: bacterial lysis; heterogeneous nucleation; Ostwald ripening; redox potential; silver nanoparticles;surface plasmon resonance; surface waters; visible light

Introduction

W idespread use of silver nanoparticles (AgNPs) inindustrial, consumer, and healthcare products makes

silver (Ag) one of the most commonly discharged metals inthe environment (Benn and Westerhoff, 2008; Chen andSchluesener, 2008; Edwards-Jones, 2009; Benn et al., 2010;Woodrow Wilson International Center for Scholars, 2010;Glover et al., 2011). Since Ag exhibits an exceptionally broadspectrum of antimicrobial and antiviral properties, it mayadversely affect aquatic biota (Ratte, 1999; Wiesner et al.,2006; Navarro et al., 2008; Panyala et al., 2008; Rai et al.,

2009; Dallas et al., 2011; Galdiero et al., 2011). AgNP-embedded products release Ag ions and nanoparticles tosurface waters, after which silver is further transformed bythe visible light and aquatic components such as bacteria,extracellular polymeric substances (EPS), natural organicmatter (NOM), chloride, and/or sulfide. For example, AgNPsundergo oxidative dissolution in surface waters, releasing Agions that have been shown, in turn, to form complexes or newnanoparticles by natural reducing agents (e.g., EPS, NOM) inthe presence or absence of light (Liu and Hurt, 2010; Hoet al., 2011; Akaighe et al., 2012; Yin et al., 2012; Adeg-boyega et al., 2013). Further, AgNPs can also form naturallyfrom macroscale Ag objects under environmentally relevantconditions (Glover et al., 2011) or by bacteria; for example,Pseudomonas stutzeri AG259 (Tanja et al., 1999), Bacilluslicheniformis (Kalimuthu et al., 2008), and Escherichia coli(Gurunathan et al., 2009a, 2009b) after extended periods of

*Corresponding author: Department of Civil and EnvironmentalEngineering, Duke University, Box 90287, 120 Hudson Hall,Durham, NC 27708-0287. Phone: 919-660-5292; E-mail: wiesner@duke.edu

ENVIRONMENTAL ENGINEERING SCIENCEVolume 31, Number 7, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/ees.2013.0366

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incubation with Ag ions in the absence of light (Klaus-Joergeret al., 2001). In wastewater-activated sludge, Ag2S nanoparticlesare formed via sulfidation (Kim et al., 2010). Ag ions are highlybioactive and inactivate/lyse bacteria and other microorganismsby deactivating key cell-bound respiratory enzymes (Russell andHugo, 1994; Ratte, 1999; Sondi and Salopek-Sondi, 2004;Yamanaka et al., 2005). Although a variety of Ag species, in-cluding AgNPs, AgCl, Ag-chlorocomplexes, and Ag2S exist inthe natural environment, the dominant species in a specificsystem depends on the solution chemistry (e.g., redox potentials,organic matter, and inorganic anions) and presence or absence oflight. At thermodynamic equilibrium, AgNPs, AgCl, and Ag2Sare predicted to exist in freshwater, whereas AgCl2

- and AgNPsexist in sea water (Levard et al., 2012). In sunlit waters, dissolvedorganic matter (DOM) can reduce Ag ions to AgNPs, whichimplies that Ag can persist as AgNPs in waters instead of existingas DOM-associated Ag ions (Wen et al., 1997). These earlierstudies have shown that the interaction of Ag ions with indi-vidual aquatic components such as NOM, bacteria, chloride, orsulfide would result in the formation of Ag species, including‘‘new’’ AgNPs, but there were no studies to date that examinedthe resulting effect of combinations of aquatic components onAg ion transformations, especially in the presence and absenceof visible light. Based on these previous reports, we hypothesizethat the mechanism of AgNP formation and dissolution areinfluenced by the components in the water, and the presence orabsence of light. The information on the resulting effect of thecomponents on released Ag ions or AgNPs would shed light ondominant Ag species, and hence on environmental persistence,bioavailability, toxicity, and mobility of Ag in sunlit waters(Chinnapongse et al., 2011; Levard et al., 2012; Lowry et al.,2012). Further questions regarding AgNP formation mecha-nisms and rates in environmental waters with and without lightare still unanswered. AgNP toxicity to bacteria has been attrib-uted to the release of Ag ions (Xiu et al., 2012), but the trans-formation of the ions upon contact with bacterial cell walls hasnot yet been studied. Such information has particular pertinencein evaluating the impacts of released Ag on environmental mi-croorganisms and associated nutrient and biogeochemical cycles(Madsen, 2011).

The overall goal of this study was to demonstrate that Agpersists as ‘‘new’’ AgNPs formed from dissolved Ag in light-illuminated oxic surface waters. For this study, Pseudomonasaeruginosa was used as a model bacterium, as these bacteriaproduce copious amounts of EPS in a short duration and arecommonly found in freshwaters, marine waters, and recreationalwaters systems (Khan et al., 2010). Commercially availablehumic and fulvic acids were mixed in a 1:1 (w/w) ratio to rep-resent the major components of NOM. Separate experimentswere also conducted with synthesized polyvinylpyrrolidone(PVP)-AgNPs in selected media to discern the AgNP dissolu-tion behavior and possible transformations of released Ag ions.The results obtained from the earlier sets of experiments wereevaluated to reveal the possible cyclic nature of AgNP forma-tion and dissolution, and possible nanoparticle persistence andbacterial lysis in light-illuminated waters. Enhanced darkfield-hyperspectral imaging (HSI) microscopy, UV-Visible spectro-photometry (UV-Vis), transmission electron microscopy (TEM),scanning electron microscopy (SEM), X-ray photoelectronspectroscopy (XPS), and inductively coupled plasma-massspectrometry (ICP-MS) were used for the characterizationand analysis of AgNPs.

Materials and Methods

Chemicals and stock suspensions

Silver nitrate (AgNO3), sodium nitrate (NaNO3), sodiumchloride (NaCl), and humic and fulvic acids were purchasedfrom Sigma-Aldrich (St. Louis, MO). Tryptone, Bacto agar,and yeast extract were purchased from Difco (Detroit, MI).Stock suspension of PVP-coated AgNPs (mean diameter*25 nm) was synthesized according to published procedures(Ma et al., 2012). Stock suspensions of 100 mg/L AgNO3,100 mg/L NOM (humic acids and fulvic acids were mixed at1:1 w/w), and 8 g/L NaCl were prepared in sterile ultrapurewater. Bacterial growth medium consisted of 10 g/L tryptone,1 g/L yeast extract, and 8 g/L NaCl (aka LB w/NaCl). Simi-larly, LB w/NaNO3 medium was prepared. All media wereprepared in ultrapure water (Barnstead NANOpure Dia-mond), and growth media were autoclaved before use.

Bacterial culture and test media

P. aeruginosa (ATCC # 47085) culture was grown in LBw/NaCl medium that was incubated at 37�C with shaking at150 rpm for 18 h overnight. Supernatant (free of bacteria andcell debris) was prepared by subjecting 10 mL aliquots ofovernight culture to sequential centrifugation, once at 6,000 gfor 5 min and twice at 12,000 g for 30 min at 4�C. The col-lected supernatant, comprising secretions of P. aeruginosa(or EPS) and the spent LB w/NaCl medium, was used as EPSmedium (this is referred to here as EPS w/NaCl). Similarly,EPS w/NaNO3 was prepared from P. aeruginosa culture thatwas grown in LB w/NaNO3 medium (to study the effect ofchloride, NaNO3 was substituted for NaCl in the growthmedia). For the purpose of this study, various test media wereprepared and are referred to here as follows: (1) overnightgrown P. aeruginosa cultures in LB broth with NaCl areidentified as ‘‘P. aeruginosa + EPS w/NaCl’’; (2) after re-moving the bacteria through centrifugation, EPS medium isidentified as ‘‘EPS w/NaCl’’; (3) overnight grown P. aeru-ginosa cultures in LB broth with NaNO3 are identified as‘‘P. aeruginosa + EPS w/NaNO3’’; (4) the P. aeruginosa +EPS w/NaNO3 medium without bacteria is identified as‘‘EPS w/NaNO3’’; (5) LB broth with NaCl is identified as‘‘LB w/NaCl’’ (this is the growth media); (6) LB broth withNaNO3 is identified as ‘‘LB w/NaNO3’’; (7) LB broth with-out NaCl is identified as ‘‘LB wo/NaCl’’; and (8) 100 mg/LNOM medium [1:1 (w/w) humic and fulvic acids that con-tains no chloride or bacteria] is identified as NOM medium.These media were stored at 4�C until use.

Experimental protocol

A typical test suspension consisted of 100 lL of 100 mg/LAgNO3, 100 lL of test medium, and 800 lL of ultrapurewater (total volume 1 mL). Two sets of 1 mL test suspensionscomprising Ag ions at 10 mg/L were prepared, in which oneset was exposed to visible light (light source and flux infor-mation is provided in the Supplementary Data; Supplemen-tary Data are available online at www.liebertpub.com/ees)and the other was kept in the dark (no visible light) for90 min. Further, the experiments in the dark were continuedfor 14 days, as no observable changes occurred within thefirst 90 min. We acknowledge that 10 mg/L Ag is not an en-vironmentally relevant concentration, but it enables the real-

AGNP FORMATION IN VISIBLE LIGHT-EXPOSED WATERS 339

time visualization of Ag ion transformations in the test mediawith or without light [via darkfield HSI microscopy] and,thus, provides information on the transformation mecha-nisms. The absorbance spectra in wavelength range of 300–800 nm were collected for samples at predetermined timeintervals using a UV-Vis spectrophotometer (Varian Cary300). In addition, a set of experiments with selected media inthe absence of light was carried out using PVP-AgNPs at anear-neutral pH of 6.8 and room temperature for a period of 4days to assess the kinetics of AgNP dissolution.

Characterization and analysis

Enhanced darkfield HSI microscopy (aka CytoViva HSIMicroscopy) was used for imaging samples in wet-form toobserve and analyze Ag ion transformations in real-time(Badireddy et al., 2012). Hyperspectral image scans onsample droplets consisted of 512 lines rastered at a step sizeof 10 nm at a spectral resolution of 1.5 nm in the visible andnear-infrared wavelengths (400–1,000 nm). TEM (FEI Tec-nai G2 Twin, Hillsboro, OR) was used to image samples driedon the Formvar side of a Carbon Type A Formvar-coatedcopper grid (Ted Pella, Redding, CA) at an acceleratingvoltage of 160 kV. SEM (FEI XL30 SEM-FEG, Hillsboro,OR) was performed on gold-sputtered samples that werefixed with 2.5% glutaraldehyde and sequentially dehydratedand washed, respectively, with graded ethanol and 1 ·phosphate-buffered saline. Inductively coupled plasma-massspectroscopy (ICP-MS; 7700 Agilent Technologies, SantaClara, CA) was used for measuring Ag ion concentration inthe test suspensions (Lowry et al., 2012). X-ray photoelectronspectroscopy (XPS) was used to collect the high-resolutionscans (step size 0.1 eV) of the samples that were air dried on asilicon wafer for verifying the presence of zerovalent silver(Ag0). Total organic carbon analyzer (TOC 5050A; Shi-madzu, Kyoto, Japan) was used for measuring the NOMconcentration. Oxidation-reduction potential (ORP) mea-surements were made on all test suspensions against a silver/silver chloride electrode in the presence and absence of lightusing an Orion ORP probe (Thermo Scientific, Waltham,MA) (see Supplementary Data for more details). Note that noattempt was made to determine which specific components ofEPS or bacteria or NOM were responsible for the Ag iontransformations; however, the experiments were designed todemonstrate the resulting effect of the combination of watercomponents on Ag ions and nanoparticles in the visible light-illuminated waters.

Results

Ag ion reduction in P. aeruginosa + EPSw/NaCl + visible light

In P. aeruginosa + EPS w/NaCl medium, a broad UV-Visabsorbance peak associated with surface plasmon resonance(SPR) of AgNPs evolved at 396 nm initially, and then itgradually shifted toward 436 nm during 90 min of irradiationwith visible light (Fig. 1a). UV-Vis spectra showed that after ashort-lag period, the growth of AgNPs occurred rapidly in themedium (Fig. 1a and Supplementary Fig. S1). An increase inthe absorbance of SPR peak and the asymmetric shape of thepeak indicates a growing number of individual AgNPs as wellas the formation of AgNP aggregates. At the end of 90 min, the

FIG. 1. Evolution of UV-Vis spectra of silver nanoparticles(AgNPs) in Pseudomonas aeruginosa + extracellular poly-meric substances (EPS) w/sodium chloride (NaCl) mediumexposed to Ag ions and visible light for a period of 0–90 min(the green colored spectrum corresponds to the sample at90 min) is shown in (a), transmission electron micros-copy (TEM) image of AgNPs in P. aeruginosa + EPS w/NaClmedium at the end of 90 min of exposure to light is shownin (b), and high-resolution X-ray photoelectron spectros-copy (XPS) spectrum depicting the signature of AgNPs inP. aeruginosa + EPS w/NaCl medium is shown in (c).

340 BADIREDDY ET AL.

average diameter of AgNPs was 25.5 – 2.5 nm (TEM imageanalysis was done using Image J) (Fig. 1b), and the identity ofAgNP in the sample was confirmed by XPS (Fig. 1c).

Further, the visual evidence for the presence of Ag nano-particulates (AgNPs and AgCl) in the medium exposed to90 min of visible light is shown Figure 2. A representative

darkfield image of P. aeruginosa in EPS w/NaCl mediumwithout Ag ions (control) is shown in Figure 2a. After ex-posure to Ag ions and visible light for 30 min, numerousbrightly colored Ag nano-particulates were observed to formin the bulk medium as well as on the bacterial cell walls;while the bacteria were catastrophically damaged as shown in

FIG. 2. CytoViva darkfield image (magnification: 40 · ) of P. aeruginosa + EPS w/NaCl media before the addition of Agions (a), lysed P. aeruginosa in EPS w/NaCl medium and AgNPs formed by the reduction of Ag ions in the medium duringexposure to visible light for 90 min (b, c), scanning electron microscopy (SEM) images of samples obtained at the end of30 min of exposure to Ag ions and visible light are shown in (d–f), zerovalent silver (Ag0) nuclei on the bacterial cell wallare shown in (d), AgNPs in the vicinity of lysed bacteria are shown in (e), and AgNPs on the poles and cell walls of thebacteria are shown in (f). The presence of AgNPs on the cells walls and the vicinity of the cells were confirmed by thehyperspectral image analysis. Additional SEM images and darkfield hyperspectral images of Ag nucleation and growth onbacterial cell walls are shown in Supplementary Figures S2 and S3, respectively, in Supplementary Data.

Table 1. Average AgNP Diameter, Binding Coefficient (n Value), and Formation

and Dissolution Rate Constants

SampleaMean ( – stderror)

diameter (nm)

n > 1Cooperative

binding

Visible Light:AgNP formation

kobs · 104/s

Dark: AgNPdissolutionkdis · 104/s

Pseudomonas aeruginosa + LB w/NaCl 26.2 ( – 1.7) 2.568 – 0.230 13.6 N/AP. aeruginosa + LB w/NaNO3 23.4 ( – 1.3) 1.185 – 0.207 8.31 N/AP. aeruginosa + EPS w/NaCl 25.4 ( – 2.5) 2.243 – 0.020 12.7 N/AEPS w/NaCl 14.9 ( – 1.2) 2.649 – 0.063 12.9 0.43EPS w/NaNO3 13.5 ( – 0.8) 1.424 – 0.036 9.74 N/ANOM 31.5 ( – 5.1) 1.863 – 0.050 9.11 0.40LB w/NaCl 27.8 ( – 1.1) 2.154 – 0.110 12.3 0.36LB w/NaNO3 22.6 ( – 3.7) 1.379 – 0.526 6.03 N/ALB wo/NaCl (or NaNO3) 19.8 ( – 1.8) 1.222 – 0.110 8.57 N/ADI water N/A N/A N/A 0.37Seawater N/A N/A N/A 0.59

aLB is a mixture of 10 g/L tryptone and 1 g/L yeast extract [i.e., LB without (wo/) NaCl or NaNO3]. LB w/NaCl is a growth medium usedfor culturing P. aeruginosa; P. aeruginosa + EPS w/NaCl is a bacterial culture grown overnight (see Materials and Methods section);P. aeruginosa + LB w/NaCl corresponds to washed bacteria (extracted from P. aeruginosa + EPS w/NaCl) resuspended in fresh LB w/NaCl.EPS w/NaCl is simply P. aeruginosa + EPS w/NaCl without P. aeruginosa, which were removed by centrifugation. NOM medium is amixture of humic and fulvic acids that contains no chloride, nitrate, or bacteria.

AgNP, silver nanoparticle; EPS, extracellular polymeric substances; NOM, natural organic matter; NaNO3, sodium nitrate; NaCl, sodiumchloride.

AGNP FORMATION IN VISIBLE LIGHT-EXPOSED WATERS 341

Figure 2b, c, e, and f (also see Supplementary Figs. S2 andS3). SEM images suggest that AgNPs may have formed fromAg ions through nucleation and growth on the cell walls (Fig.2d) and poles of the bacteria (Fig. 2e). The presence of col-ored particles is an indication of the SPR-activated AgNPsand the polydisperse nature of the nanoparticles formed.Real-time imaging and HSI analysis of the samples con-firmed that AgNPs were, indeed, present on the cell walls aswell as in the bulk medium (Supplementary Fig. S3 showsAgNPs on the cell walls).

The characteristic evolution of SPR absorbance inFigure 1a and in other test media (Supplementary Fig. S1)was nearly similar, suggesting that the mechanism ofAgNP formation from Ag ions in all the media is alsoprobably the same in the presence of visible light. Theaverage diameters of AgNPs observed in the various me-dia after 90 min of irradiation are summarized in Table 1.In NOM medium (a mixture of humics and fulvics thatcontains no chloride or bacteria), two distinct peaks at 400and 500 nm in the absorbance band between 365 and650 nm may be attributed to the formation of individualAgNPs and aggregates (Supplementary Fig. S1h), which islikely mediated by the NOM molecules bridging (orlinking) individual nanoparticles and forming larger ag-gregates (Supplementary Fig. S4). Further, as shown inFigure 3, AgNPs with the largest average diameter wereobserved in NOM medium (31.5 – 5.1 nm); whereas thesmallest ones were in EPS w/NaNO3 (13.5 – 0.8 nm) (Ta-ble 1). At the end of 90 min, it was observed that the UV-Vis absorbance maximum of each medium was different,which may imply that the rate of AgNP formation in eachmedium was also different (Table 1).

Mechanism of AgNP formation in the presenceand absence of visible light

Figure 4a shows the evolution of UV-Vis absorbance at415 nm with time in each medium. The trace of absorbanceover time was sigmoidal, starting with a slow inductionphase. The data were described by a sigmoidal function thatwas analogous to a modified form of Hills equation (seeSupplementary Data for details):

A¼A1þ(A2�A1)

1þ t50%

t

� �n (1),

where A is the absorbance (arbitrary units), A1 and A2 are theleft and right asymptotes (arbitrary units), respectively, t50% isthe time (minute) at which 50% of the nanoparticle populationis produced, and n is the binding coefficient describing co-operativity of Ag ions and AgNPs. Equation (1) provides away to quantify the binding of Ag ions to SPR-activated Ag0

cluster (or AgNP) induced by visible light. Ag ion binding toAgNP is (1) positively cooperative when n > 1, (2) negativelycooperative when n < 1, and (3) noncooperative when n = 1.The value of n for each medium was determined usingEquation (1) (Fig. 4a and Table 1). The results in Figure 4ashow that the values of n are greater than 1 for all the media inthe presence of visible light, suggesting that the binding of Agions to SPR-activated AgNPs is positively cooperative, whichfollowed a sigmoidal trend. Further, the n values were nearlytwofold higher for the media w/NaCl compared to the mediawithout NaCl, which suggests that the presence of chloride inthe media enhances the AgNP formation. The n values forNOM, LB wo/NaCl, and LB w/NaNO3 were statistically

FIG. 3. TEM images of spherical AgNPs formed in: EPS w/NaCl (a), EPS w/sodium nitrate (NaNO3) (b), natural organicmatter (NOM) (c), LB wo/NaCl (d), LB w/NaCl (e), and LB w/NaNO3 (f) at the end of 90 min of exposure to visible light.

342 BADIREDDY ET AL.

indistinguishable, which may suggest possible molecular-levelsimilarities between NOM and LB medium (with nitrate orwithout NaCl) and nitrate likely played a negligible role in Agion binding to AgNPs during 90 min of exposure to visiblelight. In the absence of light, no SPR peaks were recorded forthe test suspension during the first 90 min.

The progress of Ag ion binding to Ag particles via a re-duction reaction could be quantitatively described by thefollowing linear relationship with time (Esumi et al., 2000):

lna

1� a

� �¼ kobs · t (2),

where a is the ratio of absorbance at time t (At) and theabsorbance maximum (Amax), kobs is the observed bindingconstant (s - 1), and t is time in seconds. The results in Figure4b and Supplementary Fig. S5 show linear relationships (ln(a/(1 - a))) as a function of time with reasonably high R2

FIG. 4. Sigmoidal evolution of absorbance at 415 nm with time in various media during 90 min of exposure to visible lightis shown in (a); linear relationship of light-induced growth with time describing the rapid binding of Ag ions to Ag0 nuclei(or optimal size Ag particles) in the presence of visible light is shown in (b). The co-operative binding coefficient [n value,see Eq. (1) in the main text] is shown in parentheses in (a), and the n values are also summarized in Table 1. Also seeSupplementary Figure S5 in Supplementary Data for the other media in which additional linear relationships are shown.

FIG. 5. Evolution anddisappearance of a charac-teristic peak associated withAgNP surface plasmon reso-nance (SPR) in AgCl(s) sus-pension during exposure tovisible light for 30 min isshown in (a), CytoVivadarkfield image (magnifica-tion: 40 · ) shows the pres-ence of AgNPs and partiallyreduced AgCl precipitates inwhich the brightly coloreddots and rings are indicativeof SPR of AgNPs in the me-dium (b), TEM image showsthe AgCl aggregates beforeexposure to visible light (c),and TEM image shows thepresence of smaller ‘‘new’’AgNPs formed on the re-duced AgCl precipitates afterexposure to visible light for90 min (d). At the end of90 min of exposure to light,the white suspension in thecuvette (left) turned to agray-colored suspension (e).

AGNP FORMATION IN VISIBLE LIGHT-EXPOSED WATERS 343

values, suggesting that a light-induced heterogeneous nu-cleation and growth mechanism is likely responsible for theformation of AgNPs in the presence of visible light. The kobs

value describing the binding process in each medium wasdetermined from the slope of the Equation (2) and summa-rized in Table 1. In comparison to NOM and the media withNaNO3 (or without NaCl), the kobs of media w/NaCl were ofgreater, which implies that the presence chloride anion in themedia likely enhances the AgNP formation rate, which isconsistent with the two-fold increase in the binding coeffi-cients (n) (Table 1).

Chloride anion enhances formation of AgNPsin the presence of visible light

Evidence for AgNP formation in AgCl(s) solutions exposedto visible light is shown in Figure 5. In the initial UV-Visspectrum of AgCl suspension, a broad absorbance over theentire wavelength range of 300–800 nm was observed, withno characteristic AgNP SPR band (Fig. 5a). However, uponexposure to visible light, a broad SPR band began appearingat *415 nm. This peak gradually red-shifted, and then finallyvanished after 20 min of exposure. The presence of the SPRband is indicative of transformation of AgCl to AgNP, and agradual decrease in absorbance and disappearance of the SPRband was attributed to the aggregation of Ag nano-particu-lates (AgNP-AgCl) in the suspension. A color change fromcloudy white to gray was observed at the end of 90 min ofirradiation, which also qualitatively indicates the transfor-mation of AgCl to AgNPs (Fig. 5e placed as inset in Fig. 5a).Both HSI analysis and TEM images confirmed the presence

of AgNPs in AgCl suspensions. For instance, the brightlycolored rings around the AgCl aggregates or the nano-particles interspersed among aggregates are indicative ofSPR activity of AgNPs (Fig. 5b). In the absence of visi-ble light, the AgCl aggregates remained relatively free ofsmaller nanoparticles (Fig. 5c). However, after exposure tovisible light for 90 min, numerous smaller nanoparticlescoating the surface of AgCl aggregates were observed (Fig.5d). Further evidence for the presence of AgNPs in AgClsuspension was provided by the hyperspectral fingerprintanalysis, which confirmed that spectral peaks of colored na-noparticles were exactly matching with those of AgNPs inP. aerugionosa + EPS w/NaCl and engineered PVP-AgNPs(Fig. 6). Thus, it would appear that AgCl which is formed in amedium w/NaCl is further transformed to form AgNPs in thepresence of visible light. Thus, the results in Figures 4 and5 confirm that the presence of chloride enhances the AgNPformation in the presence of visible light.

Dissolution of AgNPs in the test media

Selected experiments were designed to specifically studythe oxidative dissolution of engineered AgNPs (PVP-AgNP)at pH 6.8 in the following media: (1) ultrapure water in theabsence and presence of visible light, and (2) LB w/NaCl,EPS w/NaCl, simulated seawater media (i.e., 0.7 M NaCl inultrapure water), and NOM in the absence of light. The dis-solution trends of PVP-AgNPs in the selected media for theduration of 24 h are shown in Figure 7, and TEM images ofsamples obtained from each medium on day 4 are shown inFigure 8. In the presence or absence of visible light, AgNP

FIG. 6. Enhanced darkfield hyperspectral images (magnification: 400 · ) of individual AgNPs formed in AgCl suspension(a, e) and P. aeruginosa (PA) + EPS w/NaCl medium (b, f) are shown here. Polyvinylpyrrolidone (PVP)-AgNPs (en-gineered nanoparticles) are shown in (c, g). The hyperspectral fingerprints of AgNPs in AgCl suspension and P. aeruginosa(PA) + EPS w/NaCl medium are perfectly matched with spectral signatures of PVP-AgNPs (d, h). AgNPs are colored due tothe visible light-induced SPR and due to their size and shape. The cross hair on each image shows the region where thespectrum was collected, and the corresponding spectra are shown in (d, h). More images of the nano-particulates andassociated spectra are also shown in Supplementary Figure S6 in Supplementary Data.

344 BADIREDDY ET AL.

dissolution rates were similar in ultrapure water. Further, thedissolution rates in ultrapure water were also similar to thoseobserved in LB w/NaCl and NOM media. However, thedissolution rate is higher in the EPS w/NaCl medium andhighest in the seawater medium. In addition, a TEM exami-

nation of the samples obtained on day 4 (Fig. 8a–f) revealedthat (1) in ultrapure water [Fig. 8a (dark) and 8b (light)] andin NOM medium (Fig. 8c), PVP-AgNPs remained free ofprecipitates likely due to the absence of light and chloride; (2)in LB w/NaCl media, Ag ions released from AgNPs likelycomplexed with chloride and formed AgCl precipitates onAgNP surfaces and in their vicinity (Fig. 8d); (3) in seawatermedium, Ag ions released from AgNPs likely formed pre-cipitates of Ag-chloro complexes, which is observed as dis-persed gray matter surrounding AgNPs (Fig. 8e); and (4) inEPS w/NaCl medium, Ag ions released from AgNPs werelikely reduced to smaller ‘‘new’’ AgNPs by the organiccomponents of the medium (Fig. 8f and Supplementary Fig.S7). The earlier results and observations suggest that dis-solved Ag ions may be transformed to (1) AgNPs and AgClin EPS w/NaCl medium, which is consistent with the re-sults from dark experiments shown in Supplementary FigureS8, and (2) soluble Ag-chloro complexes in the seawatermedium.

Discussion

Silver in surface waters exists in colloidal and particulateforms, free ions, and complexes with organic and inorganicmatter (e.g., EPS, bacteria, NOM, and chloride, and sulfide)(Chinnapongse et al., 2011). The speciation of Ag in aquaticsystems depends on the redox potentials and availability ofligands (organic molecules, Cl - , and S2 - ) (Adams andKramer, 1998; Levard et al., 2012). Ag has been demonstrated

FIG. 7. Dissolution behavior of PVP-AgNPs in seawater,EPS w/NaCl, NOM, DI water, and LB w/NaCl media over a24 h duration in the absence of visible light are shown here.The nanoparticle dissolution rates (lM/h) are shown in theparentheses in the figure.

FIG. 8. TEM images of PVP-AgNPs after dissolution for 4 days in: DI water in the absence of light (a), DI water in thepresence of visible light (b), NOM medium in the absence of light (c), LB w/NaCl medium in the absence of light (d),seawater medium in the absence of light (e), and EPS w/NaCl medium in the absence of light (f) are shown here. On closerexamination, image (f) shows smaller ‘‘new’’ AgNPs in the vicinity of larger PVP-AgNPs. These ‘‘new’’ AgNPs werepossibly formed from the reduction of dissolved Ag by EPS components. Also see Supplementary Figure S7 for imagesrelated to image (f) showing the presence of smaller ‘‘new’’ AgNPs around the larger PVP-AgNPs.

AGNP FORMATION IN VISIBLE LIGHT-EXPOSED WATERS 345

to be prevalent in the form of Ag-chloro complexes as op-posed to Ag-sulfide complexes, because sulfide concentrationis low in surface waters. Therefore, for this study, the em-phasis was placed on the interaction of Ag ions with chlorideanions. This study investigates the resulting effect of Ag ion(or AgNP) interactions with a combination of components ofwater (EPS, bacteria, NOM, nitrate, and chloride) in thepresence and absence of visible light.

Our experiments revealed that a rapid reduction of Ag ions(within minutes) to AgNPs was mediated by the watercomponents in the presence of visible light (Fig. 4). Con-clusive evidence for AgNP formation in the media was ob-tained through UV-Vis spectrophotometry, HSI microscopy,TEM, SEM, and XPS analysis. Since Ag ions can exist freely,bound to organic matter, or as soluble complexes in fresh-waters and seawaters, any exposure to visible light couldreadily transform Ag ions to AgNPs via a reduction reaction(Fabrega et al., 2011). In addition, chloride anions in theaqueous media can substantially enhance the AgNP forma-tion rates under visible light since AgCl also undergoes aphoto-reduction reaction and forms ‘‘new’’ AgNPs (Fig. 5); aprocess similar to silver halide-based photography (Wanget al., 2011). Control experiments in the absence of visiblelight and chloride showed that AgNP formation was ratherslow (order of days), which was revealed by a gradual increasein SPR-absorbance (at 415 nm) over a period of 14 days(Supplementary Fig. S8). However, the AgNP formation wasminimal and the solution remained colorless (aside from thecomplete lysis of bacteria) for the duration of 14 days in thepresence of chloride but without light. These findings suggestthat in the absence of visible light, a significant portion of Agions may remain bound to and form precipitates with chlo-ride. However, organic matter (bacteria, EPS, and/or NOM)may reduce Ag ions to Ag0 nuclei (Ishibashi et al., 1990;Maurer et al., 2012; Kang et al., 2014) in the absence ofchloride due to the differences in ORPs [Eq. (a), Table 2, andSupplementary Fig. S9]. For example, the reducing capacityof EPS w/NaCl was substantially greater than NOM mediumin the absence of light and, further, NOM medium was astronger reducing agent with light than without light (Sup-plementary Fig. S8). Ag0 nuclei formed are thermodynami-cally unstable. In a process to minimize surface energy, Ag0

nuclei thus formed as an aggregate to optimal-sized Agparticles, which may be stabilized by a layer of organicmatter in the medium. After this stage, larger particles growat the expense of smaller ones through the Ostwald ripeningprocess (Hoang et al., 2002) in which the smaller Ag particles

via oxidation dissolve in the medium by releasing Ag ions,which then migrate to the surface of larger AgNPs where theyundergo reduction via binding [Eq. (b), Table 2]. Such areduction of Ag ions at the silver surface lowers the surfaceenergy by increasing the size of the particles, which is con-sistent with the findings from the previous studies (Meisel,1979; Henglein and Meisel, 1998; Pillai and Kamat, 2004)[Eq. (4)].

It should be noted that neither AgNO3 nor the test media(colorless suspensions) exhibited any UV-Vis absorbance inthe 300–800 nm wavelength range. AgNP formation anddissolution processes are summarized in Table 2 and de-scribed here: Initially, Ag0 nuclei formation occurs in themedia, due to (1) differences in redox potential [Eq. (a)](Supplementary Fig. S8), (2) photo-thermal process [Eq.(c)] and photo-reduction of AgCl [Eq. (d)], and (3) photo-sensitized O2

� - [Eq. (e)] facilitated by OM, which includesEPS and NOM. Although bulk O2

� - concentration was low(see Supplementary Fig. S10), it was likely sufficient near thephotosensitizer (i.e., EPS and/or NOM) to initiate Ag0 nucleiformation (Jones et al., 2011). These nuclei while aggre-gating to an optimal size, via Ostwald ripening [Eq. (b)],began absorbing significant amounts of visible light due toplasmon excitation, which likely enhanced reduction poten-tial at the surface of the particles and subsequently resulted inrapid growth of AgNPs in the media [Eq. (f)]. This impliesthat SPR-activated Ag particles, covered by organic com-ponents of the media, served as heterogeneous nucleationsites favoring the formation and growth of AgNPs, via re-duction of Ag ions onto their crystalline faces (i.e., binding ofAg+ to surface of Ag particles) (Xue et al., 2008). SphericalAgNPs thus formed were stabilized by OM components ofthe media (AgNP diameters are shown in Table 1). Thus,light-induced heterogeneous nucleation, Ostwald ripening,and SPR of Ag particles play a key role in the AgNP for-mation in the presence of light. The apparent differences inthe lag period (nucleation) before AgNP growth in individualmedia could be attributed to factors, such as differences inORP potential (Supplementary Fig. S9), chloride content, andO2� - generation rates (Supplementary Fig. S10). No attempt

was made to compare the chemical nature of EPS, LB, orNOM medium, but based on previous studies it is likely thatthey share similar photoactive groups that mediate the re-duction of metal ions via photosensitized O2

� - production(Rose and Waite, 2005).

Real-time HSI microscopy observations of the interac-tions of Ag ions with the components of P. aeruginosa + EPS

Table 2. The AgNP Formation and Dissolution Processes in the Presence and Absence of Visible Light

Equation Reaction Process

a ÆOMæEhþ ÆAgþ æEh ¼ 0:799 V �!�!dark

Ag0 Dark nucleation; differences in redoxpotential

b Ag0þAg0/(Ag2)0/(Agn)0 Ostwald ripening (dark or light)

c ÆOMæþ ÆAgþ æ ��!��!hv=e�Ag0 Light nucleation; photo-thermal

d AgCl!!hvAg0þCl2 Light nucleation; photo-reduction

e ÆOMæþO2!!hvO��2 þAgþ/Ag0þO2 Photosensitization and nucleation

f Ag0þAgþ!!hv(Ag)þ2 ��!��!ÆOMæ� [(Ag)þ2 . . . ÆOMæ� ]!!!!hv

(Agn)0 . . . ÆOMæ Light-induced growth and stabilization

g (Agn)0 . . . ÆOMæþ 12

O2þ 2H þ!! Agþ þH2O Oxidation

346 BADIREDDY ET AL.

w/NaCl medium revealed that P. aeruginosa lost theirswimming ability after a few seconds of exposure to Ag ions(observed in real-time using darkfield HSI microscopy).During 30 min of exposure to Ag ions, Ag0 nucleation andgrowth occurred on the cell walls and poles, and in the bulkmedium (Fig. 2d–f and Supplementary Fig. S2) resulting inconsiderable cell damage at those regions as evidenced byhyperspectral, SEM, and TEM images (Figs. 2 and 6, andSupplementary Fig. S2). Ag0 nucleation and AgNP growthon the bacterial cell walls could be considered one of themechanisms through which cell lysis occurs in the visiblelight.

When AgNPs are discharged through human activity and/or natural processes into the surface waters, they undergooxidative dissolution, through which the release of Ag ions isfavored in the presence of dissolved oxygen and excessprotons [Table 2, Eq. (g)] (Liu and Hurt, 2010). As expected,the PVP-AgNPs were oxidized in the test media releasing Agions for the duration of the experiment (Fig. 7). Visible lighthad a negligible effect on PVP-AgNP dissolution rate in ul-trapure water (pH 6.8), as organic matter and/or chloridewere absent. However, the organic components in NOM andLB w/NaCl media showed negligible influence on the Agdissolution rate as evidenced from similar dissolution rates inultrapure water (light and dark), LB w/NaCl, and NOMmedia (Table 1). The PVP-AgNP oxidation was higher inEPS w/NaCl and highest in seawater medium (Fig. 7) whencompared with other media. The nanoparticle oxidation inEPS w/NaCl medium was higher, as EPS medium was highlyreducing for Ag ions causing the formation of smaller ‘‘new’’AgNPs around the PVP-AgNPs (Fig. 8f and SupplementaryFig. S7), and therefore likely favored increased dissolution.In addition, the dissolution was highest in seawater mediumpossibly due to the formation of soluble Ag-chloro com-plexes (Liu et al., 2010; Levard et al., 2013).

In surface waters, Ag ions are regarded as equilibriumspecies whereas AgNPs are unstable and nonpersistent spe-cies (Liu and Hurt, 2010; Liu et al., 2010; Zhang et al., 2011).Contrary to the common belief, the results of this study showthat the formation of AgNPs is a continual process, throughwhich ‘‘new’’ AgNPs form spontaneously in waters undervisible light illumination. However, in the absence of light,the nanoparticles dissolve through oxidation releasing Agions, which then transform into ‘‘new’’ AgNPs when re-ex-posed to visible light. The most likely pathways involvedduring Ag ions reduction to AgNPs (or formation) followedby re-oxidation (or dissolution) to form Ag ions are shown inFigure 9. Given the abundance of bacteria and its EPS invisible light-illuminated waters, their role in forming anddissolving metallic nanoparticles and the associated toxicityeffects should be taken into consideration when assessing theenvironmental fate, persistence, and toxicity of metallic na-noparticles, especially in sunlit waters.

Conclusions

When discharged into visible light-illuminated waters,Ag species (AgNPs, free or organic matter bound-Ag + , Ag-halides, or soluble Ag-complexes) are continually re-cycledand form ‘‘new’’ AgNPs that may be stabilized by environ-mental organic matter (e.g., EPS and NOM), which, in turn,may contribute to their persistence. EPS medium appears tobe a stronger reducing agent than NOM medium, with regardto the reduction of Ag ions to AgNPs. Organic componentsreduce Ag ions to Ag0 nuclei due to redox potential differ-ences, and Ag0 nuclei thus formed may then grow into largernanoparticles through (1) Ostwald ripening and light-inducedSPR of AgNPs, or (2) Ostwald ripening process in the ab-sence of light. The chloride in the media either enhancesAgNP formation with visible light or forms Ag precipitates

FIG. 9. Pathways throughwhich ‘‘new’’ AgNPs (Nano-Ag0) could form from dis-solved Ag (Ag + ), which maybe associated with environ-mental water componentssuch as chloride, EPS orsoluble microbial products(SMP), bacteria, and NOM(humic and fulvic acids), inthe presence of visible lightare shown here.

AGNP FORMATION IN VISIBLE LIGHT-EXPOSED WATERS 347

(or soluble-complexes) without light. Photosensitized su-peroxide radicals may also play an important role in Ag ionreduction. AgNPs formed in the visible-light illuminatedwaters stabilized by organic matter are likely to persist insurface waters, as the formation rates were higher (order ofminutes) than dissolution rates (order of days). Bacterial cellwalls may also serve as nucleation sites for Ag, throughwhich Ag ions deposit and transform into AgNPs, via themechanisms described earlier. As nanoparticles grow on thecell walls, they cause cell rupture. In addition to plasmonicactivity of AgNPs, redox and photosensitizing properties ofthe media may play a major role in the metallic nanoparticleformation and persistence in sunlit waters.

Acknowledgments

This work was funded through the Center for the En-vironmental Implications of Nanotechnology (CEINT) bythe NSF and the EPA under NSF Cooperative AgreementNumber EF-0830093. Any opinions, findings, conclusions,or recommendations expressed in this material are those ofthe author(s) and do not necessarily reflect the views of theNSF or the EPA. This work has not been subjected to EPAreview and no official endorsement should be inferred.

Author Disclosure Statement

No competing financial interests exist.

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