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Effects of Chloride and Ionic Strength on Physical Morphology, Dissolution, and Bacterial Toxicity of Silver Nanoparticles Bryant A. Chambers, A.R.M. Nabiul Afrooz, Sungwoo Bae, Nirupam Aich, Lynn E. Katz, Navid Bin Saleh, and Mary Jo Kirisits Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2013 Downloaded from http://pubs.acs.org on December 17, 2013
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Effects of Chloride and Ionic Strength on Physical
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Morphology, Dissolution, and Bacterial Toxicity of
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Silver Nanoparticles
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Bryant A. Chambers§, A.R.M. Nabiul Afrooz¶,#, Sungwoo Bae§, Nirupam Aich¶, Lynn Katz§,
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Navid B. Saleh¶, and Mary Jo Kirisits§,* §
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Department of Civil, Architectural, and Environmental Engineering, University of Texas at
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Austin, Austin, TX 78712 ¶
Department of Civil and Environmental Engineering, University of South Carolina, Columbia,
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SC 29208
10 11 12 13 14 #
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Current address:
Department of Civil, Architectural, and Environmental Engineering,
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University of Texas at Austin, Austin, TX 78712
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ABSTRACT
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In this study, we comprehensively evaluate chloride- and ionic-strength-mediated changes in
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the physical morphology, dissolution, and bacterial toxicity of silver nanoparticles (AgNPs),
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which are one of the most-used nanomaterials. The findings isolate the impact of ionic strength
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from that of chloride concentration. As ionic strength increases, AgNP aggregation likewise
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increases (such that the hydrodynamic radius [HR] increases), fractal dimension (Df) strongly
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decreases (providing increased available surface relative to suspensions with higher Df), and the
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release of Ag(aq) increases.
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reduced tolerance to AgNP exposure (i.e., toxicity increases) under higher ionic strength
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conditions. As chloride concentration increases, aggregates are formed (HR increases) but are
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dominated by AgCl0(s) bridging of AgNPs; relatedly, Df increases. Furthermore, AgNP
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dissolution strongly increases under increased chloride conditions, but the dominant, theoretical,
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equilibrium aqueous silver species shift to negatively charged AgClx(x-1)- species, which appear
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to be less toxic to E. coli. Thus, E. coli demonstrates increased tolerance to AgNP exposure
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under higher chloride conditions (i.e., toxicity decreases). Expression measurements of katE, a
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gene involved in catalase production to alleviate oxidative stress, support oxidative stress in E.
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coli as a result of Ag+ exposure. Overall, our work indicates that the environmental impacts of
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AgNPs must be evaluated under relevant water chemistry conditions.
With increased Ag+ in solution, Escherichia coli demonstrates
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INTRODUCTION
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Silver nanoparticles (AgNPs) are increasingly employed in consumer products (e.g., food
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storage bins and socks,1 catheters,2 and bandages3) because of their antimicrobial effects.4–6
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Increased AgNP usage translates to increased potential for their release to the environment.7
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AgNPs can exhibit toxicity to bacteria, and the mechanism of toxicity has been suggested to be
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based primarily on the aqueous free silver (Ag+) produced from AgNP dissolution.8–12
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Furthermore, current research suggests that localized interfacial interaction between AgNPs and
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bacteria might be an important delivery mechanism of aqueous silver (Ag(aq)) to bacteria.4,6
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Background chemistry can play a key role in modulating such interfacial interactions.
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Evaluations of AgNP toxicity to bacteria abound, but the media in which bacteria are exposed
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to AgNPs (hereafter called ‘exposure solutions’) vary widely. For instance, in a survey of
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several AgNP toxicity studies,6,10,11,13–21 ionic strength values ranged from 0.055 mM to as high
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as 171 mM, and chloride concentrations from 0 mM to as high as 171 mM (Table S1). However,
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as described below, the aqueous environment affects the stability of AgNPs, a factor that is
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tightly connected to toxicity.22–24 Water chemistry strongly impacts the physical and chemical characteristics of AgNPs;7,12,22,25–
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29
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stabilization.22,25–27 For instance, ions (e.g., Cl-, SO42-, NO3-, Ca2+) have been found to affect
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AgNP aggregation,4,22,25,27 and chloride has been linked to increased aggregation rates.22,27
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Additionally, chloride might cause the formation of AgCl0(s) shells and bridging between
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AgNPs,27 possibly reducing the available AgNP surface area and dramatically changing the
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surface characteristics. Given that physical/chemical changes occur in AgNPs due to water
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chemistry and that differences in physical parameters, such as size, have been linked to
changes in water chemistry have been found to cause aggregation, dissolution, or
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differences in toxicity,11 it follows that AgNP toxicity to bacteria also will be impacted by water
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chemistry.
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Only a few studies have directly characterized how toxicity is affected by the chemical
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conditions under which bacteria are exposed to AgNPs.22,23,25,30 The toxicity of AgNPs to
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bacteria has been shown to be impacted by the presence of specific ligands8,20,23 and oxygen9 in
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the exposure solution. Further, it is suggested that increased chloride and ionic strength enhance
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the release of Ag(aq) from AgNPs.27
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dependent on the Cl/Ag ratio, where bactericidal effects were linked to increased Ag(aq);23
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however, the experiments were conducted under conditions where ionic strength and chloride
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concentrations were changing simultaneously, such that the impact of chloride concentration was
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not separated from the impact of ionic strength on toxicity. Apart from the impact of chloride on
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AgNP dissolution, the aggregation rate of AgNPs increases with increasing chloride
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concentration and ionic strength.27,31 Thus, toxicity should be dependent on the chemistry of the
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exposure solution (e.g., chloride concentration and ionic strength), and differences in exposure
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solutions among AgNP toxicity studies likely complicate data interpretation and cross-study
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comparisons.
A recent study showed that AgNP dissolution was
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To date, no studies have comprehensively isolated the impact of variation in ionic strength
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from that of variation in chloride concentration on AgNP toxicity to bacteria. Furthermore,
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although the effect of aggregate structure on AgNP dissolution was reported recently,32 the effect
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of chloride – separate from the impact of ionic strength - on such structures is still unknown.
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The objective of the current study is to link characteristics of the exposure solution, specifically
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ionic strength and chloride concentration, to the tolerance of model bacterium Escherichia coli
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(E. coli) to AgNPs. Here tolerance is defined as the ability of a bacterium to survive an
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antimicrobial insult. Our systematic evaluation utilized three classes of ionic strength: high
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(~150 mM), medium (~40 mM), and low (~8 mM). Chloride concentrations were varied from 0
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to 140 mM to produce a variety of dominant aqueous silver-chloride species (AgClx(x-1)-).
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Particular chloride concentrations in this range are representative of wastewater treatment plant
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effluent33 and brackish groundwater.34,35 Light scattering techniques (dynamic and static) and
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microscopy were employed to obtain information about the physical attributes of the AgNPs. A
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viability-based tolerance assay was used to assess AgNP toxicity to E. coli.
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analysis was used to investigate changes in expression of an oxidative stress response gene due
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to AgNP exposure. Thus, this systematic evaluation of chloride- and ionic-strength-mediated
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physical/chemical changes in AgNPs and their impact on bacterial toxicity will be valuable in
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expanding our knowledge of the effect of water chemistry on the behavior of AgNPs.
A molecular
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MATERIALS AND METHODS
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AgNP Synthesis. Mercaptosuccinic-acid-capped AgNPs were synthesized by sodium
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borohydride reduction of silver nitrate, following the procedure in Chen and Kimura (1999).36
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Much like the capping agent citrate, MSA likely exists as carboxylate groups at the nanoparticle
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surface at near neutral pH. The protocol is summarized in the Supporting Information (SI).
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Bacterial Growth Conditions. A culture of E. coli MG4, a K-12 strain,37 was grown in
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Minimal Davis (MD) medium (700 mg/L KH2PO4, 200 mg/L K2HPO4, 500 mg/L sodium citrate,
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100 mg/L MgSO4, 1 g/L (NH4)2SO4, and 1 g/L glucose, pH 7.2) at 30 oC to ~1012 colony
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forming units (CFU)/mL and was inoculated to a 200-mL chemostat operated with MD medium
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at room temperature. Chemicals were obtained from Sigma-Aldrich and were ACS grade or
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higher. The chemostat was operated for at least 2 d before cells were removed for tolerance
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assays.
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Composition of the Exposure Solutions. The composition of the solutions in which E. coli
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was exposed to AgNPs was varied. Nine exposure solutions were designed (Table 1) to cover
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the range of ionic strength and chloride concentrations that have been used in the literature to test
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AgNP toxicity to bacteria (Table S1). Each exposure solution is denoted by a high, medium, or
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low ionic strength classification (Hµ, Mµ, and Lµ, respectively) followed by the millimolar
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chloride concentration (i.e., Hµ32 indicates an ionic strength of 154.0 mM and a chloride
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concentration of 32 mM). Hµ exposure solutions (ionic strength ~150 mM) mimic the ionic
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strength of phosphate-buffered saline (PBS) and some growth media used in AgNP toxicity
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studies (see studies listed in Table S1); Mµ solutions (ionic strength ~40 mM) mimic some
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growth media (see studies listed in Table S1); Lµ solutions (ionic strength ~8 mM) mimic
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typical freshwater. At each ionic strength, the chloride concentration was varied (0 to up to 140
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mM), using NaNO3 to maintain the chosen ionic strength. Although the range of chloride
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concentrations was selected primarily based on media used in AgNP toxicity studies, these
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chloride concentrations have environmental relevance. The lower chloride concentrations are
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consistent with wastewater treatment plant effluent (1.30 – 3.36 mM33); the higher
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concentrations are consistent with brackish groundwater.34,35 Ag(aq) speciation in each exposure
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solution was simulated using Visual MINTEQ (version 3.0, John Gustafsson, KTH, Sweden).
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Table 1. Composition of Exposure Solutions High ionic strength
Sample
Medium ionic strength
Low ionic strength
Hµ140
Hµ32
Hµ2.7
Hµ0
Mµ31
Mµ2.7
Mµ0
Lµ2.7
Lµ0
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
152.6
154.0
154.0
153.8
40.3
40.4
40.1
8.3
8.3
139.7
31.6
2.7
--
30.9
2.7
--
2.7
--
Hµ0
Mµ31
Mµ2.7
Mµ0
Lµ2.7
Lµ0
Parameter pH Ionic strength (mM) Chloride (mM)
Composition Compound (mM) NaCl KCl NaNO3 Na2HPO4 • 7 H 2O KH2PO4
Hµ140
Hµ32
Hµ2.7
137.1
28.9
--
--
28.2
--
--
--
--
2.7 --
2.7 107.9
2.7 137.1
-138.8
2.7 --
2.7 28.2
-30.6
2.7 --
-2.7
7.5
7.5
7.5
7.5
3.3
3.3
3.3
1.7
1.7
1.5
1.5
1.5
1.5
1.0
1.0
1.0
0.7
0.7
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Tolerance Studies. The tolerance of E. coli to AgNPs or dissolved silver (dosed as AgNO3) in
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the exposure solutions was tested by triplicate analyses of two biological replicates. Planktonic
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cells were removed from the chemostat and washed in the appropriate exposure solution. In a
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96-well microtiter plate, the exposure solution was dosed with 0-1 mg/L Ag as AgNPs or
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dissolved silver and inoculated to a final concentration of ~107 CFU/L. The plate was incubated
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statically for 5 h at 30 ˚C in the dark, and the viable cells remaining were enumerated in triplicate
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on Lysogeny Broth (Lennox) agar.
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Quantification and Characterization of Synthesized AgNPs. To quantify AgNP
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concentrations, 60 µL of AgNP stock were digested in 174 µL of concentrated nitric acid
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(~15M) overnight and diluted with distilled deionized water (DDI). Ag(aq) was quantified against
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a silver nitrate standard (SpexCertiPrep, Metuchen, NJ) using a Varian 710 (Agilent, Santa
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Clara, CA) inductively coupled plasma–optical emission spectrometer (ICP-OES) with an
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approximate limit of detection of 5 µg/L.
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The amount of Ag(aq) remaining in the AgNP stock after the synthesis reaction was assessed in
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two ways: dialyzing 1 mL of AgNP stock in pH 9 water with a 3.5-kDa molecular weight cut off
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(MWCO) dialysis membrane (Spectrum Labs, Rancho Dominguez, CA) for 14 h, changing the
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dialysate once after 6 h, or centrifuging the stock in an Amicon Ultra-4 centrifugal filter with a 3-
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kDa MWCO (Millipore, Bellerica, MA) for 40 min at 7,500 x g. Ag(aq) in the dialysate and
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permeate was quantified by ICP-OES.
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The size of the as-synthesized AgNPs (before suspension in an exposure solution) was
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characterized using transmission electron microscopy (TEM) (Tecnai Spirit, FEI, Hillsboro, OR).
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Samples were dropped on carbon-coated copper grids and were air dried for 10-20 min before
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analysis. Images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD,
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USA, http://imagej.nih.gov/ij), and statistical analyses were performed in Excel (Microsoft
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Corp., Redmond/WA).
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Characterization of AgNPs in Exposure Solutions. When added to an exposure solution, the
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physical/chemical AgNP characteristics were expected to change as a function of the chloride
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and ionic strength of the exposure solution. These changes were assessed as follows.
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The production of Ag(aq) in the exposure solutions was assessed. AgNPs were dosed (1 mg/L)
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to 3.4 mL of an exposure solution and incubated statically at 30 °C in the dark. Samples were
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withdrawn at 10 min and 1, 3, and 5 h and processed with an Amicon Ultra-4 3-kDa centrifugal
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filter (7,500 x g for 30 min) to separate Ag(aq) from AgNPs and aggregates. The permeate was
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acidified to a final concentration of 2% nitric acid for analysis by ICP-OES.
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Surface characteristics were examined qualitatively via surface plasmon resonance (SPR) with
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a UV-visible spectrophotometer (Agilent 8453, Santa Clara, CA). AgNPs were dispersed (1
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mg/L) in each exposure solution in a 10-cm quartz cuvette and incubated statically at 30 °C in
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the dark. Readings were taken at 10 min and 1, 3, and 5 h.
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The size and aggregate structure of the AgNPs in the exposure solutions were assessed. Light
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scattering measurements were conducted on an ALV-CGS/3 goniometer system (ALV-GmbH,
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Langen, Germany). The size of the aggregating AgNPs was measured over time via dynamic
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light scattering (DLS). The DLS protocol is described in detail elsewhere38,39 and is summarized
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in the SI. The mean hydrodynamic radius (HR) and standard deviation were computed over 1-h
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intervals (0 to 5 h) and are reported at the midpoint of each interval. Static light scattering (SLS)
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measurements were performed to describe the aggregate structure (i.e., fractal dimension) of the
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AgNPs. The SLS protocol is described in detail in Khan et al. (2013)39 and is summarized in the
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SI. Changes in AgNP morphology (e.g., independent particles versus bridged particles) and
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elemental composition were evaluated by scanning electron microscopy (SEM) and energy
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dispersive X-ray (EDX) spectroscopy, respectively, and these methods are summarized in the SI.
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Additionally, TEM was performed on AgNPs exposed to Hµ140 using an H8000 TEM (Hitachi
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High Technologies, Roselyn Heights, NY). Samples were dropped on carbon/Formvar®-coated
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copper grids and were air dried for 15-20 min before imaging.
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Reverse-Transcription, Quantitative Real-time Polymerase Chain Reaction (RT-qPCR).
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The expression of katE was studied to understand how E. coli responds to AgNPs in different
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exposure solutions. The cells were grown in batch culture in MD medium, pelleted, and washed
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in Lµ0. One-milliliter aliquots of washed cells were added to duplicate selected exposure
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solutions (Hµ0, Mµ31, and Lµ0) in the presence or absence of AgNPs at an initial cell
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concentration of 3x107 CFU/mL. Cells were incubated in the dark for 5 h with shaking at 30 °C.
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An aliquot from each flask was removed for cell enumeration by viable plate counts on
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Lysogeny Broth (Lennox) agar, and the remaining cells were pelleted for RNA isolation. RNA
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was extracted in duplicate from each flask and pooled, and cDNA was synthesized in duplicate
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reactions and pooled; detailed protocols are given in the SI.
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qPCR primers targeting a 190-bp katE fragment were designed using Primer3 software40 and
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E. coli K-12 substrain MG1655, which has a sequenced genome. qPCR was run on each cDNA
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sample in triplicate, according to the protocol in the SI. A katE standard curve was constructed
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using genomic DNA from E. coli MG4 (101-107 gene copies/reaction). A Student’s t-test was
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performed (α=0.05) in Minitab 16 (Minitab Inc., State College, PA).
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RESULTS AND DISCUSSION
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Characterization of Synthesized AgNPs. After synthesis, AgNPs in pH 9 DDI were
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characterized. TEM analysis showed spherical particles with an exponential distribution and
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average diameter of 2.9 nm (Fig. S1), in close agreement with the 2.2-nm diameter described in
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the original synthesis.36 A typical batch of AgNPs contained 220 mg/L total silver, of which 7-9
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µg/L was residual Ag(aq). When the AgNP stock was diluted for bacterial tolerance studies (≤ 1
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mg/L), the residual Ag(aq) concentration was orders of magnitude below inhibitory levels.
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Aggregation. We used SPR to generally assess AgNP aggregation under different ionic strength and chloride conditions (Fig. 1).
SPR has been used previously to qualitatively
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characterize AgNPs.24,31 AgNPs typically have an extinction peak near 400 nm,31,41 but this can
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shift based on the local refractive index and NP diameter.42–44 a)
Hµ0 Mµ0 Lµ0
0.6
0.4
0.2
300
0.6
0.4
0.2
400
500
600
300
700
210
Mµ31 Mµ0
0.6
0.8
Extinction (AU)
Extinction (AU)
c)
400
500
600
700
Wavelength (nm)
Wavelength (nm)
0.8
Hµ0 Mµ0 Lµ0
0.0
0.0
0.4
0.2
d)
Mµ31 Mµ0
0.6
0.4
0.2
0.0
0.0 300
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b)
0.8
Extinction (AU)
Extinction (AU)
0.8
400
500
600
Wavelength (nm)
700
300
400
500
600
700
Wavelength (nm)
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Figure 1. SPR spectra of AgNPs in selected exposure solutions. AgNPs were probed by SPR as a
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function of ionic strength at (a) 10 min and (b) 5 h and as a function of chloride concentration at
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(c) 10 min and (d) 5 h.
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Fig. 1a–b highlights temporal changes in the SPR spectra as a function of ionic strength at a
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constant chloride concentration (0 mM). At the lowest ionic strength (Lµ0), the original AgNP
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extinction peak intensity remained nearly constant over time, indicating AgNP stability.
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However, at increased ionic strengths (Mµ0 and Hµ0), a rapid decrease in the original AgNP
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extinction peak intensity and peak broadening occurred, which suggests the depletion of stable
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AgNPs and the formation of aggregates, respectively.22 Relatedly, the DLS data show that the
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average HR at 5 h increased from 33.6±0.7 nm for Lµ0 to 51.2±1.2 nm for Hµ0 (Fig.
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S2a). Thus, the aggregation behavior observed via SPR, which is a technique that probes
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individual nano-scale interactions, is complemented by DLS, a technique that measures average
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suspension properties. Fig. 1c–d highlights temporal changes in the SPR spectra as a function of
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chloride concentration at constant ionic strength (~40 mM); the lowest and highest chloride
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concentrations at this ionic strength (0 and 31 mM chloride, respectively) were assessed because
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the changes in the SPR spectra as a function of chloride concentration were not nearly as
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dramatic as those as a function of ionic strength. Over time, both SPR spectra (Mµ0 and Mµ31)
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show reduction in the original AgNP extinction peak intensity as well as peak broadening and
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formation of a secondary peak at 564 nm. However, the peak intensity reduction at 390 nm is
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not as significant in the Mµ31 sample, suggesting less aggregation of primary AgNPs in the
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presence of chloride.
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aggregation but cause the formation of larger overall AgNP/AgCl aggregates. This is supported
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by DLS, which interrogates the overall aggregate size (AgNP plus AgCl bridging), where the
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average HR at 5 h increased from 33.6±0.7 nm for Mµ0 to 158.4±70.5 nm for Mµ31 (Fig. S2b).
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In contrast, SPR probes individual nano-scale interactions, and it indicates little effect of chloride
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on those AgNPs. A similar trend was observed in the SPR spectra for AgNPs exposed to Hµ0
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and Hµ140 (data not shown), where the presence of chloride appeared to stabilize AgNPs
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relative to the absence of chloride. Overall, the SPR data suggest that ionic strength is a major
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factor in AgNP destabilization and chloride acts to stabilize AgNPs. Evidence of AgCl bridging
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to stabilize AgNPs is provided in the Aggregate Structure section.
That is, AgCl bridging between AgNPs might reduce AgNP-AgNP
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Dissolution. The release of Ag(aq) due to the dissolution of AgNPs under different ionic
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strength and chloride conditions was examined (Fig. 2). The release of Ag(aq) from AgNPs is
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dependent upon oxidation,8,23 and we detected Ag(aq) in the exposure solutions dosed with
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AgNPs. In the absence of chloride, ionic strength only slightly impacted the initial release (at 10
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min) of Ag(aq) (Fig. 2 inset). However, for a constant ionic strength, the initial release of Ag(aq)
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from AgNPs was strongly affected by the presence of chloride in the exposure solution (Fig. 2
249
inset).
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dissolution.23,45 We continued to monitor Ag(aq) over 5 h, which corresponds to the timeframe of
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our tolerance studies, and only saw continued AgNP dissolution at the highest chloride
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concentration (Hµ140, Fig. 2). This is consistent with previous studies that have suggested that
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chloride might act catalytically in the dissolution of AgNPs.46,47 Thus, our data indicate that
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chloride concentration is more important than ionic strength in driving the production of Ag(aq)
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from AgNP dissolution.
These data are consistent with previous literature findings that show rapid AgNP
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500 400 300
Aqueous silver conc. (µ g/L)
Aqueous silver conc. (µg/L)
600 80
60
Mµ31 Mµ2.7 Mµ0 Lµ0
4
5
40
20
0
Dissolution in first 10 min
200 100 0 0
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Hµ140 Hµ32 Hµ2.7 Hµ0
1
2
3
Time (hr)
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Figure 2. Dissolution of AgNPs (dosed at 1 mg/L) over time as a function of ionic strength and
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chloride concentration, where the inset highlights the release of Ag(aq) after 10 min. Data points
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are the average of triplicate experiments, and error bars show one standard deviation.
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Aggregate Structure. Aggregate structure formation under different ionic strength and
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chloride conditions was examined via SLS (Fig. 3). For a constant chloride concentration (0
264
mM), the fractal dimension (Df) decreased with increasing ionic strength (Fig. 3a).
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observation is consistent with classical electrostatic screening-induced fractal formation of
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colloids, as observed previously.39 Higher ionic strength reduces the electrostatic potential,
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which likely imparted ‘stickiness’ to the AgNPs and resulted in lower Df (i.e., a more fractal
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structure as compared to a more densely packed aggregate in Lµ0). On the other hand, at
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constant ionic strength (~40 mM), increased chloride concentrations showed increased Df (Fig.
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3b). This shift in structure toward a more compact form in the presence of chloride supports the
This
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bridging of AgNPs via silver chloride (AgCl0(s)) formation. Using TEM, a similar observation of
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AgCl0(s)-induced bridging of AgNPs has been observed previously.45 Hµ0 Mµ0 Lµ0
3.0
2.5
2.0
1.5
1.0
After 3 hr
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Mµ31 Mµ2.7 Mµ0
b) Fractal dimension, Df
Fractal dimension, Df
a)
3.0
2.5
2.0
1.5
1.0
After 5 hr
After 3 hr
After 5 hr
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Figure 3. Fractal dimension of AgNPs as a function of (a) ionic strength and (b) chloride
275
concentration.
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While light-scattering techniques were used to assess aggregate size and structure, SEM was
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used to provide visual evidence of morphological changes under different water chemistry
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conditions.
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concentrations, as shown for AgNPs in Hµ140 (Fig. S3c-d) as compared to AgNPs in Mµ31 and
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Hµ0 (Fig. S3a-b). Furthermore, TEM of AgNPs in Hµ140 shows a change in contrast at the
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particle-particle attachment regions (indicated by arrows in Fig. S3e), which is indicative of
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bridging through chloride precipitation. EDX confirms the presence of Ag in the pristine AgNPs
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(Fig. S4a); Na and Si peaks in the spectrum are likely due to the glass slide used for SEM, as
284
these peaks appear in the spectra for all tested exposure solutions. EDX also confirms the
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presence of Ag and Cl on the aggregated particle clusters in Mµ31 and Hµ140, further
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supporting the reaction of chloride on AgNP surfaces (Fig. S4b-c).
SEM provides support for AgNP bridging in the presence of high chloride
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Impact of Ionic Strength and Chloride on Bacterial Tolerance to AgNPs. As demonstrated
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above, the chemistry of the exposure solution impacts AgNP stability. Next we examined the
290
tolerance of E. coli to AgNPs in the nine exposure solutions; selected results are shown herein.
291
Fig. 4 demonstrates the tolerance of E. coli to AgNPs as a function of ionic strength at a
292
constant chloride concentration (0 mM). At higher AgNP concentrations (greater than 50 µg/L),
293
E. coli survival is less than 10 %. At lower AgNP concentrations (15.6 and 31.2 µg/L), E. coli
294
has the lowest tolerance to AgNPs under the highest ionic strength condition (Hµ0). This is
295
likely due to one or more of the following reasons. (1) Hµ0 induces higher AgNP dissolution as
296
compared to lower ionic strength exposure solutions (e.g., 15.3 and 5.6 µg/L Ag(aq) measured in
297
the bulk solution after a 3-h exposure of AgNPs to Hµ0 and Lµ0, respectively, Fig. 2), and Ag+
298
is biocidal to bacteria.8,48,49 (2) Hµ0 causes the most compression of the electric double layer,
299
which would facilitate closer proximity of cells to AgNPs. (3) Hµ0 induces a different aggregate
300
structure as compared to lower ionic strengths. At higher ionic strengths, AgNPs were found to
301
form more fractal structures (lower Df) as compared to more densely packed structures at lower
302
ionic strengths (Fig. 3a), which leads to more available surface area for interaction with bacteria.
303
Close interaction of bacteria with AgNPs could facilitate the delivery of Ag+ to the cells as the
304
AgNPs dissolve, resulting in reduced tolerance of E. coli to AgNPs at higher ionic strengths.
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Hµ 0 Mµ 0 Lµ 0
1.0 0.8 0.6 0.4 0.2 0.0 0
305
50
100
150
200 1000
Silver nanoparticle dosage (µg/L)
306
Figure 4. Impact of ionic strength on the tolerance of E. coli to AgNPs. Surviving cells are
307
shown as a function of AgNP dose. Data are averages of triplicate assays of biological
308
duplicates, and error bars represent one standard deviation.
309 310
Fig. 5a demonstrates the tolerance of E. coli to AgNP exposure as a function of chloride
311
concentration at constant ionic strength (~40 mM). E. coli has the highest tolerance to AgNPs
312
under the highest chloride condition shown (Mµ31). This is likely due to one or both of the
313
following reasons. (1) Higher chloride concentrations result in more AgNP dissolution (Fig. 2),
314
but they produce lower concentrations of Ag+ and the neutral species AgCl0(aq) (compare Mµ31
315
to Mµ2.7 in Fig. 5b). While some work has proposed that only the total concentration of
316
aqueous AgClx(x-1)- species, rather than their speciation, controls the overall toxicity to E. coli, 23
317
our work makes the claim that AgClx(x-1)- speciation matters. As shown in Fig. S5 (where
318
aqueous silver was dosed to E. coli), the tolerance of E. coli to Ag(aq) increases as the charge on
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the dominant AgClx(x-1)- species decreases (e.g., greater tolerance when AgCl2- is the dominant
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species as compared to when Ag+ is the dominant species). However, given the values of the
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AgClx(x-1)- stability constants, it is not possible to isolate the toxicity impact of each AgClx(x-1)-
322
species. (2) Chloride induces changes in AgNP aggregate structure. When chloride is low or
323
absent (Mµ2.7 and Mµ0, respectively), AgNPs form more fractal structures (Fig. 3b). This leads
324
to higher available surface area for interaction with bacteria, likely facilitating the delivery of
325
Ag(aq) to the cells as the AgNPs dissolve, resulting in reduced tolerance of E. coli to AgNPs at
326
lower chloride concentrations. Our results are in contrast with an earlier study23 that showed
327
reduced tolerance of E. coli to AgNPs in the presence of higher chloride concentrations, but
328
those results are likely confounded by simultaneous changes in ionic strength and chloride
329
concentration.
1.0
a)
Mµ31 Mµ2.7 Mµ0
Surviving cells, C/C0
0.5 0.08 0.06 0.04 0.02 0.00 0
331
200
400
600
800
1000
Silver nanoparticle dosage (µg/L)
Species concentration (µg/L)
330 40
b)
AgNO3 2-
AgCl3 30
-
AgCl2 AgCl + Ag
20
10
0 Mµ31
Mµ2.7
Mµ0
Exposure solution
332
Figure 5. Impact of chloride on tolerance of E. coli to AgNPs. (a) Surviving cells as a function
333
of AgNP dose; (b) theoretical equilibrium AgClx(x-1)- speciation of Ag(aq) produced 5 h after 1
334
mg/L AgNPs are dosed to a particular exposure solution, where Ag(aq) for each exposure solution
335
is shown in Fig. 2. Cell data are the averages of triplicate assays of biological duplicates, and
336
error bars represent one standard deviation.
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Oxidative Stress in E. coli due to AgNP Exposure. To further assess the effect of AgNPs on
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E. coli under different ionic strength and chloride conditions, we analyzed the expression of a
339
gene linked to oxidative stress, katE.50,51
340
hydroperoxidase II and is responsible for alleviating the effects of oxidative stress and also is
341
linked to chromate resistance.51,52 Catalase hydroperoxidase II converts hydrogen peroxide to
342
molecular oxygen and water and limits the transition-metal-facilitated formation of hydroxyl
343
radicals via a Fenton-type reaction.50 While the extent to which AgNP toxicity is linked to
344
reactive oxygen species (ROS) formation is still debated,8,12,53,54 Ag+ toxicity has been linked to
345
ROS formation;55 furthermore, this study (Fig. 5b) corroborates previous findings9,11,12 that
346
AgNP toxicity is linked to Ag+ (though other AgClx(x-1)- species also might contribute to
347
toxicity). We examined the expression of katE in the presence of a low concentration of AgNPs
348
(20 µg/L), which killed only a fraction of the exposed population (Figs. 4 and 5a).
katE encodes a component of the catalase
349
The expression of katE under the tested conditions is shown in Fig. 6. katE expression
350
increased significantly (p-value=0.001) in Hµ0 in the presence of AgNPs (Hµ0-AgNP) as
351
compared to the control (absence of AgNPs, Hµ0-Ctrl). By comparison, katE expression did not
352
increase significantly (p-value=0.194) in Lµ0 in the presence of AgNPs (Lµ0-AgNP) as
353
compared to its control (Lµ0-Ctrl). This is consistent with the AgNP dissolution data, which
354
showed greater Ag(aq) in the bulk solution when AgNPs were dosed to Hµ0 as compared to Lµ0
355
(Fig. 2 inset). Relatedly, katE expression did not increase significantly (p-value=0.077) in Mµ31
356
in the presence of AgNPs (Mµ31-AgNP) as compared to its control (Mµ31-Ctrl). This is
357
consistent with theoretical equilibrium speciation for AgClx(x-1)-, which indicates that Ag+ in the
358
Mµ31 bulk solution is inconsequential (Fig. 5b). Overall, these data support oxidative stress in
359
E. coli as a result of Ag+ exposure.
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katE transcripts/ng cDNA
8000 7000 6000
Hµ0-AgNP Hµ0-Ctrl Mµ31-AgNP Mµ31-Ctrl Lµ0-AgNP Lµ0-Ctrl
5000 4000 3000 2000 1000
Exposure solution
361 362
Figure 6. katE expression in E. coli as a function of AgNP exposure under different ionic
363
strength and chloride conditions. Gene expression was measured in three exposure solutions
364
(Hµ0, Mµ31, and Lµ0) in the presence of AgNPs and the absence of AgNPs (controls). Data are
365
averages of triplicate qPCR reactions from duplicate biological experiments. Error bars represent
366
one standard deviation.
367 368
Mechanism of Toxicity. The key mechanism underlying the observed antimicrobial activity of
369
AgNPs in this study is the role of background electrolytes in altering AgNP
370
physical/chemical properties. Both ionic strength and chloride concentration affect the physical
371
morphology, dissolution, and bacterial toxicity of AgNPs (Fig. 7). As ionic strength increases,
372
AgNP aggregation likewise increases (such that the HR increases), Df strongly decreases
373
(providing increased available surface relative to suspensions with higher Df), the release of
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Ag(aq) increases, and E. coli demonstrates reduced tolerance to the AgNP exposure (i.e., toxicity
375
increases). As chloride concentration increases, aggregates are formed but are dominated by
376
AgCl0(s) bridging of AgNPs; relatedly, the fractal dimension increases. Furthermore, AgNP
377
dissolution is strongly increased under increased chloride conditions, but the dominant aqueous
378
silver species shift to negatively charged AgClx(x-1)- species, which appear to be less toxic to E.
379
coli (Fig. S5). Thus, E. coli demonstrates increased tolerance to the AgNP exposure under
380
higher chloride conditions (i.e., toxicity decreases).
381 382
Figure 7. Model of the effects of chloride and ionic strength on physical morphology,
383
dissolution, and bacterial toxicity of AgNPs.
384
Previous work on the antimicrobial activity of AgNPs reports the complexity in assessing
385
dissolution of AgNPs in the presence of chloride ions.23 Our study is the first to postulate the
386
correlation of observed toxicity of AgNPs with their dissolution and physical characteristics (i.e.,
387
aggregation,
388
conditions. Moreover, the correlation of toxicity with Ag(aq) speciation and the use of gene
389
expression analysis to examine the role of ROS in the observed toxicity of AgNPs are important
fractal
structure,
particle
bridging)
for
a
wide
range
of
chemical
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components of our mechanistic analysis. Furthermore, the findings in this study allow us to
391
draw new conclusions about the impact of chloride, separate from the impact of ionic strength,
392
on the dissolution, physical characteristics, and toxicity of AgNPs.
393 394
Environmental Implications. With respect to bacterial toxicity testing for AgNPs, it is
395
important to recognize that dramatic changes in physical morphology and dissolution can occur
396
as a result of the chosen water chemistry conditions.
397
conditions of elevated ionic strength and chloride, but this will not typically reflect relevant
398
environmental conditions where bacteria might be exposed to AgNPs (e.g., in wastewater
399
treatment). In the case of E. coli, it is likely that the increased AgNP toxicity due to high ionic
400
strength conditions would just balance the decreased AgNP toxicity due to high chloride
401
conditions (Fig. 7), but this fortuitous occurrence might not be true for all microorganisms.
402
Thus, toxicity assessments of NPs to microorganisms should be conducted under
403
environmentally relevant water chemistry conditions. Furthermore, the environmental impacts
404
of AgNPs must consider dissolution, speciation, and aggregation for the particular water
405
chemistry of interest.
406
properties might influence the manifestation of the AgNP chemical behavior (e.g., fractal-
407
structure-dependent dissolution), which can then affect toxicity.
Toxicity testing often occurs under
For instance, environmental conditions influencing AgNP physical
408 409 410 411
ASSOCIATED CONTENT Supporting Information. Detailed information regarding methods and additional results. This material is available free of charge via the Internet at http://pubs.acs.org.
412
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AUTHOR INFORMATION
414
*Corresponding Author
415
Mary Jo Kirisits
416
The University of Texas at Austin
417
Department of Civil, Architectural, and Environmental Engineering
418
301 East Dean Keeton Street
419
Austin, Texas 78712
420
Phone: (512) 232-7120
421
Fax: (512) 471-0592
422
E-mail: [email protected]
423 424
ACKNOWLEDGMENT
425
The authors thank the National Science Foundation (CBET 0846719 to M.J. Kirisits) for
426
funding and the following people for technical assistance: Taegyu Kim (tolerance assays),
427
Colleen Lyons (TEM), and Gustavo Ochoa and Katie Speights (laboratory support).
428 429 430 431 432
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(52) Rudd, K. E. EcoGene: a genome sequence database for Escherichia coli K-12. Nucleic Acids Research 2000, 28 (1), 60–64. DOI 10.1093/nar/28.1.60.
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(53) Hwang, E. T.; Lee, J. H.; Chae, Y. J.; Kim, Y. S.; Kim, B. C.; Sang, B. I.; Gu, M. B.
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Analysis of the toxic mode of action of silver nanoparticles using stress-specific bioluminescent
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bacteria. Small 2008, 4 (6), 746–750. DOI 10.1002/smll.200700954.
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(54) Choi, O.; Hu, Z. Size dependent and reactive oxygen species related nanosilver toxicity
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to nitrifying bacteria. Environmental Science & Technology 2008, 42 (12), 4583–4588. DOI
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10.1021/es703238h.
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(55) Park, H. J.; Kim, J. Y.; Kim, J.; Lee, J. H.; Hahn, J. S.; Gu, M. B.; Yoon, J. Silver-ion-
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mediated reactive oxygen species generation affecting bactericidal activity. Water Research
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2009, 43 (4), 1027–1032. DOI 10.1016/j.watres.2008.12.002.
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Effects of Chloride and Ionic Strength on Physical Morphology, Dissolution, and Bacterial Toxicity of Silver Nanoparticles Bryant A. Chambers, A.R.M. Nabiul Afrooz, Sungwoo Bae, Nirupam Aich, Lynn E. Katz, Navid Bin Saleh, and Mary Jo Kirisits Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2013 Downloaded from http://pubs.acs.org on December 17, 2013
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Effects of Chloride and Ionic Strength on Physical
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Morphology, Dissolution, and Bacterial Toxicity of
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Silver Nanoparticles
4
Bryant A. Chambers§, A.R.M. Nabiul Afrooz¶,#, Sungwoo Bae§, Nirupam Aich¶, Lynn Katz§,
5
Navid B. Saleh¶, and Mary Jo Kirisits§,* §
6
Department of Civil, Architectural, and Environmental Engineering, University of Texas at
7 8
Austin, Austin, TX 78712 ¶
Department of Civil and Environmental Engineering, University of South Carolina, Columbia,
9
SC 29208
10 11 12 13 14 #
15
Current address:
Department of Civil, Architectural, and Environmental Engineering,
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University of Texas at Austin, Austin, TX 78712
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ABSTRACT
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In this study, we comprehensively evaluate chloride- and ionic-strength-mediated changes in
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the physical morphology, dissolution, and bacterial toxicity of silver nanoparticles (AgNPs),
20
which are one of the most-used nanomaterials. The findings isolate the impact of ionic strength
21
from that of chloride concentration. As ionic strength increases, AgNP aggregation likewise
22
increases (such that the hydrodynamic radius [HR] increases), fractal dimension (Df) strongly
23
decreases (providing increased available surface relative to suspensions with higher Df), and the
24
release of Ag(aq) increases.
25
reduced tolerance to AgNP exposure (i.e., toxicity increases) under higher ionic strength
26
conditions. As chloride concentration increases, aggregates are formed (HR increases) but are
27
dominated by AgCl0(s) bridging of AgNPs; relatedly, Df increases. Furthermore, AgNP
28
dissolution strongly increases under increased chloride conditions, but the dominant, theoretical,
29
equilibrium aqueous silver species shift to negatively charged AgClx(x-1)- species, which appear
30
to be less toxic to E. coli. Thus, E. coli demonstrates increased tolerance to AgNP exposure
31
under higher chloride conditions (i.e., toxicity decreases). Expression measurements of katE, a
32
gene involved in catalase production to alleviate oxidative stress, support oxidative stress in E.
33
coli as a result of Ag+ exposure. Overall, our work indicates that the environmental impacts of
34
AgNPs must be evaluated under relevant water chemistry conditions.
With increased Ag+ in solution, Escherichia coli demonstrates
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INTRODUCTION
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Silver nanoparticles (AgNPs) are increasingly employed in consumer products (e.g., food
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storage bins and socks,1 catheters,2 and bandages3) because of their antimicrobial effects.4–6
39
Increased AgNP usage translates to increased potential for their release to the environment.7
40
AgNPs can exhibit toxicity to bacteria, and the mechanism of toxicity has been suggested to be
41
based primarily on the aqueous free silver (Ag+) produced from AgNP dissolution.8–12
42
Furthermore, current research suggests that localized interfacial interaction between AgNPs and
43
bacteria might be an important delivery mechanism of aqueous silver (Ag(aq)) to bacteria.4,6
44
Background chemistry can play a key role in modulating such interfacial interactions.
45
Evaluations of AgNP toxicity to bacteria abound, but the media in which bacteria are exposed
46
to AgNPs (hereafter called ‘exposure solutions’) vary widely. For instance, in a survey of
47
several AgNP toxicity studies,6,10,11,13–21 ionic strength values ranged from 0.055 mM to as high
48
as 171 mM, and chloride concentrations from 0 mM to as high as 171 mM (Table S1). However,
49
as described below, the aqueous environment affects the stability of AgNPs, a factor that is
50
tightly connected to toxicity.22–24 Water chemistry strongly impacts the physical and chemical characteristics of AgNPs;7,12,22,25–
51 52
29
53
stabilization.22,25–27 For instance, ions (e.g., Cl-, SO42-, NO3-, Ca2+) have been found to affect
54
AgNP aggregation,4,22,25,27 and chloride has been linked to increased aggregation rates.22,27
55
Additionally, chloride might cause the formation of AgCl0(s) shells and bridging between
56
AgNPs,27 possibly reducing the available AgNP surface area and dramatically changing the
57
surface characteristics. Given that physical/chemical changes occur in AgNPs due to water
58
chemistry and that differences in physical parameters, such as size, have been linked to
changes in water chemistry have been found to cause aggregation, dissolution, or
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differences in toxicity,11 it follows that AgNP toxicity to bacteria also will be impacted by water
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chemistry.
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Only a few studies have directly characterized how toxicity is affected by the chemical
62
conditions under which bacteria are exposed to AgNPs.22,23,25,30 The toxicity of AgNPs to
63
bacteria has been shown to be impacted by the presence of specific ligands8,20,23 and oxygen9 in
64
the exposure solution. Further, it is suggested that increased chloride and ionic strength enhance
65
the release of Ag(aq) from AgNPs.27
66
dependent on the Cl/Ag ratio, where bactericidal effects were linked to increased Ag(aq);23
67
however, the experiments were conducted under conditions where ionic strength and chloride
68
concentrations were changing simultaneously, such that the impact of chloride concentration was
69
not separated from the impact of ionic strength on toxicity. Apart from the impact of chloride on
70
AgNP dissolution, the aggregation rate of AgNPs increases with increasing chloride
71
concentration and ionic strength.27,31 Thus, toxicity should be dependent on the chemistry of the
72
exposure solution (e.g., chloride concentration and ionic strength), and differences in exposure
73
solutions among AgNP toxicity studies likely complicate data interpretation and cross-study
74
comparisons.
A recent study showed that AgNP dissolution was
75
To date, no studies have comprehensively isolated the impact of variation in ionic strength
76
from that of variation in chloride concentration on AgNP toxicity to bacteria. Furthermore,
77
although the effect of aggregate structure on AgNP dissolution was reported recently,32 the effect
78
of chloride – separate from the impact of ionic strength - on such structures is still unknown.
79
The objective of the current study is to link characteristics of the exposure solution, specifically
80
ionic strength and chloride concentration, to the tolerance of model bacterium Escherichia coli
81
(E. coli) to AgNPs. Here tolerance is defined as the ability of a bacterium to survive an
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antimicrobial insult. Our systematic evaluation utilized three classes of ionic strength: high
83
(~150 mM), medium (~40 mM), and low (~8 mM). Chloride concentrations were varied from 0
84
to 140 mM to produce a variety of dominant aqueous silver-chloride species (AgClx(x-1)-).
85
Particular chloride concentrations in this range are representative of wastewater treatment plant
86
effluent33 and brackish groundwater.34,35 Light scattering techniques (dynamic and static) and
87
microscopy were employed to obtain information about the physical attributes of the AgNPs. A
88
viability-based tolerance assay was used to assess AgNP toxicity to E. coli.
89
analysis was used to investigate changes in expression of an oxidative stress response gene due
90
to AgNP exposure. Thus, this systematic evaluation of chloride- and ionic-strength-mediated
91
physical/chemical changes in AgNPs and their impact on bacterial toxicity will be valuable in
92
expanding our knowledge of the effect of water chemistry on the behavior of AgNPs.
A molecular
93 94
MATERIALS AND METHODS
95
AgNP Synthesis. Mercaptosuccinic-acid-capped AgNPs were synthesized by sodium
96
borohydride reduction of silver nitrate, following the procedure in Chen and Kimura (1999).36
97
Much like the capping agent citrate, MSA likely exists as carboxylate groups at the nanoparticle
98
surface at near neutral pH. The protocol is summarized in the Supporting Information (SI).
99 100
Bacterial Growth Conditions. A culture of E. coli MG4, a K-12 strain,37 was grown in
101
Minimal Davis (MD) medium (700 mg/L KH2PO4, 200 mg/L K2HPO4, 500 mg/L sodium citrate,
102
100 mg/L MgSO4, 1 g/L (NH4)2SO4, and 1 g/L glucose, pH 7.2) at 30 oC to ~1012 colony
103
forming units (CFU)/mL and was inoculated to a 200-mL chemostat operated with MD medium
104
at room temperature. Chemicals were obtained from Sigma-Aldrich and were ACS grade or
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higher. The chemostat was operated for at least 2 d before cells were removed for tolerance
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assays.
107 108
Composition of the Exposure Solutions. The composition of the solutions in which E. coli
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was exposed to AgNPs was varied. Nine exposure solutions were designed (Table 1) to cover
110
the range of ionic strength and chloride concentrations that have been used in the literature to test
111
AgNP toxicity to bacteria (Table S1). Each exposure solution is denoted by a high, medium, or
112
low ionic strength classification (Hµ, Mµ, and Lµ, respectively) followed by the millimolar
113
chloride concentration (i.e., Hµ32 indicates an ionic strength of 154.0 mM and a chloride
114
concentration of 32 mM). Hµ exposure solutions (ionic strength ~150 mM) mimic the ionic
115
strength of phosphate-buffered saline (PBS) and some growth media used in AgNP toxicity
116
studies (see studies listed in Table S1); Mµ solutions (ionic strength ~40 mM) mimic some
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growth media (see studies listed in Table S1); Lµ solutions (ionic strength ~8 mM) mimic
118
typical freshwater. At each ionic strength, the chloride concentration was varied (0 to up to 140
119
mM), using NaNO3 to maintain the chosen ionic strength. Although the range of chloride
120
concentrations was selected primarily based on media used in AgNP toxicity studies, these
121
chloride concentrations have environmental relevance. The lower chloride concentrations are
122
consistent with wastewater treatment plant effluent (1.30 – 3.36 mM33); the higher
123
concentrations are consistent with brackish groundwater.34,35 Ag(aq) speciation in each exposure
124
solution was simulated using Visual MINTEQ (version 3.0, John Gustafsson, KTH, Sweden).
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Table 1. Composition of Exposure Solutions High ionic strength
Sample
Medium ionic strength
Low ionic strength
Hµ140
Hµ32
Hµ2.7
Hµ0
Mµ31
Mµ2.7
Mµ0
Lµ2.7
Lµ0
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
7.4
152.6
154.0
154.0
153.8
40.3
40.4
40.1
8.3
8.3
139.7
31.6
2.7
--
30.9
2.7
--
2.7
--
Hµ0
Mµ31
Mµ2.7
Mµ0
Lµ2.7
Lµ0
Parameter pH Ionic strength (mM) Chloride (mM)
Composition Compound (mM) NaCl KCl NaNO3 Na2HPO4 • 7 H 2O KH2PO4
Hµ140
Hµ32
Hµ2.7
137.1
28.9
--
--
28.2
--
--
--
--
2.7 --
2.7 107.9
2.7 137.1
-138.8
2.7 --
2.7 28.2
-30.6
2.7 --
-2.7
7.5
7.5
7.5
7.5
3.3
3.3
3.3
1.7
1.7
1.5
1.5
1.5
1.5
1.0
1.0
1.0
0.7
0.7
127 128
Tolerance Studies. The tolerance of E. coli to AgNPs or dissolved silver (dosed as AgNO3) in
129
the exposure solutions was tested by triplicate analyses of two biological replicates. Planktonic
130
cells were removed from the chemostat and washed in the appropriate exposure solution. In a
131
96-well microtiter plate, the exposure solution was dosed with 0-1 mg/L Ag as AgNPs or
132
dissolved silver and inoculated to a final concentration of ~107 CFU/L. The plate was incubated
133
statically for 5 h at 30 ˚C in the dark, and the viable cells remaining were enumerated in triplicate
134
on Lysogeny Broth (Lennox) agar.
135 136
Quantification and Characterization of Synthesized AgNPs. To quantify AgNP
137
concentrations, 60 µL of AgNP stock were digested in 174 µL of concentrated nitric acid
138
(~15M) overnight and diluted with distilled deionized water (DDI). Ag(aq) was quantified against
139
a silver nitrate standard (SpexCertiPrep, Metuchen, NJ) using a Varian 710 (Agilent, Santa
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Clara, CA) inductively coupled plasma–optical emission spectrometer (ICP-OES) with an
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approximate limit of detection of 5 µg/L.
142
The amount of Ag(aq) remaining in the AgNP stock after the synthesis reaction was assessed in
143
two ways: dialyzing 1 mL of AgNP stock in pH 9 water with a 3.5-kDa molecular weight cut off
144
(MWCO) dialysis membrane (Spectrum Labs, Rancho Dominguez, CA) for 14 h, changing the
145
dialysate once after 6 h, or centrifuging the stock in an Amicon Ultra-4 centrifugal filter with a 3-
146
kDa MWCO (Millipore, Bellerica, MA) for 40 min at 7,500 x g. Ag(aq) in the dialysate and
147
permeate was quantified by ICP-OES.
148
The size of the as-synthesized AgNPs (before suspension in an exposure solution) was
149
characterized using transmission electron microscopy (TEM) (Tecnai Spirit, FEI, Hillsboro, OR).
150
Samples were dropped on carbon-coated copper grids and were air dried for 10-20 min before
151
analysis. Images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD,
152
USA, http://imagej.nih.gov/ij), and statistical analyses were performed in Excel (Microsoft
153
Corp., Redmond/WA).
154 155
Characterization of AgNPs in Exposure Solutions. When added to an exposure solution, the
156
physical/chemical AgNP characteristics were expected to change as a function of the chloride
157
and ionic strength of the exposure solution. These changes were assessed as follows.
158
The production of Ag(aq) in the exposure solutions was assessed. AgNPs were dosed (1 mg/L)
159
to 3.4 mL of an exposure solution and incubated statically at 30 °C in the dark. Samples were
160
withdrawn at 10 min and 1, 3, and 5 h and processed with an Amicon Ultra-4 3-kDa centrifugal
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filter (7,500 x g for 30 min) to separate Ag(aq) from AgNPs and aggregates. The permeate was
162
acidified to a final concentration of 2% nitric acid for analysis by ICP-OES.
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Surface characteristics were examined qualitatively via surface plasmon resonance (SPR) with
164
a UV-visible spectrophotometer (Agilent 8453, Santa Clara, CA). AgNPs were dispersed (1
165
mg/L) in each exposure solution in a 10-cm quartz cuvette and incubated statically at 30 °C in
166
the dark. Readings were taken at 10 min and 1, 3, and 5 h.
167
The size and aggregate structure of the AgNPs in the exposure solutions were assessed. Light
168
scattering measurements were conducted on an ALV-CGS/3 goniometer system (ALV-GmbH,
169
Langen, Germany). The size of the aggregating AgNPs was measured over time via dynamic
170
light scattering (DLS). The DLS protocol is described in detail elsewhere38,39 and is summarized
171
in the SI. The mean hydrodynamic radius (HR) and standard deviation were computed over 1-h
172
intervals (0 to 5 h) and are reported at the midpoint of each interval. Static light scattering (SLS)
173
measurements were performed to describe the aggregate structure (i.e., fractal dimension) of the
174
AgNPs. The SLS protocol is described in detail in Khan et al. (2013)39 and is summarized in the
175
SI. Changes in AgNP morphology (e.g., independent particles versus bridged particles) and
176
elemental composition were evaluated by scanning electron microscopy (SEM) and energy
177
dispersive X-ray (EDX) spectroscopy, respectively, and these methods are summarized in the SI.
178
Additionally, TEM was performed on AgNPs exposed to Hµ140 using an H8000 TEM (Hitachi
179
High Technologies, Roselyn Heights, NY). Samples were dropped on carbon/Formvar®-coated
180
copper grids and were air dried for 15-20 min before imaging.
181 182
Reverse-Transcription, Quantitative Real-time Polymerase Chain Reaction (RT-qPCR).
183
The expression of katE was studied to understand how E. coli responds to AgNPs in different
184
exposure solutions. The cells were grown in batch culture in MD medium, pelleted, and washed
185
in Lµ0. One-milliliter aliquots of washed cells were added to duplicate selected exposure
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solutions (Hµ0, Mµ31, and Lµ0) in the presence or absence of AgNPs at an initial cell
187
concentration of 3x107 CFU/mL. Cells were incubated in the dark for 5 h with shaking at 30 °C.
188
An aliquot from each flask was removed for cell enumeration by viable plate counts on
189
Lysogeny Broth (Lennox) agar, and the remaining cells were pelleted for RNA isolation. RNA
190
was extracted in duplicate from each flask and pooled, and cDNA was synthesized in duplicate
191
reactions and pooled; detailed protocols are given in the SI.
192
qPCR primers targeting a 190-bp katE fragment were designed using Primer3 software40 and
193
E. coli K-12 substrain MG1655, which has a sequenced genome. qPCR was run on each cDNA
194
sample in triplicate, according to the protocol in the SI. A katE standard curve was constructed
195
using genomic DNA from E. coli MG4 (101-107 gene copies/reaction). A Student’s t-test was
196
performed (α=0.05) in Minitab 16 (Minitab Inc., State College, PA).
197 198
RESULTS AND DISCUSSION
199
Characterization of Synthesized AgNPs. After synthesis, AgNPs in pH 9 DDI were
200
characterized. TEM analysis showed spherical particles with an exponential distribution and
201
average diameter of 2.9 nm (Fig. S1), in close agreement with the 2.2-nm diameter described in
202
the original synthesis.36 A typical batch of AgNPs contained 220 mg/L total silver, of which 7-9
203
µg/L was residual Ag(aq). When the AgNP stock was diluted for bacterial tolerance studies (≤ 1
204
mg/L), the residual Ag(aq) concentration was orders of magnitude below inhibitory levels.
205 206 207
Aggregation. We used SPR to generally assess AgNP aggregation under different ionic strength and chloride conditions (Fig. 1).
SPR has been used previously to qualitatively
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characterize AgNPs.24,31 AgNPs typically have an extinction peak near 400 nm,31,41 but this can
209
shift based on the local refractive index and NP diameter.42–44 a)
Hµ0 Mµ0 Lµ0
0.6
0.4
0.2
300
0.6
0.4
0.2
400
500
600
300
700
210
Mµ31 Mµ0
0.6
0.8
Extinction (AU)
Extinction (AU)
c)
400
500
600
700
Wavelength (nm)
Wavelength (nm)
0.8
Hµ0 Mµ0 Lµ0
0.0
0.0
0.4
0.2
d)
Mµ31 Mµ0
0.6
0.4
0.2
0.0
0.0 300
211
b)
0.8
Extinction (AU)
Extinction (AU)
0.8
400
500
600
Wavelength (nm)
700
300
400
500
600
700
Wavelength (nm)
212
Figure 1. SPR spectra of AgNPs in selected exposure solutions. AgNPs were probed by SPR as a
213
function of ionic strength at (a) 10 min and (b) 5 h and as a function of chloride concentration at
214
(c) 10 min and (d) 5 h.
215
Fig. 1a–b highlights temporal changes in the SPR spectra as a function of ionic strength at a
216
constant chloride concentration (0 mM). At the lowest ionic strength (Lµ0), the original AgNP
217
extinction peak intensity remained nearly constant over time, indicating AgNP stability.
218
However, at increased ionic strengths (Mµ0 and Hµ0), a rapid decrease in the original AgNP
219
extinction peak intensity and peak broadening occurred, which suggests the depletion of stable
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AgNPs and the formation of aggregates, respectively.22 Relatedly, the DLS data show that the
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average HR at 5 h increased from 33.6±0.7 nm for Lµ0 to 51.2±1.2 nm for Hµ0 (Fig.
222
S2a). Thus, the aggregation behavior observed via SPR, which is a technique that probes
223
individual nano-scale interactions, is complemented by DLS, a technique that measures average
224
suspension properties. Fig. 1c–d highlights temporal changes in the SPR spectra as a function of
225
chloride concentration at constant ionic strength (~40 mM); the lowest and highest chloride
226
concentrations at this ionic strength (0 and 31 mM chloride, respectively) were assessed because
227
the changes in the SPR spectra as a function of chloride concentration were not nearly as
228
dramatic as those as a function of ionic strength. Over time, both SPR spectra (Mµ0 and Mµ31)
229
show reduction in the original AgNP extinction peak intensity as well as peak broadening and
230
formation of a secondary peak at 564 nm. However, the peak intensity reduction at 390 nm is
231
not as significant in the Mµ31 sample, suggesting less aggregation of primary AgNPs in the
232
presence of chloride.
233
aggregation but cause the formation of larger overall AgNP/AgCl aggregates. This is supported
234
by DLS, which interrogates the overall aggregate size (AgNP plus AgCl bridging), where the
235
average HR at 5 h increased from 33.6±0.7 nm for Mµ0 to 158.4±70.5 nm for Mµ31 (Fig. S2b).
236
In contrast, SPR probes individual nano-scale interactions, and it indicates little effect of chloride
237
on those AgNPs. A similar trend was observed in the SPR spectra for AgNPs exposed to Hµ0
238
and Hµ140 (data not shown), where the presence of chloride appeared to stabilize AgNPs
239
relative to the absence of chloride. Overall, the SPR data suggest that ionic strength is a major
240
factor in AgNP destabilization and chloride acts to stabilize AgNPs. Evidence of AgCl bridging
241
to stabilize AgNPs is provided in the Aggregate Structure section.
That is, AgCl bridging between AgNPs might reduce AgNP-AgNP
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Dissolution. The release of Ag(aq) due to the dissolution of AgNPs under different ionic
244
strength and chloride conditions was examined (Fig. 2). The release of Ag(aq) from AgNPs is
245
dependent upon oxidation,8,23 and we detected Ag(aq) in the exposure solutions dosed with
246
AgNPs. In the absence of chloride, ionic strength only slightly impacted the initial release (at 10
247
min) of Ag(aq) (Fig. 2 inset). However, for a constant ionic strength, the initial release of Ag(aq)
248
from AgNPs was strongly affected by the presence of chloride in the exposure solution (Fig. 2
249
inset).
250
dissolution.23,45 We continued to monitor Ag(aq) over 5 h, which corresponds to the timeframe of
251
our tolerance studies, and only saw continued AgNP dissolution at the highest chloride
252
concentration (Hµ140, Fig. 2). This is consistent with previous studies that have suggested that
253
chloride might act catalytically in the dissolution of AgNPs.46,47 Thus, our data indicate that
254
chloride concentration is more important than ionic strength in driving the production of Ag(aq)
255
from AgNP dissolution.
These data are consistent with previous literature findings that show rapid AgNP
256
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500 400 300
Aqueous silver conc. (µ g/L)
Aqueous silver conc. (µg/L)
600 80
60
Mµ31 Mµ2.7 Mµ0 Lµ0
4
5
40
20
0
Dissolution in first 10 min
200 100 0 0
257
Hµ140 Hµ32 Hµ2.7 Hµ0
1
2
3
Time (hr)
258
Figure 2. Dissolution of AgNPs (dosed at 1 mg/L) over time as a function of ionic strength and
259
chloride concentration, where the inset highlights the release of Ag(aq) after 10 min. Data points
260
are the average of triplicate experiments, and error bars show one standard deviation.
261 262
Aggregate Structure. Aggregate structure formation under different ionic strength and
263
chloride conditions was examined via SLS (Fig. 3). For a constant chloride concentration (0
264
mM), the fractal dimension (Df) decreased with increasing ionic strength (Fig. 3a).
265
observation is consistent with classical electrostatic screening-induced fractal formation of
266
colloids, as observed previously.39 Higher ionic strength reduces the electrostatic potential,
267
which likely imparted ‘stickiness’ to the AgNPs and resulted in lower Df (i.e., a more fractal
268
structure as compared to a more densely packed aggregate in Lµ0). On the other hand, at
269
constant ionic strength (~40 mM), increased chloride concentrations showed increased Df (Fig.
270
3b). This shift in structure toward a more compact form in the presence of chloride supports the
This
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bridging of AgNPs via silver chloride (AgCl0(s)) formation. Using TEM, a similar observation of
272
AgCl0(s)-induced bridging of AgNPs has been observed previously.45 Hµ0 Mµ0 Lµ0
3.0
2.5
2.0
1.5
1.0
After 3 hr
273
Mµ31 Mµ2.7 Mµ0
b) Fractal dimension, Df
Fractal dimension, Df
a)
3.0
2.5
2.0
1.5
1.0
After 5 hr
After 3 hr
After 5 hr
274
Figure 3. Fractal dimension of AgNPs as a function of (a) ionic strength and (b) chloride
275
concentration.
276
While light-scattering techniques were used to assess aggregate size and structure, SEM was
277
used to provide visual evidence of morphological changes under different water chemistry
278
conditions.
279
concentrations, as shown for AgNPs in Hµ140 (Fig. S3c-d) as compared to AgNPs in Mµ31 and
280
Hµ0 (Fig. S3a-b). Furthermore, TEM of AgNPs in Hµ140 shows a change in contrast at the
281
particle-particle attachment regions (indicated by arrows in Fig. S3e), which is indicative of
282
bridging through chloride precipitation. EDX confirms the presence of Ag in the pristine AgNPs
283
(Fig. S4a); Na and Si peaks in the spectrum are likely due to the glass slide used for SEM, as
284
these peaks appear in the spectra for all tested exposure solutions. EDX also confirms the
285
presence of Ag and Cl on the aggregated particle clusters in Mµ31 and Hµ140, further
286
supporting the reaction of chloride on AgNP surfaces (Fig. S4b-c).
SEM provides support for AgNP bridging in the presence of high chloride
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Impact of Ionic Strength and Chloride on Bacterial Tolerance to AgNPs. As demonstrated
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above, the chemistry of the exposure solution impacts AgNP stability. Next we examined the
290
tolerance of E. coli to AgNPs in the nine exposure solutions; selected results are shown herein.
291
Fig. 4 demonstrates the tolerance of E. coli to AgNPs as a function of ionic strength at a
292
constant chloride concentration (0 mM). At higher AgNP concentrations (greater than 50 µg/L),
293
E. coli survival is less than 10 %. At lower AgNP concentrations (15.6 and 31.2 µg/L), E. coli
294
has the lowest tolerance to AgNPs under the highest ionic strength condition (Hµ0). This is
295
likely due to one or more of the following reasons. (1) Hµ0 induces higher AgNP dissolution as
296
compared to lower ionic strength exposure solutions (e.g., 15.3 and 5.6 µg/L Ag(aq) measured in
297
the bulk solution after a 3-h exposure of AgNPs to Hµ0 and Lµ0, respectively, Fig. 2), and Ag+
298
is biocidal to bacteria.8,48,49 (2) Hµ0 causes the most compression of the electric double layer,
299
which would facilitate closer proximity of cells to AgNPs. (3) Hµ0 induces a different aggregate
300
structure as compared to lower ionic strengths. At higher ionic strengths, AgNPs were found to
301
form more fractal structures (lower Df) as compared to more densely packed structures at lower
302
ionic strengths (Fig. 3a), which leads to more available surface area for interaction with bacteria.
303
Close interaction of bacteria with AgNPs could facilitate the delivery of Ag+ to the cells as the
304
AgNPs dissolve, resulting in reduced tolerance of E. coli to AgNPs at higher ionic strengths.
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Surviving cells, C/C0
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Hµ 0 Mµ 0 Lµ 0
1.0 0.8 0.6 0.4 0.2 0.0 0
305
50
100
150
200 1000
Silver nanoparticle dosage (µg/L)
306
Figure 4. Impact of ionic strength on the tolerance of E. coli to AgNPs. Surviving cells are
307
shown as a function of AgNP dose. Data are averages of triplicate assays of biological
308
duplicates, and error bars represent one standard deviation.
309 310
Fig. 5a demonstrates the tolerance of E. coli to AgNP exposure as a function of chloride
311
concentration at constant ionic strength (~40 mM). E. coli has the highest tolerance to AgNPs
312
under the highest chloride condition shown (Mµ31). This is likely due to one or both of the
313
following reasons. (1) Higher chloride concentrations result in more AgNP dissolution (Fig. 2),
314
but they produce lower concentrations of Ag+ and the neutral species AgCl0(aq) (compare Mµ31
315
to Mµ2.7 in Fig. 5b). While some work has proposed that only the total concentration of
316
aqueous AgClx(x-1)- species, rather than their speciation, controls the overall toxicity to E. coli, 23
317
our work makes the claim that AgClx(x-1)- speciation matters. As shown in Fig. S5 (where
318
aqueous silver was dosed to E. coli), the tolerance of E. coli to Ag(aq) increases as the charge on
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the dominant AgClx(x-1)- species decreases (e.g., greater tolerance when AgCl2- is the dominant
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species as compared to when Ag+ is the dominant species). However, given the values of the
321
AgClx(x-1)- stability constants, it is not possible to isolate the toxicity impact of each AgClx(x-1)-
322
species. (2) Chloride induces changes in AgNP aggregate structure. When chloride is low or
323
absent (Mµ2.7 and Mµ0, respectively), AgNPs form more fractal structures (Fig. 3b). This leads
324
to higher available surface area for interaction with bacteria, likely facilitating the delivery of
325
Ag(aq) to the cells as the AgNPs dissolve, resulting in reduced tolerance of E. coli to AgNPs at
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lower chloride concentrations. Our results are in contrast with an earlier study23 that showed
327
reduced tolerance of E. coli to AgNPs in the presence of higher chloride concentrations, but
328
those results are likely confounded by simultaneous changes in ionic strength and chloride
329
concentration.
1.0
a)
Mµ31 Mµ2.7 Mµ0
Surviving cells, C/C0
0.5 0.08 0.06 0.04 0.02 0.00 0
331
200
400
600
800
1000
Silver nanoparticle dosage (µg/L)
Species concentration (µg/L)
330 40
b)
AgNO3 2-
AgCl3 30
-
AgCl2 AgCl + Ag
20
10
0 Mµ31
Mµ2.7
Mµ0
Exposure solution
332
Figure 5. Impact of chloride on tolerance of E. coli to AgNPs. (a) Surviving cells as a function
333
of AgNP dose; (b) theoretical equilibrium AgClx(x-1)- speciation of Ag(aq) produced 5 h after 1
334
mg/L AgNPs are dosed to a particular exposure solution, where Ag(aq) for each exposure solution
335
is shown in Fig. 2. Cell data are the averages of triplicate assays of biological duplicates, and
336
error bars represent one standard deviation.
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Oxidative Stress in E. coli due to AgNP Exposure. To further assess the effect of AgNPs on
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E. coli under different ionic strength and chloride conditions, we analyzed the expression of a
339
gene linked to oxidative stress, katE.50,51
340
hydroperoxidase II and is responsible for alleviating the effects of oxidative stress and also is
341
linked to chromate resistance.51,52 Catalase hydroperoxidase II converts hydrogen peroxide to
342
molecular oxygen and water and limits the transition-metal-facilitated formation of hydroxyl
343
radicals via a Fenton-type reaction.50 While the extent to which AgNP toxicity is linked to
344
reactive oxygen species (ROS) formation is still debated,8,12,53,54 Ag+ toxicity has been linked to
345
ROS formation;55 furthermore, this study (Fig. 5b) corroborates previous findings9,11,12 that
346
AgNP toxicity is linked to Ag+ (though other AgClx(x-1)- species also might contribute to
347
toxicity). We examined the expression of katE in the presence of a low concentration of AgNPs
348
(20 µg/L), which killed only a fraction of the exposed population (Figs. 4 and 5a).
katE encodes a component of the catalase
349
The expression of katE under the tested conditions is shown in Fig. 6. katE expression
350
increased significantly (p-value=0.001) in Hµ0 in the presence of AgNPs (Hµ0-AgNP) as
351
compared to the control (absence of AgNPs, Hµ0-Ctrl). By comparison, katE expression did not
352
increase significantly (p-value=0.194) in Lµ0 in the presence of AgNPs (Lµ0-AgNP) as
353
compared to its control (Lµ0-Ctrl). This is consistent with the AgNP dissolution data, which
354
showed greater Ag(aq) in the bulk solution when AgNPs were dosed to Hµ0 as compared to Lµ0
355
(Fig. 2 inset). Relatedly, katE expression did not increase significantly (p-value=0.077) in Mµ31
356
in the presence of AgNPs (Mµ31-AgNP) as compared to its control (Mµ31-Ctrl). This is
357
consistent with theoretical equilibrium speciation for AgClx(x-1)-, which indicates that Ag+ in the
358
Mµ31 bulk solution is inconsequential (Fig. 5b). Overall, these data support oxidative stress in
359
E. coli as a result of Ag+ exposure.
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katE transcripts/ng cDNA
8000 7000 6000
Hµ0-AgNP Hµ0-Ctrl Mµ31-AgNP Mµ31-Ctrl Lµ0-AgNP Lµ0-Ctrl
5000 4000 3000 2000 1000
Exposure solution
361 362
Figure 6. katE expression in E. coli as a function of AgNP exposure under different ionic
363
strength and chloride conditions. Gene expression was measured in three exposure solutions
364
(Hµ0, Mµ31, and Lµ0) in the presence of AgNPs and the absence of AgNPs (controls). Data are
365
averages of triplicate qPCR reactions from duplicate biological experiments. Error bars represent
366
one standard deviation.
367 368
Mechanism of Toxicity. The key mechanism underlying the observed antimicrobial activity of
369
AgNPs in this study is the role of background electrolytes in altering AgNP
370
physical/chemical properties. Both ionic strength and chloride concentration affect the physical
371
morphology, dissolution, and bacterial toxicity of AgNPs (Fig. 7). As ionic strength increases,
372
AgNP aggregation likewise increases (such that the HR increases), Df strongly decreases
373
(providing increased available surface relative to suspensions with higher Df), the release of
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Ag(aq) increases, and E. coli demonstrates reduced tolerance to the AgNP exposure (i.e., toxicity
375
increases). As chloride concentration increases, aggregates are formed but are dominated by
376
AgCl0(s) bridging of AgNPs; relatedly, the fractal dimension increases. Furthermore, AgNP
377
dissolution is strongly increased under increased chloride conditions, but the dominant aqueous
378
silver species shift to negatively charged AgClx(x-1)- species, which appear to be less toxic to E.
379
coli (Fig. S5). Thus, E. coli demonstrates increased tolerance to the AgNP exposure under
380
higher chloride conditions (i.e., toxicity decreases).
381 382
Figure 7. Model of the effects of chloride and ionic strength on physical morphology,
383
dissolution, and bacterial toxicity of AgNPs.
384
Previous work on the antimicrobial activity of AgNPs reports the complexity in assessing
385
dissolution of AgNPs in the presence of chloride ions.23 Our study is the first to postulate the
386
correlation of observed toxicity of AgNPs with their dissolution and physical characteristics (i.e.,
387
aggregation,
388
conditions. Moreover, the correlation of toxicity with Ag(aq) speciation and the use of gene
389
expression analysis to examine the role of ROS in the observed toxicity of AgNPs are important
fractal
structure,
particle
bridging)
for
a
wide
range
of
chemical
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components of our mechanistic analysis. Furthermore, the findings in this study allow us to
391
draw new conclusions about the impact of chloride, separate from the impact of ionic strength,
392
on the dissolution, physical characteristics, and toxicity of AgNPs.
393 394
Environmental Implications. With respect to bacterial toxicity testing for AgNPs, it is
395
important to recognize that dramatic changes in physical morphology and dissolution can occur
396
as a result of the chosen water chemistry conditions.
397
conditions of elevated ionic strength and chloride, but this will not typically reflect relevant
398
environmental conditions where bacteria might be exposed to AgNPs (e.g., in wastewater
399
treatment). In the case of E. coli, it is likely that the increased AgNP toxicity due to high ionic
400
strength conditions would just balance the decreased AgNP toxicity due to high chloride
401
conditions (Fig. 7), but this fortuitous occurrence might not be true for all microorganisms.
402
Thus, toxicity assessments of NPs to microorganisms should be conducted under
403
environmentally relevant water chemistry conditions. Furthermore, the environmental impacts
404
of AgNPs must consider dissolution, speciation, and aggregation for the particular water
405
chemistry of interest.
406
properties might influence the manifestation of the AgNP chemical behavior (e.g., fractal-
407
structure-dependent dissolution), which can then affect toxicity.
Toxicity testing often occurs under
For instance, environmental conditions influencing AgNP physical
408 409 410 411
ASSOCIATED CONTENT Supporting Information. Detailed information regarding methods and additional results. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
414
*Corresponding Author
415
Mary Jo Kirisits
416
The University of Texas at Austin
417
Department of Civil, Architectural, and Environmental Engineering
418
301 East Dean Keeton Street
419
Austin, Texas 78712
420
Phone: (512) 232-7120
421
Fax: (512) 471-0592
422
E-mail: [email protected]
423 424
ACKNOWLEDGMENT
425
The authors thank the National Science Foundation (CBET 0846719 to M.J. Kirisits) for
426
funding and the following people for technical assistance: Taegyu Kim (tolerance assays),
427
Colleen Lyons (TEM), and Gustavo Ochoa and Katie Speights (laboratory support).
428 429 430 431 432
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