Environ. Sci. Technol.
2009, 43, 7285–7290
Silver Nanoparticle Impact on
Bacterial Growth: Effect of pH,
Concentration, and Organic Matter
JULIA FABREGA, SHONA R. FAWCETT,
JOANNA C. RENSHAW, AND
JAMIE R. LEAD*
School of Geography, Earth and Environmental Sciences,
University of Birmingham, Edgbaston,
Birmingham, B15 2TT, United kingdom
Received November 18, 2008. Revised manuscript received
May 12, 2009. Accepted May 14, 2009.
Silver nanoparticles (Ag NPs) are widely used as antibacterial
agents. This antibacterial property carries with it a potential
environmental risk once these NPs are discharged into
the environment. This study investigated the impact on
Pseudomonas fluorescens over a 24 h exposure of well
characterized Ag NPs at pH values of 6-9, in the presence
and absence of Suwannee River humic acids (SRHA). Ag NPs
were characterized by size, aggregation, morphology,
dissolution, and surface properties under all conditions.
Solubility was low (less than 2%) for all Ag NP concentrations
(2-2000 ppb) and under all conditions was less than 40 ppb
(0.38 µM). SRHA caused a partial disaggregation of Ag NP
aggregates by nanoscale film formation, with individual NPs
stabilizedbychargeandentropicallydrivenstericeffects.Dissolved
Ag reduced bacterial growth entirely at 2000 ppb (19 µM)
under all conditions and adversely affected growth at 200 ppb
(1.9 µM) under some conditions, indicating some toxicity.
The Ag NPs showed similar toxicity at 2000 ppb (19 µM) in
the absence of SRHA and at pH 9 only i.e. SRHA mitigated
bactericidal action. Solubility and interactions with SRHA indicate
that there was a specific nanoparticle effect, which could
not be explained by the effect of dissolved Ag.
Introduction
Nanoparticles (NPs) can be defined as particles with novel
and distinctive physicochemical properties with at least one
dimension within the range of 1-100 nm (1). The importance
of nanotechnology in today’s society is shown by the broad
range of scientific and technological applications using NPs
(1, 2), including imaging and medical apparatus (3), sensors
(4), fabrics, cosmetics, health products, and water reme-
diation technologies. Recently, the use of NPs in the medical
field has received special attention as a molecular tool and
method for targeted drug delivery (5). However, despite the
novelty of this technology and its rapid growth in a variety
of products, there are relatively few published accounts
assessing the potential hazards of NPs to organisms and to
the environment (6-11). The growing production and use
of NPs inevitably leads to their release into the environment
but their impact is largely unknown. Both in vitro and in vivo
studies using model organisms including fish, invertebrates,
and microorganisms (7-12) have shown that NPs can be
toxic. A limited number of studies (8, 13-15) have shown
* Corresponding author e-mail: j.r.lead@bham.ac.uk.
10.1021/es803259g CCC: $40.75  2009 American Chemical Society
Published on Web 06/08/2009
that physicochemical parameters such as size, specific surface
area, and shape are potential factors influencing toxicity,
but there are few quantitative structure-activity relation-
ships. Nevertheless, these physicochemical parameters are
known to be important in affecting both mechanisms of
uptake and toxicity and exposure dose.
Ag NPs have been found to have particularly high intrinsic
toxicities to bacteria (e.g., ref (8)) while are likely to be less
harmful to humans. Ag NPs are emerging as one of today’s
fastest growing nanomaterials; they have received consider-
able attention due to their promising antibacterial capacity
(10, 11, 16) and are present in a range of consumer goods.
However, their antibacterial effects indicate that they will be
highly toxic to natural bacterial communities if/when they
reach the environment. Studies have shown that Ag NPs
toxicity is size dependent at low concentrations. One study
(8) observed that silver NPs of 1-10 nm were preferentially
bound to cell membranes and were incorporated into
bacteria, whereas larger NPs were not, while others (14)
indicated that toxicity was shape dependent. Additionally,
it has been shown that Ag NPs caused pitting on bacterial
cell membranes, leading to increased permeability and cell
death (16). The effects were greater when bacteria were grown
on solid agar media compared with those grown in suspen-
sion in aqueous media. The difference is likely to be related
to changes in exposure dose over the experiments due to NP
aggregation and sedimentation.
To fully understand NP effects on microbial communities
it is necessary to characterize the physicochemical structure
of the NPs under relevant environmental conditions and to
determine toxicity. In this study we have fully characterized
Ag NPs as a function of pH and the presence or absence of
humic substances (HS) and quantified the effects of Ag NPs
on the growth and viability of the model aquatic bacterium
Pseudomonas fluorescens.
Materials and Methods
Preparation of Ag NPs Stock Suspension. Silver nanopowder
was purchased from Stanford Materials Corp.(Aliso Viejo,
CA). The reported mean particle size was 30-50 nm diameter,
the standardized surface area (SSA) was 5-10 m2 g-1 and the
purity 99.9%. A suspension of Ag NPs was prepared by
dispersing 5 g of silver nanopowder in 1 L sterile Milli-Q
water with 0.25 mM sodium citrate for stabilization of the
particles. The resultant suspension was sonicated (Ultrasonic
bath Fisherbrand unheated, 230 V 50 Hz) for 30 min per day
for 4 consecutive days. After the last sonication, the suspen-
sion was left to stand for 96 h. Then, the supernatant was
carefully removed and the final Ag NP concentration was
quantified by inductively coupled mass spectrometry (ICP-
MS, Agilent 7500ce Octopole Reaction System). Exposure
doses were calculated from this stock suspension and losses
measured over 24 h of exposure. Suwannee River Humic
Acid (SRHA) was purchased from International Humic
Substances Society (International Humic Substances Society,
St. Paul, MN) and used without further pretreatment. A stock
solution of 1 g L-1 was prepared and left shaking for 24 h to
fully solubilize. Stock solutions were kept at 4 °C in the dark
and discarded after four weeks.
Physico-Chemical Characterization. The physicochem-
ical properties of Ag NPs were characterized at concentrations
between 0 and 2000 ppb (0 and 19 µM) Ag NP suspension
in minimal Davies Medium (MDM (17)) at pH values of 6,
7.5, and 9, in the presence and absence of 10 mg L-1 (4.58
g C L-1) SRHA. MDM was the media used to perform bacterial
experiments, chosen as a very simple solution but with
VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7285
sufficient nutrients to allow bacterial growth. Suspensions
were prepared using the Ag NPs stock suspension, adjusted
to the required pH values, and placed on a shaking incubator
at 120 rpm and 25 °C for 24 h. The stability, agglomerate size
and changes in surface properties of Ag NPs in the medium
were investigated using zeta-potential measurements (Mal-
vern Instruments Zetasizer 1000Hs, 5-50 mW, He-Ne laser
at 633 nm, 25 °C), dynamic light scattering (DLS, Malvern
High performance particle seizer HPPS 5001, 25 °C), and
transmission electron microscopy (TEM), with energy dis-
persive X-ray spectrometry (EDX; JEOL 1200EX, 80Kv and
TEM-Tecnaif 20 EDX). For TEM and EDX analyses the samples
were ultracentrifuged at 20 000 rpm for 30 min at 25 °C using
a Beckman Coulter Avanti J-25 (Rotor Beckman JA-20) on a
Formvar coated copper grid. Particle numbers were calcu-
lated by quantitatively transferring known volumes of
suspension to the TEM grid. Particles within a minimum of
five known areas of 10 µm2 were counted and this was related
to the total surface of the sample on the TEM grid. To
determine the solubility of the Ag NPs, suspensions of 0-2000
ppb (0-19 µM) at pH 6, 7.5, and 9 were equilibrated for 24 h
and ultrafiltered (membrane pore sizes nominally 1 kDa)
and the filtrate acidified with concentrated nitric acid
for analysis by ICP-MS. Ultrafiltration of Ag NPs at pH 1 were
also performed for comparison. X-ray diffraction (XRD)
analysis using a Siemens D5000 diffractometer gave further
information about particle size and crystal structure.
Bacterial Strain and Growth Medium. The gram negative
Pseudomonas fluorescens SBW25 used in this study was obtained
from Prof. C. M. Thomas from the University of Birmingham.
The media used for routine culturing of P. fluorescens, the Ag
NP toxicity experiments, and the NP characterization was MDM.
The media contained 700 mg L-1 KH2PO4, 200 mg L-1 K2HPO4,
500 mg L-1 sodium citrate, 100 mg L-1 MgSO4, 1 g L-1 (NH4)2SO4,
and 1 g L-1 glucose as a carbon source. The ionic strength of
the media was 0.055 M, somewhat higher than typical fresh-
waters. Preliminary research performed with this media
suggests that it is adequate for NP toxicity studies in bacteria
(17), although the high salt concentrations are conducive to
aggregation All experiments were conducted at 25 °C in a
shaking incubator at 120 rpm.
Exposure of Ag NPs to P. fluorescens. All glass- and plastic-
ware was acid washed with 10% nitric acid and sterilized by
autoclaving or with 20% sodium hypochlorite solution. The
effects of Ag NPs, latex NPs (APS 21 ( 1.5 nm dia; 10 ppm
stock suspension; Nanosphere Size Standard, Duke scientific
Corp; used as negative control), and Ag+(aq) (Silver nitrate
solution 1000 mg L-1 stock solution; PerkinElmer, AAS
Calibration Standard; used as a positive control) on P.
fluorescens were studied under six different growth conditions
(pH values of 6, 7.5, and 9; with and without 10 mg L-1 SRHA
in the medium), two different exposure times (3 and 24 h),
and four different concentrations, (2 ppb (0.019 µM), 20 ppb
(0.19 µM), 200 ppb (1.9 µM), and 2000 ppb (19 µM)). P.
fluorescens was grown overnight in MDM at the appropriate
pH value (25 °C and 120 rpm) to exponential phase, collected
by centrifugation, washed twice with MDM, and resuspended
in MDM at the right pH. The cell suspension was measured
by optical density at 595 nm (OD595; Lightwave WPA diode
array spectrophotometer) and adjusted to OD595 ) 0.1 by the
addition of MDM. Aliquots of Ag NPs, latex nanoparticle or
Ag+ solutions were added to 25 mL of the bacterial suspen-
sions to give final concentrations of 2, 20, 200, or 2000 ppb.
SRHA solution was added to those treatments requiring 10
mg L-1 HS, prior to dissolved silver or NP addition. Equi-
librium was not established beforehand (at least for dissolved
Ag-SRHA interactions) allowing better discrimination be-
tween dissolved and NP silver effects. Clearly, if equilibrium
between dissolved Ag and SRHA had been established, lower
toxicity may have been observed. No metal speciation data
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FIGURE 1. Particle size distribution (nm) of primary Ag NPs in
MDM. Data pooled from particles at pH 6, 7.5, and 9 with and
without SRHA, n ) 600.
was obtained or needed, as this study investigates NP toxicity,
but dissolved silver is used as a positive control and treated
in an identical manner to the NP exposures. Control cultures
contained no Ag NPs, latex NPs or Ag+(aq). After 3 and 24 h,
the bacterial density was measured by UV-vis OD595. In
addition, the background readings (OD595) of MDM solutions
containing only latex NPs, Ag NPs, or Ag nitrate solution
were measured at each sampling time; all background
readings were essentially zero and all readings were normal-
ized appropriately. Each treatment was prepared in triplicate.
After 3 and 24 h, the bacterial density was measured by
UV-vis OD595. The effects of the treatments on bacterial
growth were analyzed by a one-way ANOVA followed by a
Tukey test (SPSS Inc., Chicago IL).
Results and Discussion
Characterization of Ag NPs in MDM. The average size of
discrete, individual Ag NPs was reported by the manufacturers
to be ca. 30-50 nm and this was broadly in agreement with
our results here from TEM and XRD showing primary particle
size to be ca. 65 ( 30 nm (TEM, mean ( standard deviation;
n ) 600) and 100 ( 20 nm (XRD, mean ( standard deviation).
The NPs were highly polydisperse, with particle sizes ranging
from 5 to 150 nm and particle size distributions (PSDs) from
TEM are shown in Figure 1. No significant difference in
primary particle size was found between conditions (ANOVA,
P > 0.05, n ) 600) and Figure 1 represents the summation
of particle counting at pH 6, 7.5, and 9 and with and without
SRHA.
TEM (Figure 2) and DLS (data not shown) measurements
showed that the dominant form of Ag NPs in MDM is as
small aggregates of several hundred nanometers. The size of
the aggregate did not vary significantly with pH value. In the
presence of SRHA two effects were noted by TEM. First, the
small Ag NP aggregates were coated and surrounded by what
appeared to be SRHA, based on morphologies of HS in the
literature and on the film only being present when HS was
added (Figure 2). Second, in the presence of SRHA, disag-
gregation of the Ag NPs occurred and fully dispersed primary
particles (verified by EDX analysis) were observed in the
presence of SRHA only (Figure 3). No such dispersed particles
were observed in the absence of SRHA. Partial disaggregation
of the Ag NP aggregates in the presence of SRHA is
unsurprising and we have seen similar effects on iron oxide
NPs (data unpublished), although there is little published
data on disaggregation due to HS (18). Qualitatively, TEM
images revealed that these single NPs in the presence of
SRHA were large in terms of particle number, although their
FIGURE 2. Transmission electron micrographs of Ag NPs at (A) pH 6, (B) pH 7.5, (C) pH 9, (D) pH 6 and 10 mg L-1 SRHA, (E) pH 7.5
and 10 mg L-1 SRHA, and (F) pH 9 and 10 mg L-1 SRHA. Line represents 100 nm.

FIGURE 3. (A) EDX spectrum of dispersed Ag NPs in MDM with 10 mg l-1 SRHA. (B) Electron micrograph of primary Ag NPs in MDM.

mass concentrations were likely to have been low. Mass
appeared to be dominated by small aggregates even in the
presence of SRHA. Based on our recent results, longer reaction
times and higher concentrations of HS would increase
disaggregation. Initially, we hypothesized that the presence
of a relatively large particle number concentration of
dispersed NPs may have increased bacterial toxicity and this
should be borne in mind in later discussions. There is thus
good agreement between TEM, DLS, and XRD data, although
slight discrepancies may have been due to sample polydis-

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TABLE 1. Amount (ppb) of Dissolved Silver (Ag+aq) Released by Silver Nanoparticles in MDM Stirred for 24 h at pH 6, 7.5, and 9
Without SRHA
AgNPs concentration (ppb) pH 6
2000 (18,670 nM) 6.00 ( 0.04 (56.0 ( 0.4 nM)
200 (1870 nM) 3.1 ( 0.4 (28.8 ( 3.4 nM)
20 (187 nM) 0.02 ( 0.03 (0.2 ( 0.3 nM)
persity and different biases of the measurements involved.
Our data show that the manufacturer’s estimates of particle
size were reasonable but were lower than their actual size.
Ag NPs were clearly negatively charged with electro-
phoretic mobilities (µe) of ca. -2 at pH 6 and ca. -3.5 cm2
V-1 s-1 at pH 7.5 and 9 (Supporting Information Figure S1).
In the presence of HS µe was more negative and greater than
-4.0 cm2 V-1 s-1 for all pH values. The change in electro-
phoretic mobility to more negative values (usually reported
as zeta potential, ζ; we do not present these here because of
the questionable physical meaningfulness of ζ for soft colloids
such as SRHA 19, 20) indicated humic acid adsorption on
the Ag NPs. Sorption and change in electrophoretic mobility
has been shown to occur on exposure of humic substances
to macroscopic surfaces (21-23) and to metal (20), metal
oxide (24), and carbon-based nanoparticles (25). The mech-
anisms for such sorption here are not clear but are likely to
be entropically driven replacement of citrate capping agent
with the polydentate SRHA (20). The presence of HS generally
improves colloidal stability by charge effects, although the
situation is often more complex (20) and steric stabilization
is likely to play a role. This sorption and increased charge
most likely accounts for the dispersion mentioned above.
The concentration of dissolved silver (Ag+) from Ag NPs
in MDM without SRHA (Ag NP concentrations 2-2000 ppb)
after 24 h was measured at pH 1, 6, 7.5, and 9 and was low
at all the exposure pH values. Concentrations at the higher
pH values are shown in Table 1. Dissolved Ag solubility was
low in all cases, always lower than 40 ppb (0.38 µM). At pH
1, more than 20% of the Ag NP dissolved but this is not relevant
to the bacterial exposures. The values of dissolved Ag are not
constant for all conditions, perhaps due to continuing
dissolution of the NPs over time. Nevertheless, minor
dissolution of between 1 and 2% of the total Ag is observed
over the time of exposure to bacteria. Quantification of Ag
NP solubility in the presence of SRHA was not performed.
This was due to the possibility of artifacts such as membrane-
SRFA interactions, leading to separations not wholly based
on size and consequent difficulties in interpretation of results.
Nominal mass concentrations used 2-2000 ppb (0.019-19
µM) are, at the lowest values, similar to current or expected
modeled concentrations of Ag NPs in the environment,
according to a recent report from the Project on Emerging
Technologies published by the Woodrow Wilson Interna-
FIGURE 4. Percentage decrease on growth from control treatments (not plotted and representing 100% growth) of planktonic
P.fluorescens cultures 3 h after exposure to silver nitrate (AgNO3), Ag nanoparticles (AgNP) and latex nanoparticles (Latex NP) at A)
pH 9 and B) pH 9 with 10 mg L-1 SRHA. Bars represent 1 standard deviation.
7288 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 19, 2009
total Ag+aq released (ppb)
pH 7.5 pH 9
18.80 ( 2.60 (175.7 ( 24.3nM) 43.2 ( 0.1 (403.7 ( 0.6 nM)
N/D 3.66 ( 0.10 (34.2 ( 1.0 nM)
N/D N/D
tional Center for Scholars (http://www.nanotechproject.org/
process/assets/files/7036/nano_pen_15_final.pdf). The higher
concentrations are not environmentally realistic but have
been used to attempt to investigate dose-response relation-
ships. Total mass concentration of Ag NPs was estimated by
ICP-MS (by mass) and TEM (by particle number). Over 24 h,
concentrations of total Ag (NP + dissolved, primarily in
nanoparticulate form, see above) changed by 40-60% in the
absence of bacteria, indicating a loss over the exposure period
to the bacteria due to unavoidable aggregation and sedi-
mentation and, possibly, to sorption to container walls. Thus
the actual exposure doses were reduced by this factor over
the 24 h of experiment and the nominal values of 2, 20, 200,
and 2000 ppb were overestimates of actual dose. Further
work on the effects of bacteria on reduction in dose and the
kinetics of loss over the exposure time period are required
in all NP studies. It is likely that in most current ecotoxicology
papers on NPs that dose is overestimated. Particle numbers
were estimated as 6 × 107 and 6 × 1010 particles mL-1 for the
nominal 2-2000 ppb (0.019-19 µM) dose.
Toxicity of Ag NPs to Pseudomonas fluorescens. Plank-
tonic P. fluorescens grown at pH 6, 7.5, or 9 were incubated
with different concentrations of Ag NPs, latex NPs, or Ag
nitrate, with and without SRHA, and their growth was
monitored after 3 and 24 h. Controls contained only bacteria
or Ag NPs, latex NPs (negative control, similar size but
expected to have little or no chemical toxicity) or Ag nitrate
(positive control) in the absence of bacteria. The results are
shown in Figures 4 and 5 and are normalized to the controls.
The effect of pH on growth in the controls was negligible,
with slightly lower growth at lower pH. However, the presence
of SRHA increased growth of the control by about 20-25%.
Toxicity after 3 h. As expected, latex beads of similar size
to the Ag NPs showed only a minor degree of toxicity. The
bactericidal effects of Ag nitrate were evident after 3 h of
exposure in all samples at nominal concentrations as low as
200 ppb (ANOVA, p < 0.05), decreasing bacterial population
density by ca. 15% and 60% at nominal concentrations of
200 and 2000 ppb (only pH 9.0 is shown in Figure 4, but
equivalent growth reduction occurred at all pH values). In
addition, the effects were independent of the presence of
SRHA (ANOVA, p < 0.05). Very few data sets are available on
dissolved Ag binding to HS, but any Ag-HS binding clearly
has no effect on bacterial growth in this study. It is possible
FIGURE 5. Percentage decrease on growth from control treatments (not plotted and representing 100% growth) of planktonic P.fluorescens
cultures 24 h after exposure to silver nitrate (AgNO3), Ag nanoparticles (AgNP) and latex nanoparticles (Latex NP) at A) pH 6, B) pH 6 with
10 mg L-1 SRHA, C) pH 7.5 D) pH 7.5 with 10 mg L-1 SRHA, E) pH 9, F) pH 9 with 10 mg L-1 SRHA. Bars represent 1 standard deviation.
that the SRHA does not substantially bind Ag, that the Ag-HS
complexes are still available to bacteria or that biological
effects are indirect and uptake is not required, although lack
of equilibrium or the metal: SRFA ratios may have contributed
to this. Other studies (26) have shown protective effects of
HS to Ag toxicity and these should be considered more reliable
in terms of dissolved silver ion toxicity; in general natural
organic matter may alter metal ion toxicity. However, under
these conditions, no such effect was observed and this allowed
discrimination between dissolved and NP silver.
At 3 h reduced growth is observed with the highest
concentration of Ag NPs in the absence of SRHA and only
significantly at pH 9 (Figure 4 A, 50% reduced growth).
However, this reduced growth at 3 h disappears in the
presence of SRFA (Figure 4 B) i.e. growth as in the control,
without Ag NPs, in the presence of SRHA. The mechanism
by which toxicity is reduced is not clear, since the toxicity
mechanism for Ag NPs is not clear from the literature, but
potentially the HS simply presents a physical barrier to cell-
NP interactions and prevents binding to important proteins
or pitting observed in previous studies. However, if Ag NPs
caused oxidative stress, a frequently cited mechanism of NP
toxicity, then the HS may act as an antioxidant by reacting
with any reactive oxygen species (ROS). A further possibility
is that the NP in contact with the bacteria is a source of
dissolved silver due to continual leaching of the NP by the
bacteria, which can still be considered as a ‘nanoparticle
effect’. We observed no increase in dissolved Ag (from the
NP) in the presence of bacteria, although if this occurs it is
likely to be only at the bacteria-NP interface. Further work
is required on mechanisms of Ag NP toxicity. Nevertheless,
there appear to be toxicity associated with the Ag NP, which
is not connected to the dissolved Ag ion. Two lines of evidence
suggest an effect of the Ag NP themselves, separate form any
dissolved Ag. First, the SRHA has an entirely different effect
on dissolved Ag and Ag NPs, with NP toxicity being removed
and no effect on dissolved Ag, under these conditions. Second,
the low solubility of the Ag NPs means that there is insufficient
dissolved Ag from the NP to cause toxicity, unless through
leaching caused by bacterial processes. Other work (27, 28)
also shows that Ag NPs also have a specific effect on bacteria
separate from the dissolved phase.
Toxicity after 24 h. In comparison to 3 h, there was no
observed effect on growth at 200 ppb of Ag nitrate in most
treatments after 24 h as shown by the low mortality of bacterial
populations at this concentration (Figure 5A-F). For the lower
concentration of dissolved Ag, slightly reduced growth was
observed at the lower pH values in the absence of SRHA (Figure
5A and C), but no consistent trends are observed; the bacteria
appear able to survive and grow in the same manner as in the
control, at this later time period, overcoming the initial reduction
in growth. After 24 h of exposure, only 2000 ppb of dissolved
Ag caused reduced growth, with the population density
decreasing by 90% or more, for all pH values and conditions
tested (Figure 5A-F, ANOVA p < 0.05). This shows that at this
concentration, dissolved Ag is toxic to P. fluorescens and the
population does not easily recover from exposure as they do
at the lower concentration. The presence of SRHA did not affect
the bactericidal properties of Ag nitrate at 2000 ppb consistent
with the 3 h exposure; any binding of Ag by the SHRA had no
effect on toxicity. The reduced growth in the presence of SRHA
is again either due to limited binding by HS under these
conditions, that the HS-Ag complex is sufficiently labile for
the silver ion to be taken up by the bacteria or that the dissolved
Ag does not need to be taken up by the bacteria and inhibits
growth by a different mechanism not requiring uptake.
Ag NPs were toxic only at a concentration of 2000 ppb at
pH 9 without SRHA (Figure 5E, ANOVA p < 0.05), causing a
90% decrease in the bacterial cell density at 24 h after
exposure. The reason for the pH related toxicity is not clear,
VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7289
but independent replicates gave the same results showing
the result to be repeatable. Given the solubility measured
previously and the difference between the effects of SRHA
on dissolved Ag and Ag NPs, the significant changes in growth
again cannot be caused by dissolved Ag from the Ag NP. The
cause of toxicity is related to the NP itself and its interactions
with the bacteria as with the 3 h data.
Further experiments in the presence of SRHA showed that
the presence of SRHA completely removed any effect on
bacterial growth of the Ag NP (Figure 5B, D, and F, ANOVA p
< 0.05). However, TEM images (Figure 1D-F) suggest that Ag
NPs disaggregate in the presence of SRHA, to give a relatively
high number concentration of very small NPs and this would
be expected a priori to increase toxicity (Figure 3). It is likely
that toxicity was mitigated due to the sorption of SRHA onto
the NP surface (Figures 2 and 3, and Supporting Information
Figure S1) and consequent changes in surface properties of the
particle. A possible mechanism is that the SRHA presents a
physical barrier, perhaps reducing dissolution, between the Ag
NP and the bacteria and prevents interaction, although
decreased interaction due to increased charge repulsion may
also play a part. Clearly, as discussed the effects are not related
to the dissolved Ag ions. Previous studies suggest that the toxic
effects of Ag NPs are caused by their interaction and uptake by
bacteria (8, 10, 28) and perhaps pitting on cell membranes
leading to cell disruption and death (28). This evidence is
consistent with our study showing the effects are mitigated by
the presence of HS, which likely forms film on the NP surface
as seen in other studies (20, 24). Our study shows two major
conclusions. First, that toxicity is caused by the intrinsic
properties of the Ag NPs, and not by dissolution of the NPs in
suspension and subsequent effects of the dissolved Ag. Second,
natural organic macromolecules such as HS can mitigate short-
term bacterial toxicity.
Acknowledgments
This work was funded by EU Marie Curie Early Stage Training
(MEST-CT-2004-504356) awarded to J.R.L.
Supporting Information Available
Additional details include one figure showing the electro-
phoretic mobility of Ag NPs in the presence and absence of
SRHA. This material is available free of charge via the Internet
at http://pubs.acs.org.
Literature Cited
(1) Moore, M. N. Do nanoparticles present ecotoxicological risks
for the health of the aquatic environment. Environ. Int. 2006,
32, 967–976.
(2) Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potential of materials at
the nanolevel. Science 2006, 311, 622–627.
(3) Warren, W. S.; Ahn, S.; Mescher, M.; Garwood, M.; Ugurbil, K.;
Richter, W.; Rizi, R. R.; Hopkins, J.; Leigh, J. S. MR imaging
contrast enhancement based on intermolecular zero quantum
coherences. Science 1998, 281, 247–251.
(4) Vieira, S. M. C.; Beecher, P.; Haneef, I.; Udrea, F.; Milne, W. I.;
Namboothiry, M. A. G.; Carroll, D. L.; Park, J.; Maeng, S. Use of
nanocomposites to increase electrical “gain” in chemical
sensors. Appl. Phys. Lett. 2007, 91, 203111–203113.
(5) Dong, Y.; Feng, S.-S. In vitro and in vivo evaluation of methoxy
polyethylene glycol-polylactide (MPEG-PLA) nanoparticles for
small-molecule drug chemotherapy. Biomaterials 2007, 28,
4154–4160.
(6) Griffitt, R. J.; Weil, R.; Hyndman, K. A.; Denslow, N. D.; Powers,
K.; Taylor, D.; Barber, D. S. Exposure to copper nanoparticles
causes gill injury and acute lethality in zebrafish (Danio rerio).
Environ. Sci. Technol. 2007, 41, 8178–8186.
(7) Lovern, S. B.; Strickler, J. R.; Klaper, R. Behavioral and
physiological changes in Daphnia magna when exposed to
nanoparticle suspensions (titanium dioxide, nano-C-60, and
C(60)HxC(70)Hx). Environ. Sci. Technol. 2007, 41, 4465–4470.
7290 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 19, 2009
(8) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri,
J. B.; Ramı́rez, J. T.; Yacaman, M. J. The bactericidal effect of
silver nanoparticles. Nanotechnology 2005, 16, 2346–2353.
(9) Roberts, A. P.; Mount, A. S.; Seda, B.; Souther, J.; Qiao, R.; Lin,
S.; Ke, P. C.; Rao, A. M.; Klaine, S. J. In vivo biomodification of
lipid-coated carbon nanotubes by Daphnia magna. Environ.
Sci. Technol. 2007, 41, 3025–3029.
(10) Shrivastava, S.; Bera, T.; Roy, A.; Singh, G.; Ramachandrarao, P.;
Dash, D. Characterization of enhanced antibacterial effects of
novel silver nanoparticles. J. Nanotechnol. 2007, 18, 225103–
225112.
(11) Yoon, K.-Y.; Hoon Byeon, J.; Park, J.-H.; Hwang, J. Susceptibility
constants of Escherichia coli and Bacillus subtilis to silver and
copper nanoparticles. Sci. Tot. Environ. 2007, 373, 572–575.
(12) Griffitt, R. J.; Weil, R.; Hyndman, K. A.; Denslow, N. D.; Powers,
K.; Taylor, D.; Barber, D. S. Exposure to copper nanoparticles
causes gill injury and acute lethality in zebrafish (Danio rerio).
Environ. Sci. Technol. 2007, 41, 8178–8186.
(13) Duffin, R.; Tran, L.; Brown, D.; Stone, V.; Donaldson, K.
Proinflammogenic effects of low-toxicity and metal nanopar-
ticles in vivo and in vitro: Highlighting the role of particle surface
area and surface reactivity. Inhalation Toxicol. 2007, 19, 849–
856.
(14) Pal, S.; Tak, Y. K.; Song, J. M. Does the antibacterial activity of
silver nanoparticles depend on the shape of the nanoparticle?
A study of the gram-negative bacterium Escherichia coli. Appl.
Environ. Microbiol. 2007, 73, 1712–1720.
(15) Poland, C. A.; Duffin, R.; Kinloch, I.; Maynard, A.; Wallace,
W. A. H.; Seaton, A.; Stone, V.; Brown, S.; Macnee, W.; Donaldson,
K. Carbon nanotubes introduced into the abdominal cavity of
mice show asbestos-like pathogenicity in a pilot study. Nat.
Nanotechnol. 2008, 3, 423–8.
(16) Shahverdi, A. R.; Fakhimi, A.; Shahverdi, H. R. Synthesis and
effect of silver nanoparticles on the antibacterial activity of
different antibiotics agains Staphylococcus aureus and Escheri-
chia coli. Nanomedicine: Nanotechnol., Biol. Med. 2007, 3, 68–
171.
(17) Lyon, D. Y.; Adams, L. K.; Falkner, J. C.; Alvarez, P. J. J.
Antibacterial activity of fullerene water suspensions: effects of
preparation method and particle size. Environ. Sci. Technol.
2006, 40, 4360–4366.
(18) Baalousha, M. Aggregation and disaggregation of iron oxide
nanoparticles: Influence of particle concentration, pH and
natural organic matter. Sci. Tot. Environ. 2009, 407 (6), 2093–
2101.
(19) Duval, J. F. L.; Wilkinson, K. J.; Van Leeuwen, H. P.; Buffle, J.
Humic substances are soft and permeable: Evidence from their
electrophoretic mobilities. Environ. Sci. Technol. 2005, 39, 6435–
6445.
(20) Diegoli, S.; Manciulea, A. L.; Begum, S.; Jones, I. P.; Lead, J. R.;
Preece, J. A. Interaction between manufactured gold nanopar-
ticles and naturally occurring organic macromolecules. Sci. Total
Environ. 2008, 402, 51–61.
(21) Assemi, S.; Hartley, P. G.; Scales, P. J.; Beckett, R. Investigation
of adsorbed humic substances using atomic force microscopy.
Colloids Surf., A 2004, 248, 17–23.
(22) Lead, J. R.; Muirhead, D.; Gibson, C. T. Characterization of
freshwater natural aquatic colloids by atomic force microscopy
(AFM). Environ. Sci. Technol. 2005, 39, 6930–6936.
(23) Neihof, R. A.; Loeb, G. I. Surface Charge of Particulate Matter
in Seawater. Limnol. Oceanogr. 1972, 17, 7–16.
(24) Baalousha, M.; Manciulea, A.; Cumberland, S.; Kendall, K.; Lead,
J. R. Aggregation and surface properties of iron oxide nano-
particles: Influence of pH and natural organic matter. Environ.
Toxicol. Chem. 2008, 27, 1875–1882.
(25) Hyung, H.; Fortner, J. D.; Hughes, J. B.; Kim, J. H. Natural organic
matter stabilizes carbon nanotubes in the aqueous phase.
Environ. Sci. Technol. 2007, 41, 179–184.
(26) Erickson, R. J.; Brooke, L. T.; Kahl, M. D.; Venter, F. V.; Harting,
S. L.; Markee, T. P.; Spehar, R. L. Effects of laboratory test
conditions on the toxicity of silver to aquatic organisms. Environ.
Toxicol. Chem. 1998, 17 (4), 572–578.
(27) Lok, C.; Ho, C.; Chen, R.; He, Q.; Yu, W.; Sun, H.; Tam, P.; Chiu,
J.; Che, C. Proteomic analysis of the mode of antibacterial action
of silver nanoparticles. J. Proteome Res. 2006, 5, 916–624.
(28) Sondi, I.; Salopek-Sondi, B. Silver nanoparticles as antimicrobial
agent: a case study on E. coli as a model for Gram-negative
bacteria. J. Colloid Interface Sci. 2004, 275, 177–182.
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