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SILVER NANOPARTICLE TOXICITY EFFECT ON GROWTH AND CELLULAR

VIABILITY OF THE AQUATIC PLANT LEMNA GIBBA


ABDALLAH OUKARROUM,y LOTFI BARHOUMI,yz LAURA PIRASTRU,y and DAVID DEWEZ*y
yDepartment of Chemistry, University of Quebec in Montreal, Quebec, Canada
zLaboratory of Integrative Physiology, University of Bizerte, Bizerte, Tunisia
(Submitted 31 May 2012; Returned for Revision 19 August 2012; Accepted 30 November 2012)
AbstractThe toxicity effect of silver nanoparticles (AgNPs) on growth and cellular viability was investigated on the aquatic plant
Lemna gibba exposed over 7 d to 0, 0.01, 0.1, 1, and 10 mg/Lof AgNPs. Growth inhibition was demonstrated by a significant decrease of
frond numbers dependent on AgNP concentration. Under these conditions, reduction in plant cellular viability was detected for 0.1, 1,
and 10 mg/L of AgNPs within 7 d of AgNPs treatment. This effect was highly correlated with the production of intracellular reactive
oxygen species (ROS). A significant increase of intracellular ROS formation was triggered by 1 and 10 mg/L of AgNP exposure. The
induced oxidative stress was related to Ag accumulation within L. gibba plant cells and with the increasing concentration of AgNP
exposure in the medium. The authors results clearly suggested that AgNP suspension represented a potential source of toxicity for L.
gibba plant cells. Due to the low release capacity of free soluble Ag from AgNP dissolution in the medium, it is most likely that the
intracellular uptake of Ag was directly from AgNPs, triggering cellular oxidative stress that may be due to the release of free Ag inside
plant cells. Therefore, the present study demonstrated that AgNP accumulation in an aquatic environment may represent a potential
source of toxicity and a risk for the viability of duckweeds. Environ. Toxicol. Chem. 2013;32:902907. # 2013 SETAC
KeywordsSilver nanoparticles Aquatic plant Lemna gibba Cellular viability Reactive oxygen species
INTRODUCTION
During recent years, the production of nanoparticles has
increased for a wide variety of applications in consumer
products; therefore, their environmental impact needs to be
assessed, especially concerning the risk of toxicity to aquatic
organisms [1]. In particular, silver nanoparticles (AgNPs)
represent one of the most widely used nanomaterials in
industrial products for medical needs due to their antibacterial
and antifungal activities; they are also added as active
compounds in detergent (www.nanotechproject.org). In aque-
ous solution, agglomeration or stabilization of metallic nano-
particles can be affected by parameters such as pH, ionic
strength, composition in chemical salts, temperature, and
nanoparticle concentration [2,3]. Concerning the bioavailability
of metallic nanoparticles, uptake and toxicity potential
within aquatic organisms are determined by the nanoparticle
physicochemical properties such as the size, shape, chemical
composition, charge at particle surface, surface structure
and area, and finally the solubility of particles [4]. These
physicochemical properties of nanoparticles need to be taken
into account in aquatic toxicological studies when done with
different organisms.
The toxicity of silver compounds has been investigated in
different terrestrial and aquatic organisms and was related to the
bioaccumulation effect of silver [5]. More recently, the toxicity
effect of AgNPs was also studied in aquatic organisms such as
algae or plants. Although AgNPs toxicity effect was partly
explained by the release of Ag

, a direct or indirect cause


of AgNPs toxicity is still in debate. It was shown that
photosynthesis of algal cells appeared to be more affected by
Ag

when Chlamydomonas reinhardtii was exposed shortly


(6 h) to AgNPs or its equal mass in silver ions [6]. However,
others studies demonstrated that the release of Ag

fromAgNPs
was the only direct cause of toxicity effects on marine algae
[7,8]. It was also shown that AgNPs had a deteriorating effect on
the physiological state of Chlorella vulgaris and Dunaliella
tertiolecta, as manifested by a strong decrease in viable algal
cells, which was related to the increase of ROS formation
and lipids peroxidation [9]. Algal cells of C. vulgaris and
D. tertiolecta, exposed 24 h to agglomerates of AgNPs,
indicated a decrease of cell growth due to AgNP toxicity [9].
Similar agglomeration was previously reported in toxicological
studies for others nanoparticles in aqueous solution. For
example, it was reported that the agglomerated form of SiO
2
and TiO
2
nanoparticles was able to interact directly with the
algal cells surface through adsorption to the cell walls [10,11]. It
was also noticed that the agglomeration of nanoparticles could
alter the acquisition of essential nutrients by clogging the cell
wall, causing the inhibition of algal cell growth [12,13].
Moreover, the aquatic plant Lemna minor exposed to 5 mg/L of
small and larger AgNP suspensions showed an inhibition of
growth [14]. It was also reported for 50-nm AgNPs in colloidal
form to cause a growth inhibition of Lemna paucicostata in
concentration range higher than 1 mg/L [15]. Therefore, AgNP
suspension in aqueous solution may be a direct source of
toxicity, and/or indirectly by releasing Ag

, which is also
dependent on the agglomeration state of nanoparticles.
In the present study, the aquatic plant Lemna gibba was used
as a model organism to evaluate the potential risk of toxicity
of AgNP suspension in aqueous solution. This plant species
represents a suitable aquatic organism for toxicological
studies because it has been shown to have a high sensitivity
to the toxicity effect of pollutants causing the inhibition of
photosynthetic processes and biomass growth [16,17]. Here, the
toxicity effect of AgNP suspension was investigated on the
* To whom correspondence may be addressed
(dewez.david@uqam.ca)
Published online 22 January 2013 in Wiley Online Library
(wileyonlinelibrary.com).
Environmental Toxicology and Chemistry, Vol. 32, No. 4, pp. 902907, 2013
# 2013 SETAC
Printed in the USA
DOI: 10.1002/etc.2131
902
growth of L. gibba plants, which was related to the change of
the physiological state of plant and the bioaccumulation of
intracellular Ag. Under these experimental stress conditions,
the physiological state of L. gibba plants was indicated by
the change of plant cellular viability and the production of
intracellular reactive oxygen species (ROS). We clearly show
that the inhibition of plant growth over 7 d was due to an induced
oxidative stress caused by the bioaccumulation of intracellular
Ag in plant cells. Therefore, the present study demonstrates that
AgNP accumulation in an aquatic environment may represent
a potential source of toxicity and a risk for the viability of
duckweeds.
MATERIALS AND METHODS
The aquatic plant L. gibba
The aquatic plant L. gibba was obtained from the Canadian
Phycological Culture Centre (formerly UTCC #310). Plants
were grown in an inorganic growth culture mediumas described
by Frankart et al. [18]. The stock cultures were maintained in
aquariums containing 400 ml of inorganic autoclaved growth
medium (pH 6.5). This medium consisted of the following:
KNO
3
, 202 mg/L; KH
2
PO
4
, 50.3 mg/L; K
2
HPO
4
, 27.8 mg/L;
K
2
SO
4
, 17.4 mg/L; MgSO
4
7 H
2
O, 49.6 mg/L; CaCl
2
,
11.1 mg/L; FeSO
4
7 H
2
O, 6 mg/L; H
3
BO
3
, 5.72 mg/L;
MnCl
2
4 H
2
O, 2.82 mg/L; ZnSO
4
, 0.6 mg/L; (NH
4
)Mo
7
O
24

4 H
2
O, 0.043 mg/L; CuCl
2
2 H
2
O, 0.078 mg/L; CoCl
2

6 H
2
O, 0.054 mg/L. Ionic strength for L. gibba culture medium
was 4.25 10
3
. Ionic strength was calculated with chemical
equilibrium model software Visual MINTEQ 3.0. Plant culture
and experiments were done in a growing chamber CONVIRON
(Controlled Environments Limited) with a light:dark cycle
of 16:8 h and temperature of 248C. A light irradiance of
100 mmol m
2
s
1
was provided by cool white fluorescent
lamps (Sylvania GRO-LUX F40/GS/WS).
AgNP characterization
Spherical silver nanopowder was purchased from MTI
Corporation. According to the manufacturer, the diameter of
AgNPs was 50 nm, purity was 99.9%, and the specific surface
area was 5 to 10 m
2
/g. We determined AgNP distribution by
dynamic light scattering (DLS) with a ZetaPlus particle sizer
(Brookhaven Instruments) using 90Plus Particles Sizing
Software Version 4.20. Stock AgNPs suspension of 100 mg/L
was prepared in L. gibba culture medium and sonicated for
2 min before use to homogenize nanoparticle suspension. Zeta
potential of AgNPs suspended in L. gibba culture medium was
determined by the electrophoretic mobility method with the
ZetaPlus system. Silver nanoparticle size was evaluated by
transmission electronic microscopy (TEM) and a suspension of
1 mg/L was prepared in L. gibba culture medium.
To determine the soluble fraction of Ag released from
AgNPs, nanoparticle suspensions of 0 to 10 mg/L were
prepared in culture medium and incubated for 24 h in the
same condition as described above for L. gibba. Before analysis,
AgNPs suspensions were centrifuged at 12,000 g for 30 min.
Before analysis, the absence of any nanoparticles was verified
using DLS with the ZetaPlus particle sizer. The quantification of
Ag in solution was done by atomic absorption spectroscopy
using a Varian SpectrAA 220 FS system.
L. gibba plants exposure to AgNPs
Triple-fronded L. gibba plants, in the exponential growth
phase, were used for experiments. Five triple-fronded L. gibba
plants in three replicates were treated over 7 d in Petri dishes
containing growth medium having initial AgNP concentrations
of 0, 0.01, 0.1, 1, and 10 mg/L. The treated medium of each
Petri dish was changed every 24 h to maintain constantly the
exposure of L. gibba plants to initial concentrations of AgNPs.
According to the Organisation for Economic Co-operation
and Development (OECD) guidelines for the testing of
chemicals using the Lemna sp. growth inhibition test [19],
the inhibition for specific growth rate in percentage (% Ir) was
determined by frond numbers using the following formula: %
Ir ((mC mT)/mC) 100, where % Ir represents the
percentage of inhibition in average specific growth rate, mC
the mean value for min the control, and mT the meanvalue for m
in the treatment group.
Determination of viable cells
Viability of cells in L. gibba plants was estimated using the
fluorescein diacetate (FDA) method according to [20]. The
FDA is a nonpolar ester that passes through cell membranes.
Once inside the cell, FDA is hydrolyzed by esterases enzymatic
activities to produce fluorescein, which accumulates only inside
viable cells and emits fluorescence under ultraviolet (UV)
light [21]. Four intact plants from each AgNP treatment and the
control were treated with 5 mM of FDA in 1 ml of solution
medium. A blue excitation light at 485 nm was used to measure
the fluorescence emission at 530 nm. Fluorescence data was
normalized by fresh weight.
Determination of reactive oxygen species formation
The production of ROS in L. gibba plants was determined
as [22,23]. At the end of 7 d of AgNP exposure, four intact
plants from each AgNP treatment and the control were washed
in L. gibba culture medium three times and placed in 1-ml new
solution medium. The formation of ROS was measured by using
the cell permeable indicator 2,7dichlorodihydrofluorescein
diacetate (H
2
DCFDA) [24]. Cellular esterases hydrolyze the
probe to the nonfluorescent compound 2,7dichlorodihydro-
fluorescein (H
2
DCF), which is better retained in the cells. In the
presence of ROS and cellular peroxidases, H
2
DCF is trans-
formed to a highly fluorescent compound, the 2,7dichloro-
fluorescein (DCF) [25]. The plants were treated with 5 mM of
H
2
DCFDA in 1 ml of solution medium for 30 min at 258C. The
fluorescence emission at 530 nm was measured by using the
excitation wavelength at 485 nm. Fluorescence data was
normalized by fresh weight. All the fluorescence data were
collected using a fluorescence plate reader (SpectraMax M2e
Multi-Mode Microplate Reader).
Determination of intracellular Ag
After 7 d of AgNPs treatment, plants from each AgNP
treatment were washed in L. gibba culture medium three times
and then washed with ethylenediaminetetraacetic acid (10 mM)
in L. gibba culture mediumfor 2 min to remove Ag bound to the
cell surface. Lemna gibba plants were dried at 1058C for 24 h,
and weighed to calculate dry weight. Then, they were placed in
an acid-washed glass tube containing 2-ml HNO
3
and 500-ml
H
2
O
2
. The digestion process was done during 48 h at 1298C
before being diluted in Nanopure purified water for ICP-AES
quantification of Ag into plant cells. Obtained soluble Ag
concentrations were normalized by dry weight.
Data analysis and statistics
Means and standard deviations were determined for each
treatment. Significant differences between control and treated
Silver nanoparticle toxicity effect on Lemna gibba Environ. Toxicol. Chem. 32, 2013 903
samples were determined by using multiple comparison
Bonferroni tests, where p values less than 0.05 were considered
to be significantly different. The median effective concentration
(EC50) was obtained by Log(inhibitor) versus response fitting
the toxicity data with Prism software, resulting in effective
concentrations given at 50%.
RESULTS
When spherical silver nanopowder having 50 nm of
diameter was suspended in L. gibba culture medium, AgNPs
formed rapidly agglomerates (Fig. 1A). The TEM images
confirmed that AgNPs were forming agglomerates in L. gibba
culture media (Fig. 1B). This agglomeration state was directly
dependent on the pH and the ionic strength of the medium,
which was stable during 24 h because we noticed no significant
differences of AgNPs agglomerates size distribution at the
beginning and the end of the 24-h period experiment. Particle-
size measurements obtained from DLS indicated that median
diameter of particle size distribution was 240 nm, and AgNPs
had a zeta potential of 34.75 2.15. These results were due to
the physicochemical properties of L. gibba culture mediumsuch
as the pH (6.5) and nutrients composition. During the 24-h
experiment, it was also noticed that very low dissolved Ag
(1%) was released from those AgNPs agglomerates (Table 1).
The effect of AgNPs on growth rates of L. gibba plants was
evaluated over 7 d by counting fronds. Within this toxicological
test period, a significant inhibition of plant growth relative to the
control was observed when L. gibba was exposed to AgNPs
(Fig. 2). After 7 d of exposure, the reduction of growth was
dependent on AgNPs concentration, and at high concentrations
of AgNPs fewer fronds were produced. When L. gibba was
exposed to AgNPs concentrations of 1 and 10 mg/L, the fronds
number decreased by 40 and 45% compared to the control,
respectively. Figure 2 shows growth inhibition (in percentage)
of L. gibba plants based on frond number in relation to AgNPs
treatments. This relation indicated that the inhibition of plant
growth occurred at all AgNPs concentrations, and was
dependent on the quantity of AgNPs suspension in aqueous
solution. For the highest AgNPs concentration (10 mg/L)
tested, inhibition of growth reached 44% compared to the
control. For this treatment condition (AgNPs concentration
range from 0.01 to 10 mg/L), the EC50 on plant growth based
on frond number was 9.36 ( 2.36) mg/L.
The accumulation of Ag into L. gibba plants exposed to
AgNPs was quantitatively analyzed by measuring total Ag
content (Fig. 3). When L. gibba plants were exposed from 0.01
to 10 mg/L, an important uptake of Ag was found at 10 mg/L of
AgNP treatment. Silver accumulation within the cells was
increased with AgNPs increasing concentration in the medium.
Total Ag content in L. gibba plants treated with 0.01, 0.1, 1, and
10 mg/L of AgNPs was 7.72 10
3
, 9.5 10
3
, 11.3 10
3
, and 17.5
10
3
mg/mg dry weight, respectively. These results indicated
that the amount of Ag uptake into L. gibba plant cells was
dependent on the quantity of AgNPs suspension in aqueous
solution.
Viability of cells in L. gibba plants evaluated by FDAshowed
a significant decrease (p < 0.05) after 7 d of treatment for 0.1, 1,
and 10 mg/L of AgNPs (Fig. 4). The decrease of cellular
viability reached 80% compared to the control for the highest
AgNP concentration (10 mg/L), indicating the cytotoxicity
effect of AgNPs on plant cells.
The toxicity effect of AgNPs was also evaluated via the
production of intracellular ROS in L. gibba plants due to
the induction of oxidative stress (Fig. 4). A high fluorescence
emission of the ROS sensor (H
2
DCFDA) compared to the
control was found when the L. gibba plant was exposed to
10 mg/L, suggesting that a strong increase in ROS formation
revealed a higher AgNP toxicity. The production of ROS
reached 340% compared to the control (p < 0.05) for the
highest AgNP concentration (10 mg/L). The relationship
between viable cells and ROS formation is shown in Figure 5.
Here, we clearly found a high correlation (r
2
0.91) between
the formation of ROS and the decrease of viable cells.
Table 1. Soluble fraction of silver (as Ag in mg/L) released from silver
nanoparticles (AgNPs) suspensions after 24 h in Lemna gibba culture
medium
AgNPs 0.01 mg/L 0.1 mg/L 1 mg/L 10 mg/L
Ag mg/L 0.83
( 0.02) 10
4
0.87
( 0.02) 10
3
1.02
( 0.04) 10
2
1.50
( 0.02) 10
1
Fig. 1. (A) Size distribution of silver nanoparticles (AgNPs) prepared in Lemna gibba medium. (B) Transmission electron microscopy image of AgNP
suspension (1 mg/ml) in L. gibba medium.
904 Environ. Toxicol. Chem. 32, 2013 Oukarroum et al.
DISCUSSION
In the present study, the toxicity effect of Ag bioaccumu-
lation was investigated in L. gibba plants exposed to AgNPs
suspension. In aqueous solution, AgNPs formed agglomer-
ations as indicated by characterization measurements of
nanoparticles suspension in culture medium. The AgNPs
Fig. 2. The change of Lemna gibba plant growth (total fronds number) and
the inhibition of growth (in %) at day 7 after the exposure to different
concentrations of silver nanoparticles (AgNPs).
Fig. 3. Total amount of intracellular Ag concentration in Lemna gibba plants
exposed 7 d to different concentrations of silver nanoparticles (AgNPs; mg/
L). Asterisk indicates statistical significance between AgNPs treatments and
control (p < 0.001).
Fig. 4. The change of viable cells and the production of reactive oxygen
species (ROS) in Lemna gibba plants exposed to silver nanoparticles
(AgNPs) over 7 d. The results are shown as the mean (n 4) with standard
deviations. Asterisk indicates statistical significance between the control and
AgNPs treatments (p < 0.05).
Fig. 5. Relationship between the change of viable cells and reactive oxygen
species (ROS) in Lemna gibba plants exposed to silver nanoparticles
(AgNPs) over 7 d.
Silver nanoparticle toxicity effect on Lemna gibba Environ. Toxicol. Chem. 32, 2013 905
suspension exhibits a zeta potential of 31.49 2.16, showing
a high stability of physicochemical properties of these
agglomerated nanoparticles. Indeed, L. gibba plants were able
to bioaccumulate Ag into their multicellular system when
exposed to agglomerated AgNPs in a concentration dependent
relation. Because the soluble fraction of Ag was very low, it is
most likely that the accumulation of intracellular Ag was
coming directly from nanoparticles, causing cellular toxicity
effects as indicated by the decrease of plant growth. This
inhibition of plant growth was dependent on AgNPs concentra-
tion (0.0110 mg/L) in suspension and the period of exposure.
The reduction of L. gibba growth was evidently caused by an
alteration of the physiological state of plant cells. The cytotoxic
effect of bioaccumulated Ag was indicated by the decrease of
viable cells, which was directly related to the increase of
intracellular ROS production. Indeed, we found the formation of
ROS to increase by more than 340% compared to the control
when L. gibba plant was exposed to high concentration of
AgNPs (10 mg/L). The production of ROS caused by the
accumulation of intracellular Ag demonstrated an evident
cellular toxicity impact as indicated by the decrease of cellular
viability. We noted here that in the control condition, ROS were
also produced, but in lower amount because the ROS production
can occur in both stressed and unstressed cells. However, it is
well known that plants have developed biochemical defense
strategies against intracellular ROS accumulation, where the
production and the elimination of ROS are in balance under
unstressed conditions [27]. It has been proposed for the toxic
mechanism of action of many nanomaterials to induce a strong
oxidative stress altering the balance between oxidant and
antioxidant cellular processes [28,29]. In the present study,
when the L. gibba plant was exposed to AgNPs, the increase of
intracellular ROS concentration was inducing an oxidative
stress, therefore disturbing cellular biochemical functions.
In the present study, the EC50 was 9.36 ( 2.36) mg/L for
the L. gibba plant exposed 7 d to AgNPs, which was similar to
that previously reported for Lemna minor L. clone St [26],
which was evaluated from the effect of soluble Ag

released
from AgNO
3
. According to our results, it is most likely that the
observed cellular toxic effect is mediated by soluble Ag released
from AgNPs inside plant cells. It was previously suggested that
the solubilization of free metal ions in aqueous solution from
nanoparticles in suspension was considered to be the most
plausible mechanisms of toxicity for several types of metallic
nanoparticles [30]. In particular, AgNPs in suspension tend to
release free Ag; thus, any AgNPs represent a potential source of
toxicity caused by the release of Ag

into aqueous solution. The


toxicity of soluble free Ag to microorganisms has been
extensively studied and was directly related to the bioaccumu-
lation effect [31]. In a previous study, it was reported that
0.1 mg/L of silver nitrate (AgNO
3
) inhibits the growth of the
microalgae Chlorella vulgaris and Chlorella VT-1 [33]. In
another study, the alteration of photosynthesis in algal cells of
alga Chlamydomonas reinhardtii exposed shortly (6 h) was
indicated by the decrease of the photosystem II quantum yield;
the authors suggested that this inhibition was caused by toxic
Ag

released from AgNPs inside the cells [6]. Furthermore,


Turner et al. [8] also proposed that AgNPs were only toxic to the
marine algae Ulva lactuca through their dissolution into Ag
ions. Therefore, we suggest here that the observed cytotoxicity
effect on L. gibba plant growth and cellular viability was
attributable to soluble free Ag originating from absorbed
nanoparticles in plant cells. This cytotoxicity effect causing
oxidative stress may result from the interaction of Ag

with
proteins and/or enzymes through specific functional groups as
thiol (-SH). It was previously found that the exposure of Ag
compounds provoked the production of ROS in algal cells of C.
reinhardtii, which was due to the indirect effect of Ag

having
high affinity with thiol groups [32].
In conclusion, the relationship between the production of
intracellular ROS and cellular viability was used to estimate the
physiological alteration induced on L. gibba plant cells by the
accumulation of intracellular Ag. Our results clearly suggest
that AgNP suspension represented a potential source of toxicity
for L. gibba plant cells. Due to the low release capacity of free
soluble Ag from AgNP dissolution in the medium, it is most
likely that the intracellular uptake of Ag was directly from
AgNPs, triggering cellular oxidative stress that may be due to
the release of free Ag inside plant cells. Furthermore, it was
evident that under these experimental conditions the production
of ROS causing an oxidative stress was responsible for the
deterioration of plant cells viability and growth over 7 d.
Therefore, the present study demonstrates that AgNP suspen-
sion accumulated in an aquatic environment represents a
potential source of toxicity and a risk for the viability of
duckweeds.
AcknowledgementD. Dewez acknowledges the financial support provided
by both the Faculty of Sciences and the Department of Chemistry at
University of Quebec in Montreal. A. Oukarroum and L. Pirastru were
collaborator research agents. L. Barhoumi was involved in this study as a
research trainee.
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