Ecotoxicology and Environmental Safety 78 (2012) 80–85

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris

and Dunaliella tertiolecta
Abdallah Oukarroum a, Sébastien Bras a,b, Franc- ois Perreault a, Radovan Popovic a,n
a
Department of Chemistry, University of Quebec in Montreal, C.P. 8888, Succ. Centre-Ville, Montreal, Quebec, Canada H3C 3P8
b
Poitiers University, Faculty of Science, Department of Chemistry, 40 Avenue de recteur Pineau, 86022 Poitiers CEDEX, France

a r t i c l e i n f o a b s t r a c t

Article history: Freshwater microalga Chlorella vulgaris and marine microalga Dunaliella tertiolecta were used to
Received 28 July 2011 investigate toxic effects induced by 50 nm silver nanoparticles (AgNPs). To induce AgNPs effect, we
Received in revised form exposed Chlorella vulgaris and Dunaliella tertiolecta for 24 h to 0–10 mg/L. We showed that growth
14 October 2011
media had different effects in AgNPs agglomerates’ formation. Cellular viability, reactive oxygen species
Accepted 15 November 2011
(ROS) formation and lipids peroxidation were employed to assess the toxic effects of AgNPs. AgNPs
Available online 3 December 2011
were able to interact directly with the Chlorella vulgaris cells surface and large aggregates were
Keywords: observed. AgNPs have a negative effect on Chlorella vulgaris and Dunaliella tertiolecta, as manifested by a
Silver nanoparticles strong decrease in chlorophyll content, viable algal cells, increased ROS formation and lipids
Chlorella vulgaris
peroxidation. The variability in sensitivity of both algae towards AgNPs was observed. We conclude
Dunaliella tertiolecta
that AgNPs have a negative effect on aquatic algae and these alterations might have serious
Reactive oxygen species
Lipids peroxidation consequences on structure and function of aquatic plant communities.
& 2011 Elsevier Inc. All rights reserved.

1. Introduction
Silver nanoparticles (AgNPs) are one of the most widely used
nanomaterials in consumer products (www.nanotechproject.org).
AgNPs are used in industrial products, for medical needs due to
their antibacterial and antifungal activities and they are added as
active compounds in detergent (Sambhy et al., 2006; Pal et al.,
2007; Rai et al., 2009). AgNPs in water may have a high mobility
and they can be easily transported to large aquatic environment
(Blaser et al., 2008). However, their environmental impact on
aquatic ecosystems is still unknown.
Algae species vary widely in their response to different toxic
chemicals (Boyle, 1984). Park et al. (2010) reported that AgNPs
have selective inhibitory effects on the harmful cyanobacterium
Microcystis aeruginosa and this alga was more sensitive to AgNPs
than green algae. Klaine et al. (2008) reported that major
differences exist in the chemical behavior of nanoparticles in
seawater compared to freshwater that will impact on the beha-
vior of nanoparticles and therefore the habitats or organisms
being exposed. Nanoparticles’ toxicity could be due to algae
cell-wall. Indeed, Perreault et al. (2011) compared aggregates’
formation in the wild type Chlamydomonas reinhardtii to its cell
wall-deficient mutant exposed 48 h to glycodendrimer-coated
n
Corresponding author. Fax: þ1 514 987 4054.
E-mail address: popovic.radovan@uqam.ca (R. Popovic).
gold NPs. They observed that the wild type Chlamydomonas
reinhardtii formed large aggregates while no aggregates were
observed when the cell-wall was lacking. Furthermore, such
aggregates’ formation has been reported for other nanoparticles.
It was reported that SiO2 and TiO2 NPs were able to interact
directly with the algal cells’ surface through adsorption to the cell
walls (Van Hoecke et al., 2008; Sadiq et al., 2011). Aggregates’
formation might reduce the light available to algal cells and thus
inhibit their growth (Navarro et al., 2008; Perreault et al., 2011),
or alter the cellular acquisition of essential nutrients by clogging
to the walls (Wei et al., 2010).
Large surface area to mass ratio of small size AgNPs provides
strong reactive interactions with cellular and intercellular com-
partments (Pal et al., 2007; Carlson et al., 2008). It is known for
AgNPs to generate free radicals in microorganisms and deterio-
rate cellular functions (Kim et al., 2007). Oxidative stress is an
important factor in nanoparticles-induced toxicity (Nel et al.,
2006). Induction of oxidative stress by AgNPs was observed in
different organisms (Kim et al., 2009; Ahamed et al., 2010; Choi
et al., 2010) and the toxicity of AgNPs to the photosystem II
quantum yield of a freshwater alga (Chlamydomonas reinhardtii)
was demonstrated (Navarro et al., 2008). The latter authors
suggested that toxicity of AgNPs to Chlamydomonas reinhardtii
presents indirect evidence that toxicity of AgNPs is mediated by
Ag þ . Therefore, better knowledge of AgNPs toxicity mechanism is
required in order to evaluate the environmental risk of their
toxicity and the possible change of ecosystem equilibrium.

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.11.012
 A. Oukarroum et al. / Ecotoxicology and Environmental Safety 78 (2012) 80–85
Toxicological studies have shown that nanoparticle size and
aggregation play an important role in determining toxicity (Sager
et al., 2007; Panessa-Warren et al., 2009). Aggregation of nano-
particles depends on particle concentration, pH, ionic strength,
ionic composition, concentration and composition of natural
organic matter, and other characteristics of the aqueous media
(Keller et al., 2010). In the present study, a freshwater microalga
Chlorella vulgaris and a marine microalga Dunaliella tertiolecta
were used to investigate toxic effects induced by 50 nm AgNPs.
Cellular viability, reactive oxygen species (ROS) formation and
lipids peroxidation were employed to assess the toxic effects of
AgNPs. The results from this study could facilitate a better
understanding of the potential toxicity risks of AgNPs in aquatic
environments and compare differential sensitivity among two
green algae.
2. Materials and methods
2.1. Algal culture
The freshwater microalga Chlorella vulgaris and the marine green alga
Dunaliella tertiolecta (CPCC-420) were obtained from the Canadian Phycological
Culture Center (CPCC, University of Waterloo, Canada). The microalga Chlorella
vulgaris was grown in sterile BG-11 liquid medium (Rippka et al., 1979) and
Dunaliella tertiolecta (CPCC-420) was cultivated in seawater growth medium
according to McLachlan (1960). The cells were grown under continuous and
constant light intensity (100 mmol m  2 s  1, SYLVANIA GRO-LUX Wide Spectrum
light F40/GRQ/AQ/WS) at 24 1C. The stock culture was aerated with bubbling air.
Aliquot of algal samples was used when cellular cultures were in their exponential
growth phase. We note here that Dunaliella tertiolecta is a cell-wall lacking alga
(Oliveira et al., 1980).
2.2. AgNPs characterization
Spherical silver nanopowder was purchased from MTI Corporation (Richmond,
CA, USA). According to the manufacturer the diameter of AgNPs was 50 nm, purity
was 99.9% and the specific surface area was 5–10 m2/g. In this study, AgNPs size
was evaluated by transmission electronic microscopy (TEM) and a suspension of
1 mg/L was prepared in: nanopure water (pH ¼6.8), BG-11 (pH¼ 7) and McLachlan
(1960) (pH¼ 6) media. AgNPs particle distribution was determined by dynamic
light scattering (DLS) with a ZetaPlus particle sizer (Brookhaven Instruments
Corporation, USA) using 90Plus Particles Sizing Software Ver. 4.20. Three suspen-
sions were prepared in: nanopure water, BG-11 and McLachlan (1960) media.
A stock suspension of 100 mg/L was prepared and sonicated before use for 2 min
with a sonicator. Zeta potential in the culture media was determined by the
electrophoretic mobility method with the ZetaPlus system.To determine the
solubility of the AgNPs, suspensions of 0–10 mg/L were prepared for 24 h and
incubated in the condition as described above for algal culture. The suspensions
were filtered on a 0.45 mM filter. 10% nitric acid was added for analysis by atomic
emission spectroscopy using a Varian SpectrAA 220 FS system.
2.3. Algal exposure to AgNPs
Aliquots of 100 mL of the algal culture (1  106 cells/mL) in growing media
(BG-11 and McLachlan (1960)) were exposed to 0, 0.01, 0.1, 1 and 10 mg/L of
AgNPs and incubated in the condition as described above for 24 h.
Fig. 1. Transmission electron microscopy (TEM) image of silver nanoparticles (1 mg/mL) in three suspensions media: nanopure water (a), BG-11 (b) and McLachlan (1960) (c).
The arrow in (c) shows AgNPs of 50 nm.
81
2.4. Algal optical microphotographs
Morphological changes in algal culture exposed to 0, 0.1 and 10 mg/L of AgNPs
for 24 h was determined using a Nikon Eclipse TS100 microscope and pictures
were recorded with a Pixelink camera.
2.5. Determination of total chlorophyll
Total chlorophyll extraction was done in 100% of methanol at 65 1C and
quantitative determination was done according to Lichtenthaler (1987).
2.6. Determination of viable cells
Fluorescein diacetate (FDA) is a non-polar ester that passes through cell
membranes. Once inside the cell, FDA is hydrolyzed by esterase (an enzyme present
in viable cells) to produce fluorescein, which accumulates inside viable cell walls
and fluoresces under UV light (Regel et al., 2002). Viability of algal cells was
estimated using the FDA method (Mayer et al., 1997). Each AgNPs’ treatment and
control was treated with 5 mM of FDA in 1 mL of solution. The fluorescence was
measured using an excitation wavelength of 485 nm and an emission wavelength of
530 nm. All the fluorescence data were collected using a fluorescence plate reader.
2.7. Determination of reactive oxygen species (ROS) formation
ROS formation was measured using the cell permeable indicator 20 ,70 -dichlor-
odihydro fluorescein diacetate (H2DCFDA) (Gerber and Dubery, 2003). Cellular
esterases hydrolyze the probe to the non-fluorescent 20 ,70 -dichlorodihydrofluor-
escein (H2DCF), which is better retained in the cells. In the presence of ROS and
cellular peroxidases, H2DCF is transformed to the highly fluorescent 20 ,70 -dichlor-
ofluorescein (DCF). Each AgNPs’ treatment and control was treated with 5 mM of
H2DCFDA in 1 mL of solution. The DCF fluorescence was measured using an
excitation wavelength of 485 nm and an emission wavelength of 530 nm. All the
fluorescence data were collected using a fluorescence plate reader.
2.8. Determination of lipids peroxidation
Lipids peroxidation was measured using the cell permeable indicator
C11-BODIPY581/591. Each AgNPs’ treatment and control was treated with 5 mM of
C11-BODIPY581/591 (Pap et al., 1999) in 1 mL of solution. The fluorescence was
measured using an excitation wavelength of 485 nm and an emission wavelength of
530 nm. All the fluorescence data were collected using a fluorescence plate reader.
2.9. Data analysis and statistics
All treatments were done in triplicates. Means and standard deviations were
calculated for each treatment. Significant differences between control samples and
AgNPs exposed algal samples were determined by analysis of variance (ANOVA)
and Tukey Honestly Significant Differences (HSD) test where p value less than 0.05
was considered to be significant.
3. Results
3.1. Characterisation of AgNPs
Transmission electronic microscopy (TEM) images confirm
that AgNPs have a spherical size of 50 nm (Fig. 1). However,
AgNPs agglomerates’ formation was observed in all tested media.
82 A. Oukarroum et al. / Ecotoxicology and Environmental Safety 78 (2012) 80–85

The results obtained indicated that when AgNPs’ were suspended Table 2
in nanopure water, BG-11 and McLachlan (1960) media, median Dissolved Silver (Ag þ , mg/L) released by AgNPs in the two different culture media.
diameter of particle size distribution determined by DLS was,
Culture 0.01 mg/L 0.1 mg/L 1 mg/L 10 mg/L
respectively, 139.6 nm, 307.3 nm and 448.9 nm, as observed by medium (10  4) (10  4) (10  4) (10  4)
TEM (Fig. 2). AgNPS are negatively charged in the three culture
media as determined by their Zeta potential. A more negative Zeta BG-11 0.22 72 0.23 72 0.0267 4 0.055 7 2
potential was observed in nanopure media (  31.49 72.16) while McLachlan 0.13 74 0.20 72.6 0.487 3 0.76 7 2
(1960)
in BG-11 and McLachlan (1960), Zeta potential was, respectively,
28.52 72.82 and  15.0273.02 (Table 1). After removal of the
particulate fraction by centrifugation and filtration, total soluble
Ag concentration increased when AgNPs’ concentration increased
(Table 2). However total soluble Ag was higher in the McLachlan
(1960) medium compared to BG-11 medium. At 10 mg/L AgNPs’
total soluble Ag was 0.05572  10  4 mg/L and 0.7672  10  4 mg/L,
respectively, in BG-11 and McLachlan (1960) media.

3.2. Cell aggregates’ formation

The effects of AgNPs on Chlorella vulgaris and Dunaliella

tertiolecta were found to be different. For both Chlorella vulgaris
and Dunaliella tertiolecta, algal cultures exposed to 0.1 mg/L
AgNPs for 24 h showed cell aggregates’ formation compared to
control (0 mg/L) (Fig. 3). We noted here that large aggregates
were observed in Chlorella vulgaris exposed to 10 mg/L.

3.3. Chlorophyll content

When Chlorella vulgaris and Dunaliella tertiolecta were exposed

to AgNPs (from 0.01 to10 mg/L) for 24 h, a reduction of the total
chlorophyll in the treatments was observed (Fig. 4). Chlorophyll
content decreased in Chlorella vulgaris by 34% and 51% compared
to control (p o0.05), respectively, at 1 and 10 mg/L AgNPs.

Fig. 3. Morphological changes in Chlorella vulgaris and Dunaliella tertiolecta alga

culture exposed to 0, 0.1 and 10 mg/L of AgNPs for 24 h compared to control.

Fig. 2. Log-normal size distribution of silver nanoparticles prepared in nanopure Fig. 4. Decrease of chlorophyll content in Chlorella vulgaris and Dunaliella
water, BG-11 and McLachlan (1960) media. A stock suspension of 100 mg/L was tertiolecta alga exposed to silver nanoparticles for 24 h. The experiments were
prepared and sonicated before use for 2 min with a sonicator. conducted in triplicate and results are shown as the mean with standard
deviations.

Table 1
Effect of culture medium on Zeta potential (mV)
In Dunaliella tertiolecta chlorophyll content decreased, respectively,
of AgNPs.
at 1 and 10 mg/L by 44% and 75% compared to control (po0.05).
Culture medium Zeta potential (mV)
3.4. Viable cells
Nanopure water  31.49 7 2.16
BG-11  28.52 7 2.82
McLachlan (1960)  15.027 3.02
Algal cells’ viability, evaluated by fluorescein diacetate indi-
cator (FDA), revealed a reduction in viable cell after 24 h treatment.
 A. Oukarroum et al. / Ecotoxicology and Environmental Safety 78 (2012) 80–85 83

Exposure of algae to 1–10 mg/L AgNPs resulted in a significant

reduction of viable cells compared to the control (p o0.05)
(Fig. 5). 1 mg/L AgNPs’ exposure induced a 44% and 33% decrease
of viable cells, respectively, for Dunaliella tertiolecta and Chlorella
vulgaris compared to the control (po0.05). Reduction of viable
cells reached 96% and 88% at 10 mg/L AgNPs’, respectively, for
Dunaliella tertiolecta and Chlorella vulgaris.

3.5. Reactive oxygen species production (ROS) after

exposure to AgNPs

Exposure of algal culture to AgNPs’ induced an increase in

intracellular ROS concentrations. ROS formation increased,
respectively, in Chlorella vulgaris and Dunaliella tertiolecta by
44% and 123% compared to control (p o0.05) at 1 AgNPs
(Fig. 6). At 10 mg/L AgNPs’ ROS formation increased by 7 times
for Chlorella vulgaris and 25 times for Dunaliella tertiolecta com-
pared to control (p o0.05). Therefore, AgNPs can lead to toxico-
logical injury through the production of ROS.

3.6. Lipids peroxidation

Algal cells exposed to AgNPs, using the C11-BODIPY581/591

indicator approach, indicated that AgNPs increased lipids perox-
idation (Fig. 6). The data showed that at 1 mg/L AgNPs lipids
peroxidation increased by 45% and 48%, respectively, in Chlorella
and Dunaliella tertiolecta. For the highest AgNPs concentration
(10 mg/L) lipids peroxidation increased by 4 times in Chlorella
vulgaris and 15 times in Dunaliella tertiolecta compared to control
(po0.05).
The relationship between ROS production and lipids peroxida-
tion is shown in Fig. 7. A positive relationship is observed.
Dunaliella tertiolecta, which had the highest ROS formation, also
exhibited a higher membrane peroxidation relative to the control
during AgNPs’ exposure. However, no difference was observed
between the two algae species during 0.01–1 mg/L of AgNPs, but
Dunaliella tertiolecta was more sensitive to 10 mg/L of AgNPs than
Chlorella vulgaris. Fig. 6. The change of ROS/viable cells and lipids peroxidation/viable cells ratios in
Chlorella vulgaris and Dunaliella tertiolecta alga exposed to silver nanoparticles for
24 h. The experiments were conducted in triplicate and results are shown as the
mean with standard deviations.
4. Discussion

The physicochemical properties of nanoparticles are attribu-

table to their small size, large surface area, chemical composition,
surface reactivity, charge, shape and media interactions
(Oberdorster et al., 2005). In our study, the use of three different

Fig. 5. The change of viable cells in Chlorella vulgaris and Dunaliella tertiolecta alga Fig. 7. Relationship between ROS production and membrane peroxidation in
exposed to silver nanoparticles for 24 h. The experiments were conducted in Chlorella vulgaris and Dunaliella tertiolecta alga exposed to silver nanoparticles
triplicate and results are shown as the mean with standard deviations. during 24 h.
84 A. Oukarroum et al. / Ecotoxicology and Environmental Safety 78 (2012) 80–85
aqueous media (nanopure water, BG-11 and McLachlan (1960))
led to different behaviors of AgNPs in terms of agglomerates’
formation. AgNPs’ characterization using TEM and DLS techniques
showed that nanoparticles aggregated more in McLachlan (1960)
than in BG-11 medium. In the seawater growth medium
(McLachlan, 1960) AgNPs formed large agglomerates
( 4400 nm) as showed by TEM and DLS (Figs. 1 and 2). Handy
et al. (2008) reported that aggregation of nanoparticles in sea-
water is more likely than in freshwater and that the pH of the
water may also influence the aggregation rate depending on the
surface charge of the particles involved. In nanopure water
(pH¼6.8) and BG-11 medium (pH¼7) the AgNPs’ suspension
exhibits  31.4972.16 and  28.52 72.82 of Zeta potential,
respectively, (Table 1). In these aqueous media AgNPs present a
high stability. However in McLachlan (1960) medium (pH¼6)
Zeta potential (  15.0273.02) indicates moderate stability. Then
the AgNPs’ suspensions have different behavior in different pH
media. The pH will alter aggregation chemistry, and are expected
to influence toxicity (Handy et al., 2008). The negative charge of
AgNPs in BG-11 medium might increase the attachment of
nanoparticles to the cell membrane of Chlorella vulgaris.
AgNPs have the potential to cause toxicity in Chlorella vulgaris
microalga and the marine cell wall-lacking alga Dunaliella tertio-
lecta (Figs. 3–6). However, both algae were affected differently.
Chlorella vulgaris exposed to 1 and 10 mg/L AgNPs formed large
aggregates while, at 10 mg/L AgNPs, these aggregates were less
formed in Dunaliella tertiolecta. Therefore, the interaction between
Chlorella vulgaris and AgNPs was considered to be more important
than in Dunaliella tertiolecta. This difference in responses of
exposure to 24 h AgNPs between the two algae could be due to
the cell-wall. Indeed, algal cells’ aggregation was observed in the
wild type Chlamydomonas reinhardtii while no aggregates were
observed when the cell-wall was lacking when algal cells were
exposed for 48 h to glycodendrimer coated gold NPs (Perreault
et al., 2011). Furthermore, such aggregates’ formation has been
reported for other nanoparticles. It was reported that SiO2 and
TiO2 NPs were able to interact directly with the algal cells’ surface
through adsorption to the cell walls (Van Hoecke et al., 2008;
Sadiq et al., 2011). The diameter of pores across the cell wall has a
thickness ranging from 5 to 20 nm that determines its sieving
properties (Navarro et al., 2008). However 50 nm AgNPs were not
able to pass through the cell wall but might act as a binding agent
between algal cells. Therefore aggregates’ formation might inhibit
algal cells’ growth (Navarro et al., 2008; Wei et al., 2010; Perreault
et al., 2011). Such effect was observed in our study by strong
reduced chlorophyll content and viable cells (Figs. 4 and 5). Cells’
aggregation may have participated in the toxicological effects
of AgNPs.
Our study demonstrated that AgNPs induce a strong ROS
formation in both algae. It is known that the high levels of ROS
induction can lead to cell structure damage and may involve lipid
peroxidation (Halliwell and Gutteridge, 1999). High lipids perox-
idation was observed also in both algae exposed to 24 h AgNPs.
Such effect can lead to impaired cellular function and alterations
in physico-chemical properties of cell membranes, which disrupt
vital functions (Rikans and Hornbook, 1997) and reduced viable
cells (Fig. 5). We noted here that cell wall-lacking alga Dunaliella
tertiolecta was more affected and then sensitive to AgNPs in terms
of ROS induction and lipids peroxidation than Chlorella vulgaris
(po0.05). The ability to generate ROS and oxidant injury may
provide an assay to compare the toxic potential of AgNPs. The
release of Ag þ might contribute to algae toxicity. Indeed, the
characteristics of medium affect the toxicological effects of
AgNPs. Solubilization of the NPs into free metal ions in the media
is considered as the most common mechanisms of toxicity for
several types of NPs (Franklin et al., 2007). Turner et al. (2011)
reported that AgNPs are only indirectly toxic to marine algae Ulva
lactuca through the dissolution of Ag þ ions into bulk sea water. It
is known that Ag þ is one of the most phytotoxic metal ion (Ratte,
1999; Hiriart-Baer et al., 2006) because of its cationic nature and
its strong association with various ligands in natural waters. The
toxicity of Ag þ ions depends largely on the strength and amount
of the ligands present in the medium (Ratte, 1999). In the
presence of chloride (present in seawater growth medium more
than BG-11 medium) aqueous toxicity may be augmented by the
formation of the moderately hydrophobic, neutral chloro complex
(Reinfelder and Chang, 1999). Therefore Dunaliella tertiolecta
(CPCC-420) cultivated in seawater growth medium appeared
sensitive to AgNPs compared to freshwater microalga Chlorella
vulgaris.
The use of unicellular microalgae has advantages compared to
higher plant since algae have bigger surface contact to pollutants.
The observation of Dunaliella tertiolecta sensitivity compared to
Chlorella vulgaris gives the possibility that the difference in
toxicity may be due to different physiological effects. Conse-
quently, we may suggest for toxicity bioassays that Dunaliella
tertiolecta is an excellent toxicity test organism and valuable
biomarker of stress induced by AgNPs.
5. Conclusion
In conclusion the variability in sensitivity of both algae
towards AgNPs was observed. From these results we concluded
that AgNPs have a negative effect on aquatic algae, as manifested
by the decrease in viable algal cells. AgNPs in terms of agglom-
erates’ formation have different behaviors dependent on the
culture media, leading to physical and chemical alterations and
therefore different toxicity effects. The theme of ROS production,
lipids peroxidation injury and decrease of viable cells has become
an established endpoint for AgNPs’ toxicity. Alga communities are
involved in maintaining oxygen production as well as food chain
and these alterations might have serious consequences on the
overall functioning of the aquatic ecosystem.
Acknowledgments
This work was supported by the Natural Sciences and Engi-
neering Research Council (NSERC, Canada) grants awarded to
R. Popovic. F. Perreault was supported by an NSERC Ph.D. fellowship.
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