International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)

Volume-1 , Issue-1 , August 2012

7

EFFECT OF SILVER ANOPARTICLE (SNPs) ON PROTEIN AND
DNA CONTENT TO TOMATO SEED (L.esculentum),
CUCUMBER (Cucumis sativus) and MAIZE (Zea mays)
Mamta Kuamri
1
, Amitava Mukherjee
1
, N, Chandrasekaran
1
1
VIT University , Vellore India
mamtapoddar@gmail.com



Abstract

Phytotoxic effects of manufactured or
commercially available Silver
Nanoparticles (SNPs) on L.esculentum
(tomato), Cucumis sativus (cucumber) and
Zea mays (maize) seeds were investigated
by means of increase in root length, seed
germination percentage and protein
concentration. There was concentration
dependent decrease in root length,
germination percent and protein
concentration and DNA content. The R
2

for root length of seeds treated with
commercially available SNPs was 0.9282
(L.esculentum), 0.9625 (Z.mays) and
0.7329 (C.sativus). Concentration
dependent decrease in germination
percent was observed when SNPs was
increased. Control seeds showed 100%
germination. Seeds treated with 500ppm
of SNPs showed a maximum germination
of 43.3% for tomato, 30% for cucumber
and 50% for maize. DNA content
decreased in control and treated sample as
(2.2 to 0.64) cucumber, (2.19 to1.2)
tomato and for maize (2.46-1.1)g/g,
respectively.

Key words: Silver nanoparticles,
phytotoxicity, L.esculentum (tomato),
Cucumis sativus (cucumber), Zea mays
(maize) seeds, dissolution mechanism.

1. INTRODUCTION

Nanotechnology is often described as an
emerging technology-one that not only
holds promises for society, but also is
capable of revolutionizing our approaches
to common problems. The value of
nanomaterials in many technology areas is
very high because of their versatile
properties. Today some nanomaterials are
already being used commercially. For
example, some companies are using TiO2
nanoparticles in sunscreen lotions.
Nanomaterials can also be found in
sporting equipment, clothing, and
telecommunication infrastructure.
Nanotechnology has direct beneficial
applications for medicine and the
environment, but like all technologies it
may have unintended effects that can
adversely impact the environment, both
within the human body and within the
natural ecosystem. While taking advantage
of this new technology for health,
environmental, and sustainability benefits,
science needs to examine the
environmental and health implications.
Recently, nanotechnology has received
considerable attention from the media.
Most of the initial reports have been
positive; however, scientists should not
forget that not all nanomaterials will be
benign. Therefore, it is very important to
identify the negative aspects of the
technology before we introduce it to the
marketplace.
International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
8

Nanometer-sized particles have
shown special toxicity and are usually
more toxic than the bulk material of larger
size [11]. When inhaled as single particles,
particles having less than 50 nm diameter
proved to be highly toxic [10].
Nanoparticles like fullerene, carbon
nanotubes and metal oxides pose toxicity
to human cells, bacteria and rodents [3].
Data on potential toxicity of nanoparticles
to ecological terrestrial test species is still
limited [22]. Few studies have reported
both positive and negative effects of
nanoparticles on higher plants. Nanoscale
SiO
2
and TiO
2
enhanced nitrate reductase
activity in soybean, and apparently
hastened its germination and growth [4].
Nano-TiO
2
promoted photosynthesis and
nitrogen metabolism, and improved
growth of spinach [8] [13]. Silver
nanoparticles have been proved to cause
be genotoxic effects in the root cells of
Allium cepa [14]. There are around 800
consumer products where silver
nanoparticles are being used [5]. The
antimicrobial properties of silver
nanoparticles are being increasingly
exploited in consumer products like
deodorants, clothing materials, bandages,
and also in cleaning solutions and sprays
[24]. In the near future there is a risk of
enhanced bioavailability of the
nanoparticles in the environment.
Historically, plants have been used
as indicator organisms, to study toxicity of
substances. Plant systems have a variety of
well defined end points in phytotoxicity
testing, like seed germination toxicity test,
root length; shoot length, biochemical
parameters etc. The reports from few
previous studies have advanced our
understanding of nanotoxicology for
several types of nanomaterials. There are
still many unresolved issues and
challenges concerning the biological
effects of nanoparticles. Therefore, the
present study is designed to investigate
toxicological effect of Silver Nanoparticles
(SNPs) to Tomato seeds (L.esculentum),
Cucumber (C. sativus) and Maize (Z.
mays).

2. MATERIAL AND METHODS
2.1. CHEMICALS
All the chemicals were procured from
Sigma Aldrich, USA. The physical
characteristics of the nanoparticles as
reported by the suppliers are as follows:
size of the particle (near 100 nm), surface
area (5.0 m2g-1) and density (10.4 g cc-1).
What about your analysis?
2.2. CHARACTERIZATION OF THE
SILVER NANOPARTICLES

The morphological features of the SNPs
were characterized using Transmission
Electron Microscopy (Tecnai G-20, FEI,
Netherlands) and Scanning Electron
Microscopy (FEI Sirion, Eindhoven,
Netherlands).
2.3. PREPARATION OF
NANOPARTICLE DISPERSION
The SNPs were suspended in deionized
water and were dispersed using ultrasonic
vibrations (100 W, 30 kHz) for 30 min to
produce four different concentrations,
namely 10ppm, 100ppm, 500ppm. All
SNPs concentrations were selected
arbitrarily.
2.4. SEED EXPERIMENT
Seeds of L.esculentum (tomato),
Cucumis sativus (cucumber) and Zea mays
(maize) were used for the study. The
seeds were procured from a nursery
(Vellore, Tamilnadu) and were stored in
the dark under room temperature. All the
seeds were first checked for their viability
by suspending them in deionized water.
The seeds which settled to the bottom were
selected for further study. The seeds were
then soaked for 10 min in a 10% sodium
International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
9

hypochlorite solution, which acts as a
surface sterilizing agent [17].
After surface sterilization, the seeds
were rinsed in deionized water thrice and
were then stirred for 2 h in SNPs
dispersion (10ppm, 100ppm, 500ppm)
using a magnetic stirrer. Whatmann No.1
filter paper was then placed into each Petri
dish (100 mm x 15 mm) and 5 ml of the
respective particle suspensions were added
using a Pasteur pipette. The seeds were
then transferred to the Petri dish, with 10
seeds per dish and they were placed
equidistant from one another. The dishes
were covered and sealed with sealing tape
and placed in dark condition.
2.5. SEED GERMINATION TEST
This test was conducted following the
standard method [18]. The test was
performed on the three seeds (C. sativus,
Z. mays and L. esculentum). Result has
been shown in the Table 1, 2 and 3. The
Relative Seed Germination rate (RSG) and
Relative Root Growth (RRG) were
calculated using the Eqs.

(1) and (2). Germination Index (GI) was
also determined using Eq. (3). In addition,
50% Effective Concentration and its 95%
confidence level of nanoparticles (IC50)
was determined by using Probit software
computer program [19].
Relative Seed Germination rate = Ss/Sc
100 (1)
Relative Root Growth = Rs/Rc 100 (2)
Germination Index = RSG RRG/100 (3)
Where Ss is the no. of seed germinated in
sample; Sc is the no. of seed germinated in
control; Rs is the average root length in
sample; Rc is the average root length in
control.
2.6. ACCUMULATION OF SILVER
ROOTS
To determine silver accumulation in
plant root tissue, after 72-82 hours, all
plants were washed thoroughly with
distilled water to remove the test medium.
Samples for AAS were prepared by
dissolving 0.1 g of dried plant in 2 mL of
concentrated HNO3 and 2 mL of deionised
water at 90
o
C for 2h. This solution was
filtered through a glass frit and made up to
a specified volume in a volumetric flask.
Absorption was measured at 328.1 nm
with a Varian atomic absorption
spectrophotometer (AA240). Standard
AgNO3 in 0.1 M HNO
3
was used to
determine the curve. Total silver
concentrations are reported here as a
weight percentage on a dry plant tissue
basis.
2.7. TEST PARAMETERS
The endpoints for the phytotoxicity
experiments were the germination (%) of
the seeds, change in the root lengths of the
germinated seeds under the test conditions
[23].
The biochemical assay involved
determination of the protein content of the
samples by the Lowrys method [18] and
DNA content of the sample by standard
protocol of DNA isolation and
quantification [6].
2.8. STATISTICAL ANALYSIS
The statistical significance of the data
was determined by Students t-test. p value
at 0.05 was taken as significant. The
results were expressed as mean values
SD.



3. RESULTS
International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
10

3.1. CHARACTERIZATION OF THE
SILVER NANOPARTICLES
Fig. 1(a) (b) shows TEM and SEM
image of the commercially available
nanoparticles. The commercially available
SNPs mostly were spherical and near
spherical with a size of nearly 100 nm

Fig 1(a) Transmission electron micrograph
of commercially available silver
nanoparticles (SNPs)


Fig 1(b) Scanning electron micrograph of
commercially available silver
nanoparticles (SNPs)

3.2. EFFECTS OF NANOPARTICLES
TO ROOT LENGTH, GERMINATION
AND PROTEIN CONCENTRATION
AND DNA CONTENT
In the case of control seeds the root
length measured 5.59 cm (L.esculentum),
2.98 cm (Cucumis sativus) and 3.49 cm
(Zea mays) compared to a maximum of
5.18 cm for 10 ppm of SNPs treated seeds.
For 100, 500ppm treated SNPs the
maximum root length measured was for
L.esculentum (4.4 cm, 2.4 cm), Cucumis
sativus (4.1 cm, 0.63 cm) and Zea mays
(3.24 cm, 1.85 cm) respectively. Root
length decreased when concentration of
nanoparticles was increased, R2 = 0.9282
(L.esculentum), R2 = 0.9625 (Z.mays) and
R2 = 0.7339 (C.sativus) (Figure 2).
Control seeds showed 100 % germination.
There was a concentration dependent
decrease in the germination percent for
commercially available nanoparticles from
a mean of 83.3 %, 57%, and 43% for
L.esculentum, 80%, 50% and 30% for
Cucumis sativus, 93.3%, 80% and 50% for
Zea mays. The germination percent and
root length of seeds are shown in Fig. 2
(a) (b) and table I, II and III., protein
content and DNA content decreased on
increasing the concentration of
nanoparticles are shown in. 3 (a) (b).
The protein concentr ation, DNA content
in seeds decreased when SNPs was
increased for seed samples.
3.4. ACCUMULATION OF SILVER IN
ROOTS
Uptake studies showed maximum uptake
bCucumis sativus than Lycopersicum
esculentum and Zea mays. It may be due
to increase surface area of root in Cucumis
sativus. Silver uptake in C .sativus (2.04
mg/L for 10 mg/L), (12.4 mg/L for 100
mg/L) and (26.7mg/L for 500mg/L), in L
esculentum (1.67mg/L for 10mglL),
(8.50.35 for 100 mg/L) and (19.2 mg/L
for 500 mg/L) and in Zea mays (1.2mg/L
for 10mg/L), (7.23 for 100 mg/L) and
(16.9mg/L for 500mg/L).
International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
11

0
20
40
60
80
100
120
Control 10ppm 100ppm 500ppm
Concentration
G
e
r
m
i
n
a
t
i
o
n

(
%
)

%
)
C.sativus
Z.mays
L.esculentum

Fig 2 (a) Relationship between silver
nanoparticles (SNPs) concentrations and
Root length (cm)

0
1
2
3
4
5
6
7
Control 10ppm 100ppm 500ppm
Concentration
R
o
o
t

l
e
n
g
t
h

(
c
m
)
m
)
C.sativus
Z.mays
L.esculentum

Fig 2 (b) Effect of silver nanoparticles on
percentage germination of different seed
systems
0
50
100
150
200
250
300
350
400
450
Control 10ppm 100ppm 500ppm
Concentration
P
r
o
t
e
i
n

c
o
n
t
e
n
t

(

g
/
g
)

.
L.esculentum
C.sativus
Z.mays

Fig 3 (a) Dose dependent effect of silver
nanoparticles (SNPs) on protein content
(g/g) in roots of different seed system

0
0.5
1
1.5
2
2.5
3
Control 10ppm 100ppm 500ppm
Concentration
D
N
A

c
o
n
t
e
n
t

(

g
/
g
)

.
.
.
L.esculentum
Z.mays
C.sativus

Fig 3(b) Dose dependent effects of silver
nanoparticles (SNPs) on DNA content
(g/g) in roots of different seed system
3.3. EFFECT OF BULK METAL
(AGNO
3
) ON GERMINATION
AgNO3 showed maximum toxicity at
lowest concentration. Nanoparticles
available concentration in the water was
found to be 4, 41 and 168 mg/L for 10,
100 and 500 mg/L. AgNO3 showed
maximum toxicity at 4 mg/L, it was found
to be 30% growth for Lycopersicum
esculentum, 20% for Cucumis sativus and
50% for Zea mays. It showed there was no
growth at 41 and 168 mg/L.

Table I: Effect of SNPs on root length cm) and germination (%) of L. esculentum
Control 10 g ml
-1
100 g ml
-1
500 g ml
-1

A B C A1 B1 C1 A2 B2 C2
6.00 8.50 6.50 11.00 3.50 3.00 6.10 4.90 5.50 6.20
International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
12



Table II: Effect of SNPs on root length cm) and germination (%) of Z. mays

Table III: Effect of SNPs on root length cm) and germination (%) of C. sativus
9.50 9.50 8.00 6.50 2.50 4.50 5.50 5.50 6.10 5.10
8.00 6.40 9.50 11.50 7.50 3.10 7.10 3.70 4.20 6.10
7.00 9.50 7.40 2.50 4.80 2.50 3.50 2.10 2.20 3.10
4.50 9.00 12.50 7.10 1.00 7.20 2.90 1.90 0.00 0.00
5.50 2.50 6.50 9.00 2.00 0.00 0.00 0.00 0.00 0.00
0.40 3.00 0.40 1.50 3.00 0.00 0.00 0.00 0.00 0.00
6.50 0.50 1.00 0.50 0.00 0.00 0.00 0.00 0.00 0.00
1.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
7.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Mean 5.59 4.89 5.18 4.96 2.43 2.03 2.51 1.81 1.80 2.05
SD 2.81 4.10 4.50 4.59 2.42 2.49 2.90 2.19 2.53 2.77
SE 0.45 0.36 0.31 0.45 0.27 0.25 0.29 0.22 0.21 0.25
RSG 100 90 80 80 70 50 50 50 40 40
RRG 100 87.47 92.66 88.7 43.47 44.54 51.87 39.17 45.25 49.55
GI 100 102.89 86.39 99.19 161.03 112.2 96.39 127.6 88.39 80.7
Germination% 100 90 80 80 70 50 50 50 40 40

Control 10 g ml
-1
100 g ml
-1
500 g ml
-1

A B C A1 B1 C1 A2 B2 C2
4.2 0.5 5 7 2.5 2.6 3.1 3.5 2 3.5
5.4 5.9 4.5 6.3 3 3.1 3.2 3 3 2
3.5 6 5.5 5.5 3.2 3.3 4.2 3.5 3 3.5
3.1 6 4.5 5.4 4.1 3.5 2.5 5.5 2 3
3.5 5.5 5 6 2.6 2.1 2.6 3.5 2 3
2.9 2.5 3 4 2.7 2.5 2.7 3.5 2.5 2
3.6 5.5 1.4 3 3.1 2.6 3.1 3.5 2 2.5
4.8 4.5 6.4 4 2.9 3.1 3 1 3 1.5
5.4 4 5.5 0 0 0 0 0 0 0
3.2 4.5 0 0 0 0 0 0 0 0
Mean 3.49 3.24 3.12 3.24 2.37 2.28 2.44 1.9 1.69 1.85
SD 0.9 1.78 1.67 2.48 1.34 1.27 1.36 1.7 1.11 1.35
SE 0.31 0.57 0.53 0.73 0.43 0.4 0.43 0.54 0.35 0.42
RSG 100 100 100 80 70 60 60 60 50 50
RRG 100 92.8 89.3 92.8 67.9 36.4 69.9 54.4 48.4 47.4
GI 100 107.7 111.9 86.2 103.09 164.8 85.8 110.2 103.3 104.8
Germination% 100 100 100 80 70 60 60 60 50 50

International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
13







4. DISCUSSION
Silver is known for its toxic behavior to
cells [21]. Moreover, it is known for its
antimicrobial properties [9]. In our
experiments we have showed that an
increase in the concentration of SNPs
reduces the root length and germination
percentage. Increased uptake of silver by
plants with corresponding increase in the
substrate metal concentration was reported
[1]. This parallels our hypothesis that
there could be uptake of nano silver
through seeds and this could in turn affect
germination percent, root length and
protein concentration. This could have
been due to the entry of nano silver into
the cell and could have caused damage to
DNA [2]; or it might have also been due to
the inhibition of DNA synthesis at S-phase
[19]. Ultimately, root length, germination
percent and protein concentration reduced
with an increasing concentration of SNPs.
Decrease in germination percent and root
length could also be due to the silver
nanoparticles which directly provoked
alterations of membranes and other cell
structures and molecules, as well as
protective mechanisms [20]. Indirect
effects of nanoparticles depend on their
chemical and physical properties and may
include physical restraints (clogging
effects),
SNPs shape, size, surface area, and
surface charge, as well as the adsorption
properties of the material could be the
reason for toxicity of these particles to
plant system. Abiotic factors such as pH,
ionic strength, water hardness and the
presence of organic matter would alter
Control 10 g ml
-1
100 g ml
-1
500 g ml
-1

A B C A1 B1 C1 A2 B2 C2
3.6 4.2 4 3.1 2.6 1.5 1.6 2.1 2.5 2.2
4.9 3.7 2.6 4.4 1.3 1.8 1.5 2.5 2.5 2.5
3.7 5.7 3.1 5 1.7 2.1 1.3 2.2 1.5 2.5
4.5 0 5 3.3 2.1 1.3 2.1 2.5 0.5 0.5
3.9 4.5 4.3 2.8 2.1 1.6 3.8 0.5 3.5 1.5
5.4 5 2.5 4 2 3.5 2 1.5 2.1 1.7
4 4.2 3.7 4.1 1.5 0 0 0 0 0
3.8 0 3 3.9 0 0 0 0 0 0
3.7 0 0 0 0 0 0 0 0 0
3.6 0 0 0 0 0 0 0 0 0
Mean 4.11 3.24 3.72 3.87 1.9 1.49 1.59 1.05 1.21 1.02
SD 0.44 1.91 0.93 1.37 0.87 0.77 0.61 0.64 0.78 0.79
SE 0.15 0.6 0.29 0.43 0.27 0.24 0.19 0.2 0.25 0.25
RSG 100 90 100 90 80 80 80 60 60 60
RRG - 78.8 90.5 94.1 48.4 36.2 38.7 25.5 29.4 24.8
GI 100 114.2 110.4 95.64 165.2 220.9 206.7 235.3 204.08 241.9
Germination% 100 90 100 90 80 80 80 60 60 60

International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
14

aggregation chemistry; and are expected to
influence toxicity [17] [18] have also
observed that nanoparticles (Zinc oxide)
inhibited seed germination and root growth
after 2 hrs of exposure. Silver
nanoparticles could penetrate plant system
and may interfere with intracellular
components, causing damage to cell
division. The cell division was arrested, at
metaphase stage, showing Chromatin
Bridge, stickiness and chromosomal
breaks (14). Therefore, SNPs can cause
toxicity to tomato seeds, and moreover it
can also cause toxicity to other plant
system as well.
5. CONCLUSION
SNPs are being used in many household
products and industries. The major
household product would be the washing
machine and refrigerators. Nano Silver
used in washing machines and consumer
products is thought to release nano silver
directly into the waste water systems.
There is always a chance of SNPs to reach
the plant system.
This would in turn hamper the terrestrial
ecosystem. Moreover, nano silver is a
powerful anti-bacterial agent. Samsungs
own advertising claims that its nano silver
products will sterilize over 650 types of
bacteria. There is a risk that effluent
containing nano silver could kill beneficial
bacteria too and disrupt the ecosystem
functioning by disturbing nitrogen fixing
bacteria for plants. Therefore, there are
more direct and indirect effects of SNPs to
plants.
The present study investigated the
potential effect of engineered NPs on plant
system. Commercially available SNPs
showed toxic effects on the tomato seeds.
Lycopersicum esculentum, Cucumis
sativus and Zea mays were exposed to
different concentration of Ag
nanoparticles. The germination rate and
growth rate of plants were inhibited as a
result of exposure to nanoparticles. DNA
content and protein content were also
decreased on increasing the concentration
of NPs. Solubilization studies have been
done, and by using bulk metal (AgNO3)
analysis it has been proved that toxicity
was not due to ions released from
nanoparticles, since it was in minute
concentration, bulk metal analysis showed
the maximum toxicity at lowest
concentration (4 mg/L). Available
concentration was found to be (4.040.15
mg/L for 10 mg/L) and (168.70.17 mg/L
for 500 mg/L), this studies showed toxicity
was not due to input concentration of
nanoparticles, it was due to available
concentration of nanoparticles. The EC50s
along with 95% confidence limit value
were as follows: for L.esculentum
(459.15), C.sativus (281.72) and Z.mays
(538.29) mg/L, respectively. The amount
of Ag ions released (0.3mg/L for 10mg/L)
and (1.4 mg/L for 500mg/L). The
accumulation of Ag nanoparticles was
dependent on the exposure concentration.
Uptake studies showed the amount of
silver uptake in C.sativus (2.04mg/L for
10mg/L) and (26.7mg/L for 500mg/L), in
L esculentum (1.67mg/L for 10mglL) and
(19.2mg/L for 500mg/L) and in Z.mays
(1.2mg/L for 10mg/L) and (16.9mg/L for
500mg/L).
ACKNOWLEDGEMENTS
Authors thank Management, VIT
University, for providing us with funding
towards nanotoxicological research.



REFERENCES:

International Journal of Human Genetics Medical Biotechnology and Microbiological Studies (IJHGMBMS)
Volume-1 , Issue-1 , August 2012
15

[1] A. T. Harris and R. Bali, On the formation and
extent of uptake of silver nanoparticles by live plants, J
Nanopart Res, 2008; vol. 10, pp. 691695.
[2] ATSDR (Agency for toxic substances and Disease
Registry), 1990. Toxicological profile for Silver.
Prepared by Clement International Corporation, under
Contract 205-88-0608). U.S. Public Health Service.
ATSDR/TP-90-24.
[3] T.J. Brunner, P. Wick, P. Manser, P. Spohn, R. ,N.
Grass, L.K. Limbach, A. Ruinink and W.J. Stark, In
vitro cytotoxicity of oxide nanoparticles: comparison to
asbestos, silica, and the effect of particle solubility,
Environ. Sci. Technol., 2006; vol.40, pp. 4374.
[4] C.M Lu, C.Y. Zhang, J. Q. Wen, G. R Wu and M. X.
Tao, Research of the effect of nanometer materials on
germination and growth enhancement of Glycine max
and its mechanism, Soybean Sci, 2002; vol. 21, pp. 168-
172 (in Chinese).
[5] D. A. J. Maynard, R. B. Aitken, C. Tilman, D. Vicki,
O. Ken, A. Gnter, P. Martin, R. John, S. Anthony, S.
Vicki, S. Sally, T. Lang, J. Nigel and B. David Warheit,
Safe handling of nanotechnology, Nature, 2006; vol.
444, pp. 267-269.
[6] D. M. Hoisington, Khairallah and D. Gonzales de
leon (1994). Laboratory protocols: CIMMYT Applied
Biotechnology Center, 2nd (ed.), Mexixo, CIMMYT.
[7] D. Lin and B. Xing, Phytotoxicity of Nanoparticles:
Inhibition of seed germination and root growth. Environ
Pol, 2007; vol. 150, pp. 243-250.
[8] F.S. Hong, J. Zhou, C. Liu, F. Yang, C. Wu, L. Zheng
and P.Yang, Effect of nano-TiO
2
on photochemical
reaction of chloroplasts of spinach, Biol. Trace Elem.
Res., 2005; vol. 105, pp. 269.
[9] F. Q. Zhang, W. J. She and Y. F. Fu, Comparison of
the toxicity in vitro among six types of nanosilver base
inorganic antibacterial agents, Zhonghua kou Qiang Yi
Xue Za Zhi, 2005; vol. 40, pp. 504-507.
[10] G., E. Oberdorster, Oberdorster and J. Oberdorster,
Nanotoxicology: an emerging discipline evolving from
studies of ultrafine particles, Environ. Health Perspect.,
2005; vol. 113, pp. 823839.
[11] K. Donaldson, V. Stone and W. MacNee (1999).
The toxicology of ultrafine particles, In: Maynard L,
Howards CV (eds), Particulate matter properties and
effects upon health, Oxford Bios, pp. 115.
[12] L. Geoff, F. Nies Loring, F.Tarco Ronald, John W.
Bickham, Maria S Sepulveda. The effect of silver
nanoparticles on fathead minnow (Pimephales promelas)
embryos. Ecotoxicology 2009.
[13] L. Yang and D. J. Watts, Particle surface
characteristics may play an important role in
phytotoxicity of alumina nanoparticles Toxicol Lett,
2005; vol. 158, pp. 122-132.
[14] M. Kumari, A. Mukherjee and N. Chandrasekaran,
Genotoxicity of Silver Nanoparticles, Science of The
total Environment, 2009; vol. 407, pp. 5243-5246.
[15] NanoVate. Nanotechnology industry news and
views, No.2. 2007.
[16] O.H. Lowry,. N.J. Rosbrough, A.L. Farr and R.J.
Randall Protein Measurement with the Folin Phenol
Reagent, J. Biol. Chem., 1951; vol. 193, pp. 267-275.
[17] R. D. Handy, F. V. D. Kammer, J. R. Lead, M.
Hassello, R. Owen and M. Crane, The ecotoxicology
and chemistry of manufactured nanoparticles,
Ecotoxicology, 2008; vol. 17, pp. 287314.
[18] R. D. Handy, R. Owen and E. Valsami, The
ecotoxicology of nanoparticles and nanomaterials current
status, knowledge gaps, challenges, and future needs,
Ecotoxicology, 2008; vol. 17(5), pp. 315-325.
[19] R. Sudhakar, Gowda N, Venu G. Mitotic
abnormalities induced by silk dyeing Industry Effluents
in the cells of Allium cepa. Cytologia 2001; 66: 235
239.
[20] S. A. Blaser, M. Scheringer, M. Macleod and K.
Hungerbhler, Estimation of cumulative aquatic
exposure and risk due to silver, contribution of nano-
functionalized plastics and textiles, Sci Tot Environ,
2008; vol. 390, pp. 396490.
[21] U.S. Environmental Protection Agency; Ecological
Effects Test Guidelines; 1996.
[22] U.S. Environmental Protection Agency (2005).
Nanotechnology White Paper e External Review Draft.
Available from: <http://www.epa.gov/osa/pdfs/
EPA_nanotechnology_white_paper_external_review_dra
ft_12-02-2005.

[23] US Environmental Protection Agency. Toxicity data
analysis-software EMHL. Cincinnati, OH. USA; 1994.
[24] W. Y. Chen, H. Cui, J. Bao, X. Zhou and Q. Shu,
A simplified rice DNA extraction Protocol for PCR
analysis, Rice science, 2006;
http://www.ricescience.org. vol. 13(1), pp. 67-70.
[25] W.M Lee, Y. H. Yoon and H. Kweon, Toxicity
and Bioavailability of copper nanoparticles to the
terrestrial plants bean (Triticum aestivum): plant agar test
for water-insoluble nanoparticles, Environment
toxicology and chemistry, 2008; vol. 27, pp. 1915-1921.