J. Biomedical Science and Engineering, 2011, 4, 248-254 JBiSE
doi:10.4236/jbise.2011.44034 Published Online April 2011 (http://www.SciRP.org/journal/jbise/).
Published Online April 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Antimicrobial silver nanoparticle induces organ deformities in
the developing Zebrafish (Danio rerio) embryos
Rajaretinam Rajesh Kannan1, Arockya Jeyabalan Avila Jerley2, Muthiah Ranjani2,
Vincent Samuel Gnana Prakash1
1International Centre for Nanobiotechnology (ICN), Centre for Marine Science and Technology Campus, Manonmaniam Sundaranar
University, Kanyakumari, India.
2Department of Microbiology, N.M.S.S. Vellaichamy Nadar College, Madurai, India.
First two authors are equally contributed.
Received 12 February 2011; revised 26 February 2011; accepted 3 March 2011.
ABSTRACT
Silver Nanoparticles were synthesized by Esche-
richia coli using Silver nitrate in the growth me-
dium and characterized in X-Ray Diffraction, UV-
Vis Spectrophotometer and Scanning Electron Mi-
croscope. They exhibited antimicrobial activity ag ains t
human pathogens except Escherichia Coli. Nano-
particles were impregnated in yarn and analyzed
for their inhibition in the broth culture. The Mini-
mal Inhibitory Concentratio was calculated for the
human pathogens in Microtitre plate. The toxicity
assessment of the nanoparticles in the embryonic
Zebrafish showed many organogensis deformities
like cardiac malformations, eye and head edema,
tail and trunk flexure were observed in the organ
system of the developing embryos for 1 to 5 day
post fertilization in different concentrations of Ag
Nanoparticles. The Organogenesis disruptive effects
were found in 14 - 20 ng/ml of silver nanoparticles
but the inhibition was found in 4-10ng/ml for the
pathogens in vitro and 10ng/ml in embryos. Never-
theless, in Cardiac assay, the Heart Beat rates were
calculated as 42 - 45 for 15 Sec in the concentra-
tions ranging from 10 - 20 ng/ml of Silver nano-
particles. The blood flows, rhythmicity, contractil-
ity of heart beat rates were observed normal. The
Mean value of blood Cell counting did not showed
any notable effects in the Nanoparticle treated Ze-
brafish embryos and control. The LC50 value for
the Biosynthesized nanoparticle was at 22 ng/ml in
all the developmental stages of the embryos. Our
results shows silver nanoparticles disrupts the nor-
mal organogenesis during development and further
detailed studies are needed to prove silver nano-
partcles are an antimicrobial agent for use in hu-
mans.
Keywords: Biocidal Effect; X-ray Diffraction;
Scanning Electron Microscope; Organogenesis;
Deformities; Cardiac Assay
1. INTRODUCTION
Microorganisms such as bacteria, yeast and fungi play
an important role in remediation of toxic metals through
reduction of metal ions; this is considered interesting as
nanofactories [1]. Nanoparticles has a significant poten-
tial for a wide range of biological applications such as
antifungal agents, antibacterial agents for antibiotic re-
sistant bacteria, preventing infections, healing wounds
and anti-inflammatory [2]. The new technology of im-
pregnation of silver nanoparticles are now commercially
available and silver coated dressings are used extensively
for wound management, particularly in burn wounds [3,4],
Chronic leg ulcers [5], diabetic wounds [6] and trau-
matic injuries. The dressing component are also varies,
as nylon, mesh, and hydrocolloid or methyl cellulose.
The interactions of silver nanoparticles with biosystems
are just beginning to be understood, and these particles
are increasingly being used as microbicidal agents. A
new generation of dressing with antimicrobial agents
like silver and biopolymer was developed to reduce or
prevent infections.
Zebrafishes have unique applications over other ver-
tebrate model system (mouse, rat) [7]. The metabolism,
physiology and development of Zebrafish are compara-
ble to humans and therefore Zebrafish is highly relevant
model for toxicity, safety and efficacy testing. Genetic
screens of Zebrafish phenotypes show similarities in
human diseases and protein sequences of drug binding
proteins [8]. Zebrafish have served as a vital model sys-
tem for screening drug targets for curing human diseases.
Large numbers of embryos can be generated rapidly at
low cost, which can serve as an ideal in vivo assay for

R. R. Kannan et al. / J. Biomedical Science and Engineering 4 (2011) 248-254 249
screening biocompatibility, pharmacological efficacy, and
toxicity of nanoparticle. This study was aimed at synthe-
sis of microbial Silver nanoparticles and assay its antim-
icrobial properties and evaluating its human compatibil-
ity.
2. MATERIALS AND METHODS
2.1. Biosynthesis and Characterization of Ag
Nanoparticles
The silver seeds were prepared by rapidly injecting 0.5
ml of 10 mM NaBH4 into an aqueous solution contain-
ing 0.5 ml of 0.01 M AgNO3 and 20 ml of 0.001 M So-
dium citrate. It was stirred for 5 min and incubated for
90 minutes at 37˚C. The spherical silver hydrosols were
prepared by adding 3 ml of the silver seed solution to the
aqueous solution of sodium citrate and silver nitrate so-
lution (100 ml, 0.001 M). This solution was inoculated
with 10 ml of E. coli culture broth and incubated at 37˚C
for 24 hrs until the color changes to greenish yellow. The
solution was filtered in 0.22 µm filter to remove the mi-
crobes and the nanoparticles were washed with nanopure
water using centrifugation to remove the chemicals in-
volved in nanoparticle synthesis. The synthesized silver
nanoparticles were characterized by UV-Vis Spectros-
copy, X-Ray Diffraction and Scanning Electron Micro-
scope for estimation of crystalline structure, mean size
and morphology. The nanoparticle pellets were resus-
pended in nanopure water for assaying Antimicrobial
properties and the physiological studies in Zebrafish
embryos.
2.2. Antimicrobial Activity a nd M inimal
Inhibitory Concentration
The nutrient broth was prepared, sterilized and inocu-
lated with fresh culture of human pathogens (Figure 1)
and incubated at 37˚C for 24 hours. After incubation the
cultures were centrifuged at 12 000 rpm and the super-
natant was used for further experiments. Nutrient broth
plates were supplemented with 1 - 15 ng/ml concentra-
tion of Silver nano particles and tested with pathogens
for its antimicrobial property. Antimicrobial activity was
determined by the microtiter broth dilution method [9].
Dilutions of the Silver nanoparticles (1 - 15 ng/ml) were
added in 1% DMSO to each well of the 96 well micro-
titer plates containing fixed volume of Nutrient broth.
Each well was inoculated with bacteria (105 CFU ml–1)
and incubated at 37˚C for 24 h. The MIC was calculated
at which no growth of bacteria was observed for all the
microbial pathogens shown in Figure 1.
2.3. Prepara t i on of Silv e r Impregnated Dress ings
and Antimicr obial Analysis
10 g of white degreased yarn was cut into many pieces of
Figure 1. Minimal Inhibitory Concentrations (MIC) of Ag
Nanoparticles in the Human pathogens.
1 cm2 each. This was immersed in the 10 ng/ml Ag
Nanoparticles solution and it was squeezed and dried in
hot air oven. The yarn was further washed twice with
Nanopure water. The Ag coated dressing was placed in
to the sterile vial containing 80 µl of Nanopure water for
10 minutes and 2.2 ml of nutrient broth was added to
each vial to make the volume up to 3 ml. 10 µl of bacte-
rial suspension of the selected pathogens were inocu-
lated into the test tubes containing antimicrobial silver
dressing. These test tubes were incubated at 35˚C for 24
hours. After incubation the bacteria suspensions were
spread on the blood agar and nutrient agar plates, incu-
bated overnight at 37˚C and the growth of the organisms
were observed.
2.4. Breeding and Maintenance of Zebrafish
Embryos
Zebrafishes were bred and maintained in Fish Culture
facility of International Centre for Nanobiotechnology,
CMST, M. S. University. Zebrafishes were maintained in
30 L tanks at 28˚C with 14 h:10 h light/dark cycle. Fol-
lowing successful breeding eggs fell through the mesh,
and was subsequently collected from the bottom of tanks.
Zebrafishes were maintained according to Westerfield,
1989 [10]. Zebrafish embryos were raised in E3 medium
(5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2 and 0.16
mM MgSO4). For Zebrafish embryo chemical experi-
ments 1% DMSO was used as a vehicle for small mole-
cules to permeate the embryo. Eggs containing dead or
obviously poor quality embryos were removed. The re-
maining embryos were used for Organogenesis and
Physiological effects [11].
2.5. Effect of Ag Nanoparticles in the
Organogenesis of Zebrafish Embryonic
Development
10 µl of the human pathogens and 1 - 25 ng/ml of Ag

R. R. Kannan et al. / J. Biomedical Science and Engineering 4 (2011) 248-254 250
Nanoparticles were treated in the 1 - 5 dpf (days post
fertilization) Zebrafish embryos in the 48 well microtitre
plates to observe the Organogenesis and Physiological
effects. A parallel control was made in all the in vivo
studies. The Biocompatibility and the physiological ef-
fects of the silver Nanoparticles were monitored in the
developing embryos. The Heart beat rates and the blood
flow levels were analyzed in the Image and the Video.
The highest non lethal concentration was also reported.
The RBC and WBC counting of the adult Zebrafish em-
bryos were carried out by cutting the tail of the embryos
by sharp blade under Microscope (Motic) and 0.5 - 1 µl
of blood was pipetted out in 0.5 - 10 µl Eppendorf
Micropipette and diluted in RBC and WBC dilution fluid
and counted in Hemocytometer.
3. RESULTS
3.1. Synthesis and Characterization of Ag
Nanoparticles
All diffraction peaks correspond to the characteristic
face centered cubic (FCC) silver lines. The analysis was
carried out in 2θ range 20˚ - 80˚, with step size 0.05˚ in a
Rigaku D-Max B diffractometer. These diffraction lines
observed at 2θ angle 38.1˚, 44.3˚, 64.4˚, and 77.5˚ re-
spectively, have been indexed as (111), (200), (220) and
(311) respectively. X-Ray Diffraction patterns were ana-
lyzed to determine peak intensity, position and width.
Full width at half-maximum (FWHM) data was used
with the scherrer’s formula to determine mean particle
size. Scherrer’s equation is given by
09
con
.
d




Where ‘d’ is the mean diameter of the nanoparticles
‘λ’ is wavelength of X-ray radiation source, θ is the an-
gular FWHM of the XRD peak at the diffraction angle θ.
The mean size of nanoparticles estimated by XRD was
13 nm and shown in Figure 2. The SEM micrograph of
Figure 2. XRD analysis of Ag nanoparticles produced by E.
coli.
bacterial cell biomass treated with silver nanoparticles in
which silver has been found adhered to the bacterial cell
wall surface and shown in Figure 3. The UV-Vis Spec-
trophotometer of Ag nanoparticles showed the value of
430 nm.
3.2. In vitro Antimicr obial Assay o f Ag N anopar-
ticle
Minimum Inhibitory Concentration of the Silver nano-
particles for the human pathogens was ranging from 4 -
10 ng/ml and is shown in Figure 1 and in the spread
plate method. The silver nanoparticle impregnated yarn
and control yarn is shown in Figures 4(a) and 4(b). The
bactericidal effect of the silver impregnated yarn showed
no microbial growth was observed in the microbial plate
culture and is shown in Figure 5 and Figure 6. Thus all
silver-impregnated dressings investigated for antagonis-
tic property, on all the human pathogen at the concentra-
tion of 10 ng/ml had inhibiting property. Interestingly,
the synthesized nanoparticle did not inhibit the growth of
E. coli.
3.3. Physiological and Organogenesis Effect of
Silver Nanoparticles
3.3.1. O rganogene sis Effect
To determine the effect of different doses of Ag nanopar-
Figure 3. SEM analysis—Ag nanoparticles.
Figure 4. (a) Control yarn, (b) Ag nanoparticles impregnated
yarn.

R. R. Kannan et al. / J. Biomedical Science and Engineering 4 (2011) 248-254 251
Figure 5. (a) and (b) Showing the antimicrobial properties of
Ag nanopartcles for Streptococcus mutans.
Figure 6. (a) and (b) Showing the antimicrobial properties of Ag
nanoparticles for Bacillus Subtilis ((a) No growth observed in
Ag impregnated yarn, (b) Growth observed in the control yarn).
ticles on embryonic development, we treated Ag nano-
particles in stages 1 dpf to 5 dpf Zebrafish embryos and
carefully monitored the Organogenesis in the embryos
(24, 48, 72, 96, and 120 hpf), the results are shown in
Figures 7(a)-7(j). Different organogenesis effects were
observed in the developing embryos. The different phe-
notypic deformities were observed in the concentration
of the 14 - 20 ng/ml of Ag Nanoparticles treated em-
bryos. Many physiological changes were observed in the
embryos from 24 hpf to 120 hpf Figures 7(a)-7(j). The
organogenesis effect like finfold abnormalities, tail flex-
ure and spinal truncation, cardiac malformation, yolk sac
edema, head edema, eye deformity, blood accumulation
and tumor formations in zebrafish embryos were ob-
served from 1 - 5 dpf embryos treated Ag nanoparticles.
Multiple organogenesis deformities were observed in the
16 - 18 ng/ml and the LC50 value of the embryos were
observed at 20 ng/ml to 22 ng/ml of Ag Nanoparticles.
They lead to the death of the embryos in all the 5 stages
(1 dpf to 5 dpf) after 36 - 50 hrs of the treatment. 96 Mi-
crotitre plates with embryos were inoculated with 5 µl of
pathogens and 10 ng/ml of silver Nanoparticles were
added in the Embryo Rearing Solutions and the inhibi-
tion concentration at in vivo studies are similar to the
inhibition concentration of in vitro studies.
Figure 7. Organogenesis effects of Ag Nanoaparticles from 1
dpf to 5 dpf of Zebrafish embryos ((a) to (i)—10x, (j)—40x
and (k) and (l)—10x) (a) 1 dpf showing eye deformity at 15
ng/ml; (b) 2dpf showing trunk and spinal cord flexure 16 ng/ml;
(c) 2 dpf showing tail flexure and yolk edema 14 ng/ml; (d) 3
dpf showing cardiac malformation and yolk edema 15 ng/ml;
(e) 3 dpf showing trunk braekage at 23 ng/ml, 16ng/ml; (f) 3
dpf showing tail flexure and necrosis effect in the yolk region
15 ng/ml; (g) 4 dpf showing pericardial bulging, cardiac mal-
formation and head edema 16 ng/ml; (h) 5 dpf howing blood
accumulation and damage in the blood vessels at 20 ng/ml; (i)
5 dpf abnormality in the cardiac and yolk edema leads to
death 20 ng/ml; (j) 5 dpf tumour formation and blood accu-
mulationin the 20 ng/ml of the nanoparticles; (k) 3 dpf control
embryos; (l) 5 dpf control embryos.
3.3.2. Cardiac Assay and Blood Cell Counting
The cardiac malformations did not affect the Heart Beat
Rates (HBR) and the normal heart beat ranges from 42 -
43 and similar mean value of heart beat rate of 43 for 15
seconds was recorded. The heart beat rates were normal
and in the malformed heart of the nanoparticle treated
embryos. The rhythmicity and blood flow level were
monitored in the simple microscope and the effects were
observed in 15 - 17 ng/ml of the Ag Nanoparticles. The
blood flow level, contractility and rhythmicity were
normal in all the abnormal embryos like Yolk sac edema,
tail and spinal cord flexure, finfold abnormality, cardiac
malformation and head edema. These findings suggest
that phenotypic deformities of Ag Nanoparticles did not
affect the Hematopoiesis process and vasculogenesis at
the concentration of 14 - 20 ng/ml, but the vasculogene-
sis and rhythmicity of blood flow were affected above
the concentration of 20 µg/ml, tumor formation and
blood accumulation were observed and shown in Figure
7(j). The Blood cell counting was carried out in the
Hemocytometer in all the defected embryos from 3 dpf -
5 dpf and the mean results were tabulated in Figures 8(a)
and 8(b). The mean value of WBC and RBC counts
were seemed to be normal and showed very minor dif-

R. R. Kannan et al. / J. Biomedical Science and Engineering 4 (2011) 248-254 252
ferences in the blood cell count in 10 - 16 ng/ml concen-
tration in the Zebrafish embryos.
4. DISCUSSION
Nanotechnology involves the tailoring of materials at
atomic level to attain unique properties, which can be
suitably manipulated for the desired applications [12].
The new age drugs are nanoparticle which can fight hu-
man pathogens [13]. In recent years, extensive studies
have been undertaken on the use of antimicrobial prop-
erties of silver, incorporated within medical devices. The
aim of this study was to prepare a formulation contain-
ing silver ion, which could be applied for wound dress-
ing. In Nanoparticle preparation appearance of light
brown color in solution indicates the formation of silver
nanoparticles [14]. Thus, it was evident that the metabo-
lites excreted by the culture exposed to silver could re-
duce silver ions, clearly indicating that the reduction of
the ions occur extracellularly through reducing agents
released into the solution by E. coli. The silver nanopar-
ticles were characterized by UV-Visible spectroscopy
and this technique has proved to be a very useful tech-
(a)
(b)
Figure 8. (a) Level of WBC Count in different concentration
of Ag Nanoaparticles in 3 - 5 dpf embryos. (WBC Count ×
103/cu.mm), (b) Level of RBC Count in different concentration
of Ag Nanoaparticles in 3 - 5 dpf embryos. (RBC Count ×
106/cu.mm).
nique for the analysis of nanoparticles. This event
clearly indicated that the reduction of the ions occured
extracellularly through reducing agents released into the
solution by E. coli, as it showed a strong and broad, sur-
face peak at 430 nm. The solution was extremely stable
with no evidence of flocculation of the particles even
several weeks after reaction. This showed that the silver
cations were highly reactive and tend to bind strongly to
electron donor groups containing sulfur, oxygen or ni-
trogen [15] and this indicated that E. coli can synthesize
silver nanoparticles extracellularly (Figure 3).
Silver nanoparticles are the important product in the
nanotechnology industries for many clinical applications,
but the risk factors of toxicity was least studied [16]. In
this work we have analyzed the organogenesis effect of
antimicrobial Ag Nanoparticles. Nanoparticles of silver
have been studied as coating materials [17]. Hence in
this present work a silver nanoparticle were impregnated
in the dressing yarn and was found to have antimicrobial
effects, proved the inhibition of human pathogens as
shown in Figure 1. This approach was a simple method
for the synthesis and coating of silver nano particles in
the yarn, so that the yarn would inhibit the microbial
growth in the wound. It was found that the nanoparticles
were very novel for its antimicrobial activity against the
human pathogens. The bactericidal property of these
nanoparticles depends on their stability in the growth
medium, since this imparted greater retention time for
bacterium-nanoparticle interaction and the stability was
confirmed by UV-Vis Spectrophotometer. The purified
Ag nanoparticles had no effect against E. coli. There was
no reduction in the growth at Microtitre plate. This
opens up a field of interesting, as how E. coli resisted the
Ag Nanoparticles. These were correlated to the result, in
which the process of synthesis of Ag Nanoparticles did
not inhibit the growth of E. coli in the broth. Zebrafish is
an ideal organism to study the organogenesis effect.
Hence, to determine the effect of dose dependent and
organogenesis we have analyzed the physiological ef-
fects in the embryos for the Ag Nanoparticles and simi-
lar dose dependant studies in Zebrafish embryos were
also carried out for Ag Nanoparticles, but it showed
toxic effects in lesser concentration [18]. In the present
study, the toxicity was observed from 14 ng/ml of the
silver nano particles but 10 ng/ml of the Ag nanoparti-
cles inhibited the bacterial growth in vitro and in vivo in
the Zebrafish embryonic model. The results of the de-
formities were shown in Figures 7(a)-7(j) and similar
deformities were also observed in the embryonic devel-
opment of Zebrafish [18]. The Nano-ZnO toxicity and
deformities were also studied in the Zebrafish [19].The
control embryos which showed the pigmentation that the
Ag Nanoparticles treated embryos, hence it was con-
firmed that the Ag nanoparticles were influencing the

R. R. Kannan et al. / J. Biomedical Science and Engineering 4 (2011) 248-254 253
pigment formations. The cardiac malformations and yolk
edema observed in this study is similar to the zebrafish
treated DCA and cadmium [20,21]. The lethality and the
phenotypic deformalities of the Ag Nanoparticles are
higher in the Zebrafish embryos were reported in the
earlier studies [18]. The shrunken ventricular myocar-
dium observed in cardiac malformed zebrafish induced
by nanoparticles were similar to the observation in ze-
brafish treated with Ag Nanopartcles and TCDD [18,22].
Thus it was proving that the toxic effects of the Ag
nanoparticles are similar to TCDD and chemically syn-
thesized nanoparticle [18]. The preclinical studies of the
silver nanoparticles and the physiological effects were
analysed in the Zebrafish embryos and it was proving
that if the Ag nanoparticles yarn formulation would be
applied to the pregnant women that might affect fetus
Organogenesis. However thorough studies have to be
mde in higher vertebrates to prove organ deformities.
This study proves the Biological synthesis and Chemical
synthesis [18] of silver nanoparticles are toxic to the
developing embryos by this embryonic model study in
Zebrafish. The blood cell counting (WBC & RBC) of the
Zebrafish did not show notable effects in blood cell for-
mations and the blood vessels. The blood cell count was
normal in both the Ag nanoparticles treated embryos and
in the control. In the cardiac assay the Heart beat rates
and the rhythmicity (atrial and ventricular) were ob-
served in the microscope, only the pericardial edema and
the malformation was observed in the heart, but the
Heart Beat Rates were normal in both the control and the
treated embryos. It proved that the Ag nanoparticle af-
fected the organogenesis process of cardiac muscle for-
mation without affecting the Heart Beat Rates, and
proving that the transcriptional regulatory mechanism
for the myocardiac muscle formation was affected and
this will be studied by functional genomic approach.
This is the first report on the Ag Nanoparticles which did
not affect the Hematopoietic process and Heart Baet
Rates, earlier there was no in vivo studies in Zebrafish
models. In the similar ay the antimicrobial properties of
the anti MRSA molecule and the compound toxicity for
cardiac assay and pathogenic infection study was ana-
lyzed in the Zebrafish embryos [11]. Also Silver-coated
nanoparticles were the most effective among all the
nanoparticles without significant cytotoxicity, suggesting
their use as antimicrobial additives in the process of fab-
rication of ambulatory and nonambulatory medical de-
vices [23]. In this work the Minimum Inhibitory concen-
tration (4 - 10 ng/ml) of Ag nanoparticle for microor-
ganisms are non toxic to the physiology and organo-
genesis of the Zebrafish embryos, but the Inhibitory
Concentration level of the Ag Nanoparticles and the
nanotoxic level was assessed in vivo in the fish model.
The lethal and non lethal concentration was reported in
the in vivo studies, confirmed that the silver nanoparti-
cles did not affect the heart beat rates and Hematopoiesis
process. The stability of the nanoparticles were checked
in the Embryo Rearing Solution (ERS) by UV-Vis spec-
troscopy showing the same wavelength (420 nm and 430
nm). The Biosynthesized silver nanoparticles does not
showed any notable different toxic effect in the Zebraf-
ish embryonic model at the Minimum Inhibitory Con-
centration of microbes but if it exceeds the level, Ag
Nanoparticle will be more toxic to the developing em-
bryos and shown in Figure 7. Further studies on higher
vertebrate models (mouse, dog, monkey, etc.) are needed
for the dose of silver nanoparticle which does not cause
organ deformities in embryos.
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