ARTICLE

pubs.acs.org/est

Acute and Chronic Toxicity Effects of Silver Nanoparticles

(NPs) on Drosophila melanogaster
Ales Panacek,† Robert Prucek,† Dana Safarova,‡ Milan Dittrich,§ Jana Richtrova,†
Katerina Benickova,† Radek Zboril,† and Libor Kvitek*,†
†
Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science,
Palacky University, 17. Listopadu 12, 77146 Olomouc, Czech Republic
‡
Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
§
Department of Pharmaceutical Technology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203,
50005 Hradec Kralove, Czech Republic
bS Supporting Information
ABSTRACT: The use of nanoscaled materials is rapidly
increasing, however, their possible ecotoxicological effects are
still not precisely known. This work constitutes the first com-
plex study focused on in vivo evaluation of the acute and chronic
toxic effects and toxic limits of silver nanoparticles (NPs) on the
eukaryotic organism Drosophila melanogaster. For the purpose
of this study, silver NPs were prepared in the form of solid
dispersion using microencapsulation method, where mannitol
was used as an encapsulation agent. This newly prepared solid
dispersion with a high concentration of silver NPs was exploited
to prepare the standard Drosophila culture medium at a silver concentration range from 10 mg 3 L
1 to 100 mg 3 L
1 of Ag in the case
of the acute toxicity testing and at a concentration equal to 5 mg 3 L
1 in the case of the chronic toxicity testing. The acute toxic effect
of silver NPs on Drosophila melanogaster was observed for the silver concentration equal to 20 mg 3 L
1. At this silver concentration,
50% of the tested flies were unable to leave the pupae, and they did not finish their developmental cycle. Chronic toxicity of silver
NPs was assessed by a long-term exposure of overall eight filial generations of Drosophila melanogaster to silver NPs. The long-term
exposure to silver NPs influenced the fertility of Drosophila during the first three filial generations, nevertheless the fecundity of flies
in subsequent generations consequently increased up to the level of the flies from the control sample due to the adaptability of flies to
the silver NPs exposure.

’ INTRODUCTION properties. Silver NPs are increasingly becoming a part of our

Nanoparticles (NPs) and their unique properties are currently
not only the subject of the scientific research, but they are being
implemented and used in chemical industry and everyday human
life as commercial products. Various nanomaterials and nano-
technologies are now commonly used, for example, in areas
including electrical engineering, construction, cosmetics, food,
healthcare, and disinfection preparations.1
3 On the other hand,
the research and knowledge of side effects or adverse physical and
chemical interactions of nanomaterials with living organisms and
the environment is not as far advanced as compared to basic and
applied research of nanomaterials production. Nanoparticles of
various compounds can normally occur in nature, however,
engineered nanomaterials are not of natural origin, and they
can, contrary to their bulk form or to normally occurred
nanoparticles, induce totally different interactions within living
organisms.
Silver NPs, which are ranked among the most intensively
studied nanomaterials, are used in various commercial products
as a disinfection agent due to their extraordinary antimicrobial
everyday life, which considerably increases the risk of contam-
ination of the environment. Hence, it is necessary to pay a greater
attention to the ecotoxicological properties of silver NPs. In the
past few years, several studies have been published dealing with
the ecotoxicity of silver NPs, particularly to aquatic organisms
such as crustaceans,4,5 protozoan ciliate,6 fish,4,7
10 or algae,11
and also to soil organisms12,13 and plants.14 However, the
research in this area is still at the beginning and it is necessary
to further extend the knowledge on the ecotoxicity of Ag NPs in
more comprehensive studies.
The fruit fly (Drosophila melanogaster), a commonly occurring
organism in nature, is characterized by a short life cycle with
distinct developmental stages. Thanks to its easy manipulation
and cultivation, numerous offspring and possibility to induce
Received: December 17, 2010
Accepted: April 28, 2011
Revised: April 24, 2011
Published: May 09, 2011

r 2011 American Chemical Society 4974 dx.doi.org/10.1021/es104216b | Environ. Sci. Technol. 2011, 45, 4974–4979
Environmental Science & Technology
Figure 1. (A) Silver nanoparticle solid dispersion and (B) microscopic image of mannitol microparticles dispersed in linen oil.
mutations, it is commonly used as a model organism for many
biological processes including toxicity testing. Drosophila mela-
nogaster has been exploited in testing the toxicity of several
nanomaterials such as carbon nanotubes,15 cerium oxide NPs,16
or silver NPs.12,13 In the case of toxicity testing of silver NPs with
respect to Drosophila melanogaster, the molecular mechanism of
toxic action was investigated by Ahamed and co-workers.12 This
study revealed that silver NPs at high silver concentrations equal
to 50 and 100 mg 3 L
1 of Ag induce heat shock protein 70,
oxidative stress and apoptosis in Drosophila melanogaster. On the
other hand, dose-dependent study evaluating acute and chronic
toxic limits of silver NPs to Drosophila melanogaster, have not
been investigated yet. Therefore, the subject of this study was to
determine the acute toxic limits and chronic toxicity effects of
silver NPs to developmental stages of Drosophila melanogaster. In
the case of acute toxicity testing, numbers of larvae, pupae, and
hatched adult individuals have been determined together with
the phenotype changes in the Drosophila organism. In the case of
chronic toxicity testing, the fecundity of adult flies and hatching
of individuals have been observed.
’ EXPERIMENTAL SECTION
Preparation of the Solid Dispersion of Silver NPs. For the
purpose of toxicity testing of silver NPs at high silver concentra-
tions, stable and highly concentrated silver NPs dispersion had to
be prepared. Therefore, a solid dispersion of silver NPs was found
to be the most suitable form to achieve the goal of this study.
Primarily an aqueous dispersion of silver NPs was synthesized
by the well established modified Tollens process,17 and conse-
quently, it was concentrated following the previously published
dialysis based procedure.6 Briefly, aqueous dispersion of silver
NPs has been prepared by the reduction of complex cation
[Ag(NH3)2]þ by D-maltose in alkaline media.17 Utilizing this
procedure, nearly monodisperse 29 ( 4 nm sized spherical silver
NPs were prepared as determined by DLS method using a
Zetasizer Nano ZS (Malvern, U.K.). The size and morphology
of the prepared silver NPs have been verified by the TEM
method using a JEM 2010 (Jeol, Japan) (Supporting Information
Figure S1). The zeta potential of the prepared silver NPs was
(
29 ( 5) mV as determined by electrophoretic technique using
a Zetasizer Nano ZS instrument.
The initially prepared silver NPs dispersion with a concentra-
tion of silver equal to 108 mg 3 L
1 was subsequently concen-
trated by the 7 dialysis tubing membranes containing 1 g of the
superabsorbing copolymer polyacrylate-polyalcohol during 48 h
to a final silver concentration of 270 mg 3 L
1 as determined by
atomic absorption spectrometry method (AAS).
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The highly concentrated solid dispersion of silver NPs was
then prepared using spray drying of primarily preconcentrated
silver NPs aqueous dispersion with addition of a mannitol as an
encapsulating agent. To enhance the aggregation stability of the
silver NPs during the whole microencapsulation procedure, bovine
serum albumin was added into the preconcentrated dispersion at
a final concentration of 0.01% (w/w). Subsequently, mannitol
was added into the stabilized dispersion of silver NPs at a final
concentration of 5% (w/w) and it was homogenized for one hour
at 25 °C. After homogenization, the dispersion of silver NPs was
dried in a spray dryer at 105 °C. The final product was a solid
dispersion consisting of mannitol microparticles with an average
size of 3 μm, in which silver NPs were incorporated (Figure 1).
The concentration of silver in final solid dispersion was deter-
mined by AAS at a value of 2.5 mg of Ag per 1 g of solid
dispersion. This concentration was found to be sufficient for the
proposed toxicity study with regard to achieving the highest silver
concentration in the culture medium equal to 100 mg 3 L
1.
Acute and Chronic Toxicity Assay. Toxicity of silver NPs was
determined for the following silver concentrations: 10, 20, 40, 60,
80, and 100 mg 3 L
1 for acute toxicity assay and 5 mg 3 L
1 for
chronic toxicity assay. Silver NPs, having the form of solid
dispersion, were added under stirring into heated culture media
(45 °C) at desired amounts in order to achieve the final silver
concentrations. When the solid dispersion of silver NPs was
added into the standard culture medium, the flask was closed
with a sterile paper stopper. When the culture medium cooled
down to the laboratory temperature, the water condensed on the
flask wall was dried using a sterile filtering paper. All handling
associated with the preparation of culture medium was carried
out under sterile conditions using a laminar flow-box.
For the purpose of acute toxicity assay, ten freshly hatched
(not more than 4 h old individuals) virginal females and ten
males of Drosophila melanogaster were placed into the flasks with
culture medium containing silver NPs. Each flask with tested flies
was placed into a thermostat adjusted at 20 °C. After 10 days
when larvae became pupae, parental adults were let out from the
flasks. The same procedure was used in the case of negative
control where flies were cultivated on the standard culture
medium without silver NPs.
The acute toxicity of silver NPs was determined with regard to
developmental stages of Drosophila melanogaster and the toxic
manifestations of silver NPs were also observed on the hatched
adult flies. During the developmental stages, the following
parameters were monitored: number of larvae, pupation ability,
number of pupae, and development time. Acute toxicity index
(LC50) was determined from the dose dependent curve as the
concentration of silver leading to death of 50% tested organisms
dx.doi.org/10.1021/es104216b |Environ. Sci. Technol. 2011, 45, 4974–4979
Environmental Science & Technology
Table 1. Toxic Effects of Silver NPs at Different Concentra-
tions Evaluated As Number of Individuals for Each Devel-
opmental Stage of Drosophila melanogaster
number of
silver concentration development number number adults
(mg 3 L
1) time (days) of larvae of pupae (males/females)
0 (negative control) 14 216 208 208 (103/105)
10 14 214 204 203 (100/103)
20 14 208 195 104 (57/47)
40 16 206 196 24 (10/14)
60 unfinished 108 68
80 unfinished 22 6 0
100 unfinished 6 0
(dead organism means not hatched individuals). The toxic effects
of silver NPs were also monitored on adult flies where the
number of adults, the number of females and males, and changes
in the phenotype of adult flies were considered. All obtained
results including the toxic effects of silver NPs on Drosophila
melanogaster were compared with those obtained in the case of
negative control.
Chronic toxicity assay was initiated by placing ten freshly
hatched (not more than 4 h old individuals) virginal females and
ten males of Drosophila melanogaster into the flasks with culture
media containing silver NPs with a concentration of 5 mg 3 L
1 of
Ag. This generation of flies entitled as “parental generation, P”
gave rise to the first filial generation, F1 generation. Conse-
quently, the parental generation was let out from the flasks after
10 days. After the hatching of new imagoes from F1 generation,
ten randomly selected freshly hatched females and ten males
were transferred into the flasks with fresh culture media contain-
ing silver NPs where the individuals from F1 generation gave rise
to the new generation, F2 generation. This procedure was
repeated until F8 generation.
All the experiments evaluating the toxicity of silver NPs on
Drosophila melanogaster were performed in parallel three flasks
and were repeated three times. Thus, the acquired results are
expressed as the average values of nine toxicity assays in total.
’ RESULTS AND DISCCUSSION
Acute Toxicity Assay. The acute toxic effects of silver NPs on
developmental stages of Drosophila melanogaster were investi-
gated for six different concentrations of silver ranging from 10
to 100 mg 3 L
1. The observed numbers of larvae, pupae and
hatched individuals are summarized in Table 1. The lowest tested
concentration of silver equal to 10 mg 3 L
1, sufficient for killing
bacteria,18 did not show any acute toxic effects against any of the
developmental stages and it did not prolong the Drosophila's
development time either. The numbers of larvae, pupae and
hatched individuals were comparable with those observed for the
control sample. Only very slight reduction in pigmentation of adult
flies was observed. Silver NPs at concentration of 20 mg 3 L
1,
comparable to concentration cytotoxic to human fibroblasts,19
showed an acute toxic effect manifested by the decrease in the total
number of hatched individuals. Hatching of larvae and pupae was
not affected at these concentrations, their numbers were compa-
rable with those observed for the control sample. The acute toxic
effects of silver NPs were found just at the stage of hatching adults,
which were not able to leave the pupae (Supporting Information
ARTICLE
Figure S2). At a concentration of 20 mg 3 L
1 of silver, a 50%
decrease in the number of hatched individuals was observed,
nevertheless the development time was not prolonged. All the
hatched adult flies had highly reduced body pigmentation at this
concentration (Figure 2). At a concentration of 40 mg 3 L
1
of silver, the development time was prolonged from 14 days to
16 days compared to that found for the control sample and
decrease in the number of hatched individuals was almost 88%
compared to the negative control sample. Silver concentrations
equal to 60, 80, and 100 mg 3 L
1 significantly influenced devel-
opmental stages in the phase of larvae development and conse-
quently at the stage of pupae hatching. Such high concentrations of
0 silver induced a strong toxic effect leading to a significant decrease
in the number of larvae and pupae. Drosophila development cycle
0 was not finished in such high silver concentrations and the
individuals were unable to leave the pupae cases. At 100 mg 3 L
1
concentration, 97% of larvae were dead, and no pupae were formed.
Silver NPs did not affect only the developmental stages of
Drosophila flies, but also affected the physical characteristics of
the hatched flies, especially their body color and body size. A
slight reduction in the intensity of body pigmentation emerged
already at a concentration of 10 mg 3 L
1 of silver. Reduced
pigmentation persisted throughout all life of imago and was
observed for both males and females. With increasing the silver
concentration, the number of individuals with reduced pigmen-
tation increased and the intensity of the reduced pigmentation
also increased. Changes in eye pigmentation were not observed.
The reason for the reduction of pigmentation may lie in the
inhibition of body pigment synthesis pathways (for a detailed
information, see the discussion part concerning the chronic
toxicity). Another change observed on the adult flies that were
cultivated during their larval stages on medium containing silver
NPs was a decrease in the body proportions (Figure 2). The
weight loss of adult flies hatched on medium containing silver
NPs at a silver concentration of 20 mg 3 L
1 was 24% for both sexes
(150 male and female individuals were weighted separately) when
compared to the body weight of adult flies in the control sample.
Body weights of flies hatched on medium without silver NPs were
0.11 g for males and 0.124 g for females. Body weights of male and
female individuals hatched on medium containing silver NPs were
0.084 g (males) and 0.094 g (females).
Analyzing the results obtained within the acute toxicity assays,
it is evident that silver NPs negatively influenced the develop-
mental stages of Drosophila at the silver concentrations from 20
to 100 mg 3 L
1. At a concentration of 20 mg 3 L
1, a half of the
total number of individuals did not finished the development
cycle. Therefore, this concentration might be considered as a
concentration leading to the death of 50% of individuals: LC50 =
20 mg 3 L
1 of silver. Toxicity index, LC100, was achieved at a
concentration equal to 60 mg 3 L
1 of silver. As proved in an
earlier study considering the toxicity of silver NPs against
Drosophila melanogaster, exposure to such high lethal concentra-
tions of silver NPs (20 mg 3 L
1 and higher) induces heat shock
stress and also generation of free oxygen radicals in larvae of
Drosophila melanogaster. This oxidative stress subsequently re-
sults in DNA damage and ultimately apoptosis 12 which leads to
the death of the tested organism.
Chronic Toxicity Assay. On the basis of the results obtained
within the acute toxicity assay, it was proved that the concentra-
tion of 10 mg 3 L
1 of silver did not cause the acute toxic effect
against any developmental stages of Drosophila. Therefore, a
lower concentration equal to 5 mg 3 L
1 of silver was chosen for
4976 dx.doi.org/10.1021/es104216b |Environ. Sci. Technol. 2011, 45, 4974–4979
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Figure 2. Comparison of body pigmentation and body proportion of flies hatched on culture medium without silver NPs (on the left) and with silver
NPs at a concentration of 20 mg 3 L
1 of silver (on the right).
Figure 3. Total numbers of Drosophila individuals hatched on standard
culture medium without (b) and with silver NPs (O) at the silver
concentration equal to 5 mg 3 L
1 in each filial generation. Total number
of flies at the outset held of each generation was constant (10 males and
10 females).
the chronic toxicity assay. Overall eight filial generations of
Drosophila were consecutively exposed to silver NPs included
in the culture medium. The flies from the first filial generation F1
cultivated on the medium containing silver NPs were not
influenced by silver NPs and did not show any changes in the
total number of hatched individuals (Figure 3). Only a slight
decrease in the body pigmentation of adult flies was observed.
Nevertheless, the second filial generation F2 was already influ-
enced by silver NPs included in the culture medium. A decrease
in the total number of hatched individuals was 25% in compar-
ison with number registered for the negative control sample and a
decrease in pigmentation was observed again. The development
time of the second filial generation was 1 day prolonged in
comparison with the value found for the negative control sample.
In the third filial generation, only a slight decrease in the total
number of hatched individuals compared to F2 generation was
observed and in the fourth filial generation, the number of
hatched individuals was stable with regard to the F3 generation
(Figure 3). All hatched individuals belonging to the third and
fourth filial generations exhibited a decreased pigmentation and
development time was still 1 day prolonged in comparison with
the respective value for the control sample.
A considerable change in total number of hatched individuals
was observed from F5 to F7 generations. The total number of
hatched individuals gradually increased and in the case of F7
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generation it was even at the same level as that in the control
sample. Development time of F5 and F6 generations was not
prolonged in comparison with the control sample, on the
contrary, the development time of F7 generation was shortened
by 2 days in comparison with the control sample. In the last filial
generation F8, the total number of hatched individuals was found
to be comparable with that of the control sample, but develop-
ment time was shortened by 3 days. Body pigmentation of flies
from F4 to F8 generations was still reduced in comparison with
the flies from the control samples. A decrease in the body pig-
mentation was more apparent for females than for males (Sup-
porting Information Figure S3).
Taking into account the obtained data, it is evident that
Drosophila is negatively influenced by the repeated exposure to
low concentrations of silver NPs especially between F2 and
F4 generations. Because such low concentrations of silver
(5 mg 3 L
1 of Ag) did not affect the developmental stages of
Drosophila, the decrease in the total number of the hatched
individuals might be caused by a decrease in fecundity of flies that
ingested nutrient containing silver NPs. This statement can be
supported by the fact, that the total number of the hatched flies
from the F1 generation was not affected, because their parental
generation P was not influenced by silver NPs. Decreasing the
total number of hatched individuals was observed in the case of
flies from the F2 generation the parental generation of which was
flies from the F1 generation: that means the flies exposed to silver
NPs during their developmental stages. Decreased fecundity of
flies from the F1 generation was verified by the subsequent
experiment, in which the flies from the F1 generation were placed
on the standard culture medium without silver NPs. Their
fecundity was still decreased, which was proved by a decrease
in the total number of their offspring (F20 generation not exposed
to the silver NPs) by 18% in comparison with the control sample.
However, such a decrease in fecundity does not persist within the
following generations not exposed to the silver NPs and dis-
appears in the second generation of F1 generation’s offspring
(F30 generation not exposed to the silver NPs) for which the
number of offspring was identical to the number of individuals in
the control sample. Therefore, a negative impact of silver can
transfer to the first generation of offspring not exposed to the
silver NPs, but not to the second and next generations. Based on
these results, it can be concluded that silver NPs did not
introduce any heritable changes in the Drosophila organism.
Decrease in the pigmentation and fecundity of flies exposed to
the low concentrations of the silver NPs can be caused by
dx.doi.org/10.1021/es104216b |Environ. Sci. Technol. 2011, 45, 4974–4979
Environmental Science & Technology
Figure 4. (A) Weight of one hundred females of Drosophila individuals and (B) one hundred males of Drosophila individuals hatched on standard
culture medium without (b) and with silver NPs (O) at the silver concentration equal to 5 mg 3 L
1 in each filial generation.
oxidative and heat stress, which are induced by silver NPs.9 These
stress conditions induced during the developmental stages of
Drosophila can be the reason for disturbance of metabolic
synthesis pathways of biogenic amines and several hormones
regulating Drosophila reproduction. It has been demonstrated
that the metabolic systems involved in the stress reaction in
Drosophila are those of dopamine (DA), octopamine (OA),
juvenile hormone (JH), and ecdysteroids.20
28 Metabolic synth-
esis of biogenic amines such as DA and OA is important for
further metabolic processes and also for regulation of gonado-
tropins secretion. Since DA is, beside all, the major precursor for
a melanization of body cuticle,29 affecting its metabolic synthesis
via oxidative and heat shock stress induced by silver NPs can lead
to the disturbance of metabolic synthesis of melanine which may
result in a decrease in the pigmentation of adult flies. The JH
metabolic system in females was shown to respond to stress with
a decrease in JH degradation,30,31 and the ecdysteroid system
responded to heat stress with an increase in 20-hydroxyecdysone
(20HE) levels.32 A dramatic fertility decrease was the result of the
changes in the JH and 20HE metabolic systems.30 Another work
reported that heat stress in wild-type females of Drosophila
results in oocyte maturation delays, degradation of early vitello-
genic egg chambers, inhibition of yolk protein gene expression in
follicle cells, and accumulation of mature oocytes.21
Decrease in fecundity of flies exposed to the silver NPs from
F1 to F3 generations was stopped at F4 generation and from F5
to F8 generation the flies’ fecundity subsequently increased up to
the level of the flies from the control sample. Fecundity can be
restored because of the adaptability of flies to the exposure to the
silver NPs. Adaptation can be indirectly encouraged based on the
shortening of the development time observed in F7 and F8
generations. Drosophila larvae strive to minimize the ingestion
time of nutrient containing silver NPs in order to minimize their
negative impact. This way of adaptation on the negative impact of
silver NPs was consequently exerted in a decrease of flies’ weight
and body proportion due to the shortening of ingestion time of
nutrient. The weight of hatched flies from F5 to F8 generations
decreased by 15% and 20% for females and males, respectively, in
comparison with the flies from the control sample (Figure 4).
Comparison of body proportion of the control flies and flies from
F8 generation hatched on the medium containing the silver NPs
is shown in Supporting Information Figure S3.
Silver NPs are a subject of the scientific research especially
because of their antimicrobial properties which appear at silver
concentrations equal to units of milligram per liter. Nevertheless,
4978
ARTICLE
the knowledge of their toxic properties with respect to the higher
eukaryotic organisms is more than desirable regarding rapid
increases in exploitation of medical products containing silver
NPs and especially commercial products containing the silver
NPs. In this work, the acute toxicity of silver NPs on the develop-
mental stages of Drosophila melanogaster was proved for the silver
concentrations higher than 20 mg 3 L
1. Such high concentra-
tions of silver NPs are one order higher than the minimum
bactericidal concentrations of silver NPs which are equal to
several units of mg 3 L
1 of silver.17,18 From these results, it is
evident that the silver NPs kill prokaryotic bacterial organisms
more efficiently than higher eukaryotic organisms. On the other
hand, low concentration of silver NPs equal to 5 mg 3 L
1, which
is sufficient for killing the bacteria, showed the chronic toxicity
effect against the tested organism of Drosophila melanogaster
manifesting by a decrease in the fecundity of this organism.
However, Drosophila organisms exposed to low concentrations
of silver successively adapted to this long-term toxic treatment of
silver NPs and their fecundity returned back to the normal level
due to their adaptation. Beyond the positive findings that silver
NPs evoke acute toxic effects against Drosophila melanogaster at
high concentrations of silver above 20 mg 3 L
1, it is necessary to
pay a great attention to the manipulation and liquidation of
materials containing the silver NPs considering their possible
chronic toxicity impact in the environment.
’ ASSOCIATED CONTENT
bS Supporting Information. Materials and methods, TEM
images of silver NPs (Figure S1), unfinished Drosophila’s devel-
opment cycle (Figure S2) and comparison of body proportion
and pigmentation of flies hatched on standard culture medium
with and without silver NPs (Figure S3). This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: þ420585634420. Fax: þ420585634425. E-Mail:
libor.kvitek@upol.cz.
’ ACKNOWLEDGMENT
The authors gratefully acknowledge the supports by the Operational
Program Research and Development for Innovations—European
dx.doi.org/10.1021/es104216b |Environ. Sci. Technol. 2011, 45, 4974–4979
Environmental Science & Technology
Social Fund (project CZ.1.05/2.1.00/03.0058). The authors also
gratefully acknowledge the supports by the Ministry of Education,
Youth and Sports of the Czech Republic (MSM6198959218,
MSM6198959201, MSM0021620822), Czech Science Foundation
(GAP304/10/1316), and Academy of Sciences of the Czech Republic
(KAN115600801).
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