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RESEARCH ARTICLE
Investigation of ZnO nanoparticles’ecotoxicological effects
towards different soil organisms
Sonia Manzo &Annamaria Rocco &Rita Carotenuto &
Fabiano De Luca Picione &Maria Lucia Miglietta &
Gabriella Rametta &Girolamo Di Francia
Received: 21 June 2010 / Accepted: 18 November 2010
#Springer-Verlag 2010
Abstract
Introduction Nanomaterials have widespread applications
in several industrial sectors. ZnO nanoparticles (NPs) are
among the most commonly used metal oxide NPs in
personal care products, coating and paints. However, their
potential toxicological impact on the environment is largely
unexplored.
Materials and methods The aim of this work was to
evaluate whether ZnO nanoparticles exert toxic and geno-
toxic effects upon terrestrial organisms: plants (Lepidium
sativum,Vicia faba), crustaceans (Heterocyipris incon-
gruens), insects (Folsomia candida). To achieve this
purpose, organisms pertaining to different trophic levels of
the soil ecosystem have been exposed to ZnO NPs. In
parallel, the selected soil organisms have been exposed to the
same amount of Zn in its ionic form (Zn
2+
)andtheeffects
have been compared.
Results The most conspicuous effect, among the test battery
organisms, was obtained with the ostracod H. incongruens,
which was observed to be the most sensitive organism to
ZnO NPs. The root elongation of L. sativum was also mainly
affected by exposure to ZnO NPs with respect to ZnCl
2
,
while collembolan reproduction test produced similar results
for both Zn compounds. Slight genotoxic effects with V. f a b a
micronucleus test were observed with both soils.
Conclusion Nanostructured ZnO seems to exert a higher
toxic effect in insoluble form towards different terrestrial
organisms with respect to similar amounts of zinc in ionic
form.
Keywords ZnO nanoparticles .Soil .Toxicity test .
Genotoxicity
1 Introduction
A nanoparticle may be defined as any solid state material
that has at least one dimension below 100 nm and whose
properties are different from its bulk counterpart (Auffan et
al. 2009). Due to their novel and remarkable properties,
nanomaterials are finding widespread applications in
several industrial sectors. This poses an urgent need for
the assessment of their potential toxicological impact on
human health and environment. Indeed, several papers have
reported on its toxicology in relation to human health
(Auffan et al. 2009). In contrast, the effect of nanoparticles
(NPs) released in the environment is still largely unexplored
although some reviews have been already published (Baun
et al. 2008; Kahru et al. 2008; Ruffini Castiglione and
Cremonini 2009). Two different mechanisms compete to
control the NPs toxic action (Brunner et al. 2006): (1) a
chemical effect based on the chemical composition, e.g.,
release of (toxic) ions; and (2) stress or stimuli caused by
the surface, size and/or shape of the nanoparticle itself.
These stimuli can be either due to a mechanical hindrance
to biological functions or to a different interaction of the
chemical compound in the nanostructured form with the
biological environment. In fact, the peculiar properties of
the nanostate, with special attention to the atypical surface
structure and reactivity, may modify the biophysico-
Responsible editor: Vera Slaveykova
S. Manzo (*):A. Rocco :R. Carotenuto :F. De Luca Picione
ENEA Research Centre Portici, UTTP ChiA,
Napoli, Italy
e-mail: sonia.manzo@enea.it
M. L. Miglietta :G. Rametta :G. Di Francia
ENEA Research Centre Portici, UTTP-MDB,
P.le E. Fermi 1 80055-Portici,
Napoli, Italy
Environ Sci Pollut Res
DOI 10.1007/s11356-010-0421-0
chemical interactions of these materials with biological
environment, enhancing processes such as dissolution,
redox reaction, generation of reactive oxygen species,
affecting the NPs reactivity towards the environment (Nel
et al. 2009; Carlson et al. 2008; Xia et al. 2006). Thus,
when investigating the toxicity of a compound in relation
to its nanometric nature, it is important to verify which is
the actual size of the particles dispersed in the testing
media.
In this frame, ZnO are among the most widely used NPs
since they have applications in a large variety of sectors
ranging from personal care products to coatings and
catalysts in environmental remediation (Choopun et al.
2009; Kamat and Meisel 2003; Wang 2004).
The toxicity of ZnO nanoparticles in freshwater has been
thoroughly investigated and the EC
50
and LC
50
levels have
been registered for several aquatic organisms: for Vib r io
fischeri EC
50
is1.9mgl
−1
;forDaphnia magna,LC
50
is
3.2 mg l
−1
;forTamnocephalus platyurus,LC
50
is 0.18 mg l
−1
(Heinlaan et al. 2008); and for the freshwater alga Pseudo-
kirchneriella subcapitata, the estimated 72-h EC
50
value is as
low as 0.07 mg l
−1
(Franklin et al. 2007). ZnO nanoparticles
exposition has been found to induce effects on humans as
well. For instance, they can cause gastroduodenal corrosive
injury (Liu et al. 2006) and pulmonary toxicity (Moos et al.
2010). In most cases, soluble zinc ions (Zn
2+
)fromZnO
seem to be the main source for the (eco)toxicity (Aruoja et
al. 2009;Heinlaanetal.2008). For T. platyurus,the
toxicity is attributed to the soluble, dissociated Zn ions,
and the LC
50
and NOEC values for D. magna are up to
threefold lower for ZnO NPs than for bulk ZnO, suggest-
ing a clear toxic effect of the nano ZnO due to the
peculiarity of the nanometric size.
In comparison to freshwater, much less data have been
reported on the NPs toxicity effects in soils although the soil
solid phase is the primary, large sink for any human waste.
As far as elemental zinc is concerned, it is well known
that it is one of the most important soil contaminant and
furthermore a number of key processes are likely to affect
its fate and availability in this environment. Nevertheless
scarce data have been published on the effects produced by
zinc based nanomaterials in soil. To date, phytotoxic effects
of nanomaterials have been mainly investigated (Lin and
Xing 2007). Goodman et al. (2004) have reported a clear
reduction in seed germination of corn and in root growth of
corn, cucumber, lettuce, radish, mustard rape, and ryegrass
upon exposure to functionalized gold NPs.
In this study, we investigated the ZnO nanopowder toxic
effect on different terrestrial organisms. Since most of the cited
literature agrees on the involvement of the ion Zn
2+
in the toxic
action of zinc based nanoparticles, in this work the impact
of ZnO NPs on terrestrial organisms has been investigated
and compared to that of the same amount of ionic zinc.
As no single test or species of living organism shows
uniform sensitivity to all chemical compounds, a biotest
battery with different sensitivity profiles is often recom-
mended and used to perform the ecotoxicological assess-
ment. In addition, due to the complexity of the ecosystems,
the toxicological hazard assessment is more informative/
predictive if the battery involves organisms of different
trophic levels (Blaise 1998). According to these general
criteria, in this study a battery of toxicity contact tests with
different organisms: plants (Lepidium sativum), crustaceans
(Heterocyipris incongruens), insects (Folsomia candida),
together with a genotoxicity test with plant seeds (Vicia
faba), were performed.
2 Materials and methods
ZnCl
2
with purity >98% was purchased from Sigma-Aldrich
(CAS Number: 20,808-6). ZnO nanopowder was purchased
from Sigma-Aldrich (CAS Number: 1314-13-2), with a
nominal primary particle size of less than 100 nm (i.e.,
r
p
≤50 nm).
Pure water (conductivity: 0.056 μScm
−1
; TOC <5 ppb)
was prepared with Milli-Q Gradient A10 system, Millipore.
OECD (Organisation for Economic Co-operation and
Development) standard soil was purchased from ECOTOX
LDS srl (Milan, Italy).
A ZnCl
2
stock solution was prepared at a concentration
of 10 g l
−1
in pure water.
ZnO nanopowder (10 mg) was finely dispersed in 35 g
of OECD dry standard soil. The 10 g soil aliquots for
toxicity assays were sampled from the whole solid mixture
through a Fritsch rotary sample divider Laborette 27.
The specific surface (SS) of both the soil samples and ZnO
NPs was analyzed using the Brunauer, Emmett and Teller
(BET) method on a Quantachrome, Autosorb-1instrument,
recording N
2
adsorption/desorption isotherms at 77 K
(Brunauer et al. 1938).
Aqueous extracts of soil samples were obtained by
mixing 2.5 g of soil with 15 ml of pure water and
vigorously stirring the mixtures for 30 min.
Measurements of particles size were performed with a
Zetasizer Nano system (Malvern Instruments, U.K.). Unfil-
tered aqueous extracts were placed in clean disposable
cuvettes and measured, at least in triplicate, at 25°C. At
first, the presence of very large sedimenting particles
negatively affected the measurements. After particles
settling, instrument’s internal quality criteria were passed
and data were recorded. All tests were conducted at least in
triplicate.
The nanoparticles dissolution was assessed by measuring
and comparing the Zn content in unfiltered aqueous extracts
and in samples filtered through an alumina Whatman
Environ Sci Pollut Res
Anotop filter 0.02 μm. Samples were diluted tenfold with
pure water prior to the inductively coupled plasma-mass
spectrometry (ICP-MS) analysis. Measurements were per-
formed on a spectrometer ICP-MS Elan 6000 (Perkin-
Elmer) equipped with a cross-flow nebulizer.
SEM analysis was performed, on a LEO 1530, over soils
samples with two different concentration of zinc oxide, at
0.03% and 10%. Small soil aliquots were wetted with few
millilitres of pure water and the obtained suspensions
casted over a clean silica wafer and finally dried onto a
hot plate.
2.1 Experimental design
To compare the toxicity due to the different chemical and
structural forms of zinc, a battery of toxicity tests was
performed on artificial standard soil (OECD 2000) samples
spiked with ZnO NPs and ZnCl
2
, both containing the same
Zn amount. A screening dose of Zn (i.e., 230 mg kg
soil
−1
)
was used for spiking the dry soil. This concentration was
chosen to satisfy two requirements: (1) for NPs, the Zn
concentration is well above the solubility of the oxide, so
that ZnO can be considered as only negligibly dissociated
into ions (Zn
2+
and/or Zn(OH)
+
)eveninwettedsoil
matrices; (2) for ZnCl
2
testing, the metal ion concentration
is capable of producing clear, marked effects on the
selected organisms for toxicity tests (Aramba et al. 1995;
Lock and Janssen 2003).
According to the OECD guidelines, standard artificial
soil, composed by sphagnum peat (10%), kaolin clay (20%)
and quartz sand (70%), was used both as control soil and as
matrix for Zn contamination. For spiking of the soil with
ZnO and ZnCl
2
, two different routes were followed: zinc
oxide nanopowder was finely dispersed in the dry solid
matrix, while the zinc chloride was administered as aqueous
solution to the dry soil. The use of different administration
routes was aimed to supply the contaminants in the specific
structural/chemical form to be tested (i.e., nanopowder in
its primary particle size and zinc chloride in its ionic,
dissociated form).
Ten milligrams of zinc oxide nanopowder was directly
added and thoroughly mixed with 35 g dry weight (d.w.) of
the standard soil to obtain a concentration of 286 mg kg
−1
d.w. (corresponding to 230 mg kg
−1
d.w. of Zn). In order to
obtain representative samples of this contaminated soil to
be employed in toxicity assays, soil aliquots (10 g) were
collected by sampling them from a stream of the solid
mixture, taking further care of sampling the entire cross
section of the stream for many times. Different aliquots of
pure water were then added to the spiked soil in agreement
with the different assays to be performed. The same
nominal zinc concentration was reached by adding a ZnCl
2
aqueous solution to the soil. The spiked soil obtained was
then wetted, for the test, taking into account the water
added with the ZnCl
2
solution.
2.2 Characterization of ZnO spiked soil
If an ensemble of NPs is dispersed in an external matrix,
under the assumption that no interaction occurs between the
NPs and the matrix itself, the dispersion specific surface
(SSd) can be considered as the weighted mean of the
external matrix and the NPs specific surfaces, SSem and
SSnp, respectively:
SSd ¼1
#
ðÞSSem þ
#
SSnp ð1Þ
where χis the ratio of the NP weight over the dispersion
weight.
Rearranging Eq. 1, it turns out that:
SSd ¼SSnp SSemðÞ
#
þSSem ð2Þ
Therefore, if SSd is measured vs. χand the relation is
found to be linear, then the NPs ensemble and the external
matrix can be considered non-interacting and, once SSnp is
evaluated from the best fit, the average NP radius in the
dispersion, r
p
can be calculated by means of Eq. 3(Roelofs
and Vogelsberger 2004).
SSnp ¼N»a0¼3=rp»rð3Þ
where a
0
=4*π*r
p
2
is the surface of a single NP (assumed
spherical for simplicity), r
p
is the NP radius and Nis the
total number of NPs per unit of mass.
In Table 1, the dispersion specific surfaces are reported
vs. χin our case, in which the external matrix is the
standard soil and the NPs ensemble consists of ZnO
nanoparticles.
In our experimental conditions, the relative error on SSd
is less than ±0.2%. It is easily found that data follow very
accurately a linear fit and, from the slope and the intercept,
an NP specific surface (SSnp) of 9.89 m
2
/g is extracted.
Substituting this value into Eq. 3, we find: r
p
=53 nm,
which is in good agreement with the actual radius of ZnO
NPs used in our experiments. We can therefore conclude
that ZnO NPs do not form aggregates and that the ensemble
chemical and physical properties are the result of the
χ(%) SSd (m
2
/g)
0 0.78
0.1 1.19
0.2 2.62
0.2 2.49
a
1 9.9
Table 1 Weight ratio of NPs in
soil dispersions χand the spe-
cific surface area of soil samples
measured via BET method
a
Wetted sample
Environ Sci Pollut Res
superimposition of the chemical and physical properties of
each of the ZnO NP comprising the ensemble itself.
In addition, soil samples, both contaminated and not,
were extracted with pure water in order to investigate the
ZnO solubility under the experimental conditions and to
characterize the size distribution of suspended matter in the
aqueous extracts. The tests were performed over 2.5 g
aliquots of OECD standard soil and contaminated soil
suspended in 15 ml of pure water. The extracts were rested
to settle and the surnatant was collected for particle sizing
via dynamic light scattering (DLS) and Zn analysis via ICP-
MS. Aqueous extracts of soil samples have pH 6.2–6.5
values.
2.3 Toxicity tests
2.3.1 Plants
Seed germination and root elongation test According to
EPA (1996) and OECD (2003) guidelines, ten L. sativum
seeds were exposed to 10 g of contaminated soil and
control soil (OECD) and wetted with 5 ml of deionized
water. After incubation in darkness (25± 2°C, 72 h), the
germinated seeds were counted and the root lengths
measured. The results were expressed as percent effect
with respect to the control (percent effect).
V. faba micronucleus test Five V. faba seeds were exposed
to 20 g of contaminated soil and control soil, both saturated
with 6 ml deionized water. OECD soil was used as negative
control while positive control was prepared by saturating
OECD soil with 6 ml of a 10 mg l
−1
K
2
Cr
2
O
7
solution.
After incubation in darkness (20 ± 1°C, 96 h), according to
Kanaya et al. (1994) the primary roots were cut, fixed and
then stained by the Feulgen technique. Squash preparations
were produced in 45% acetic acid and the slides made
permanent in Histovitrex (Carlo Erba, Milan, Italy). Two
hundred cells per tip were scored in order to evaluate
micronucleus frequency.
2.3.2 Crustaceans
H. incongruens growth and mortality test Following the
Chial and Persoone (2002a,b), ten freshly hatched ostracods
were exposed to 1 g of contaminated soil and control soil
(OECD) previously added with 4 ml of standard solution
(EPA Standard Freshwater). One millilitre of algal suspen-
sion (P. subcapitata 10
7
cells ml
−1
) was added to each well as
food supply. After incubation in darkness (25±2°C, 6 days),
mortality rates were recorded and the lengths of organisms
that survived were measured. The results are expressed as
mortality and growth percentage with respect to the control.
2.3.3 Insects
F. candida reproduction test Following ISO 11267 (1999),
ten juvenile collembolans (F. candida) aged 10–12 days
were exposed to 30 g of control and spiked soils saturated
with 9 ml of deionized water and incubated under
controlled conditions (light/dark cycle 16/8 h; 20 ± 2°C).
After 28 days of exposure, during which insects were fed
weekly with 2 mg of dry yeast, test chambers were floated
to allow survived adult and neonate count and to evaluate
mortality and reproduction endpoints.
F. candida avoidance test Following Aldaya et al. (2006),
7.5 g of standard soil as control and 7.5 g of contaminated
soil, both saturated with 2.25 ml of deionized water, were
placed in sterile polystyrene Petri dishes (55 mm diameter,
10 mm height), leaving a 2-mm space line between the
soils. At the centre of the line an individual of F. candida
was deposited and its position was recorded every 20 min
up to 100 min. The test was performed over five replicates.
During the observation period, the Petri dishes were kept
under fluorescent light at 20±2°C. A blank experiment
using control soil at both sides of the Petri dishes was
performed to check for any light gradient effect. The five
counts over the 100-min observation period for each Petri
dish was used to calculate the average position of a single
individual.
2.4 Statistic analysis
All toxicity test results are expressed as mean ± standard
error (SE). The toxicity data obtained for both ZnO NP and
ZnCl
2
treatments were analyzed with Student’st-test using
the software SigmaStat v. 3.5. Levels of p<0.05 and p<
0.01 were considered statistically significant and highly
significant, respectively.
3 Results and discussion
The BET characterization of the spiked soil samples
indicate that the ZnO NPs dispersed in the soil are still in
their pristine state and no aggregation has occurred. Data in
Table 1show that the addition of ZnO NPs to standard soil
gives rise to a linear increase in the specific surface area
with the NPs weight fraction for the contaminated soil,
while the calculated BET radius of the NP itself remains
unchanged. The adopted spiking procedure seems therefore
to preserve the NP size. The dispersion state of the NPs
remains unchanged after the wetting procedure of the soil
itself, carried out in the toxicity tests, as could be observed
by measuring the SSd in the wetted soil. SEM analysis of
Environ Sci Pollut Res
the contaminated soil sample (i.e., 230 mg
Zn
kg
soil
−1
,
corresponding to 0.03 wt.%) is not very informative. In
fact, due to the very low concentration of nanoparticles,
even after a very careful examination of this soil sample, it
was not possible to identify any particle, agglomerate or
mass portion that could be ascribed to the ZnO (Fig. 1). It is
interesting to note that a bimodal distribution of particles is
observed only in the aqueous extract of a 10 wt.% of ZnO
contaminated soil, for which the DLS analysis show well
resolved peaks at 470 and 103 nm. Other test soils (i.e., 0
and 0.03 wt.%) have shown monomodal distributions.
Therefore, the peak at 103 nm can be ascribed to the ZnO
NPs, whereas the other peak is assigned, by comparison, to
the soil component.
Among the test battery organisms, the most striking
effect of ZnO NP spiked soil was obtained with the
ostracod H. incongruens, which was observed to be the
most sensitive organism to ZnO NPs (Fig. 2). H. incon-
gruens is an ostracod recently introduced as a test organism
for soil toxicity assessment (Chial and Persoone 2002a,b)
which allows handlers to evaluate both acute and chronic
endpoints. ZnO NPs spiked soil exerted a lethal effect
(100% mortality) after 6 days of exposure. On the contrary,
ZnCl
2
spiked soil (soluble Zn) caused only moderate, acute
(21%) and chronic (34%) effects. The noticeable difference
between the toxic action exerted by ZnO NPs and ionic zinc
against these organisms could be ascribed to an interference
with some vital processes of the oxide conveyed by the
nanodimension. According to Franklin et al. (2007), a
similar, indirect effect through food depletion, i.e., algae P.
subcapitata, due to soluble zinc, may be taken into account
for both the experiments. This study shows that the 72-
hIC
50
values against P. subcapitata for ZnO bulk, ZnO
nano and ZnCl
2
range from 60 to 69 μgl
−1
, and that the
toxic action can be attributable to the solely dissolved zinc.
In our experiment, a much higher concentration of ionic
zinc (as ZnCl
2
) was administered to the soil (at 57.5 mg
l
−1
), and hence, to the feeding organism P. subcapitata.
Analogously, the same toxic action on the green alga has to
be considered in the evaluation of the ZnO NPs effects. In
fact, even if the ICP-MS analysis have shown that the
concentration of zinc in the aqueous extracts corresponds to
less than two order of magnitude (i.e., 1.6 mg kg
soil
−1
) with
respect to the ionic zinc concentration derived by ZnCl
2
(230 mg kg
soil
−1
), still the total amount of zinc in the water
phase (290 μgl
−1
) exceeds the toxicity value for the total
Zn observed by Franklin. Following the filtration of the raw
extract with a 0.02-μm filter, thus separating the ZnO NPs
from the aqueous solution, the zinc concentration is only
slightly decreased to 104 μgl
−1
that is, still higher than the
IC
50
value already reported. Therefore, it is clear that in our
experiment, the soluble zinc ions do exert toxic effects
against the feeding organisms, P. subcapitata, and that this
toxic action may, in turn, affect the overall toxicity against
H. incongruens resulting in acute and chronic responses up
to 21% and 34%, respectively. However, in order to explain
the 100% of mortality observed we should hypothesize
some other toxic mechanism. It has been reported, indeed,
that NPs may adsorb on phytoplankton (Rhee and Thomson
1992) and onto the algal cell surface (Navarro et al. 2008)
or even that adhesion of NPs aggregates to the exoskeleton
of crustaceans (Baun et al. 2008) may cause physical effects
and/or loss of mobility. This study is, to the best of our
knowledge, the first to report the effect of NPs upon
ostracods.
L. sativum seeds showed a 100% germination (no effect
with respect to control) with both the soil contaminants. A
clear difference between ZnO NPs and soluble Zn can be
observed instead for root elongation; in particular, ZnO NPs
Fig. 1 SEM image of a ZnO contaminated soil sample (230 mg
Zn
kg
soil
−1
). Inset: SEM image of pure ZnO NPs
-100
-75
-50
-25
0
25
50
75
100
Effect (%)
ZnO NPs
ZnCl2
L. sativum H. incongruens (M) H. incongruens (G) F. candida
*
a
a
Fig. 2 ZnO NP toxicity. Mean percent effect of mortality (M) and
body growth (G) measured at Zn concentration of 230 mg g
−1
d.w.,
tested both as ZnO NPs and ZnCl
2
.(
a
) Statistically significant
difference with p<0.05 between the two treatments; (*) no data could
be measured because of 100% mortality
Environ Sci Pollut Res
spiked soil exerted a moderate toxic effect, while ZnCl2
spiked soil produced a 35% biostimulation (Fig. 2). This
biostimulation can be attributed to an hormetic effect. In
fact, the OECD standard soil is devoid of Zn and, as this
metal is an essential element for many plants, a certain
amount of promptly available ionic Zn might have a
biostimulation effect (Paschke et al. 2006). The ZnO
nanoparticles in the contaminated soil exerted a mild toxic
effect on the root elongation. This result is in agreement
with the significant inhibition effect of ZnO NPs, mainly on
root growth, of six plant species observed by Lin and Xing
(2007), who suggested a toxic action mainly due to a purely
mechanical effect based on the production of ‘holes’into
the cell walls. This hypothesis is further supported by
considering that, in our case, the soil contaminant is mainly
constituted by nanoparticles of ZnO in their pristine size
and that its soluble fraction cannot account for the
experimental observation. As a consequence, the toxic
action observed can be related to the surface properties of
the NPs, whereas a chemical toxic action induced by
dissolution processes can be ruled out.
Zinc seemed to exhibit no adverse effect upon collembolan
reproduction at the tested concentration (230 mg kg
−1
)
regardless of the form. In contrast, both ZnO NPs and ZnCl
2
spiked soils produce a clear biostimulation (106% and 94%)
with respect to the control (OECD soil). This should not
surprise. Zinc is an essential element, and the observed effect
could be the result of exposed organisms needs, in the
control soil. When assessing the environmental risks of
essential metals such as zinc, both deficiency and toxicity
levels should be in fact, considered (Calabrese and Baldwin
2003;Locketal.2001). Similar effects have also been
shown to occur in the springtails Protaphorura armata and
Orchesella cincta during feeding experiments (Posthuma and
van Straalen 1993; van Straalen et al. 1989). It is interesting
to note that a chronic zinc toxicity for F. candida (EC
50
)in
standard artificial soil has been found at concentration much
higher than that used in our investigation (from 487 mg kg
−1
d.w. [Smit and Van Gestel 1998] to 900 mg kg
−1
d.w.
[Sandifer and Hopkin 1997]). However, in short time tests
with collembolans (avoidance test), ZnO NP spiked soil
produced only a 16% avoidance during the 100-min
observation time with respect to a 76% avoidance for ZnCl
2
probably due to oral uptake.
Slight genotoxic effects with V. faba micronucleus test
(Fig. 3) were appraised with both spiked soils. The
micronucleus frequencies observed for ZnO NPs and ZnCl
2
contaminated soils are quite similar (5.2 and 5.6 micro-
nuclei 1000 cell
−1
, respectively).
The V. faba micronucleus test has proved to be a very
sensitive and useful method: micronuclei are, in fact, the
result of chromosome breaks (or mitotic anomalies) that
require a passage through mitosis to be recognizable.
Although the molecular mechanism of DNA breakage is
not yet clearly understood, different toxic mechanisms have
been proposed for Zn
2+
and for ZnO NPs. Zn ions could
interfere with DNA repair process similarly to what has
been suggested to happen in mammals (Scicchitano and
Pegg 1987; Yang et al. 1996), while it has been supposed
that ZnO NPs can damage DNA by inducing lipid
peroxidation and oxidative stress (Xia et al. 2006).
In this study, micronucleus tests performed with meri-
stematic root tip cells of V. faba suggested the same, mild,
genotoxic effect with both spiked soils. It could be
presumed that the observed effects result from different
interaction pathways of the tested materials: soluble Zn ions
can easily penetrate directly into meristematic cell mem-
branes while the ZnO NPs can penetrate the cell walls
through a mechanical action (Lin and Xing 2007). Also, in
this last case, the contribution of dissociated ionic Zn is so
low that it can be neglected.
4 Conclusion
In this work we have reported the evidence of toxic effects
of ZnO NPs towards different terrestrial organisms. Unlike
most of the results reported in literature, which address to
soluble fraction of the ZnO NPs (i.e., the Zn
2+
ion) the
(eco)toxic actions, here we show that, for some organisms,
ZnO NPs exert a higher toxic effect in its insoluble form
compared to that of the same amount of ionic zinc. Thus,
the NPs toxic action can be linked to a chemical effect and/
or stress or stimuli caused by the peculiar physical
characteristics of the nanostate.
Negative control Pos itive control ZnO NP ZnCl
0
2
4
6
8
10
12
Micronuclei (nr/1000 cells)
2
a B A B
Fig. 3 Mean values of V. faba micronucleus frequencies due to ZnO
NPs and ZnCl
2
spiked soils. OECD standard soil was used as negative
control while the same standard soil saturated with K
2
Cr
2
O
7
solution
(10 mg l
−1
) was used as positive control. Differences between each
treatment and the negative control were statistically significant with
(a) p<0.05 and (A) p< 0.01; for positive control they were statistically
significant with (B) p<0.01
Environ Sci Pollut Res
A physical interaction of ZnO nanoparticles on plant
root elongation has been, in fact, observed. A high toxic
action against ostracods, the most sensitive organisms, is
observed and is ascribed to the ZnO nanoparticles. Besides,
the genotoxic effect with V. faba can be linked to the
nanoparticulate form, even if it is comparable to that
exerted by soluble Zn. As far as the different tests utilized
did not produce an univocal response, a nanoparticle-
dependent toxicity has been indeed observed for the
investigated NPs.
Due to the complexity of the soil ecosystem and of the
considered test organisms, these findings constitute a
preliminary insight into the comprehension of the
biophysico-chemical interactions of nanomaterials with the
biological environment. Then, for a proper evaluation of
ecotoxicological risk of nanoparticles in terrestrial environ-
ment, it will be necessary to increase the battery of toxicity
test utilizing more soil dwelling organisms and evaluating
more different endpoints.
Acknowledgements The authors are grateful to A. Salluzzo for ICP-
MS measurements and to A. De Girolamo Del Mauro and V. La
Ferrara for their support in SEM analysis.
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