Ecotoxicology and Environmental Safety 144 (2017) 543–551

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Ecotoxicology and Environmental Safety

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

Bioavailability of coated and uncoated ZnO nanoparticles to cucumber in MARK

soil with or without organic matter
⁎
Sahar Moghaddasia, Amir Fotovata, , Amir Hossein Khoshgoftarmaneshb, F. Karimzadehc,
Hamid Reza Khazaeid, Reza Khorassania
a
Department of Soil Science, Ferdowsi University of Mashhad, 91775-1163 Mashhad, Iran
b
Department of Soil Science, College of Agriculture, Isfahan University of Technology, 84156-83111 Isfahan, Iran
c
Department of Materials Engineering, Isfahan University of Technology, 84156-83111 Isfahan, Iran
d
Department of Crop Science, Ferdowsi University of Mashhad, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: There is a gap of knowledge for the fate, effects and bioavailability of coated and uncoated ZnO nanoparticles
Zinc oxide nano particles (NPs) in soil. Moreover, little is known about the effects of soil properties on effects of NPs on plants. In this
Soil fractionation study, the availability ZnO NPs in two soils with different organic matter content (one treated with cow manure
Uptake (CM) and the other as untreated) was compared with their bulk particles. Results showed that coated and
uncoated ZnO NPs can be more bioaccessible than their bulk counterpart and despite their more positive effects
at low concentration (< 100 mg kg−1), they were more phytotoxic for plants compared to the bulk ZnO particles
at high concentration (1000 mg kg−1) in the soil untreated with CM. The concentration of 1000 mg kg−1 of ZnO
NPs, decreased shoot dry biomass (52%) in the soil untreated with CM but increased shoot dry biomass (35%) in
CM-treated soil compared to their bulk counterpart. In general, plants in the CM-treated soil showed higher Zn
concentration in their tissues compared with those in untreated soil. The difference in shoot Zn concentration
between CM-treated and untreated soil for NPs treatments was more than bulk particles treatment. This different
percentage at 100 mg kg−1 of bulk particles was 20.6% and for coated and uncoated NPs were 37% and 32%,
respectively. Generally, the distribution of ZnO among Zn fractions in soil (exchangeable, the metal bound to
carbonates, Fe-Mn oxides, organic matter and silicate minerals and the residual fraction) changed based on
applied Zn concentration, Zn source and soil organic matter content. The root tip deformation under high
concentration of NPs (1000 mg kg−1 treatment) was observed by light microscopy in plants at the soil untreated
with CM. It seems that root tip deformation is one of the specific effects of NPs which in turn inhibits plant
growth and nutrients uptake by root. The transmission electron microcopy image showed the aggregation of NPs
inside the plant cytoplasm and their accumulation adjacent to the cell membrane.

1. Introduction on their properties, dose and method of application and plant species
(Lin and Xing, 2007; Zhang et al., 2007; Torney et al., 2007). Dissolu-
Nanoparticles (NPs) are suggested to be used as a source of nutrients tion, aggregation and change in the surface properties of ZnO NPs in
required for plant growth (Monreal et al., 2015). Available data suggest solid matrices might modify bioactivity of these particles (Moghaddasi
that NPs are more bioreactive than bulk counterpart due to their et al., 2015). The main reactions of NP in soil-plant systems seems to be
smaller size and greater surface area (Dietz and Herth, 2011). These dissolution into ions, aggregation of individual NPs into the larger size
properties can also increase the NPs risks for environment health. units, the dissolution of aggregation in soils, interaction with root
Previous studies have shown both positive and negative effects of NPs exudates or labile soil organic matter and adsorption on root surfaces
on plants (Moghaddasi et al., 2013; Zhang et al., 2007). Adverse effects (Monreal et al., 2015).
of copper oxide NPs i.e., growth retardation, reduced biomass pro- To date, a great number of studies have been conducted on artificial
duction, lower root length, shrinking of root tip, and high collapse of plant growth media while the final destination of all engineered NPs is
root epidermal and cortical cells of Arabidopsis thaliana, have been in soil. In addition, several factors in soil influence the fate and effects
reported (Nair and Chung, 2014). The effect of NPs on plants depends of NP. For example soil contains a wide variety of solutes and colloidal

⁎
Corresponding author.
E-mail address: [email protected] (A. Fotovat).

http://dx.doi.org/10.1016/j.ecoenv.2017.06.074
Received 3 January 2017; Received in revised form 26 June 2017; Accepted 30 June 2017
0147-6513/ © 2017 Published by Elsevier Inc.
S. Moghaddasi et al. Ecotoxicology and Environmental Safety 144 (2017) 543–551

materials including dissolved organic carbon, which may coat en- NPs (coated and non-coated) obtained by randomly sampling 50 par-
gineered nanomaterials (NMs) and thus, alter their interactions with ticles in their corresponding TEM images. The sample was prepared by
soil and plant (Lowry et al., 2010). The translocation factor of cerium in placing a droplet of the colloidal suspension (200 mg L−1) on a Cu
the nano-CeO2 exposed plants grown in organic matter enriched soil mesh TEM grid and left to air drying. The zeta potential and hydro-
was twice as the plants grown in low organic matter soil (Majumdar dynamic (agglomerate) particle sizes of NP aqueous suspensions were
et al., 2016). The absence of ZnO NPs in shoot of wheat may be ex- measured by dynamic light scattering (DLS) method using Zetasizer
plained by their attachment to the soil particle (Dimkpa et al., 2013). In Nano ZS system (Malvern Instruments Ltd.).
addition, plant uptake of metals from soil depends on both the soluble
fraction of total metal and the capability of soil solid pools to release the
metals into solution (Backes et al., 1995). Metal availability to plant is, 2.2. Soil preparation and cucumber cultivation
therefore, considerably affected by soil properties particularly the dis-
tribution of metal among different fractions. To better understand the A bulk soil (100 kg) with silty clay loam texture was collected from
metal phytoavailability, knowledge on the metal fractionation in soil surface layer (0–20 cm) of the field of Research Station of Isfahan
solid phase in relation with plant uptake is useful. The method of University of Technology, Isfahan, Iran. Soil samples were air-dried and
Tessier et al. (1979) to estimate the bioavailability of heavy metals in sieved through a 1 mm mesh prior to experimental use. Half of the oven
soil to plants, provides a potential useful approach to evaluate the dried soil was thoroughly mixed with 1% fully matured cow manure.
bioavailability of engineered NPs. This 5-steps sequential examination The rest of the soil was considered as un-amended soil. Cow manure
method separates the metal of interest into five fractions i.e., the ex- was obtained from a local farm with pH 7.3, C:N 3.1, % OC and total N
changeable, water/acid soluble metal (F1), the metal bound to carbo- 6.6 and 2.2 respectively. Before adding to the soil, collected manure
nates (F2), the metal bound to Fe-Mn oxides (F3), the metal bound to was air dried for 3 days and sieved through a 2 mm sieve. The soil
organic matter (F4), and finally the metal bound to silicate minerals and texture was determined using the wet sieve analysis and hydrometer
the residual fraction (F5). test (Bouyoucos, 1962). Soil pH was measured potentiometrically in
ZnO NPs are one of the most widely used NPs. These NPs are used in 1 M KCl after 24 h in the liquid/soil ratio of 2.5. The total nitrogen (Nt)
personal care products e.g., sunscreens as well as in coatings and paints was determined by the Kjeldahls’ method. The concentration of K, Mg,
due to their ultra violet (UV) absorption efficiency and transparency Ca, and Na were determined according to procedures by Van Reeuwijk,
(Lin and Xing, 2007). On the other hand, the potential adverse effects of (2002). Total organic carbon (TOC) was determined by TOC-VCSH
NPs on ecosystems as well as human health have increased. (SHIMADZU) with Solid Sample Module SSM-5000. Selected char-
Moghaddasi et al. (2013) showed the positive effects of NPs having ZnO acteristics of both untreated and CM treated soils are described in
at low concentration (1–25 mg kg−1) on cucumber was grown in hy- Table 1.
droponic and negative effects of them at higher concentration Triplicate subsamples (20, 200 and 2000 g) of coated and uncoated
(250 mg kg−1). According to previous research and more effects of NPs and bulk ZnO were sonicated for 30 min (25 °C, 100 W, 40 kHz)
elements on hydroponic systems the concentration of Zn in this study and applied through spraying (100 ml) to the soils to achieve final
were chosen. Cucumber plant was selected for this experiment due to concentrations of 10, 100, 1000 mg ZnO per kg soil. The control
high growth speed and good response to Zn concentration. treatment was pots without ZnO application.
The aim of the present study was to evaluate (1) uptake and toxicity Seeds of Cucumis sativus L. cv. Dominus were placed in rows on wet
of coated and uncoated ZnO NPs in comparison with bulk ZnO on plant blotting paper to germinate for three days. Uniform seedlings were
growth, (2) the fractionation and bioavailability of ZnO NPs in com- transplanted into plastic pots filled with 2 kg soil and continued their
parison with bulk counterpart (3) the impact of organic matter on growth for 30 days under greenhouse conditions. The daily photoperiod
translocation of Zn supplied in the form of ZnO NPs and their coun- in the greenhouse was 12 h, with average daily maximum and
terpart. minimum temperature of 30 °C and 18 °C, respectively. During culti-
vation, soil moisture was kept near field capacity by weighting the pots.
2. Materials and methods At harvest (30 days after seeding), shoot and roots were separated,
washed with deionized water, dried at 70 °C for 48 h and weighed.
2.1. ZnO and its Nanoparticles Plant materials digested by 2 M HCl and Zn concentration in the ex-
tracts, were measured by atomic absorption spectrophotometer (AAS)
Bulk ZnO powder (99.9%, nominal diameter < 1 µm) was pur- (Perkin Elmer, Model 3030).
chased from Sigma –Aldrich. ZnO NPs were synthesized according to
the method presented by Hosseini et al. (2011). According to the at-
mospheric pressure solution evaporation method, 5 g of zinc acetate
[Zn (CH3COO)2·2H2O] with 99.5% purity, was heated to 80 °C tem-
Table 1
perature. After temperature stabilization of the precursor solution,
Physiochemical properties of the study soils. n/a: not available. All values are mean ± SD
450 ml stoichiometric ethylene glycol solution (90% purity), was added (n = 3). All measurement had tree replicate.
to the suspension on a drop-by-drop basis. The stirring and heating of
the reaction mixture was continued for 3–4 h. The suspension was dried Parameters CM-treated soil Untreated soil Unit
in an oven at 100 °C for 12 h, and then crystallized at 600 °C for 1 h.
Coordinates 32°29′N, 52°10'E 32°29′N, 52°10'E –
For coating NPs, humic acid (HA) solution (500 mg L−1) was ob- pH(CaCl2) 5.6 ± 0.3 6.8 ± 0.2 –
tained by dissolving solid HA (HA technical, purchased SIGMA- Carbonate concentration 0.5 ± 0.06 0.4 ± 0.04 g/100 g dw
ALORICH) in 0.1 M NaOH. The solution pH was adjusted to 5.0 with Total N 0.5 ± 0.02 n/a g/100 g dw
0.1 M HCl. Nano-ZnO particles (5 g) were mixed with1L HA solution in Chloride concentration 15 ± 0.6 5.5 ± 0.04 mg/kg dw
Sulfate concentration 8 ± 0.7 n/a mg/kg g dw
a bottle and shaken for 2 days, after which the suspension was cen- Sulfur concentration 530 ± 28 75 ± 9 mg/kg dw
trifuged at 3500 rpm for 30 min. Wet material due to precipitation was Total organic carbon 6.5 ± 0.9 0.9 ± 0.01 g/100 g dw
rinsed with deionized water, freeze-dried (Model FD-4, Pishtaz en- Water holding capacity 35 ± 3 31 ± 2 %
gineering co., Iran), and ground gently (Yang et al., 2009). Character- Calcium concentration 21.6 ± 19 3.2 ± 0.7 Cmol(+)/kg
Magnesium concentration 4.2 ± 0.05 1.3 ± 0.01 Cmol(+)/kg
ization of the NPs has been presented in a study by Moghaddasi et al.
Potassium concentration 0.9 ± 0.02 0.1 ± 0.02 Cmol(+)/kg
(2017). Transmission electron microscopy (TEM) (Model EM10C-80KV, Sodium concentration 0.1 ± 0.01 < 0.1 ± 0.01 Cmol(+)/kg
Germany) was used to examine the primary particle size distributions of

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Fig. 1. Root (A, B, C) and shoot (D, E, F) dry biomass percentage of cucumber plants grown in CM-treated or untreated soil treated with 10, 100, 1000 mg of Zn per kilogram soil. The
error bars present standard error (n = 3). Different letters in root or shoot are separated, in lower case and upper case represent significant differences between the treatments in reached
and unreached soil respectively (< 0.05).

2.3. Microscopic observations procedure (Ni et al., 2005). The cucumber roots were prefixed in 2–4%
glutaraldehyde, washed in 0.1 mol/L pH 7.0 phosphate buffer, profixed
The effect of ZnO NPs on root tips of cucumber was observed by in 1% osmium tetrooxide, and dehydrated in acetone. Cross sections
light microscopy (LM) (Model E100, German). At harvest, the first 4 cm (65 nm thick) of the root tips were cut for TEM (Model EM10C-80KV,
plant rootlets were longitudinally sliced (500 nm thick), washed with Germany) using a microtome with a diamond knife.
distilled water and prefixed with alcohol (70%) and glycerin. Sliced
root samples were washed in sodium hypochlorite (20%) and acetic
acid and embedded in carmine for 15 min. The samples were then 2.4. Zn fractionation in soil
washed with deionized water and embedded in green methyl for 30 s.
Root and stem samples of cucumber plants from all treatments were After plant harvesting, rhizosphere soil samples were collected from
washed by running tap water and rinsed with deionized water before each pot, air dried and submitted to sequential extraction. The dis-
scanning Electron Microscopy (SEM) observations. Small cross section tribution of Zn in different soil solid fractions was determined according
roots and stems were dehydrated in an acetone series. SEM photographs to the method described by Tessier et al. (1979) with minor modifica-
were carried for the samples using Philips XL30 Robeahi attached with tions. For this purpose, 1 g of soil was used for initial extraction and
energy dispersive X-ray unit, with an accelerating voltage of 20 kV. after that, in each step the residue sample of last step was used for next
Root samples were also prepared for TEM according to a standard extraction. The extracted solutions were stored in 50 ml centrifuge
tubes and supernatants were separated from the solid fraction by

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Fig. 2. Root (A, B, C) and shoot (D, E, F) Zn uptake of cucumber plants grown in CM-treated or untreated soil treated with 10, 100, 1000 mg of Zn per kilogram soil. The error bars present
standard error (n = 3). Different letters in root or shoot are separated in lower case and upper case represent significant differences between the treatments in reached and unreached soil
respectively (< 0.05).

centrifugation (15 min at 10,000 g). Water and exchangeable soluble fraction were digested with a mixture of 5 ml concentrated HNO3
were extracted at room temperature for 1 h with 10 ml of distilled (HNO3, 70% w/w), 10 ml of hydrofluoric acid (HF, 40% (w/w)) and
water and magnesium chloride solution (1.0 M) respectively. The re- 10 ml of perchloric acid (HCLO4, 60% w/w). The overall recovery of
sidue solution were extracted at room temperature for 1 h with 10 ml of zinc at the end of the multi-step procedure was about 90%, and losses
sodium acetat (1.0 M) at pH 5 and named carbonate bound fraction. For were probably due to adsorption to centrifuge tubes, pipettes and
extracting Fe/Mn bound, the residual solution were extracted by 10 ml funnels. Concentration of Zn in the extracted samples was measured by
0.4 M NH2OH.HCL in 25% (v/v) acetic acid (pH 2.0) with agitation at AAS (Perkin Elmer, Model 3030).
96 °C in water bath for 5 h. For extracting organic bound, the residual
solution were extracted by 7.5 ml of 30% (v/v) hydrogen peroxide with 2.5. Statistical analysis
pH 2 that was heated 85 °C for 5 h. Then 2.5 ml of 3.2 M ammonium
acetate in 20% (v/v) nitric acid was added, shaken for 0.5 h and cen- The pot experiment was set up in a completely randomized design
trifuged. For extracting residual bound fraction, residue from last with three treatments in triplicates. Treatments effects were analyzed

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Fig. 3. Relative distribution of ZnO in sequential extraction fractions

CM-treated and untreated soils were spiked with either coated, un-
coated NPs and their bulk counterparts. The error bars present standard
error (n = 3). Identical letters indicate treatments that do not differ
significantly (P > 0.05).

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by analysis of variance using the GLM procedure. Means were com- In general, plants at the CM-treated soil had higher Zn concentration
pared using least significant differences (LSD) at P < 0.05 (25). in their tissues compared with those at the untreated soil (except at
10 mg kg−1 Zn level in the form of coated ZnO NPs). The difference in
3. Results shoot Zn concentration between CM-treated and untreated soil for NPs
treatments was more than bulk particles treatment. This different per-
3.1. Size and ζ-potential of ZnO NPs centage at 100 mg kg−1 of bulk particles was 20.6% and for coated and
uncoated NPs were 37% and 32%, respectively. In CM-treated soil, at
The size of ZnO NPs estimated by TEM was 75 nm. The size of ZnO most of Zn levels, the increase in root Zn was greater from the NPs than
NPs increased from 75 nm to 100 nm after coating with HA while hy- bulk counterpart. The total shoot Zn uptake treated with NPs was
drodynamic diameter of NPs was decreased by coating from 441 to higher than bulk particles at 10 and 100 mg kg−1 unreached soil and
328 nm. The ζ-potential of non-coated ZnO NPs (20 mg/L ZnO NPs) was 100 and 1000 mg kg−1 reached soil. In soil untreated with CM at
+3.1 while it was −15.4 for ZnO NPs coated with HA. 1000 mg kg−1, plants grown at the bulk particles treatment had higher
Zn concentration in root and shoot compared with those at the NPs
3.2. Root and shoot dry mass treatments. As shown in Fig. 2, no significant difference in reached soil
was found between the coated and uncoated NPs treatments in root and
The results revealed an inverse U-shape response of root and shoot shoot Zn concentration.
to Zn concentrations (Fig. 1). Both 0 mg kg−1 and high (1000 mg kg−1)
Zn concentrations resulted in inhibition of plant growth due to Zn de- 3.4. Zn fractionation in soil
ficiency and toxicity, respectively. Increasing Zn concentration in soil,
up to 10 or 100 mg kg−1, increased root and shoot dry mass of cu- The actual concentrations of Zn in each individual fraction of soil
cumber, however, application of higher Zn levels had no significant are illustrated in Fig. 3. Generally, the distribution of ZnO among these
effect or even decreased plant growth. five fractions changed based on applied Zn concentration, Zn source
At high concentration of Zn (1000 mg kg−1) in the soil untreated and soil organic matter content. Regardless of applied Zn source, up to
with CM, phytotoxicity of Zn from NPs (coated and uncoated) was concentration of 100 mg Zn kg−1, the residual fraction was the domi-
higher as compared with bulk counterpart. Application of nant fraction of Zn in the solid phase. While at 1000 mg kg−1, Zn bound
1000 mg kg−1 coated and uncoated ZnO NPs in the soil, untreated with to Fe and Mn oxides was the dominant fraction. Approximately 5% of
CM, resulted in more than % reduction of shoot growth compared with added Zn remained bound with soil carbonate but this value increased
control. The shoot growth reduction in comparison with the maximum up to 70% by application of 1000 mg kg−1 Zn compared with the
shoot biomass which was obtained at the 10 or 100 mg kg−1 ZnO, was 100 mg kg−1 Zn treatment. Unlike what was seen for F1 and F4, the
110% and 230%, respectively (Fig. 1). In contrast to NPs, bulk ZnO at value of Zn bound with Fe and Mn, increased extremely by increasing
the Zn concentration of 1000 mg kg−1, resulted in 21% higher dry ZnO concentration (1000 mg kg−1) in soil.
biomass compared with control. In the soil untreated with CM, no significant difference was found
In the soil untreated with CM, the maximum root growth at the between the coated and uncoated NPs and bulk particle treatments in
coated NPs treatments was more than the uncoated NPs and bulk par- the exchangeable Zn fraction. At the CM-treated soil, distribution of Zn
ticle treatments. However in CM-treated soil, no significant difference in the exchangeable fraction was greater at 10 and 100 mg kg−1 Zn
was found between the coated and uncoated NPs treatment for root dry concentrations in the form of NPs (coated and uncoated) compared
biomass. In both CM-treated and untreated soils, also no significant with that in the form of bulk ZnO. At 1000 mg kg−1 Zn, exchangeable
difference in root dry biomass was found between the uncoated NPs and Zn fraction was greater at the coated NPs treatment than that by un-
their bulk counterparts except at 1000 mg kg−1 treatment. At high Zn coated NPs treatment.
rate (1000 mg kg−1), the values of root growth in CM-treated soil were In both CM-treated and untreated soils, the source of ZnO (coated
significantly more from the source of ZnO NPs compared to bulk and uncoated NPs and bulk particles) had no significant effect on dis-
counterparts. In contrast, in untreated soil, root growth reduction from tribution of Zn in the carbonate-bound fraction.
NPs was more than their bulk counterparts. In CM-treated soil, no significant difference was found between NPs
Regardless of soil organic matter content, no significant difference and their bulk counterparts (except at 1000 mg kg−1 Zn level) in the
was found between the coated and uncoated NP treatments for the organically bound fraction of Zn. At the soil untreated with CM and Zn
maximum and minimum shoot biomass, although shoot growth in- rate of 100 mg kg−1, the concentration of organic matter bound Zn in
crease at low Zn concentration was greater from NPs (coated and un- coated NPs was more than uncoated NPs and bulk particles. At the
coated) than from the bulk ZnO. The stimulating effect of NPs on shoot untreated soil with CM, the fraction extracted with hydroxylamine
growth at < 100 mg kg−1 was more than bulk ZnO (32% in untreated (easily reducible material, e.g. Mn/Fe oxides) was larger when
soil with CM and 25% in treated soil with CM). The higher con- 100 mg kg−1Zn was applied in the form of NPs (coated and uncoated)
centration of ZnO NPs (coated and uncoated) (1000 mg kg−1) de- than when the same amount of Zn was applied in the form of bulk ZnO
creased shoot dry biomass (about 52%) when the soil was untreated particles. In general, the CM-treated soil contained higher Zn in the
with CM, however increased it (about 35%) for the CM-treated soil, oxidizable fraction (F3) than untreated soil.
compared to their bulk counterparts. At CM-treated soil, no significant difference in the residual fraction
of Zn was found between NPs and their bulk counterparts while at the
3.3. Zn uptake by root and shoot soil untreated with CM, at all Zn levels (except 100 mg kg−1), the re-
sidual fraction of Zn for the coated NPs treatment was greater than that
The effect of ZnO on Zn uptake by cucumber varied upon the soil of uncoated and bulk particles treatments. The highest distribution of
organic matter content and the source of Zn (Fig. 2). In the soil un- Zn in the residual fraction was observed for uncoated NPs treatment at
treated with CM, irrespective of Zn source, by increasing Zn levels to the 100 and 1000 mg kg−1 Zn concentrations,.
100 mg kg−1, its concentration in root and shoot increased. By in-
creasing Zn level to 1000 mg kg−1, based on Zn source, no change (by 3.5. Microscopy observations
the bulk ZnO) or significant decrease (by the coated and uncoated NPs)
in tissue Zn concentration occurred. In the CM-treated soil, addition of The TEM image (Fig. 4) showed the aggregation of NPs inside the
≥10 mg kg−1 Zn in the form of ZnO NPs resulted in higher root and plant cytoplasm and their accumulation adjacent to the cell membrane.
shoot Zn concentration (Fig. 2). The presence of nano-particles in cucumber stem indicated the possible

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Fig. 4. TEM image of root (NPs of ZnO treatment (A) bulk ZnO (B) and, control (C)) and shoot cells (NPs of ZnO treatment (a), bulk (b) and control (c)) indicates the penetration of ZnO
NPs into the root and shoot cells and precipitation on cell wall.

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transport of NPs from the root to the shoot (Fig. 4); although a small functional groups and they can form complexes with NPs, which may
part of ZnO NPs seems to be transported from root-to-shoot. These dark also contribute to reduction of their bioavailability (Lee et al., 2011).
NPs were observed in all tree replicates of nano treatments but not seen Jos´ko and Oleszczuk (2013) showed that ZnO did not cause any in-
under either the control and bulk ZnO treatments. hibition of root growth of Lolium sativum in soil with higher con-
centration of total organic carbon. In the present study, higher Zn up-
4. Discussion take by plants at the CM-treated soil in comparison with those at the
untreated soil indicated the role of organic matter in increasing Zn
Decreasing hydrodynamic size of nanoparticle after coating with availability for plants. Therefore, other mechanisms resulted in lower
humic acid (HA) can be explained by more negative charge of coated phytotoxicity of HM-coated ZnO NPs than uncoated particles. Trapping
NPs. Adsorption of humic acid on the surface of ZnO nanoparticles ZnO NPs by HA and enhancing accumulation of Zn in root apoplastic
leads to increased electrostatic repulsion. The decrease in the ζ-poten- spaces or soil particles surfaces can explain lower phytotoxicity of
tial of NPs after coating with HA could be attributed to the negative coated NPs than uncoated NPs. Navarro et al. (2012) reported that
charges of adsorbed HA on ZnO surfaces. Ligand exchange of hydroxyl humic acid enhanced accumulation of synthesized NPs onto the root
groups on surface of ZnO NPs with HA, which may provide less hy- surface of Arabidopsis thaliana however the mechanism is not clear yet.
droxyl groups for protonation, could also be partly responsible for de- On the other hand, coating with HA increased the zeta potential (in-
crease in ζ-potential (Tombacz et al., 1999). In addition, organic anions creasing negative charge), dispersion, ionization and uptake bioavail-
could increase the negative charge density adjacent to mineral particle ability of ZnO. Zeta potential is a factor determining the fate of NPs
surface and cause a shift in position of the shear plane further away because it significantly affects their tendency to agglomeration.
from the surface, which could lead to decrees in ζ-potential of nano- Therefore, HA and other organic matters as dispersion agents have
oxide (Yang et al., 2009). However, our data showed that the zeta more effects on NPs compared to their bulk counterparts. This sug-
potential still fell within the range of flocculation (− 30 to + 30), gested that dispersion of NPs not only increases ionization of these
therefore the higher negative change alone might not be able to explain particles but also affects their penetration into the plant root cells. It
less aggregation of coated ZnO NPs; other factors such as enfluence of can also explain higher root and shoot growth and Zn uptake by cu-
steric forces due to the coating might be involved. For example, Leong cumber under the coated NPs treatments than the uncoated ones
(1997) proposed a quantitative particle-pair intractions model in- In the CM-treated soil, the presence of relatively high organic matter
corporating steric and hydrophobic intractions for the behavior of ZrO2 content decreased specific effects of nanoparticles on plant and higher
suspensions containing organic acids. dry biomass was produced by plants supplied with NPs ZnO than those
Although previous studies provided significant valuable information supplied with bulk ZnO. In contrast, in the soil untreated with CM,
on the fate and effects of NPs (Lin and Xing, 2007) in hydroponic sys- specific effects of coated and uncoated NPs at the 1000 mg kg−1 Zn
tems, there is still little information on the fate and bioavailability of level resulted in lower dry biomass production at the ZnO NPs com-
these particles to terrestrial plants in soil. In addition to the type and pared to the bulk ZnO. In both CM-treated and untreated soils at low
concentration of NPs, various soil characteristics (e.g. soil pH, organic concentration of Zn (≤ 100 mg kg−1), ZnO NPs produced higher shoot
matter content and EC) affect availability of the NPs in soil. In this dry biomass of cucumber than bulk particles. Greater plant shoot dry
study, the availability ZnO NPs in two soils with different organic biomass and Zn uptake at the CM-treated soil compared to the un-
matter content (one treated with CM and the other as untreated) was treated soil can be due to nutrient balance, easier Zn transport, uptake
compared with their bulk particles. Results showed that coated and and improved soil properties in presence of organic matter. Lower Zn
uncoated ZnO NPs can be more bio-accessible than their bulk coun- uptake from the ZnO NPs compared to the ZnO bulk particles can be
terpart and despite their more positive effects in low concentration due to root shrank induced by NPs. It seems that root destruction at
(< 100 mg kg−1), they were more phytotoxic for plants compared to 1000 mg kg−1 NPs was more than bulk counterparts and it is in line
the bulk ZnO particles at high concentration (1000 mg kg−1) in the soil with obtained dry biomass. At this Zn concentration, root and shoot dry
untreated with CM. Similar to this result, Stampoulis et al. (2009) found biomass were greatly lower at the NPs treatments compared to the bulk
that ZnO NPs were more toxic than their bulk particles and greater ion treatment. Moghaddasi et al. (2013) showed that in hydroponic culture,
dissolution from the NPs only partly explained the toxicity. The root tip shoot Zn concentration of cucumber plant treated with synthesized NPs
deformation under high concentration of NPs (1000 mg kg−1 treat- at 125 mg kg−1 was lower than 25 mg kg−1of Zn concentration due to
ment) was observed by LM in plants at the soil untreated with CM. It root deformation at high Zn level.
seems that root tip deformation is one of the specific effects of NPs that Greater shoot Zn uptake from NPs than their bulk counterparts can
inhibits plant growth and nutrients uptake. At the highest Zn con- be due to higher solubility of Zn in soil or more penetration of NPs into
centration (1000 mg kg−1), ZnO NPs, despite the higher phytotoxicity, root cells. The TEM images of cucumber roots indicated the presence of
resulted the same or even lower Zn accumulation in plant tissues ZnO NPs inside the root endodermal cells (Fig. 4). Current studies have
compared with the ZnO bulk particles. This indicates that plants growth shown that nanoparticles can be taken up by plant roots as intact
inhibition by NPs at high Zn concentration (1000 mg kg−1) was not particles, with potential for release in planta (Monreal et al., 2015, Lin
only associated with Zn2+ toxicity but also due to specific effects of NPs and zing, 2008, Ghafariyan et al., 2013; Moghaddasi et al., 2013).
on plant. Pokhrel and Dubey (2013) showed that exposure to ZnO NPs However the NPs penetration and transport mechanisms are not quite
at concentration of 1000 mg kg−1, caused 'tunneling-like effect' in the clear and further evidences are required to clarify this aspect. Appar-
primary root tip of maize. They suggested that the tender cells of epical ently, both NPs and their aggregates were taken up into plant root and
meristem upon contact with ZnO NPs suspension led to cell dissolution shoot (Fig. 4). Nanoparticles can be potentially taken up by plant root
from cellular structural disintegration. and transport to shoots through vascular systems depending upon the
Higher organic matter content in the CM-treated soil resulted in composition, dose, shape, and size of nanoparticles and plant species
smaller plant growth retardation in the presence of ZnO NPs at the (Ma et al., 2010).
1000 mg kg−1 Zn level. This is possibly at least in part due to decrease According to this study results, coating with HA, size, and con-
of NPs specific effects on plant. It has been reported that the phyto- centration of NPs and soil characteristics were significant factors af-
toxicity of NPs even at high applied doses in soil could be negated, fecting the fractionation of ZnO NPs in soil. Low solubility of bulk ZnO
dependent on soil properties (Watson et al., 2015). Lee et al. (2011) and strong adsorption of ZnO NPs on the surface of soil particles are
showed that covering the surface of NPs by organic matter prevents the possible reasons for small distribution of Zn in exchangeable fraction
homo-aggregation (repulsive forces) of NPs, but it also reduces their (F1). Different hydrodynamic size of ZnO particles at different applied
availability to living organisms. Organic compounds are rich in various concentrations may also affect distribution of Zn among various

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S. Moghaddasi et al. Ecotoxicology and Environmental Safety 144 (2017) 543–551

fractions in the solid phase (Zhang et al., 2015). According to our re- of natural organic matter. J. Nanopart. Res. 13, 3051–3061.
Lin, D.H., Xing, B.S., 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ.
sults, differences in soil Zn fractionation between NPs and bulk ZnO Sci. Technol. 42, 5580–5585.
sources at the applied level of 1000 mg kg−1 was more than other Lin, D., Tian, X., Wu, F., Xing, B., 2010. Fate and transport of engineered nanomaterials in
contractions. In most soils, the surface charges of quartz and feldspars, the environment. J. Environ. Qual. 39, 1896–1908.
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which are the main components of sand and silt, are negative (Yin and tion and root elongation. Environ. Pollut. 150, 243–250.
Drelich, 2008). Fe-Mn oxides, considered as secondary minerals and Lowry, G.V., Hotze, E.M., Bernhardt, E.S., Dionysiou, D.D., Pedersen, J.A., Wiesner, M.R.,
exist primarily in clay (Fields and Swindale, 1954). So it suggests that Xing, B., 2010. Environmental occurrences, behavior, fate, and ecological effects of
nanomaterials: an introduction to the special series. J. Environ. Qual. 39, 1867–1874.
different zeta potential of particles caused different electrostatic ad- Ma, X., Geisler-Lee, J., Deng, Y., Kolmakov, A., 2010. Interactions between engineered
sorption on soil different components. Higher percentage of coated NPs nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci. Total
in Fe-Mn oxides compared to uncoated NPs and their bulk counterparts Environ. 408, 3053–3061.
Majumdar, S., Peralta-Videa, J.R., Trujillo-Reyes, J., Sun, Y., Barrios, A.C., Niu, G., Flores-
at high concentration of Zn in untreated soil can be due to higher ne-
Margez, J.P., Gardea-Torresdey, J.L., 2016. Soil organic matter influences cerium
gative charges of these particles. Lin et al. (2010) showed that natural translocation and physiological processes in kidney bean plants exposed to cerium
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charge and steric stabilization. Zhang et al. (2015) showed that the Monreal, C.M., Derosa, M., Mallubhotla, S.C., Bindeaban, P.S., Dimkpa, C., 2015.
Nanotechnologies for increasing the crop use efficiency of fertilizer-micronutrients.
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loamy sand was consistent with organic matter. Soil organic matter is Moghaddasi, S., Fotovat, A., Karimzadeh, F., Khazaei, H.R., Khorassani, R., Lakzian, A.,
important factor affecting the soil fractionation and subsequently 2017. Effects of coated and non-coated ZnO nano particles on cucumber seedlings
grown in gel chamber. Arch. Agron. Soil Sci. 63, 1108–1120.
bioavailability of ZnO NPs to plants. Increasing organic fraction espe- Moghaddasi, S., Khoshgoftarmanesh, A.H., Karimzadeh, F., Chaney, R.L., 2015. Fate and
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