Zinc Oxide Nanoparticles Induced

Oxidative Stress and Changes in the

Photosynthetic Apparatus in Fenugreek
(Trigonella foenum graecum L.)

Hajer Chemingui, Moez Smiri, Takwa

Missaoui & Amor Hafiane

Bulletin of Environmental
Contamination and Toxicology

ISSN 0007-4861

Bull Environ Contam Toxicol

DOI 10.1007/s00128-019-02590-5

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https://doi.org/10.1007/s00128-019-02590-5

Zinc Oxide Nanoparticles Induced Oxidative Stress and Changes

in the Photosynthetic Apparatus in Fenugreek (Trigonella foenum
graecum L.)
Hajer Chemingui1,3 · Moez Smiri1,2 · Takwa Missaoui1 · Amor Hafiane1

Received: 4 December 2018 / Accepted: 8 March 2019

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
The aim of this work was to study the toxicity of nanosheet zinc oxide nanoparticle with the size of 45 nm. The penetration
of nanoparticles at an exposure by a localized spray does not make it possible to understand the mechanism of transport
and bioavailability of the nanoparticles. In contrast, nanoparticles penetrated, via the roots, as a function of their diameter,
the smaller ones having caused leaf stress (by translocation) at low concentrations. So that the choice of method of root
application. Plants treated with 50 mg L−1 of ZnO-NPs presented disturbance in leaf due to changes in chlorophyll’s bio-
synthesis. The highest value of the photosynthetic pigments was recorded at 5 mg L−1 of ZnO-NPs. However, the treatment
with 50 mg L−1 of ZnO-NPs caused a decrease in the levels of chlorophyll a and b. Moreover, ZnO-NPs leaves significantly
enhanced antioxidant enzymes activities.

Keyword  Co-precipitation · Zinc oxide nanoparticles · Photosynthetic · Oxidative stress · Trigonella foenum graecum L

Nanoparticles (NPs) have received great attention due to associated with agro-chemicals production and distribution
their unique properties and beneficial applications in agricul- (Koller 2004). However, minimizing the environmental risk
ture and related sectors. Zinc oxide (ZnO) is one of the most often involves yield losses that discourage the farmer. Mod-
important nanoparticles because of its interesting and unique ern agriculture is ,therefore, looking for new technologies
characteristics, including wide band gap (3.37 eV), nontox- or biotechnologies that would reduce the use of chemical
icity, and high photosensitivity, piezoelectric and pyroelec- without affecting crop yields or farmers’ incomes (Koller
tric properties (Wang et al. 2009). Furthermore, owing to its 2004). Plant nanotoxicology has been introduced as a disci-
interesting physical and chemical properties, ZnONPs has pline that studies the effects and mechanisms of nanoparti-
been used in many areas such as catalysis (Akir et al. 2016), cle toxicity in plants, such as transport, surface interactions,
solar cells (Suliman et al. 2007), sunscreens (Lewicka et al. and plant-specific responses to the nature of nanoparticles
2013), and biomedical application (Zhang et al. 2013). (Dietz and Herth 2011). Among the mechanisms behind
The decrease in fertilizer and biocide applications on the phytotoxicity of nanoparticles, oxidative stress is often
crops would limit the risk of soil, groundwater, and water mentioned. However, the induction of oxidative stress in
pollution, and reduce the energy and environmental costs plants exposed to nanoparticles is a field of research that
remains little exploited (Dietz and Herth 2011). Like many
* Moez Smiri abiotic and biotic stressors, one of the most common conse-
[email protected] quences following nanoparticulate exposure appears to be
the development of oxidative stress by a production of reac-
1
Laboratory of Water, Membranes and Environment tive oxygen species and peroxidation processes (Oberdörster
Biotechnology (LEMBE), Technopole of Borj Cedria
(CERTE), 2050 Hammam‑Lif, Tunisia et al. 2007). Oxidative stress in response to nanoparticles
2 has been observed in different animal tissues but remains
Department of Biology, University of Carthage, Higher
Institute of Environmental Science and Technology of Borj largely unexplored in plants (Dietz and Herth 2011). Con-
Cedria, B.P. no. 1003, 2050 Hammam‑Lif, Tunisia versely, Rico et al. (2013) noted that the amounts of ROS
3
University of Tunis El Manar, Campus Universitaire El produced in rice samples exposed to 62.5 and 125 mg L−1
Manar II ‑ B.P. 94 Poste, 1068 Romena, Tunisia of ­CeO2NPs are reduced, compared to unexposed plants, in

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all leaves and roots. However, further studies are needed to and this analysis demonstrated an average particle size of
clarify this phenomenon (Rico et al. 2013). For example, 47 ± 2.1 nm in the 50 µg/L stock ZnO-NPs suspension. The
Sageer et al. (2009) studied the effects of ­TiO2NPs on wil- powder obtained was placed into a crucible and calcinated at
low cuttings and found limited effects on growth in terms of 300 °C in a muffle furnace without any atmospheric condi-
water use and transpiration. A study on the effects of ZnO tion. X-ray powder diffraction data (XRD) were obtained on
nanoparticles on ryegrass (Lolium perenne) indicated that aX‘Pert Pro Panalytical diffractometer with CuKα radiation
the phytotoxicity cannot be explained by the dissolution of (λ = 0.154 nm). The XRD measurements were carried out by
the ZnO nanoparticles (ZnO-NPs) from the raw material applying a step scanning method (range from 10 to 85), the
(Lin and Xing 2008). Similarly, the phytotoxicity of ZnO- scanning rate is 0.02 s−1 and the step time is 1 s (Fig. 1a).
NPs in Arabidopsis was much stronger than that of solutions The obtained powder of ZnO-NPs was placed in a smooth
containing the same concentration of soluble Zn, highlight- agate mortar and mixed thoroughly with 2.5  mg of dry
ing the most important effects due to the nanoparticle form potassium bromide (KBr) using a pestle. Infrared spectrum
(Lee et al. 2010). In this context, our work aims to study has been recorded at room temperature (26 °C ± 1 °C) by
ZnO nanoparticle risk on fenugreek growth for a period of a Perkin-Elmer (FTIR 2000) spectrometer in the region of
18 days. We hypothesize that ZnO nanosheets will regulate 4000–400 cm−1 (Fig. 1b).
seedling growth. The morphology was examined by drying 6 µL of the
ZnO stock suspension on a glass slide and examining the
suspension using scanning electron microscopy (SEM)
Materials and Methods (Philips XL30 SFEG, Fig. 2a). A fine powder of ZnO-NPs
environ 1 mg was mixed with 2 mL of Ethanol. The size
Zinc acetate dehydrate (Zn ­(CH 3COO) 2·2H 2O, 99%), distribution was characterized using transmission electron
Ammonium ­(NH4OH) were all purchased from Sigma- microscopy (TEM) measurement in a TECHNAI 20-Philips
Aldrich, United States). Zinc acetate dehydrates and N
­ H4OH instrument (Fig. 2b).
were used as precursor and reducing agent respectively to Seeds of fenugreek (Trigonella foenum-graecum L.) were
synthesize ZnO nanoparticles via co-precipitation method. purchased from the Space green company, Tunisia. They
50 mL of freshly prepared aqueous Zn·(CH3COO)2·2H2O were disinfected with 2% of sodium hypochlorite for 10 min
(0.2M) was taken as a precursor and 50 mL of N ­ H4OH (2M) and rinsed thoroughly to remove the disinfectant and soaked
solution was added to it slowly in drops under continuous in distilled water at 4 °C for 24 h to obtain an initial stage
stirring for 3 h. The powder obtained was placed into a cru- (Reid 1971). The same number of seeds (20 seeds per 9 cm
cible and calcinated at 300 °C in a muffle furnace without Petri dishes) was germinated in the dark at 24–26 °C over
any special atmospheric condition. two sheets of filter paper imbibed with 10 mL of distilled
Particle size quantification of ZnO-NPs stock suspen- water for 3 days. Seedlings were then transferred in pots
sion was investigated by dynamic light scattering (DLS) containing Hoagland’s nutrient solution containing or not
using a Malvern (zetasizer nano-ZS) particle analyser, two concentrations of ZnO NPs (5 or 50 mg L−1) (Hoagland

Fig. 1  a Xray Diffraction b Fourier transforms infrared spectrum of ZnO Nanoparticles

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Fig. 2  a Scanning electronic microscopy image b Transmission electronic microscopy image

and Arnon 1950) and exposed for 15 days to ZnO NPs treat- The choice of these concentrations was taken from lit-
ments. Controls are growing plants without ZnONPs and the erature (Siani et al. 2016). The remaining seedlings were
first day of treatment considered as day 0. Growth condi- then divided into leaves and stem individually and kept at
tions were as follows: 25 °C, relative humidity of 70% and a − 80 °C in an ultradeep freezer for measurements of pigment
light: Dark cycles of 16:8 h. Nutrient solutions were changed contents (Cha,Chb and ChT) (mg g− 1 FW), anti-oxidative
every 4 days during the experiment (scheme 1). During the enzyme activities (GPOX and APX), and NADH oxidase
greenhouse stage, an experiment was conducted with five ­ g−1
activities (expressed as units per gram of fresh weight U
replicas, and each pot contained 40 plants (Missaoui et al. FW) using an Ultraviolet–visible spectrophotometer (Lamba
2017, 2018). 2, PerkinElmer).The experimental data were analyzed using

Scheme 1  Germination and greenhouse stage of Fenugreek

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two-way ANOVA and differences were considered signifi-

cant when they reached the 0.05 level of probability.

Results and Discussions

The X-ray diffraction (XRD) spectrum of ZnO is shown in

Fig. 1a. As indicated the XRD patterns of the as-obtained
sample shows that all diffraction peaks can be perfectly
indexed to the hexagonal wurtzite structure of ZnO (JCPDS
Card No.36-1451). The results indicate that the powder con-
sist of pure phase without any impurities detected indicat-
ing that ZnO crystalline phase with high purity could be
obtained using this synthetic process (Chen et al. 2011). The
average crystallite size D, of the ZnO as-synthesized, was
calculated using the Debye–Scherer equation Eq. (1).
D = Kλ∕(β cos θ)
where K is the Scherer constant (K = 0.89), λ is the incident
X-ray wavelength (λ = 0.154056 nm), β is the peak width
at half maximum, and θ is the Bragg diffraction angle. The
average crystallite size has been found to be about 45 nm.
As seen in Fig.  1b the broad peak in the range of
3000–3500 cm−1 is present in both the figures which can be
attributed to the vibrational mode of hydroxyl groups O–H
(variation of water molecules) (Moulahi et al. 2012). The
appearance of a broad band of O–H on annealed material
at 300 °C can be related to the amount of O–H from the
­NH4OH reacts with the Zn(CH3COO)2·2H2O at high tem-
peratures. In fact, many investigations presented the appear-
ance of the peaks of O–H mode of vibration annealed at high
temperatures (Kayani et al. 2015; Hassan et al. 2015).
In fact, the situated band at 539 cm−1 is attributed to the
vibrations of elongation of the vibratory Zn–O vibration of
ZnO (Wu et al. 2005). The Fig. 2a shows the agglomerated Fig. 3  Root length (a), stem length (b) and leaf area (c) of fenugreek
in hydroponic media from control,5 and 50 mgL−1of ZnO NPs treat-
nanoparticles of pure ZnO. It is clear that the morphology ment. The values are the averages of six repetitions (± SE)
of the material synthesized is quite like flowers. From this
figure one can find that the microstructure consisted of many
nanosheets shaped crystalline particles. Figure 2b illustrates effect on stem growth. The treatment with 5 and 50 mg L−1
typical TEM images of nanosheets of ZnO, with average size showed an increase in the length of the stems by about 40%
particle lower than 50 nm which matches with XRD result- compared to the control (Fig. 3b). The results of Fig. 3c
ing. The uniform, regular shape of NPs may directly affect showed that there is significant effect for treatment with
the efficiency of nanoparticles in their applications. Nano- ZnO-NPs (50 mg L−1) on the leaf area compared with the
particles are part of a new generation of solutions for farmers control, and no changes in water content (Fig. 4). Although
to move towards sustainable agriculture that can meet the scientific investigations on intercellular and intracellular
growing food needs of humankind. They help produce more, penetration and the accumulation of nanoparticles in plants
while reducing the use of inputs with limited resources and are still rudimentary, new publications have supplemented
the impact on the environment. Figure 3a showed the varia- this area of research (penetration, behavior, accumulation,
tion in the lengths of the fenugreek roots. ZnO nanoparticles toxicity) (Monica and Cremonini 2009). Several studies have
stimulated root growth. The treatments with ZnO-NPs shown the tissue penetration of different nanoparticles in
(50 mg L−1) had the longest roots that reach 7 cm after higher plants following root or leaf exposure. The penetra-
15 days of exposure. A significant effect on root growth tion and transport of nanoparticles after exposure by a local-
compared to control (about 66%). ZnO-NPs had significant ized spray in the aerial parts of the plant show that only the

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Fig. 4  Water content in stem (a) and leaves (b) of fenugreek plant

after in hydroponic media from control, 5 and 50 mg L−1 of ZnO NPs
treatment. The values are the averages of six repetitions (± SE)

exposed samples containing nanoparticles in the cells of the

epidermis of the petiole. In cells, the nanoparticles appeared
isolated, non-aggregated and no nanoparticles were clearly
detected in the neighboring cells of the epidermis. This does
not make it possible to understand the mechanism of trans-
port and bioavailability of the nanoparticles. In contrast,
nanoparticles penetrated, via the roots, as a function of their Fig. 5  Chlorophyll a (a), b (b) and total chlorophyll contents (c) in
fenugreek plant after 18  days of development in hydroponic media
diameter, the smaller ones having caused leaf stress (by from Control, ZnO (5  mg  L−1, square point) and ZnO (50  mg  L−1,
translocation) at low concentrations (Battke et al. 2008). Em dash) treatment. The values are the averages of 6 repetitions
Dietz and Herth (2011) and Lin and Xing (2008) analyzed (± SE)
the internalization and translocation of ZnO nanoparticles
in Lolium perenne (ryegrass). Adsorbed on the surface of the
roots, they were then observed in the apoplast. Their trans- (NP-Fe3O4) on Cucurbita maxima (pumpkin) grown in liq-
location factor observed from root to shoot was very slow. uid medium, highlighting their adsorption and accumulation
Similarly, Battke et al. (2008) analyzed the penetration of in plant tissues, unlike Phaseolus limensis (lima beans) has
palladium nanoparticles (Pd-NP) by Hordeum vulgare (bar- not been able to absorb and mobilize these nanoparticles.
ley) and their behavior in the nutrient solution used for plant The addition of the nanoparticles improves the photosyn-
growth. Pd-NPs, of different sizes, were tested and the thetic parameters: accumulation of water and chlorophylls
results show that the Pd-NPs penetrated, via the roots, as a and increase of leaf area (Figs. 3, 4, 5). These responses
function of their diameter, the smaller ones having caused varied depending on the dose. We recorded the highest
stress at the leaf level (by translocation) to low concentra- responses for the highest doses of NPs-ZnO. The morpho-
tions. Some authors have focused their research on the logical responses observed in the first part could be due to
behavior of iron nanoparticles (Fe-NP. Similarly, Zhu et al. these variations in photosynthetic activities in the presence
(2008) studied the effects of nanoparticles of magnetite and absence of nanoparticles. We recorded positive dose

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effect in roots and negative in stems. We can suggest that Similarly, by ensuring better absorption of certain nutrients,
there is a specificity of the responses for each organ. These stimulants contribute to the preservation of non-renewable
responses could be due to the anatomical and functional resources. This is the case for example of certain micro-
properties of the various organs tested. The stimulants are organisms solubilizing phosphorus. Stimulatory effects have
defined first by their function, even before the active sub- been shown in some studies to lead to increased tolerance to
stances are specified. Indeed, feeding plants is not just a salt stress (Mazhar et al. 2016), corn (Hazman et al. 2015),
matter of nutrient intake. Considering these global interac- pepper (Çimrin et al. 2010) and pistachio. One of the goals
tions around the plant, we can classify among the stimulants has been to limit impacts by reducing fertilizer consumption
all the products that promote or improve at least one of a and nutrient losses. Today, thanks to the use of stimulants,
functions: (1) The absorption of nutrients, (2) The bioavail- and with less fertilizer, we have also improved the quality of
ability and the efficiency of the assimilation of nutrients, (3) our plants. The effect is very clear on the stimulation of
Abiotic stress tolerance, (4) Crop quality or crop yield, (5) growth, especially at the root level. Many studies on the
Maintenance of growth and production levels, under non- toxicity of nanoparticles in plants have focused on certain
optimal conditions and (6) Stimulation and fortification of parameters such as germination, growth through elongation
plants. Characterized first by their function, stimulants can of different parts of the plant (Khodakovskaya et al. 2009)
be (1) Micro-organisms, such as, mycorrhizal fungi, bacte- and biomass (Zheng et al. 2005; Feng et al. 2013). In the
ria, (2) Natural substances or substances of natural origin roots, the results of Fig. 6a–c show that GPOX activity was
(e.g. humic acids, amino acids, algae extracts, plant extracts), significantly decreased compared with controls for both
and (3) Synthetic substances such as nanoparticles. The doses 5 and 50 mg L−1, but increased in stem and leaves
stimulants can be used alone, by supplying the soil or in the after ZnO-NPs treatment. The addition of ZnO-NPs stimu-
culture medium, by spraying on the plants or by coating lated APX activity in root but not activities from stems and
seeds. Because of the often-small quantities to be added, the leaves organs Fig. 6d–f. The stimulation varied depending
use of stimulants is frequently combined with the addition with ZnO-NPs doses. These results suggest that enzymatic
of other fertilizers: this is already the case of widespread use antioxidant system responses (GPOX and APX) are made
of culture media enriched with mycorrhizae. By promoting by specific isoforms of each compartment. The stimulation
a better use of nutrients by plants, stimulants have a very of the defense system after application of ZnO NPs doses
positive impact on the environment and the preservation of could also explain the improvement of fenugreek plant
resources. They contribute to the reduction of greenhouse growth. Antioxidant system responses are controlled by spe-
gas emissions (via the better use of nitrogen, for example). cific isoforms of each organ. Among the mechanisms behind

Fig. 6  GPOX activities (a; Root, b; Stem and c; Leaves) and APX (5 mg L−1, square point) and ZnO (50 mg L−1, Em dash) treatment.
(d; Root, e; Stem and f; Leaves) activities in fenugreek plant after The values are the averages of six repetitions (± SE)
18  days of development in hydroponic media from Control, ZnO

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the phytotoxicity of nanoparticles, oxidative stress is often (enzymatic and non-enzymatic). Shaw and Hossain (2013)
mentioned (Fig. 7): NADH oxidase activities increased in studied the toxicity of CuO nanoparticles on Oryza sativa
root, stem and leaves exposed for 15 days–5 an 50 mg L−1 cv. Swarna (rice). They have shown that the nanoparticles
of ZnO-NPs (3–6 times in roots; 50%-4 times in the stem of CuO have causedsignificant oxidative stress with H ­ 2O 2
and 20%–40% in the leaves). The stimulation varied depend- production and proline production. Similarly, they found a
ing with ZnO-NPs doses. The high activity of NADH oxi- modulation of enzymes involved in oxidative stress such as
dase explains the installation of the oxidative stress in ZnO- ascorbates peroxidases (APOX), superoxide dismutases
NPs poisoned tissues of fenugreek plants. The induction of (SOD), monodehydroascorbate reductases (MDHAR) and
oxidative stress in plants exposed to nanoparticles is an area glutathione reductases (GR). Finally, levels of malondialde-
of research that remains little exploited (Dietz and Herth hyde have also been induced. Similarly, Faisal et al. (2013)
2011). Other markers of oxidative stress have also been stud- studied the effects of nickel oxide nanoparticles (NiO-NPs)
ied, such as lipid lipoperoxidation and antioxidant systems on oxidative stress. They showed an increase in the activity
of catalases (CAT), SOD and an increase in glutathione
(GSH) and lipoperoxidation membrane which confirms an
induced oxidative stress. Previous studies have focused on
the dissolution of nanoparticles in the exhibition environ-
ment and made possible to underline the importance of this
phenomenon. Two categories can be distinguished: the
nanoparticles soluble in the exposure media (Zn, Cu, Ag, Fe,
etc.), part of whose effects can be attributed to ions and
insoluble nanoparticles ­(CeO2, ­TiO2, carbon nanotubes, etc.)
(Larue 2011). A study on the effects of ZnO nanoparticles
on ryegrass (Lolium perenne) indicated that phytotoxicity
can only be explained by the dissolution of ZnO nanoparti-
cles (ZnO-NPs) from raw material (Lin and Xing 2008).
Similarly, the phytotoxicity of ZnO-NPs in Arabidopsis was
much greater than that of solutions containing the same con-
centration of soluble Zn, showing greater effects due to the
nanoparticle form (Lee et al. 2010). Indeed, studies on the
effects of nanoparticles on plants highlight beneficial effects
and harmful effects (Larue 2011). The different results on
the toxicity of nanoparticles on plants depend on the type of
nanoparticles (Feng et al. 2013) and their properties (Dietz
and Herth 2011), but also plant species (Zhu et al. 2008) and
their stage of development, time, doses and exposure meth-
ods. Research has been conducted to highlight the impor-
tance of considering the methods of preparing exposure
solutions. Finally, in a review on the effects of ZnO nano-
particles, Ma et al. confirm the importance of the various
parameters mentioned above by asserting that the toxic
effects of ZnO nanoparticles in aquatic and terrestrial spe-
cies strongly depend on the exposure conditions and their
characteristics. Studies of the toxic effects of nanoparticles
in plants are beginning to develop. It has been demonstrated
that nanoparticles can be toxic to plant tissues due to chemi-
cal and/or physical effects. Nanoparticles interact with bio-
logical systems through five main modes (Dietz and Herth
2011): (1) The chemical effects of metal ions that can be
dissolved in the solution; (2) mechanical effects due to hard
Fig. 7  NADH oxidase activities (a; Root, b; Stem and c; Leaves) in spheres and defined interfaces; (3) catalytic effects on sur-
fenugreek plant after 18 days of development in hydroponic media
faces; (4) surface effects, due to the binding of proteins to
from Control, ZnO (5  mg  L−1, square point) and ZnO (50  mg  L−1,
Em dash) treatment. The values are the averages of six repetitions the surface, either by non-covalent or covalent mechanisms
(± SE) or oxidative effects and (5) changes in the chemical

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Acknowledgements  Financial support for this work was received from Mazhar R, Ilyas N, Saeed M et al (2016) Biocontrol and salinity tol-
the Tunisian Ministry of Higher Education, Scientific Research, and erance potential of Azospirillum lipoferum and its inoculation
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org/10.17957​/IJAB/15.0115
Missaoui T, Smiri M, Chemingui H, Hafian A (2017) Effects of nano-
Compliance with Ethical Standards  sized titanium dioxide on the photosynthetic metabolism of fenu-
greek (Trigonella foenum-graecum L.). C R Biol 340:1–13
Conflict of interest  The authors declare that they have no competing Missaoui T, Smiri M, Chemingui H et al (2018) Regulation of mito-
interest. chondrial and cytosol antioxidant systems of Fenugreek (Trigo-
nella foenum graecum L.) exposed to nanosized titanium dioxide.
Bull Environ Contam Toxicol 101:326
Monica RC, Cremonini R (2009) Nanoparticles and higher plants.
Caryologia 62(2):161–165
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