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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2017) 41: 575-586© TÜBİTAKdoi:10.3906/biy-1611-27

Comparative phytotoxicity of undoped and Er-doped ZnO nanoparticles onLemna minor L.: changes in plant physiological responses

Samaneh TORBATI1,*, Alireza KHATAEE2,3, Shabnam SAADI2

1Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran2Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry,

Faculty of Chemistry, University of Tabriz, Tabriz, Iran3Department of Materials Science and Nanotechnology Engineering, Near East University, Nicosia, North Cyprus

* Correspondence: samaneh.torbati@yahoo.com

1. IntroductionIn the last decade, widespread use of nanoparticles (NPs) in different fields has inevitably led to the release and entrance of these materials into ecosystems (Mishra et al., 2014; Mousavi Kouhi et al., 2015). Although there is no clear evidence showing damage due to the low discharge levels of nanomaterials, it is well recognized that there is a gap in our understanding of the behavior and fate of nanomaterials in the environment (Djurišić et al., 2015). Therefore, their potential hazards to biological systems and their interactions with organisms should be understood. Consideration of such interactions is necessary for choosing and designing nanomaterials with minimum adverse impacts.

In recent years, the toxicological effects of nanomaterials have been widely studied in different micro- and macroorganisms (Reddy et al., 2007; Baun et al., 2008; Heinlaan et al., 2008; Zhu et al., 2008; Aruoja et al., 2009; Hu et al., 2013). Plants, as an indispensable and essential part of ecosystems, may be subjected to nanomaterial pollutants and therefore may be involved in

their fate and importance in the food chains (Ma et al., 2010; Ghodake et al., 2011). The aquatic environment is the ultimate destination of released NPs, and many studies have focused on the toxicity of NPs in aquatic organisms such as aquatic plants (Kumari et al., 2011; Li et al., 2013; Mousavi Kouhi et al., 2015). Among different plant species, the family Lemnaceae has found broad applications in ecotoxicological studies as model organisms (Radic et al., 2010; Torbati, 2016) and the use of duckweed in nanotoxicology investigations has been reported by some previous studies (Juhel et al., 2011; Hu et al., 2013; Li et al., 2013). Due to the simple structure and morphology, high growth rate, and small size of Lemna species, these plants have many applications in ecotoxicological studies. Their sensitivity to different classes of pollutants and easy cultivation make them suitable for such investigations (Zezulka et al., 2013; Torbati, 2015).

Among many types of NPs, zinc oxide (ZnO) NPs have a wide range of applications, including sunscreens, solar cell coatings, pharmaceutical processes, food additives, and semiconductors (Yoo et al., 2005; Yoon et al., 2014;

Abstract: The present study is the first research on the phytotoxicological effects of Er-doped zinc oxide nanoparticles (NPs) on duckweed (Lemna minor L.), as a model aquatic floating macrophyte. It concentrated on the comparison of the physiological effects of different concentrations of undoped and Er-doped zinc oxide NPs on L. minor. Pure and Er-doped zinc oxide samples were synthesized through a sonochemical method and their morphology and chemical composition were studied by scanning electron microscopy and energy dispersive X-ray analysis. Plant growth, photosynthetic pigment contents, and antioxidant enzyme activities were investigated as indices to assess the effects and toxicity of the NPs on L. minor. Moreover, the dissolution of Zn2+ from the suspensions of NPs was investigated to clearly determine whether the Zn2+ released from the NPs was the main source of their toxicity to the plant. The results showed significant changes in the physiological parameters of the plant in response to the treatments. The negative effects of treatments on the growth of L. minor, in order, were Zn2+ >> ZnO-NPs >> EZO-NPs >> bulk-ZnO. The activity of superoxide dismutase was remarkably increased by increasing the concentration of the contaminants in the plant.

Key words: Duckweed, phytotoxicity, doped nanoparticles, zinc oxide nanoparticles, environmental pollution, risk assessment, plant physiological responses

Received: 10.11.2016 Accepted/Published Online: 21.02.2017 Final Version: 24.08.2017

Research Article

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Chen et al., 2016). In recent years, the ultrasonic method, coupled with semiconductor oxides, has been introduced as a possible and effective method for wastewater treatment and its successful application has been reported for the degradation of organic pollutants (Khataee et al., 2014, 2015b). ZnO-NPs as semiconductors have desirable physical, chemical, and optical properties, such as high UV absorption potential and high exciting binding energy (Zamiri et al., 2014; Khataee et al., 2015b). However, pure ZnO as a nanocatalyst has some drawbacks, such as the fast recombination of the produced electron-hole pairs (Ansari et al., 2012; Khataee et al., 2015b). In this regard, rare earth metals like erbium (Er) have received a great deal of attention and have been introduced as possible doping agents to improve the catalytic activity of ZnO-NPs (Anandan et al., 2007; Khatamian et al., 2012).

Er-doped ZnO (EZO) NPs have been studied for their optical and dielectric properties (Zamiri et al., 2014) and their sonocatalytic activities for degradation of textile dyes (Khataee et al., 2015a). To the best of the authors’ knowledge, this is the first report on their toxicological aspects and their impacts on living organisms, mainly focused on the evaluation of EZO-NP effects on Lemna minor L. in comparison with ZnO-NPs, bulk-ZnO, and Zn2+. Various plant physiological responses, such as growth, photosynthetic pigment contents, and activities of some antioxidant enzymes (peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD)), were investigated. EZO-NPs and pure ZnO-NPs were synthesized by the sonochemical method. Moreover, the influence of Zn2+ dissolution from EZO-NP and ZnO-NP suspensions on NP toxicity was determined.

2. Material and methods 2.1. Methods of zinc oxide nanoparticle synthesis and zinc(II) oxideFor the sonochemical synthesis of undoped and Er-doped ZnO nanoparticles, a prespecified amount of Er(CH3COO)3.H2O was added to an aqueous solution of ZnCl2. The pH value of the solution was kept constant around 10 by adding NaOH (1 M). In the next step, the solution was sonicated by a bath-type sonicator (Sonica

2200 EP; Soltec, Milan, Italy) with a frequency of 50–60 kHz for 3 h. Subsequently, the prepared white solid was washed three times with absolute ethanol (C2H6O) and double-distilled water and dried at 80 °C for 12 h. Er(CH3COO)3.H2O as the precursor of Er+3, ethanol (99%), ZnCl2, and NaOH were purchased from Merck (Darmstadt, Germany) and their chemical characterizations are illustrated in Table 1. Zinc(II) oxide was purchased from Merck and used as ZnO bulk. The microstructure and the chemical compositions of the synthesized samples were studied using a scanning electron microscope (SEM), model Mira3 FEG–SEM (Tescan, Kohoutovice, Czech Republic), with an energy dispersive X-ray (EDX) analyzer attached (acceleration voltage of 10 kV). Microstructure distance measurement software (Microstructure Measurement, Version 1.0) was utilized to determine the size distribution of the synthesized nanoparticles.2.2. Plant cultivation and treatment methodsPlant materials were gathered from Ali Jan near Bostanabad, in the northwest of Iran, and their surfaces, including shoots and roots, were washed carefully using distilled water. Plants were acclimatized for 1 week in Steinberg culture medium (Steinberg, 1946) under laboratory conditions, with a temperature of 25 °C and a photoperiod of 16 h/8 h (light/dark). The initial pH of the culture medium was 6.8–7. The culture medium contained 1.25 mM Ca(NO3)2, 3.46 mM KNO3, 0.66 mM KH2PO4, 0.41 mM MgSO4, 0.072 mM K2HPO4, 1.94 µM H3BO3, 0.63 µM ZnSO4, 2.81 µM FeCl3, 0.18 µM Na2MoO4, 0.91 µM MnCl2, and 4.03 µM EDTA (all from Merck). All chemicals were of analytical grade and applied without extra purification.

Three stock suspensions of 500 mg/L bulk-ZnO, ZnO-NPs, and EZO-NPs were obtained by their addition to the culture medium. The suspensions were treated by sonication (Soniprep 150, MSE, London, UK; 50 Hz, 10-s pulse and 5-s interval) for 10 min and used to prepare the various concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs (0, 1, 10, and 50 mg/L).

Initial experiments evaluating the toxic effects of the three examined contaminant groups on L. minor showed that the ZnO-NP treatment had more negative effects on

Table 1. The characterizations of the chemical constituents that were used for the nanomaterials’ synthesis.

Material CAS number Purity (%) Formula weight (g/mol)

Er(CH3COO)3.H2O 207234-04-6 99.9 344.39C2H6O 64-17-5 ≥99.5 46.07ZnCl2 7646-85-7 98.0 136.30NaOH 1310-73-2 ≥99.0 40

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the plant. Plant growth was reduced up to 50% after the 50 mg/L ZnO NP treatment and 50 mg/L was selected as the highest concentration in the experiments. Therefore, in all of the experiments, the plants (2 g) were transferred to 250-mL beakers containing 200 mL of the culture medium with different concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs (0, 1, 10, and 50 mg/L). The temperature was kept constant in the incubator (Sanyo, Ogawa Seiki Co., Okubu, Japan) during the experiments. A positive control experiment was also done using 3.5 mg/L ZnSO4 (Zn2+) exposure.2.3. Physiological analysis 2.3.1. Growth rateIn order to determine the plant growth rate, relative frond number (RFN) and relative growth rate (RGR) were applied as suitable indicators of potential toxicity. RFN was measured using Eq. (1) (Mitsou et al., 2006):

RFN = [(frond number at day n – frond number at day 0) / frond number at day 0] (1)

Here, n = 0, 4, 8, 12, 16, 20.RGR was quantified according to the increase in the

plant fresh weight (FW) after 20 days of experiment using Eq. (2) (Radic et al., 2010):

RGR (day–1) = [(ln(final weight) – ln(initial weight)]/day (2)2.3.2. Photosynthetic pigments contentFreshly sampled leaves (100 mg) were ground in 100% acetone for the extraction of photosynthetic pigments. Chlorophyll and carotenoid contents were measured spectrometrically at 662, 645, and 470 nm for the maximum absorption of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids, respectively. Pigment content was measured according to the equations described by Lichtenthaler (1987).2.3.3. Enzyme activity assayThe plants were treated with different concentrations of bulk-ZnO, ZnO-NP, and EZO-NP suspensions (0, 1, 10, and 50 mg/L) in the nutrient solution for 7 days to investigate the effect of the materials on antioxidant enzyme activities and compare them with the control. To obtain the crude extract, 0.25 g of fresh plant tissues was homogenized in 3 mL of 0.1 mol/L phosphate buffer solution (pH 7) containing 0.2% polyvinylpyrrolidone. The homogenates were centrifuged at 4000 rpm for 15 min at 4 °C and the resulting supernatants were used to measure antioxidant enzyme activities and protein content.

SOD activity was assayed by measuring the inhibition of the photochemical reduction rate of nitroblue tetrazolium (NBT) by the plant extract (Winterbourn et al., 1976). The reaction buffer contained 2.65 mL of 67 mmol/L potassium phosphate buffer solution (pH 7.8), 0.1 mL of 1.5 mmol/L NBT, 0.2 mL of 0.1 mmol/L EDTA solution containing 0.3

mmol/L sodium cyanide, 50 µL of 0.12 mmol/L riboflavin, and a suitable aliquot of enzyme extract. The reaction mixture was illuminated for 15 min at a light intensity of 5000 lx. The absorbance was measured at 560 nm. One unit of SOD was the amount of the enzyme catalyzing 50% inhibition of NBT photochemical reduction. The control assay was done in the absence of any plant extract to prevent possible autoxidation of the substrates.

POD activity was assayed using the method reported by Chance and Maehly (1955). The reaction mixture contained 0.1 mol/L citrate-phosphate-borate buffer (pH 7.5), 50 µL of 15 mmol/L guaiacol, 50 µL of 3.3 mmol/L H2O2, and 25 µL of enzyme extract. The polymerization of guaiacol was initiated by adding H2O2, and the increase in absorbance at 470 nm was recorded for 3 min. The activity was calculated using the extinction coefficient of 26.6 mM–1 cm–1 for guaiacol.

CAT activity was measured by following the dismutation of H2O2 at 240 nm for 3 min using the UV absorbance method. The reaction mixture contained 1.50 mL of 0.1 mol/L citrate-phosphate-borate buffer solution (pH 7.5), 50 µL of enzyme extract, and 13 µL of 10 mmol/L H2O2. One unit of CAT was the amount of enzyme for the dismutation of 1µ mol/L H2O2 per minute. The extinction coefficient for H2O2 at 240 nm was 39.4 M–1 cm–1 (Obinger et al., 1997).

Protein content was determined according to the method developed by Bradford (1976), using bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) as a standard protein.2.4. Determination of Zn2+ ion dissolutionDissolution of Zn2+ ions from bulk-ZnO, ZnO-NP, and EZO-NP suspensions was determined to evaluate their effects on the plant. The prepared suspensions of bulk-ZnO, ZnO-NPs, and EZO-NPs at different concentrations were centrifuged at 4000 rpm for 10 min and filtered with a 0.22-µm sterilized filter (Kumari et al., 2011). Finally, the Zn2+ concentrations in the supernatants were measured by atomic absorption spectrometry (novAA 400, Analytik Jena, Jena, Germany).2.5. Statistical analysisData with four replicates were statistically analyzed by one-way analysis of variance, which was followed by Tukey–Kramer multiple comparisons test using GraphPad software (GraphPad Software, Inc., La Jolla, CA, USA). The results were described as mean ± standard deviation (SD). Significant difference was reported when the probability was less than 0.05.

3. Results and discussion 3.1. Characterization of the synthesized nanoparticlesSEM images of Er-doped and undoped ZnO nanoparticles are shown in Figures 1a and 1b. According to Figure 1,

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Er-doped ZnO particles were smaller than undoped ZnO particles. The growth of ZnO particles was hindered upon Er doping, which was attributed to the Er-O-Zn generation on the surface of the doped samples, subsequently preventing the growth of crystal grains. It is clear from Figures 1c and 1d that the synthesized samples are of nanometer size and the major particle size distributions of Er-doped ZnO and undoped ZnO are in the range of 20–40 nm and 60–80 nm, respectively.

Figures 2a and 2b demonstrate the EDX analysis of undoped and Er-doped ZnO samples. The Zn and O peaks can be easily observed in the obtained EDX results of undoped ZnO (Figure 2a). Moreover, Figure 2b confirms the presence of Er in the doped ZnO particles. The high purity of the obtained products was confirmed by the absence of unexpected extra peaks in nanocrystals. A detailed physicochemical characterization of EZO-NPs and ZnO-NPs can be found in the authors’ previous published work (Khataee et al., 2015a).3.2. Effects on the growth of the plantFigure 3 illustrates the RFN and RGR of L. minor during 20 days in the presence of various concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs. RFN decreased with

increasing concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs; according to the results, high concentrations of the materials had notable negative effects on plant growth. For instance, RFN significantly decreased to 49.7%, 62.4%, and 53% after 20 days of exposure to 50 mg/L of bulk-ZnO, ZnO-NPs, and EZO-NPs, respectively. Nevertheless, the 20-day exposure of the plant to 3.5 mg/L ZnSO4 introduced more negative effects and RFN was reduced by approximately 75.5% (Figure 3a).

RGR decreased significantly with increasing concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs. Between the two groups of nanoparticles, it seemed that EZO-NPs had a less negative effect on plant growth as compared with ZnO-NPs. The negative effects of ZnO-NPs were remarkable when compared with bulk-ZnO and EZO-NP treatments, as well as with the control. For instance, the 1 mg/L ZnO-NP treatment led to a significant reduction of RGR as compared with the control (P < 0.05). Additionally, RGR was reduced up to 43.1%, 61.2%, and 52% after 20 days of treatment with 50 mg/L of bulk-ZnO, ZnO-NPs, and EZO-NPs, respectively (Figure 3b). Twenty days of 3.5 mg/L of ZnSO4 treatment led to RGR decline of up to 69.7% as compared with the control (P < 0.001).

Figure 1. SEM images of a) Er-doped ZnO-NPs and b) ZnO-NPs, and size distributions of c) Er-doped ZnO-NPs and d) ZnO-NPs.

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Figure 2. EDX analysis of a) undoped and b) Er-doped ZnO-NPs.

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Moreover, increasing concentrations in each examined group caused significant impacts on RGR in that group. For instance, after 20 days of treatment with 1 mg/L of ZnO-NP, RGR showed a significant difference compared with the two other used concentrations of ZnO-NP (10 and 100 mg/L). The negative effects of the NPs on plants could be related to the release of ions from them and/or their direct interactions with plants (Perreault et al., 2014; Rao and Shekhawat, 2014). However, the obtained results were in agreement with the fact that the excess Zn2+ can cause delayed growth by the decrease or inhibition of photosynthesis and imperfections of many enzyme activities, leading to metabolic disorders (Rosen et al., 1977; Hossain et al., 2012).

In addition to RFN and RGR, root elongation is an important indicator of environmental toxicity and its inhibition is one of the disorders seen under Zn toxicity (Marschner, 1995). Table 2 and Figure 4 show the effects of different concentrations of nanomaterials, including ZnO-NP and EZO-NP, on root length after 10 days of exposure. As shown in Table 2 and Figure 4, after 10 days of treatment

with all concentrations of the nanomaterials, only the 50 mg/L ZnO-NP and EZO-NP treatments led to significant decreases in root length compared with the control (P < 0.001). In contrast, after 20 days of treatment, plant root length was inhibited by all concentrations of ZnO-NP and EZO-NP except 1 mg/L of EZO-NP (Table 2), and root length decreased with increasing concentrations of nanomaterials. In accordance with the obtained results, the negative effects of Zn on root and shoot growth of different plants have been reported by many previous studies (Lin and Xing, 2008; Mousavi Kouhi et al., 2015). According to the literature, NPs can possibly adhere to the root surface of plants and inhibit root growth. Plant root growth inhibition can subsequently block water transport pathways and decrease mineral absorption, thus affecting the growth of the whole plant (Lee et al., 2013).3.3. The effect on photosynthetic pigments contentPigment content was determined after 7 days of exposure to 1, 10, and 50 mg/L of bulk-ZnO, ZnO-NP, and EZO-NP. According to the results, after 7 days of exposure to all concentrations of the contaminants, Chl a, Chl b, and

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Figure 3. Effect of three concentrations of EZO-NP, ZnO-NP, and bulk-ZnO (1, 10, and 50 mg/L) on a) relative frond number (RFN) and b) relative growth rate (RGR) of L. minor (mean ± SD, n = 4). *: Significant difference at P < 0.05. **: Significant difference at P < 0.01. ***: Significant difference at P < 0.001.

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Chl a + b amounts were significantly decreased (Figure 5). Carotenoid content was reduced to 36% and 42% at 10 and 50 mg/L of ZnO-NP, respectively, whereas there were no statistically significant differences between carotenoid contents of the control plants and the plants treated with different concentrations of bulk-ZnO and EZO-NP (P > 0.05) (Figure 5). Moreover, in each group

of the examined contaminants (ZnO-NP, bulk-ZnO, and EZO-NP), a significant difference in chlorophyll and carotenoid contents was observed in plants treated with 1 and 50 mg/L of the contaminants as the lowest and highest concentrations of each group. In each group of the examined contaminants, a 50-fold increase in the concentration of the contaminants (1 mg/L versus 50

Table 2. Effect of three concentrations of EZO-NP and ZnO-NP (1, 10, and 50 mg/L) on root length of L. minor after 10 and 20 days of treatment (mean ± SD). *: Significant difference with the control sample at P < 0.05. ***: Significant difference with the control sample at P < 0.001.

Treatment concentration (mg/L)

Root length (cm)

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Control 0 4.8 ± 0.06 6.4 ± 0.09

ZnO-NP1 4.5 ± 0.13 5.7 ± 0.20*10 4.1 ± 0.53 4.8 ± 0.13***50 2.3 ± 0.06*** 2.8 ± 0.12***

EZO-NP1 4.6 ± 0.06 6.1 ± 0.0910 4.4 ± 0.04 5.6 ± 0.17***50 3.1± 0.07*** 3.6 ± 0.14***

Zn 2+ 3.5 1.3 ± 0.04*** 1.7 ± 0.11***

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ZnO-NP (1 mg/L) ZnO-NP (10 mg/L) ZnO-NP (50 mg/L)ZnSO4 (3.5 mg/L)

Figure 4. Effect of different concentrations of ZnO-NP and EZO-NP on plant root length after 10 days of treatment.

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mg/L) caused significant reductions in the photosynthetic pigment contents. There was no significant difference in photosynthetic pigment contents in the plants treated with 10 mg/L of each examined contaminant relative to the two other concentrations (1 and 50 mg/L). In addition, the 3.5 mg/L ZnSO4 treatment led to a significant reduction of chlorophylls and carotenoid content as compared with the control. By considering that the dissolution of Zn ions may play a significant role in the toxicity of NPs, some previous studies have also reported the inadequacy of chloroplasts and the reduced number of thylakoids in response to surplus Zn2+ (Ebbs and Uchil, 2008; Wang et al., 2009). Accordingly, Zn, by damaging chloroplasts and reducing photosynthetic pigment contents, could produce chlorosis as a common symptom of Zn toxicity (Wang et al., 2009).3.4. Enzymatic analysisIt has been established that nanomaterials induce toxicity mediated by reactive oxygen species (ROS) and consequent production of oxidative stress in many biological systems. ROS overproduction is regarded as one of the principal causes of cellular damage (Fu et al., 2014; He et al., 2015). ROS induce the biological defense system to reactivate intermediates or repair the damages and lead to antioxidant responses. The activities of antioxidant enzymes such as SOD, POD, and CAT usually change in response to oxidative stress (Xia et al., 2008). In the present study, these enzyme activities were assayed under different concentrations of bulk-ZnO, ZnO-NP, and EZO-NP. The effects of their different concentrations on SOD, POD, and CAT activities are illustrated in Figure 6.

According to Figure 6a, the 10 and 50 mg/L ZnO NP and EZO NP treatments significantly increased SOD activity as compared with the control. However, only 50 mg/L of bulk-ZnO could significantly induce enzyme activity after 7 days of exposure. Moreover, the ZnSO4 treatment (3.5 mg/L) notably increased SOD activity to near 72.6%. A similar trend was previously reported in plant species treated with Zn2+ (Radic et al., 2010; Hu et al., 2013; Rao and Shekhawat, 2014). SOD neutralizes reactive superoxide radicals to less destructive hydrogen peroxides, which are then detoxified by CAT, POD, or other antioxidative enzymes (Radic et al., 2010). Additionally, it was revealed that enhancement in the concentrations of the contaminants in each of three examined groups (bulk-ZnO, ZnO-NPs, and EZO-NPs) led to notable and statistically significant increases in SOD activity in that group. This means that there were significant differences in SOD activity among the three obtained amounts according to the three various contaminant concentration treatments in each group.

As shown in Figure 6b, there were no significant changes in POD activity after 7 days of 1 mg/L bulk-ZnO, ZnO-NP, and EZO-NP treatment as compared to the control. POD activity was increased by 18.2%, 52.3%, and 39.8% after 7 days of exposure to 10 mg/L bulk-ZnO, ZnO-NP, and EZO-NP, respectively (P < 0.001) (Figure 6b). In contrast, the high concentration of the treated compounds (50 mg/L) led to a significant reduction of POD activity, which was similar to the effect of the 3.5 mg/L of ZnSO4 treatment. CAT activity showed a pattern similar to that of POD (Figure 6c). The 10 mg/L bulk-ZnO, ZnO-NP, and

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Figure 5. Chlorophyll a, chlorophyll b, and total carotenoid contents (mg g–1 FW) in control L. minor plants and plants exposed to 1, 10, and 50 mg/L of ZnO-NP, EZO-NP, and bulk-ZnO for 7 days (mean ± SD, n = 4). ***: Significant difference at P < 0.001.

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EZO-NP treatments significantly increased its activity (P < 0.001), but exposure to 50 mg/L of the aforementioned compounds caused a decline in CAT activity to near 48.7%, 51.3%, and 57.9%, respectively. Improved POD and CAT activities after the augmentation of SOD activity by 10 mg/L of the treated materials probably reflected the high demand for the detoxification of the produced H2O2 in the treatments with such concentrations of bulk-ZnO, ZnO-NP, and EZO-NP. Increased POD and CAT activities in plants treated with Zn2+ have been previously reported in the literature (Hu et al., 2013; Mousavi Kouhi et al., 2015). In contrast with the 10 mg/L bulk-ZnO, ZnO-NP, and EZO-NP treatments, POD and CAT activities were suppressed in L. minor treated with 50 mg/L of the materials. Possibly, the accumulation of ROS in the high concentrations of Zn2+ damaged subcellular organelles (Wang et al., 2009). This could be due to the weakness of the plant defense system in scavenging ROS produced in high concentrations of Zn2+. 3.5. Zn2+ ion dissolution Various mechanisms of nanotoxicity have been proposed and published in different nanotoxicological studies in recent years. Oxidative stress via ROS overproduction is regarded as one of the major causes of toxicity and cellular damage. Other possible mechanisms include dissolution of metal ions and/or direct interactions of the nanomaterials

(He et al., 2015). Previous studies on toxicity of ZnO-NPs have been criticized for not precisely distinguishing between the biological effects of ZnO-NPs and dissolved Zn2+ (Mudunkotuwa et al., 2012). Hence, these studies include contradictory information about the impacts of ZnO-NPs and dissolved Zn2+. Some of the studies suggest that the toxicity of nanoparticles is due to soluble Zn ions released from nanoparticles. For instance, ZnO-NP toxicity studies on different organisms, including human and rat cell lines (Brunner et al., 2006; Kao et al., 2012), bacteria such as Escherichia coli and Bacillus subtilis (Kim and An, 2012), and various species of the family Lemnaceae (Hu et al., 2013; Chen et al., 2016), confirmed that dissolved Zn plays a central role in causing ZnO-NP toxicity. However, according to other reports, ZnO-NP toxicity was not due to dissolved Zn2+ alone and there was a close relationship between the toxicity of NPs and their dissolved portion (Franklin et al., 2007; Ji et al., 2011; Kumari et al., 2011). In contrast, some other studies indicated that Zn dissolution plays a minor role in causing toxicity (Lin and Xing, 2008). To investigate these discrepancies in the literature, ZnO-NP dissolution was quantified in the present study. The concentrations of total Zn2+ in the culture media and in the treated suspensions after 24 h are shown in Table 3. The medium of the control provided Zn2+ as one of the essential nutrients for the normal growth of L. minor at

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a

Figure 6. a) SOD, b) POD, and c) CAT activities in control L. minor plants and plants exposed to different concentrations of ZnO-NP, EZO-NP, and bulk-ZnO for 7 days (mean ± SD, n = 4). *: Significant difference at P < 0.05. **: Significant difference at P < 0.01. ***: Significant difference at P < 0.001.

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a concentration of 0.05 mg/L. According to Table 3, the concentration of Zn2+ dissolved from 50 mg/L of ZnO-NP was more than that of other groups, and the amount of Zn2+ in such groups was closer to the Zn2+ concentration in the positive control group (3.5 mg/L). On the other hand, the aforementioned groups (50 mg/L ZnO-NP and 3.5 mg/L ZnSO4) had the same effects on the growth of the plant and some other physiological indicators. In contrast, different concentrations of EZO-NP and bulk-ZnO had less negative effects on the investigated physiological parameters. The obtained results show that the soluble part of the treatment compounds (bulk-ZnO, ZnO-NP, and EZO-NP) had a significant function in the toxicity of the treated compounds, leading to different biochemical and physiological responses in L. minor.

We investigated the biochemical and plant physiological responses of L. minor to EZO-NPs and ZnO-NPs. The obtained results confirmed that the negative effects of EZO-NPs on L. minor were not as severe as those of ZnO-NPs. Zn2+ ions released from the NPs play a substantial role in the toxicity of the treatment compounds, leading to the different biochemical and physiological responses in L.

minor. Chl a, Chl b, and Chl a + b contents were notably reduced after 7 days of exposure to all concentrations of the contaminants. The reduction in pigment content could be one of the important reasons for other negative responses, such as growth reduction. RFN and RGR decreased significantly with increasing concentrations of bulk-ZnO, ZnO-NPs, and EZO-NPs. SOD activity increased remarkably with increasing concentrations of the contaminants, thereby verifying its significance in plant sensitivity to the contaminants. POD and CAT activities were increased in 10 mg/L bulk-ZnO, ZnO-NP, and EZO-NP treatments, but their activities declined after 7 days of exposure to 50 mg/L of the aforementioned compounds. Research on examined NP aggregation in the natural aquatic environment, sediment toxicity, and their bioaccumulation may provide additional valuable information.

AcknowledgmentThe authors thank Urmia University and the University of Tabriz, Iran, for providing support during the present study.

Table 3. Dissolved free Zn2+ ions released from different concentrations of ZnO-NP, EZO-NP, and bulk-ZnO suspensions in L. minor culture medium after 24 h.

Treatment concentration (mg/L)

Free Zn2+ ion concentration (mg/L)

ZnO-NP

1 0.8

10 1.05

50 1.74

EZO-NP

1 0.6

10 0.83

50 1.6

Bulk-ZnO

1 0.4

10 0.7

50 0.82

Culture medium 0.05

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