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Effects of foliar applications of ceria nanoparticles and CeCl3 on common bean (Phaseolus vulgaris)
Environmental Pollution 250 (2019) 530e536
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Effects of foliar applications of ceria nanoparticles and CeCl3 on common bean (Phaseolus vulgaris)* Changjian Xie a, b, 1, Yuhui Ma a, 1, Jie Yang a, Boxin Zhang c, Wenhe Luo a, b, Sheng Feng a, Junzhe Zhang a, b, Guohua Wang a, b, Xiao He a, Zhiyong Zhang a, d, * a
Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China School of Chemistry and Chemical Engineering, University of the Chinese Academy of Sciences, Beijing, 100049, China c International Department, Beijing National Day School, Beijing, 100049, China d School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 100049, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2018 Received in revised form 14 February 2019 Accepted 8 April 2019 Available online 11 April 2019
In this study, comparative effects of foliar application of ceria nanoparticles (NPs) and Ce3þ ions on common bean plants were investigated. Soil grown bean seedlings were exposed to ceria NPs and Ce3þ ions at 0, 40, 80, and 160 mg Ce$L1 every other day at the vegetative growth stage for 17 d. The plants were harvested 47 d after the last treatment. Performed analyses involved growth, physiological and biochemical parameters of the plants and nutritional quality of the pods. Ceria NPs at 40 mg Ce$L1 increased dry weight of the plants by 51.8% over the control. Neither ceria NPs nor Ce3þ ions significantly affected other vegetative growth parameters. Pod yields and nutrient contents except for several mineral elements were also not significantly different among groups. Compared to control, pods from ceria NPs at 80 mg Ce$L1 had significantly less S and Mn. At 40 and 80 mg Ce$L1, ceria NPs reduced pod Mo by 27% and 21%, while Ce3þ ions elevated Mo contents by 20% and 18%, respectively, compared with control. Ce3þ ions at 80 and 160 mg Ce$L1 significantly increased pod Zn by 25% and 120%, respectively, compared with control. At the end of the experiment, Ce3þ ions at 40, 80, and 160 mg Ce$L1 increased contents of malondialdehyde (MDA) by 46%, 65%, and 82% respectively as compared with control. While ceria NPs led to a significant increase of MDA level only at the highest concentration. X-ray absorption near edge structure (XANES) analysis of the leaf samples revealed that both ceria NPs and Ce3þ ions kept their original chemical species after foliar applications, suggesting the observed effects of ceria NPs and Ce3þ ions on the plants were probably due to their nano-specific properties and ionic properties respectively. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Ceria nanoparticles Ce3þ ions Foliar application Nutritional quality Common bean
1. Introduction Cerium, a member of the lanthanides, is the most abundant element of the series in the earth's crust. A unique property of Ce is that it has a stable tetravalent state in contrast to the trivalent state which is dominant for the lanthanides (Ju-Nam and Lead, 2008). Cerium dioxide (ceria) is the most important lanthanide oxides, especially in a nanoparticle formulation, and has been widely used
*
This paper has been recommended for acceptance by Bernd Nowack. * Corresponding author. School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 100049, China. E-mail address: [email protected] (Z. Zhang). 1 The two authors contributed equally to this article. https://doi.org/10.1016/j.envpol.2019.04.042 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
in catalysis, optical and sensor technologies, and medicine (Xu and Qu, 2014). Therefore, ceria NPs are expected to enter the environment deliberately or accidently, where they may pose a potential risk to aquatic and terrestrial organisms (Johnson and Park, 2012). A comprehensive understanding of the interactions between plants and ceria NPs is of extreme importance for assessing the toxicity and possible trophic transfer of these particles (Miralles et al., 2012; Zuverza-Mena et al., 2017). Phytotoxicity studies on ceria NPs were mainly performed by root exposure on plants grown in soil and other media. Numerous studies have documented negative, positive, or no effect of ceria NPs on plant growth (Dahle and Arai, 2015). Ma et al. (2010) found that ceria NPs inhibited root elongation of lettuce but showed no effect on oilseed rape, radish, pez-Moreno wheat, cabbage, tomato and cucumber. Similarly, Lo
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et al. (2010) observed that 2000 mg L1 ceria NPs significantly reduced germination rates of corn, tomato and cucumber but that of alfalfa was unaffected. Priester et al. (2012) reported significant reductions of both plant growth and pod yield of soybean plants cultivated in farm soil amended with ceria NPs. Moreover, Zhao et al. (2014) noticed that exposure to ceria NPs at 400 and 800 mg kg1 soil led to a reduced nutritional quality of cucumber fruits. Similar studies of the same group demonstrated that ceria NPs could modify the nutritional value of two important cereal crops, rice and wheat, which may have a long-term negative effect on food quality (Rico et al., 2013a, 2014). Conversely, Wang et al. (2012) noted ceria NPs at the concentrations of 0.1e10 mg L1 had either an inconsequential or a slightly positive effect on plant growth and tomato production. Recently, ceria NPs were shown to be able to alleviate detrimental effects of abiotic stresses in plants (Rossi et al., 2017; Tassi et al., 2017). Ceria NPs are now widely used as a fuel additive to reduce particulate matter emissions from diesel engines. It can be expected that more and more ceria NPs will be released into the atmosphere (Majestic et al., 2010). Plant leaves are a major sink of aerosol and €sa €nen et al., 2017). Exposure airborne particles (Burkhardt, 2010; Ra of plants to ceria NPs may occur by both wet and dry deposition. However, the impact of ceria NPs on plants after foliar application is only rarely studied. Birbaum et al. (2010) reported that ceria NPs applied as aerosol (0.178 g m3) or suspension (10 mg L1) could be adsorbed on and incorporated in leaves but were not able to translocate within maize plants. However, Hong et al. (2014) found that foliar applied ceria NPs (0, 1, 2, 8, and 8 mg per plant) to cucumber leaves translocated to stems, roots, and flowers of the plants. Moreover, the activities of dehydroascorbate reductase (DHAR), catalase (CAT), and ascorbate peroxidase (APX) in plant tissues were modified. In another study, these authors exposed cucumber seedlings to ceria NPs by foliar application (3.1, 6.2, and 12.5 mg per plant) and found that ceria NPs altered the nutritional status of cucumber fruits and impacted cucumber photosynthetic parameter in seedlings at the highest dose (Hong et al., 2016). Compared with control, net photosynthesis rate, and transpiration rate were decreased by 22% and 11%, respectively. Salehi et al. (2018) reported that foliar spray of ceria NPs altered stomatal density and length, photosynthesis and electron transport chain biochemical machinery of bean seedlings at relatively high doses (16.7, 33.3, 66.7, and 133 mg per plant). Conversely, Adisa et al. (2018) found that foliar application of ceria NPs at 1.25 mg per plant significantly reduced Fusarium wilt severity of tomato plants by 57%. Clearly, more studies are needed, such as those focusing on the species, dosimetry, chronic toxicity, and life cycle assessment, to better understand the effects of ceria NPs on plants after foliar application. This study aimed to investigate physiological and biochemical responses of the common bean plants to ceria NPs and Ce3þ ions after repeated foliar applications. Cerium chloride (CeCl3) was used as an ionic control for comparison. Agronomic and yield characteristics, oxidative stress and antioxidant defense indicators, gas exchange and photosynthetic parameters, and nutritional quality of bean pods were assessed. Additionally, contents and chemical species of Ce in plant tissues were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) and X-ray absorption near edge structure (XANES), respectively. 2. Materials and methods
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G2 20 S-Twin, FEI, Japan) was 6.9 ± 0.4 nm (Fig. S1). The hydrodynamic diameter and zeta-potential of the NPs in ultrapure water (20 mg L1) that determined by dynamic light scattering (DLS, ZetaSizer, Malvern Instruments, UK) were 40.2 ± 7.2 nm and 32.9 ± 8.5 mV, respectively. Ceria NPs were suspended in ultrapure water at 40, 80, and 160 mg Ce$L1 and homogenized by sonication for 30 min before use. CeCl3$7H2O purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai China) was dissolved in ultrapure water at concentrations of 0.29, 0.58, and 1.16 mM (corresponding to ceriumequivalent mass concentrations 40, 80, and 160 mg Ce$L1, respectively). Seeds of common bean (Phaseolus vulgaris L. Ningxing 81e6) purchased from Pingluo Xiongnong Seeds & Seedling Co., Ltd. (Ningxia, China) were surface-sterilized with 3% H2O2 for 20 min and washed thoroughly with ultrapure water. Then the seeds were sown in a plug tray with 50 cells (55 mL/cell) filled with a commercial substrate and germinated in a greenhouse. On day 16 after sowing, uniform seedlings were transferred to 4-L sterile polypropylene pots (one seedling per pot) filled with a substrate. The substrate was prepared with local loam sand soil (13% clay, 55% silt, 32% sand, and 1.1% organic matter content, pH 7.85) and MiracleGro potting mix at a ratio of 1:1 (v/v) by hand mixing with a spatula. The background concentration of Ce in the substrate was 44.4 mg kg1. Thirty-five plants were divided randomly into 7 groups of 5 plants each. One group served as a control and the other 6 experimental groups were exposed to either nanoparticulate or ionic cerium at different concentrations. Ceria NP suspensions or cerium chloride solutions at 40, 80, and 160 mg Ce$L1 were sprayed on the upper surface of each leaf of each plant at 15 d after transplanting. The treatments were applied using 100 mL aerosol hand sprayers and the nozzles were directed toward the leaves from about 5 cm. It could accurately target spray of the leaves with little or even no run-off. Control plants were sprayed with ultrapure water. The neighbor plants and soil were protected with blotting paper to prevent contamination. The foliar application was ended before flowering. A total volume of 435 mL ceria NP suspensions or cerium chloride solutions per treatment were sprayed on the leaves every other day for 17 d. The dosages correspond to approximately 0, 3.5, 7.0 and 13.9 mg Ce per plant, respectively. Transpiration rate (E), stomatal conductance (gs), and net photosynthesis (Pn) were measured with an open gas exchange system (LI-6400XT; Li-Cor Environmental, Lincoln, NE, USA) in 60d old plants. Chlorophyll content in the leaves were analyzed from fully expanded leaves (similar leaves in all groups) using a chlorophyll meter (Konic Minolta SPAD-502 Plus, Japan). Five weeks after the last foliar spray, fully developed pods were harvested and weighed. Then 12 d later, plants were carefully removed from the soil. Physical measurements were recorded such as plant height, leaf length, leaf count, and biomass. Shoots and roots were separated, thoroughly washed with tap water, immersed in 10 mM CaCl2 for 20 min, again twice with ultrapure water. This washing procedure was adapted from Hong and his colleagues’ work (Hong et al., 2014). They found that CaCl2 solution could remove more ceria NPs from the leaf surface compared to HNO3 or water. Leaf samples (3 discs of 1 cm diameter each) were taken from the 4 uppermost fully expanded leaves on each plant, frozen with liquid nitrogen and were stored at 80 C for enzyme assay and lipid peroxidation measurement. Dry weight (DW) of shoots and roots was determined after oven-drying at 60 C for 2 d.
2.1. Experimental design 2.2. Enzyme assay and lipid peroxidation determination Ceria NPs were synthesized using a precipitation method as reported previously and described in detail in Supplementary data (Zhang et al., 2011). The primary size characterized by TEM (Tecnai
Leaf and root samples were homogenized with PBS (50 mM, pH 7.8) under ice bath, and then centrifuged at speed of 10000g and
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4 C for 10 min. The supernatants were kept for analyses of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and malondialdehyde (MDA) contents using the assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The data was collected from 5 replicates/treatment. 2.3. Nutritional quality assessment Nutrient compositions of the pods were determined according to previously established methods, i.e. starch content by the anthrone method of Jayaraman, (1981); reducing sugar by dinitrosalicylic acid method of Miller, (1959); the total soluble sugars were estimated by the methods of DuBois (DuBois et al., 1956). The water-soluble protein was determined by the method of Lowry (Lowry et al., 1951). Vitamin C content was determined using the method of Deutsch and Weeks (Deutsch and Week, 1965). Total phenols of the pods were extracted and estimated according to Malik and Singh (1971). 2.4. Quantification of Ce, macro- and micronutrient contents The dry samples of leaf, root, and pod were ground into fine powders and digested with a mixture of HNO3 and H2O2 (4:1 by volume) on a heating plate. After decomposition, the samples were diluted to 10 mL with 2% (v/v) nitric acid. Ce contents in all samples and 8 mineral element contents in pod samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Elemental X7) or inductively coupled plasma-optical emission spectroscopy (ICP-OES; PerkinElmer). Indium of 20 ng/ mL was used as an internal standard to compensate for the matrix suppression and signal drifting. Analytical runs include calibration verification samples, spike recovery samples and duplicate dilutions. The linearity was from 0.1 to 500 mg L1 of Fe, Mn, Mo, Cu, Zn, and Ce. The linearity was from 0.1 to 20 mg L1 of Ca, P, and S. Standard reference materials GBW07602 and GBW07603 were also digested and analyzed as samples to examine the recovery. The recoveries for all elements were between 84.9% and 128.9% with the relative standard deviation of <1.5% (Table S1).
3. Results and discussion 3.1. Distribution and chemical species Ce in plant tissues Ce contents in the roots, leaves, and pods of the common bean plants are shown in Fig. 1. In the control plants, Ce contents of plant parts varied in the order of root > leaf > pod. This is consistent with the general rare earth elements distribution in plants of the highest concentrations in roots, intermediate values in the above-ground shoots and leaves, and lowest levels in the edible seeds and fruits (Hu et al., 2004). In the present study, the highest Ce contents among all treatments were found in the leaves due to foliar application. Leaves of the plants exposed to ceria NPs or Ce3þ ions at 40, 80, and 160 mg Ce$L1 had 467, 1094, 2999, and 128, 163, 391 mg of Ce per kg DW, respectively. Ceria NPs at all concentrations exhibited significantly higher leaf Ce contents compared with the respective Ce3þ ions. After foliar spray, ceria NPs or Ce3þ either adsorbed to the leaf surface or incorporated into the leaf structure (Birbaum et al., 2010). Using electron microscopy, Birbaum et al. (2010) found that agglomerated ceria NPs on the surface of maize leaves were integrated into surface wax. Therefore, removing of ceria NPs from the leaf surface was difficult than soluble Ce3þ ions. For Ce content in the roots, there was no significant difference between the control and all the treated plants. The pod is the edible part of common bean. Only the highest concentration of Ce3þ (160 mg Ce$L1) produced a significant increase of Ce content in the
2.5. Ce speciation analysis Chemical species of Ce in the roots and leaves of the plants that treated with ceria NPs and CeCl3 at 160 mg L1 Ce were analyzed by synchrotron radiation based X-ray absorption near edge structure. Leaves and roots were lyophilized, ground into fine powders, and pressed into slices with a diameter of 10 mm and a thickness of 2 mm for analyses. Ce LIII edge (5723 eV) spectra of the samples were recorded at ambient temperature in fluorescence mode at beamline 1W1B of the Beijing Synchrotron Radiation Facility (BSRF). The storage ring energy during the collection was 2.5 GeV with current intensity of 50 mA. Spectra of ceria and CePO4 were collected at transmission mode as standard references. Linear combination fitting (LCF) analyses of the XANES spectra were performed on software program ATHENA to identify and quantify Ce species. 2.6. Statistical analysis The data processing was performed on Statistical Packages for the Social Sciences (SPSS) 10.0. All the data were expressed as mean ± standard deviation (n ¼ 5). Statistical significance of differences among treatments were determined using one-way analysis of variance and covariance (ANOVA) with least significant difference (LSD) test or Kruskal-Wallis H ANOVA with MannWhitney U test for multiple comparison. p 0.05 were considered statistically significant.
Fig. 1. Ce contents in the leaves, roots, and pods of the common bean plants that foliarly treated with ceria NPs (indicated as N40, N80, and N160) and Ce3þ (indicated as I40, I80, and I160). Data are average of five replicates±standard deviations. Different letters stand for statistical differences at p 0.05.
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pod when compared with control (2.18 fold). Phaseolus vulgaris L., the plant species used in this study has stomata on both upper and lower leaf surfaces. Several studies have confirmed the foliar uptake nanoparticles by passing through the stomata (Hong et al., 2014; Larue et al., 2014; Abdel-Aziz et al., 2016; Hong et al., 2016; rez-de-Luque, 2017). It was supposed that ceria NPs deposited on Pe the leaves might translocate from the stomatal pathway, pass through the phloem sieve tubes, and finally reach other parts of the plants (Wang et al., 2013). However, the results of this study does not exclude the possible foliar uptake of ceria NPs since natural background of Ce in plant tissues hindered the quantitative determination of translocation of Ce at relatively small amount. Fig. 2 shows the Ce LIII XANES spectra of two Ce model compounds, Ce(IV)O2 and Ce(III)PO4 and leaves that treated with ceria NPs or Ce3þ ions at 160 mg Ce$L1. The spectrum of Ce in the leaves exposed to ceria NPs only shows the feature of Ce(IV) oxidation state. As confirmed by LCF, almost 100% of the Ce in the sample was present as CeO2. Chemical species of Ce in the leaves that treated with Ce3þ also remained unchanged as Ce(III). Unfortunately, Ce contents in the roots and pods were too low to carry out a reliable XANES analysis. We previously found that ceria NPs could undergo transformation at the root surface of hydroponic plants and the released Ce3þ ions caused the phytotoxicity to lactuca plants (Zhang et al., 2015). Moreover, once entered plant tissues, ceria NPs could not be further reduced, even inside the roots (Ma et al., 2015). Biogenic reducing substances (e.g. ascorbic acid, and catechol, etc.) and organic acids from root exudates might play critical roles in the transformation process (Zhang et al., 2012; Rui et al., 2015). Similarly, several reports also stated transformation of ceria NPs in plants cultivated in soil amended with ceria NPs (HernandezViezcas et al., 2013; Majumdar et al., 2014; Rico et al., 2018). Transformation of ceria NPs from Ce(IV) to Ce(III) undergoes a reductive reaction which occurs predominantly in acidic media and needs a reductant (Schwabe et al., 2014). Accordingly, this chemical reaction is unlikely to take place in the aerial parts of a plant. 3.2. Effects of foliar treatments on plant growth and photosynthetic parameters None of the treatments significantly affected plant height, number of leaves, leaf length, and dry matter percentage, compared to those of the control (Table 1). However, ceria NPs at 40 mg Ce$L1
Fig. 2. XANES normalized Ce LIII edge spectra of reference compounds (CePO4 and CeO2) and samples. Straight and dotted lines marked the feature of Ce (Ⅲ) and Ce (Ⅳ) compounds, respectively.
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significantly increased dry weight by 51.8% relative to the control. Moreover, both fresh and dry weights of the plants that treated with ceria NPs at 40 mg Ce$L1 were higher than that of the plants treated with Ce3þ ions at the same concentration. There is few report about the effects of foliar-applied ceria NPs on agronomic characteristics of crop plants. Several studies have demonstrated that the addition of ceria NPs in culture media can improve plant biomass production (Priester et al., 2012; Rico et al., 2014; Cao et al., 2017). The possible mechanisms were discussed in 3.3. Chlorophyll content and photosynthetic parameters such as E, gs, and Pn are considered indicators of photosynthetic responses of green plants under environmental stress. There were no significant differences among treatments for these parameters (Table S1). Liu et al. (2019) found that atmospheric fine particle could block stomata, thus reducing photosynthetic activity. Hong et al. (2014) demonstrated that at a dose of 12.5 mg ceria per plant which was a little lower than the highest dose used here, ceria NPs significantly decreased Pn and E in cucumber leaves. The differences between studies may reflect differential responses to ceria NPs based on plant species or other conditions. 3.3. Effects of foliar treatments on antioxidant enzymes and lipid peroxidation in plant tissues Primary indicators of stress including SOD, POD, CAT, and MDA in the leaves (Figs. 3 and 4) and roots (Fig. S2) were measured to examine the effects of ceria NP and Ce3þ ion treatments on the antioxidant system of common bean plants. In the roots, neither ceria NP nor Ce3þ ion treatments induced significant changes in these indicators compared to the control. In the leaves, the plants exposed to ceria NPs showed no evidence of changes in SOD activity, while plants exposed to 80 and 160 mg L1 Ce3þ showed increases in SOD activity as compared to that of control plants. Lipid peroxidation, an effective indicator of cellular oxidative damage, was measured as MDA levels in our study. In the plants treated with the ceria NPs, the contents of MDA in the leaves showed a tendency of dose-dependent increase, but only the highest Ce level (160 Ce mg$L1) produced a statistically significant elevation. While Ce3þ ions at 40, 80, and 160 mg Ce$L1 increased MDA levels by 46%, 65%, and 82% respectively, as compared with control. Lipid peroxidation causes destruction of lipid components of membrane by membrane impairment and leakage (Rico et al., 2013b). At the end of the experiment, foliar chlorosis and necrosis were observed on the plants treated with Ce3þ ions in a dose-dependent way, especially the older leaves (Fig. S3). The symptoms might be due to an increased cell or tissue damage estimated by MDA production. No obvious chlorosis or necrosis was observed in the plants treated by ceria NPs, which agrees with MDA analysis. Ceria NPs are reported to have both beneficial and toxic effects on biological systems (Yokel et al., 2014; Walkey et al., 2015). The particles can exert a pro-oxidative effect by producing reactive oxygen species (ROS) which damage lipids, proteins, DNA, and cause cell death. On the other hand, ceria NPs have been shown to be excellent free radical scavengers and therefore protecting cells from oxidative damages (Dahle and Arai, 2015). It has been suggested that subcellular localization determines the biological effect of ceria NPs in animal cells. Li et al. (2015) demonstrated that ceria NPs could become a long-acting superoxide scavenger in cytosol. While Asati et al. (2010) showed that in lysosomes, ceria NPs became toxic since the acidic microenvironment activated the oxidase activity of ceria NPs. In the present study, ceria NPs led to the biomass increase of common bean plants at 40 mg Ce$L1, while induced an elevation of MDA level in the leaves at 160 mg Ce$L1. More studies at the cellular level are needed to elucidate the mechanism of biological effects of ceria NPs on plants.
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Table 1 Effects of ceria NPs and Ce3þ ions on vegetative growth parameters of common bean. Treatments
Plant height (cm)
No. of leaves/plant
Leaf length
Fresh weight (g)
Dry weight (g)
Dry matter (%)
Control N 40 N 80 N 160 I 40 I 80 I 160
31.0 ± 9.4 28.4 ± 4.6 27.8 ± 12.3 31.0 ± 1.8 29.4 ± 4.8 28.8 ± 6.6 33.2 ± 6.0
23.0 ± 1.7 ab 29.3 ± 2.9 a 21.4 ± 10.9 ab 18.6 ± 3.9 ab 23.3 ± 2.9 ab 30.0 ± 6.5 a 15.8 ± 2.2 b
12.3 ± 0.8 ab 13.1 ± 0.9 a 10.9 ± 0.5 b 11.6 ± 0.1 ab 11.6 ± 0.4 ab 11.5 ± 1.7 ab 11.6 ± 1.1 ab
27.1 ± 4.5 ab 37.9 ± 8.9 a 24.9 ± 12.0 b 26.7 ± 7.1 b 26.7 ± 9.3 b 28.7 ± 10.4 b 23.1 ± 6.6 b
5.02 ± 1.29 b 7.62 ± 2.17 a 4.30 ± 2.57 b 4.78 ± 0.06 ab 4.97 ± 2.09 b 5.64 ± 2.40 ab 4.40 ± 1.38 b
18.3 ± 1.9 19.9 ± 1.1 16.5 ± 2.4 18.7 ± 0.5 18.3 ± 2.0 19.2 ± 1.6 18.4 ± 0.3
Data are average of 5 replicates ± standard deviations. Values in a column followed by different letters have significant difference (p 0.05).
Fig. 4. Effects of ceria NPs and Ce3þ treatments on the contents of MDA in the leaves. Data are average of five replicates ± standard deviations. Different letters stand for statistical differences between treatments at p 0.05.
Fig. 3. Effects of ceria NPs and Ce3þ ions treatments on the activities of SOD, POD, and CAT in the leaves. Data are average of five replicates±standard deviations. Different letters stand for statistical differences between treatments at p 0.05.
Ce3þ ions also have been reported to have both favorable and adverse effects on plants (Hu et al., 2004). From 1970s to 1990s, mixtures of soluble chlorides or nitrates of Ce and other light rare earth elements (La, Pr, and Nd) had been widely used as microfertilizers in China (Hu et al., 2004). Several studies demonstrated that foliar application of appropriate concentrations (54e109 mg RE$L1) of rare earth fertilizers once or twice not only increased plant biomass, but also improved crop quality and resistance against abiotic stresses (He et al., 1998; Niu, 2004; Duan et al., 2007). However, there are also studies showing no such stimulating effect and some even suggest the opposite. For instance, Diatloff et al. (1999) reported that foliar application of rare earth fertilizer once at about 70e2720 mg RE$L1 to maize and mungbean did not increase plant growth, but rather was toxic at concentrations higher than 1360 mg RE$L1. Precise mechanisms of the
physiological and biochemical effects of Ce3þ ions on plants are still poorly understood. The ionic radii of Ce3þ (1.03 Å) is similar to that of Ca2þ (0.99 Å), but its affinity to the Ca2þ binding sites is much higher due to its higher charge density. It is suggested that Ce3þ might interfere with the biological effects of Ca2þ by inhibiting or replacing Ca2þ at the cellular level (Hu et al., 2004). On the other hand, some researchers attribute this controversy effects to hormesis phenomenon which is a dose-response relationship characterized by low-dose stimulation and high-dose inhibition (Pagano et al., 2015). In our study, common bean plants were repeatedly exposed to Ce3þ ions at 40, 80, and 160 mg Ce$L1 for 9 times and resulted relatively high cumulative doses. Consequently, Ce3þ ions produced toxic symptoms on the leaves, especially at high concentrations. 3.4. Effects of foliar treatments on yield and nutritional qualities of bean pods Common bean is a good source of protein, some vitamins and minerals, as well as complex carbohydrates in many parts of the world (de Mejía et al., 2003). Table 2 shows pod yield, number of pod per plant, as well as soluble protein, soluble sugar, reducing sugar, starch, vitamin C, and total phenol contents in the pods harvested from the plants treated with ceria NPs or Ce3þ. None of the treatments significantly affected yields and the nutrient contents of the pods. The influence of ceria NPs and Ce3þ ions on contents of some macronutrients (P, S, and Ca) and micronutrients (Mn, Fe, Cu, Zn, and Mo) in the common bean pods is shown in Table 3. Neither ceria NPs nor Ce3þ ions significantly modified P, Ca, Fe, and Cu
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Table 2 Yields and nutrient contents of the common bean pods harvested from plants foliarly treated with ceria NPs and CeCl3 suspensions/solutions. Data are average of 5 replicates. Treatments
Pod number
Fresh weight (g)
Soluble protein (mg,g1 FW)
Soluble sugar (mg,g1 FW)
Reducing sugar (mg,g1 FW)
Starch (mg,g1 FW).
Vitamin C (mg,g1 FW)
Total phenol (mg,g1 DW)
Control N 40 N 80 N 160 I 40 I 80 I 160
5.4 6.0 3.8 5.2 5.8 6.0 5.4
21.2 26.6 14.7 18.9 24.0 25.2 19.8
18.1 20.7 24.0 18.7 24.0 20.6 24.7
70.8 85.2 58.9 61.6 82.7 85.1 97.2
30.7 33.4 34.2 31.6 34.3 32.0 33.7
11.3 11.8 12.6 15.0 10.9 10.9 12.7
74.6 92.3 78.6 114.2 81.3 77.9 74.6
7.52 7.87 7.40 7.18 7.68 6.54 6.89
Table 3 Mineral contents of the common bean pods harvested from plants treated with ceria NPs and CeCl3 suspensions/solutions. Treatments
P
S
Ca
Mn
Fe
Ctrl N-40 N-80 N-160 I-40 I-80 I-160
4554 ab 4036 b 4403 ab 5076 a 4001 b 5042 a 4433 ab
1229 ab 1355 ab 885 c 1308 ab 1230 b 1531 a 1252 ab
5198 ab 4801 ab 4668 ab 5389 ab 5190 ab 5557 a 4304 b
19.5 a 16.4 ab 14.4 b 18.8 a 17.0 ab 20.5 a 17.7 ab
45.8 79.5 45.5 64.1 76.0 87.1 82.3
a a a a a a a
Cu
Zn
Mo
3.11 ab 3.37 a 2.64 b 3.68 ab 3.09 b 3.47 a 3.32 a
28.0 b 28.6 b 25.6 b 28.3 b 28.8 b 34.9 a 61.5 a
0.66 bc 0.48 e 0.52 de 0.76 ab 0.79 a 0.78 a 0.62 cd
P, S, and Ca were determined by ICP-MS; Mn, Fe, Cu, Zn, and Mo were determined by ICP-OES. Data are average of 5 replicates. Values in a column followed by different letters have significant difference (p 0.05).
contents of the pods when compared with the control. However, the contents of S, Mn, Mo, and Zn were differentially affected by ceria NPs and Ce3þ ions and most of the data did not show any dose dependency. Ceria NPs at 40 and 80 mg Ce L1 significantly reduced pod Mo by 27% and 21%, respectively, compared to controls; meanwhile, S and Mn were reduced by 80 mg L1 Ce Ceria NPs by 28% and 26%, respectively, compared to controls. Ce3þ ions at 40 and 80 mg L1 elevated Mo contents by 20% and 18%, respectively, compared to controls. At 80 and 160 mg L1, Ce3þ ions significantly increased Zn by 25% and 120%, respectively, compared to controls. It has been proved that application of Ce3þ ions as fertilizers can modify mineral contents of cereals, fruits and vegetables but the exact mechanism of this is unknown (Hu et al., 2004). Competition with Ca2þ-binding sites on the cell membranes that influences the ionic fluxes into cells is a possible reason. Recently, several authors reported that both soil and foliar application of ceria NPs affected mineral contents in plant tissues. Hong et al. (2016) found foliar application of ceria NPs at 200 mg L1 reduced cucumber fruit Zn by 25%.21 Peralta-Videa et al. (2014) noted that soybean pods from the plants grown in 500 mg kg1 ceria NPs amended soil had significantly less Ca but more P and Cu compared to control. Rico et al. (2014) reported that ceria NPs reduced S and Mn accumulations in wheat grains. It is speculated that ceria NPs may directly or indirectly interact with certain ion transporters, and thus affect mineral accumulations in plants. More studies are required to understand the causes and mechanism of the mineral accumulations influenced by ceria NPs and Ce3þ ions.
damages in the leaves at the end of the experiment and Ce3þ ions were significantly more toxic than equimolar ceria NPs. Ceria NPs and Ce3þ ions kept their original chemical species after foliar applications, which suggests that the observed effects of ceria NPs and Ce3þ ions on common bean plants were probably due to their nanospecific properties and ionic properties respectively. To have a better understanding of the influence of ceria NPs after foliar exposure, detailed studies on physiological and biochemical effects of ceria NPs on crop plants at different growth stages should be carried out. Declaration of interest The authors declare no competing financial interest. Acknowledgments The authors thank Mr. Tong Zhang, and Ms. Ying Guo for assistance with growing the plants. This work was financially supported by National Natural Science Foundation of China (Grant Nos. 11875267, 11675190, and 11575208) and the Ministry of Science and Technology of China (Grant No. 2016YFA0201604). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.04.042. References
4. Conclusions The effects of ceria NPs on plants after foliar exposure have only been investigated by a few studies, although this pathway is very likely to occur. This comparative study demonstrated that neither ceria NPs nor Ce3þ ions had adverse effect on vegetative growth of common bean plants after repeatedly foliar applications. Chemical constituents of the pods such as soluble protein, soluble sugar, reducing sugar, starch, vitamin C, and total phenol of the pods were not significantly changed, except for several mineral elements. However, both treatments provoked dose-dependent oxidative
Abdel-Aziz, H.M.M., Hasaneen, M.N.A., Omer, A.M., 2016. Nano chitosan-NPK fertilizer enhances the growth and productivity of wheat plants grown in sandy soil. Span. J. Agric. Res. 14, e0902. Adisa, I.O., Reddy Pullagurala, V.L., Rawat, S., Hernandez-Viezcas, J.A., Dimkpa, C.O., Elmer, W.H., White, J.C., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2018. Role of cerium compounds in Fusarium wilt suppression and growth enhancement in tomato (Solanum lycopersicum). J. Agric. Food Chem. 66, 5959e5970. Asati, A., Santra, S., Kaittanis, C., Perez, J.M., 2010. Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles. ACS Nano 4, 5321e5331. Birbaum, K., Brogioli, R., Schellenberg, M., Martinoia, E., Stark, W.J., Günther, D., Limbach, L.K., 2010. No evidence for cerium dioxide nanoparticle translocation in maize plants. Environ. Sci. Technol. 44, 8718e8723. Burkhardt, J., 2010. Hygroscopic particles on leaves, nutrients or desiccants? Ecol.
536
C. Xie et al. / Environmental Pollution 250 (2019) 530e536
Monogr. 80, 369e399. Cao, Z., Stowers, C., Rossi, L., Zhang, W., Lombardini, L., Ma, X., 2017. Physiological effects of cerium oxide nanoparticles on the photosynthesis and water use efficiency of soybean (Glycine max (L.) Merr.). Environ. Sci.: Nano 4, 1086e1094. Dahle, J.T., Arai, Y., 2015. Environmental geochemistry of cerium, applications and toxicology of cerium oxide nanoparticles. Int. J. Environ. Res. Public Health 12, 1253. n-Maldonado, S.H., Acosta-Gallegos, J.A., Reynosode Mejía, E.G., Guzma ndez, J.L., Gonz Camacho, R., Ramírez-Rodríguez, E., Pons-Herna alezChavira, M.M., Castellanos, J.Z., Kelly, J.D., 2003. Effect of cultivar and growing location on the trypsin inhibitors, tannins, and lectins of common beans (Phaseolus vulgaris L.) grown in the semiarid highlands of Mexico. J. Agric. Food Chem. 51, 5962e5966. Deutsch, M.J., Week, C.E., 1965. Microfluorometric assay for vitamin C. J. Assoc. Off. Anal. Chem. 48, 1248e1256. Diatloff, E., Asher, C.J., Smith, F.W., 1999. Foliar application of rare earth elements to maize and mungbean. Aust. J. Exp. Agric. 39, 189e194. Duan, L., Wang, J., Xing, S., 2007. Effects of rare earth micro fertilizer on hot pepper yield and the content of nitrogen, phosphorus and potassium in the plant. Chin. J. Soil Sci. 38, 613e615. DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350e356. He, Y., Wang, J., Fang, N., Gan, W., Zhao, G., 1998. Effects of rare earth micro-fertilizer on plant physiological indexes and yield of hot pepper. Chin. Rare Earth 12, 36e40. Hernandez-Viezcas, J.A., Castillo-Michel, H., Andrews, J.C., Cotte, M., Rico, C., PeraltaVidea, J.R., Ge, Y., Priester, J.H., Holden, P.A., Gardea-Torresdey, J.L., 2013. In situ synchrotron X-ray fluorescence mapping and speciation of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max). ACS Nano 7, 1415e1423. Hong, J., Peralta-Videa, J.R., Rico, C., Sahi, S., Viveros, M.N., Bartonjo, J., Zhao, L., Gardea-Torresdey, J.L., 2014. Evidence of translocation and physiological impacts of foliar applied CeO2 nanoparticles on cucumber (Cucumis sativus) plants. Environ. Sci. Technol. 48, 4376e4385. Hong, J., Wang, L., Sun, Y., Zhao, L., Niu, G., Tan, W., Rico, C.M., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2016. Foliar applied nanoscale and microscale CeO2 and CuO alter cucumber (Cucumis sativus) fruit quality. Sci. Total Environ. 563e564, 904e911. Hu, Z., Richter, H., Sparovek, G., Schnug, E., 2004. Physiological and biochemical effects of rare Earth elements on plants and their agricultural significance, a review. J. Plant Nutr. 27, 183e220. Jayaraman, J., 1981. Laboratory Manual in Biochemistry. Wiley Eastern, New Delhi. Johnson, A.C., Park, B., 2012. Predicting contamination by the fuel additive cerium oxide engineered nanoparticles within the United Kingdom and the associated risks. Environ. Toxicol. Chem. 31, 2582e2587. Ju-Nam, Y., Lead, J.R., 2008. Manufactured nanoparticles, an overview of their chemistry, interactions and potential environmental implications. Sci. Total Environ. 400, 396e414. cillon, L., Bureau, S., Barthe s, V., Larue, C., Castillo-Michel, H., Sobanska, S., Ce re, M., Sarret, G., 2014. Foliar exposure of the crop Lactuca Ouerdane, L., Carrie sativa to silver nanoparticles, Evidence for internalization and changes in Ag speciation. J. Hazard Mater. 264, 98e106. Li, Y., He, X., Yin, J.J., Ma, Y., Zhang, P., Li, J., Ding, Y., Zhang, J., Zhao, Y., Chai, Z., Zhang, Z., 2015. Acquired superoxide-scavenging ability of ceria nanoparticles. Angew Chem. Int. Ed. Engl. 54, 1832e1835. Liu, H.L., Zhou, J., Li, M., Hu, Y.M., Liu, X., Zhou, J., 2019. Study of the bioavailability of heavy metals from atmospheric deposition on the soil-pakchoi (Brassica chinensis L.) system. J. Hazard Mater. 362, 9e16. pez-Moreno, M.L., de la Rosa, G., Herna ndez-Viezcas, J.A., Peralta-Videa, J.R., Lo Gardea-Torresdey, J.L., 2010. X-ray absorption spectroscopy (XAS) corroboration of the uptake and storage of CeO2 nanoparticles and assessment of their differential toxicity in four edible plant species. J. Agric. Food Chem. 58, 3689e3693. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement. J. Biol. Chem. 193, 265e275. Ma, Y., Kuang, L., He, X., Bai, W., Ding, Y., Zhang, Z., Zhao, Y., Chai, Z., 2010. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78, 273e279. Ma, Y., Zhang, P., Zhang, Z., He, X., Zhang, J., Ding, Y., Zhang, J., Zheng, L., Guo, Z., Zhang, L., Chai, Z., Zhao, Y., 2015. Where does the transformation of precipitated ceria nanoparticles in hydroponic plants take place? Environ. Sci. Technol. 49, 10667e10674. Majestic, B.J., Erdakos, G.B., Lewandowski, M., Oliver, K.D., Willis, R.D., Kleindienst, T.E., Bhave, P.V., 2010. A review of selected engineered nanoparticles in the atmosphere, sources, transformations, and techniques for sampling and analysis. Int. J. Occup. Environ. Health 16, 488e507. Majumdar, S., Peralta-Videa, J.R., Bandyopadhyay, S., Castillo-Michel, H., HernandezViezcas, J.-A., Sahi, S., Gardea-Torresdey, J.L., 2014. Exposure of cerium oxide nanoparticles to kidney bean shows disturbance in the plant defense mechanisms. J. Hazard Mater. 278, 279e287. Malik, C.P., Singh, M.B., 1971. Extraction and Estimation of Total Phenols. Kalyani Publication, New Delhi. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426e428. Miralles, P., Church, T.L., Harris, A.T., 2012. Toxicity, uptake, and translocation of
engineered nanomaterials in vascular plants. Environ. Sci. Technol. 46, 9224e9239. Niu, C., 2004. The influence of rare earths on the strong bearing fruit Chinese date. Chin. J. Biol. 21, 40e41. Pagano, G., Guida, M., Tommasi, F., Oral, R., 2015. Health effects and toxicity mechanisms of rare earth elementsdknowledge gaps and research prospects. Ecotoxicol. Environ. Saf. 115, 40e48. Peralta-Videa, J.R., Hernandez-Viezcas, J.A., Zhao, L.J., Diaz, B.C., Ge, Y., Priester, J.H., Holden, P.A., Gardea-Torresdey, J.L., 2014. Cerium dioxide and zinc oxide nanoparticles alter the nutritional value of soil cultivated soybean plants. Plant Physiol. Biochem. 80, 128e135. rez-de-Luque, A., 2017. Interaction of nanomaterials with plants, what do we Pe need for real applications in agriculture? Front. Environ. Sci. 5 (12). Priester, J.H., Ge, Y., Mielke, R.E., Horst, A.M., Moritz, S.C., Espinosa, K., Gelb, J., Walker, S.L., Nisbet, R.M., An, Y.J., Schimel, J.P., Palmer, R.G., HernandezViezcas, J.A., Zhao, L., Gardea-Torresdey, J.L., Holden, P.A., 2012. Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption. Proc. Natl. Acad. Sci. U. S. A. 109, E2451eE2456. €sa €nen, J.V., Leskinen, J.T.T., Holopainen, T., Joutsensaari, J., Pasanen, P., Ra €enp€ €, M., 2017. Titanium dioxide (TiO2) fine particle capture and BVOC Kivima aa emissions of Betula pendula and Betula pubescens at different wind speeds. Atmos. Environ. 152, 345e353. Rico, C.M., Morales, M.I., Barrios, A.C., McCreary, R., Hong, J., Lee, W.Y., Nunez, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2013. Effect of cerium oxide nanoparticles on the quality of rice (Oryza sativa L.) grains. J. Agric. Food Chem. 61, 11278e11285. Rico, C.M., Morales, M.I., McCreary, R., Castillo-Michel, H., Barrios, A.C., Hong, J., Tafoya, A., Lee, W.Y., Varela-Ramirez, A., Peralta-Videa, J.R., GardeaTorresdey, J.L., 2013. Cerium oxide nanoparticles modify the antioxidative stress enzyme activities and macromolecule composition in rice seedlings. Environ. Sci. Technol. 47, 14110e14118. Rico, C.M., Lee, S.C., Rubenecia, R., Mukherjee, A., Hong, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2014. Cerium oxide nanoparticles impact yield and modify nutritional parameters in wheat (Triticum aestivum L.). J. Agric. Food Chem. 62, 9669e9675. Rico, C.M., Johnson, M.G., Marcus, M.A., 2018. Cerium oxide nanoparticles transformation at the rootesoil interface of barley (Hordeum vulgare L.). Environ. Sci.: Nano 5, 1807e1812. Rossi, L., Zhang, W., Ma, X., 2017. Cerium oxide nanoparticles alter the salt stress tolerance of Brassica napus L. by modifying the formation of root apoplastic barriers. Environ. Pollut. 229, 132e138. Rui, Y., Zhang, P., Zhang, Y., Ma, Y., He, X., Gui, X., Li, Y., Zhang, J., Zheng, L., Chu, S., Guo, Z., Chai, Z., Zhao, Y., Zhang, Z., 2015. Transformation of ceria nanoparticles in cucumber plants is influenced by phosphate. Environ. Pollut. 198, 8e14. Salehi, H., Chehregani, A., Lucini, L., Majd, A., Gholami, M., 2018. Morphological, proteomic and metabolomic insight into the effect of cerium dioxide nanoparticles to Phaseolus vulgaris L. under soil or foliar application. Sci. Total Environ. 616e617, 1540e1551. Schwabe, F., Schulin, R., Rupper, P., Rotzetter, A., Stark, W., Nowack, B., 2014. Dissolution and transformation of cerium oxide nanoparticles in plant growth media. J. Nanoparticle Res. 16, 2668. Tassi, E., Giorgetti, L., Morelli, E., Peralta-Videa, J.R., Gardea-Torresdey, J.L., Barbafieri, M., 2017. Physiological and biochemical responses of sunflower (Helianthus annuus L.) exposed to nano-CeO2 and excess boron, Modulation of boron phytotoxicity. Plant Physiol. Biochem. 110, 50e58. Walkey, C., Das, S., Seal, S., Erlichman, J., Heckman, K., Ghibelli, L., Traversa, E., McGinnis, J.F., Self, W.T., 2015. Catalytic properties and biomedical applications of cerium oxide nanoparticles. Environ. Sci.: Nano 2, 33e53. Wang, Q., Ma, X., Zhang, W., Pei, H., Chen, Y., 2012. The impact of cerium oxide nanoparticles on tomato (Solanum lycopersicum L.) and its implications for food safety. Metallomics 4, 1105e1112. Wang, W.-N., Tarafdar, J.C., Biswas, P., 2013. Nanoparticle synthesis and delivery by an aerosol route for watermelon plant foliar uptake. J. Nanoparticle Res. 15, 1417. Xu, C., Qu, X., 2014. Cerium oxide nanoparticle, a remarkably versatile rare earth nanomaterial for biological applications. NPG Asia Mater. 6, e90. Yokel, R.A., Hussain, S., Garantziotis, S., Demokritou, P., Castranova, V., Cassee, F.R., 2014. The yin, an adverse health perspective of nanoceria, uptake, distribution, accumulation, and mechanisms of its toxicity. Environ. Sci.: Nano 1, 406e428. Zhang, Z., He, X., Zhang, H., Ma, Y., Zhang, P., Ding, Y., Zhao, Y., 2011. Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics 3, 816e822. Zhang, P., Ma, Y., Zhang, Z., He, X., Zhang, J., Guo, Z., Tai, R., Zhao, Y., Chai, Z., 2012. Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 6, 9943e9950. Zhang, P., Ma, Y., Zhang, Z., He, X., Li, Y., Zhang, J., Zheng, L., Zhao, Y., 2015. Speciesspecific toxicity of ceria nanoparticles to Lactuca plants. Nanotoxicology 9, 1e8. Zhao, L., Peralta-Videa, J.R., Rico, C.M., Hernandez-Viezcas, J.A., Sun, Y., Niu, G., Servin, A., Nunez, J.E., Duarte-Gardea, M., Gardea-Torresdey, J.L., 2014. CeO2 and ZnO nanoparticles change the nutritional qualities of cucumber (Cucumis sativus). J. Agric. Food Chem. 62, 2752e2759. ndez, D., Du, W., Hernandez-Viezcas, J.A., BonillaZuverza-Mena, N., Martínez-Ferna pez-Moreno, M.L., Kom Bird, N., Lo arek, M., Peralta-Videa, J.R., GardeaTorresdey, J.L., 2017. Exposure of engineered nanomaterials to plants, Insights into the physiological and biochemical responses-A review. Plant Physiol. Biochem. 110, 236e264.
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Effects of foliar applications of ceria nanoparticles and CeCl3 on common bean (Phaseolus vulgaris)* Changjian Xie a, b, 1, Yuhui Ma a, 1, Jie Yang a, Boxin Zhang c, Wenhe Luo a, b, Sheng Feng a, Junzhe Zhang a, b, Guohua Wang a, b, Xiao He a, Zhiyong Zhang a, d, * a
Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China School of Chemistry and Chemical Engineering, University of the Chinese Academy of Sciences, Beijing, 100049, China c International Department, Beijing National Day School, Beijing, 100049, China d School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 100049, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2018 Received in revised form 14 February 2019 Accepted 8 April 2019 Available online 11 April 2019
In this study, comparative effects of foliar application of ceria nanoparticles (NPs) and Ce3þ ions on common bean plants were investigated. Soil grown bean seedlings were exposed to ceria NPs and Ce3þ ions at 0, 40, 80, and 160 mg Ce$L1 every other day at the vegetative growth stage for 17 d. The plants were harvested 47 d after the last treatment. Performed analyses involved growth, physiological and biochemical parameters of the plants and nutritional quality of the pods. Ceria NPs at 40 mg Ce$L1 increased dry weight of the plants by 51.8% over the control. Neither ceria NPs nor Ce3þ ions significantly affected other vegetative growth parameters. Pod yields and nutrient contents except for several mineral elements were also not significantly different among groups. Compared to control, pods from ceria NPs at 80 mg Ce$L1 had significantly less S and Mn. At 40 and 80 mg Ce$L1, ceria NPs reduced pod Mo by 27% and 21%, while Ce3þ ions elevated Mo contents by 20% and 18%, respectively, compared with control. Ce3þ ions at 80 and 160 mg Ce$L1 significantly increased pod Zn by 25% and 120%, respectively, compared with control. At the end of the experiment, Ce3þ ions at 40, 80, and 160 mg Ce$L1 increased contents of malondialdehyde (MDA) by 46%, 65%, and 82% respectively as compared with control. While ceria NPs led to a significant increase of MDA level only at the highest concentration. X-ray absorption near edge structure (XANES) analysis of the leaf samples revealed that both ceria NPs and Ce3þ ions kept their original chemical species after foliar applications, suggesting the observed effects of ceria NPs and Ce3þ ions on the plants were probably due to their nano-specific properties and ionic properties respectively. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Ceria nanoparticles Ce3þ ions Foliar application Nutritional quality Common bean
1. Introduction Cerium, a member of the lanthanides, is the most abundant element of the series in the earth's crust. A unique property of Ce is that it has a stable tetravalent state in contrast to the trivalent state which is dominant for the lanthanides (Ju-Nam and Lead, 2008). Cerium dioxide (ceria) is the most important lanthanide oxides, especially in a nanoparticle formulation, and has been widely used
*
This paper has been recommended for acceptance by Bernd Nowack. * Corresponding author. School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 100049, China. E-mail address: [email protected] (Z. Zhang). 1 The two authors contributed equally to this article. https://doi.org/10.1016/j.envpol.2019.04.042 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
in catalysis, optical and sensor technologies, and medicine (Xu and Qu, 2014). Therefore, ceria NPs are expected to enter the environment deliberately or accidently, where they may pose a potential risk to aquatic and terrestrial organisms (Johnson and Park, 2012). A comprehensive understanding of the interactions between plants and ceria NPs is of extreme importance for assessing the toxicity and possible trophic transfer of these particles (Miralles et al., 2012; Zuverza-Mena et al., 2017). Phytotoxicity studies on ceria NPs were mainly performed by root exposure on plants grown in soil and other media. Numerous studies have documented negative, positive, or no effect of ceria NPs on plant growth (Dahle and Arai, 2015). Ma et al. (2010) found that ceria NPs inhibited root elongation of lettuce but showed no effect on oilseed rape, radish, pez-Moreno wheat, cabbage, tomato and cucumber. Similarly, Lo
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et al. (2010) observed that 2000 mg L1 ceria NPs significantly reduced germination rates of corn, tomato and cucumber but that of alfalfa was unaffected. Priester et al. (2012) reported significant reductions of both plant growth and pod yield of soybean plants cultivated in farm soil amended with ceria NPs. Moreover, Zhao et al. (2014) noticed that exposure to ceria NPs at 400 and 800 mg kg1 soil led to a reduced nutritional quality of cucumber fruits. Similar studies of the same group demonstrated that ceria NPs could modify the nutritional value of two important cereal crops, rice and wheat, which may have a long-term negative effect on food quality (Rico et al., 2013a, 2014). Conversely, Wang et al. (2012) noted ceria NPs at the concentrations of 0.1e10 mg L1 had either an inconsequential or a slightly positive effect on plant growth and tomato production. Recently, ceria NPs were shown to be able to alleviate detrimental effects of abiotic stresses in plants (Rossi et al., 2017; Tassi et al., 2017). Ceria NPs are now widely used as a fuel additive to reduce particulate matter emissions from diesel engines. It can be expected that more and more ceria NPs will be released into the atmosphere (Majestic et al., 2010). Plant leaves are a major sink of aerosol and €sa €nen et al., 2017). Exposure airborne particles (Burkhardt, 2010; Ra of plants to ceria NPs may occur by both wet and dry deposition. However, the impact of ceria NPs on plants after foliar application is only rarely studied. Birbaum et al. (2010) reported that ceria NPs applied as aerosol (0.178 g m3) or suspension (10 mg L1) could be adsorbed on and incorporated in leaves but were not able to translocate within maize plants. However, Hong et al. (2014) found that foliar applied ceria NPs (0, 1, 2, 8, and 8 mg per plant) to cucumber leaves translocated to stems, roots, and flowers of the plants. Moreover, the activities of dehydroascorbate reductase (DHAR), catalase (CAT), and ascorbate peroxidase (APX) in plant tissues were modified. In another study, these authors exposed cucumber seedlings to ceria NPs by foliar application (3.1, 6.2, and 12.5 mg per plant) and found that ceria NPs altered the nutritional status of cucumber fruits and impacted cucumber photosynthetic parameter in seedlings at the highest dose (Hong et al., 2016). Compared with control, net photosynthesis rate, and transpiration rate were decreased by 22% and 11%, respectively. Salehi et al. (2018) reported that foliar spray of ceria NPs altered stomatal density and length, photosynthesis and electron transport chain biochemical machinery of bean seedlings at relatively high doses (16.7, 33.3, 66.7, and 133 mg per plant). Conversely, Adisa et al. (2018) found that foliar application of ceria NPs at 1.25 mg per plant significantly reduced Fusarium wilt severity of tomato plants by 57%. Clearly, more studies are needed, such as those focusing on the species, dosimetry, chronic toxicity, and life cycle assessment, to better understand the effects of ceria NPs on plants after foliar application. This study aimed to investigate physiological and biochemical responses of the common bean plants to ceria NPs and Ce3þ ions after repeated foliar applications. Cerium chloride (CeCl3) was used as an ionic control for comparison. Agronomic and yield characteristics, oxidative stress and antioxidant defense indicators, gas exchange and photosynthetic parameters, and nutritional quality of bean pods were assessed. Additionally, contents and chemical species of Ce in plant tissues were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) and X-ray absorption near edge structure (XANES), respectively. 2. Materials and methods
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G2 20 S-Twin, FEI, Japan) was 6.9 ± 0.4 nm (Fig. S1). The hydrodynamic diameter and zeta-potential of the NPs in ultrapure water (20 mg L1) that determined by dynamic light scattering (DLS, ZetaSizer, Malvern Instruments, UK) were 40.2 ± 7.2 nm and 32.9 ± 8.5 mV, respectively. Ceria NPs were suspended in ultrapure water at 40, 80, and 160 mg Ce$L1 and homogenized by sonication for 30 min before use. CeCl3$7H2O purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai China) was dissolved in ultrapure water at concentrations of 0.29, 0.58, and 1.16 mM (corresponding to ceriumequivalent mass concentrations 40, 80, and 160 mg Ce$L1, respectively). Seeds of common bean (Phaseolus vulgaris L. Ningxing 81e6) purchased from Pingluo Xiongnong Seeds & Seedling Co., Ltd. (Ningxia, China) were surface-sterilized with 3% H2O2 for 20 min and washed thoroughly with ultrapure water. Then the seeds were sown in a plug tray with 50 cells (55 mL/cell) filled with a commercial substrate and germinated in a greenhouse. On day 16 after sowing, uniform seedlings were transferred to 4-L sterile polypropylene pots (one seedling per pot) filled with a substrate. The substrate was prepared with local loam sand soil (13% clay, 55% silt, 32% sand, and 1.1% organic matter content, pH 7.85) and MiracleGro potting mix at a ratio of 1:1 (v/v) by hand mixing with a spatula. The background concentration of Ce in the substrate was 44.4 mg kg1. Thirty-five plants were divided randomly into 7 groups of 5 plants each. One group served as a control and the other 6 experimental groups were exposed to either nanoparticulate or ionic cerium at different concentrations. Ceria NP suspensions or cerium chloride solutions at 40, 80, and 160 mg Ce$L1 were sprayed on the upper surface of each leaf of each plant at 15 d after transplanting. The treatments were applied using 100 mL aerosol hand sprayers and the nozzles were directed toward the leaves from about 5 cm. It could accurately target spray of the leaves with little or even no run-off. Control plants were sprayed with ultrapure water. The neighbor plants and soil were protected with blotting paper to prevent contamination. The foliar application was ended before flowering. A total volume of 435 mL ceria NP suspensions or cerium chloride solutions per treatment were sprayed on the leaves every other day for 17 d. The dosages correspond to approximately 0, 3.5, 7.0 and 13.9 mg Ce per plant, respectively. Transpiration rate (E), stomatal conductance (gs), and net photosynthesis (Pn) were measured with an open gas exchange system (LI-6400XT; Li-Cor Environmental, Lincoln, NE, USA) in 60d old plants. Chlorophyll content in the leaves were analyzed from fully expanded leaves (similar leaves in all groups) using a chlorophyll meter (Konic Minolta SPAD-502 Plus, Japan). Five weeks after the last foliar spray, fully developed pods were harvested and weighed. Then 12 d later, plants were carefully removed from the soil. Physical measurements were recorded such as plant height, leaf length, leaf count, and biomass. Shoots and roots were separated, thoroughly washed with tap water, immersed in 10 mM CaCl2 for 20 min, again twice with ultrapure water. This washing procedure was adapted from Hong and his colleagues’ work (Hong et al., 2014). They found that CaCl2 solution could remove more ceria NPs from the leaf surface compared to HNO3 or water. Leaf samples (3 discs of 1 cm diameter each) were taken from the 4 uppermost fully expanded leaves on each plant, frozen with liquid nitrogen and were stored at 80 C for enzyme assay and lipid peroxidation measurement. Dry weight (DW) of shoots and roots was determined after oven-drying at 60 C for 2 d.
2.1. Experimental design 2.2. Enzyme assay and lipid peroxidation determination Ceria NPs were synthesized using a precipitation method as reported previously and described in detail in Supplementary data (Zhang et al., 2011). The primary size characterized by TEM (Tecnai
Leaf and root samples were homogenized with PBS (50 mM, pH 7.8) under ice bath, and then centrifuged at speed of 10000g and
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4 C for 10 min. The supernatants were kept for analyses of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and malondialdehyde (MDA) contents using the assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The data was collected from 5 replicates/treatment. 2.3. Nutritional quality assessment Nutrient compositions of the pods were determined according to previously established methods, i.e. starch content by the anthrone method of Jayaraman, (1981); reducing sugar by dinitrosalicylic acid method of Miller, (1959); the total soluble sugars were estimated by the methods of DuBois (DuBois et al., 1956). The water-soluble protein was determined by the method of Lowry (Lowry et al., 1951). Vitamin C content was determined using the method of Deutsch and Weeks (Deutsch and Week, 1965). Total phenols of the pods were extracted and estimated according to Malik and Singh (1971). 2.4. Quantification of Ce, macro- and micronutrient contents The dry samples of leaf, root, and pod were ground into fine powders and digested with a mixture of HNO3 and H2O2 (4:1 by volume) on a heating plate. After decomposition, the samples were diluted to 10 mL with 2% (v/v) nitric acid. Ce contents in all samples and 8 mineral element contents in pod samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Elemental X7) or inductively coupled plasma-optical emission spectroscopy (ICP-OES; PerkinElmer). Indium of 20 ng/ mL was used as an internal standard to compensate for the matrix suppression and signal drifting. Analytical runs include calibration verification samples, spike recovery samples and duplicate dilutions. The linearity was from 0.1 to 500 mg L1 of Fe, Mn, Mo, Cu, Zn, and Ce. The linearity was from 0.1 to 20 mg L1 of Ca, P, and S. Standard reference materials GBW07602 and GBW07603 were also digested and analyzed as samples to examine the recovery. The recoveries for all elements were between 84.9% and 128.9% with the relative standard deviation of <1.5% (Table S1).
3. Results and discussion 3.1. Distribution and chemical species Ce in plant tissues Ce contents in the roots, leaves, and pods of the common bean plants are shown in Fig. 1. In the control plants, Ce contents of plant parts varied in the order of root > leaf > pod. This is consistent with the general rare earth elements distribution in plants of the highest concentrations in roots, intermediate values in the above-ground shoots and leaves, and lowest levels in the edible seeds and fruits (Hu et al., 2004). In the present study, the highest Ce contents among all treatments were found in the leaves due to foliar application. Leaves of the plants exposed to ceria NPs or Ce3þ ions at 40, 80, and 160 mg Ce$L1 had 467, 1094, 2999, and 128, 163, 391 mg of Ce per kg DW, respectively. Ceria NPs at all concentrations exhibited significantly higher leaf Ce contents compared with the respective Ce3þ ions. After foliar spray, ceria NPs or Ce3þ either adsorbed to the leaf surface or incorporated into the leaf structure (Birbaum et al., 2010). Using electron microscopy, Birbaum et al. (2010) found that agglomerated ceria NPs on the surface of maize leaves were integrated into surface wax. Therefore, removing of ceria NPs from the leaf surface was difficult than soluble Ce3þ ions. For Ce content in the roots, there was no significant difference between the control and all the treated plants. The pod is the edible part of common bean. Only the highest concentration of Ce3þ (160 mg Ce$L1) produced a significant increase of Ce content in the
2.5. Ce speciation analysis Chemical species of Ce in the roots and leaves of the plants that treated with ceria NPs and CeCl3 at 160 mg L1 Ce were analyzed by synchrotron radiation based X-ray absorption near edge structure. Leaves and roots were lyophilized, ground into fine powders, and pressed into slices with a diameter of 10 mm and a thickness of 2 mm for analyses. Ce LIII edge (5723 eV) spectra of the samples were recorded at ambient temperature in fluorescence mode at beamline 1W1B of the Beijing Synchrotron Radiation Facility (BSRF). The storage ring energy during the collection was 2.5 GeV with current intensity of 50 mA. Spectra of ceria and CePO4 were collected at transmission mode as standard references. Linear combination fitting (LCF) analyses of the XANES spectra were performed on software program ATHENA to identify and quantify Ce species. 2.6. Statistical analysis The data processing was performed on Statistical Packages for the Social Sciences (SPSS) 10.0. All the data were expressed as mean ± standard deviation (n ¼ 5). Statistical significance of differences among treatments were determined using one-way analysis of variance and covariance (ANOVA) with least significant difference (LSD) test or Kruskal-Wallis H ANOVA with MannWhitney U test for multiple comparison. p 0.05 were considered statistically significant.
Fig. 1. Ce contents in the leaves, roots, and pods of the common bean plants that foliarly treated with ceria NPs (indicated as N40, N80, and N160) and Ce3þ (indicated as I40, I80, and I160). Data are average of five replicates±standard deviations. Different letters stand for statistical differences at p 0.05.
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pod when compared with control (2.18 fold). Phaseolus vulgaris L., the plant species used in this study has stomata on both upper and lower leaf surfaces. Several studies have confirmed the foliar uptake nanoparticles by passing through the stomata (Hong et al., 2014; Larue et al., 2014; Abdel-Aziz et al., 2016; Hong et al., 2016; rez-de-Luque, 2017). It was supposed that ceria NPs deposited on Pe the leaves might translocate from the stomatal pathway, pass through the phloem sieve tubes, and finally reach other parts of the plants (Wang et al., 2013). However, the results of this study does not exclude the possible foliar uptake of ceria NPs since natural background of Ce in plant tissues hindered the quantitative determination of translocation of Ce at relatively small amount. Fig. 2 shows the Ce LIII XANES spectra of two Ce model compounds, Ce(IV)O2 and Ce(III)PO4 and leaves that treated with ceria NPs or Ce3þ ions at 160 mg Ce$L1. The spectrum of Ce in the leaves exposed to ceria NPs only shows the feature of Ce(IV) oxidation state. As confirmed by LCF, almost 100% of the Ce in the sample was present as CeO2. Chemical species of Ce in the leaves that treated with Ce3þ also remained unchanged as Ce(III). Unfortunately, Ce contents in the roots and pods were too low to carry out a reliable XANES analysis. We previously found that ceria NPs could undergo transformation at the root surface of hydroponic plants and the released Ce3þ ions caused the phytotoxicity to lactuca plants (Zhang et al., 2015). Moreover, once entered plant tissues, ceria NPs could not be further reduced, even inside the roots (Ma et al., 2015). Biogenic reducing substances (e.g. ascorbic acid, and catechol, etc.) and organic acids from root exudates might play critical roles in the transformation process (Zhang et al., 2012; Rui et al., 2015). Similarly, several reports also stated transformation of ceria NPs in plants cultivated in soil amended with ceria NPs (HernandezViezcas et al., 2013; Majumdar et al., 2014; Rico et al., 2018). Transformation of ceria NPs from Ce(IV) to Ce(III) undergoes a reductive reaction which occurs predominantly in acidic media and needs a reductant (Schwabe et al., 2014). Accordingly, this chemical reaction is unlikely to take place in the aerial parts of a plant. 3.2. Effects of foliar treatments on plant growth and photosynthetic parameters None of the treatments significantly affected plant height, number of leaves, leaf length, and dry matter percentage, compared to those of the control (Table 1). However, ceria NPs at 40 mg Ce$L1
Fig. 2. XANES normalized Ce LIII edge spectra of reference compounds (CePO4 and CeO2) and samples. Straight and dotted lines marked the feature of Ce (Ⅲ) and Ce (Ⅳ) compounds, respectively.
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significantly increased dry weight by 51.8% relative to the control. Moreover, both fresh and dry weights of the plants that treated with ceria NPs at 40 mg Ce$L1 were higher than that of the plants treated with Ce3þ ions at the same concentration. There is few report about the effects of foliar-applied ceria NPs on agronomic characteristics of crop plants. Several studies have demonstrated that the addition of ceria NPs in culture media can improve plant biomass production (Priester et al., 2012; Rico et al., 2014; Cao et al., 2017). The possible mechanisms were discussed in 3.3. Chlorophyll content and photosynthetic parameters such as E, gs, and Pn are considered indicators of photosynthetic responses of green plants under environmental stress. There were no significant differences among treatments for these parameters (Table S1). Liu et al. (2019) found that atmospheric fine particle could block stomata, thus reducing photosynthetic activity. Hong et al. (2014) demonstrated that at a dose of 12.5 mg ceria per plant which was a little lower than the highest dose used here, ceria NPs significantly decreased Pn and E in cucumber leaves. The differences between studies may reflect differential responses to ceria NPs based on plant species or other conditions. 3.3. Effects of foliar treatments on antioxidant enzymes and lipid peroxidation in plant tissues Primary indicators of stress including SOD, POD, CAT, and MDA in the leaves (Figs. 3 and 4) and roots (Fig. S2) were measured to examine the effects of ceria NP and Ce3þ ion treatments on the antioxidant system of common bean plants. In the roots, neither ceria NP nor Ce3þ ion treatments induced significant changes in these indicators compared to the control. In the leaves, the plants exposed to ceria NPs showed no evidence of changes in SOD activity, while plants exposed to 80 and 160 mg L1 Ce3þ showed increases in SOD activity as compared to that of control plants. Lipid peroxidation, an effective indicator of cellular oxidative damage, was measured as MDA levels in our study. In the plants treated with the ceria NPs, the contents of MDA in the leaves showed a tendency of dose-dependent increase, but only the highest Ce level (160 Ce mg$L1) produced a statistically significant elevation. While Ce3þ ions at 40, 80, and 160 mg Ce$L1 increased MDA levels by 46%, 65%, and 82% respectively, as compared with control. Lipid peroxidation causes destruction of lipid components of membrane by membrane impairment and leakage (Rico et al., 2013b). At the end of the experiment, foliar chlorosis and necrosis were observed on the plants treated with Ce3þ ions in a dose-dependent way, especially the older leaves (Fig. S3). The symptoms might be due to an increased cell or tissue damage estimated by MDA production. No obvious chlorosis or necrosis was observed in the plants treated by ceria NPs, which agrees with MDA analysis. Ceria NPs are reported to have both beneficial and toxic effects on biological systems (Yokel et al., 2014; Walkey et al., 2015). The particles can exert a pro-oxidative effect by producing reactive oxygen species (ROS) which damage lipids, proteins, DNA, and cause cell death. On the other hand, ceria NPs have been shown to be excellent free radical scavengers and therefore protecting cells from oxidative damages (Dahle and Arai, 2015). It has been suggested that subcellular localization determines the biological effect of ceria NPs in animal cells. Li et al. (2015) demonstrated that ceria NPs could become a long-acting superoxide scavenger in cytosol. While Asati et al. (2010) showed that in lysosomes, ceria NPs became toxic since the acidic microenvironment activated the oxidase activity of ceria NPs. In the present study, ceria NPs led to the biomass increase of common bean plants at 40 mg Ce$L1, while induced an elevation of MDA level in the leaves at 160 mg Ce$L1. More studies at the cellular level are needed to elucidate the mechanism of biological effects of ceria NPs on plants.
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Table 1 Effects of ceria NPs and Ce3þ ions on vegetative growth parameters of common bean. Treatments
Plant height (cm)
No. of leaves/plant
Leaf length
Fresh weight (g)
Dry weight (g)
Dry matter (%)
Control N 40 N 80 N 160 I 40 I 80 I 160
31.0 ± 9.4 28.4 ± 4.6 27.8 ± 12.3 31.0 ± 1.8 29.4 ± 4.8 28.8 ± 6.6 33.2 ± 6.0
23.0 ± 1.7 ab 29.3 ± 2.9 a 21.4 ± 10.9 ab 18.6 ± 3.9 ab 23.3 ± 2.9 ab 30.0 ± 6.5 a 15.8 ± 2.2 b
12.3 ± 0.8 ab 13.1 ± 0.9 a 10.9 ± 0.5 b 11.6 ± 0.1 ab 11.6 ± 0.4 ab 11.5 ± 1.7 ab 11.6 ± 1.1 ab
27.1 ± 4.5 ab 37.9 ± 8.9 a 24.9 ± 12.0 b 26.7 ± 7.1 b 26.7 ± 9.3 b 28.7 ± 10.4 b 23.1 ± 6.6 b
5.02 ± 1.29 b 7.62 ± 2.17 a 4.30 ± 2.57 b 4.78 ± 0.06 ab 4.97 ± 2.09 b 5.64 ± 2.40 ab 4.40 ± 1.38 b
18.3 ± 1.9 19.9 ± 1.1 16.5 ± 2.4 18.7 ± 0.5 18.3 ± 2.0 19.2 ± 1.6 18.4 ± 0.3
Data are average of 5 replicates ± standard deviations. Values in a column followed by different letters have significant difference (p 0.05).
Fig. 4. Effects of ceria NPs and Ce3þ treatments on the contents of MDA in the leaves. Data are average of five replicates ± standard deviations. Different letters stand for statistical differences between treatments at p 0.05.
Fig. 3. Effects of ceria NPs and Ce3þ ions treatments on the activities of SOD, POD, and CAT in the leaves. Data are average of five replicates±standard deviations. Different letters stand for statistical differences between treatments at p 0.05.
Ce3þ ions also have been reported to have both favorable and adverse effects on plants (Hu et al., 2004). From 1970s to 1990s, mixtures of soluble chlorides or nitrates of Ce and other light rare earth elements (La, Pr, and Nd) had been widely used as microfertilizers in China (Hu et al., 2004). Several studies demonstrated that foliar application of appropriate concentrations (54e109 mg RE$L1) of rare earth fertilizers once or twice not only increased plant biomass, but also improved crop quality and resistance against abiotic stresses (He et al., 1998; Niu, 2004; Duan et al., 2007). However, there are also studies showing no such stimulating effect and some even suggest the opposite. For instance, Diatloff et al. (1999) reported that foliar application of rare earth fertilizer once at about 70e2720 mg RE$L1 to maize and mungbean did not increase plant growth, but rather was toxic at concentrations higher than 1360 mg RE$L1. Precise mechanisms of the
physiological and biochemical effects of Ce3þ ions on plants are still poorly understood. The ionic radii of Ce3þ (1.03 Å) is similar to that of Ca2þ (0.99 Å), but its affinity to the Ca2þ binding sites is much higher due to its higher charge density. It is suggested that Ce3þ might interfere with the biological effects of Ca2þ by inhibiting or replacing Ca2þ at the cellular level (Hu et al., 2004). On the other hand, some researchers attribute this controversy effects to hormesis phenomenon which is a dose-response relationship characterized by low-dose stimulation and high-dose inhibition (Pagano et al., 2015). In our study, common bean plants were repeatedly exposed to Ce3þ ions at 40, 80, and 160 mg Ce$L1 for 9 times and resulted relatively high cumulative doses. Consequently, Ce3þ ions produced toxic symptoms on the leaves, especially at high concentrations. 3.4. Effects of foliar treatments on yield and nutritional qualities of bean pods Common bean is a good source of protein, some vitamins and minerals, as well as complex carbohydrates in many parts of the world (de Mejía et al., 2003). Table 2 shows pod yield, number of pod per plant, as well as soluble protein, soluble sugar, reducing sugar, starch, vitamin C, and total phenol contents in the pods harvested from the plants treated with ceria NPs or Ce3þ. None of the treatments significantly affected yields and the nutrient contents of the pods. The influence of ceria NPs and Ce3þ ions on contents of some macronutrients (P, S, and Ca) and micronutrients (Mn, Fe, Cu, Zn, and Mo) in the common bean pods is shown in Table 3. Neither ceria NPs nor Ce3þ ions significantly modified P, Ca, Fe, and Cu
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Table 2 Yields and nutrient contents of the common bean pods harvested from plants foliarly treated with ceria NPs and CeCl3 suspensions/solutions. Data are average of 5 replicates. Treatments
Pod number
Fresh weight (g)
Soluble protein (mg,g1 FW)
Soluble sugar (mg,g1 FW)
Reducing sugar (mg,g1 FW)
Starch (mg,g1 FW).
Vitamin C (mg,g1 FW)
Total phenol (mg,g1 DW)
Control N 40 N 80 N 160 I 40 I 80 I 160
5.4 6.0 3.8 5.2 5.8 6.0 5.4
21.2 26.6 14.7 18.9 24.0 25.2 19.8
18.1 20.7 24.0 18.7 24.0 20.6 24.7
70.8 85.2 58.9 61.6 82.7 85.1 97.2
30.7 33.4 34.2 31.6 34.3 32.0 33.7
11.3 11.8 12.6 15.0 10.9 10.9 12.7
74.6 92.3 78.6 114.2 81.3 77.9 74.6
7.52 7.87 7.40 7.18 7.68 6.54 6.89
Table 3 Mineral contents of the common bean pods harvested from plants treated with ceria NPs and CeCl3 suspensions/solutions. Treatments
P
S
Ca
Mn
Fe
Ctrl N-40 N-80 N-160 I-40 I-80 I-160
4554 ab 4036 b 4403 ab 5076 a 4001 b 5042 a 4433 ab
1229 ab 1355 ab 885 c 1308 ab 1230 b 1531 a 1252 ab
5198 ab 4801 ab 4668 ab 5389 ab 5190 ab 5557 a 4304 b
19.5 a 16.4 ab 14.4 b 18.8 a 17.0 ab 20.5 a 17.7 ab
45.8 79.5 45.5 64.1 76.0 87.1 82.3
a a a a a a a
Cu
Zn
Mo
3.11 ab 3.37 a 2.64 b 3.68 ab 3.09 b 3.47 a 3.32 a
28.0 b 28.6 b 25.6 b 28.3 b 28.8 b 34.9 a 61.5 a
0.66 bc 0.48 e 0.52 de 0.76 ab 0.79 a 0.78 a 0.62 cd
P, S, and Ca were determined by ICP-MS; Mn, Fe, Cu, Zn, and Mo were determined by ICP-OES. Data are average of 5 replicates. Values in a column followed by different letters have significant difference (p 0.05).
contents of the pods when compared with the control. However, the contents of S, Mn, Mo, and Zn were differentially affected by ceria NPs and Ce3þ ions and most of the data did not show any dose dependency. Ceria NPs at 40 and 80 mg Ce L1 significantly reduced pod Mo by 27% and 21%, respectively, compared to controls; meanwhile, S and Mn were reduced by 80 mg L1 Ce Ceria NPs by 28% and 26%, respectively, compared to controls. Ce3þ ions at 40 and 80 mg L1 elevated Mo contents by 20% and 18%, respectively, compared to controls. At 80 and 160 mg L1, Ce3þ ions significantly increased Zn by 25% and 120%, respectively, compared to controls. It has been proved that application of Ce3þ ions as fertilizers can modify mineral contents of cereals, fruits and vegetables but the exact mechanism of this is unknown (Hu et al., 2004). Competition with Ca2þ-binding sites on the cell membranes that influences the ionic fluxes into cells is a possible reason. Recently, several authors reported that both soil and foliar application of ceria NPs affected mineral contents in plant tissues. Hong et al. (2016) found foliar application of ceria NPs at 200 mg L1 reduced cucumber fruit Zn by 25%.21 Peralta-Videa et al. (2014) noted that soybean pods from the plants grown in 500 mg kg1 ceria NPs amended soil had significantly less Ca but more P and Cu compared to control. Rico et al. (2014) reported that ceria NPs reduced S and Mn accumulations in wheat grains. It is speculated that ceria NPs may directly or indirectly interact with certain ion transporters, and thus affect mineral accumulations in plants. More studies are required to understand the causes and mechanism of the mineral accumulations influenced by ceria NPs and Ce3þ ions.
damages in the leaves at the end of the experiment and Ce3þ ions were significantly more toxic than equimolar ceria NPs. Ceria NPs and Ce3þ ions kept their original chemical species after foliar applications, which suggests that the observed effects of ceria NPs and Ce3þ ions on common bean plants were probably due to their nanospecific properties and ionic properties respectively. To have a better understanding of the influence of ceria NPs after foliar exposure, detailed studies on physiological and biochemical effects of ceria NPs on crop plants at different growth stages should be carried out. Declaration of interest The authors declare no competing financial interest. Acknowledgments The authors thank Mr. Tong Zhang, and Ms. Ying Guo for assistance with growing the plants. This work was financially supported by National Natural Science Foundation of China (Grant Nos. 11875267, 11675190, and 11575208) and the Ministry of Science and Technology of China (Grant No. 2016YFA0201604). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.04.042. References
4. Conclusions The effects of ceria NPs on plants after foliar exposure have only been investigated by a few studies, although this pathway is very likely to occur. This comparative study demonstrated that neither ceria NPs nor Ce3þ ions had adverse effect on vegetative growth of common bean plants after repeatedly foliar applications. Chemical constituents of the pods such as soluble protein, soluble sugar, reducing sugar, starch, vitamin C, and total phenol of the pods were not significantly changed, except for several mineral elements. However, both treatments provoked dose-dependent oxidative
Abdel-Aziz, H.M.M., Hasaneen, M.N.A., Omer, A.M., 2016. Nano chitosan-NPK fertilizer enhances the growth and productivity of wheat plants grown in sandy soil. Span. J. Agric. Res. 14, e0902. Adisa, I.O., Reddy Pullagurala, V.L., Rawat, S., Hernandez-Viezcas, J.A., Dimkpa, C.O., Elmer, W.H., White, J.C., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2018. Role of cerium compounds in Fusarium wilt suppression and growth enhancement in tomato (Solanum lycopersicum). J. Agric. Food Chem. 66, 5959e5970. Asati, A., Santra, S., Kaittanis, C., Perez, J.M., 2010. Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles. ACS Nano 4, 5321e5331. Birbaum, K., Brogioli, R., Schellenberg, M., Martinoia, E., Stark, W.J., Günther, D., Limbach, L.K., 2010. No evidence for cerium dioxide nanoparticle translocation in maize plants. Environ. Sci. Technol. 44, 8718e8723. Burkhardt, J., 2010. Hygroscopic particles on leaves, nutrients or desiccants? Ecol.
536
C. Xie et al. / Environmental Pollution 250 (2019) 530e536
Monogr. 80, 369e399. Cao, Z., Stowers, C., Rossi, L., Zhang, W., Lombardini, L., Ma, X., 2017. Physiological effects of cerium oxide nanoparticles on the photosynthesis and water use efficiency of soybean (Glycine max (L.) Merr.). Environ. Sci.: Nano 4, 1086e1094. Dahle, J.T., Arai, Y., 2015. Environmental geochemistry of cerium, applications and toxicology of cerium oxide nanoparticles. Int. J. Environ. Res. Public Health 12, 1253. n-Maldonado, S.H., Acosta-Gallegos, J.A., Reynosode Mejía, E.G., Guzma ndez, J.L., Gonz Camacho, R., Ramírez-Rodríguez, E., Pons-Herna alezChavira, M.M., Castellanos, J.Z., Kelly, J.D., 2003. Effect of cultivar and growing location on the trypsin inhibitors, tannins, and lectins of common beans (Phaseolus vulgaris L.) grown in the semiarid highlands of Mexico. J. Agric. Food Chem. 51, 5962e5966. Deutsch, M.J., Week, C.E., 1965. Microfluorometric assay for vitamin C. J. Assoc. Off. Anal. Chem. 48, 1248e1256. Diatloff, E., Asher, C.J., Smith, F.W., 1999. Foliar application of rare earth elements to maize and mungbean. Aust. J. Exp. Agric. 39, 189e194. Duan, L., Wang, J., Xing, S., 2007. Effects of rare earth micro fertilizer on hot pepper yield and the content of nitrogen, phosphorus and potassium in the plant. Chin. J. Soil Sci. 38, 613e615. DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350e356. He, Y., Wang, J., Fang, N., Gan, W., Zhao, G., 1998. Effects of rare earth micro-fertilizer on plant physiological indexes and yield of hot pepper. Chin. Rare Earth 12, 36e40. Hernandez-Viezcas, J.A., Castillo-Michel, H., Andrews, J.C., Cotte, M., Rico, C., PeraltaVidea, J.R., Ge, Y., Priester, J.H., Holden, P.A., Gardea-Torresdey, J.L., 2013. In situ synchrotron X-ray fluorescence mapping and speciation of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max). ACS Nano 7, 1415e1423. Hong, J., Peralta-Videa, J.R., Rico, C., Sahi, S., Viveros, M.N., Bartonjo, J., Zhao, L., Gardea-Torresdey, J.L., 2014. Evidence of translocation and physiological impacts of foliar applied CeO2 nanoparticles on cucumber (Cucumis sativus) plants. Environ. Sci. Technol. 48, 4376e4385. Hong, J., Wang, L., Sun, Y., Zhao, L., Niu, G., Tan, W., Rico, C.M., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2016. Foliar applied nanoscale and microscale CeO2 and CuO alter cucumber (Cucumis sativus) fruit quality. Sci. Total Environ. 563e564, 904e911. Hu, Z., Richter, H., Sparovek, G., Schnug, E., 2004. Physiological and biochemical effects of rare Earth elements on plants and their agricultural significance, a review. J. Plant Nutr. 27, 183e220. Jayaraman, J., 1981. Laboratory Manual in Biochemistry. Wiley Eastern, New Delhi. Johnson, A.C., Park, B., 2012. Predicting contamination by the fuel additive cerium oxide engineered nanoparticles within the United Kingdom and the associated risks. Environ. Toxicol. Chem. 31, 2582e2587. Ju-Nam, Y., Lead, J.R., 2008. Manufactured nanoparticles, an overview of their chemistry, interactions and potential environmental implications. Sci. Total Environ. 400, 396e414. cillon, L., Bureau, S., Barthe s, V., Larue, C., Castillo-Michel, H., Sobanska, S., Ce re, M., Sarret, G., 2014. Foliar exposure of the crop Lactuca Ouerdane, L., Carrie sativa to silver nanoparticles, Evidence for internalization and changes in Ag speciation. J. Hazard Mater. 264, 98e106. Li, Y., He, X., Yin, J.J., Ma, Y., Zhang, P., Li, J., Ding, Y., Zhang, J., Zhao, Y., Chai, Z., Zhang, Z., 2015. Acquired superoxide-scavenging ability of ceria nanoparticles. Angew Chem. Int. Ed. Engl. 54, 1832e1835. Liu, H.L., Zhou, J., Li, M., Hu, Y.M., Liu, X., Zhou, J., 2019. Study of the bioavailability of heavy metals from atmospheric deposition on the soil-pakchoi (Brassica chinensis L.) system. J. Hazard Mater. 362, 9e16. pez-Moreno, M.L., de la Rosa, G., Herna ndez-Viezcas, J.A., Peralta-Videa, J.R., Lo Gardea-Torresdey, J.L., 2010. X-ray absorption spectroscopy (XAS) corroboration of the uptake and storage of CeO2 nanoparticles and assessment of their differential toxicity in four edible plant species. J. Agric. Food Chem. 58, 3689e3693. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement. J. Biol. Chem. 193, 265e275. Ma, Y., Kuang, L., He, X., Bai, W., Ding, Y., Zhang, Z., Zhao, Y., Chai, Z., 2010. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78, 273e279. Ma, Y., Zhang, P., Zhang, Z., He, X., Zhang, J., Ding, Y., Zhang, J., Zheng, L., Guo, Z., Zhang, L., Chai, Z., Zhao, Y., 2015. Where does the transformation of precipitated ceria nanoparticles in hydroponic plants take place? Environ. Sci. Technol. 49, 10667e10674. Majestic, B.J., Erdakos, G.B., Lewandowski, M., Oliver, K.D., Willis, R.D., Kleindienst, T.E., Bhave, P.V., 2010. A review of selected engineered nanoparticles in the atmosphere, sources, transformations, and techniques for sampling and analysis. Int. J. Occup. Environ. Health 16, 488e507. Majumdar, S., Peralta-Videa, J.R., Bandyopadhyay, S., Castillo-Michel, H., HernandezViezcas, J.-A., Sahi, S., Gardea-Torresdey, J.L., 2014. Exposure of cerium oxide nanoparticles to kidney bean shows disturbance in the plant defense mechanisms. J. Hazard Mater. 278, 279e287. Malik, C.P., Singh, M.B., 1971. Extraction and Estimation of Total Phenols. Kalyani Publication, New Delhi. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426e428. Miralles, P., Church, T.L., Harris, A.T., 2012. Toxicity, uptake, and translocation of
engineered nanomaterials in vascular plants. Environ. Sci. Technol. 46, 9224e9239. Niu, C., 2004. The influence of rare earths on the strong bearing fruit Chinese date. Chin. J. Biol. 21, 40e41. Pagano, G., Guida, M., Tommasi, F., Oral, R., 2015. Health effects and toxicity mechanisms of rare earth elementsdknowledge gaps and research prospects. Ecotoxicol. Environ. Saf. 115, 40e48. Peralta-Videa, J.R., Hernandez-Viezcas, J.A., Zhao, L.J., Diaz, B.C., Ge, Y., Priester, J.H., Holden, P.A., Gardea-Torresdey, J.L., 2014. Cerium dioxide and zinc oxide nanoparticles alter the nutritional value of soil cultivated soybean plants. Plant Physiol. Biochem. 80, 128e135. rez-de-Luque, A., 2017. Interaction of nanomaterials with plants, what do we Pe need for real applications in agriculture? Front. Environ. Sci. 5 (12). Priester, J.H., Ge, Y., Mielke, R.E., Horst, A.M., Moritz, S.C., Espinosa, K., Gelb, J., Walker, S.L., Nisbet, R.M., An, Y.J., Schimel, J.P., Palmer, R.G., HernandezViezcas, J.A., Zhao, L., Gardea-Torresdey, J.L., Holden, P.A., 2012. Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption. Proc. Natl. Acad. Sci. U. S. A. 109, E2451eE2456. €sa €nen, J.V., Leskinen, J.T.T., Holopainen, T., Joutsensaari, J., Pasanen, P., Ra €enp€ €, M., 2017. Titanium dioxide (TiO2) fine particle capture and BVOC Kivima aa emissions of Betula pendula and Betula pubescens at different wind speeds. Atmos. Environ. 152, 345e353. Rico, C.M., Morales, M.I., Barrios, A.C., McCreary, R., Hong, J., Lee, W.Y., Nunez, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2013. Effect of cerium oxide nanoparticles on the quality of rice (Oryza sativa L.) grains. J. Agric. Food Chem. 61, 11278e11285. Rico, C.M., Morales, M.I., McCreary, R., Castillo-Michel, H., Barrios, A.C., Hong, J., Tafoya, A., Lee, W.Y., Varela-Ramirez, A., Peralta-Videa, J.R., GardeaTorresdey, J.L., 2013. Cerium oxide nanoparticles modify the antioxidative stress enzyme activities and macromolecule composition in rice seedlings. Environ. Sci. Technol. 47, 14110e14118. Rico, C.M., Lee, S.C., Rubenecia, R., Mukherjee, A., Hong, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2014. Cerium oxide nanoparticles impact yield and modify nutritional parameters in wheat (Triticum aestivum L.). J. Agric. Food Chem. 62, 9669e9675. Rico, C.M., Johnson, M.G., Marcus, M.A., 2018. Cerium oxide nanoparticles transformation at the rootesoil interface of barley (Hordeum vulgare L.). Environ. Sci.: Nano 5, 1807e1812. Rossi, L., Zhang, W., Ma, X., 2017. Cerium oxide nanoparticles alter the salt stress tolerance of Brassica napus L. by modifying the formation of root apoplastic barriers. Environ. Pollut. 229, 132e138. Rui, Y., Zhang, P., Zhang, Y., Ma, Y., He, X., Gui, X., Li, Y., Zhang, J., Zheng, L., Chu, S., Guo, Z., Chai, Z., Zhao, Y., Zhang, Z., 2015. Transformation of ceria nanoparticles in cucumber plants is influenced by phosphate. Environ. Pollut. 198, 8e14. Salehi, H., Chehregani, A., Lucini, L., Majd, A., Gholami, M., 2018. Morphological, proteomic and metabolomic insight into the effect of cerium dioxide nanoparticles to Phaseolus vulgaris L. under soil or foliar application. Sci. Total Environ. 616e617, 1540e1551. Schwabe, F., Schulin, R., Rupper, P., Rotzetter, A., Stark, W., Nowack, B., 2014. Dissolution and transformation of cerium oxide nanoparticles in plant growth media. J. Nanoparticle Res. 16, 2668. Tassi, E., Giorgetti, L., Morelli, E., Peralta-Videa, J.R., Gardea-Torresdey, J.L., Barbafieri, M., 2017. Physiological and biochemical responses of sunflower (Helianthus annuus L.) exposed to nano-CeO2 and excess boron, Modulation of boron phytotoxicity. Plant Physiol. Biochem. 110, 50e58. Walkey, C., Das, S., Seal, S., Erlichman, J., Heckman, K., Ghibelli, L., Traversa, E., McGinnis, J.F., Self, W.T., 2015. Catalytic properties and biomedical applications of cerium oxide nanoparticles. Environ. Sci.: Nano 2, 33e53. Wang, Q., Ma, X., Zhang, W., Pei, H., Chen, Y., 2012. The impact of cerium oxide nanoparticles on tomato (Solanum lycopersicum L.) and its implications for food safety. Metallomics 4, 1105e1112. Wang, W.-N., Tarafdar, J.C., Biswas, P., 2013. Nanoparticle synthesis and delivery by an aerosol route for watermelon plant foliar uptake. J. Nanoparticle Res. 15, 1417. Xu, C., Qu, X., 2014. Cerium oxide nanoparticle, a remarkably versatile rare earth nanomaterial for biological applications. NPG Asia Mater. 6, e90. Yokel, R.A., Hussain, S., Garantziotis, S., Demokritou, P., Castranova, V., Cassee, F.R., 2014. The yin, an adverse health perspective of nanoceria, uptake, distribution, accumulation, and mechanisms of its toxicity. Environ. Sci.: Nano 1, 406e428. Zhang, Z., He, X., Zhang, H., Ma, Y., Zhang, P., Ding, Y., Zhao, Y., 2011. Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics 3, 816e822. Zhang, P., Ma, Y., Zhang, Z., He, X., Zhang, J., Guo, Z., Tai, R., Zhao, Y., Chai, Z., 2012. Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 6, 9943e9950. Zhang, P., Ma, Y., Zhang, Z., He, X., Li, Y., Zhang, J., Zheng, L., Zhao, Y., 2015. Speciesspecific toxicity of ceria nanoparticles to Lactuca plants. Nanotoxicology 9, 1e8. Zhao, L., Peralta-Videa, J.R., Rico, C.M., Hernandez-Viezcas, J.A., Sun, Y., Niu, G., Servin, A., Nunez, J.E., Duarte-Gardea, M., Gardea-Torresdey, J.L., 2014. CeO2 and ZnO nanoparticles change the nutritional qualities of cucumber (Cucumis sativus). J. Agric. Food Chem. 62, 2752e2759. ndez, D., Du, W., Hernandez-Viezcas, J.A., BonillaZuverza-Mena, N., Martínez-Ferna pez-Moreno, M.L., Kom Bird, N., Lo arek, M., Peralta-Videa, J.R., GardeaTorresdey, J.L., 2017. Exposure of engineered nanomaterials to plants, Insights into the physiological and biochemical responses-A review. Plant Physiol. Biochem. 110, 236e264.