http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, Early Online: 1–8 ! 2013 Informa UK Ldt. DOI: 10.3109/17435390.2013.855829
ORIGINAL ARTICLE
Species-specific toxicity of ceria nanoparticles to Lactuca plants Peng Zhang1, Yuhui Ma1, Zhiyong Zhang1, Xiao He1, Yuanyuan Li1, Jing Zhang2, Lirong Zheng2, and Yuliang Zhao1 1
Key Laboratory of Nuclear Analytical Techniques, Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Beijing, China and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China
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Abstract
Keywords
Species-specific differences in the toxicity of manufactured nanoparticles (MNPs) have been reported, but the underlying mechanisms are unknown. We previously found that CeO2 NPs inhibited root elongation of head lettuce, whereas no toxic effect was observed on other plants (such as wheat, cucumber and radish). In this study, interactions between Lactuca plants and three types of CeO2 NPs (lab-synthesized 7 and 25 nm CeO2 NPs, and a commercial CeO2 NPs) were investigated. It was found that CeO2 NPs were toxic to three kinds of Lactuca genus plants and different CeO2 NPs showed different degrees of toxicity. The results of X-ray absorption near edge fine structure indicate that small parts of CeO2 NPs were transformed from Ce(IV) to Ce(III) in roots of the plants that were treated with CeO2 NPs during the seed germination stage. But the high sensitivity of Lactuca plants to the released Ce3þ ions caused the species-specific phytotoxicity of CeO2 NPs. Differences in sizes and zeta potentials among three types of CeO2 NPs resulted in their different degrees of biotransformation which accounted for the discrepancy in the toxicity to Lactuca plants. This study is among the few, and may indeed the first, that addresses the relation between the physicochemical properties of nanoparticles and its species-specific phytotoxicity.
Biotransformation, CeO2, Lactuca plants, nanoparticles, phytotoxicity
Introduction Recent advances in nanoscience and nanotechnology have led to the dramatic growth of the global nanotechnology industry. Many new nanomaterials with unique physical and chemical properties have been synthesized and incorporated into a variety of industrial and commercial products. The release of these nano-sized particles into the environment is inevitable and their potential impacts on the environment and human health have aroused great concern (Handy et al., 2008; Nel et al., 2006; Zhao et al., 2008). Terrestrial plants interact directly with the soil, water and atmospheric environmental compartments, all of which can be routes of MNP distribution. Therefore, they are more easily exposed to the MNPs and harmed. Moreover, concern has been expressed regarding the possibility of trophic transfer of plant accumulated NPs to herbivorous consumers (Judy et al., 2011; Holbrook et al., 2008). Studies pertaining to the impact of MNPs on plants are mostly based on the common toxicity endpoints (e.g. germination rate, root elongation and biomass; Lin & Xing, 2007; Zhang et al., 2012a). Phytotoxicity of MNPs at cellular and genetic level was also studied recently (Ghosh et al., 2010; Liu et al., 2010). However, there are always controversial results and no general patterns could be concluded. The phytotoxicity of MNPs is influenced by various factors, such as chemical composition, shape, surface charge, aggregation and solubility of the
Correspondence: Prof. Zhiyong Zhang, Key Laboratory of Nuclear Analytical Techniques, Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China. Tel: +86-1088233215; Fax: +86-10-88235294. E-mail: [email protected]
History Received 6 June 2013 Revised 5 September 2013 Accepted 25 September 2013 Published online 20 November 2013
nanomaterials, as well as the plant species in question (Rico et al., 2011). Species-specific differences in the phytotoxicity of several MNPs have been reported. Lee et al. (2008) found mungbean was more sensitive to the toxicity of copper NPs than wheat. Graphene with increasing concentration significantly inhibited the seedling growth of cabbage, tomato and red spinach while showed no toxicity to lettuce (Begum et al., 2011). It is considered that discrepancy of root structure (xylem and phloem) in different plant species (monocot or dicot plants) may lead to different uptake of NPs and account for the different toxicity across different plant species. However, no conclusions on the mechanism can be made (Ma et al., 2010a). More research should be carried out on diverse plant species to provide more data for the risk assessment of nanoparticles. CeO2 NPs have been widely used for various applications such as catalytic converters for automobile exhaust, ultraviolet absorber, and electrolyte in fuel cells (Cassee et al., 2011). It is among the MNPs on the OECD (organization for economic cooperation and development) list that are required immediate test of their toxicity. Priester et al. (2012) found that CeO2 NPs diminished the plant growth and pod yield of the soil-grown soybeans and shut the nitrogen fixation down at high concentration. Wang et al. (2012) reported that CeO2 NPs at concentrations of 0.1–10 mg/L had either an inconsequential or a slightly positive effect on plant growth and tomato production. In a previous work, we found that 2000 mg/L CeO2 NPs significantly inhibited the root elongation of head lettuce at the germination stage while showed no effect to the other six tested plant species (Ma et al., 2010b). To explore the mechanisms involved in the toxicity of CeO2 NPs to head lettuce, in this study, we tested the following hypotheses: (1) CeO2 NPs are not only toxic to head lettuce, but also to other Lactuca plants; (2) different CeO2 NPs
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show different degrees of toxicity; and (3) the phytotoxicity of CeO2 NPs is caused by release of Ce3þ ions instead of nanoeffect. Effects of three types of CeO2 NPs (lab-synthesized 7 and 25 nm CeO2 NPs, and a commercial CeO2 NPs) on three kinds of Lactuca plants were investigated. Ce species in the plant roots were analyzed by synchrotron based X-ray absorption spectroscopy. Toxicity of Ce3þ to lettuce and other plants were also evaluated for comparison.
Methods
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Chemicals and seeds CeO2 NPs with the sizes of 7 and 25 nm were synthesized by precipitation methods as described previously (Zhang et al., 2011). Commercial CeO2 NPs were purchased from Sigma Aldrich (USA). Seeds of three kinds of Lactuca genus plants: head lettuce (Lactuca sativa var. capitata L.), romaine lettuce (Lactuca sativa L. var. longifolia Lam), asparagus lettuce (Lactuca sativa Linn. var. angustata Irish ex Bremer) and seeds of cucumber (Cucumis sativus L.), wheat (Triticum aestivum L.) and radish (Raphanus sativus L.), were purchased from the Chinese Academy of Agricultural Sciences and kept in a refrigerator at 4 C for use. Average germination rates of the seeds were examined to be greater than 85%. Characterization of nanoparticles Transmission electron microscopy (TEM, JEM-2010, Japan) was applied to characterize the morphology and the average sizes of the CeO2 NPs. X-ray diffraction (XRD, X’pert PRO MPD, Holland) was used to determine their crystal forms. Suspensions of CeO2 NPs (20 mg/L) were prepared for measurement of hydrodynamic sizes and zeta potential (Nicomp 380 ZLS Zeta potential/Particle system, Santa Barbara, CA, USA). Seed germination and NPs treatment Uniform seeds were selected and immersed in a 10% sodium hypochlorite solution for 10 min and rinsed with deionized water to ensure surface sterility. CeO2 NP suspensions of 2000 mg/L in deionized water were prepared and ultrasonically dispersed for 15 min under water bath. Then the seeds were soaked in deionized water (for control) or NP suspensions for 2 h. One piece of filter paper was place in a 100 mm 15 mm Petri dish and added with 5 mL of the test media. Fourteen seeds were then arrayed in each dish with a certain distance between them. All the Petri dishes were sealed with tapes and then placed in the dark under 25 C in a climate incubator. For each treatment, four replicates were set. At the fifth day, the germination was halted. Germination rates were calculated. Root lengths were measured by a meter ruler. Stress response of head lettuce to CeO2 NPs After 5 days germination, roots of head lettuce were excised, homogenized with PBS (50 mM, pH 7.8) under ice bath, and then centrifuged at speed of 10 000g and 4 C for 10 min. The supernatants were kept for analyses of SOD, POD activities and MDA contents using the assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). H2O2 accumulation in roots of head lettuces treated with 2000 mg/L CeO2 NPs were analyzed by means of ROS-sensitive dye DAB (Tarasenko et al., 2012). H2O2 can be localized in roots by imaging the insoluble dark brown formazan produced by reaction of H2O2 with DAB. After being washed with deionized water carefully, root segments of 20 mm from the apices were excised and incubated in 0.1 mg/L DAB (diaminobenzidine) solution (pH 3.2) at room temperature for 30 min in the absence of
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light. Then the root segments were rinsed thoroughly with deionized water, and the images were photographed using a Digital Single Lens Reflex (Olympus, Japan). For quantitative assessment, the root segments were rinsed with deionized water thoroughly after incubation in DAB solution, grinded in a mixture of KOH and DMSO at a ratio of 1:1.167 (v/v) and then centrifuged at 10 000 g for 10 min. The absorbance of the supernatants was recorded at 700 nm on a Multimode Microplate Reader (SpectraMax M2, USA). Root cell deaths of head lettuce treated with 2000 mg/L CeO2 NPs were evaluated by Evan’s blue staining (Baker & Mock, 1994). Root segments of 20 mm from the apices were excised and incubated in 0.25% (w/v) aqueous solution of Evan’s blue for 5 h and then rinsed with deionized water thoroughly. The images were photographed using a Digital Single Lens Reflex (Olympus, Japan). For quantitative assessment, after Evan’s blue staining, root segments were thoroughly washed with deionized water and then immersed in 1% (w/v) SDS in 50% (v/v) methanol at 50 C for 15 min, and the absorbance was measured at 600 nm. Determination of Ce contents in head lettuce After 5 days germination, roots and shoots of head lettuce were separated, washed thoroughly with deionized water and lyophilized with a freeze dryer (Alpha 1-2 LD plus, Christ, Germany). The dried tissues were digested with a mixture of concentrated plasma–pure HNO3 and H2O2 (vol/vol, 4:1) on a heating plate. The obtained residual solutions were then diluted with deionized water and analyzed by ICP-MS (Thermo X7, USA). A standard reference (bush branches and leaves, GBW07602) was also digested and analyzed by ICP-MS to examine the recovery. 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 50 ng/mL, Recovery from GBW07602 was 99%. Spike recovery was 102%. Relative standard deviation was 2.5%. Detection limit is 0.01 ng/mL. X-ray absorption near edge fine structure spectroscopy of head lettuce roots After 5 days germination, roots of head lettuces were separated from the seedlings and lyophilized. The dried roots were grinded to powder and pressed into slices with a diameter of 10 mm and a thickness of 2 mm for the X-ray absorption near edge fine structure spectroscopy (XANES) analysis. XANES spectra were collected on beamline 1W1B at Beijing Synchrotron Radiation Facility (BSRF). The ring storage energy of the synchrotron radiation accelerator during data collection was 2.5 GeV with current intensity of 50 mA. CeLIII-edge (5723 eV) spectra were collected at ambient temperature in the fluorescence mode. CePO4 and Ce(CH3COO)3 as well as the three types of CeO2 NPs were used as the standard compounds. Data processing and the linear combination fitting (LCF) of the collected spectra were performed in the software program ATHENA. Samples of cucumber and wheat treated with the three types of CeO2 NPs (2000 mg/L) were also prepared for the XANES analysis. Impact of Ce3þ ions on root growth of different plant species According to LCF analyses of the XNAES spectra, we calculated the total quantities of Ce(III) in the roots and converted them into Ce3þ concentrations in each petri dish. A calculation method was shown in Supplementary Material (ESM). In each petri dish, the estimated concentrations of Ce3þ were 0.883, 0.464 and
Toxicity of ceria NPs to Lactuca plants
DOI: 10.3109/17435390.2013.855829
0.058 mg/L for 7 nm, 25 nm and Sigma CeO2 NPs, respectively. Based on the calculation, seeds of head lettuce, wheat, cucumber and radish were exposed to 0.5, 1, 5, 10 and 20 mg/L Ce3þ [as Ce(NO3)3] to determine the sensitivity of different plant species to Ce3þ ions. After 5 days germination, root lengths were measured by a rule meter.
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Transformation of the CeO2 NPs in simulated reaction solutions Organic acids and reducing substances in the rhizosphere are required for the transformation of CeO2 (Zhang et al., 2012b). Herein, four reaction solutions: citric acid þ ascorbic acid (Vc), citric acid þ catechol, EDTA þ Vc and EDTA þ catechol, which are composed of an organic acid and a reducing substances, were prepared. Citric acid is an organic acid widely existing in plants; EDTA is a commonly used organic acid with high complex ability to metal ions; Vc and catechol are reducing substances commonly existing in plants. The final concentrations of all the components were 1 mM and the pH were adjusted to 5.5. CeO2 NPs were added into the solutions with stirring and ultrasonication for 15 min. The final concentrations of CeO2 NPs were 2000 mg/L. After 5 days incubation, the solutions were centrifuged at 10 000 g for 15 min and the supernatants were filtrated through 0.2 mm polytetrafluoroethylene filters. The obtained clear solutions were diluted with 2% HNO3 and Ce concentrations were determined by ICP-MS. Statistical analysis The data processing was performed on Statistical Packages for the Social Sciences (SPSS, Chicago, IL) 10.0. All the data were expressed as mean SD (standard deviation). One-way ANOVA and S–N–K test were performed to examine the statistic difference. p50.05 was considered to be a significant difference.
Results
Effect of CeO2 NPs on root elongation of Lactuca plants Seed germination of head lettuce, romaine lettuce and asparagus lettuce was not affected by the CeO2 NPs exposure (Supplemental Figure S1). Figure 2(A and B) shows the root length of the three Lactuca plants that exposed to 2000 mg/L 7 nm, 25 nm and Sigma CeO2. It was observed that root growths of the three Lactuca plants were significantly inhibited by the three types of CeO2 NPs, except for the Sigma CeO2 NP treated romaine lettuce. Among all the CeO2 NPs tested, 7 nm CeO2 showed the most severe toxic sign, while 25 nm CeO2 caused more inhibitory effect to the root growth of romaine lettuce than Sigma CeO2. Statistical analysis between different species under same CeO2 NP treatment was performed as well and no significant difference was observed. Therefore, only head lettuce was used as a representative to explore the mechanism of the phytotoxicity in the following studies. Stress response of Lactuca plants to CeO2 NPs In Figure 3(A, inset), CeO2 NPs treated lettuce roots clearly exhibited dark brown color compared to the control, indicating an over accumulation of H2O2. Quantitative results show that 7 nm CeO2 NPs triggered a significant increase of H2O2 accumulation in roots as compared to the control. We further determined whether there were cell deaths occurred in the roots using Evan’s blue as a cell death marker. Compared to the control, deep blue color in the zone above the root tips was observed for the 7 nm CeO2 treatment and light blue for the 25 nm and sigma CeO2 treatments, which indicating the cell membrane damage and cell death of the roots (Figure 3B). The high dye retention in the roots indicates the loss of plasma membrane integrity. As can be seen from the quantitative data of the extracted Evan’s blue, CeO2
Table 1. Physicochemical properties of CeO2 NPs.
Physicochemical characteristics of CeO2 NPs TEM images of the CeO2 NPs used in this study are shown in Figure 1; their physicochemical properties are described in Table 1. The average sizes of the lab-synthesized CeO2 NPs are 6.9 0.5 and 25.2 2.3 nm, respectively. Commercial CeO2 NPs (called ‘‘Sigma CeO2’’ in the following text) contain particles of diameters ranging from 6.2 up to 48 nm, with the centre of the distribution at 16.3 nm. From the data of zeta potential and hydrodynamic size, we can see that 7 and 25 nm CeO2 NP suspensions were more stable and dispersive than the Sigma CeO2 NPs.
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7 nm CeO2
25 nm CeO2
a
a
Sigma CeO2
Particle size (nm) 6.9 0.5 25.2 2.3 25b Crystal form Cubic fluoritec Cubic fluoritec Cubic fluoritec Purity (%) 99.98%d 99.99%d 99.9%b Hydrodynamic size (nm)e 38.3 6.5 103.0 15.8 149.1 23.5 Zeta potential (mV)e 32.9 2.8 30.9 3.5 13.8 4.0 a
Data from TEM. Data provided by producer. c Data from XRD (data not shown). d Data from ICP-MS. e Data measured by DLS of 20 mg/L CeO2 NP suspensions in deionized water. b
Figure 1. TEM images of 7 nm CeO2 NPs (A), 25 nm CeO2 NPs (B) and Sigma CeO2 NPs (C).
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Figure 2. (A) Root lengths of the Lactuca plants treated with 2000 mg/L CeO2 NPs for 5 days; (B) Image of head lettuce seedlings treated with 2000 mg/L CeO2 NPs for 5 days. All the values were expressed as mean SD of four replicates with 14 seeds each. Different letters show significant differences (p50.05).
NPs-treated roots showed significant (for 7 and 25 nm CeO2 treatment) or non-significant (for Sigma CeO2 NPs treatment) increase of absorbance compared to the control. This suggests that the CeO2 NPs exposure caused membrane damage and finally the cell death of the roots. Plants have developed a series of defense mechanisms including enzymatic and non-enzymatic antioxidant system when encounter the external stress including oxidative stress. As seen from Figure 3(C), SOD activities were significantly upregulated by CeO2 NPs exposure compared to the control. Similar to SOD, POD activities in the roots were also up-regulated by CeO2 NPs and showed a significant increase for 7 nm CeO2 NPs treatment compared to the control. These suggest that lettuce roots increased the production of antioxidant enzymes in order to counteract the oxidative stress caused by the CeO2 NP exposure. Over accumulation of H2O2 may cause cell membrane damage. Compared to the control, statistically significant increase of MDA content was observed for the 7 nm CeO2 NP treatment, indicating the membrane damage of the root cells. Combining the above-mentioned data, we can see that lettuce increased the antioxidant enzyme activities to defend the oxidative stress induced by CeO2 NPs; however, it cannot timely eliminate the excess of ROS and finally failed to avoid the membrane damage and cell death. We can also find that 7 nm CeO2 with the smallest size caused the most severe damage, but the difference between 25 nm CeO2 and Sigma CeO2 is uncertain. This discrepancy will be further discussed in the following text. Contents and chemical species of Ce in head lettuce From Figure 4, Ce concentrations in the roots of head lettuce were much higher than that in the shoots. Lettuce plants that treated with 7 nm and 25 nm CeO2 showed significant higher Ce concentrations in the roots than that treated with Sigma CeO2 NPs. However, the translocation factor (TF) of Sigma CeO2 is higher than the two others (Supplemental Figure S2). Ce species in the roots of head lettuce that treated with CeO2 NPs were determined by XANES. In Figure 5, the low-energy feature a and high-energy features b and c are respectively attributed to the Ce(III) and Ce(IV) compounds. Compared with the standard references, the spectra of lettuce roots present mainly the same feature as CeO2 NPs, but a slight difference still can be seen at the low energy feature a, indicating the existence of Ce(III) oxidation state. To obtain the quantitative information of Ce species, LCF was performed on the normalized spectra of samples using CeO2 NPs, CePO4 and Ce(CH3COO)3 as the standard compounds.
Ce(CH3COO)3 was chosen here as the representation of the cerium carboxylates which possibly formed in the plants. The fitted lines and fitting parameters indicate that the results are satisfying and convincible (Supplemental Figure S3). In the root samples, Ce species presented as 93.8% CeO2 and 6.2% cerium carboxylates in lettuce treated with 7 nm CeO2, 96.4% CeO2 and 3.6% cerium carboxylates in lettuce treated with 25 nm CeO2, and 98.2% CeO2 and 1.2% cerium carboxylates in lettuce treated with Sigma CeO2. Effects of Ce3þ on root elongation Root elongation of head lettuce was significantly inhibited by Ce3þ ions at concentrations even as low as 0.5 mg/L (Figure 6). However, Ce3þ ions had no impact on root elongation of cucumber, wheat and radish until up to 20 mg/L. Differences in transformation of the three types of CeO2 NPs by simulation studies We further examined the transformation of three types of CeO2 NPs in a series of simulated solutions. In Table 2, 7 nm CeO2 released more Ce3þ than the other two types of CeO2 NPs, exhibiting much higher reactivity. This was also confirmed by the color changes of the solutions once mixed with the CeO2 NPs, which attributed to the oxidation of Vc and catechol by CeO2 NPs (Supplemental Figure S4). 7 nm CeO2 showed the quickest change of color once being added into the reaction solution. The amount of released Ce3þ decreased in the order of 7 nm CeO24Sigma CeO2425 nm CeO2 (Table 2).
Discussion Root elongation at the germination stage was recommended by US EPA (1996b) as an indicator of phytotoxicity of pollutants and has been applied to test the phytotoxicity of nanomaterials. The nanoparticles can be reported with minimal toxicity on test plants if it has no negative effect on seed germination and root growth at 2000 mg/L according to the US EPA guidelines (US EPA, 1996a). We previously found that CeO2 NPs (7 nm) significantly inhibited root elongation of head lettuce at 2000 mg/L (Ma et al., 2010b). In this study, we further demonstrated that CeO2 NPs are also toxic to other Lactuca genus plants and different CeO2 NPs show different degrees of toxicity. Several mechanisms can cause NP toxicity in organisms, but it is generally accepted that most intracellular and in vivo toxicities from NPs arise from the production of excess reactive
Toxicity of ceria NPs to Lactuca plants
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Figure 3. H2O2 localization (A), cell death (B), antioxidant enzyme activities and MDA content (C) in roots of head lettuce treated with CeO2 NPs.
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Table 2. Released Ce3þ of different CeO2 NPs in the four reaction mixtures (mg/L).
7 nm CeO2 25 nm CeO2 Sigma CeO2
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Figure 4. Ce concentrations in roots and shoots of head lettuce treated with 2000 mg/L CeO2 NPs. All the values were expressed as mean SD of four replicates with 14 seeds each. Different letters show significant differences at (p50.05).
Figure 5. XANES CeLIII-edge spectra (5723 eV) in roots of head lettuce treated with CeO2 NPs. The dotted line indicates the feature a, b and c.
Figure 6. Root length of head lettuce, cucumber, wheat and radish treated with Ce3þ ions for 5 days. All the values were expressed as mean SD of four replicates with 14 seeds each. Significant difference compared to the control were marked with * (p50.05).
Citric acid þ Vc
Citric acid þ catechol
EDTA þ Vc
EDTA þ catechol
123.4 9.1 25.4 0.4 49.6 2.7
41.7 3.4 5.4 0.9 20.8 1.5
179.7 11.6 16.8 0.3 44.5 1.0
42.0 5.4 5.9 0.6 13 0.4
oxygen species (ROS; Sharifi et al., 2012). We observed increases of H2O2 content, MDA formation, antioxidant enzyme activities such as SOD and POD, and cell death in CeO2 treated head lettuce roots in comparison with controls. These results suggest that the phytotoxic effects of CeO2 NPs in Lactuca plants may be achieved by an enhanced production of ROS and subsequent lipid peroxidation. In this experiment, we confirmed our first and second hypothesis, that (1) CeO2 NPs are not only toxic to head lettuce, but also to other Lactuca plants and (2) CeO2 NPs with different sizes show different degrees of toxicity. For a comparison, stress responses to 7 nm CeO2 NPs (2000 mg/L) in wheat, radish and cucumber plants were also investigated. H2O2 and MDA levels were not significantly altered (Supplemental Figure S5 and S6). As for the antioxidative enzymes, only SOD activities were increased (Supplemental Figure S6). These data indicate that the plants increased the antioxidant enzyme production and defended the toxicity successfully. CeO2 NPs are generally recognized as stable in biological or environmental systems and therefore are used as a model material to study the behavior and mechanisms of toxicity of NPs compared with other soluble NPs such as ZnO NPs (Xia et al., 2008). However, in a recent work, we proved for the first time that CeO2 NPs can be transformed to CePO4 and cerium carboxylates in hydroponic cucumber plants (Zhang et al., 2012b). In this study, the results of XANES indicate that small parts of CeO2 NPs were transformed from Ce(IV) to Ce(III) not only in the roots of lettuce, but also in cucumber, and wheat at the germination stage (Supplemental Figure S7 and Table S1). Plants can create a microenvironment around the roots called ‘‘rhizosphere’’ by secreting large amounts of substances that include organic acids and reducing substances, which can assist the transformation of CeO2 NPs (Zhang et al., 2012b). Release of heavy metal ions is an important transformation pathway of metal based NMs in biological and natural environment, and has been considered as a critical factor that accounts for the toxicity of many nanoparticles (Lin & Xing, 2008; Xia et al., 2008). We have proved that dissolution of La2O3 and Yb2O3 NPs induced by the organic acids exuded from plant roots played an important role in the phytotoxicity of the two rare earth oxide nanoparticles (Ma et al., 2011; Zhang et al., 2012a). However, in this study, biotransformation of CeO2 NPs to similar extents with that in head lettuce was also found in wheat and cucumber seedlings that treated with 2000 mg/L CeO2 NPs. Why are CeO2 NPs speciesspecific toxic to Lactuca plants? In order to answer this question, we compared the phytotoxicity of Ce3þ on lettuce and other plant species and found that lettuce was more vulnerable to the toxicity of Ce3þ ions than the other plant species. Stress responses of the head lettuce to Ce3þ were also tested. Ce3þ induced a concentration dependent over accumulation of H2O2 in roots of head lettuce (Supplemental Figure S8). SOD activities showed a significant increase at 0.5 mg/L Ce3þ treatment but no difference at other concentrations, as compared to the control (Supplemental Figure S9). POD activities increased significantly at 10 and 20 mg/L. MDA content significantly increased with the concentration and to a similar extent at 0.5 mg/L with the 7 nm CeO2 NP
Toxicity of ceria NPs to Lactuca plants
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treatments. To further prove the role of released Ce3þ in the toxicity, effect of EDTA on the toxicity of 7 nm CeO2 NPs and Ce3þ to Lactuca plants were evaluated. Results show that EDTA significantly relieved the inhibition of root elongation by CeO2 NPs and Ce3þ (Supplemental Figures S10 and S11). EDTA could chelate the released Ce3þ and thus limit the interactions between Ce3þ and the roots, therefore reduced the toxicity. These results suggest that the specific toxicity of CeO2 NPs to the Lactuca plants was mainly caused by the high sensitivity of Lactuca plants to the released Ce3þ ions, although we cannot ignore the role of particular effect of CeO2. It should be noted that the estimated concentrations based on XANES results are the conservative values only in roots. More Ce3þ ions may be released because the released Ce3þ ions outside the roots were not taken into account. Different biotransformation of the three types of CeO2 NPs has been proved by LCF analyses of XANES spectra and account for their discrepancy in the phytotoxicity. Among the three types of CeO2 NPs, 7 nm CeO2 with smallest size released the highest concentration of Ce3þand accordingly caused the most severe toxicity. Particles with smaller size have higher specific surface area and can be expected to show higher reactivity. In the simulated studies, we examined the different reactivity of the three types of CeO2 NPs. Seen from the released Ce3þ concentrations, it is obvious that the order of the reactivity is as follows: 7 nm CeO24Sigma CeO2425 nm CeO2. Sigma CeO2 released more Ce3þ than the 25 nm CeO2, which mainly because that Sigma CeO2 with non-uniform size contained a large number of small particles less than 25 nm. Interestingly, this is inconsistent with the LCF results that more Ce(III) was found in the lettuce treated with 25 nm CeO2 than in those treated with Sigma CeO2. During their life, plant roots release organic compounds that contain many negative charges (Lin & Xing, 2008). The nanoparticles used in this study were positively charged and thus displayed an electrostatic attraction toward the negatively charged root surfaces. As shown in Supplemental Figure S12, CeO2 NPs were absorbed on the epidermis of the roots. Sigma CeO2 NPs had a relatively lower zeta potential and larger hydrodynamic diameter than the 25 nm CeO2. Therefore, the amount of Sigma CeO2 NPs that adsorbed by the roots should be less than the 25 nm CeO2. This was proved by the ICP-MS results that Ce content in the roots of Sigma CeO2 treated lettuce was much lower than that in 25 nm CeO2 treated lettuce. This limited the direct contact of the Sigma CeO2 with the roots and resulted in the less dissolution actually occurred than the 25 nm CeO2, since the root rhizosphere played an important role in the dissolution of NPs (Zhang et al., 2012a). These results proved that differences in sizes and zeta potentials among three types of CeO2 NPs resulted in their different degrees of biotransformation which accounted for the discrepancy in the toxicity to Lactuca plants.
Conclusion This study for the first time reports the species-specific toxicity of CeO2 NPs to Lactuca plants. CeO2 NP exposure caused oxidative damage and finally inhibited the root growth. Different types of CeO2 NPs showed different degrees of toxicity. The toxicity was probably attributed to the biotransformation of CeO2 NPs and the high sensitivity of Lactuca plants to the released Ce3þ ions. Although a high exposure concentration (2000 mg/L) was used, we believe the result of this study is not a special case and not only shows the importance of the plant species in the phytotoxicity of NPs, but also provides us with a deep insight of the mechanism involved in the toxicity. The role of biotransformation in the toxicity of NPs should be paid more attention in future studies.
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Declaration of interest This study was financially supported by the Ministry of Science and Technology of China (Grant No. 2011CB933400, 2013CB932703), Ministry of Environmental Protection of China (Grant No. 201209012) and National Natural Science Foundation of China (Grant No. 10905062, 11005118, 11275215, 11275218).
References Baker JC, Mock NM. 1994. An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell Tiss Org 39:7–12. Begum P, Ikhtiari R, Fugetsu B. 2011. Graphene phytotoxicity in the seedling stage of cabbage, tomato, red spinach, and lettuce. Carbon 49: 3907–19. Cassee FR, Van Balen EC, Singh C, Green D, Muijser H, Weinstein J, Dreher K. 2011. Exposure, health and ecological effects: review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive. Crit Rev Toxicol 41:213–29. Ghosh M, Bandyopadhyay M, Mukherjee A. 2010. Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: plant and human lymphocytes. Chemosphere 81:1253–62. Handy RD, Owen R, Valsami-Jones E. 2008. The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology 17:315–25. Holbrook RD, Murphy KE, Morrow JB, Cole KD. 2008. Trophic transfer of nanoparticles in a simplified invertebrate food web. Nat Nanotechnol 3:352–5. Judy JD, Unrine JM, Bertsch PM. 2011. Evidence for biomagnification of gold nanoparticles within a terrestrial food chain. Environ Sci Technol 45:776–81. Lee WM, An YJ, Yoon H, Kweon HS. 2008. Toxicity and bioavailability of copper nanoparticles to the terrestrial plants mung bean (Phaseolus radiatus) and wheat (Triticum aestivum): plant agar test for waterinsoluble nanoparticles. Environ Toxicol Chem 27:1915–21. Lin DH, Xing BS. 2007. Phytotoxicity of nanoparticles: inhibition of seed germination and root growth. Environ Pollut 150:243–50. Lin DH, Xing BS. 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ Sci Technol 42:5580–5. Liu Q, Zhao Y, Wan Y, Zheng J, Zhang X, Wang C, et al. 2010. Study of the inhibitory effect of water-soluble fullerenes on plant growth at the cellular level. ACS Nano 4:5743–8. Ma X, Geiser-Lee J, Deng Y, Kolmakov A. 2010a. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci Total Environ 408:3053–61. Ma YH, Kuang LL, He X, Bai W, Ding YY, Zhang ZY, et al. 2010b. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78:273–9. Ma YH, He X, Zhang P, Zhang ZY, Guo Z, Tai RZ, et al. 2011. Phytotoxicity and biotransformation of La2O3 nanoparticles in a terrestrial plant cucumber (Cucumis sativus). Nanotoxicology 5: 743–53. Nel A, Xia T, Ma¨dler L, Li N. 2006. Toxic potential of materials at the nano level. Science 311:622–7. Priester JH, Ge Y, Mielke RE, Horst AM, Moritz SC, Espinosa K, et al. 2012. Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption. Proc Natl Acad Sci USA 109:2451–6. Rico CM, Majumdar S, Duarte-Gardea M, Peralta-Videa JR, GardeaTorresdey JL. 2011. Interaction of nanoparticles with edible plants and their possible implications in the food chain. J Agric Food Chem 59: 3485–98. Sharifi S, Behzadi S, Laurent S, Forrest ML, Stroeve P, Mahmoudi M. 2012. Toxicity of nanomaterials. Chem Soc Rev 41:2323–43. Tarasenko VI, Garnik EY, Shmakov VN, Konstantinov, YM. 2012. Modified alternative oxidase expression results in different reactive oxygen species contents in Arabidopsis cell culture but not in whole plants. Biol Plantarum 56:635–40. US EPA.1996a. Ecological effects test guidelines OPPTS 8504150 Terrestrial Plant Toxicity, Tier I. vegetative Vigor. Washington, DC: EPA 712-C-96-163 Public Draft. US EPA. 1996b. Ecological effects test guidelines: seed germination/root elongation toxicity test (7101). Prevention, pesticides and toxic substances. Washington, DC.
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YbCl3 to cucumber Cucumis sativus. Environ Sci Technol 46: 1834–41. Zhang P, Ma YH, Zhang ZY, He X, Zhang J, Guo Z, et al. 2012b. Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 6:9943–50. Zhang ZY, He X, Zhang HF, Ma YH, Zhang P, Ding YY, Zhao YL. 2011. Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics 3:751–854. Zhao Y, Xing G, Chai Z. 2008. Nanotoxicology: are carbon nanotubes safe? Nat Nanotechnol 3:191–2.
ORIGINAL ARTICLE
Species-specific toxicity of ceria nanoparticles to Lactuca plants Peng Zhang1, Yuhui Ma1, Zhiyong Zhang1, Xiao He1, Yuanyuan Li1, Jing Zhang2, Lirong Zheng2, and Yuliang Zhao1 1
Key Laboratory of Nuclear Analytical Techniques, Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Beijing, China and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China
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Abstract
Keywords
Species-specific differences in the toxicity of manufactured nanoparticles (MNPs) have been reported, but the underlying mechanisms are unknown. We previously found that CeO2 NPs inhibited root elongation of head lettuce, whereas no toxic effect was observed on other plants (such as wheat, cucumber and radish). In this study, interactions between Lactuca plants and three types of CeO2 NPs (lab-synthesized 7 and 25 nm CeO2 NPs, and a commercial CeO2 NPs) were investigated. It was found that CeO2 NPs were toxic to three kinds of Lactuca genus plants and different CeO2 NPs showed different degrees of toxicity. The results of X-ray absorption near edge fine structure indicate that small parts of CeO2 NPs were transformed from Ce(IV) to Ce(III) in roots of the plants that were treated with CeO2 NPs during the seed germination stage. But the high sensitivity of Lactuca plants to the released Ce3þ ions caused the species-specific phytotoxicity of CeO2 NPs. Differences in sizes and zeta potentials among three types of CeO2 NPs resulted in their different degrees of biotransformation which accounted for the discrepancy in the toxicity to Lactuca plants. This study is among the few, and may indeed the first, that addresses the relation between the physicochemical properties of nanoparticles and its species-specific phytotoxicity.
Biotransformation, CeO2, Lactuca plants, nanoparticles, phytotoxicity
Introduction Recent advances in nanoscience and nanotechnology have led to the dramatic growth of the global nanotechnology industry. Many new nanomaterials with unique physical and chemical properties have been synthesized and incorporated into a variety of industrial and commercial products. The release of these nano-sized particles into the environment is inevitable and their potential impacts on the environment and human health have aroused great concern (Handy et al., 2008; Nel et al., 2006; Zhao et al., 2008). Terrestrial plants interact directly with the soil, water and atmospheric environmental compartments, all of which can be routes of MNP distribution. Therefore, they are more easily exposed to the MNPs and harmed. Moreover, concern has been expressed regarding the possibility of trophic transfer of plant accumulated NPs to herbivorous consumers (Judy et al., 2011; Holbrook et al., 2008). Studies pertaining to the impact of MNPs on plants are mostly based on the common toxicity endpoints (e.g. germination rate, root elongation and biomass; Lin & Xing, 2007; Zhang et al., 2012a). Phytotoxicity of MNPs at cellular and genetic level was also studied recently (Ghosh et al., 2010; Liu et al., 2010). However, there are always controversial results and no general patterns could be concluded. The phytotoxicity of MNPs is influenced by various factors, such as chemical composition, shape, surface charge, aggregation and solubility of the
Correspondence: Prof. Zhiyong Zhang, Key Laboratory of Nuclear Analytical Techniques, Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China. Tel: +86-1088233215; Fax: +86-10-88235294. E-mail: [email protected]
History Received 6 June 2013 Revised 5 September 2013 Accepted 25 September 2013 Published online 20 November 2013
nanomaterials, as well as the plant species in question (Rico et al., 2011). Species-specific differences in the phytotoxicity of several MNPs have been reported. Lee et al. (2008) found mungbean was more sensitive to the toxicity of copper NPs than wheat. Graphene with increasing concentration significantly inhibited the seedling growth of cabbage, tomato and red spinach while showed no toxicity to lettuce (Begum et al., 2011). It is considered that discrepancy of root structure (xylem and phloem) in different plant species (monocot or dicot plants) may lead to different uptake of NPs and account for the different toxicity across different plant species. However, no conclusions on the mechanism can be made (Ma et al., 2010a). More research should be carried out on diverse plant species to provide more data for the risk assessment of nanoparticles. CeO2 NPs have been widely used for various applications such as catalytic converters for automobile exhaust, ultraviolet absorber, and electrolyte in fuel cells (Cassee et al., 2011). It is among the MNPs on the OECD (organization for economic cooperation and development) list that are required immediate test of their toxicity. Priester et al. (2012) found that CeO2 NPs diminished the plant growth and pod yield of the soil-grown soybeans and shut the nitrogen fixation down at high concentration. Wang et al. (2012) reported that CeO2 NPs at concentrations of 0.1–10 mg/L had either an inconsequential or a slightly positive effect on plant growth and tomato production. In a previous work, we found that 2000 mg/L CeO2 NPs significantly inhibited the root elongation of head lettuce at the germination stage while showed no effect to the other six tested plant species (Ma et al., 2010b). To explore the mechanisms involved in the toxicity of CeO2 NPs to head lettuce, in this study, we tested the following hypotheses: (1) CeO2 NPs are not only toxic to head lettuce, but also to other Lactuca plants; (2) different CeO2 NPs
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show different degrees of toxicity; and (3) the phytotoxicity of CeO2 NPs is caused by release of Ce3þ ions instead of nanoeffect. Effects of three types of CeO2 NPs (lab-synthesized 7 and 25 nm CeO2 NPs, and a commercial CeO2 NPs) on three kinds of Lactuca plants were investigated. Ce species in the plant roots were analyzed by synchrotron based X-ray absorption spectroscopy. Toxicity of Ce3þ to lettuce and other plants were also evaluated for comparison.
Methods
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Chemicals and seeds CeO2 NPs with the sizes of 7 and 25 nm were synthesized by precipitation methods as described previously (Zhang et al., 2011). Commercial CeO2 NPs were purchased from Sigma Aldrich (USA). Seeds of three kinds of Lactuca genus plants: head lettuce (Lactuca sativa var. capitata L.), romaine lettuce (Lactuca sativa L. var. longifolia Lam), asparagus lettuce (Lactuca sativa Linn. var. angustata Irish ex Bremer) and seeds of cucumber (Cucumis sativus L.), wheat (Triticum aestivum L.) and radish (Raphanus sativus L.), were purchased from the Chinese Academy of Agricultural Sciences and kept in a refrigerator at 4 C for use. Average germination rates of the seeds were examined to be greater than 85%. Characterization of nanoparticles Transmission electron microscopy (TEM, JEM-2010, Japan) was applied to characterize the morphology and the average sizes of the CeO2 NPs. X-ray diffraction (XRD, X’pert PRO MPD, Holland) was used to determine their crystal forms. Suspensions of CeO2 NPs (20 mg/L) were prepared for measurement of hydrodynamic sizes and zeta potential (Nicomp 380 ZLS Zeta potential/Particle system, Santa Barbara, CA, USA). Seed germination and NPs treatment Uniform seeds were selected and immersed in a 10% sodium hypochlorite solution for 10 min and rinsed with deionized water to ensure surface sterility. CeO2 NP suspensions of 2000 mg/L in deionized water were prepared and ultrasonically dispersed for 15 min under water bath. Then the seeds were soaked in deionized water (for control) or NP suspensions for 2 h. One piece of filter paper was place in a 100 mm 15 mm Petri dish and added with 5 mL of the test media. Fourteen seeds were then arrayed in each dish with a certain distance between them. All the Petri dishes were sealed with tapes and then placed in the dark under 25 C in a climate incubator. For each treatment, four replicates were set. At the fifth day, the germination was halted. Germination rates were calculated. Root lengths were measured by a meter ruler. Stress response of head lettuce to CeO2 NPs After 5 days germination, roots of head lettuce were excised, homogenized with PBS (50 mM, pH 7.8) under ice bath, and then centrifuged at speed of 10 000g and 4 C for 10 min. The supernatants were kept for analyses of SOD, POD activities and MDA contents using the assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). H2O2 accumulation in roots of head lettuces treated with 2000 mg/L CeO2 NPs were analyzed by means of ROS-sensitive dye DAB (Tarasenko et al., 2012). H2O2 can be localized in roots by imaging the insoluble dark brown formazan produced by reaction of H2O2 with DAB. After being washed with deionized water carefully, root segments of 20 mm from the apices were excised and incubated in 0.1 mg/L DAB (diaminobenzidine) solution (pH 3.2) at room temperature for 30 min in the absence of
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light. Then the root segments were rinsed thoroughly with deionized water, and the images were photographed using a Digital Single Lens Reflex (Olympus, Japan). For quantitative assessment, the root segments were rinsed with deionized water thoroughly after incubation in DAB solution, grinded in a mixture of KOH and DMSO at a ratio of 1:1.167 (v/v) and then centrifuged at 10 000 g for 10 min. The absorbance of the supernatants was recorded at 700 nm on a Multimode Microplate Reader (SpectraMax M2, USA). Root cell deaths of head lettuce treated with 2000 mg/L CeO2 NPs were evaluated by Evan’s blue staining (Baker & Mock, 1994). Root segments of 20 mm from the apices were excised and incubated in 0.25% (w/v) aqueous solution of Evan’s blue for 5 h and then rinsed with deionized water thoroughly. The images were photographed using a Digital Single Lens Reflex (Olympus, Japan). For quantitative assessment, after Evan’s blue staining, root segments were thoroughly washed with deionized water and then immersed in 1% (w/v) SDS in 50% (v/v) methanol at 50 C for 15 min, and the absorbance was measured at 600 nm. Determination of Ce contents in head lettuce After 5 days germination, roots and shoots of head lettuce were separated, washed thoroughly with deionized water and lyophilized with a freeze dryer (Alpha 1-2 LD plus, Christ, Germany). The dried tissues were digested with a mixture of concentrated plasma–pure HNO3 and H2O2 (vol/vol, 4:1) on a heating plate. The obtained residual solutions were then diluted with deionized water and analyzed by ICP-MS (Thermo X7, USA). A standard reference (bush branches and leaves, GBW07602) was also digested and analyzed by ICP-MS to examine the recovery. 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 50 ng/mL, Recovery from GBW07602 was 99%. Spike recovery was 102%. Relative standard deviation was 2.5%. Detection limit is 0.01 ng/mL. X-ray absorption near edge fine structure spectroscopy of head lettuce roots After 5 days germination, roots of head lettuces were separated from the seedlings and lyophilized. The dried roots were grinded to powder and pressed into slices with a diameter of 10 mm and a thickness of 2 mm for the X-ray absorption near edge fine structure spectroscopy (XANES) analysis. XANES spectra were collected on beamline 1W1B at Beijing Synchrotron Radiation Facility (BSRF). The ring storage energy of the synchrotron radiation accelerator during data collection was 2.5 GeV with current intensity of 50 mA. CeLIII-edge (5723 eV) spectra were collected at ambient temperature in the fluorescence mode. CePO4 and Ce(CH3COO)3 as well as the three types of CeO2 NPs were used as the standard compounds. Data processing and the linear combination fitting (LCF) of the collected spectra were performed in the software program ATHENA. Samples of cucumber and wheat treated with the three types of CeO2 NPs (2000 mg/L) were also prepared for the XANES analysis. Impact of Ce3þ ions on root growth of different plant species According to LCF analyses of the XNAES spectra, we calculated the total quantities of Ce(III) in the roots and converted them into Ce3þ concentrations in each petri dish. A calculation method was shown in Supplementary Material (ESM). In each petri dish, the estimated concentrations of Ce3þ were 0.883, 0.464 and
Toxicity of ceria NPs to Lactuca plants
DOI: 10.3109/17435390.2013.855829
0.058 mg/L for 7 nm, 25 nm and Sigma CeO2 NPs, respectively. Based on the calculation, seeds of head lettuce, wheat, cucumber and radish were exposed to 0.5, 1, 5, 10 and 20 mg/L Ce3þ [as Ce(NO3)3] to determine the sensitivity of different plant species to Ce3þ ions. After 5 days germination, root lengths were measured by a rule meter.
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Transformation of the CeO2 NPs in simulated reaction solutions Organic acids and reducing substances in the rhizosphere are required for the transformation of CeO2 (Zhang et al., 2012b). Herein, four reaction solutions: citric acid þ ascorbic acid (Vc), citric acid þ catechol, EDTA þ Vc and EDTA þ catechol, which are composed of an organic acid and a reducing substances, were prepared. Citric acid is an organic acid widely existing in plants; EDTA is a commonly used organic acid with high complex ability to metal ions; Vc and catechol are reducing substances commonly existing in plants. The final concentrations of all the components were 1 mM and the pH were adjusted to 5.5. CeO2 NPs were added into the solutions with stirring and ultrasonication for 15 min. The final concentrations of CeO2 NPs were 2000 mg/L. After 5 days incubation, the solutions were centrifuged at 10 000 g for 15 min and the supernatants were filtrated through 0.2 mm polytetrafluoroethylene filters. The obtained clear solutions were diluted with 2% HNO3 and Ce concentrations were determined by ICP-MS. Statistical analysis The data processing was performed on Statistical Packages for the Social Sciences (SPSS, Chicago, IL) 10.0. All the data were expressed as mean SD (standard deviation). One-way ANOVA and S–N–K test were performed to examine the statistic difference. p50.05 was considered to be a significant difference.
Results
Effect of CeO2 NPs on root elongation of Lactuca plants Seed germination of head lettuce, romaine lettuce and asparagus lettuce was not affected by the CeO2 NPs exposure (Supplemental Figure S1). Figure 2(A and B) shows the root length of the three Lactuca plants that exposed to 2000 mg/L 7 nm, 25 nm and Sigma CeO2. It was observed that root growths of the three Lactuca plants were significantly inhibited by the three types of CeO2 NPs, except for the Sigma CeO2 NP treated romaine lettuce. Among all the CeO2 NPs tested, 7 nm CeO2 showed the most severe toxic sign, while 25 nm CeO2 caused more inhibitory effect to the root growth of romaine lettuce than Sigma CeO2. Statistical analysis between different species under same CeO2 NP treatment was performed as well and no significant difference was observed. Therefore, only head lettuce was used as a representative to explore the mechanism of the phytotoxicity in the following studies. Stress response of Lactuca plants to CeO2 NPs In Figure 3(A, inset), CeO2 NPs treated lettuce roots clearly exhibited dark brown color compared to the control, indicating an over accumulation of H2O2. Quantitative results show that 7 nm CeO2 NPs triggered a significant increase of H2O2 accumulation in roots as compared to the control. We further determined whether there were cell deaths occurred in the roots using Evan’s blue as a cell death marker. Compared to the control, deep blue color in the zone above the root tips was observed for the 7 nm CeO2 treatment and light blue for the 25 nm and sigma CeO2 treatments, which indicating the cell membrane damage and cell death of the roots (Figure 3B). The high dye retention in the roots indicates the loss of plasma membrane integrity. As can be seen from the quantitative data of the extracted Evan’s blue, CeO2
Table 1. Physicochemical properties of CeO2 NPs.
Physicochemical characteristics of CeO2 NPs TEM images of the CeO2 NPs used in this study are shown in Figure 1; their physicochemical properties are described in Table 1. The average sizes of the lab-synthesized CeO2 NPs are 6.9 0.5 and 25.2 2.3 nm, respectively. Commercial CeO2 NPs (called ‘‘Sigma CeO2’’ in the following text) contain particles of diameters ranging from 6.2 up to 48 nm, with the centre of the distribution at 16.3 nm. From the data of zeta potential and hydrodynamic size, we can see that 7 and 25 nm CeO2 NP suspensions were more stable and dispersive than the Sigma CeO2 NPs.
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7 nm CeO2
25 nm CeO2
a
a
Sigma CeO2
Particle size (nm) 6.9 0.5 25.2 2.3 25b Crystal form Cubic fluoritec Cubic fluoritec Cubic fluoritec Purity (%) 99.98%d 99.99%d 99.9%b Hydrodynamic size (nm)e 38.3 6.5 103.0 15.8 149.1 23.5 Zeta potential (mV)e 32.9 2.8 30.9 3.5 13.8 4.0 a
Data from TEM. Data provided by producer. c Data from XRD (data not shown). d Data from ICP-MS. e Data measured by DLS of 20 mg/L CeO2 NP suspensions in deionized water. b
Figure 1. TEM images of 7 nm CeO2 NPs (A), 25 nm CeO2 NPs (B) and Sigma CeO2 NPs (C).
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Figure 2. (A) Root lengths of the Lactuca plants treated with 2000 mg/L CeO2 NPs for 5 days; (B) Image of head lettuce seedlings treated with 2000 mg/L CeO2 NPs for 5 days. All the values were expressed as mean SD of four replicates with 14 seeds each. Different letters show significant differences (p50.05).
NPs-treated roots showed significant (for 7 and 25 nm CeO2 treatment) or non-significant (for Sigma CeO2 NPs treatment) increase of absorbance compared to the control. This suggests that the CeO2 NPs exposure caused membrane damage and finally the cell death of the roots. Plants have developed a series of defense mechanisms including enzymatic and non-enzymatic antioxidant system when encounter the external stress including oxidative stress. As seen from Figure 3(C), SOD activities were significantly upregulated by CeO2 NPs exposure compared to the control. Similar to SOD, POD activities in the roots were also up-regulated by CeO2 NPs and showed a significant increase for 7 nm CeO2 NPs treatment compared to the control. These suggest that lettuce roots increased the production of antioxidant enzymes in order to counteract the oxidative stress caused by the CeO2 NP exposure. Over accumulation of H2O2 may cause cell membrane damage. Compared to the control, statistically significant increase of MDA content was observed for the 7 nm CeO2 NP treatment, indicating the membrane damage of the root cells. Combining the above-mentioned data, we can see that lettuce increased the antioxidant enzyme activities to defend the oxidative stress induced by CeO2 NPs; however, it cannot timely eliminate the excess of ROS and finally failed to avoid the membrane damage and cell death. We can also find that 7 nm CeO2 with the smallest size caused the most severe damage, but the difference between 25 nm CeO2 and Sigma CeO2 is uncertain. This discrepancy will be further discussed in the following text. Contents and chemical species of Ce in head lettuce From Figure 4, Ce concentrations in the roots of head lettuce were much higher than that in the shoots. Lettuce plants that treated with 7 nm and 25 nm CeO2 showed significant higher Ce concentrations in the roots than that treated with Sigma CeO2 NPs. However, the translocation factor (TF) of Sigma CeO2 is higher than the two others (Supplemental Figure S2). Ce species in the roots of head lettuce that treated with CeO2 NPs were determined by XANES. In Figure 5, the low-energy feature a and high-energy features b and c are respectively attributed to the Ce(III) and Ce(IV) compounds. Compared with the standard references, the spectra of lettuce roots present mainly the same feature as CeO2 NPs, but a slight difference still can be seen at the low energy feature a, indicating the existence of Ce(III) oxidation state. To obtain the quantitative information of Ce species, LCF was performed on the normalized spectra of samples using CeO2 NPs, CePO4 and Ce(CH3COO)3 as the standard compounds.
Ce(CH3COO)3 was chosen here as the representation of the cerium carboxylates which possibly formed in the plants. The fitted lines and fitting parameters indicate that the results are satisfying and convincible (Supplemental Figure S3). In the root samples, Ce species presented as 93.8% CeO2 and 6.2% cerium carboxylates in lettuce treated with 7 nm CeO2, 96.4% CeO2 and 3.6% cerium carboxylates in lettuce treated with 25 nm CeO2, and 98.2% CeO2 and 1.2% cerium carboxylates in lettuce treated with Sigma CeO2. Effects of Ce3þ on root elongation Root elongation of head lettuce was significantly inhibited by Ce3þ ions at concentrations even as low as 0.5 mg/L (Figure 6). However, Ce3þ ions had no impact on root elongation of cucumber, wheat and radish until up to 20 mg/L. Differences in transformation of the three types of CeO2 NPs by simulation studies We further examined the transformation of three types of CeO2 NPs in a series of simulated solutions. In Table 2, 7 nm CeO2 released more Ce3þ than the other two types of CeO2 NPs, exhibiting much higher reactivity. This was also confirmed by the color changes of the solutions once mixed with the CeO2 NPs, which attributed to the oxidation of Vc and catechol by CeO2 NPs (Supplemental Figure S4). 7 nm CeO2 showed the quickest change of color once being added into the reaction solution. The amount of released Ce3þ decreased in the order of 7 nm CeO24Sigma CeO2425 nm CeO2 (Table 2).
Discussion Root elongation at the germination stage was recommended by US EPA (1996b) as an indicator of phytotoxicity of pollutants and has been applied to test the phytotoxicity of nanomaterials. The nanoparticles can be reported with minimal toxicity on test plants if it has no negative effect on seed germination and root growth at 2000 mg/L according to the US EPA guidelines (US EPA, 1996a). We previously found that CeO2 NPs (7 nm) significantly inhibited root elongation of head lettuce at 2000 mg/L (Ma et al., 2010b). In this study, we further demonstrated that CeO2 NPs are also toxic to other Lactuca genus plants and different CeO2 NPs show different degrees of toxicity. Several mechanisms can cause NP toxicity in organisms, but it is generally accepted that most intracellular and in vivo toxicities from NPs arise from the production of excess reactive
Toxicity of ceria NPs to Lactuca plants
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Figure 3. H2O2 localization (A), cell death (B), antioxidant enzyme activities and MDA content (C) in roots of head lettuce treated with CeO2 NPs.
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Table 2. Released Ce3þ of different CeO2 NPs in the four reaction mixtures (mg/L).
7 nm CeO2 25 nm CeO2 Sigma CeO2
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Figure 4. Ce concentrations in roots and shoots of head lettuce treated with 2000 mg/L CeO2 NPs. All the values were expressed as mean SD of four replicates with 14 seeds each. Different letters show significant differences at (p50.05).
Figure 5. XANES CeLIII-edge spectra (5723 eV) in roots of head lettuce treated with CeO2 NPs. The dotted line indicates the feature a, b and c.
Figure 6. Root length of head lettuce, cucumber, wheat and radish treated with Ce3þ ions for 5 days. All the values were expressed as mean SD of four replicates with 14 seeds each. Significant difference compared to the control were marked with * (p50.05).
Citric acid þ Vc
Citric acid þ catechol
EDTA þ Vc
EDTA þ catechol
123.4 9.1 25.4 0.4 49.6 2.7
41.7 3.4 5.4 0.9 20.8 1.5
179.7 11.6 16.8 0.3 44.5 1.0
42.0 5.4 5.9 0.6 13 0.4
oxygen species (ROS; Sharifi et al., 2012). We observed increases of H2O2 content, MDA formation, antioxidant enzyme activities such as SOD and POD, and cell death in CeO2 treated head lettuce roots in comparison with controls. These results suggest that the phytotoxic effects of CeO2 NPs in Lactuca plants may be achieved by an enhanced production of ROS and subsequent lipid peroxidation. In this experiment, we confirmed our first and second hypothesis, that (1) CeO2 NPs are not only toxic to head lettuce, but also to other Lactuca plants and (2) CeO2 NPs with different sizes show different degrees of toxicity. For a comparison, stress responses to 7 nm CeO2 NPs (2000 mg/L) in wheat, radish and cucumber plants were also investigated. H2O2 and MDA levels were not significantly altered (Supplemental Figure S5 and S6). As for the antioxidative enzymes, only SOD activities were increased (Supplemental Figure S6). These data indicate that the plants increased the antioxidant enzyme production and defended the toxicity successfully. CeO2 NPs are generally recognized as stable in biological or environmental systems and therefore are used as a model material to study the behavior and mechanisms of toxicity of NPs compared with other soluble NPs such as ZnO NPs (Xia et al., 2008). However, in a recent work, we proved for the first time that CeO2 NPs can be transformed to CePO4 and cerium carboxylates in hydroponic cucumber plants (Zhang et al., 2012b). In this study, the results of XANES indicate that small parts of CeO2 NPs were transformed from Ce(IV) to Ce(III) not only in the roots of lettuce, but also in cucumber, and wheat at the germination stage (Supplemental Figure S7 and Table S1). Plants can create a microenvironment around the roots called ‘‘rhizosphere’’ by secreting large amounts of substances that include organic acids and reducing substances, which can assist the transformation of CeO2 NPs (Zhang et al., 2012b). Release of heavy metal ions is an important transformation pathway of metal based NMs in biological and natural environment, and has been considered as a critical factor that accounts for the toxicity of many nanoparticles (Lin & Xing, 2008; Xia et al., 2008). We have proved that dissolution of La2O3 and Yb2O3 NPs induced by the organic acids exuded from plant roots played an important role in the phytotoxicity of the two rare earth oxide nanoparticles (Ma et al., 2011; Zhang et al., 2012a). However, in this study, biotransformation of CeO2 NPs to similar extents with that in head lettuce was also found in wheat and cucumber seedlings that treated with 2000 mg/L CeO2 NPs. Why are CeO2 NPs speciesspecific toxic to Lactuca plants? In order to answer this question, we compared the phytotoxicity of Ce3þ on lettuce and other plant species and found that lettuce was more vulnerable to the toxicity of Ce3þ ions than the other plant species. Stress responses of the head lettuce to Ce3þ were also tested. Ce3þ induced a concentration dependent over accumulation of H2O2 in roots of head lettuce (Supplemental Figure S8). SOD activities showed a significant increase at 0.5 mg/L Ce3þ treatment but no difference at other concentrations, as compared to the control (Supplemental Figure S9). POD activities increased significantly at 10 and 20 mg/L. MDA content significantly increased with the concentration and to a similar extent at 0.5 mg/L with the 7 nm CeO2 NP
Toxicity of ceria NPs to Lactuca plants
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DOI: 10.3109/17435390.2013.855829
treatments. To further prove the role of released Ce3þ in the toxicity, effect of EDTA on the toxicity of 7 nm CeO2 NPs and Ce3þ to Lactuca plants were evaluated. Results show that EDTA significantly relieved the inhibition of root elongation by CeO2 NPs and Ce3þ (Supplemental Figures S10 and S11). EDTA could chelate the released Ce3þ and thus limit the interactions between Ce3þ and the roots, therefore reduced the toxicity. These results suggest that the specific toxicity of CeO2 NPs to the Lactuca plants was mainly caused by the high sensitivity of Lactuca plants to the released Ce3þ ions, although we cannot ignore the role of particular effect of CeO2. It should be noted that the estimated concentrations based on XANES results are the conservative values only in roots. More Ce3þ ions may be released because the released Ce3þ ions outside the roots were not taken into account. Different biotransformation of the three types of CeO2 NPs has been proved by LCF analyses of XANES spectra and account for their discrepancy in the phytotoxicity. Among the three types of CeO2 NPs, 7 nm CeO2 with smallest size released the highest concentration of Ce3þand accordingly caused the most severe toxicity. Particles with smaller size have higher specific surface area and can be expected to show higher reactivity. In the simulated studies, we examined the different reactivity of the three types of CeO2 NPs. Seen from the released Ce3þ concentrations, it is obvious that the order of the reactivity is as follows: 7 nm CeO24Sigma CeO2425 nm CeO2. Sigma CeO2 released more Ce3þ than the 25 nm CeO2, which mainly because that Sigma CeO2 with non-uniform size contained a large number of small particles less than 25 nm. Interestingly, this is inconsistent with the LCF results that more Ce(III) was found in the lettuce treated with 25 nm CeO2 than in those treated with Sigma CeO2. During their life, plant roots release organic compounds that contain many negative charges (Lin & Xing, 2008). The nanoparticles used in this study were positively charged and thus displayed an electrostatic attraction toward the negatively charged root surfaces. As shown in Supplemental Figure S12, CeO2 NPs were absorbed on the epidermis of the roots. Sigma CeO2 NPs had a relatively lower zeta potential and larger hydrodynamic diameter than the 25 nm CeO2. Therefore, the amount of Sigma CeO2 NPs that adsorbed by the roots should be less than the 25 nm CeO2. This was proved by the ICP-MS results that Ce content in the roots of Sigma CeO2 treated lettuce was much lower than that in 25 nm CeO2 treated lettuce. This limited the direct contact of the Sigma CeO2 with the roots and resulted in the less dissolution actually occurred than the 25 nm CeO2, since the root rhizosphere played an important role in the dissolution of NPs (Zhang et al., 2012a). These results proved that differences in sizes and zeta potentials among three types of CeO2 NPs resulted in their different degrees of biotransformation which accounted for the discrepancy in the toxicity to Lactuca plants.
Conclusion This study for the first time reports the species-specific toxicity of CeO2 NPs to Lactuca plants. CeO2 NP exposure caused oxidative damage and finally inhibited the root growth. Different types of CeO2 NPs showed different degrees of toxicity. The toxicity was probably attributed to the biotransformation of CeO2 NPs and the high sensitivity of Lactuca plants to the released Ce3þ ions. Although a high exposure concentration (2000 mg/L) was used, we believe the result of this study is not a special case and not only shows the importance of the plant species in the phytotoxicity of NPs, but also provides us with a deep insight of the mechanism involved in the toxicity. The role of biotransformation in the toxicity of NPs should be paid more attention in future studies.
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Declaration of interest This study was financially supported by the Ministry of Science and Technology of China (Grant No. 2011CB933400, 2013CB932703), Ministry of Environmental Protection of China (Grant No. 201209012) and National Natural Science Foundation of China (Grant No. 10905062, 11005118, 11275215, 11275218).
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