Environmental Pollution 198 (2015) 8e14
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Transformation of ceria nanoparticles in cucumber plants is influenced by phosphate Yukui Rui a, **, 1, Peng Zhang b, 1, Yanbei Zhang a, Yuhui Ma b, Xiao He b, Xin Gui a, Yuanyuan Li b, Jing Zhang c, Lirong Zheng c, Shengqi Chu c, Zhi Guo d, Zhifang Chai b, Yuliang Zhao b, Zhiyong Zhang b, * a
College of Resources and Environmental Sciences, China Agricultural University, Beijing 100091, China Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Key Laboratory of Nuclear Radiation and Nuclear Energy Technology, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China c Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China d Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2014 Received in revised form 3 December 2014 Accepted 9 December 2014 Available online
Transformation is a critical factor that affects the fate and toxicity of manufactured nanoparticles (NPs) in the environment and living organisms. This paper aims to investigate the effect of phosphate on the transformation of CeO2 NPs in hydroponic plants. Cucumber seedlings were treated with 2000 mg/L CeO2 NPs in nutrient solutions with or without adding phosphate (þP or eP) for 3 weeks. Large quantities of needle-like CePO4 was found outside the epidermis in the þP group. While in the eP group, CePO4 only existed in the intercellular spaces and vacuole of root cells. X-ray absorption near edge spectroscopy (XANES) indicates that content and percentage of Ce-carboxylates in the shoots of eP group (418 mg/kg, 67.5%) were much higher than those in the þP group (30.1 mg/kg, 21%). The results suggest that phosphate might influence the transformation process of CeO2 NPs in plants and subsequently their ultimate fate in the ecosystem. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Transformation Translocation CeO2 CePO4 Nanoparticles Plant
1. Introduction CeO2 NPs are increasingly used in a wide variety of diverse applications such as fuel-borne catalysts, UV-blockers, ceramics, solid-state fuel cells and polishing agents due to their unique properties including high oxygen storage and release ability, UV absorption ability, and high hardness and reactivity (Cassee et al., 2011). The estimated production of CeO2 NPs is around 1000 tons per year in the world (Piccinno et al., 2012). The environmental concentration of CeO2 will inevitably increase with their continuing use. Harmful effects of CeO2 NPs to terrestrial plants and plantassociated soil bacteria have been reported by a number of studies recently (Ma et al., 2010; Zhang et al., 2013; Rico et al., 2013;
* Corresponding author. P.O. Box 918, Beijing 100049, China. ** Corresponding author. E-mail addresses: [email protected] (Y. Rui), [email protected] (Z. Zhang). 1 The two authors contributed equally to this article. http://dx.doi.org/10.1016/j.envpol.2014.12.017 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
pez-Moreno et al., 2010; Bandyopadhyay et al., 2012; Antisari Lo et al., 2011). Plant is the essential component of the environment and food source for animals and human beings. Accumulation of ENPs may not only impair the plant growth, but also potentially contaminate the food chain, posing a threat to environmental safety and human health (Judy et al., 2011). Uptake and accumulation of CeO2 NPs in plants including edible tissues were reported in recent studies (Zhang et al., 2012, 2013; pez-Moreno et al., 2010; Bandyopadhyay et al., Rico et al., 2013; Lo 2012; Wang et al., 2012). However, there is only limited information on the chemical species of accumulated Ce in plants. Actually, NPs may undergo various kinds of physical, chemical, or biological transformations in environmental and biological conditions (Lowry et al., 2012). These transformations will alter the fate, transport, and toxicity of the NPs. Since CeO2 NPs are generally considered to be highly stable, transformation of CeO2 NPs is rarely concerned. However, in a previous study, we found that nano-sized Ce(IV)O2 was reduced to Ce(III) in cucumber plants under hydroponic conditions and biogenic reducing substances and organic acids were
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the key factors involved in the biotransformation process (Zhang et al., 2012). Transformation of CeO2 NPs in agar and soil cultivated plants was also reported (Zhao et al., 2012; Cui et al., 2014). Recently, we found that the high sensitivity of Lactuca plants to the released Ce3þ ions caused the species-specific phytotoxicity of CeO2 NPs (Zhang et al., 2013). These results indicate that the behavior of CeO2 NPs in plants is highly related to their transformation processes. It can be expected that the alteration of the transformation may affect the uptake, translocation and toxicity in plants. Phosphates widely exist in environment and phosphorus is one of the essential elements for plant growth. It is also a basic component of buffer solution and culture media in laboratory studies. In particular, phosphate is a strong precipitant for many metals including rare earth elements (CePO4: Ksp ¼ 1024) (Byrne et al., 1996). For instance, phosphates can induce the speciation and structural transformation of ZnO NPs by forming Zn3(PO4)2 in aqueous environment (Lv et al., 2012). As described earlier, rare earth (RE) oxide NPs (La2O3, Yb2O3 and CeO2) can transform to REPO4 in cucumber plants (Ma et al., 2011; Zhang et al., 2011a; 2012). This paper aims to investigate whether phosphate can affect the transformation and subsequent translocation of the transformation products in hydroponic cucumber plants. Distribution and chemical species of Ce in the plants cultured in nutrient solution with and without phosphate were compared. Multiple analytical methods including transmission electron microscopy (TEM), synchrotronbased scanning transmission soft X-ray microscopy (STXM), and X-ray absorption near edge spectroscopy (XANES) were used. This study will provide an insightful understanding of the transformation and translocation of NPs in plants. 2. Materials and methods 2.1. Chemicals and seeds All the commercial chemicals were analytical grade. Cucumber (Cucumis sativus) seeds (Zhongnong NO.8) were purchased from Chinese Academy of Agricultural Sciences.
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0.25 mM to compensate for the reduced potassium nutrient (Stewart et al., 2001). CeO2 NPs were then added into the þP NS and eP NS to a concentration of 2000 mg/L followed by ultrasonic pretreatment for 15 min. A high exposure concentration can lead to a proper accumulative concentration of Ce in plant shoots (>10 ppm) which is necessary for collection of XANES spectra with a good signal quality (Zhang et al., 2012). The seedlings were then transferred into the beakers with CeO2 suspensions and allowed to grow in the chamber for three weeks. Each beaker contained 100 mL CeO2 NP suspension with one seedling. A constant volume (100 mL) in each beaker was maintained by replenishing with fresh nutrient solution every 2 days. To maintain the required phosphors for the normal growth of plants, 1 mM KH2PO4 solution (~10 mL for each plant) was sprayed on foliage of plants in eP groups every two days. Plants in þP and eP NS without CeO2 NPs were set as controls. 2.4. Biomass and Ce content determination After treatment for 21 days, plants were harvested and washed by flowing tap water and then deionized water thoroughly. Roots and shoots were separated, lyophilized and weighed. The dry samples were then ground to fine powders and digested with a mixture of HNO3 and H2O2 on a heating plate (80 C for 1 h, 120 C for 3 h, and 160 C for 0.5 h). The digestive residues were diluted with deionized water and the Ce contents were analyzed by ICP-MS (Thermo X7, USA). Bush branches and leaves (GBW07602) were also digested and analyzed by ICP-MS as standard references. Indium of 20 ng/mL was used as an internal standard to compensate for the matrix suppression and signal drifting. The linearity ranged from 0.1 to 50 ng/mL, Recovery from the standard reference was 99.1%. Spike recovery was 101%. Relative standard deviation was 1.5% and the detection limit is 0.01 ng/mL. Translocation factors (TF) of Ce in plants were calculated by comparing Ce contents in shoots to that in roots. 2.5. TEM observation
CeO2 NPs were synthesized by a precipitation method (Zhang et al., 2011b). TEM (JEM 200CX, Japan) was used to determine the particle morphology and size. Hydrodynamic size and Zeta potential of the CeO2 NPs suspension (20 mg/L) in deionized water and nutrient solution were measured by a dynamic light scattering (DLS) system (Malvern, UK).
After growing for 21 days, plant roots were washed with tap water and deionized water thoroughly and the root apexes were cut and fixed in 2.5% glutaraldehyde solution. Then they were dehydrated and embedded in Spurr's resin. Ultrathin sections of 90 nm were cut by an UC6i ultramicrotome (Leica, Austria) with a diamond knife and collected on copper grids. To avoid the image illusion that may be induced by high metal stain, uranyl acetate and lead citrate that are commonly used in TEM sample preparation were not used here. Sections were observed on a JEM-1230 (JEOL, Japan) transmission electron microscope operating at 80 kV.
2.3. Seedling culture and nanoparticle application
2.6. In situ speciation of Ce in roots by STXM
Seeds were sterilized by 10% NaClO solution for 10 min and rinsed with deionized water thoroughly. Then the seeds were arrayed on moist filter papers in Petri dishes and placed in an artificial climatic chamber (PRX-450C, Saifu, China) at 25 C in darkness. After 3 days, uniform seedlings were selected and each seedling was anchored by a plastic foam and transferred into a 250 mL beaker containing 100 mL modified 1/4 strength Hoagland solution. Six replicates were set for each treatment. The seedlings were allowed to grow in the climate chamber with 16-h photoperiod (light intensity of 1.76 104 mmol/m2 s), 25 C/18 C day/night temperature and 50%/70% day/night humidity for 10 days before CeO2 NP exposure. Modified 1/4 strength Hoagland nutrient solution with 0.25 mM 3 PO3 4 (called “þP NS”) and without PO4 (called “eP NS”) were prepared. KCl was added in eP solution to a concentration of
Root sections of cucumber were analyzed by STXM to determine the chemical species of Ce in situ. STXM analyses were performed on the beamline BL08U1 at Shanghai Synchrotron Radiation Facility. CeO2 NPs, CePO4 and Ce(CH3COO)3 were chosen as the reference materials. The reference materials were ultrasonically dispersed in ethanol and deposited on a TEM grid. Root sections with a thickness of 1.5 mm were prepared by the same protocols as the TEM sample preparation. For STXM analyses, a dual-energy method was performed on the chosen regions of the sample and a Ce element map was derived by calculation to ensure the existence of the Ce-component in the regions. Then, image sequences (called “stack”) were acquired at energies spanning the relevant element absorption edge (from 884 to 915 eV for Ce M4,5 edge) and aligned via a spatial cross correlation analysis method. Finally, XANES spectra were
2.2. Synthesis and characterization of CeO2 NPs
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extracted from pixels that have similar absorption features within the image sequences using the software IDL package aXis2000. 2.7. Bulk XANES spectroscopy Bulk XANES were used to determine the Ce chemical species in plant tissues. The fine powders of the roots and shoots were pressed into thin slices. XANES spectra were collected at beamline 1W1B, Beijing Synchrotron Radiation Facility. The ring storage energy of the synchrotron radiation accelerator during spectra collection was 2.5 GeV with current intensity of 50 mA. CeO2 NPs, CePO4, and Ce(CH3COO)3 as well as Ce2(C2O4)3 were used as standards and analyzed using transmission mode. CeLIII-edge spectra of root samples were collected using fluorescence mode by Lytle detector. A 19-element germanium array solid detector at fluorescence mode was used for shoot samples due to the low Ce contents. Athena software was used to process the normalization and linear combination fitting (LCF) of the XANES spectra. 2.8. Simulation studies on the transformation of CeO2 NPs In a previous work, we have demonstrated that biogenic reducing substances and organic acids play critical roles in the transformation of CeO2 NPs in plants (Zhang et al., 2012). Here, two reaction solutions composed of a biogenic organic acid and two different reducing substances were prepared as follows: (1) citric acid (Citr) þ ascorbic acid (Vc), and (2) citric acid þ catechol (Cat). For the same reaction solution, two sub-treatments were prepared. Only in one sub-treatment KH2PO4 was added, and another one was used for a comparison. Three replicates were set for each sample. The final concentrations of all the components were set as 1 mM, and the pH were adjusted to 5.5 which were same as the pH of nutrient solution. CeO2 NPs were added to the solutions to a concentration of 2000 mg/L with stirring and ultrasonication. After a 21 d static incubation, the suspensions were centrifuged at 10,000 g and the Ce3þ in the supernatants were analyzed by ICPMS. The pellets were washed with deionized water ultrasonically for 3 times and lyophilized under 50 C. The dry powders were quantitatively mixed with KBr, finely grinded, and pressed into transparent slices. The infrared spectra (IR) were recorded on an IR spectrometer (Tensor 27, Bruker, Germany) at the range of 400e4000 cm1. 2.9. Statistical analyses Data were expressed as mean ± standard deviation (n ¼ 6). Statistical analyses were performed on SPSS 18.0 statistical
Fig. 1. TEM image of the CeO2 NPs.
Table 1 Hydrodynamic sizes and Zeta potentials of CeO2 NPs.
Deionized water þP NS P NS
Hydrodynamic size (nm)
Zeta potential (mV)
122.6 ± 20.9 1484.5 ± 254.5 1235.8 ± 393.4
34.3 ± 5.1 8.0 ± 3.2 6.8 ± 0.1
software for Windows. Student's t test was applied to examine the significant difference. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of CeO2 NPs The TEM image shows that CeO2 NPs exhibit octahedral morphology and uniform size distribution (Fig. 1). Particle size calculated from TEM is 25.2 ± 2.3 nm. CeO2 NPs in deionized water was stable evidenced by the Zeta potential and small hydrodynamic size (Table 1). While in the nutrient solution, Zeta potential turned negative and hydrodynamic size became larger than that in deionized water, indicating the aggregation of the particles (Table 1). CeO2 NPs in the þP and eP NS have similar hydrodynamic size and Zeta potential. The high ionic strength in the nutrient solution compressed the double electric layer on the particle surface, and resulted in low surface charge and aggregation of NPs. 3.2. Ce contents in plant tissues The biomass production of plant in the þP and eP NS without CeO2 showed no significant difference (Fig. 1S). CeO2 NP exposure also had no significant effects on the biomass production under both the þP and P conditions (Fig. 1S). However, the uptake of Ce in the plants is different under the two conditions. As shown in Fig. 2, Ce contents in the roots were similar between the þP and P conditions, but Ce contents in the shoots of the P group were significantly higher than those in the þP group. Translocation factor (TF) of Ce in the plants of the P group (TF ¼ 0.0107 ± 0.0007) is almost four times higher than that of the þP treatment (TF ¼ 0.0028 ± 0.0005). It has been reported that phosphate precipitation is an important factor which can control the accumulation and fractionation of REE ions in plant roots (Ding et al., 2005, 2006). Here, a question thus arises as to whether the difference is also related to the phosphate precipitation.
Fig. 2. Contents of Ce in plants treated with CeO2 NPs. Data were expressed as mean ± SD (n ¼ 6). Asterisk (*) indicate significant difference (p < 0.05).
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Fig. 3. TEM images of root sections of cucumber treated with CeO2 NPs in the þP (A and B) and P (CeF) groups. (A) and (B) indicate epidermis and intercellular regions of cucumber roots. (C) cucumber roots in the P group. D-F was respectively the magnification of the arrowed regions 1e3 in C. ep: epidermis; cw: cell wall.
Fig. 4. STXM stack images and XANES spectra of cucumber roots. (A) and (B) are the stack images of Ce-components on epidermis and in intercellular regions of the roots in the þP treatment. (C) TEM image of the roots in the P treatment. Inner color map is stack image of the rectangular area in panel C; Panels D, E and F are respectively the XANES spectra extracted from the color image sequences in panel A, B and C. The black line spectra above belong to the standard compounds and the colored spectra below belong to the root samples. Vertical dotted line and dash line respectively denote the characteristic peak of CePO4 and CeO2. Different colors represent the different amounts of Ce components. CW: cell wall; IS: intercellular spaces; V: vacuole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Distribution and speciation of cerium in roots TEM technique was applied to localize Ce in the roots (Fig. 3). In the þP group, CeO2 NP aggregates located on the outside of the
epidermis of the roots. There were needle-like clusters with totally different morphology and size from the pristine CeO2 NPs among the aggregates (indicated by the rectangle area in Fig. 3A). Similar needle-like clusters in the intercellular regions were also observed
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Fig. 5. XANES spectra of roots, shoots and standard references.
(Fig. 3B). In the case of the eP treatment, CeO2 NP aggregates were found on the outside of the root epidermis as same as that in the þP group; however, there were no needle-like clusters among the aggregates (Fig. 3C and D). Interestingly, we found much more needle-like clusters in the intercellular regions of the P group than that in the þP group (Fig. 3C and E). Moreover, needle-like clusters were also found in the vacuole (Fig. 3C and F). Similar needle-like clusters were reported in roots of cucumber treated with 7 nm CeO2 NPs and were verified to be CePO4 (Zhang et al., 2012). In this study, the needle-like clusters might also be CePO4. To confirm it, STXM was used to determine the species of Ce in these needle-like clusters. On the root epidermis of the plants in the þP group, XANES spectra clearly show a mixed feature of CeO2 and CePO4, indicating the transformation of CeO2 NPs (Fig. 4A and D). However, in the intercellular regions, XANES spectra show exactly the same feature as CePO4 (Fig. 4B and E). As for the P group, XANES spectra of Ce components in the intercellular regions and vacuole exhibit the same feature belongs to CePO4 as well (Fig. 4C and F). This is the first evidence of transformation products of NPs in plant vacuole. Combining the results of STXM and TEM, we substantiated that the needle-like clusters were CePO4. Obviously, the transformation of CeO2 NPs and distribution of transformation products were different between the þP and P groups.
roots of the þP and eP groups exhibit the similar features, most of which present as Ce(IV). However, in the shoots, XANES spectra exhibit mainly the feature of Ce(III) in the eP group but present mainly the feature of Ce(IV) in the þP group. To quantify the different Ce species, CeO2 NPs, Ce(CH3COO)3, Ce2(C2O4)3 and CePO4 were used as the standard references to perform LCF analyses on the XANES spectra (Table 2). Ce(CH3COO)3 and Ce2(C2O4)3 were chosen here because trivalent rare earth ions tended to form complexes with carboxyl groups in plants (Jiang et al., 2008). To explore whether the LCF results are influenced by the type of organic carboxyl acids, the following two groups: 1) CeO2 NPs, Ce(CH3COO)3 and CePO4; 2) CeO2 NPs, Ce2(C2O4)3 and CePO4 were also used as standards to perform the LCF analyses. Results show that the percentages of cerium carboxylates (Table S1 and S2) are basically identical with that in Table 2, indicating the standard materials chosen here are reasonable and the LCF result is not influenced by the types of organic acids we chose. By comparison of the þP and eP groups (Table 2), we can see that the percentages of Ce species in the roots are similar, and Ce species present mainly as CeO2 and CePO4, accompanied with a small part of cerium carboxylates. However, in the shoots, Ce species present mainly as CeO2 in þP treatment but as cerium carboxylates in P treatment. Contents of Ce(IV) and Ce(III) species were calculated by multiplying the total Ce contents with percentage of the corresponding Ce species (Table S3). Interestingly, the content of Ce(IV) in the shoots of the eP group is 1.5 times as higher as that in the þP group; however, the content of Ce(III) species in the shoots of the eP group is almost 12 times as higher as that in the þP group.
3.4. Quantitative speciation of Ce in roots and shoots Chemical species of Ce in the roots and shoots were quantitatively analyzed by XANES. As shown in Fig. 5, XANES spectra in the
Table 2 Percentages of Ce species in the shoots and roots by LCF analyses of XANES spectra.
þP-shoot P-shoot þP-root P-root
CeO2
Ce(CH3COO)3
81.3 32.5 74.4 74.1
21 67.5 2.1
Ce2(C2O4)3
1.3 4.5
CePO4
R-factor
Chi-square
20.4 19.1
0.00007 0.00176 0.000333 0.000117
0.0008 0.0219 0.05219 0.01780
Fig. 6. IR spectra of CeO2 NPs (A) and Ce3þ concentration (B) in the supernatant of reaction solution after 21 days incubation in different reaction solutions.
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The above results imply that the higher contents of Ce in the shoots of the eP group than that of the þP group is mainly attributed to the high translocation efficiency of Ce(III) species. Translocation of NPs from root to shoot is considered to be driven by the plant transpiration (Miralles et al., 2012). However, only single particles or small aggregates that are absorbed onto the root surface may enter into plant roots and be translocated with the water flows (Zhang et al., 2011b). Apparently, this translocation process will be influenced by the absorption and aggregation state of NPs on the root surface. In the present study, the hydrodynamic sizes and Zeta potentials of CeO2 NPs in both the þP and eP nutrient solutions are close (Table 1), it can be expected that the aggregation and absorption behavior on the root surface will be similar. Therefore, similar uptake and translocation of particulate CeO2 between the two treatments can be explained. But the translocation of Ce(III) species was significantly influenced by phosphate. In the þP group, the roots were immersed in the nutrient solution abundant with phosphate. Most of the released Ce(III) ions from CeO2 NPs were precipitated by phosphate on the root surface. The others were combined with organic acids (cerium carboxylates) and uptaken by the roots via an apoplastic pathway. Part of the cerium carboxylates transformed to CePO4 in the intercellular spaces by reacting with PO3 4 while the others were translocated upward to the shoots. However, in the plants of the eP group, the Ce carboxylates can easily enter into the roots and be translocated upward instead of being immobilized as CePO4 on the root surface. Consequently, much more CePO4 and Ce carboxylates were found in the intercellular regions of the roots and shoots of the eP group than those in the þP group, respectively. Moreover, in the eP group, CePO4 precipitations were even found within cytoplasm and mainly in vacuole which was known as “phosphorus pool” (Bieleski, 1973). Storage of heavy metal ions in vacuole is one of the detoxification mechanisms for plants to counter the phytotoxicity of heavy metals (Zenk, 1996). Storage of CePO4 in the vacuole in this study may also be a detoxification process to counter the excessive Ce3þ that entered into plants of the P group. Effects of phosphate on the transformation and translocation of CeO2 NPs in ryegrass, a monocotyledon were also investigated and similar results were observed (Figs. S2eS5, Table S4), indicating that phosphate can potentially affect the behavior of CeO2 in other plant species. 4. Transformation of CeO2 NPs in simulated solutions XANES results have suggested that there are different transformation products in cucumber plants. To further understand this process, we investigated the transformation of CeO2 NPs in different simulated reaction solutions with or without phosphate. As shown in Fig. 6A, typical feature of CePO4 at 1043 cm1 only can be seen in the þP groups. With the assistance of citric acid and reducing agents, CeO2 NPs were partially dissolved. In the þP groups, most of the released Ce3þ ions were precipitated by phosphate, a stronger ligand than citric acid (logK ¼ 7.54) (Smith and Martell, 1989). If there is no phosphate added in the solution, Ce3þ will complex with citric acid, as shown in Fig. 6B. On the other hand, Vc has a much lower redox potential (0.015 V) (Sevanian et al., 1991) than catechol (0.53 V) (Jovanovic et al., 1996), therefore, more CePO4 formed in the þP groups and more Ce3þ released in the eP groups when Vc was involved. Due to the coexistence of phosphate and various organic ligands in plants, the released Ce3þ will not only be precipitated as CePO4 but also coordinated as cerium carboxylates or others. As a conclusion, phosphate can affect the transformation process of CeO2 NPs and the subsequent translocation of Ce species in hydroponic plants. There are many factors that affect the
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environmental fate of NPs. In consideration of the high affinity of phosphate to many metal ions and its’ widely existence in the organisms and natural environment, phosphate may also influence the transformation of other metal based NPs under different conditions. Notes The authors declare no competing financial interest. Acknowledgments This work was financially supported by the Key project of National Natural Science Foundation of China (No. 41130526), 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 (No. 11005118, 11275215, 11275218, 11375009, and 41371471). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2014.12.017. References Antisari, L.V., Carbone, S., Fabrizi, A., Gatti, A., Vianello, G., 2011. Response of soil microbial biomass to CeO2 nanoparticles. J. Environ. Qual. 7, 1e16. -Yacama n, M., GarBandyopadhyay, S., Peralta-Videa, J.R., Plascencia-Villa, G., Jose dea-Torresdey, J.L., 2012. Comparative toxicity assessment of CeO2 and ZnO nanoparticles towards Sinorhizobium meliloti, a symbiotic alfalfa associated bacterium: use of advanced microscopic and spectroscopic techniques. J. Hazard. Mat. 241, 379e386. Bieleski, R., 1973. Phosphate pools, phosphate transport, and phosphate availability. Annu. Rev. Plant Physiol. 24, 225e252. Byrne, R.H., Liu, X., Schijf, J., 1996. The influence of phosphate coprecipitation on rare earth distributions in natural waters. Geochim. Cosmochim. Acta 60, 3341e3346. Cassee, F.R., van Balen, E.C., 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, 213e229. Cui, D., Zhang, P., Ma, Y.H., He, X., Li, Y.Y., Zhang, J., Zhao, Y.C., Zhang, Z.Y., 2014. Effect of cerium oxide nanoparticles on asparagus lettuce cultured in an agar medium. Environ. Sci. Nano 1 (5), 459e465. Ding, S.M., Liang, T., Zhang, C.S., Yan, J.C., Zhang, Z.L., 2005. Accumulation and fractionation of rare earth elements (REEs) in wheat: controlled by phosphate precipitation, cell wall absorption and solution complexation. J. Exp. Bot. 56, 2765e2775. Ding, S.M., Liang, T., Zhang, C.S., Huang, Z.C., Xie, Y.N., Chen, T.B., 2006. Fractionation mechanisms of rare earth elements (REEs) in hydroponic wheat: an application for metal accumulation by plants. Environ. Sci. Technol. 40, 2686e2691. Jiang, W.J., Li, Z.J., Zhang, Z.Y., Zhang, J., Liu, T., Yu, M., Zhou, Y.L., Chai, Z.F., 2008. Distribution in internodal cells of chara and the bonding states with the cell wall of lanthanum. Acta Chim. Sin. 66, 1740e1744. Jovanovic, S.V., Steenken, S., Hara, Y., Simic, M.G., 1996. Reduction potentials of flavonoid and model phenoxyl radicals. Which ring in flavonoids is responsible for antioxidant activity? J. Chem. Soc. Perkin Trans. 2 (11), 2497e2504. Judy, J.D., Unrine, J.M., Bertsch, P.M., 2011. Evidence for biomagnification of gold nanoparticles within a terrestrial food chain. Environ. Sci. Technol. 45, 776e781. 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, G.V., Gregory, K.B., Apte, S.C., Lea, J.R., 2012. Transformations of nanomaterials in the environment. Environ. Sci. Technol. 46, 6893e6899. Lv, J.T., Zhang, S.Z., Luo, L., Han, W., Zhang, J., Yang, K., Christie, P., 2012. Dissolution and microstructural transformation of ZnO nanoparticles under the influence of phosphate. Environ. Sci. Technol. 46, 7215e7221. Ma, Y.H., Kuang, L.L., He, X., Bai, W., Ding, Y.Y., Zhang, Z.Y., Zhao, Y.L., Chai, Z.F., 2010. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78, 273e279.
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nanoparticles on tomato (Solanum lycopersicum L.) and its implications for food safety. Metallomics 4, 1105e1112. Zenk, M.H., 1996. Heavy metal detoxification in higher plants-a review. Gene 179, 21e30. Zhang, P., Ma, Y.H., Zhang, Z.Y., He, X., Guo, Z., Tai, R.Z., Ding, Y.Y., Zhao, Y.L., Chai, Z.F., 2011a. Comparative toxicity of nanoparticulate/bulk Yb2O3 and YbCl3 to cucumber (Cucumis sativus). Environ. Sci. Technol. 46, 1834e1841. Zhang, P., Ma, Y.H., Zhang, Z.Y., He, X., Zhang, J., Guo, Z., Tai, R.Z., Zhao, Y.L., Chai, Z.F., 2012. Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 6, 9943e9950. Zhang, P., Ma, Y.H., Zhang, Z.Y., He, X., Li, Y.Y., Zhang, J., Zheng, L.R., Zhao, Y.L., 2013. Species-specific toxicity of ceria nanoparticles to Lactuca plants. Nanotoxicology. http://dx.doi.org/10.3109/17435390.2013.855829. Zhang, Z.Y., He, X., Zhang, H., Ma, Y.H., Zhang, P., Ding, Y.Y., Zhao, Y.L., 2011b. Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics 3, 751e854. Zhao, L.J., Peralta-Videa, J.R., Varela-Ramirez, A., Castillo-Michel, H., Li, C., Zhang, J.Y., Aguilera, R.J., Keller, A.A., Gardea-Torresdey, J.L., 2012. Effect of surface coating and organic matter on the uptake of CeO2 NPs by corn plants grown in soil: insight into the uptake mechanism. J. Hazard. Mat. 225-226, 131e138.
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Transformation of ceria nanoparticles in cucumber plants is influenced by phosphate Yukui Rui a, **, 1, Peng Zhang b, 1, Yanbei Zhang a, Yuhui Ma b, Xiao He b, Xin Gui a, Yuanyuan Li b, Jing Zhang c, Lirong Zheng c, Shengqi Chu c, Zhi Guo d, Zhifang Chai b, Yuliang Zhao b, Zhiyong Zhang b, * a
College of Resources and Environmental Sciences, China Agricultural University, Beijing 100091, China Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Key Laboratory of Nuclear Radiation and Nuclear Energy Technology, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China c Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China d Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2014 Received in revised form 3 December 2014 Accepted 9 December 2014 Available online
Transformation is a critical factor that affects the fate and toxicity of manufactured nanoparticles (NPs) in the environment and living organisms. This paper aims to investigate the effect of phosphate on the transformation of CeO2 NPs in hydroponic plants. Cucumber seedlings were treated with 2000 mg/L CeO2 NPs in nutrient solutions with or without adding phosphate (þP or eP) for 3 weeks. Large quantities of needle-like CePO4 was found outside the epidermis in the þP group. While in the eP group, CePO4 only existed in the intercellular spaces and vacuole of root cells. X-ray absorption near edge spectroscopy (XANES) indicates that content and percentage of Ce-carboxylates in the shoots of eP group (418 mg/kg, 67.5%) were much higher than those in the þP group (30.1 mg/kg, 21%). The results suggest that phosphate might influence the transformation process of CeO2 NPs in plants and subsequently their ultimate fate in the ecosystem. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Transformation Translocation CeO2 CePO4 Nanoparticles Plant
1. Introduction CeO2 NPs are increasingly used in a wide variety of diverse applications such as fuel-borne catalysts, UV-blockers, ceramics, solid-state fuel cells and polishing agents due to their unique properties including high oxygen storage and release ability, UV absorption ability, and high hardness and reactivity (Cassee et al., 2011). The estimated production of CeO2 NPs is around 1000 tons per year in the world (Piccinno et al., 2012). The environmental concentration of CeO2 will inevitably increase with their continuing use. Harmful effects of CeO2 NPs to terrestrial plants and plantassociated soil bacteria have been reported by a number of studies recently (Ma et al., 2010; Zhang et al., 2013; Rico et al., 2013;
* Corresponding author. P.O. Box 918, Beijing 100049, China. ** Corresponding author. E-mail addresses: [email protected] (Y. Rui), [email protected] (Z. Zhang). 1 The two authors contributed equally to this article. http://dx.doi.org/10.1016/j.envpol.2014.12.017 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
pez-Moreno et al., 2010; Bandyopadhyay et al., 2012; Antisari Lo et al., 2011). Plant is the essential component of the environment and food source for animals and human beings. Accumulation of ENPs may not only impair the plant growth, but also potentially contaminate the food chain, posing a threat to environmental safety and human health (Judy et al., 2011). Uptake and accumulation of CeO2 NPs in plants including edible tissues were reported in recent studies (Zhang et al., 2012, 2013; pez-Moreno et al., 2010; Bandyopadhyay et al., Rico et al., 2013; Lo 2012; Wang et al., 2012). However, there is only limited information on the chemical species of accumulated Ce in plants. Actually, NPs may undergo various kinds of physical, chemical, or biological transformations in environmental and biological conditions (Lowry et al., 2012). These transformations will alter the fate, transport, and toxicity of the NPs. Since CeO2 NPs are generally considered to be highly stable, transformation of CeO2 NPs is rarely concerned. However, in a previous study, we found that nano-sized Ce(IV)O2 was reduced to Ce(III) in cucumber plants under hydroponic conditions and biogenic reducing substances and organic acids were
Y. Rui et al. / Environmental Pollution 198 (2015) 8e14
the key factors involved in the biotransformation process (Zhang et al., 2012). Transformation of CeO2 NPs in agar and soil cultivated plants was also reported (Zhao et al., 2012; Cui et al., 2014). Recently, we found that the high sensitivity of Lactuca plants to the released Ce3þ ions caused the species-specific phytotoxicity of CeO2 NPs (Zhang et al., 2013). These results indicate that the behavior of CeO2 NPs in plants is highly related to their transformation processes. It can be expected that the alteration of the transformation may affect the uptake, translocation and toxicity in plants. Phosphates widely exist in environment and phosphorus is one of the essential elements for plant growth. It is also a basic component of buffer solution and culture media in laboratory studies. In particular, phosphate is a strong precipitant for many metals including rare earth elements (CePO4: Ksp ¼ 1024) (Byrne et al., 1996). For instance, phosphates can induce the speciation and structural transformation of ZnO NPs by forming Zn3(PO4)2 in aqueous environment (Lv et al., 2012). As described earlier, rare earth (RE) oxide NPs (La2O3, Yb2O3 and CeO2) can transform to REPO4 in cucumber plants (Ma et al., 2011; Zhang et al., 2011a; 2012). This paper aims to investigate whether phosphate can affect the transformation and subsequent translocation of the transformation products in hydroponic cucumber plants. Distribution and chemical species of Ce in the plants cultured in nutrient solution with and without phosphate were compared. Multiple analytical methods including transmission electron microscopy (TEM), synchrotronbased scanning transmission soft X-ray microscopy (STXM), and X-ray absorption near edge spectroscopy (XANES) were used. This study will provide an insightful understanding of the transformation and translocation of NPs in plants. 2. Materials and methods 2.1. Chemicals and seeds All the commercial chemicals were analytical grade. Cucumber (Cucumis sativus) seeds (Zhongnong NO.8) were purchased from Chinese Academy of Agricultural Sciences.
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0.25 mM to compensate for the reduced potassium nutrient (Stewart et al., 2001). CeO2 NPs were then added into the þP NS and eP NS to a concentration of 2000 mg/L followed by ultrasonic pretreatment for 15 min. A high exposure concentration can lead to a proper accumulative concentration of Ce in plant shoots (>10 ppm) which is necessary for collection of XANES spectra with a good signal quality (Zhang et al., 2012). The seedlings were then transferred into the beakers with CeO2 suspensions and allowed to grow in the chamber for three weeks. Each beaker contained 100 mL CeO2 NP suspension with one seedling. A constant volume (100 mL) in each beaker was maintained by replenishing with fresh nutrient solution every 2 days. To maintain the required phosphors for the normal growth of plants, 1 mM KH2PO4 solution (~10 mL for each plant) was sprayed on foliage of plants in eP groups every two days. Plants in þP and eP NS without CeO2 NPs were set as controls. 2.4. Biomass and Ce content determination After treatment for 21 days, plants were harvested and washed by flowing tap water and then deionized water thoroughly. Roots and shoots were separated, lyophilized and weighed. The dry samples were then ground to fine powders and digested with a mixture of HNO3 and H2O2 on a heating plate (80 C for 1 h, 120 C for 3 h, and 160 C for 0.5 h). The digestive residues were diluted with deionized water and the Ce contents were analyzed by ICP-MS (Thermo X7, USA). Bush branches and leaves (GBW07602) were also digested and analyzed by ICP-MS as standard references. Indium of 20 ng/mL was used as an internal standard to compensate for the matrix suppression and signal drifting. The linearity ranged from 0.1 to 50 ng/mL, Recovery from the standard reference was 99.1%. Spike recovery was 101%. Relative standard deviation was 1.5% and the detection limit is 0.01 ng/mL. Translocation factors (TF) of Ce in plants were calculated by comparing Ce contents in shoots to that in roots. 2.5. TEM observation
CeO2 NPs were synthesized by a precipitation method (Zhang et al., 2011b). TEM (JEM 200CX, Japan) was used to determine the particle morphology and size. Hydrodynamic size and Zeta potential of the CeO2 NPs suspension (20 mg/L) in deionized water and nutrient solution were measured by a dynamic light scattering (DLS) system (Malvern, UK).
After growing for 21 days, plant roots were washed with tap water and deionized water thoroughly and the root apexes were cut and fixed in 2.5% glutaraldehyde solution. Then they were dehydrated and embedded in Spurr's resin. Ultrathin sections of 90 nm were cut by an UC6i ultramicrotome (Leica, Austria) with a diamond knife and collected on copper grids. To avoid the image illusion that may be induced by high metal stain, uranyl acetate and lead citrate that are commonly used in TEM sample preparation were not used here. Sections were observed on a JEM-1230 (JEOL, Japan) transmission electron microscope operating at 80 kV.
2.3. Seedling culture and nanoparticle application
2.6. In situ speciation of Ce in roots by STXM
Seeds were sterilized by 10% NaClO solution for 10 min and rinsed with deionized water thoroughly. Then the seeds were arrayed on moist filter papers in Petri dishes and placed in an artificial climatic chamber (PRX-450C, Saifu, China) at 25 C in darkness. After 3 days, uniform seedlings were selected and each seedling was anchored by a plastic foam and transferred into a 250 mL beaker containing 100 mL modified 1/4 strength Hoagland solution. Six replicates were set for each treatment. The seedlings were allowed to grow in the climate chamber with 16-h photoperiod (light intensity of 1.76 104 mmol/m2 s), 25 C/18 C day/night temperature and 50%/70% day/night humidity for 10 days before CeO2 NP exposure. Modified 1/4 strength Hoagland nutrient solution with 0.25 mM 3 PO3 4 (called “þP NS”) and without PO4 (called “eP NS”) were prepared. KCl was added in eP solution to a concentration of
Root sections of cucumber were analyzed by STXM to determine the chemical species of Ce in situ. STXM analyses were performed on the beamline BL08U1 at Shanghai Synchrotron Radiation Facility. CeO2 NPs, CePO4 and Ce(CH3COO)3 were chosen as the reference materials. The reference materials were ultrasonically dispersed in ethanol and deposited on a TEM grid. Root sections with a thickness of 1.5 mm were prepared by the same protocols as the TEM sample preparation. For STXM analyses, a dual-energy method was performed on the chosen regions of the sample and a Ce element map was derived by calculation to ensure the existence of the Ce-component in the regions. Then, image sequences (called “stack”) were acquired at energies spanning the relevant element absorption edge (from 884 to 915 eV for Ce M4,5 edge) and aligned via a spatial cross correlation analysis method. Finally, XANES spectra were
2.2. Synthesis and characterization of CeO2 NPs
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extracted from pixels that have similar absorption features within the image sequences using the software IDL package aXis2000. 2.7. Bulk XANES spectroscopy Bulk XANES were used to determine the Ce chemical species in plant tissues. The fine powders of the roots and shoots were pressed into thin slices. XANES spectra were collected at beamline 1W1B, Beijing Synchrotron Radiation Facility. The ring storage energy of the synchrotron radiation accelerator during spectra collection was 2.5 GeV with current intensity of 50 mA. CeO2 NPs, CePO4, and Ce(CH3COO)3 as well as Ce2(C2O4)3 were used as standards and analyzed using transmission mode. CeLIII-edge spectra of root samples were collected using fluorescence mode by Lytle detector. A 19-element germanium array solid detector at fluorescence mode was used for shoot samples due to the low Ce contents. Athena software was used to process the normalization and linear combination fitting (LCF) of the XANES spectra. 2.8. Simulation studies on the transformation of CeO2 NPs In a previous work, we have demonstrated that biogenic reducing substances and organic acids play critical roles in the transformation of CeO2 NPs in plants (Zhang et al., 2012). Here, two reaction solutions composed of a biogenic organic acid and two different reducing substances were prepared as follows: (1) citric acid (Citr) þ ascorbic acid (Vc), and (2) citric acid þ catechol (Cat). For the same reaction solution, two sub-treatments were prepared. Only in one sub-treatment KH2PO4 was added, and another one was used for a comparison. Three replicates were set for each sample. The final concentrations of all the components were set as 1 mM, and the pH were adjusted to 5.5 which were same as the pH of nutrient solution. CeO2 NPs were added to the solutions to a concentration of 2000 mg/L with stirring and ultrasonication. After a 21 d static incubation, the suspensions were centrifuged at 10,000 g and the Ce3þ in the supernatants were analyzed by ICPMS. The pellets were washed with deionized water ultrasonically for 3 times and lyophilized under 50 C. The dry powders were quantitatively mixed with KBr, finely grinded, and pressed into transparent slices. The infrared spectra (IR) were recorded on an IR spectrometer (Tensor 27, Bruker, Germany) at the range of 400e4000 cm1. 2.9. Statistical analyses Data were expressed as mean ± standard deviation (n ¼ 6). Statistical analyses were performed on SPSS 18.0 statistical
Fig. 1. TEM image of the CeO2 NPs.
Table 1 Hydrodynamic sizes and Zeta potentials of CeO2 NPs.
Deionized water þP NS P NS
Hydrodynamic size (nm)
Zeta potential (mV)
122.6 ± 20.9 1484.5 ± 254.5 1235.8 ± 393.4
34.3 ± 5.1 8.0 ± 3.2 6.8 ± 0.1
software for Windows. Student's t test was applied to examine the significant difference. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of CeO2 NPs The TEM image shows that CeO2 NPs exhibit octahedral morphology and uniform size distribution (Fig. 1). Particle size calculated from TEM is 25.2 ± 2.3 nm. CeO2 NPs in deionized water was stable evidenced by the Zeta potential and small hydrodynamic size (Table 1). While in the nutrient solution, Zeta potential turned negative and hydrodynamic size became larger than that in deionized water, indicating the aggregation of the particles (Table 1). CeO2 NPs in the þP and eP NS have similar hydrodynamic size and Zeta potential. The high ionic strength in the nutrient solution compressed the double electric layer on the particle surface, and resulted in low surface charge and aggregation of NPs. 3.2. Ce contents in plant tissues The biomass production of plant in the þP and eP NS without CeO2 showed no significant difference (Fig. 1S). CeO2 NP exposure also had no significant effects on the biomass production under both the þP and P conditions (Fig. 1S). However, the uptake of Ce in the plants is different under the two conditions. As shown in Fig. 2, Ce contents in the roots were similar between the þP and P conditions, but Ce contents in the shoots of the P group were significantly higher than those in the þP group. Translocation factor (TF) of Ce in the plants of the P group (TF ¼ 0.0107 ± 0.0007) is almost four times higher than that of the þP treatment (TF ¼ 0.0028 ± 0.0005). It has been reported that phosphate precipitation is an important factor which can control the accumulation and fractionation of REE ions in plant roots (Ding et al., 2005, 2006). Here, a question thus arises as to whether the difference is also related to the phosphate precipitation.
Fig. 2. Contents of Ce in plants treated with CeO2 NPs. Data were expressed as mean ± SD (n ¼ 6). Asterisk (*) indicate significant difference (p < 0.05).
Y. Rui et al. / Environmental Pollution 198 (2015) 8e14
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Fig. 3. TEM images of root sections of cucumber treated with CeO2 NPs in the þP (A and B) and P (CeF) groups. (A) and (B) indicate epidermis and intercellular regions of cucumber roots. (C) cucumber roots in the P group. D-F was respectively the magnification of the arrowed regions 1e3 in C. ep: epidermis; cw: cell wall.
Fig. 4. STXM stack images and XANES spectra of cucumber roots. (A) and (B) are the stack images of Ce-components on epidermis and in intercellular regions of the roots in the þP treatment. (C) TEM image of the roots in the P treatment. Inner color map is stack image of the rectangular area in panel C; Panels D, E and F are respectively the XANES spectra extracted from the color image sequences in panel A, B and C. The black line spectra above belong to the standard compounds and the colored spectra below belong to the root samples. Vertical dotted line and dash line respectively denote the characteristic peak of CePO4 and CeO2. Different colors represent the different amounts of Ce components. CW: cell wall; IS: intercellular spaces; V: vacuole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Distribution and speciation of cerium in roots TEM technique was applied to localize Ce in the roots (Fig. 3). In the þP group, CeO2 NP aggregates located on the outside of the
epidermis of the roots. There were needle-like clusters with totally different morphology and size from the pristine CeO2 NPs among the aggregates (indicated by the rectangle area in Fig. 3A). Similar needle-like clusters in the intercellular regions were also observed
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Fig. 5. XANES spectra of roots, shoots and standard references.
(Fig. 3B). In the case of the eP treatment, CeO2 NP aggregates were found on the outside of the root epidermis as same as that in the þP group; however, there were no needle-like clusters among the aggregates (Fig. 3C and D). Interestingly, we found much more needle-like clusters in the intercellular regions of the P group than that in the þP group (Fig. 3C and E). Moreover, needle-like clusters were also found in the vacuole (Fig. 3C and F). Similar needle-like clusters were reported in roots of cucumber treated with 7 nm CeO2 NPs and were verified to be CePO4 (Zhang et al., 2012). In this study, the needle-like clusters might also be CePO4. To confirm it, STXM was used to determine the species of Ce in these needle-like clusters. On the root epidermis of the plants in the þP group, XANES spectra clearly show a mixed feature of CeO2 and CePO4, indicating the transformation of CeO2 NPs (Fig. 4A and D). However, in the intercellular regions, XANES spectra show exactly the same feature as CePO4 (Fig. 4B and E). As for the P group, XANES spectra of Ce components in the intercellular regions and vacuole exhibit the same feature belongs to CePO4 as well (Fig. 4C and F). This is the first evidence of transformation products of NPs in plant vacuole. Combining the results of STXM and TEM, we substantiated that the needle-like clusters were CePO4. Obviously, the transformation of CeO2 NPs and distribution of transformation products were different between the þP and P groups.
roots of the þP and eP groups exhibit the similar features, most of which present as Ce(IV). However, in the shoots, XANES spectra exhibit mainly the feature of Ce(III) in the eP group but present mainly the feature of Ce(IV) in the þP group. To quantify the different Ce species, CeO2 NPs, Ce(CH3COO)3, Ce2(C2O4)3 and CePO4 were used as the standard references to perform LCF analyses on the XANES spectra (Table 2). Ce(CH3COO)3 and Ce2(C2O4)3 were chosen here because trivalent rare earth ions tended to form complexes with carboxyl groups in plants (Jiang et al., 2008). To explore whether the LCF results are influenced by the type of organic carboxyl acids, the following two groups: 1) CeO2 NPs, Ce(CH3COO)3 and CePO4; 2) CeO2 NPs, Ce2(C2O4)3 and CePO4 were also used as standards to perform the LCF analyses. Results show that the percentages of cerium carboxylates (Table S1 and S2) are basically identical with that in Table 2, indicating the standard materials chosen here are reasonable and the LCF result is not influenced by the types of organic acids we chose. By comparison of the þP and eP groups (Table 2), we can see that the percentages of Ce species in the roots are similar, and Ce species present mainly as CeO2 and CePO4, accompanied with a small part of cerium carboxylates. However, in the shoots, Ce species present mainly as CeO2 in þP treatment but as cerium carboxylates in P treatment. Contents of Ce(IV) and Ce(III) species were calculated by multiplying the total Ce contents with percentage of the corresponding Ce species (Table S3). Interestingly, the content of Ce(IV) in the shoots of the eP group is 1.5 times as higher as that in the þP group; however, the content of Ce(III) species in the shoots of the eP group is almost 12 times as higher as that in the þP group.
3.4. Quantitative speciation of Ce in roots and shoots Chemical species of Ce in the roots and shoots were quantitatively analyzed by XANES. As shown in Fig. 5, XANES spectra in the
Table 2 Percentages of Ce species in the shoots and roots by LCF analyses of XANES spectra.
þP-shoot P-shoot þP-root P-root
CeO2
Ce(CH3COO)3
81.3 32.5 74.4 74.1
21 67.5 2.1
Ce2(C2O4)3
1.3 4.5
CePO4
R-factor
Chi-square
20.4 19.1
0.00007 0.00176 0.000333 0.000117
0.0008 0.0219 0.05219 0.01780
Fig. 6. IR spectra of CeO2 NPs (A) and Ce3þ concentration (B) in the supernatant of reaction solution after 21 days incubation in different reaction solutions.
Y. Rui et al. / Environmental Pollution 198 (2015) 8e14
The above results imply that the higher contents of Ce in the shoots of the eP group than that of the þP group is mainly attributed to the high translocation efficiency of Ce(III) species. Translocation of NPs from root to shoot is considered to be driven by the plant transpiration (Miralles et al., 2012). However, only single particles or small aggregates that are absorbed onto the root surface may enter into plant roots and be translocated with the water flows (Zhang et al., 2011b). Apparently, this translocation process will be influenced by the absorption and aggregation state of NPs on the root surface. In the present study, the hydrodynamic sizes and Zeta potentials of CeO2 NPs in both the þP and eP nutrient solutions are close (Table 1), it can be expected that the aggregation and absorption behavior on the root surface will be similar. Therefore, similar uptake and translocation of particulate CeO2 between the two treatments can be explained. But the translocation of Ce(III) species was significantly influenced by phosphate. In the þP group, the roots were immersed in the nutrient solution abundant with phosphate. Most of the released Ce(III) ions from CeO2 NPs were precipitated by phosphate on the root surface. The others were combined with organic acids (cerium carboxylates) and uptaken by the roots via an apoplastic pathway. Part of the cerium carboxylates transformed to CePO4 in the intercellular spaces by reacting with PO3 4 while the others were translocated upward to the shoots. However, in the plants of the eP group, the Ce carboxylates can easily enter into the roots and be translocated upward instead of being immobilized as CePO4 on the root surface. Consequently, much more CePO4 and Ce carboxylates were found in the intercellular regions of the roots and shoots of the eP group than those in the þP group, respectively. Moreover, in the eP group, CePO4 precipitations were even found within cytoplasm and mainly in vacuole which was known as “phosphorus pool” (Bieleski, 1973). Storage of heavy metal ions in vacuole is one of the detoxification mechanisms for plants to counter the phytotoxicity of heavy metals (Zenk, 1996). Storage of CePO4 in the vacuole in this study may also be a detoxification process to counter the excessive Ce3þ that entered into plants of the P group. Effects of phosphate on the transformation and translocation of CeO2 NPs in ryegrass, a monocotyledon were also investigated and similar results were observed (Figs. S2eS5, Table S4), indicating that phosphate can potentially affect the behavior of CeO2 in other plant species. 4. Transformation of CeO2 NPs in simulated solutions XANES results have suggested that there are different transformation products in cucumber plants. To further understand this process, we investigated the transformation of CeO2 NPs in different simulated reaction solutions with or without phosphate. As shown in Fig. 6A, typical feature of CePO4 at 1043 cm1 only can be seen in the þP groups. With the assistance of citric acid and reducing agents, CeO2 NPs were partially dissolved. In the þP groups, most of the released Ce3þ ions were precipitated by phosphate, a stronger ligand than citric acid (logK ¼ 7.54) (Smith and Martell, 1989). If there is no phosphate added in the solution, Ce3þ will complex with citric acid, as shown in Fig. 6B. On the other hand, Vc has a much lower redox potential (0.015 V) (Sevanian et al., 1991) than catechol (0.53 V) (Jovanovic et al., 1996), therefore, more CePO4 formed in the þP groups and more Ce3þ released in the eP groups when Vc was involved. Due to the coexistence of phosphate and various organic ligands in plants, the released Ce3þ will not only be precipitated as CePO4 but also coordinated as cerium carboxylates or others. As a conclusion, phosphate can affect the transformation process of CeO2 NPs and the subsequent translocation of Ce species in hydroponic plants. There are many factors that affect the
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environmental fate of NPs. In consideration of the high affinity of phosphate to many metal ions and its’ widely existence in the organisms and natural environment, phosphate may also influence the transformation of other metal based NPs under different conditions. Notes The authors declare no competing financial interest. Acknowledgments This work was financially supported by the Key project of National Natural Science Foundation of China (No. 41130526), 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 (No. 11005118, 11275215, 11275218, 11375009, and 41371471). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2014.12.017. References Antisari, L.V., Carbone, S., Fabrizi, A., Gatti, A., Vianello, G., 2011. Response of soil microbial biomass to CeO2 nanoparticles. J. Environ. Qual. 7, 1e16. -Yacama n, M., GarBandyopadhyay, S., Peralta-Videa, J.R., Plascencia-Villa, G., Jose dea-Torresdey, J.L., 2012. Comparative toxicity assessment of CeO2 and ZnO nanoparticles towards Sinorhizobium meliloti, a symbiotic alfalfa associated bacterium: use of advanced microscopic and spectroscopic techniques. J. Hazard. Mat. 241, 379e386. Bieleski, R., 1973. Phosphate pools, phosphate transport, and phosphate availability. Annu. Rev. Plant Physiol. 24, 225e252. Byrne, R.H., Liu, X., Schijf, J., 1996. The influence of phosphate coprecipitation on rare earth distributions in natural waters. Geochim. Cosmochim. Acta 60, 3341e3346. Cassee, F.R., van Balen, E.C., 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, 213e229. Cui, D., Zhang, P., Ma, Y.H., He, X., Li, Y.Y., Zhang, J., Zhao, Y.C., Zhang, Z.Y., 2014. Effect of cerium oxide nanoparticles on asparagus lettuce cultured in an agar medium. Environ. Sci. Nano 1 (5), 459e465. Ding, S.M., Liang, T., Zhang, C.S., Yan, J.C., Zhang, Z.L., 2005. Accumulation and fractionation of rare earth elements (REEs) in wheat: controlled by phosphate precipitation, cell wall absorption and solution complexation. J. Exp. Bot. 56, 2765e2775. Ding, S.M., Liang, T., Zhang, C.S., Huang, Z.C., Xie, Y.N., Chen, T.B., 2006. Fractionation mechanisms of rare earth elements (REEs) in hydroponic wheat: an application for metal accumulation by plants. Environ. Sci. Technol. 40, 2686e2691. Jiang, W.J., Li, Z.J., Zhang, Z.Y., Zhang, J., Liu, T., Yu, M., Zhou, Y.L., Chai, Z.F., 2008. Distribution in internodal cells of chara and the bonding states with the cell wall of lanthanum. Acta Chim. Sin. 66, 1740e1744. Jovanovic, S.V., Steenken, S., Hara, Y., Simic, M.G., 1996. Reduction potentials of flavonoid and model phenoxyl radicals. Which ring in flavonoids is responsible for antioxidant activity? J. Chem. Soc. Perkin Trans. 2 (11), 2497e2504. Judy, J.D., Unrine, J.M., Bertsch, P.M., 2011. Evidence for biomagnification of gold nanoparticles within a terrestrial food chain. Environ. Sci. Technol. 45, 776e781. 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, G.V., Gregory, K.B., Apte, S.C., Lea, J.R., 2012. Transformations of nanomaterials in the environment. Environ. Sci. Technol. 46, 6893e6899. Lv, J.T., Zhang, S.Z., Luo, L., Han, W., Zhang, J., Yang, K., Christie, P., 2012. Dissolution and microstructural transformation of ZnO nanoparticles under the influence of phosphate. Environ. Sci. Technol. 46, 7215e7221. Ma, Y.H., Kuang, L.L., He, X., Bai, W., Ding, Y.Y., Zhang, Z.Y., Zhao, Y.L., Chai, Z.F., 2010. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78, 273e279.
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