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Journal of Biomedical Nanotechnology Vol. 7, 677–684, 2011

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Hong-Xuan Ren1 2 † , Ling Liu3 † , Chong Liu4 , Shi-Ying He1 , Jin Huang5 , Jun-Li Li5 , Yu Zhang1 , Xing-Jiu Huang6 ∗ , and Ning Gu1 ∗ 1

School of Biological Science and Medical Engineering, Southeast University, Nanjing 210098, PR China 2 National Center for Nanoscience and Technology of China, Beijing 100080, PR China 3 College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, PR China 4 Institute of Agricultural Sciences of Jiangsu Coastal District, Yancheng 224002, PR China 5 School of Mechanical and Electronic Engineering and College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, China 6 Research Center for Biomimetic Functional Materials and Sensing Devices, Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230031, PR China While few publications have documented systematic physiological effects of nanoparticles on plant cells and tissues, this is the first study describing a detailed evidence of impact of -Fe2 O3 magnetite nanoparticles (MNPs) on Chinese mung bean plants by measuring the physiological parameters such as germination, root activity, activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), content of chlorophyll, soluble protein, and content of malondialdehyde (MDA). Our results will help answer the question on how both positive and negative or inconsequential effects take place in plants after treatment by the nutrient solution containing nanoparticles.

Keywords: -Fe2 O3 Magnetite Nanoparticles (MNPs), Physiological Investigation, Plants, Cells and Tissues.

1. INTRODUCTION Nanomaterials, such as carbon nanotubes and nanoparticles, show signs of toxicity.1–2 Until now, most of studies on the adverse outcomes of nanomaterials were limited to animal cells or whole organisms. The impact of nanomaterials on plants has scantly been examined in the current literature. In the past two years, the impact of nanomaterials on plants is attracting increasing attention. For example, Xing et al.3–5 studied the effects of multi-walled carbon nanotube, aluminum, alumina, zinc, and zinc oxide on seed germination and root growth of radish, rape, ryegrass, lettuce, corn, and cucumber, and found that seed germination was not affected except for the inhibition of nanoscale zinc (nano-Zn) on ryegrass and zinc oxide (nano-ZnO) on corn at 2000 mg L−1 , and the inhibition occurred during the seed incubation process rather than seed soaking stage. An et al.6 found that Cu nanoparticles can cross the cell membrane and might agglomerate in the cells of plant species were Phaseolus radiatus (mung bean) and Triticum ∗ †

Authors to whom correspondence should be addressed. Two authors have the equivalent contribution to this work.

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aestivum (wheat). Shah et al.7 studied the short term influence of Si, Pd, Au and Cu nanoparticles on the germination of lettuce seeds at two different concentrations of nanoparticles. Trapp et al.8 found that manufactured TiO2 nanoparticles have low toxic effects on willow trees. Font et al.9 studied the risk of the release to the environment of Fe3 O4 , Ag and Au nanoparticles by seed germination test (cucumber and lettuce), anaerobic toxicity test, and bioluminescent test (Photobacterium phosphoreum). In all cases low or zero toxicity was observed at the assayed concentrations. White et al.10 investigated the effects of five nanomaterials (multiwalled carbon nanotubes, Ag, Cu, ZnO, Si) and their corresponding bulk counterparts on seed germination, root elongation, and biomass of Cucurbita pepo (zucchini). Zhang et al.11 reported the phytotoxicity of four rare earth oxide nanoparticles, nano-CeO2 , nano-La2 O3 , nano-Gd2 O3 and nano-Yb2 O3 on seven higher plant species (radish, rape, tomato, lettuce, wheat, cabbage, and cucumber) by means of root elongation experiments. Gardea-Torresdey et al.12 studied the fate, transport, and possible toxicity of cerium oxide nanoparticles (nanoceria, CeO2  on seeds of alfalfa (Medicago sativa), corn (Zea mays), cucumber (Cucumis sativus), and tomato (Lycopersicon esculentum).

1550-7033/2011/7/677/008

doi:10.1166/jbn.2011.1338

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Smalle et al.13 described uptake and distribution of the ultrasmall anatase TiO2 in the plant model system Arabidopsis. Ke et al.14–15 characterized the dynamic uptake, compartment distribution, and transformation of fullerene C70 in rice plants and have detected the transmission of C70 to the progeny through seeds. Alvarez et al.16 investigated the effects of aluminum oxide (nAl2 O3 , silicon dioxide (nSiO2 , magnetite (nFe3 O4 , and zinc oxide (nZnO) on the development of Arabidopsis thaliana (Mouse-ear cress) from three toxicity indicators (seed germination, root elongation, and number of leaves). Very recently, Ma et al.17 reviewed the current knowledge on the phytotoxicity and interactions of engineered nanoparticles (ENPs) with plants at seedling and cellular levels and discussed the information gap and some immediate research needs to further our knowledge on this topic. Kumar et al.18 reviewed the delivery of nanoparticulate materials to plants and their ultimate effects which could provide some insights for the safe use of this novel technology for the improvement of crops. Obviously, Most of these studies were focused on the study of potential toxicity (seed germination and root growth) of ENPs to plants via several standard methods and both positive and negative or inconsequential effects have been reported. However, little is known about systematic physiological effects of nanoparticle on plants. Iron oxide magnetite nanoparticles (MNPs), e.g., Fe3 O4 , possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools.19–27 A few studies have been conducted to assess the effects of iron oxide MNPs on ecological terrestrial species such as plants. The first study was by Jin and co-workers,28 who demonstrated significant uptake of Fe3 O4 MNPs by pumpkin plants and their subsequent translocation and accumulation in various tissues. However, fundamental questions remain regarding the systematic physiological effects on plant cells and tissues, and the impact of these processes on plant reproduction. Here, we provide a detailed evidence of impact of -Fe2 O3 MNPs on Chinese mung bean plants by measuring the physiological parameters such as germination, root activity, activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and content of chlorophyll, soluble protein, and content of malondialdehyde (MDA). The data presented in this article suggest the potential impact of nanomaterial exposure on plant development and the food chain, and prompt further investigation into the genetic consequences through plant nanomaterial interactions.

2. MATERIALS AND METHODS 2.1. Synthesis of -Fe2 O3 MNPs Fe3 O4 MNPs with diameters of approximately 9 nm and 18 nm were first prepared according to the chemical 678

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co-precipitation method.29 The synthesized Fe3 O4 particles were diluted to 5.0 L and the pH of the solution was adjusted to 3 with 2 M HCl under nitrogen gas protection and vigorous stirring using nonmagnetic stirrer. Then the Fe3 O4 nanoparticles were transferred into a double-layer glass reaction vessel to react with oxygen through bubbling air under continuous stirring at 90  C. After stirring for 5 h, the obtained -Fe2 O3 nanoparticles precipitate was separated from the reaction medium by magnetic field, and washed with 200 mL deionized water four times. With this synthesis, once the synthetic conditions are fixed, the quality of the magnetite nanoparticles is fully reproducible. The prepared -Fe2 O3 nanoparticles were dispersed in deionized water, and the pH of the solution was adjusted to 2.7. 1.365 g DMSA dissolved in 50 mL DMSO was added to the dispersion with continuous stirring. After the reaction for 5 h at room temperature, the products were collected with a magnet and were dispersed in (CH3 4 NOH solution, and the pH of the solution was adjusted to 10. The stable magnetic fluids were obtained after the pH of the solution was adjusted to neutral and reverse osmosis. 2.2. Superoxide Dismutase (SOD) Activity Assay A 0.5 g of fresh samples were ground in a mortar with 0.01 g PVP, 5 mL of 0.1 mol · L−1 phosphate buffer containing 0.2 mM EDTA and 0.4 mM -mercaptoethanol (pH 7.8). After extraction in an ice bath, the homogenate was centrifuged at 1200 rpm for 10 min. The supernatant was collected for further measurements. Take 50 L supernatant and add to 1.5 mL of 0.05 M phosphate buffer (pH 7.8), 0.3 mL of 130 mM methionine, 0.3 mL of 750 M Nitrotetrazolium blue chloride, 0.3 mL of 100 M EDTA-Na, 0.3 mL 20 M lactochrome and 0.25 mL of distilled water. Another two control samples were prepared using buffer instead of enzyme solution. After uniform mixing, one control test was put in the dark, and others were illuminated under fluorescence lamp for 20 min. Activity was measured by the changes in absorbance at 560 nm. 2.3. Peroxidase (POD) Activity Assay Peroxidase activity was determined by guaiacol oxidation. POD reaction mixture was first prepared by the addition of 28 L guaiacol into 50 mL of 100 mM phosphate buffer (pH 7.0), when completely dissolving and cooling at room temperature, 19 l of 30% H2 O2 was introduced (Note: such mixture was required to freshly prepared before use, and stored in fridge). The assay mixture contained in 100 L of the diluted supernatant, 2 mL POD reaction mixture and 1 mL of 0.2 M potassium dihydrophospate buffer. The hydrogen peroxide can oxidize guaiacol and produce dark brown substance in the presence of peroxidase. The conversion of the dye was monitored by the measurement of the changes in absorbance at 470 nm per J. Biomed. Nanotechnol. 7, 677–684, 2011

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

30 sec. The POD activity was expressed as the change of absorbance per minute (A470 /min/gFW).

Catalase (CAT) activity was determined by the addition of 100 L supernatant to 3 ml mixture containing 0.1% H2 O2 and 100 mM phosphate buffer (pH 7.0). The results were monitored by the measurements of the changes in absorbance at 240 nm per 30 sec. The activity was expressed as the change of absorbance per minute (A240 /min/g FW (fresh weigh)). 2.5. Soluble Protein Content Assay Soluble protein content was analyzed using dying method with Coomasie Brilliant Fluka G-250. Protein standard solution was firstly prepared and stored in the refrigerator. Coomassie Brilliant Blue G-250 solution was prepared by weighing coomassie brilliant blue G-250 100 mg, adding 50 mL of 95% ethanol and 100 mL of 85% (w/v) H3 PO4 to the final volume with distilled water 1000 mL at room temperature. The test solution was prepared by weighing a dried sample 0.5 g, adding a small amount of quartz sand with distilled water, the mixture was ground into a homogenate. After standing 10–30 min (3–5 min, shaking time) via a filter (such as active carbon containing pigment available), the volume of the solution was set to 100 mL for further use. 2.6. Malondialdehyde Content Assay Take 1 ml supernatant and add to 5 mL of 20% trichloroacetic acid (TCA) containing 0.5 M thiobarbituric acid (TBA), such a mixture was then manually agitated. The container was placed in a boiling water bath for 30 min. After cooling down to room temperature, it was centrifuged at 4000 rpm for 10 min. Using 20% TCA as a reference, the supernatant was collected for measurements of the changes in absorbance at 450, 532, and 600 nm. 2.7. Chlorophyl Content Assay 0.1 g of fresh leaves were ground and transferred to a test-tube. Chlorophyl was extracted with 10 ml mixture of ethanol and acetone (1:1 v/v) under a controlled environment of room temperature and 24 h dark photoperiod. Data was collected by the changes in absorbance at 645 and 663 nm. 2.8. Determination of Root Activity The root activity was estimated by triphenyl tetrazolium chloride (TTC) reduction.30 Control roots were boiled for 10 min in distilled water to insure that enzymes were denatured. All roots were cut into 1 cm pieces, submerged in J. Biomed. Nanotechnol. 7, 677–684, 2011

3. RESULTS AND DISCUSSION Chinese mung bean was selected as a model plant because it’s a very popular plant and widely cultivated in China. As in the case of the potential biological effects of nanoparticles in the green agriculture, the physiological investigation of magnetic iron oxide nanoparticles could be of enormous benefit. In this study, a complex ecosystem in a greenhouse was constructed to model the growth environment of Chinese mung bean for further measuring the behaviour of -Fe2 O3 magnetic nanoparticles (Fig. 1(A)). Considering that the properties of nanoscale materials are often dependent on size and nanoparticles have the potential to pass across physiological barriers or target specific cells and organs and administer small quantities of drugs, and most importantly, individual -Fe2 O3 particles of 20 nm can be existed in the suspension, -Fe2 O3 MNPs with size of 9 and 18 nm were chosen and prepared for further experiments. The -Fe2 O3 MNPs appeared spherical and homogeneous and were of the expected size (Figs. 1(B–C)). After immersing into -Fe2 O3 MNPs nutrient solution, Chinese mung bean seeds were transferred into separate temporary containers filled with natural silica sediment to allow the growth till the germination. These seedlings were then transferred into plastic pots (filled with silica sediment) and supplemented with 1/2 Hoagland solution containing -Fe2 O3 MNPs everyday till the appearance of leaf. Two different concentrations of 10 and 20 mg · L−1 were prepared for the investigation. Together with a control test, each treatment was conducted with three replicates, and the results were statistical analysis and presented as mean ±SD (standard deviation). The details can be found in Methods. Figure 2(A) shows the results of germination of Chinese mung bean seeds after treatment for 10 days by the nutrient solution containing 9 and 18 nm -Fe2 O3 MNPs (10 and 20 mg · L−1 , respectively. In this test, 100 seeds were selected for the experiments. Seed germinations were affected by the concentration of nanoparticles in nutrient solution. As seen, the germination of seeds was inhibited by the lower concentration of nanoparticles, 10 mg · L−1 . The percentage is 56% and 52% for size 9 and 18 nm of nanoparticles, respectively. It is quite lower than the normal case, 72%, in the absence of nanoparticles. However, 679

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2.4. Catalase (CAT) Activity Assay

3 mL of 0.6% (w/v) 2,3,5-triphenyl tetrazolium chloride in 0.05 M Na2 HPO4 -KH2 PO4 (pH 7.4) + 005% wetting agent (Triton X-100), and vacuum-infiltrated for 5 min to insure infiltration of TTC. Samples were incubated at 30  C for 24 h, rinsed twice with distilled water, and extracted four times in 4 mL of 95% (v/v) ethanol for 5 min in a waterbath at 85  C. The total solution extracted was brought up to a volume of 25 mL and measured with a spectrophotometer (Shimadzu UV160U, Kyoto, Japan) at 490 nm.

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Fig. 1. Experimental setup. (A) Growth of Chinese mung bean modelling the natural environment was maintained in a greenhouse. Chinese mung bean is shown growing in the sediment pots. Each pot contained natural silica sediment and received a certain dose of -Fe2 O3 MNPs nutritive medium. (B–C) TEM images of -Fe2 O3 magnetic iron oxide nanopartiles from deionized water used in this study. (B) 18 nm; (C) 9 nm.

the germination was promoted by the higher concentration of nanoparticles, 20 mg · L−1 . The percentage is 78% for both 9 nm and 18 nm of nanoparticles. The results are quite different to the situation which ryegrass and corn were inhibited by nano-Zn and nano-ZnO. This might be due to that Magnetic -Fe2 O3 nanoparticles are generally considered to be biologically and chemically inert. Magnetic -Fe2 O3 nanoparticles have been coated with metal catalysts or conjugated with enzymes, to combine the separating power of the magnetic properties with the catalytic (A)

(B)

activity of the metal surface or enzyme conjugate. It is concluded that the higher concentration has a promotion impact on the seed germination. We therefore chose 20 mg · L−1 of nutrient solution containing 9 and 18 nm -Fe2 O3 MNPs to investigate the length of sprout during the growth, as presented in Figure 2(B). In contrast to the control test (that is, in the absence of nanoparticles), -Fe2 O3 MNPs nutrient solution can accelerated the growth of sprout. This indicates that the existing of -Fe2 O3 MNPs helps root cell open (C)

Fig. 2. Effect of -Fe2 O3 magnetic nanoparticles on the germination of Chinese mung bean seeds. (A) Percentage of germination of Chinese mung bean seeds inclubated in size 9/18 nm of 10 and 20 mg · L−1 -Fe2 O3 magnetic nanoparticles nutritive medium. (B) Length of sprout as a function of growth time under different sizes of nanoparticle. Concentration: 20 mg · L−1 . (C) Length of sprout as a function of growth time under different concentrations. -Fe2 O3 MNPs: 18 nm.

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(A)

(B)

water channels and effectively promotes cell better adsorption to water, inorganic ions, and other nutritional components, and that the influence of smaller nanoparticle (size of 9 nm) is more obvious than that of bigger one (size of 18 nm). The nanoparticles with small size readily permeate through the plant cell wall driven by a concentration gradient. This contribution is excellent consistent with the effect of -Fe2 O3 MNPs on the root activity, as will be discussed in the following section. Figure 2(C) is the results of length of sprout as a function of growth time under different concentrations. It is seen that the growing of sprout is well at lower concentration (10 mg · L−1 . This phenomenon might be attributed to that the higher concentration can form clusters and tends to block the porous plant cell wall. The absorption of plant roots is an active organ and synthetic organs, root growth and vigor of shoot directly (A)

(B)

(C)

Fig. 4. Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2 O3 magnetic nanoparticles of various concentrations. (A) Activity of catalase (CAT). (B) Activity of peroxidase (POD). (C) Activity of superoxide dismutase (SOD).

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Fig. 3. (A) Root activity in the presence of 9 nm and 18 nm -Fe2 O3 magnetic nanoparticles of various concentrations. (B) Root activity versus concentrations (0, 10, 20, 40, 60, 80, and 100 mg · L−1  in the presence of 9 nm -Fe2 O3 magnetic nanoparticles.

affect the level of the nutritional status and production levels. Figure 3 shows the root activity of Chinese mung bean seedlings in the presence of 9-nm and 18-nm -Fe2 O3 MNPs of various concentrations. As seen from Figure 3(A), no changes were observed for 18 nm MNPs at 20 mg · L−1 compared to the control. At the same concentration, 9 nm -Fe2 O3 MNPs have a positive effect; it proposed an increase of 50.3 percent for root activity. The best root activity can be seen in the case of 9 nm MNPs of 10 mg · L−1 ; an increase of 117.2 percent is obtained. For both of 9 nm and 18 nm nanoparicles, root activity influenced by lower concentration is more obvious than that influenced by higher concentration. This experiment gives a supportive evidence for the growth of sprout shown in Figures 2(B)–(C). Figure 3(B) shows the root activity under different concentrations in the presence of 9 nm -Fe2 O3 MNPs. It is seen that as increasing the concentration to 40 mg·L−1 the root activity sharply decreases and consequently again increases to normal case (see dotted line). This might be explained either by the significant aggregation of those MNPs at high concentration, or by the accumulation of many -Fe2 O3 nanoparticles on the root surface (i.e., the accumulation inhibits the transmission of water and other nutritional components). Peroxide accumulation may cause changes in plant metabolism in several ways. They may oxidize sulflhydryl groups, and in combination with superoxides they can form hydroxyl radicals which may be involved in the aging process. H2 O2 may be involved also in the oxidative breakdown of indoleacetic acid. It has been shown that increased H2 O2 levels inactivate indoleacetic acid. This inactivation was reversed upon the introduction of catalase. Figure 4 shows the activity of a group of enzymes, such as tatalase, peroxidase, and superoxide dismutase, in the presence of 9 nm and 18 nm -Fe2 O3 magnetic nanoparticles

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

of various concentrations. Figure 4(A) shows the effect of -Fe2 O3 MNPs on the function of the enzyme catalase, which is found in all plant tissues. As seen, a nanoparticle addition in nutrient solution results in a decrease of activity of catalase, while the effect of nanoparticles in these plants showed no significant concentration or size dependence. A decrease in catalase activity could lead to the observed accumulation of H2 O2 in tissue since there is an increase in peroxidase activity (Fig. 4(B)) to compensate for the H2 O2 removal by catalase, even though the activity is still lower than normal levels. Taking 20 mg · L−1 of -Fe2 O3 MNPs solution as an example, the catalase activity treated by 18 nm MNPs is a litter higher than that treated by 9 nm (Fig. 4(A)). Peroxidase activity is opposite to this result (Fig. 4(B)). Similar result can also be found in the case of 10 mg L−1 -Fe2 O3 MNPs solution. As seen from Figure 4(C), the activity of superoxide dismutase was inhibited by the suspension of -Fe2 O3 MNPs, and shows a size and concentration dependence. A significant effect was found at higher concentration (20 mg · L−1  and larger size (18 nm). Superoxide dismutase (SOD) in plant catalyzes the destruction of the O2− free radical, the decrease of activity of SOD result in the accumulation of O2− free radical in plant leaf. Consequently, these free radicals react with H2 O2 produced by chloroplast to form OH• free radicals which might result in the degradation of chlorophyll. However, we observed a quite different phenomenon from the test of chlorophyl content, as shown in Figure 5(A). As can be seen from Figure 5(A), a slight increasing on the content of chlorophyll was observed after treated by -Fe2 O3 MNPs. It is obvious that the introduction of nanoparticles is contributed to a synthesis of chlorophyll. However, it is not clear whether this is due to a decrease in peroxidase activity, intrinsic peroxidase-like activity of -Fe2 O3 nanoparticles possess, or both. Content of soluble (A)

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protein was highly correlated with the age of plants, i.e., content of soluble protein decreases with the increasing of the age. The results shown in Figure 5(B) reveal that -Fe2 O3 MNPs can stimulate the growth of plants, especially for those samples treated using 18 nm of 10 mg · L−1 nanoparticle suspension. Soluble protein was also strong relative to the Content of chlorophyl of plant leaf. With the increase of content of chlorophyl, photosynthesis of plants increases, resulting in the accumulation of soluble protein. Malondialdehyde (MDA) is an important lipid peroxidation product when plant is in stress conditions of aging or injured. Its content is closely related to plant senescence and stress injury. The extent of the damage of plant membrane system and plant resistance can be known by measuring the MDA level of lipid peroxidation. The low content of MDA is useful for protecting the structure and function of cell membrane. As depicted in Figure 5(C), the content of MDA decreases in the present conditions except the treatment using 9 nm of 20 mg · L−1 nanoparticles, indicating that the proxidation of unsaturated fatty acid in cell membrane is weaker than that of the control. This situation should be due to the increase of the content of SOD, CAT, and POD in theory. It is quite different from the above mentioned experimental results. From TEM image, we are sure that -Fe2 O3 MNPs were introduced to plant (as will be discussed later); suggesting that there may have another mechanism. Maybe one of these three enzymes or both/three of them was/were replaced by -Fe2 O3 MNPs. The unusual result obtained under 9 nm of 20 mg · L−1 nanoparticles is likely owing to the glomeration of small particles. Larger particles cannot easily penetrate the cell wall and membrane, resulting in the lower utilization efficient and consequently affect the synthesis of chlorophyll and increasing of MDA content. Figure 6 shows TEM (Transmission electron microscopy, JEM-2010 microscope equipped with Oxford (C)

Fig. 5. Physiological parameters in the presence of 9 nm and 18 nm -Fe2 O3 magnetic nanoparticles of various concentrations. (A) Content of chlorophyl. (B) Content of soluble protein (SP). (C) Content of malondialdehyde (MDA).

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Fig. 6. TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg · L−1 -Fe2 O3 MNPs suspension. (C–D) Detection of nanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg · L−1 -Fe2 O3 MNPs suspension.

INCA EDS operated at 200 kV accelerating voltage) images of the plant tissues after treating in 9 nm of 20 mg · L−1 -Fe2 O3 MNPs suspension. Figure 6(A) is under treatments of control of epidermal cell of Chinese mung bean root. However, the cristae of mitochondria becomes rough and the color of the cytoplasm gets lighter (Fig. 6(B)). The tumefaction of mitochondria cannot be observed, indicating that the membrane of mitochondria was not damaged by the nanoparticles. Taking account of above mentioned results, CAT, POD, and SOD content of plant tissue decrease after treating by -Fe2 O3 nanoparticles, this observation demonstrates that the peroxidation of the membrane does not occur. Black aggregates were frequently found in the cytoplasm (Figs. 6(C and D)), indicating that the sequence of nanoparticle uptake was from the plant seeds and roots to the stems and leaves.

4. CONCLUSIONS This manuscript reports the first study describing a detailed evidence of impact of -Fe2 O3 magnetite nanoparticles J. Biomed. Nanotechnol. 7, 677–684, 2011

(MNPs) on Chinese mung bean plants by measuring the physiological parameters such as germination, root activity, activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), content of chlorophyll, soluble protein, and content of malondialdehyde (MDA). It should be noted that, although the nanoparticles used in this study have a diameter size within the nanometre range, aggregates of different sizes were formed in the plant cells. Thus, it can be concluded that the -Fe2 O3 nanoparticles could enter into the tissues or cells from the roots. Even though more works need further attention in order to obtain aspects of mechanism including uptake and translocation and the interactions between the particles with plant tissue at the cellular level, this research nevertheless provides convincing evidence that plant uptake is a potential transport pathway of nanoparticles in the environment. Another main significance of this study could provide a guideline for the production of so-called selenium (Se)-riched rice in China. After our inspection, selenium (Se)-riched rice were produced in two ways. One is manual spraying of Se solution during rice growing, and then through bio-transformation, the inorganic selenium 683

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into organic selenium, and stored in the rice, so absorbed by the body. Another is the local soil rich in selenium content, the rice produced natural selenium. Our results will help answer the question on how significant physiological changes of plant occur. Acknowledgments: X.-J. Huang thanks “One Hundred Person Project” of the Chinese Academy of Sciences, China, and N. Gu thanks State Key Development Program for Basic Research of China (Grant No. 2006CB933200) and National Natural Science Funds for Distinguished Young Scholar (NSFC-60725101), for their financial supports.

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Received: 10 June 2011. Accepted: 20 June 2011.

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