Root Uptake and Phytotoxicity of ZnO Nanoparticles - Environmental

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Environ. Sci. Technol. 2008, 42, 5580–5585

Root Uptake and Phytotoxicity of ZnO Nanoparticles D A O H U I L I N †,‡ A N D B A O S H A N X I N G * ,‡ Department of Environmental Science, Zhejiang University, Hangzhou, 310028, China, and Department of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, Massachusetts 01003

Received February 11, 2008. Revised manuscript received May 19, 2008. Accepted May 20, 2008.

Increasing application of nanotechnology highlights the need to clarify nanotoxicity. However, few researches have focused on phytotoxicity of nanomaterials; it is unknown whether plants can uptake and transport nanoparticles. This study was to examine cell internalization and upward translocation of ZnO nanoparticles by Lolium perenne (ryegrass). The dissolution of ZnO nanoparticles and its contribution to the toxicity on ryegrass were also investigated. Zn2+ ions were used to compare and verify the root uptake and phytotoxicity of ZnO nanoparticles in a hydroponic culture system. The root uptake and phytotoxicity were visualized by light, scanning electron, and transmission electron microscopies. In the presence of ZnO nanoparticles, ryegrass biomass significantly reduced, root tips shrank, and root epidermal and cortical cells highly vacuolated or collapsed. Zn2+ ion concentrations in bulk nutrient solutions with ZnO nanoparticles were lower than the toxicity threshold of Zn2+ to the ryegrass; shoot Zn contents under ZnO nanoparticle treatments were much lower than that under Zn2+ treatments. Therefore, the phytotoxicity of ZnO nanoparticles was not directly from their limited dissolution in the bulk nutrient solution or rhizosphere. ZnO nanoparticles greatly adhered onto the root surface. Individual ZnO nanoparticles were observed present in apoplast and protoplast of the root endodermis and stele. However, translocation factors of Zn from root to shoot remained very low under ZnO nanoparticle treatments, and were much lower than that under Zn2+ treatments, implying that little (if any) ZnO nanoparticles could translocate up in the ryegrass in this study.

Introduction Nanoparticles, with at least one dimension of 100 nanometers or less, fall in the transitional zone between individual atoms or molecules and the corresponding bulk material, which can drastically modify the physicochemical properties of the material and may generate adverse biological effects in organisms (1). Nanotoxicity, an emerging concept, is receiving increasing attention with the fast development of nanotechnology (2, 3). However, to date, plants, as important ecological receptors, have not received enough nanotoxicity research. Limited phytotoxicity studies reported both positive and negative effects of nanoparticles on higher plants. TiO2 nanoparticles were reported to promote photosynthesis and nitrogen * Corresponding author phone.: (413) 545-5212; fax: (413) 5453958; e-mail: [email protected]. † Zhejiang University. ‡ University of Massachusetts. 5580

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metabolism, and then improve growth of spinach at an optimal concentration (4, 5). Alumina nanoparticles showed no adverse effect on the growth of California red kidney bean and ryegrass (6, 7); however, they were reported to inhibit root elongation of corn, cucumber, soybean, cabbage, and carrot (8). High concentration of nanosize ferrophase particles inhibited popcorn growth (9). Our previous study showed that ZnO nanoparticles greatly halted root elongation of ryegrass, radish, and rape (7). These studies increased our knowledge on phytotoxicity of nanoparticles. However, so far phytotoxicity mechanism remains unknown, and no information on the potential uptake of nanoparticles by plants and their subsequent fate within food chains is available. Nanoparticles may increase lipid membrane peroxidation upon contact to cells due to the reactive oxygen species (ROS) (1). More severe subsequence, such as genotoxicity, may happen after nanoparticles entering into cells. Therefore, investigations are increasingly focusing on cell uptake of nanoparticles and the subsequent damage. Mammalian or bacterial cell internalization of polymeric nanoparticles (10), carbon nanotubes (11, 12), and metal-based nanoparticles including ZnO (13–18) has been reported. Severe damages by nanoparticles in cells were verified, such as disruption of cytoskeleton organization of human fibroblasts by iron nanoparticles (13), and DNA damage of mouse embryonic stem cells by carbon nanotubes (12). However, to our knowledge, no information is available on plant cell internalization of nanoparticles or other particles. Cell culture studies indicated that plasma membrane internalization of exotic macromolecules with features resembling that of endocytosis or nonendocytic penetration in mammalian cells occurs in plant cells (19). However, plant cells are surrounded by cell walls in natural settings, thus, exotics need to penetrate through the cell wall prior to the membrane invagination. Plant cell wall is primarily composed of carbonhydrate polymers, and is semipermeable (20). The limited size of pores in plant cell walls through which molecules may freely pass has been determined to be less than several nm (21). Thus, exotics with diameters larger than these pores would be restricted in their ability to penetrate such a wall of an active plant cell. However, metal-based nanoparticles, including ZnO, have been reported to increase permeability and even create “holes” in bacterial cell walls (16–18) with pore size similar to plant cell walls (21). Therefore, nanoparticles may also penetrate into plant cell, which warrants further research. Dissolution of metal-based nanoparticles was reported to be a possible cause for nanotoxicity, e.g., oxide nanoparticles to a human mesothelioma and rodent fibroblast cell line (22) and ZnO to freshwater microalga (23). However, several investigations excluded dissolution from the main mechanisms regulating the toxicity of metal-based nanoparticles (1, 14, 24). We believe that toxicity of nanoparticles depends on their property, test organism species, and surrounding solution conditions. If a test organism is very susceptible to a metal ion, the toxicity of metal-based nanoparticles could be overwhelmed by the dissolved metal ions. Therefore, more research is needed to clarify the contribution of dissolution to the toxicity of metal-based nanoparticles. ZnO nanoparticles are being used in personal care products, and coating and paints, on account of their UV absorption and transparency to visible light. Thus, their potential harm to human health have attracted attention. Acute toxicity of ZnO nanoparticles has been observed to bacteria (16, 25, 26). Our previous study showed phytotoxicity of ZnO nanoparticles (7). However, the experiment was performed in Petri dishes to examine the inhibition of ZnO 10.1021/es800422x CCC: $40.75

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nanoparticles on seedling root elongation, and plant uptake and rhizosphere dissolution of the ZnO were not investigated. In this study, we used a hydroponic culture system to examine plant cell internalization and possible upward translocation of ZnO nanoparticles. The dissolution of ZnO nanoparticles and its contribution to the phytotoxicity were also investigated. Ryegrass (Lolium perenne) was used as a model plant for its wide distribution and common use in phytotoxicity studies (27, 28).

Materials and Methods Characterization of ZnO Nanoparticles. ZnO nanoparticles were purchased from Hongchen Material Sci & Tech Co., Zhejiang, China, with a purity of 99.5%, particle size of 20 ( 5 nm and a surface area of 50 ( 10 m2/g. The surface area of the ZnO nanoparticles was further determined using the multipoint Brunauer-Emmett-Teller (BET) method (29). The morphology of the ZnO nanoparticles was examined using transmission electron microscopy (TEM, JEOL 100CX, Japan). Hydroponic Culture. Seeds of ryegrass were germinated in moist gauze for about 2 weeks after sterilization and watersoaking (7). Uniform seedlings were selected and transplanted to 1000 mL beakers containing 1000 mL of nutrient solution. Compositions of the nutrient solution (1 strength Hoagland solution) were 20 ppm (NH4)2SO4, 10 ppm NH4NO3, 3.1 ppm NaH2PO4, 40 ppm K2SO4, 15 ppm CaCl2.2H2O, 0.35 ppm EDTA · FeNa · 3H2O, 25 ppm MgSO4 · 3H2O, 20 ppm Al2(SO4)3 · 18H2O, 0.1 ppm ZnSO4 · 7H2O, 0.1 ppm H3BO3, 0.025 ppm CuSO4 · 5H2O, 1 ppm MnSO4 · H2O, and 0.05 ppm Na2MoO4 · 2H2O. All of these chemicals were analytical grade with a purity of >98%. The pH of the nutrient solution was adjusted to near neutral. The beakers were wrapped with paper to keep out light. Each beaker was covered by a plastic sheet with three holes on the top. Three bundles of seedlings, each containing three seedlings, were cultured in the solutions through the three holes, respectively. The open areas between plastic and seedlings were sealed with sponge. Roots of the seedlings were submerged into the nutrient solution. The seedlings were allowed to grow in the nutrient solution for 1 week before the phytotoxicity study. Phytotoxicity experiment included three treatments. Treatment 1 was set as control without ZnO nanoparticles or Zn2+ ions added into the nutrient solution. Treatment 2 was set to study the phytotoxicity of ZnO nanoparticles which were added into the nutrient solution followed by water bath ultrasonic treatment (25 °C, 100 W, 40 kHz) for 1 h before being planted. Treatment 3 was set to study the phytotoxicity of Zn2+ ions obtained by dissolving ZnSO4 · 7H2O into the nutrient solution. Initial concentrations of the ZnO nanoparticles or Zn2+ ions were 10, 20, 50, 100, 200, and 1000 mg/L. All beakers of the three treatments were randomly placed together in a growth chamber with temperature at about 25-30 °C in daytime (16 h) and 15-20 °C in night (8 h). The phytotoxicity experiment lasted for 12 days. The suspensions were stirred with a glass rod three times per day with an 8 h interval. Characterization of ZnO Nanoparticles in Nutrient Solutions. Two types of bulk nutrient solutions in the beakers with the nanoparticles were collected. They were the bulk nutrient solutions just after stirring and after settling for 8 h without stirring by air and the glass rod, respectively. The rhizosphere solutions (root adsorbed) were also sampled by slightly shaking the seedlings after being pulled out from the nutrient solutions which had already settled for 8 h without stirring. Rhizosphere solution could contain root exudates which may change property and behavior of nanoparticles. Zeta potentials and colloid sizes of the ZnO nanoparticles in the bulk nutrient and the rhizosphere solutions were

measured with a ZetaSizer (Malvern Instrument Ltd., UK). The pH of the solutions was measured. Biomass and Zinc Concentration Determination. At the end of experiment, the seedlings were washed with flowing tap water ca. 1 minuters followed by rinsing with deionized water three times. Shoots and roots were separated, and their biomass was measured after drying at 70 °C for 24 h. Zn concentrations in the supernatants of the hydroponic solutions in treatment 2 after centrifugation (3000g for 1 h) were determined by an inductively coupled plasma optical emission spectrometer (ICP-OES) (Perkin-Elmer, Optima 4300DV, USA) at 206.2 nm wavelength (7). Zn contents in the shoots and roots were also measured by the ICP-OES after HNO3 digestion. Microscopy Observations. Fresh ryegrass roots were thoroughly washed with deionized water. The second 2 cm rootlets were cut and coated with gold for 60 s (ca. 1 nm thickness of gold) by a Sputter Coater (Cressington model 108, Ted Pella Inc., U.S.), then observed by a scanning electron microscopy (SEM, JEOL 6320FXV, Japan). The cell division states of the root tips under the three types of treatments were observed with a light microscopy (LM, Nikon Eclipse E600, Japan). The root tissues were further observed by TEM to see if ZnO nanoparticles entered the plant cells. Samples for LM and TEM were prepared following standard procedures (30). Ryegrass root samples were prefixed in 2-4% glutaraldehyde, washed in 0.1 mol/L pH 7.0 phosphate buffer, postfixed in 1% osmium tetrooxide, dehydrated in acetone, and infiltrated and embedded in epoxy resin. The first 5 mm root tips were longitudinally sliced (500 nm thick) and stained with 0.05% toluidine blue O in 0.5% sodium borate for LM, and the cross sections (65 nm thick) below the root tips were cut for TEM using a microtome with a diamond knife. Statistical Analysis. Each concentration point of the three treatments was conducted triplicately (three beakers with nine seedlings each). The results were presented as mean (SD (standard deviation) of the three beakers (nine seedlings pooled together for each beaker). The statistical analysis of experimental data utilized the Student’s t-test. Experimental data of treatments 2 and 3 were compared to their corresponding control (treatment 1). Statistical significance was accepted when the probability of the results assuming the null hypothesis (p) was less than 0.05.

Results Characterization of ZnO Nanoparticles. The surface area of ZnO nanoparticles was measured to be 58 m2/g. A typical TEM image (Figure 1A) of ZnO nanoparticles from water suspension shows aggregations due to drying on the TEM grid or aggregation in the suspension. However, individual particles in near-spherical to cuboid shape were observed. The size distribution of ZnO nanoparticles was measured and shown (Figure S1 in the Supporting Information), with a size range of 9-37 nm and a mean size of 19 ( 7 nm (n ) 152), being almost the same as that from the producer. ZnO nanoparticles appear to concentrate in the rhizosphere solution. After standing for 8 h, ZnO nanoparticles settled down in the bulk nutrient solution with a nearly transparent supernatant, whereas the turbidity of the rhizosphere solution was still similar to, or even higher than, that of the bulk nutrient solution just after being extensively stirred (Supporting Information Figure S2). The pH of the bulk nutrient and the rhizosphere solutions was the same, around 6.6. Zeta potentials of ZnO nanoparticles in the bulk nutrient and rhizosphere solutions at the end of experiment were -1.8 ( 0.2 mv and -2.9 ( 0.3 mV, respectively. As in deionized water, ZnO nanoparticles greatly aggregated in the bulk nutrient and rhizosphere solutions. A representative TEM image of ZnO nanoparticles in the rhizosphere solutions at 1000 mg/L ZnO treatment was shown in Figure 1B. Only a VOL. 42, NO. 15, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. TEM images of ZnO nanoparticles from deionized water (A) and the nutrient solution collected from the rhizosphere (B). The panel B is enlarged from the area inside the white rectangle in the insert. The length of the bar in the insert is 300 nm.

FIGURE 2. Ryegrass biomass reduction (A) and root/shoot Zn contents (B) under the treatments of ZnO nanoparticles or Zn2+. few individual ZnO nanoparticles could be identified. The mean size of ZnO nanoparticle aggregates in the nutrient and the rhizosphere solutions with initial ZnO concentration of 1000 mg/L at the end of experiment, measured by the ZetaSizer, was 900 ( 300 nm and 500 ( 80 nm respectively. Zinc concentrations (Supporting Information Figure S3) in the supernatants of the bulk nutrient solutions under ZnO nanoparticle treatments increased with increasing concentration of ZnO nanoparticles, but all lower than 8 mg/L. Our previous study observed the presence of sparse ZnO nanoparticles in the supernatants (7). Thus, the truly dissolved Zn2+ from the ZnO nanoparticles in the bulk nutrient solutions would be less than the total measured Zn in the supernatants. The dissolution of ZnO nanoparticles in this study was comparable to our previous study, but much less than the reported data by Franklin et al. (23), which indicated that 19% of the ZnO nanoparticles (ca. 30 nm in diameter) at 100 mg/L was dissolved at pH 7.5. Toxicity of ZnO Nanoparticles and Zn2+ to Ryegrass. The toxicity of ZnO nanoparticles and Zn2+ ions to the ryegrass seedlings were evident and increased with increasing concentration of both ZnO nanoparticles and Zn2+, which could be easily observed by visual examination (Supporting Information Figure S4). The seedling growth in both treatments, especially with concentrations of ZnO nanoparticles or Zn2+ higher than 50 mg/L, was retarded with shorter roots and shoots than the control. Toxic symptoms seem more severe by Zn2+ than ZnO nanoparticles. The shoots became yellow in the presence of Zn2+ with concentrations higher than 50 mg/L, and the seedlings almost withered to death with Zn2+ concentration up to 1000 mg/L. The dose-response curves of ZnO nanoparticles and Zn2+ to the ryegrass biomass are shown in Figure 2A. There appeared a concentration threshold of both treatments, below which no significant toxic symptoms were observed. However, the seedling biomass decreased with increasing dose after the threshold. The threshold of Zn2+ for both shoot and root of ryegrass was ca. 20 mg/L, whereas it was around 10 5582

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and 50 mg/L of ZnO nanoparticles for ryegrass shoots and roots, respectively. The 100% biomass inhibitory concentrations (IC100) of both ZnO nanoparticles and Zn2+ were arbitrarily taken to be near 200 mg/L, after which the biomass kept nearly unchanged with increasing concentrations. The 50% biomass inhibitory concentration (IC50) was defined in this study as the concentration at which the biomass equals the mean biomasses of blank and at IC100. IC50 of Zn2+ was estimated to be ca. 38 mg/L, which was lower than that of ZnO nanoparticles (ca. 64 ZnO mg/L, 51 mg Zn/L). Phytotoxicity of ZnO nanoparticles would diminish under environmentally relevant conditions (e.g., due to sorption to soil particles and coating by organic matter and clays). IC50 of ZnO nanoparticles to the ryegrass seedlings in this study is about 3 times higher than that of the newly germinated ryegrass seedlings in our previous study (ca. 20 mg/L) (7), indicating that the susceptibility of a plant to nanoparticles is growth stage-dependent. Toxic symptoms of ZnO nanoparticles and Zn2+ to the ryegrass were further examined by LM of the longitudinally sectioned primary root tips (Figure 3). In the control, root tips developed very well with the usual three tissue systems (epidermis, cortex, and vascular cylinder) and an intact rootcap at the apex observed (Figure 3A); longitudinally and transversely dividing cells were evident (Supporting Information Figure S5A). However, shrank morphology of the root tips (Figure 3B and 3C, respectively) indicates the severe impact of ZnO nanoparticles and Zn2+ ions. In the presence of 1000 mg/L ZnO nanoparticles or Zn2+, the epidermis and rootcap were broken, the cortical cells were highly vacuolated and collapsed, and the vascular cylinder also shrank. No living cells in the root tips could be observed in the presence of 1000 mg/L Zn2+ (Supporting Information Figure S5C), whereas part of the vascular cells seems still alive with 1000 mg/L ZnO nanoparticles, though not active as the control (Supporting Information Figure S5B). Uptake of ZnO Nanoparticles by Ryegrass. Figure 2B shows total Zn contents of the ryegrass roots and shoots

FIGURE 3. Light microscopic observation of longitudinal sections of ryegrass primary root tips under treatments of control (A); 1000 mg/L ZnO nanoparticles (B); 1000 mg/L Zn2+(C). rc: rootcap; ep: epidermis; ct: cortex; vs: vascular cylinder. under the treatments of ZnO nanoparticles and Zn2+ ions. Both treatments increased total Zn in the ryegrass tissues, but with different trends. There was no significant difference of root Zn contents between the two treatments with concentrations lower than 100 mg/L. When the concentrations of ZnO nanoparticles or Zn2+ in the nutrient solution were higher than 100 mg/L, root Zn content reduced with increasing Zn2+ concentration, however, increased with increasing ZnO concentration. Root Zn content in the presence of 1000 mg/L ZnO nanoparticles was 3.6 times higher than that of the 1000 mg/L Zn2+. Shoot Zn contents remained low under the ZnO nanoparticle treatments (0.25-1.36 mg/kg), and were much lower than that under the Zn2+ treatments (0.25-19.1 mg/kg). Translocation factor (TF) of Zn, defined as Zn content ratio of shoot to root (31), were very low (0.02-0.01) under the ZnO nanoparticle treatments, showing a decreasing tendency with increasing concentration of ZnO. However, under the Zn2+ treatments, TF (0.03-0.50) increased with increasing concentration of Zn2+. A much (1.4-50 times) lower Zn TF under the ZnO treatments than Zn2+ treatments indicates that the increasing root Zn under the ZnO treatments could be mainly from the increasing adsorption and uptake of ZnO nanoparticles by the ryegrass roots and little ZnO nanoparticles could (if any) be transported to the shoots. Figure 4 shows SEM images of the ryegrass roots under different treatments. Root surface in the control and Zn2+ treatments were free of particle adherence (Figure 4A and C). However, adsorption of ZnO nanoparticles and their aggregations on the root surface was evident (Figure 4B), and the coverage increased with increasing ZnO dose (Figure

FIGURE 4. SEM images of ryegrass root surface under treatments of control (A), 1000 mg/L ZnO nanoparticles (B), and 1000 mg/L Zn2+ (C). S6). The particles were observed filled in the epidermal crypt or adhered onto the surface. TEM images of the cross sections of ryegrass roots show the presence of dark dots (particles) in the endodermis and vascular cylinder under the ZnO treatments (Figure 5). The dark dots distributed in the apoplast, cytoplasm, and even nuclei (Figure 5B). One or several nanoparticles could be identified in the dark dots covered by cytoplast as showed by higher magnification TEM image (the insert of Figure 5C). The size of these nanoparticles were measured to be 19 ( 6 nm (n ) 89), identical to the size of ZnO nanoparticles. Such dark dots (i.e., particles) were not observed under either the control or Zn2+ treatments (Supporting Information Figure S7). Thus, it can be concluded that the ZnO nanoparticles could enter into the endodermis and vascular cylinder of the ryegrass roots.

Discussion Due to their small size and huge surface energy, nanoparticles are prone to aggregation in aqueous phase, which may influence their bioavailability and toxicity. ZnO nanoparticles aggregated in the nutrient solution with a colloid size up to several hundred nanometers. These aggregates would deposit quickly without stirring the solutions. However, our previous study (7) and this VOL. 42, NO. 15, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. TEM images showing the presence of nanoparticles covered by cytoplasm in the endodermal and vascular cells of the ryegrass root under the treatment of ZnO nanoparticles. A: part of a vascular cell enlarged from the upper rectangle area of B; B: part of a cross section of a ryegrass root; C: part of an endodermal cell enlarged from the lower rectangle area of B; the insert of panel C is magnified from the upper rectangle area of C. nu: nucleus; ne: nuclear envelop; np: nanoparticles covered by cytoplasm; vs: vascular cylinder with highly vacuolated metaxylem cells; ed: endodermis; ct: cortex; cw: cell wall. one (Figure 1) indicate that there still were individual nanoparticles stabilized in the solutions, which may be bioavailable. Meanwhile, the highly concentrated ZnO nanoparticles in the rhizosphere solution and root surface could potentially impact the ryegrass growth. Root tips and hairs can secrete large amounts of mucilage coating the root surface (20). This mucilage is a highly hydrated polysaccharide, probably a pectic substance which might contribute to the adsorption of ZnO nanoparticles to the root surface. Lolium perenne is reported to exude sugars, proteins, phenolic acids, and amino acids (32). These exudates, especially those macromolecules, might account for the stabilization of ZnO nanoparticles in the rhizosphere solution. Zn is an essential element for organisms, but it is toxic at high levels. Many previous studies showed phytotoxicity of Zn2+ (33, 34). The IC50 of Zn2+ ions was reported varying from 43 to 996 mg Zn/L to various plant species (35), which is comparable to that in this study. The general symptom of Zn2+ phytotoxicity is retardation of growth, with plants being stunted (33). This symptom was observed in the ryegrass under both ZnO nanoparticle and Zn2+ treatments in this study. However, the 5584

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yellow and withered shoots at the higher Zn2+ concentrations and the lower IC50 indicate that Zn2+ may be more toxic to the ryegrass than ZnO nanoparticles. Hence, one may wonder if the phytotoxicity of ZnO nanoparticles came from their dissolution in the nutrient solutions. ZnO is often classified as practically insoluble in water (36), but appreciable dissolution (from 1 mg/L to several thousands mg/L) of ZnO in water was reported, which was size- and pH-dependent (23). The soluble Zn in the ZnO nanoparticle-treated nutrient solutions in this study was less than 8 mg/L, which was lower than the toxic threshold of Zn2+ to the ryegrass. Thus, the phytotoxicity of ZnO nanoparticles could not directly result from their dissolution in the bulk nutrient solution. ZnO nanoparticles had different zeta potentials and aggregate sizes between the rhizosphere and bulk nutrient solutions, indicating that the root exudates may be able to change the property and behavior of ZnO nanoparticles. Thus, one may think if the phytotoxicity of ZnO nanoparticles came from their dissolution in the rhizosphere solution or on the root surface. If this was true, it is reasonable to assume that the accumulated Zn2+ from the dissolution of ZnO nanoparticles in the rhizosphere would increase the shoot Zn content just as in the Zn2+ treatments. However, shoot Zn contents in the ZnO nanoparticle treatments were lower than 1.36 mg/kg which was close to the shoot Zn content at the Zn2+ treatment with a concentration of about 25 mg/L. This Zn2+ ion concentration was lower than the IC50 and close to the threshold of Zn2+, thus, could not account for the phytotoxicity of ZnO nanoparticles. Therefore, it can be concluded that the phytotoxicity of ZnO nanoparticles could not directly result from its dissolution in the rhizosphere or on the root surface. The phytotoxicity mechanism of ZnO nanoparticles may come from their physical and chemical interferences of the root growth, which warrants further research. From the LM images, it is clear that ZnO nanoparticles severely damaged the epidermal and cortical cells and even impaired the endodermal and vascular cells, which may be a direct reason for the ryegrass growth inhibition. Nanoparticles may be able to pass through the epidermis and cortex via apoplastic passway. They have to cross the protoplast of the endodermal cells to enter into the vascular cylinder because the casparian strip between the endodermal cells would block any apoplastic transport. We observed the presence of ZnO nanoparticles in the endodermal and vascular cells, indicating that the ZnO nanoparticles could enter the ryegrass cells and move to the stele. A schematic diagram of the ryegrass cell internalization is presented and transport of ZnO nanoparticles is speculated (Supporting Information Figure S8). ZnO nanoparticles were assumed able to increase the permeability of plant cell walls and create “holes” in the walls, and then enter into the cells by permeating through the “holes”. After entering the cells, the nanoparticles may be able to transport between cells via plasmodesmata, which are microscopic channels of plants traversing the cell walls and enabling transport and communication between cells. Plasmodesmata or intercellular bridges were reported to be cylindrical channels with ∼40 nm in diameter (37). The individual ZnO nanoparticles with ca. 19 nm in diameter may enter and transport in the ryegrass cells through the plasmodesmata. However, it seems that little (if any) ZnO nanoparticles transported from root to shoot in this study considering the TF < 0.02. This may be because that most of the root ZnO nanoparticles aggregated on the outer surface, and only a few individual ones could move into the stele and were available for the upward transport. Another possible reason for the low TF is that ZnO nanoparticles were more difficult to be transported than Zn2+, and could hardly move to the shoot within the experiment duration (12 days). In summary, ZnO nanoparticles were found able to concentrate in the rhizosphere, enter the root cells, and

inhibit seedling growth of ryegrass; the phytotoxicity of ZnO nanoparticles could not primarily come from their dissolution in the bulk nutrient solution or the rhizosphere. Different types of nanoparticles and plant species need to be examined to clarify nanoparticle uptake by plant and the subsequent fate within food chains.

Acknowledgments This work was supported by the Massachusetts Agricultural Experiment Station (MAS 0090), Massachusetts Water Resources Research Center (2007MA73B), Zhejiang Provincial Natural Science Foundation of China (Z507093), and National Natural Science Foundation of China (20507015, 20737002).

Supporting Information Available Size distribution of ZnO nanoparticles (Figure S1); Photo of ZnO nanoparticles in nutrient solutions (Figure S2); Zn concentrations in the supernatants of ZnO suspensions (Figure S3); Photos of ZnO/Zn2+ treated ryegrass seedlings (Figure S4); LM images of ryegrass roots (Figure S5); SEM images of the ryegrass roots (Figure S6); TEM images of ryegrass root cells (Figure S7); Schematic diagram of plant cell internalization and transport of ZnO nanoparticles (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org.

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