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Phytotoxicity and uptake of nanoscale zero-valent iron (nZVI) by two plant species
Science of the Total Environment 443 (2013) 844–849
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Phytotoxicity and uptake of nanoscale zero-valent iron (nZVI) by two plant species Xingmao Ma a,⁎, Arun Gurung a, Yang Deng b a b
Civil and Environmental Engineering, Southern Illinois University Carbondale, IL 62901, United States Earth and Environmental Studies, Montclair State University, NJ 07403, United States
H I G H L I G H T S ► ► ► ►
nZVI may exert phytotoxic effects on plants at concentrations (> 200 mg/L) often encountered in site remediation practices. nZVI deposits on plant root surface as aggregates and some could internalize in plant root cells. Plant uptake and accumulation of nZVI are plant species-dependent. Upward transport from roots to shoots was not observed.
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Article history: Received 31 July 2012 Received in revised form 20 November 2012 Accepted 20 November 2012 Available online xxxx Keywords: nZVI Phytotoxicity Plant uptake
a b s t r a c t Use of nano-scale zero valent iron (nZVI) for the treatment of various environmental pollutants has been proven successful. However, large scale introduction of engineered nanomaterials such as nZVI into the environment has recently attracted serious concerns. There is an urgent need to investigate the environmental fate and impact of nZVI due to the scope of its application. The goal of this study was to evaluate the toxicity and accumulation of bare nZVI by two commonly encountered plant species: cattail (Typha latifolia) and hybrid poplars (Populous deltoids × Populous nigra). Plant seedlings were grown hydroponically in a greenhouse and dosed with different concentrations of nZVI (0–1000 mg/L) for four weeks. The nZVI exhibited strong toxic effect on Typha at higher concentrations (> 200 mg/L) but enhanced plant growth at lower concentrations. nZVI also significantly reduced the transpiration and growth of hybrid poplars at higher concentrations. Microscopic images indicated that large amount of nZVI coated on plant root surface as irregular aggregates and some nZVI penetrated into several layers of epidermal cells. Transmission electron microscope (TEM) and scanning transmission electron microscope (STEM) confirmed the internalization of nZVI by poplar root cells but similar internalization was not observed for Typha root cells. The upward transport to shoots was minimal for both plant species. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The scope of subsurface contamination and the hefty cost associated with the subsurface remediation require more efficient and costeffective remediation technologies. Nanoremediation, a new technology with the potential to drastically improve subsurface remediation effectiveness, involves the application of engineered nanomaterials (manmade materials with the size of 1–100 nm) in field site remediation to greatly increase the efficiency of contaminant transformation and detoxification. Since 1990 when the nano-scale zero valent iron (nZVI) was first synthesized, many different types of nanomaterials such as titanium oxide nanoparticles and carbon nanotubes have been synthesized and used in subsurface remediation (Vinu and
⁎ Corresponding author at: Department of Civil and Environmental Engineering, MC6603, Southern Illinois University Carbondale, 1230 Lincoln Drive, Carbondale, IL 62901, United States. Tel.: +1 618 453 7774; fax: +1 618 453 3044. E-mail address: [email protected] (X. Ma). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.11.073
Madras, 2010). However, nZVI remains the most predominant form of nanoremediation. nZVI was first applied in field scale in 2000 and after that, the site number remediated with nZVI has increased rapidly. Most of the sites contained some types of chlorinated compounds. However, nZVI seems not only effective for chlorinated compound remediation, but also effective for a wide variety of heavy metals such as As (Kanel et al., 2005). nZVI primarily breaks down chlorinated compounds through reductive dechlorination after receiving the electron pairs produced during the corrosion process when Fe 0 is oxidized to Fe 2+ (Li et al., 2006). Even though the coupling of reductive dechlorination and iron oxidation is energetically highly favorable, direct application of bare nZVI in the field is problematic due to their strong aggregation before and after field application (Karn et al., 2009). To enhance the reactivity and facilitate the transport of nZVI in field conditions, nZVI has been modified with various novel metals to form bimetallic nZVI or passivated with various coating materials to enhance transport from the injection point (Reddy, 2010). These nZVI have been used in at
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least forty contaminated sites and appeared not to have caused any serious environmental problems (Karn et al., 2009). However, the potential ecotoxicological impact of large scale nZVI application has undergone limited scrutiny in the past and only very recently, more attention was paid to assess the potential ecotoxicology of nZVI in the environment, after mounting evidences suggesting that nanoscale materials could carry significant ecotoxicological risks (Peralta-Videa et al., 2011). Auffan et al. found that nZVI is more toxic to Escherichia coli (E. coli) than other iron-based nanoparticles at different redox states such as maghemite and magnetite (Auffan et al., 2008). nZVI could induce cytotoxicity to E. coli at 100 mg/L while maghemite (Fe2O3) did not show any signs of toxicity until the concentration was raised up to 700 mg/L. The heightened cytotoxicity of nZVI to E. coli was attributed to more efficient generation of reactive oxygen species (ROS) by nZVI through Fenton-like reactions (Auffan et al., 2008). When the impact of nZVI on the bacterial community from a river water sample was evaluated, Barnes et al. (2010) found that 100 mg/L of nZVI affects neither the microbial community structure nor the evenness of the community. However, another group of researchers found that 1500 mg/L nZVI significantly shifted the microbial community of soil microcosms, favoring the growth of sulfate reducing bacteria and methanogens (Kirschling et al., 2010). Both research groups showed that the addition of nZVI led to a rapid decrease of the oxidation–reduction potential of the local environment. In order to effectively treat some contaminated sites including the source zones, nZVI at much higher concentrations (e.g. ~ 17% iron by weight) than mentioned above have been used (Quinn et al., 2005). The introduction of this magnitude of nZVI is concerning due to their potential toxicity to local microorganisms. A large body of literature also suggested that engineered nanomaterials at high concentrations are toxic to plants (Ma et al., 2010). Yet information on the toxicity of nZVI to plants is still scant. Plant is an essential component of our ecosystem, regulating the carbon and nitrogen cycling and is an important food source for humans and wild lives. The objective of this study was to determine the phytotoxicity of bare nZVI to two plant species as well as the uptake and accumulation of nZVI by these plants. Even though bare nZVI is rarely used in field conditions, bare nZVI is the core of all modified nZVI and previous research has indicated that the chemical composition of nanomaterial cores plays a significant role on their ecotoxicity (Griffitt et al., 2008). Therefore, we focused our investigation on bare nZVI in this study and will extend our investigation in the future to other commonly used forms of nZVI. 2. Materials and methods 2.1. Synthesis and characterization of nZVI Bare nZVI was synthesized through reductive precipitation of FeCl3 with NaBH4 as described in the literature with minor modifications (Wang and Zhang, 1997). Briefly, 15 mL of NaBH4 in DI water (0.3 M) was added dropwise into 15 mL of FeCl3 solution in 10 −4 HCl (0.1 M) while shaking continuously in a vortex shaker at 1000 rpm. Both the DI water and HCl solution were sparged with N2 for at least 2 min before their mixture. To ensure proper mixing, the mixture was shaken in a vortex shaker for additional 2 min at 2000 rpm. The nZVI so produced was sonicated in an ultrasonic water bath for 2 h at room temperature (~22 °C). The mixture was then centrifuged for 20 min at 1700 rpm to precipitate the particles. The supernatant was decanted and was replaced with same volume of N2-sparged DI water to remove the residues left in the solution after synthesis. The new mixture was shaken on a vortex shaker for 2 min at 2500 rpm to synthesize 50 mM nZVI in DI water. To characterize the NPs, the size and morphology were determined using a Hitachi S2460N scanning electron microscope (SEM). Size distribution of the particles was determined manually after
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measuring the diameters of 265 randomly selected particles with ImageJ. Specific surface area was estimated based on the average size, with an assumption that the particles are spherical and has the density of Fe 0 (7.68 g/cm 3) (Nurmi et al., 2005). 2.2. Plants Woody hybrid poplars (Populous deltoids × Populous nigra, DN34) and herbaceous cattail (Typha latifolia), both with widespread popularity and geographic availability, were used. 9 in. poplar cuttings were purchased from Segal Ranch (WA) and Typha seedlings were purchased from Hydra Aquatica (Tijeras, NM). The Typha plant seedlings were nursery grown before being shipped. The Typha seedlings were shipped and delivered in 4 in.2 containers and were transplanted to 6″ pots shortly after their receipt for continued growth. 2.3. Experimental setup Plants were grown hydroponically in a research greenhouse at Southern Illinois University Carbondale. Healthy Typha seedlings collected from the 6″ pots were placed into 64 mL brown bottles containing N2-sparged solution with different concentrations of nZVI. Each bottle contained two seedlings. The concentrations of nZVI used in this study were 0, 25, 50, 200, 500 and 1000 mg/L respectively. Four plant replicates were grown for each concentration. The plant seedlings were weighed and their respective root and shoot length were measured before placed inside the bottle. The solutions were replaced once a week with new solutions containing freshly prepared nZVI of the same concentration. The bottles were then immediately sealed at the top with aluminum foil in order to prevent evaporation and air movement. Four weeks after the initiation of the experiment, plants were carefully taken out of the bottles. Plant roots were blotted dry with a tissue paper to remove the excessive amount of moisture. Afterwards, the length of the roots and shoots were measured with a ruler and the wet weight and dry weight of the tissues were measured. The experimental setup for hybrid poplars was similar except that only one cutting was included in each bottle. Three replicates were prepared for each treatment. Shoot length was not measured for poplars because that new sprouts of this plant species were developed from the side. After four weeks of exposure to the same concentrations of nZVI as described above, plant biomass was separated into roots, stems and leaves and weighed separately. The dry weight of roots and leaves was also determined. Plant stems were kept in separate containers for further analysis. As described above, the solutions were replaced each week and after each replacement, the total weight of the reactor was measured. The water transpiration was determined as the water loss from each solution replacement to the following replacement. The accumulative transpiration of hybrid poplars during the experiment is summarized in Supplementary Fig. 2. 2.4. Scanning electron microscope (SEM) Fresh root tip obtained from 200 mg/L nZVI containing bottles was cut into small pieces (with one side being no larger than 1 mm) using a razor blade, and put into a fixative composed of 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at room temperature. The specimens were then rinsed in distilled water and were dehydrated in an ethanol series (25, 50, 75, 95, and 100%). Afterwards, they were transferred to a Tousimis SamDry II Critical Point Dryer and dried using liquid carbon dioxide. The dried specimens were then transferred onto sticky, conductive carbon tabs on aluminum stubs. Some of the specimens were coated with carbon and examined in a Hitachi S2460N SEM using a Robinson backscattered electron (BSE) detector. Other specimens were coated with 45 nm Pd/Au using a Denton Desk II sputter coating device. Examination of specimens was conducted with a Hitachi S570 SEM using a standard Everhart–Thornley secondary
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electron detector. A dispersive X-ray analysis (EDX) was also conducted to determine the composition of the deposits on root surface.
is comparable to SSA measured by the BET gas adsorption (Nurmi et al., 2005).
2.5. Transmission electron microscope (TEM)
3.2. Phytotoxicity to Typha
Samples for TEM images were cut and fixed similarly as mentioned above under SEM section. Specimens were then rinsed in buffer solution 3 × 1 h each and then placed overnight into buffer solution. Next day, specimens were dehydrated in an ethanol series (25, 50, 75, 95, and 100%) for 1 h each and then overnight in a 1:1 mixture of 100% ethanol and LR White (acrylic) embedding medium. Next day, specimens were transferred through two changes of pure LR White resin (2 h each change) and then into gelatin capsules. The sealed capsules were polymerized for 48 h at 60 °C. Thin (60–80 nm) sections of the specimens were obtained using a Leica UC6 ultramicrotome and sections were placed onto standard 200 mesh grids of copper. In order to stabilize the specimens in the electron beam, the grids with sections were coated with a 4–8 nm coating of carbon in a high vacuum evaporator (Denton DV 502). The grids were then examined with a Hitachi H-7650 II TEM. The images were recorded at various magnifications using an AMT Digital Camera. The TEM was operated at 60 kV accelerating voltage.
After four weeks of dosing, Typha plants that were exposed to higher levels of nZVI (>200 mg/L) clearly demonstrated some signs of toxic effects. The plants were markedly shorter than the controls. Plants subjected to lower concentration of nZVI (b 50 mg/L) grew better than controls. Plants dosed with over 200 mg/L of nZVI had many dry leaves and had lower biomass than the initial plants (Fig. 1A). For example, plants treated with 1000 mg/L of nZVI were on average 6.93% lighter than their initial weight due to these dry leaves. The roots of the plants at higher dose were very dark in color while the roots of control plants showed the natural healthy color of roots. It could be established by a visual observation that there was heavy deposition of materials on the root surface. In contrast to the shoot development, plant roots treated with higher dose elongated more than the root in lower dosed plants. The relative changes of plant weight and shoot height after four weeks are shown in Fig. 1B.
2.6. Scanning transmission electron microscope (STEM)
To determine whether the black coating on root surfaces was nZVI, SEM images of root samples were acquired. In the SEM images, aggregates of different sizes and morphologies were noticed on the root surface treated with 200 mg/L of bare nZVI. EDX analysis of the aggregates confirmed that these aggregates are iron (Fig. 2). The back scattering electron image highlighted the nZVI on the surface of root sample grown in 200 mg/L media. SEM image of control roots showed no deposits of nZVI. To investigate whether nZVI would penetrate through cell wall and internalize in plant cells, TEM images of root tip from the control and 200 mg/L nZVI treated plants were acquired (Fig. 3). TEM image indicated that there was no internalization of nZVI. Both the control and treated plants showed similar cellular structure and presence of foreign objects was not discovered.
TEM sample plates were also examined with a FEI 450FE STEM. Dark field images were acquired (where image appears in negative contrast compared to the TEM) to enhance contrast of the nanoparticles. After the image was obtained, X-ray analysis was performed using an Oxford INCA Energy SEM350 system equipped with an X-Max 50 silicon drift detector. 2.7. Vibrating sample magnetometer (VSM) analysis of poplar cuttings Quantum Design VSM was used for the magnetic analysis of poplar cuttings to determine the presence of iron in plant stems. The stem was hand cut into pieces with dimensions of 5 mm × 3 mm × 1 mm (Length × breadth × height). Following the cut, stem samples about 2 cm above the submerged section without direct contact with nZVI were weighed and carefully glued to the center of the paddle shaped sampler. For additional support, Teflon tape was wrapped around the sample. The sample holder was then placed at the center of the detection coil inside the VSM for measurement. The automatic centering was achieved by applying a small magnetic field of 0.1 T. Before the samples were run, a blank measurement was performed to evaluate the background magnetic signals. The background signal was used to calculate the net magnetic moment of each sample. The samples was fed into the VSM and oscillated at a predetermined frequency and amplitude. A magnetic field varying from −3 T (−30,000 Oestard) to 3 T (30,000 Oestard) was applied and the moment induced was measured as a function of the applied field. The whole experiment was performed at room temperature (300 K). 3. Results 3.1. Characterization of nZVI Scanning electron microscope (SEM) images of nZVI shown in Supplementary Fig. 1 revealed that most of the nanoparticles were spherical in shape, forming long chains and clusters due to aggregation. The average and median diameter of 265 randomly chosen particles are 366.8 nm and 344.8 nm respectively. The size distribution of bare ZVI nanoparticles is also shown in Supplementary Fig. 1. Over 50% of the nanoparticles fell in the size range of 300–400 nm. The estimated average specific surface area of nZVI is approximately 0.27 m 2/g. Previous study indicated that the calculated SSA of nZVI
3.3. Microscopic observation of nZVI accumulation in plant issues
3.4. Phytotoxicity to hybrid poplars Bare nZVI had somewhat similar effects on poplar cuttings as in Typha. Poplars dosed with high doses of bare nZVI demonstrated little growth of leaves and the older leaves grown before dosing died. The roots in the plants dosed with higher concentration were dark in color. In contrast, control plants and plants exposed to lower doses (b50 mg/L) showed healthy roots and leaves. Plant biomass determined at the termination showed concentration dependent growth. The fresh biomass was actually reduced at 1000 mg/L due to the death of initial leaves. The biomass result is consistent with the plant water usage. The accumulative transpiration rate in plants indicated that transpiration was highest for controls and gradually decreased as the concentration of nZVI increased (Supplementary Fig. 2). The water usage was reduced by approximately 63% after four weeks of exposure to 200 and 500 mg/L of nZVI. Plants treated with 1000 mg/L of nZVI had a 78% reduction of water usage compared with controls. Visual observation and SEM analysis on poplar roots revealed similar information as Typha roots that nZVI exposed roots accumulated large amorphous iron aggregates on the surface while roots from controls did not (data not shown). However, TEM image on the ultrastructure of poplar roots showed highly interesting results (Fig. 3). As expected, No nZVI was found in the control root tissue sample. But the images of treated poplar root sample contained many dark dots in the cell wall and the adjacent region of the membrane. It appeared that some of the nZVI penetrated through the membrane and was internalized in the poplar root cells. To confirm the presence of nZVI in plant roots,
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Fig. 1. A. Image of Typha seedlings four weeks after their exposure to different concentrations of nZVI; B. Percentage of plant weight and shoot height change after four weeks of exposure. Error bars represent standard deviation, n = 4.
scanning transmission electron microscope (STEM) images of the same sample were acquired (Fig. 4). Bright substances were shown in the dark field image within cell wall and inside the cell. The control root sample image was dark and did not have any highlights. EDX analysis confirmed that the bright spots are iron (Fig. 4). Following the detection of nZVI in poplar root tissues, we further investigated whether poplars will transport nZVI up to the shoots. nZVI in plant stems were measured with a vibrating samples magnetometer (VSM). VSM result indicated that there was limited uptake of the bare nZVI by poplars because no hysteresis curve was observed when a treated sample was run in the VSM machine (Supplementary Fig. 3). nZVI exposed stems generated similar curves as control stems.
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4. Discussion The results indicated that the nZVI is phytotoxic to plants at concentrations often used in the field. Several mechanisms could be attributed to the phytotoxicity of nZVI. The formation of black coating on root surface could effectively block the root membrane pores and interfere with the water and nutrient uptake process. Earlier literature has documented that under reduced condition, ferrous iron (Fe 2 +), a byproduct of iron oxidation, could be further oxidized to its less soluble form of ferric iron (Fe 3 +) by the oxidative agents that are released from plant roots, thus creating a cover of an insoluble Fe 3 + compound on the root surface. The
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Fig. 2. Scanning electron microscope images showing the deposition of nZVI on root surface. A. SEM backscatter electron image of control plants; B SEM backscatter electron image of Typha root exposed to 200 mg/L of nZVI. C and D are SEM secondary electron images of control and Typha roots exposed to 200 mg/L of nZVI. E and F are EDX analyses indicating that the aggregates are primarily composed of iron.
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A
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Fig. 3. Transmission electron microscope images. A: control root of Typha, B: Typha roots exposed to 200 mg/L of nZVI, C: control roots of hybrid poplars; D: poplar roots exposed to 200 mg/L of nZVI, and E and F are higher resolution images of hybrid poplar roots of the same samples of C and D. Scale bars shown on A–D are 500 nm and scale bars shown on E–F are 200 nm.
black coating on plant roots could also result from the direct deposition of nZVI on the surface. It is likely that the black materials were a combination of both ferric iron oxides and zero valent
irons. Regardless of the nature of the black plaque, it could block the membrane pores and substantially reduce the effectiveness of root uptake of water and nutrients.
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B
C
D
Cell wall
Fe
Fig. 4. Scanning transmission electron microscope images of poplar roots. A: Root tissues from control plants, B: root tissues exposed to 200 mg/L of nZVI, and C and D: elemental spectrum of highlighted regions on A and B.
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The introduction of nZVI could shift the redox condition in the local environment and affect the oxygen release rate of plant roots Kirschling et al., 2010. The oxygen release rates were highest for intermediate reducing condition (i.e. −250 mVb Eh b −150 mV), whereas for extremely reducing rhizospheric condition having Ehb −250 mV and for slightly less reducing condition of Eh > −150 mV, the release of oxygen was lower. In a study carried out by Kludze and DeLaune (1996), they tested the effects of soil redox intensity on the oxygen exchange and growth of Typha and Sawgrass. Their study indicated that maximum plant growth rate was recorded at slightly reducing soil condition, suggesting that nZVI could also affect plant growth through its impact on local environment. Our observation on plant biomass development dosed with bare nZVI was somewhat similar to the observations made by Stampoulis et al. (2009). These researchers reported that exposure of zucchini plants to 1000 and 500 mg/L silver nanoparticles caused a reduction of biomass by 71% and 57% respectively compared to controls and plants exposed to corresponding bulk counterparts. Their results also suggested that at concentrations lower than 100 mg/L there was no significant difference in plant biomass from controls. In this study, a positive effect on Typha biomass was observed when plants were dosed with 25 and 50 mg/L of nZVI. No such effect was noted for poplars. Their data presented that transpiration volume of zucchini exposed to 100, 500 and 1000 mg/L of silver nanoparticles were reduced by 41%, 78% and 79% respectively. Similar decrease in the transpiration was also observed in our case for the poplars. And again the reason behind the decrease in transpiration rate may be the formation of iron plaque on the root surface. The other reason could be less number of leaves in highly dosed plants as transpiration depends, to a great extent, upon the surface area of leaves. Clearly, introduction of nZVI in the environment at thousands of mg/L could affect plant growth but the extent of inhibition is dependent on plant species. The effect of plant species was also noticed in the uptake and accumulation of nZVI in plant tissues. It was found that nZVI was able to move into the root cells of poplar plants while such internalization was absent in the case of Typha. nZVI somehow managed to pass through the cell wall and membrane of poplar roots yet failed to do the same for the Typha root cells. It is unknown why poplar root cells and Typha root cells demonstrated different capabilities to fend off nZVI. A possible reason could be the differences in the structure and composition of the root cells of these two plant species. The structural, functional and composition diversity of plant cell walls has long been noticed even though the molecular and genetic mechanisms are unknown (Fangel et al., 2012). However, it appears certain that pectin, the gel-like polysaccharides contained in monocots (e.g. Typha) are very different in mechanical strength, porosity and stiffness with those in dicots (e.g. hybrid poplars) (Burton et al., 2010). In addition, aquatic plants generally contained higher contents of lignin, which is important for structural support, making plants strong enough to resist gravity in terrestrial plants and hydrodynamic forces in case of aquatic plants (Dawes et al., 1987). Lignin also helps plants to transport water and nutrients and defend from microorganisms (Campbell and Sederoff, 1996). Lignin can act as a barrier to reduce the permeability of foreign materials in cells. This could be a reason why nZVI were able to enter the root cells of the poplar plants, while relatively high lignin in the cell wall of Typha prevented nZVI from getting through it. In both cases, upward transport to the shoots was insignificant. It is possible that the aggregate of nZVI might be too large for the xylem tissues to transport. Alternatively, it is possible that nZVI might not have reached the xylem tissues in plant roots. If nZVI is synthesized and applied at the typically reported size of 35–60 nm, it is possible that they can be transported upwards. Lin et al. (2009) showed that 70 nm fullerene nanoparticles can be taken up by rice roots and transported to shoots. As a result, when nanoremediation with nZVI is selected as the remediation methods,
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it is important to consider both the remedial benefits and potential fate and toxicity of nZVI. In summary, this study reported the first study on the fate and phytotoxicity of bare nZVI to two commonly encountered plant species. The results suggested that nZVI at the concentrations used in field conditions could lead to phytotoxic effects on plants and the extent of toxicity is dependent upon plant species. Even though this study used bare nZVI and did not depict the exact influences of modified nZVI, the result indicated that large scale introduction of nZVI to the environmental could lead to serious environmental consequences and the environmental impact of such application warrants further attention. Acknowledgment The authors acknowledge the help of Dr. Saikat Talapatra in the Department of Physics at Southern Illinois University Carbondale for his help with the VSM measurement. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.scitotenv.2012.11.073. References Auffan M, Achouak W, Rose J, Roncato MA, Chaneac C, Waite DT, et al. Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ Sci Technol 2008;42:6730–5. Barnes RJ, van der Cast CJ, Riba O, Lehtovirta LE, Prosser JI, Dobson PJ, Thompson IP. The impact of zero-valent iron nanoparticles on a river water bacterial community. J Hazard Mater 2010;184:73–80. Burton RA, Gidley MJ, Fincher GB. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol 2010;6:724–32. Campbell MM, Sederoff RR. Variation in lignin content and composition: mechanisms of control and implications for the genetic improvement of plants. Plant Physiol 1996;110:3-13. Dawes C, Chan M, Chinn R, Koch EW, Lazar A, Tomasko D. Proximate composition, photosynthetic and respiratory responses of the seagrass Halophila engelmannii from Florida. Aquat Bot 1987;27:195–201. Fangel JU, Ulvskov P, Knox JP, Mikkelsen MD, Harholt J, Popper ZA, et al. Cell wall evolution and diversity. Front Plant Sci. 2012. http://dx.doi.org/10.3389/fpls.2012.00152. Griffitt RJ, Luo J, Gao J, Bonzongo J, Barber DS. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ Toxicol Chem 2008;27:1972–8. Kanel SR, Manning B, Charlet L, Choi H. Removal of arsenic (III) from groundwater by nanoscale zero-valent iron. Environ Sci Technol 2005;39:1291–8. Karn B, Kuiken T, Otto M. Nanotechnology and in situ remediation: a review of the benefits and potential risks. Environ Health Perspect 2009;117:1813–31. Kirschling TL, Gregory KB, Minkley Jr EG, Lowry GV, Tilton RD. Impact of nanoscale zero valent iron on geochemistry and microbial populations in trichloroethylene contaminated aquifer materials. Environ Sci Technol 2010;44:3474–80. Kludze HK, DeLaune RD. Soil redox intensity effects on oxygen exchange and growth of cattail and sawgrass. Soil Sci Soc Am J 1996;60:616–21. Li X, Elliott DW, Zhang W. Zero valent iron nanoparticles for abatement of environmental pollutants: materials and engineering aspect. Crit Rev Solid State Mater Sci 2006;31:111–22. Lin S, Reppert J, Hu Q, Hudson JS, Reid ML, Ratnikova TA, et al. Uptake, translocation and transmission of carbon nanomaterials in rice plants. Small 2009;5:1128–32. Ma X, Geisler-Lee J, Deng Y, Kolmakov A. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci Total Environ 2010;408:3053–61. Nurmi JT, Tratnyek PG, Sarathy V, Baer DR, Amonette JE, Pecher K, et al. Characterization and properties of metallic iron nanoparticles: spectroscopy, electrochemistry and kinetics. Environ Sci Technol 2005;39:1221–30. Peralta-Videa JR, Zhao L, Lopez-Moreno ML, Rosa GD, Hong J, Gardea-Torresdey JL. Nanomaterials and the environment: a review for the biennium 2008–2010. J Hazard Mater 2011;186:1-15. Quinn J, Geiger C, Clausen C, Brooks K, Coon C, Ohare S, et al. Field demonstration of DNAPL dehalogenation using emulsified zero-valent iron. Environ Sci Technol 2005;39:1309–18. Reddy KR. Nanotechnology for site remediation: dehalogenation of organic pollutants in soils and groundwater by nanoscale iron nanoparticles. 6th International Congress on Environmental Geotechnics, New Delhi, India; 2010. p. 165–82. Stampoulis D, Sinha SK, White JC. Assay-dependent phytotoxicity of nanoparticles to plants. Environ Sci Technol 2009;43:9473–9. Vinu R, Madras G. Environmental remediation by photocatalysis. J Indian Inst Sci 2010;90:189–230. Wang C, Zhang W. Nanoscale metal particles for dechlorination of PCE and PCBs. Environ Sci Technol 1997;31:2154–6.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Phytotoxicity and uptake of nanoscale zero-valent iron (nZVI) by two plant species Xingmao Ma a,⁎, Arun Gurung a, Yang Deng b a b
Civil and Environmental Engineering, Southern Illinois University Carbondale, IL 62901, United States Earth and Environmental Studies, Montclair State University, NJ 07403, United States
H I G H L I G H T S ► ► ► ►
nZVI may exert phytotoxic effects on plants at concentrations (> 200 mg/L) often encountered in site remediation practices. nZVI deposits on plant root surface as aggregates and some could internalize in plant root cells. Plant uptake and accumulation of nZVI are plant species-dependent. Upward transport from roots to shoots was not observed.
a r t i c l e
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Article history: Received 31 July 2012 Received in revised form 20 November 2012 Accepted 20 November 2012 Available online xxxx Keywords: nZVI Phytotoxicity Plant uptake
a b s t r a c t Use of nano-scale zero valent iron (nZVI) for the treatment of various environmental pollutants has been proven successful. However, large scale introduction of engineered nanomaterials such as nZVI into the environment has recently attracted serious concerns. There is an urgent need to investigate the environmental fate and impact of nZVI due to the scope of its application. The goal of this study was to evaluate the toxicity and accumulation of bare nZVI by two commonly encountered plant species: cattail (Typha latifolia) and hybrid poplars (Populous deltoids × Populous nigra). Plant seedlings were grown hydroponically in a greenhouse and dosed with different concentrations of nZVI (0–1000 mg/L) for four weeks. The nZVI exhibited strong toxic effect on Typha at higher concentrations (> 200 mg/L) but enhanced plant growth at lower concentrations. nZVI also significantly reduced the transpiration and growth of hybrid poplars at higher concentrations. Microscopic images indicated that large amount of nZVI coated on plant root surface as irregular aggregates and some nZVI penetrated into several layers of epidermal cells. Transmission electron microscope (TEM) and scanning transmission electron microscope (STEM) confirmed the internalization of nZVI by poplar root cells but similar internalization was not observed for Typha root cells. The upward transport to shoots was minimal for both plant species. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The scope of subsurface contamination and the hefty cost associated with the subsurface remediation require more efficient and costeffective remediation technologies. Nanoremediation, a new technology with the potential to drastically improve subsurface remediation effectiveness, involves the application of engineered nanomaterials (manmade materials with the size of 1–100 nm) in field site remediation to greatly increase the efficiency of contaminant transformation and detoxification. Since 1990 when the nano-scale zero valent iron (nZVI) was first synthesized, many different types of nanomaterials such as titanium oxide nanoparticles and carbon nanotubes have been synthesized and used in subsurface remediation (Vinu and
⁎ Corresponding author at: Department of Civil and Environmental Engineering, MC6603, Southern Illinois University Carbondale, 1230 Lincoln Drive, Carbondale, IL 62901, United States. Tel.: +1 618 453 7774; fax: +1 618 453 3044. E-mail address: [email protected] (X. Ma). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.11.073
Madras, 2010). However, nZVI remains the most predominant form of nanoremediation. nZVI was first applied in field scale in 2000 and after that, the site number remediated with nZVI has increased rapidly. Most of the sites contained some types of chlorinated compounds. However, nZVI seems not only effective for chlorinated compound remediation, but also effective for a wide variety of heavy metals such as As (Kanel et al., 2005). nZVI primarily breaks down chlorinated compounds through reductive dechlorination after receiving the electron pairs produced during the corrosion process when Fe 0 is oxidized to Fe 2+ (Li et al., 2006). Even though the coupling of reductive dechlorination and iron oxidation is energetically highly favorable, direct application of bare nZVI in the field is problematic due to their strong aggregation before and after field application (Karn et al., 2009). To enhance the reactivity and facilitate the transport of nZVI in field conditions, nZVI has been modified with various novel metals to form bimetallic nZVI or passivated with various coating materials to enhance transport from the injection point (Reddy, 2010). These nZVI have been used in at
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least forty contaminated sites and appeared not to have caused any serious environmental problems (Karn et al., 2009). However, the potential ecotoxicological impact of large scale nZVI application has undergone limited scrutiny in the past and only very recently, more attention was paid to assess the potential ecotoxicology of nZVI in the environment, after mounting evidences suggesting that nanoscale materials could carry significant ecotoxicological risks (Peralta-Videa et al., 2011). Auffan et al. found that nZVI is more toxic to Escherichia coli (E. coli) than other iron-based nanoparticles at different redox states such as maghemite and magnetite (Auffan et al., 2008). nZVI could induce cytotoxicity to E. coli at 100 mg/L while maghemite (Fe2O3) did not show any signs of toxicity until the concentration was raised up to 700 mg/L. The heightened cytotoxicity of nZVI to E. coli was attributed to more efficient generation of reactive oxygen species (ROS) by nZVI through Fenton-like reactions (Auffan et al., 2008). When the impact of nZVI on the bacterial community from a river water sample was evaluated, Barnes et al. (2010) found that 100 mg/L of nZVI affects neither the microbial community structure nor the evenness of the community. However, another group of researchers found that 1500 mg/L nZVI significantly shifted the microbial community of soil microcosms, favoring the growth of sulfate reducing bacteria and methanogens (Kirschling et al., 2010). Both research groups showed that the addition of nZVI led to a rapid decrease of the oxidation–reduction potential of the local environment. In order to effectively treat some contaminated sites including the source zones, nZVI at much higher concentrations (e.g. ~ 17% iron by weight) than mentioned above have been used (Quinn et al., 2005). The introduction of this magnitude of nZVI is concerning due to their potential toxicity to local microorganisms. A large body of literature also suggested that engineered nanomaterials at high concentrations are toxic to plants (Ma et al., 2010). Yet information on the toxicity of nZVI to plants is still scant. Plant is an essential component of our ecosystem, regulating the carbon and nitrogen cycling and is an important food source for humans and wild lives. The objective of this study was to determine the phytotoxicity of bare nZVI to two plant species as well as the uptake and accumulation of nZVI by these plants. Even though bare nZVI is rarely used in field conditions, bare nZVI is the core of all modified nZVI and previous research has indicated that the chemical composition of nanomaterial cores plays a significant role on their ecotoxicity (Griffitt et al., 2008). Therefore, we focused our investigation on bare nZVI in this study and will extend our investigation in the future to other commonly used forms of nZVI. 2. Materials and methods 2.1. Synthesis and characterization of nZVI Bare nZVI was synthesized through reductive precipitation of FeCl3 with NaBH4 as described in the literature with minor modifications (Wang and Zhang, 1997). Briefly, 15 mL of NaBH4 in DI water (0.3 M) was added dropwise into 15 mL of FeCl3 solution in 10 −4 HCl (0.1 M) while shaking continuously in a vortex shaker at 1000 rpm. Both the DI water and HCl solution were sparged with N2 for at least 2 min before their mixture. To ensure proper mixing, the mixture was shaken in a vortex shaker for additional 2 min at 2000 rpm. The nZVI so produced was sonicated in an ultrasonic water bath for 2 h at room temperature (~22 °C). The mixture was then centrifuged for 20 min at 1700 rpm to precipitate the particles. The supernatant was decanted and was replaced with same volume of N2-sparged DI water to remove the residues left in the solution after synthesis. The new mixture was shaken on a vortex shaker for 2 min at 2500 rpm to synthesize 50 mM nZVI in DI water. To characterize the NPs, the size and morphology were determined using a Hitachi S2460N scanning electron microscope (SEM). Size distribution of the particles was determined manually after
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measuring the diameters of 265 randomly selected particles with ImageJ. Specific surface area was estimated based on the average size, with an assumption that the particles are spherical and has the density of Fe 0 (7.68 g/cm 3) (Nurmi et al., 2005). 2.2. Plants Woody hybrid poplars (Populous deltoids × Populous nigra, DN34) and herbaceous cattail (Typha latifolia), both with widespread popularity and geographic availability, were used. 9 in. poplar cuttings were purchased from Segal Ranch (WA) and Typha seedlings were purchased from Hydra Aquatica (Tijeras, NM). The Typha plant seedlings were nursery grown before being shipped. The Typha seedlings were shipped and delivered in 4 in.2 containers and were transplanted to 6″ pots shortly after their receipt for continued growth. 2.3. Experimental setup Plants were grown hydroponically in a research greenhouse at Southern Illinois University Carbondale. Healthy Typha seedlings collected from the 6″ pots were placed into 64 mL brown bottles containing N2-sparged solution with different concentrations of nZVI. Each bottle contained two seedlings. The concentrations of nZVI used in this study were 0, 25, 50, 200, 500 and 1000 mg/L respectively. Four plant replicates were grown for each concentration. The plant seedlings were weighed and their respective root and shoot length were measured before placed inside the bottle. The solutions were replaced once a week with new solutions containing freshly prepared nZVI of the same concentration. The bottles were then immediately sealed at the top with aluminum foil in order to prevent evaporation and air movement. Four weeks after the initiation of the experiment, plants were carefully taken out of the bottles. Plant roots were blotted dry with a tissue paper to remove the excessive amount of moisture. Afterwards, the length of the roots and shoots were measured with a ruler and the wet weight and dry weight of the tissues were measured. The experimental setup for hybrid poplars was similar except that only one cutting was included in each bottle. Three replicates were prepared for each treatment. Shoot length was not measured for poplars because that new sprouts of this plant species were developed from the side. After four weeks of exposure to the same concentrations of nZVI as described above, plant biomass was separated into roots, stems and leaves and weighed separately. The dry weight of roots and leaves was also determined. Plant stems were kept in separate containers for further analysis. As described above, the solutions were replaced each week and after each replacement, the total weight of the reactor was measured. The water transpiration was determined as the water loss from each solution replacement to the following replacement. The accumulative transpiration of hybrid poplars during the experiment is summarized in Supplementary Fig. 2. 2.4. Scanning electron microscope (SEM) Fresh root tip obtained from 200 mg/L nZVI containing bottles was cut into small pieces (with one side being no larger than 1 mm) using a razor blade, and put into a fixative composed of 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at room temperature. The specimens were then rinsed in distilled water and were dehydrated in an ethanol series (25, 50, 75, 95, and 100%). Afterwards, they were transferred to a Tousimis SamDry II Critical Point Dryer and dried using liquid carbon dioxide. The dried specimens were then transferred onto sticky, conductive carbon tabs on aluminum stubs. Some of the specimens were coated with carbon and examined in a Hitachi S2460N SEM using a Robinson backscattered electron (BSE) detector. Other specimens were coated with 45 nm Pd/Au using a Denton Desk II sputter coating device. Examination of specimens was conducted with a Hitachi S570 SEM using a standard Everhart–Thornley secondary
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electron detector. A dispersive X-ray analysis (EDX) was also conducted to determine the composition of the deposits on root surface.
is comparable to SSA measured by the BET gas adsorption (Nurmi et al., 2005).
2.5. Transmission electron microscope (TEM)
3.2. Phytotoxicity to Typha
Samples for TEM images were cut and fixed similarly as mentioned above under SEM section. Specimens were then rinsed in buffer solution 3 × 1 h each and then placed overnight into buffer solution. Next day, specimens were dehydrated in an ethanol series (25, 50, 75, 95, and 100%) for 1 h each and then overnight in a 1:1 mixture of 100% ethanol and LR White (acrylic) embedding medium. Next day, specimens were transferred through two changes of pure LR White resin (2 h each change) and then into gelatin capsules. The sealed capsules were polymerized for 48 h at 60 °C. Thin (60–80 nm) sections of the specimens were obtained using a Leica UC6 ultramicrotome and sections were placed onto standard 200 mesh grids of copper. In order to stabilize the specimens in the electron beam, the grids with sections were coated with a 4–8 nm coating of carbon in a high vacuum evaporator (Denton DV 502). The grids were then examined with a Hitachi H-7650 II TEM. The images were recorded at various magnifications using an AMT Digital Camera. The TEM was operated at 60 kV accelerating voltage.
After four weeks of dosing, Typha plants that were exposed to higher levels of nZVI (>200 mg/L) clearly demonstrated some signs of toxic effects. The plants were markedly shorter than the controls. Plants subjected to lower concentration of nZVI (b 50 mg/L) grew better than controls. Plants dosed with over 200 mg/L of nZVI had many dry leaves and had lower biomass than the initial plants (Fig. 1A). For example, plants treated with 1000 mg/L of nZVI were on average 6.93% lighter than their initial weight due to these dry leaves. The roots of the plants at higher dose were very dark in color while the roots of control plants showed the natural healthy color of roots. It could be established by a visual observation that there was heavy deposition of materials on the root surface. In contrast to the shoot development, plant roots treated with higher dose elongated more than the root in lower dosed plants. The relative changes of plant weight and shoot height after four weeks are shown in Fig. 1B.
2.6. Scanning transmission electron microscope (STEM)
To determine whether the black coating on root surfaces was nZVI, SEM images of root samples were acquired. In the SEM images, aggregates of different sizes and morphologies were noticed on the root surface treated with 200 mg/L of bare nZVI. EDX analysis of the aggregates confirmed that these aggregates are iron (Fig. 2). The back scattering electron image highlighted the nZVI on the surface of root sample grown in 200 mg/L media. SEM image of control roots showed no deposits of nZVI. To investigate whether nZVI would penetrate through cell wall and internalize in plant cells, TEM images of root tip from the control and 200 mg/L nZVI treated plants were acquired (Fig. 3). TEM image indicated that there was no internalization of nZVI. Both the control and treated plants showed similar cellular structure and presence of foreign objects was not discovered.
TEM sample plates were also examined with a FEI 450FE STEM. Dark field images were acquired (where image appears in negative contrast compared to the TEM) to enhance contrast of the nanoparticles. After the image was obtained, X-ray analysis was performed using an Oxford INCA Energy SEM350 system equipped with an X-Max 50 silicon drift detector. 2.7. Vibrating sample magnetometer (VSM) analysis of poplar cuttings Quantum Design VSM was used for the magnetic analysis of poplar cuttings to determine the presence of iron in plant stems. The stem was hand cut into pieces with dimensions of 5 mm × 3 mm × 1 mm (Length × breadth × height). Following the cut, stem samples about 2 cm above the submerged section without direct contact with nZVI were weighed and carefully glued to the center of the paddle shaped sampler. For additional support, Teflon tape was wrapped around the sample. The sample holder was then placed at the center of the detection coil inside the VSM for measurement. The automatic centering was achieved by applying a small magnetic field of 0.1 T. Before the samples were run, a blank measurement was performed to evaluate the background magnetic signals. The background signal was used to calculate the net magnetic moment of each sample. The samples was fed into the VSM and oscillated at a predetermined frequency and amplitude. A magnetic field varying from −3 T (−30,000 Oestard) to 3 T (30,000 Oestard) was applied and the moment induced was measured as a function of the applied field. The whole experiment was performed at room temperature (300 K). 3. Results 3.1. Characterization of nZVI Scanning electron microscope (SEM) images of nZVI shown in Supplementary Fig. 1 revealed that most of the nanoparticles were spherical in shape, forming long chains and clusters due to aggregation. The average and median diameter of 265 randomly chosen particles are 366.8 nm and 344.8 nm respectively. The size distribution of bare ZVI nanoparticles is also shown in Supplementary Fig. 1. Over 50% of the nanoparticles fell in the size range of 300–400 nm. The estimated average specific surface area of nZVI is approximately 0.27 m 2/g. Previous study indicated that the calculated SSA of nZVI
3.3. Microscopic observation of nZVI accumulation in plant issues
3.4. Phytotoxicity to hybrid poplars Bare nZVI had somewhat similar effects on poplar cuttings as in Typha. Poplars dosed with high doses of bare nZVI demonstrated little growth of leaves and the older leaves grown before dosing died. The roots in the plants dosed with higher concentration were dark in color. In contrast, control plants and plants exposed to lower doses (b50 mg/L) showed healthy roots and leaves. Plant biomass determined at the termination showed concentration dependent growth. The fresh biomass was actually reduced at 1000 mg/L due to the death of initial leaves. The biomass result is consistent with the plant water usage. The accumulative transpiration rate in plants indicated that transpiration was highest for controls and gradually decreased as the concentration of nZVI increased (Supplementary Fig. 2). The water usage was reduced by approximately 63% after four weeks of exposure to 200 and 500 mg/L of nZVI. Plants treated with 1000 mg/L of nZVI had a 78% reduction of water usage compared with controls. Visual observation and SEM analysis on poplar roots revealed similar information as Typha roots that nZVI exposed roots accumulated large amorphous iron aggregates on the surface while roots from controls did not (data not shown). However, TEM image on the ultrastructure of poplar roots showed highly interesting results (Fig. 3). As expected, No nZVI was found in the control root tissue sample. But the images of treated poplar root sample contained many dark dots in the cell wall and the adjacent region of the membrane. It appeared that some of the nZVI penetrated through the membrane and was internalized in the poplar root cells. To confirm the presence of nZVI in plant roots,
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Fig. 1. A. Image of Typha seedlings four weeks after their exposure to different concentrations of nZVI; B. Percentage of plant weight and shoot height change after four weeks of exposure. Error bars represent standard deviation, n = 4.
scanning transmission electron microscope (STEM) images of the same sample were acquired (Fig. 4). Bright substances were shown in the dark field image within cell wall and inside the cell. The control root sample image was dark and did not have any highlights. EDX analysis confirmed that the bright spots are iron (Fig. 4). Following the detection of nZVI in poplar root tissues, we further investigated whether poplars will transport nZVI up to the shoots. nZVI in plant stems were measured with a vibrating samples magnetometer (VSM). VSM result indicated that there was limited uptake of the bare nZVI by poplars because no hysteresis curve was observed when a treated sample was run in the VSM machine (Supplementary Fig. 3). nZVI exposed stems generated similar curves as control stems.
A
4. Discussion The results indicated that the nZVI is phytotoxic to plants at concentrations often used in the field. Several mechanisms could be attributed to the phytotoxicity of nZVI. The formation of black coating on root surface could effectively block the root membrane pores and interfere with the water and nutrient uptake process. Earlier literature has documented that under reduced condition, ferrous iron (Fe 2 +), a byproduct of iron oxidation, could be further oxidized to its less soluble form of ferric iron (Fe 3 +) by the oxidative agents that are released from plant roots, thus creating a cover of an insoluble Fe 3 + compound on the root surface. The
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Fig. 2. Scanning electron microscope images showing the deposition of nZVI on root surface. A. SEM backscatter electron image of control plants; B SEM backscatter electron image of Typha root exposed to 200 mg/L of nZVI. C and D are SEM secondary electron images of control and Typha roots exposed to 200 mg/L of nZVI. E and F are EDX analyses indicating that the aggregates are primarily composed of iron.
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A
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Fig. 3. Transmission electron microscope images. A: control root of Typha, B: Typha roots exposed to 200 mg/L of nZVI, C: control roots of hybrid poplars; D: poplar roots exposed to 200 mg/L of nZVI, and E and F are higher resolution images of hybrid poplar roots of the same samples of C and D. Scale bars shown on A–D are 500 nm and scale bars shown on E–F are 200 nm.
black coating on plant roots could also result from the direct deposition of nZVI on the surface. It is likely that the black materials were a combination of both ferric iron oxides and zero valent
irons. Regardless of the nature of the black plaque, it could block the membrane pores and substantially reduce the effectiveness of root uptake of water and nutrients.
A
B
C
D
Cell wall
Fe
Fig. 4. Scanning transmission electron microscope images of poplar roots. A: Root tissues from control plants, B: root tissues exposed to 200 mg/L of nZVI, and C and D: elemental spectrum of highlighted regions on A and B.
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The introduction of nZVI could shift the redox condition in the local environment and affect the oxygen release rate of plant roots Kirschling et al., 2010. The oxygen release rates were highest for intermediate reducing condition (i.e. −250 mVb Eh b −150 mV), whereas for extremely reducing rhizospheric condition having Ehb −250 mV and for slightly less reducing condition of Eh > −150 mV, the release of oxygen was lower. In a study carried out by Kludze and DeLaune (1996), they tested the effects of soil redox intensity on the oxygen exchange and growth of Typha and Sawgrass. Their study indicated that maximum plant growth rate was recorded at slightly reducing soil condition, suggesting that nZVI could also affect plant growth through its impact on local environment. Our observation on plant biomass development dosed with bare nZVI was somewhat similar to the observations made by Stampoulis et al. (2009). These researchers reported that exposure of zucchini plants to 1000 and 500 mg/L silver nanoparticles caused a reduction of biomass by 71% and 57% respectively compared to controls and plants exposed to corresponding bulk counterparts. Their results also suggested that at concentrations lower than 100 mg/L there was no significant difference in plant biomass from controls. In this study, a positive effect on Typha biomass was observed when plants were dosed with 25 and 50 mg/L of nZVI. No such effect was noted for poplars. Their data presented that transpiration volume of zucchini exposed to 100, 500 and 1000 mg/L of silver nanoparticles were reduced by 41%, 78% and 79% respectively. Similar decrease in the transpiration was also observed in our case for the poplars. And again the reason behind the decrease in transpiration rate may be the formation of iron plaque on the root surface. The other reason could be less number of leaves in highly dosed plants as transpiration depends, to a great extent, upon the surface area of leaves. Clearly, introduction of nZVI in the environment at thousands of mg/L could affect plant growth but the extent of inhibition is dependent on plant species. The effect of plant species was also noticed in the uptake and accumulation of nZVI in plant tissues. It was found that nZVI was able to move into the root cells of poplar plants while such internalization was absent in the case of Typha. nZVI somehow managed to pass through the cell wall and membrane of poplar roots yet failed to do the same for the Typha root cells. It is unknown why poplar root cells and Typha root cells demonstrated different capabilities to fend off nZVI. A possible reason could be the differences in the structure and composition of the root cells of these two plant species. The structural, functional and composition diversity of plant cell walls has long been noticed even though the molecular and genetic mechanisms are unknown (Fangel et al., 2012). However, it appears certain that pectin, the gel-like polysaccharides contained in monocots (e.g. Typha) are very different in mechanical strength, porosity and stiffness with those in dicots (e.g. hybrid poplars) (Burton et al., 2010). In addition, aquatic plants generally contained higher contents of lignin, which is important for structural support, making plants strong enough to resist gravity in terrestrial plants and hydrodynamic forces in case of aquatic plants (Dawes et al., 1987). Lignin also helps plants to transport water and nutrients and defend from microorganisms (Campbell and Sederoff, 1996). Lignin can act as a barrier to reduce the permeability of foreign materials in cells. This could be a reason why nZVI were able to enter the root cells of the poplar plants, while relatively high lignin in the cell wall of Typha prevented nZVI from getting through it. In both cases, upward transport to the shoots was insignificant. It is possible that the aggregate of nZVI might be too large for the xylem tissues to transport. Alternatively, it is possible that nZVI might not have reached the xylem tissues in plant roots. If nZVI is synthesized and applied at the typically reported size of 35–60 nm, it is possible that they can be transported upwards. Lin et al. (2009) showed that 70 nm fullerene nanoparticles can be taken up by rice roots and transported to shoots. As a result, when nanoremediation with nZVI is selected as the remediation methods,
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it is important to consider both the remedial benefits and potential fate and toxicity of nZVI. In summary, this study reported the first study on the fate and phytotoxicity of bare nZVI to two commonly encountered plant species. The results suggested that nZVI at the concentrations used in field conditions could lead to phytotoxic effects on plants and the extent of toxicity is dependent upon plant species. Even though this study used bare nZVI and did not depict the exact influences of modified nZVI, the result indicated that large scale introduction of nZVI to the environmental could lead to serious environmental consequences and the environmental impact of such application warrants further attention. Acknowledgment The authors acknowledge the help of Dr. Saikat Talapatra in the Department of Physics at Southern Illinois University Carbondale for his help with the VSM measurement. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.scitotenv.2012.11.073. References Auffan M, Achouak W, Rose J, Roncato MA, Chaneac C, Waite DT, et al. Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ Sci Technol 2008;42:6730–5. Barnes RJ, van der Cast CJ, Riba O, Lehtovirta LE, Prosser JI, Dobson PJ, Thompson IP. The impact of zero-valent iron nanoparticles on a river water bacterial community. J Hazard Mater 2010;184:73–80. Burton RA, Gidley MJ, Fincher GB. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol 2010;6:724–32. Campbell MM, Sederoff RR. Variation in lignin content and composition: mechanisms of control and implications for the genetic improvement of plants. Plant Physiol 1996;110:3-13. Dawes C, Chan M, Chinn R, Koch EW, Lazar A, Tomasko D. Proximate composition, photosynthetic and respiratory responses of the seagrass Halophila engelmannii from Florida. Aquat Bot 1987;27:195–201. Fangel JU, Ulvskov P, Knox JP, Mikkelsen MD, Harholt J, Popper ZA, et al. Cell wall evolution and diversity. Front Plant Sci. 2012. http://dx.doi.org/10.3389/fpls.2012.00152. Griffitt RJ, Luo J, Gao J, Bonzongo J, Barber DS. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ Toxicol Chem 2008;27:1972–8. Kanel SR, Manning B, Charlet L, Choi H. Removal of arsenic (III) from groundwater by nanoscale zero-valent iron. Environ Sci Technol 2005;39:1291–8. Karn B, Kuiken T, Otto M. Nanotechnology and in situ remediation: a review of the benefits and potential risks. Environ Health Perspect 2009;117:1813–31. Kirschling TL, Gregory KB, Minkley Jr EG, Lowry GV, Tilton RD. Impact of nanoscale zero valent iron on geochemistry and microbial populations in trichloroethylene contaminated aquifer materials. Environ Sci Technol 2010;44:3474–80. Kludze HK, DeLaune RD. Soil redox intensity effects on oxygen exchange and growth of cattail and sawgrass. Soil Sci Soc Am J 1996;60:616–21. Li X, Elliott DW, Zhang W. Zero valent iron nanoparticles for abatement of environmental pollutants: materials and engineering aspect. Crit Rev Solid State Mater Sci 2006;31:111–22. Lin S, Reppert J, Hu Q, Hudson JS, Reid ML, Ratnikova TA, et al. Uptake, translocation and transmission of carbon nanomaterials in rice plants. Small 2009;5:1128–32. Ma X, Geisler-Lee J, Deng Y, Kolmakov A. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci Total Environ 2010;408:3053–61. Nurmi JT, Tratnyek PG, Sarathy V, Baer DR, Amonette JE, Pecher K, et al. Characterization and properties of metallic iron nanoparticles: spectroscopy, electrochemistry and kinetics. Environ Sci Technol 2005;39:1221–30. Peralta-Videa JR, Zhao L, Lopez-Moreno ML, Rosa GD, Hong J, Gardea-Torresdey JL. Nanomaterials and the environment: a review for the biennium 2008–2010. J Hazard Mater 2011;186:1-15. Quinn J, Geiger C, Clausen C, Brooks K, Coon C, Ohare S, et al. Field demonstration of DNAPL dehalogenation using emulsified zero-valent iron. Environ Sci Technol 2005;39:1309–18. Reddy KR. Nanotechnology for site remediation: dehalogenation of organic pollutants in soils and groundwater by nanoscale iron nanoparticles. 6th International Congress on Environmental Geotechnics, New Delhi, India; 2010. p. 165–82. Stampoulis D, Sinha SK, White JC. Assay-dependent phytotoxicity of nanoparticles to plants. Environ Sci Technol 2009;43:9473–9. Vinu R, Madras G. Environmental remediation by photocatalysis. J Indian Inst Sci 2010;90:189–230. Wang C, Zhang W. Nanoscale metal particles for dechlorination of PCE and PCBs. Environ Sci Technol 1997;31:2154–6.