Article Cite This: Environ. Sci. Technol. 2018, 52, 2888−2897
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CuO Nanoparticle Dissolution and Toxicity to Wheat (Triticum aestivum) in Rhizosphere Soil Xiaoyu Gao,†,§ Astrid Avellan,†,§ Stephanie Laughton,†,§ Rucha Vaidya,†,§ Sónia M. Rodrigues,‡ Elizabeth A. Casman,§,⊥ and Gregory V. Lowry*,†,§ †
Department of Civil and Environmental Engineering, §Center for Environmental Implications of NanoTechnology (CEINT), and Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ‡ Centre for Environmental and Marine Studies (CESAM), Department of Chemistry, Universidade de Aveiro, 3810-193 Aveiro, Portugal Downloaded via IOWA STATE UNIV on January 28, 2019 at 01:20:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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S Supporting Information *
ABSTRACT: It has been suggested, but not previously measured, that dissolution kinetics of soluble nanoparticles such as CuO nanoparticles (NPs) in soil affect their phytotoxicity. An added complexity is that such dissolution is also affected by the presence of plant roots. Here, we measured the rate of dissolution of CuO NPs in bulk soil, and in soil in which wheat plants (Triticum aestivum) were grown under two soil NP dosing conditions: (a) freshly added CuO NPs (500 mg Cu/kg soil) and (b) CuO NPs aged for 28 d before planting. At the end of the plant growth period (14 d), available Cu was measured in three different soil compartments: bulk (not associated with roots), loosely attached to roots, and rhizosphere (soil firmly attached to roots). The labile Cu fraction increased from 17 mg/kg to 223 mg/kg in fresh treatments and from 283 mg/kg to 305 mg/kg in aged treatments over the growth period due to dissolution. Aging CuO NPs increased the toxicity to Triticum aestivum (reduction in root maximal length). The presence of roots in the soil had opposite and somewhat compensatory effects on NP dissolution, as measured in rhizosphere soil. pH increased 0.4 pH units for fresh NP treatments and 0.6 pH units for aged NPs. This lowered CuO NP dissolution in rhizosphere soil. Exudates from T. aestivum roots also increased soluble Cu in pore water. CaCl2 extractable Cu concentrations increaed in rhizosphere soil compared to bulk soil, from 1.8 mg/kg to 6.2 mg/kg in fresh treatment and from 3.4 mg/kg to 5.4 mg/kg in aged treatments. Our study correlated CuO NP dissolution and the resulting Cu ion exposure profile to phytotoxicity, and showed that plantinduced changes in rhizosphere conditions should be considered when measuring the dissolution of CuO NPs near roots.
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INTRODUCTION The anticipated benefits of nanoenabled agrochemicals include slow and controlled release of micronutrients, plant tissuespecific targeted release of micronutrients or pesticides, reduced amounts of agrochemicals being required, and generally lower toxicity compared to more soluble products.1,2 Copper-based nanoparticles (NPs) are already on the agrochemical market.3,4 Copper is an essential crop micronutrient. Deficiency may lead to reduced disease resistance5 and decreased crop yields.6 However, at high concentrations, Cu can also be toxic to plants,7 the surrounding microbial communities,8 and soil invertebrates.9 Because of its relatively slow dissolution, CuO NPs have been studied as a potential candidate for agrochemical use. It behaves differently from dissolved Cu2+ in soil, potentially affecting copper bioavailability, the release of Cu ions over time, and potential associated risks.10−12 However, the connection between NP dissolution, the resulting dose of Cu ions, and its toxicity to terrestrial plants, and the role of root exudates on this process have not been well elucidated due to a lack of appropriate characterization of the dissolution of the NPs in soil. Ideally, application rates of these novel materials should be based on their fate and effects in the terrestrial environment, their bioavailability, and potential toxicity to plants. The toxic effect of Cu species is reflected in © 2018 American Chemical Society
physiological changes in plant roots and shoots, such as decreased root length, increased root compactness, change in root color, shorter leaf length, and decreased shoot biomass.13−15 Hyperspectral imaging has been used to visualize NPs in plants and to confirm macroscopic evidence of NP toxicity.16,17 Previous studies of the toxicity of CuO NPs to terrestrial plants assumed, but did not measure, dissolution behavior of CuO NPs in soil. This has led to conflicting conclusions on the toxicity of CuO NPs. While some studies attributed the toxic effect of CuO NPs to released ionic Cu,15,18,19 others concluded the opposite.20 For example, Servin et al. chose a Cu ion control concentration for the Cu ion control treatment based on the assumption that only 10% of the CuO NPs would dissolve in soil, the same fraction that dissolved in pure sand, rather than measuring CuO dissolution in soil. They concluded that dissolution of CuO NPs could not fully explain the plant toxicity because the plant responses differed from their Cu ion control.20 Much more than 10% CuO NPs could have dissolved Received: Revised: Accepted: Published: 2888
November 14, 2017 January 15, 2018 January 31, 2018 January 31, 2018 DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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Environmental Science & Technology
interact with soil for nutrient uptake,42,43 a better understanding of how the roots impact NP dissolution and metal availability in the rhizosphere is needed to design nanoenabled agrichemicals with optimal properties for delivering nutrients. The objectives of this study are to quantify the influence of time and near-root chemical conditions on dissolution and lability of CuO NPs in rhizosphere soil, and to determine the influence of this dissolution on the toxicity of CuO NPs to Triticum aestivum during a 14-day plant growth period in soil. Wheat (Triticum aestivum) was used in this study because it is the second most cultivated plant in the world, and it is sensitive to Cu deficiency44 or excess.45 To evaluate the toxicity of CuO NPs to plants, we measured the dissolution behavior of CuO NPs in soil in the presence of plants with an emphasis on the soil−plant interface (rhizosphere) where roots interact with soil. The toxicity of Cu was evaluated by physiological changes in plant roots and shoots.
in soil because soil organic matter (SOM) acts as a Cu sink, increasing the amount of CuO NPs that can be dissolved.11 This weakens their conclusions about a NP-specific effect. Similar problems occurred in other studies.15,18,21−23 Breaking with this trend, Dimkpa et al. (2013) evaluated the total CuO NPs dissolved in soil using a water-extraction method.24 Unfortunately, the water-extraction does not extract Cu bound to the soil solid matrix which accounts for most of the dissolved Cu in soils.25−27 Thus, their assertion of a CuO NP-specific toxicity in soil is confounded by the potential that more Cu had dissolved than was assumed or measured. Recently Qiu et al. found that the toxicity of CuO NPs, CuO bulk particles, and soluble Cu (Cu(OAC)2) depends on their solubility in soil, and that the distinction in solubility diminished after a 90-day aging period. However, the actual dissolution during the incubation periods (1 day vs 90 day) was not quantified. They successfully correlated the toxicity of NPs to roots of Hordeum vulgare L. (5day root elongation experiment) with “free Cu ions” in soil pore water measured at a single time point before seeding;19 though convincing, it should be noted that the dissolution during the 5-day toxicity test was not considered. While the relatively slow dissolution of CuO NPs may result in unobservable impacts on toxicity during a relatively short 5day toxicity test, dissolution at this rate would probably affect toxicity of NPs in longer tests. The dissolution of CuO NPs is a dissolution rate-limited process. Experimental approaches, such as extraction with CaCl2 or with diethylenetriaminepentaacetic acid (DTPA), have been used to predict the bioavailability or toxicity of metals in soil.28−31 CaCl2 extracts the Cu ions in soil pore water that are considered “readily available,” while DTPA extracts the “labile” fraction including dissolved Cu in soil pore water (free Cu2+ and Cu2+ complexed with soluble ligands such as dissolved organic matter (DOM)), but also the Cu2+ associated with soil solid phases, such as soil organic matter (SOM), clay particles, and iron oxide minerals.29−31 Whereas CaCl2 extracts metals that are “readily available” to plants,29 DTPA extracts this pool as well as the pool that may eventually become bioavailable in soil, the so-called “potentially available” fraction.32 One problem with using these extraction methods to predict the bioavailability of Cu-based nanomaterials is that a single time point extraction does not capture the temporal dynamics of the CuO NP dissolution process. Our recent study used extraction methods at different times to monitor the kinetics of release of Cu ions from CuO NPs in soil. In that study, the increase in DTPA extractable Cu over 30 days in soils was used to estimate the dissolved pool of Cu in soil.11 The availability of Cu ions increased with time over a 30 d period, which may explain why previous efforts to correlate the extractable metals in metal-based NP-amended soils with their bioavailability or toxicity have generally failed.33−35 Plants also may affect the dissolution behavior and availability of CuO NP in soil, especially in the rhizosphere. Previous studies using extraction methods to predict the bioavailability or toxicity of metal-based ENMs or the dissolution of ENMs in soil did not typically consider the impact of roots on Cu availability.11,12,33−35 Plant roots exude organic acids36−38 that may affect the pH in the rhizosphere.39,40 Although soil pH and organic carbon are known to be important factors influencing the dissolution behavior of CuO NPs in soil,11,12 and previous studies have proposed that exudates from plant roots may affect the dissolution of CuO NPs in the rhizosphere,41 no studies have quantified this. Given that the rhizosphere is where plants
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METHODS AND MATERIALS Chemicals. Calcium chloride (≥99.0%, ACS grade) and hydrogen peroxide (30%, certified ACS) were purchased from Fisher Scientific. DTPA (>99%) and triethanolamine (TEA, ≥99.0% (GC)) were purchased from Sigma-Aldrich. Trace metal grade nitric acid (65%−70%) was purchased from VWR. Triticum aestivum seeds (Pembroke 2014) were bred by Dr. David Van Sanford (Department of Plant and Soil Sciences, University of Kentucky). Nanoparticles and Characterization of Nanoparticle Properties. CuO NPs (∼40 nm primary particle size), were purchased from Sigma-Aldrich. The primary size of particles, zeta potential, isoelectric point, and hydrodynamic diameter have been characterized and reported in our previous study.11 The details of characterization methods can be found in the Supporting Information (SI). Soils and Characterization of Soil Properties. Standard Lufa 2.2 soil (loamy sand) was purchased from Lufa Speyer, Germany. Lufa 2.2 soil contains 1.6 wt % soil organic matter, and little total and available Cu (see SI, Table S1 and Table S2, control treatment). Using a well-characterized standard soil allows comparisons between studies. The high carbon organic content (about 1.6%) of Lufa 2.2 makes this soil good for agricultural studies. Soil was air-dried and sieved 0.05). The DTPA extractable Cu in the aged CuO NP treatment was statistically significantly higher than both fresh and aged CuSO4 treatment (ANOVA test followed by Fisher’s LSD test for multiple comparison, P ≤ 0.05) (Figure 1c). The CaCl2 extractable Cu revealed a different order, with fresh CuSO4 treatment having the highest CaCl2 extractable Cu, followed by the aged CuO NP treatment and the aged CuSO4 treatment, with the fresh CuO NP treatment having the lowest amount of CaCl2 extractable Cu. The CaCl2 extractable copper represents the “readily available” Cu in the pore water. 2891
DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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root hairs or root tips (Figure 3 a,c−i). For the concentration of Cu in roots, all Cu treatments were significantly higher than the control treatment. The Cu concentration in roots (577 mg/kg, s.d. = 46 mg/kg, 6 replicates) was statistically significantly higher in the freshly amended CuO NP treatment than in the aged CuO NP treatment (400 mg/kg, s.d. = 60 mg/kg, 6 replicates) or either ionic treatment (278 mg/kg, s.d. = 51 mg/ kg, 6 replicates for fresh CuSO4 and 442 mg/kg, s.d. = 67 mg/ kg, 6 replicates for aged CuSO4) (Figure S7,a). For the shoot concentrations, no statistically significant differences were found for all Cu treatments (53 mg/kg to 88 mg/kg) (Figure S7,b). Effect of near-Root Environment on Cu Availability from CuO NP Treatment. Figure 4 shows the differences in extractable Cu in rhizosphere soil, loosely attached soil, and bulk soil for fresh and aged CuO NP treatments. For the CaCl2 extraction in both fresh and aged CuO NP treatments (Figure 4a,b), the extractability of Cu in the rhizosphere soil was significantly higher than the extractability of Cu in the loosely attached soil or bulk soil (ANOVA test, P ≤ 0.05). There were no statistically significant differences among DTPA extractable Cu measurements from rhizosphere soil, loosely attached soil, and bulk soil in the freshly amended CuO NP treatment (Figure 4c). However, the DTPA extractable Cu in the rhizosphere soil in the aged CuO treatment was significantly lower than the DTPA extractable Cu in bulk soil, but similar to that measured in loosely attached soil (Figure 4d). In control experiments (Na2SO4), the CaCl2 extractable Cu was below the detection limit (0.08 mg/kg in soil, 4 μg/L for the diluted samples) in all soil samples. Aging increased the concentration of CaCl2 extractable Cu and DTPA extractable Cu in loosely attached soil and bulk soil, and increased the concentration of DTPA extractable Cu in rhizosphere soil (t test, P < 0.05). But aging did not change the CaCl2 extractable Cu in rhizosphere soil (t test, P > 0.05). Soil pH in Bulk Soil, Rhizosphere Soil, and Loosely Attached Soil. For all CuO NP treatments and the control treatment (no addition), the pH of the rhizosphere soils was significantly higher than the pH of the loosely attached and bulk soils (Figure 5a,b,c). In freshly amended CuO NP treatments and control treatments, the pH of the loosely attached soils were not statistically significantly different than the pH in the bulk soils. However, in aged CuO NP treatments, the pH of the loosely attached soil was statistically significantly higher than the pH in bulk soil. In bulk soil, the pH was the highest in freshly amended CuO NP treatments, followed by aged CuO NP treatment, followed by the control treatment, followed by the aged CuSO4 treatment, with freshly amended CuSO4 treatment having the lowest soil pH (Figure 5d).
observed in Cytoviva images. In comparison to the roots exposed to CuSO4 (SI, Figure S6), the roots exposed to CuO NP (fresh or aged) did not present the same damaged physiology. Roots exposed to CuSO4 (both fresh and aged) showed a brown damaged (necrotic) zone that was not found on any of the CuO NP exposed roots. No effects of Cu on the shoots (leaf length, biomass) were observed for the CuO NP treatments. Both the freshly amended and aged CuSO4 treatments resulted in shorter third leaves (shortened by 5.4 and 4.0 cm compared to the control for fresh and aged CuSO4 treatments, respectively). The freshly amended CuSO4 treatment also had less total shoot biomass compared to the control treatment. Some indication of toxicity was evident in all treatments except for the fresh CuO NP treatment. The aging of CuSO4 decreased its toxicity to Triticum aestivum, while the aging of CuO NP increased its toxicity. Overall, the two CuSO4 treatments showed more toxic effects to Triticum aestivum compared with the two CuO NP treatments, even though the CuSO4 was added at a significantly lower Cu concentration (300 mg/kg for CuSO4 treatments vs 500 mg/kg for CuO NP treatment). Cu Root Association and Cu Uptake. The presence of CuO NPs associated with the roots after 2 weeks of plant growth in both fresh and aged CuO NP amended soils was investigated using enhanced dark-field hyperspectral imaging (DF-HSI) as shown in Figure 3. The pixels containing CuO NP have been highlighted in red. In both fresh and aged CuO NP amended soils (Figure 3), CuO NPs were found associated with specific locations on the roots, either to the root tip mucilage (Figure3a,b,f,g), or to soil aggregates attached to the
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DISCUSSION CuO NP Dissolution Is Linked to Toxicity. Compared to Cu ions, the dynamic dissolution process of CuO NP in soil led to a very different Cu exposure profile for plants. At the end of the two growth periods, the DTPA-extracted Cu concentrations in CuO NP treatments were similar or even higher than in the CuSO4 treatment. However, a decreasing trend in DTPA extractable Cu on CuSO4 treatments during the two plant growth periods was observed. This decrease can be attributed to the soil−organic matter interactions, solid-state diffusion of Cu ions into iron minerals, or metal (co)precipitates.52−54 Conversely, an increase in DTPA-extractable Cu over time was shown in CuO NP treatments (fresh treatment and aged
Figure 3. Hyperspectral imaging of plant roots grown in soil with freshly amended CuO NPs (a−e) or after aging (f−i). The b, c, and g views are magnified views from images a and f. Pixels containing the reflectance spectra specific to CuO NPs are highlighted in red. CuO NPs and their aggregates were found associated with mucilage, root tissues, and root hairs (red arrow), and to soil aggregates attached to those locations (yellow arrows). Scale bars: 25 μm. 2892
DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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Figure 4. CaCl2 and DTPA extractable Cu in fresh (left side) and aged (right side) CuO NP amended rhizosphere soil, loosely attached soil, and bulk soil. Error bars show ±1 SD. Capital letters indicate significant differences between groups (one-way ANOVA test followed by Fisher’s LSD test for multiple comparisons, P ≤ 0.05).
Figure 5. Mean ± SD of soil pH (measured using CaCl2 extraction) in rhizosphere soil, loosely attached soil, and bulk soil in (a) soil freshly amended with CuO NP, (b) aged CuO NP treatments, and (c) control soil. (d) Comparison of pH of bulk soil among all treatments. Capital letters indicate significant differences (ANOVA test followed by Fisher’s LSD test for multiple comparison, P ≤ 0.05).
These findings suggest that when evaluating the chemical availability or toxic effect of metal-based NPs in soil, single-time point chemical extractions at the end of the plant growth period cannot capture the dissolution process of NPs in soil, and thus may fail to predict the toxicity or bioavailability of NPs.11,12,55
treatment) during the two plant growth periods and the aging period. This can be attributed to the dissolution of CuO NP.11,12 Thus, the plants in the freshly amended CuO NP soil were exposed to lower amounts of labile Cu compared to either the two CuSO4 treatments or the aged CuO NP treatment. 2893
DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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compared to bulk soil in our study indicates that the rhizosphere region was indeed influenced by the plant roots. Excretion of organic acid (dissociated ions) by plant roots, nitrogen uptake, and ionic exchanges by plant roots may explain the higher pH of the rhizosphere soil compared to the pH of bulk soil.39,40,57,58 The observed pH change in rhizosphere soil was not likely a result from the presence of CuO NP, as similar pH changes occurred with both the fresh CuO NP treatment and the negative control treatment (0.4 pH unit, ANOVA test, P > 0.05). However, in the aged CuO NP treatment, the pH increase was higher (0.6 pH unit, ANOVA test, P < 0.05), potentially because the release of Cu2+ from CuO NP attached to the roots triggered more root responses. A previous study has shown that a high concentration of ionic Cu can increase root exudation.59 The other factor that may affect CuO NP dissolution besides pH is the organic acids released by plants. Although we did not measure them here, their release is a well-known mechanism by which Triticum aestivum increases the availability of nutrients and decreases the toxic effects of metal ions such as Cu and Al.36−38 Influences of rhizosphere soil pH and root exudates on the dissolution of the CuO NPs and on the lability of Cu derived from CuO NP were revealed by the two extraction methods (Figure 4). Though rhizosphere soil exhibited higher pH (which should reduce the concentration of free Cu ions), more Cu was extracted by CaCl2 from rhizosphere soil relative to bulk soil, indicating a greater amount of complexed Cu ions in rhizosphere soil pore water. This complexation is likely a result of small organic acids released by plants and their microbiomes in the rhizosphere region. The influence of the roots on the extent of dissolution of CuO NPs was shown by the extraction of “labile” Cu (DTPA). For the aged CuO NP treatment, the DTPA extractable Cu in rhizosphere soil was lower than for the bulk soil. DTPA extraction has been shown to extract most of the labile Cu species in soil (∼80%), but it cannot extract CuO NP11(SI, Figure S1). Thus, a reduction in DTPA extractable Cu suggests diminished CuO NP dissolution in rhizosphere soil. This is consistent with the slightly higher measured pH in rhizosphere soil compared to bulk soil (Figure 5), which decreases the dissolution rate and solubility of CuO NP.10,12 This difference is less evident in the fresh CuO NP treatment than in the aged CuO NP treatment, consistent with the slightly lower rhizosphere soil pH in the fresh CuO NP treatment compared to that in the aged CuO NP treatment (Figure 5). For CuO NP treatment, the DTPA extractable Cu in all soil compartments (rhizosphere soil, loosely attached soil, and bulk soil) increased from the fresh treatment to the aged treatment as a result of CuO NP dissolution. This dissolution also resulted in higher “readily available” CaCl2 extractable in loosely attached soil and bulk soil. However, in rhizosphere soil, the impact of CuO NP dissolution on the “readily available” Cu was less significant, possibly because complexation by root exudates played a more important role in increasing the “readily available” Cu in the rhizosphere soil. The interaction between plant, soil, and Cu was limited to the rhizosphere soil region during the 14-d plant growth period, as suggested from the measured pH and extractable Cu in different soil zones. This finding is consistent with a previous study showing that the pH was affected by durum wheat roots only within a few millimeters.60 Even the loosely attached soil collected in this study remained mostly unaffected by the presence of plants. However, the spatial extent where root
Considering that it is not feasible to uniformly dose Cu ions into soil over time to precisely mimic the dosing rate from NP dissolution, toxicity studies with soluble NPs should measure the dissolution rate in soil and monitor the behavior of soluble ions in soil, and interpret their results in light of the different dosing conditions that manifest. A significantly higher toxicity in CuSO4 treatments compared to that of the fresh CuO NP treatment is explained by the higher exposure of roots to labile Cu species, even though the CuO NP treatment had a higher total Cu concentration. Also, dissolution of CuO NPs over time gradually increased the available Cu in soil, leading to higher toxicity in the aged treatment. The opposite has been observed with CuSO4, where the available Cu in soil decreased over time, leading to lower toxicity to the plants in the aged treatment. The effects of time on toxicity of CuO NP and CuSO4 have already been observed.19 The authors attributed this to the dissolution behavior of CuO NP, although without quantification. Here, we clearly showed that in order to correlate the chemical availability of CuO NPs with toxicity, the dissolution kinetics, that is, predicting the total Cu released to soil during the growth period, should be considered. The dissolution kinetics can be modeled as first-order dissolution, with the rate constant fit to the extractable Cu over time,11 and the total amount of Cu ion released from CuO NPs can be estimated by integrating the expression relating the change of extractable Cu over time. This observation is relevant to NP formulations of fungicides and micronutrients, so the release rate of the active ingredients can be better timed to the plant’s needs. CaCl2 Extractable Cu Correlates with Toxicity of CuO NP to Wheat. Although DTPA extractable Cu gives a better indication of CuO NP dissolution because it extracts most of the Cu species in soil, CaCl2 extractable Cu is better for correlating toxicity, since it measures dissolved Cu in pore water that can directly interact with plant roots. DTPA extraction would predict the toxicity of the aged CuO NP to be higher than the CuSO4 treatment (Figure 1a,b). However, this was not the case. The aged CuO NP had lower toxicity compared to both the aged and the fresh CuSO4 treatment, indicating that the DTPA extraction cannot accurately predict toxicity for the CuO NPs. The CaCl2 extraction ranked them correctly (Figure 1d). Considering that the extractable Cu in CuO NP amended soil increased over time while the extractable Cu in CuSO4 amended soil decreased over time in Lufa 2.2 soil (SI, Figure S1), the wheat plants were exposed to a lower overall “readily available” Cu (i.e., concentration × time) in CuO NP treatments compared to the CuSO4 treatments. The lower CaCl2 extractable Cu in aged CuO NP treatment is a result of higher soil pH in aged CuO NP treatment compared to that of the fresh and aged CuSO4 treatment. Higher soil pH has been previously shown to lower Cu concentration in soil pore water.27,56 Root-Associated CuO NP Modulates Toxicity. In the freshly amended CuO NP soil, although being exposed to a lower concentration of labile Cu, the roots of Triticum aestivum were actually exposed to higher total Cu concentration (Figure S7) than the other treatments. This is mainly due to CuO NPs’ association with plant roots (Figure 3b). This exposure to CuO NPs did not lead to any detected toxic effects, indicating a low or de minimiz level of toxic effects from the particle itself over the 14d growth period. Root Exudates Affect CuO NP Dissolution and Availability. The increase in the pH of rhizosphere soil 2894
DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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Conceivably, nanoenabled, slow-release fertilizers could thus be designed to be applied at high concentration but low toxicity, to last for years without reapplication. This would save energy and labor, incentives to growers to adopt those new technologies. Research is still needed to determine the rate of delivery that can provide its function effectively, but without invoking toxicity.
exudates may affect the dissolution of CuO NPs over longer growth periods needs more investigation. The changes in soil pH and the release of plant exudates changed the chemical availability and the dissolution rate of CuO NPs in the rhizosphere soil. Thus, measuring the changes of CaCl2 extractable Cu over time in rhizosphere soil may be the best way to correlate CuO NP dissolution with its toxicity. Further study is needed to quantitatively evaluate the impact of root exudates and changes in pH on dissolution behavior of CuO NP in rhizosphere soil over time to decide whether rhizosphere soil should routinely be used in extraction tests to predict the toxicity of CuO NPs to terrestrial plants. Bacteria in root rhizosphere may also play a role in CuO NP availability, dissolution, and uptake via exuding chelating compounds like pyoverdines (siderophores produced by certain pseudomonads that can complex with metal ions). Future studies needs to elucidate the role that these bacteria have in the rhizosphere region.61,62 However, these experiments indicate that measurements of Cu availability in bulk soils will not likely provide an accurate representation of the Cu availability to the plants. Triticum aestivum Regulated Cu Uptake. Although we have shown a strong correlation between CuO NP dissolution and the toxicity of CuO NP to Triticum aestivum, there was no such correlation between CuO NP dissolution and Cu translocation in Triticum aestivum shoots. Despite being exposed to different concentrations of total and labile Cu species during the growth periods among all treatments, as shown in Figure 1a,b, Triticum aestivum tended to take up similar amounts of Cu (Figure S7). This suggests that the uptake of Cu was regulated by Triticum aestivum, consistent with a previous study that found low Cu uptake by wheat even when the labile Cu concentration in soil was high.63 When Cu uptake is regulated by a plant, the bioavailable Cu in soil measured by extraction cannot be correlated to Cu uptake. Agricultural Implications. Our study indicates some potential benefits of using nano-CuO as a micronutrient amendment or fungicide rather than the soluble Cu salt. First, CuO NPs were less toxic than CuSO4, despite being applied at a much higher dose to the soils. The slow dissolution of CuO NPs reduced the maximum concentration of CaCl2-extractable Cu experienced by plants, providing a continuous release of Cu over 14 days without showing any visual toxic effect. After 28 d of aging, some Cu phytotoxic effects were observed. However, the dissolution rate of a “nanoenabled” fertilizer or fungicide could likely be tuned to provide sustained release of the Cu ions at a rate in which the concentration of Cu ions would not exceed the phytotoxic concentration.22 This tuning could potentially be accomplished through surface modification, the addition of other phases (e.g., via doping), using mixtures of different sized particles, or adjusting their size or aspect ratio. Second, we have shown that CuO NPs have higher affinity to plant roots than Cu ions. Thus, “targeting” the NPs to plant roots could be another potential benefit for a nanoenabled fertilizer. In a calcareous soil with high pH, the dissolution of CuO NP in bulk soil would be very low, but plant exudates could still potentially enhance the dissolution behavior of CuO NP and the availability of Cu in the rhizosphere soil, avoiding the toxicity of excess of Cu. This may also reduce Cu accumulation in soil, a problem with many Cu-containing fertilizers/pesticides. The improvements in nutrient uptake efficiency or antifungal properties due to this “targeting” requires more investigation.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b05816. Total Cu concentration (mg/kg) in soil for each treatment, DTPA extractable Cu in the control treatments, additional method details, changes in CaCl2 extractable Cu and DTPA extractable Cu for CuO NP and CuSO4 treatments (without plants) over 30 days, different soil regions defined in this study, spectral library of soil, CuO NP spectral libraries, and the spectral angular mapping (SAM) results to test their specificity on negative controls, DTPA extractable Cu on bulk soil and bulk bottom soil, plants used in DTPA and CaCl2 extractions for rhizosphere soil and loosely attached soil, representative photos showing Cu toxicity including shortened roots and root compactness, hyperspectral imaging of plant roots grown in soil with CuO-NP, CuSO4, or Na2SO4 (control) freshly amended or after aging, and mean concentration of Cu (mg/kg) in wheat tissue (dry weight) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Xiaoyu Gao: 0000-0002-4072-9969 Astrid Avellan: 0000-0001-6081-4389 Elizabeth A. Casman: 0000-0001-5617-6521 Gregory V. Lowry: 0000-0001-8599-008X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the NSF and EPA funding under NSF Cooperative Agreement EF-1266252, Center for the Environmental Implications of NanoTechnology (CEINT), and CBET1530563 (NanoFARM). S.M.R. acknowledges the financial support from Portuguese Foundation for Science and Technology (FCT) (Project IF/01637/2013) and from the Fulbright Scholar Program (sponsored by the U.S. Department of State). The authors acknowledge that the wheat seeds used in this study were bred by Dr. David Van Sanford (Department of Plant and Soil Sciences, University of Kentucky). The authors also acknowledge that Jieran Li (Department of Plant and Soil Sciences, University of Kentucky) measured the field capacity of Lufa 2.2 soil used in this study.
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REFERENCES
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DOI: 10.1021/acs.est.7b05816 Environ. Sci. Technol. 2018, 52, 2888−2897
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