Elucidating the Effects of Cerium Oxide Nanoparticles and Zinc Oxide

Zachry Department of Civil Engineering, Texas A&M University, TAMU 3136, ... of Civil and Environmental Engineering, Southern Methodist University, 31...
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Elucidating the Effects of Cerium Oxide Nanoparticles and Zinc Oxide Nanoparticles on Arsenic Uptake and Speciation in Rice (Oryza sativa) in a Hydroponic System Xiaoxuan Wang,† Wenjie Sun,‡ Sha Zhang,‡ Hamidreza Sharifan,† and Xingmao Ma*,† †

Zachry Department of Civil Engineering, Texas A&M University, TAMU 3136, College Station, Texas 77843-3136, United States Department of Civil and Environmental Engineering, Southern Methodist University, 3101 Dyer Street, Dallas, Texas 75205, United States

Environ. Sci. Technol. Downloaded from pubs.acs.org by UNIV PIERRE ET MARIE CURIE on 08/19/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: The accumulation of arsenic (As) in rice grains depends greatly on the redox chemistry in rice rhizosphere. Intentional or accidental introduction of strong oxidizing or reducing agents, such as metallic engineered nanoparticles (ENPs) into the plant−soil ecosystem, can change As speciation and plant uptake. However, investigation on the effects of ENPs on plant uptake of co-occurring redox sensitive heavy metals and their speciation in plant tissues is scarce. We investigated the mutual effects of two commonly encountered ENPs, cerium oxide nanoparticles (CeO2 NPs) and zinc oxide nanoparticles (ZnO NPs), and two inorganic species of As on their uptake and accumulation in rice seedlings in a hydroponic system. Rice seedlings were exposed to different combinations of 1 mg/L of As(III) or As(V) and 100 mg/L of CeO2 NPs and ZnO NPs for 6 days about 40 days after germination. ZnO NPs significantly reduced the accumulation of As(III) in rice roots by 88.1 and 96.7% and in rice shoots by 71.4 and 77.4% when the initial As was supplied as As(III) and As(V), respectively. ZnO NPs also reduced As(V) in rice roots by 68.3 and 52.3% when the As was provided as As(III) and As(V), respectively. However, the As(V) in rice shoots was unaffected by ZnO NPs regardless of the initial oxidation state of As. Neither the total As nor the individual species of As in rice tissues was significantly changed by CeO2 NPs. The co-presence of As(III) and As(V) increased Ce in rice shoots by 6.5 and 2.3 times but did not affect plant uptake of Zn. The results confirmed the active interactions between ENPs and coexisting inorganic As species, and the extent of their interactions depends on the properties of ENPs as well as the initial oxidation state of As.

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INTRODUCTION Arsenic (As) is a serious environmental and food safety concern due to its carcinogenic and mutagenic effects on humans, its high propensity for accumulation in rice grains, and the popularity of rice as a staple food in the world.1 As is considered as one of the most problematic substances on the 2013 Priority List of Hazardous Substances developed by the U.S. Agency for Toxic Substances and Disease Registry (ATSDR) (http://www.atsdr.cdc.gov/spl). Both anthropogenic and natural sources contribute to the elevated As in water and soil. The average concentration of As in the U.S. soil was 5 mg/kg, with the fifth and 95th percentile ranging from 3 to 13 mg/kg, higher than the action levels of several other countries.2 As exists mostly at oxidation states of As(III) or As(V) in the environment as both organic (mostly methylated) and inorganic As, although inorganic As is generally more dominant.3 The abundance of different As species depends on the redox conditions and the microbial community in the environment.4 Even though As is a nonessential nutrient to plants and is often toxic, it displays a high propensity for © XXXX American Chemical Society

accumulation into rice tissues, most likely due to its structural similarities to several essential elements rice needs, such as silicon (Si). A recent report suggested that an uptake of as low as 0.3 μg of inorganic As/kg body weight per day may increase risks for lung, skin, and bladder cancer.5 Under a reducing condition, inorganic As is predominantly in the form of undissociated arsenous acid (H3AsO3, As(III)) at circumneutral pH. Because of its similarity with silicic acid (H4SiO4), As(III) is primarily taken up by rice roots through Si influx transporters, such as OsLsi1.6 Under an oxidizing condition, As is primarily in the form of arsenate (As(V)) and behaves as a phosphate analogue. Therefore, inorganic As(V) is taken up by rice roots mainly through phosphate transporters (e.g., OsPHT 1;1).7 Once As enters into plant root cells, both As(III) and As(V) may be transported up to the shoots and then grains. Received: Revised: Accepted: Published: A

March 28, 2018 May 25, 2018 August 3, 2018 August 3, 2018 DOI: 10.1021/acs.est.8b01664 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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MATERIALS AND METHODS Engineered Nanoparticles. CeO2 NP and ZnO NP dispersions were purchased from US Research Nanomaterials, Inc. (Houston, TX). The size and shape of these two NPs were determined by a Tecnai G2 F20 transmission electron microscope (TEM). The TEM images of these two ENPs are shown in Figure S1. Both ENPs were primarily spherical, even though ENPs with triangular or other irregular shapes could be seen in the images. Most CeO2 NPs were 6−17 nm in diameter, with an average size of 10.5 nm. The average diameter of ZnO NPs was about 68.1 nm, ranging from 15 to 137 nm. The average particle size was obtained by measuring over 100 nanoparticles for each ENP with ImageJ (ver 1.51). Both ENPs aggregated considerably in solution. The hydrodynamic size measured with a dynamic light scattering (DLS) was 961.83 ± 94.39 nm for CeO2 NPs and 621.08 ± 7.63 nm for ZnO NPs in 100 mg/L water solution. Their zeta potential was about −36.55 ± 4.72 and −28.80 ± 2.04 mV, respectively. The co-presence of As displayed little effect on the hydrodynamic size and zeta potential of these ENPs. Other Chemicals. High purity As(V) (Na2HAsO4·7H2O > 98%) and As(III) (NaAsO2 > 90%) were purchased from Sigma-Aldrich (St. Louis, MO). Zinc sulfate heptahydrate (ZnSO4·7H2O > 99%) was obtained from Acros Organics (Geel, Belgium). Hoagland solution mixture was purchased from Phyto Technology Laboratories (Lenexa, KS). Plant Cultivation and Harvest. Rice seeds were provided by the Texas A&M AgriLife Research Center at Beaumont. The procedures for seed germination and seedling development followed the reported protocols in a previous study.25 Briefly, seeds were sterilized using 1.25% sodium hypochlorite solution for about 10 min and then rinsed with deionized water thoroughly. The sterilized seeds were germinated on a moistened filter paper in a Petri dish for 10 days. Seedlings of similar size were transferred to 15 mL polypropylene centrifuge tubes with 15 mL of quarter strength Hoagland solution for 5 days. Afterward, the seedlings were transferred to 50 mL polypropylene centrifuge tubes with 50 mL of quarter strength Hoagland solution. They were then incubated in a growth cart with a 16 h/8 h light/dark cycle for 35 days. The temperature was controlled at room temperature (23−25 °C). The Hoagland solution in the tubes was replaced every other day to avoid algae growth. After the incubation period, the seedlings were transferred to new 50 mL centrifuge tubes with tap water for 2 days to remove the Hoagland solution from root surfaces. Then, the tap water was replaced with different treatments containing different combinations of As and ENPs. Specifically, the seedlings were treated with 1 mg/L of As(III) or As(V), or 100 mg/L of CeO2 NPs or ZnO NPs, or different combinations of As and ENPs at the same concentrations. The concentrations of these chemicals were selected so that none of the chemicals would cause toxicity alone to rice seedlings and yet, they are high enough to be detected in plant tissues. Altogether, eight treatments were prepared, in addition to a negative control. Each treatment had at least three replicates. For treatments containing different oxidation states of arsenic, six replicates were prepared, three were used for total As and ENP elemental analysis, and three were used for As speciation analysis. The treatment solutions were replenished daily with tap water. After 6 days of growth, the seedlings were removed from the solution and rinsed with DI water thoroughly. Roots and

The literature suggests that the same phosphate transporters are involved in the xylem loading of As(V); however, a new set of transporters such as OsLsi2 are more responsible for As(III) xylem loading for long distance transport.7 As a mechanism of detoxification, plants tend to sequester hazardous materials in the vacuoles of plant root cells. Only As(III) can be stored into the vacuoles in rice root cells after it complexes with phytochelatins (PCs). There is a sophisticated apparatus in rice root cells that reduces As(V) to As(III), primarily by arsenate reductase (AR).5 Therefore, regardless of the forms of As species taken up by rice roots, both As(III) and As(V) are found in rice tissues even though their relative abundance can vary. Due to the different uptake pathways involved for As(III) and As(V), the uptake and accumulation of different As species in rice tissues depend heavily on the As speciation in rice rhizosphere. A suite of environmental parameters, such as dissolved oxygen,3 nitrate,8 and natural organic matter (NOM),9 can alter the speciation of As in the environment. Recently, the rapid advancement of nanotechnology has introduced an increasing amount of engineered nanoparticles (ENPs) into agricultural soils.10,11 Cerium oxide nanoparticles (CeO2 NPs) and zinc oxide nanoparticles (ZnO NPs) are two commonly encountered metallic ENPs in a variety of industrial and commercial products. For instance, ZnO NPs have been explored as an effective nanofertilizer12−14 because Zn is an essential plant nutrient that has a global deficit in many agriculture soils. ZnO NPs also display strong antimicrobial properties.15,16 Numerous studies have been carried out to evaluate the environmental fate and impact of both ENPs, including their impact on plant physiological and biochemical processes and accumulation in plant tissues.17−20 In addition to the direct interactions with plants, several studies have reported the impact of these ENPs on the plant uptake and accumulation of coexisting organic and inorganic pollutants.21 For example, CeO2 NPs were shown to change the plant uptake and accumulation of coexisting Cd in soybeans in both hydroponic and soil systems.22,23 Several underlying mechanisms have been explored with regard to the alteration of Cd plant uptake and accumulation by CeO2 NPs, including the adsorption of Cd on CeO2 NPs so that CeO2 NPs may function as a carrier of Cd; the altered chemistry in plant rhizosphere, such as elevated excretion of root exudates; and plant root anatomical structure changes in the co-presence of Cd and CeO2 NPs.23 However, the impact of ZnO NPs on the plant uptake of coexisting heavy metals is rare.19,20,24 In addition, none of the previous investigations have focused on redox sensitive chemicals, such as As. Considering the potential redox reactions on some ENP surfaces (e.g., CeO2 NPs) and their possible generation of reactive oxygen species (ROS) that can trigger redox reactions with different As species, these ENPs may play a significant role in controlling As speciation and, consequently, their plant uptake and accumulation. Therefore, a key objective of this study was to determine the impact of CeO2 NPs and ZnO NPs on the As accumulation and speciation in rice. A hydroponic system was used in this initial effort to avoid the compounding effects of soil and the microorganisms in soil. The second objective of this study was to evaluate whether inorganic As species, including both As(III) and As(V), may modify the plant uptake and accumulation of the metal elements of coexisting CeO2 NPs and ZnO NPs. B

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Figure 1. Fresh biomass of rice tissues under different treatments. (a, b) Root and shoot biomass of rice seedlings treated with different combinations of 1 mg/L of As(III) and 100 mg/L of CeO2 NPs or ZnO NPs. (c, d) Root and shoot biomass of rice plants exposed to different combinations of 1 mg/L of As(V) and 100 mg/L of CeO2 NPs or ZnO NPs. Values represent mean ± SD (n = 3 or 6). Different letters indicate significant differences (p ≤ 0.05) according to one-way ANOVA followed by Tukey’s test.

predigestion. They were then further digested using a SC100 HotBlock digestion system (Environmental Express, Charleston, SC, USA) at 100 °C for 2 h until all remaining residual tissues were fully dissolved. The tube was centrifuged at 4000 rpm for 10 min and the liquid phase was filtered through a 0.45 μm membrane filter. The filtrate was then analyzed by ion chromatography (Dionex, Sunnyvale, CA) and inductively coupled plasma spectrometry (Thermo Fisher Scientific, Waltham, MA) (IC-ICP-MS). Detailed quality assurance and quality control procedures on (IC)-ICP-MS analysis are included in the Supporting Information. Statistical Analysis. All results were subjected to the analysis of variance, means, and standard deviation. Minitab 18 (Minitab Inc., State College, PA) was used to perform one-way analysis of variance (ANOVA). One-way ANOVA analysis detects statistical differences between the means of three or more independent groups. Values were considered significantly different if p ≤ 0.05.

shoots were separated and weighed to obtain their fresh weight. Three rice seedlings from each treatment containing As were freeze-dried for As speciation analysis. The remaining three replicates from these treatments and seedlings exposed to ENPs alone were oven-dried at 75 °C for 72 h for total As and Ce and Zn element analysis. Total As, Ce, and Zn Analysis in Plant Tissues. The total As, Ce, and Zn in plant tissues were determined by strong acid digestion, following EPA method 3050b, as previously reported by Ebbs et al.26 Approximately 0.3 g of dry root and 0.65 g of dry shoot were added into a 5 mL solution of nitric acid (70% by volume) and sat overnight at room temperature for predigestion. They were then further digested using a DigiPREP MS hot block digester (SCP science, Clark Graham, Canada) at 95 °C for 4 h until all remaining residual tissues were fully dissolved. The digestate was cooled to room temperature and further mixed with 2 mL of 30% (w/v) H2O2 and heated in a hot block at 95 °C for another 2 h. This solution was then analyzed through an inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer mod. DRCII, Waltham, MA). As Speciation Analysis. The As species in plant tissues were determined by a modified EPA method 3050b, as previously reported by Narukawa et al.27 Approximately 0.1 g of freeze-dried root or shoot tissues was accurately weighed into a 50 mL perfluoroalkoxy alkanes (PFA) tube with 20 mL solution containing 0.15 mol/L nitric acid and 5 mg/L of Ag+. The mixture was sat overnight at room temperature for

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RESULTS Biomass. The fresh biomass of rice roots and shoots from different treatments are shown in Figure 1. Neither As nor ENPs alone at the used concentrations affected the fresh root and shoot biomass of rice seedlings compared with the controls, except that CeO2 NPs significantly increased the shoot biomass in the study with As(III). However, the coexposure of ZnO NPs with As(III) or As(V) significantly reduced the rice root biomass compared with the control C

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Figure 2. Total arsenic in plant tissues. (a, b) Total As in rice roots and shoots exposed to 1 mg/L As(III) alone or 1 mg/L As(III) and 100 mg/L of CeO2 NPs or ZnO NPs. (c, d) Total As in rice roots and shoots exposed to 1 mg/L As (V) alone or 1 mg/L As(V) and 100 mg/L of CeO2 NPs or ZnO NPs. Reported values represent mean ± SD (n = 3), with different letters indicating significant differences (p ≤ 0.05) according to one-way ANOVA followed by Tukey’s test. The letters are only reported when differences among means are statistically significant.

Table 1. Summary of Total Arsenic and Arsenic Species in Rice Tissues Grown with and without Exposure to Coexisting ENPsa treatment

rice compartment

As(III) As(III) + CeO2 As(III) + ZnO As(III) As(III) + CeO2 As(III) + ZnO As(V) As(V)+CeO2 As(V) + ZnO As(V) As(V) + CeO2 As(V) + ZnO

shoot shoot shoot root root root shoot shoot shoot root root root

total As (mg/kg dry weight) 22.279 22.588 15.780 217.059 217.797 60.219 19.858 18.872 9.297 150.217 126.562 48.149

± ± ± ± ± ± ± ± ± ± ± ±

0.957 A 3.937 A 5.551 A 15.976 a 28.424 a 16.407 b 6.000 A 1.535 AB 2.402 B 7.326 a 11.885 a 14.507 b

sum of As species (mg/kg dry weight) 17.555 17.861 7.912 184.440 166.360 40.973 14.693 18.107 5.960 95.484 106.936 25.107

± ± ± ± ± ± ± ± ± ± ± ±

1.895 A 2.661 A 1.932 B 9.387 a 23.390 a 2.891 b 1.933 A 3.207 A 1.700 B 6.711 a 15.896 a 5.657 b

As(III) (mg/kg dry weight) 13.100 13.785 3.745 88.573 89.320 10.560 10.307 10.040 2.324 45.891 66.911 1.467

± ± ± ± ± ± ± ± ± ± ± ±

1.370 A 1.778 A 0.368 B 13.991 a 18.500 a 3.118 b 1.023 A 4.252 A 0.921 B 5.471 a 18.316 a 0.860 b

As(V) (mg/kg dry weight) 4.455 4.076 4.167 95.867 77.040 30.413 4.387 8.067 3.636 49.593 40.025 23.640

± ± ± ± ± ± ± ± ± ± ± ±

0.602 A 0.904 A 1.654 A 6.986 a 4.960 b 0.969 c 0.983 A 3.038 A 0.795 A 4.994 a 14.273 ab 6.128 b

ratio of As(III)/As(V) 2.956 3.432 1.014 0.933 1.152 0.348 2.394 2.369 0.623 0.933 1.594 0.062

± ± ± ± ± ± ± ± ± ± ± ±

0.243 0.343 0.435 0.212 0.174 0.108 0.323 1.742 0.139 0.161 0.268 0.044

A A B a a b A A A ab a b

The reported values are the average of three replicates ± standard deviation. Letters at the end of the reported values indicate significant differences for different As species in different rice tissues between treatments.

a

plants, or plants treated with these chemicals alone. The coexposure to ZnO NPs and As(III) or As(V) decreased the fresh root biomass by 19 and 29%, respectively, compared to seedlings exposed to ZnO NPs alone. The coexposure of As with CeO2 NPs did not cause any significant changes in rice root and shoot biomass compared to the controls. However, coexposure of rice seedlings to CeO2 NPs and As(III) resulted in significantly smaller fresh root and shoot biomass compared with seedlings exposed to CeO2 NPs alone. Interestingly, the

dry biomass of both rice roots and shoots was unaffected by the treatments (Figure S2), but the total water consumption was consistent with the fresh biomass changes (Figure S3). Total Arsenic accumulation. Concentrations of total As in rice root and shoot tissues from different treatments are shown in Figure 2. Regardless of the initial As oxidation state, the co-presence of CeO2 NPs displayed minimum effect on the total As in plant tissues. However, the total As in rice tissues was significantly reduced by the co-presence of ZnO NPs D

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Figure 3. Concentrations of element Ce in plant tissues. (a) Concentrations of Ce in rice roots exposed to 100 mg/L of CeO2 NPs alone or in combination with 1 mg/L of As(III) or As(V), and (b) concentrations of Ce in rice shoots exposed to 100 mg/L of CeO2 NPs alone or in combination with 1 mg/L of As(III) or As(V). Reported values represent mean ± SD (n = 3), with different letters indicating significant differences (p ≤ 0.05) according to one-way ANOVA followed by Tukey’s test.

Figure 4. Concentrations of element Zn in plant tissues. (a) Concentrations of Zn in rice roots exposed to 100 mg/L of ZnO NPs alone or in combination with 1 mg/L of As(III) or As(V), and (b) concentrations of Zn in rice shoots exposed to 100 mg/L of ZnO NPs alone or in combination with 1 mg/L of As(III) or As(V). Reported values represent mean ± SD (n = 3), with different letters indicating significant differences (p ≤ 0.05) according to one-way ANOVA followed by Tukey’s test. The letters are only reported when differences among means are statistically significant.

compared with plants exposed to As(III) or As(V) alone. The only exception was the concentration of total As in rice shoots, which was unaffected by the joint exposure to As(III) and ZnO NPs. The total As in rice roots after exposure to ZnO NPs + As(III) and ZnO NPs + As(V) for 6 days was 72 and 68% lower than those exposed to the same concentration of As(III) or As(V) alone. Arsenic Speciation. In all treatments, organic As species in rice tissues were negligible. As in plant tissues was predominantly inorganic As. The sum of the total inorganic As species in plant tissues was comparable to the total As in plant tissues obtained through strong acid digestion (52− 112%). Irrespective of the initial As oxidation state, both As(III) and As(V) were detected in rice tissues in all treatments, Table 1. In the co-presence of As(III) and CeO2 NPs, CeO2 NPs displayed little impact on the As(III) species in rice roots, but significantly reduced As(V) species by about 20%, compared to plants treated with As(III) alone. Both As(III) and As(V) species in rice shoots were unaffected by coexisting CeO2 NPs. In contrast, coexisting ZnO NPs and As(III) significantly decreased both As(III) and As(V) species by 88 and 68% in rice root tissues, leading to a much smaller As(III)/As(V) ratio (0.348) compared with rice seedlings exposed to As(III) alone (0.924). In rice shoots, ZnO NPs did

not affect the concentration of As(V), but significantly reduced the As(III) concentration by 71%, again leading to a much smaller As(III)/As(V) ratio (0.899), compared with seedlings exposed to the same concentration of As(III) alone (2.956). When As was introduced as As(V), the total concentration as well as the individual species of As(III) and As(V) in plant tissues were generally lower than when As was introduced as As(III). An exception is the As(V) concentration in rice shoots, which was similar regardless of the initial oxidation state of As. However, As(III) concentration in rice shoot was reduced by 30% when the initial As was provided as As(V) rather than As(III). The concentrations of both As(III) and As(V) in rice roots was decreased by about 50% when As(V) was the initial state of As. Interestingly, the As(III)/As(V) ratio in rice roots was almost the same, regardless of the initial As oxidation states. Similar to what was observed in the treatment of CeO2 NPs and As(III), the coexposure to CeO2 NPs and As(V) had limited effects on the As species in rice tissues, compared with plants exposed to the same concentration of As(V) alone. ZnO NPs again significantly reduced all As species in rice tissues when they were co-present with As(V), except for the As(V) species in rice shoots. The most drastic effect of ZnO NPs was observed for As(III) in rice roots, which was lowered by almost 97% compared to plants E

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transport of As(V)? Our previous studies have shown that CeO2 NPs at the size used in this study contained about 10% reduced Ce ions on the nanoparticle surface and the redox couple of Ce(III) and Ce(IV) can actively participate in the redox reactions, endowing strong reactivity and antioxidative power to CeO2 NPs.30,31 However, the reported reduction potential of soluble Ce(IV)/Ce(III) was in the range of 1.6− 1.75 V, while the redox potential of As(V)/As(III) was only 0.139 V. Therefore, direct oxidation of As(III) to As(V) can occur by Ce on nanoparticle surface.32 It is possible that part of the As were adsorbed on CeO2 NPs surfaces and cotransported upward along with the nanoparticles. During this transport process, certain degree of oxidation of As(III) might have occurred, leading to relatively higher As(V) but lower As(III) in rice shoots. The differences in As(III) and As(V) uptake and transport could be a result of affected As transporters by CeO2 NPs. However, this was unlikely because the root uptake and xylem loading of As(V) involves the same type of phosphate transporters. If the phosphate transporters were upregulated for xylem loading, higher As(V) in the root cells could also be expected, but this was inconsistent with the generally low As(V) in rice roots in the presence of CeO2 NPs. ZnO NPs exhibited a stronger impact on plant As uptake. The most pronounced impact of ZnO NPs was in the concentration of As(III) in rice roots, regardless of the initial oxidation state of As. Even though ZnO NPs also decreased As(V) uptake in rice roots, the impact was much smaller compared to As(III). In comparison, the lowering of As species in rice shoots by ZnO NPs was much less severe than in the roots, and only As(III) was lowered by ZnO NPs, indicating that ZnO NPs had a greater impact on the root uptake of both As species than on the transport of these species from roots to shoots. It is unclear why ZnO NPs displayed such a strong effect on As speciation and uptake on the rice roots. One possible reason might be because of the negative phyto-effect of co-occurring ZnO NPs and As(III) or As(V) on root development, leading to a potentially poor performance of As transporters. As shown in Figures 1 and S3, the joint treatment of ZnO NPs with both As species led to a significantly smaller root biomass and lower water uptake. If this is true, then ZnO NPs would have a much stronger effect on aquaporins used for As(III) uptake than the phosphate transporters.19,20 Alternatively, the greater decrease of As(III) in rice roots could be attributed to some chemical processes triggered by the inclusion of ZnO NPs. One possibility is that ZnO NPs strongly inhibited the reduction of As(V) to As(III) outside plant root tissues. In the absence of ZnO NPs, plant root exudates can function as an electron donor to support the As(V) to As(III) conversion.33,34 However, in the presence of ZnO NPs that might have induced ROS,35−37 these ROS are strong oxidants and quickly oxidized all root exudates, depleting the electrons required for As reduction. Another possibility can be the lowered reduction of As(V) inside plant root cells. A comparison of the As(III)/As(V) ratio indicated that the presence of ZnO NPs have drastically lowered this ratio inside rice root cells. Because rice roots have the tendency to reduce As(V) to As(III) as a way to reduce toxicity, the low ratio of As(III)/As(V) suggested that the reduction of As(V) to As(III) had been interrupted. Because AR is the primary enzyme catalyzing the reduction inside plant root cells, we hypothesize that the ROS generated by ZnO NPs might have lowered the function of this particular enzyme, leading to significantly lower As(III) in rice roots. One thing worth

treated with As(V) alone. The significantly lower As(III) in rice roots led to a much smaller As(III)/As(V) ratio (0.062) in rice roots in the co-presence of ZnO NPs and As(V). In rice shoots, ZnO NPs decreased the concentrations of As(III) by 77%. Concentrations of Cerium and Zinc in Plant Tissues. The concentrations of Ce in plant tissues are shown in Figure 3. Neither As(III) nor As(V) significantly changed the total Ce associated with rice roots compared with plants exposed to CeO2 NPs alone. However, Ce in rice roots was significantly higher from the CeO2 NPs + As(V) treatment than from the CeO2 NPs + As(III) treatment. As(III) and As(V) led to 6.5 and 2.3 times higher Ce in rice shoots compared to the seedlings treated with CeO2 NPs alone, but the difference was only significant for the As(III) + CeO2 NPs treatment. The concentrations of element Zn in plant tissues are shown in Figure 4. Total Zn associated with plant rice tissues was slightly increased by the joint exposure to both As(III) and As(V). However, none of the increases were significant.

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DISCUSSION Rice seedlings contained high concentrations of inorganic As(III) and As(V) in their tissues irrespective of the initial As oxidation states. The lack of organic As species is probably because of the scarcity of microorganisms capable of methylating As in the hydroponic system.4,28 As expected, As(V) is less bioavailable than As(III), as indicated by the significantly lower concentrations of As in rice tissues exposed to As(V). Interestingly, the As(III)/As(V) in rice roots was close to 1.0 in both scenarios. It is not clear whether this was just a coincidence or it is a strategy for rice seedlings to minimize the impact of As and its accumulation. Total As and its speciation in rice tissues are significantly affected by ENPs. The extent of effects depends on the initial As oxidation state, the physiochemical properties of ENPs and specific rice tissues. When As was supplied as As(III), CeO2 NPs significantly reduced the As(V) concentration in rice roots but had no impact on the concentrations of As species in the shoots. However, when As was supplied as As(V), the effect of CeO2 NPs on the As species was insignificant. Taken together, the coexisting CeO2 NPs promoted the transport of As(V) from roots to shoots, but somehow inhibited the transport of As(III). The reason for the relatively higher As(III) and lower As(V) in plant root tissues but not in the shoot tissues in the co-presence of CeO2 NPs is unclear. The relatively higher transport efficiency of As(V) from rice roots to shoots is also poorly understood. We postulate that the coexposure of As(V) and CeO2 NPs might have resulted in greater excretion of root exudates, which can donate electrons to trigger redox reactions in the presence of a strong oxidizing agent, such as As(V). In a previous study, enhanced root exudation was reported in the co-presence of CeO2 NPs and Cd.22 The reduction of As(V) could result in higher As(III) in root region and higher concentration of As(III) in the rice root tissues. The assumption of higher root exudation is supported by the high Ce concentration in rice shoots in the co-presence of CeO2 NPs and As, in particular, the As(III). Even though both CeO2 NPs and dissolved Ce (Ce(III)) can be taken up by plants apoplastically,11 dissolved Ce is much more efficiently transported from roots to shoots and elevated root exudation is a primary mechanism to increase the dissolution of CeO2 NPs into dissolved Ce ion.29 What happened then during the transport process that resulted in a seemingly more efficient F

DOI: 10.1021/acs.est.8b01664 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

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mentioning, is that pH in the rhizosphere is typically 1−2 units lower than that in the bulk media. Considering its role in As speciation and ENP surface properties, its role in the altered As speciation in the presence of ENPs needs further investigation. From a food safety perspective, because inorganic As(III) is often the primary concern in rice grains, the drastically reduced As(III) in rice roots and shoots suggests that ZnO NPs may be an effective agent to reduce As(III) accumulation in rice grains. However, the coexposure of ZnO NPs and As did lead to some toxicity at the concentrations used in this study, and additional tests are needed to find an optimal concentration of ZnO NPs. The generally mild effect of As on the plant uptake of coexisting ENPs, especially Zn, might be attributed to the specific pathways of As uptake by rice roots. It is likely that neither the CeO2 NPs nor ZnO NPs, or their ions, are taken up through the specific As transporters, therefore, no competition was exerted by either As(III) or As(V). In summary, we have observed strong effects of coexisting ENPs on total As accumulation and speciation in rice tissues. The specific effects of ENPs on As depend on the unique properties of ENPs and initial oxidation state of As. Several physiological and chemical processes might have contributed to the altered As speciation and uptake by rice in the presence of CeO2 NPs and ZnO NPs. Additional investigation is needed to determine whether similar interactions in a soil-plant system can be observed. Interestingly, Zn is an essential nutrient for plants, and the strongly reduced As accumulation in rice by ZnO NPs suggests that ZnO NPs may be used as an effective agrichemical to simultaneously enhance plant growth and limit As accumulation in rice. However, a Zn dose response curve is needed to find the optimal concentration of ZnO NPs which does not cause phytotoxicity. Additional studies are needed to gain more mechanistic insights into the interactions of these two ENPs and As, including a comparative study with the bulk particles and their ionic counterparts.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b01664. TEM images of CeO2 NPs and ZnO NPs, dry biomass of rice roots and shoots exposed to different treatments, accumulative water transpiration of rice seedlings at different treatments, and information on the quality control and important operational parameters of ICPMS (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 979-862-1772; Fax: 979-862-1542; E-mail: xma@ civil.tamu.edu. ORCID

Hamidreza Sharifan: 0000-0002-6990-0635 Xingmao Ma: 0000-0003-4650-2455 Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.est.8b01664 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.8b01664 Environ. Sci. Technol. XXXX, XXX, XXX−XXX