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Uptake, accumulation and in-planta distribution of co-existing cerium oxide nanoparticles and cadmium in Glycine max (L.) Merr. Lorenzo Rossi, Weilan Zhang, Arthur Paul Schwab, and Xingmao Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03363 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017
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Environmental Science & Technology
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Uptake, accumulation and in-planta distribution of co-existing cerium oxide
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nanoparticles and cadmium in Glycine max (L.) Merr.
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Lorenzo Rossi1,a, Weilan Zhang1, Arthur Paul Schwab2, Xingmao Ma1,*
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Station, TX 77843-3136, USA
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TX 77843-2474, USA
Zachry Department of Civil Engineering, Texas A&M University, TAMU 3136, College
Department of Soil and Crop Sciences, Texas A&M University, TAMU 2474, College Station,
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*For correspondence:
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Dr. Xingmao Ma
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Zachry Department of Civil Engineering
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Texas A&M University
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TAMU 3136
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College Station, TX 77843-3136 USA
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a
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University of Science & Technology, Rolla, MO 65409, USA
Current Address: Dept. of Civil, Architectural and Environmental Engineering, Missouri
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Environmental Science & Technology
Abstract
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Agricultural soils are likely to be polluted by both conventional and emerging
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contaminants at the same time. Understanding the interactions of co-existing engineered
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nanoparticles (ENPs) and trace-elements (a common source of abiotic stress) is critical to gain
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insights into the accumulation of these two groups of chemicals by plants. The objectives of this
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study were to determine the uptake and accumulation of co-existing ENPs and trace-elements by
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soybeans and to gain insights into the physiological mechanisms resulting in different plant
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accumulation of these materials. The combinations of three cadmium levels: 0 (control) and 0.25
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and 1 mg kg-1 dry soil, and two CeO2NPs concentrations: 0 (control) and 500 mg kg-1 dry soil
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were investigated. Measurements of the plant biomass and physiological parameters indicated
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that CeO2NPs led to higher Fv/Fm ratio, suggesting that CeO2NPs enhanced the plant light energy
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use efficiency by photosystem II. In addition, the presence of CeO2NPs did not affect Cd
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accumulation in soybean, but Cd significantly increased the accumulation of Ce in plant tissues,
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especially in roots and older leaves. The altered Ce in-planta distribution was partially associated
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with the formation of root apoplastic barriers in the co-presence of Cd and CeO2NPs.
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Keywords: cerium oxide nanoparticles, cadmium, soybean, Glycine max, root barriers
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Introduction Trace-elements are hazardous for the environment and can be carcinogenic to humans 1, 2
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after prolonged exposure
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concentrations of these elements can be increased to harmful levels to both plants and animals by
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human activities
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fuels, use of fertilizers and pesticides, manufacturing of batteries and other metal products,
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sewage sludge and municipal waste disposal 5, can lead to higher concentrations of trace-
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elements in the ecosystem. Among these trace-elements, cadmium (Cd) is of great concern
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because of its toxicity to animals and humans. Cd can accumulate in plants at concentrations that
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are not phytotoxic but might be toxic to animals eating the plants 6. Cd toxicity affects humans
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more than animals, because of their longevity and the accumulation of Cd in their organs by
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eating Cd-contaminated food 7.
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. Although trace-elements are naturally present in the soil, the
. Anthropic activities such as metal mining and smelting, burning of fossil
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Cadmium is present in soil as Cd2+ and its concentration on the earth’s crust has most
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often been reported from 0.1 to 0.5 mg kg-1, but much higher and lower values have also been
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reported 8. In uncontaminated soils, Cd ranges from 0.1 to 1.0 mg kg-1 8. Numerous studies have
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been conducted aiming to better understand the interactions between Cd and plants because Cd
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uptake from food consumption has been identified as a primary source of human exposure to Cd
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due to its highly efficient soil-to-plant transfer compared with other trace-elements 9. Moreover,
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a World Health Organization study estimated that food accounts for about 90% of Cd exposure
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in the general non-smoking population 10.
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Meanwhile, the rapid advancement of nanotechnology significantly increases the
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manufacturing, application and release of engineered nanoparticles (ENPs) into the environment,
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leading to a potential increase in human and environmental exposure to ENPs 4 ACS Paragon Plus Environment
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. Among a
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plethora of available ENPs nowadays, cerium oxide nanoparticles (CeO2NPs) are widely used in
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the production of catalysts, sunscreen creams, fuel additives, microelectronics and polishing
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agents due to their unique catalytic and optic properties
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frequent occurrence of CeO2NPs in the environment, with agricultural soil as an important sink
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agricultural crops
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well as the impact of various abiotic stresses 22, 23 and growing conditions 24 on the accumulation
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of CeO2NPs in plants have been investigated. Notably, environmental stresses play a crucial role
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in the plant accumulation of CeO2NPs and often lead to higher accumulation of Ce in plant
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tissues
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particularly alleviating the oxidative stresses at leaf level. For instance, CeO2NPs led to changes
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in canola (Brassica napus L.) growth and physiology improving the plant salt stress response 22.
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In a different study, Rico, et al.
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stress enzymes in rice (Oryza sativa L.) seedlings, leading to an overall better plant physiology.
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. These applications have led to the
. As a result, the physiological and biochemical impacts of CeO2NPs on a wide variety of
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15-18
, the extent and mechanisms of CeO2NPs accumulation in crops
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, as
. On the other hand, CeO2NPs conferred protection against abiotic stresses,
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found that CeO2NPs modified the activities of anti-oxidative
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Metal stress is common in agricultural soils. However, the impact of trace-element on
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plant accumulation of CeO2NPs, or other ENPs, has not been explored. In a few studies which
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examined the plant interactions with co-existing trace-elements and ENPs, emphasis was placed
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on the physiological effects of ENPs in the presence of trace-elements and the altered trace-
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element uptake and accumulation by ENPs
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metabolism in several plant species exposed to different trace-elements
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antioxidative defense, detoxification, and signaling systems. TiO2NPs and ZnONPs increased the
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. For example, TiO2NPs modulated the energy
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plant uptake of cadmium and other pollutants from the soil
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shown to reduce trace-element uptake by wheat seedlings
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, enhancing the
. However, Fe3O4NPs were
. The contradicting effects of ENPs
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on plant metal uptake may arise from the different ENPs and plant species studied. So far no
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studies have been conducted with regard to the interactions between plants and co-existing
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CeO2NPs and Cd.
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Given the high probability of Cd and CeO2NPs co-occurrence in agricultural soils and
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their potential interactions, an investigation into the impacts of the co-presence of Cd and
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CeO2NPs would yield important results. In this experiment, Glycine max (L.) Merr. is employed
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as a representative crop due to its considerable significance for global agriculture, contributing
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about 40% of annual oilseed crop yield in the world
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each year.
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and about $30 billion to the global market
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The objectives of this study were: (i) to determine the uptake, accumulation and in-planta
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distribution of co-existing Cd and Ce and (ii) to examine to underlying physiological and root
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anatomical changes in the co-presence of Cd and CeO2NPs.
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Material and Methods
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CeO2NPs and Cd
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CeO2NPs coated with Poly Vinyl Pyrrolidone (PVP) dispersion were purchased from the
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US Research Nanomaterials, Inc. (Houston, TX). The Transmission Electron Microscopy (TEM)
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image of the CeO2NPs used in this study was reported elsewhere
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CeO2NPs is 41.7 + 5.2 nm, determined by measuring the diameter of over 100 nanoparticles on
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the TEM image with ImageJ. The zeta potential of the CeO2NPs dispersed in water at 500 mg
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L−1 was −51.57 mV, as measured by dynamic light scattering (Zetasizer Nano ZS90, Malvern
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Instruments Ltd., Worcestershire, UK). A CeO2NPs concentration of 500 mg kg-1 dry sand was 6 ACS Paragon Plus Environment
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. The average size of the
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chosen because the majority of the previous studies on the toxicity of CeO2NPs to terrestrial
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plants used concentrations in the range of 1−1000 mg L-1 and our previous studies also indicated
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that CeO2NPs at this concentration affect plant physiology but is not lethal to plants 32.
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Cd sulfate (CdSO4) was purchased from Fisher Scientific Int. (Pittsburgh, PA) and was
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totally dissolved in the nutrient solution to reach the targeted concentrations (0.25 and 1.0 mg kg-
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1
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represented the background Cd concentrations in many agricultural soils 8, 33.
dry sand) at the beginning of the experiment. Cd concentrations were chosen because they
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Plant species and growth conditions
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Glycine max (L.) Merr. (soybean) cv. ‘Tohya’ seeds were purchased from Johnny’s
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Selected Seeds (Winslow, ME). Soybean seeds were germinated in moist sand for 5 days. After
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germination, young seedlings were individually transplanted into 9.5 cm diameter × 12 cm
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height plastic pots filled with 500 g of sand (Quikrete, Atlanta, GA). The sand was saturated with
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25% Hoagland solution
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made CeO2NPs stock solution (3125, mg L-1) and/or known amount of cadmium sulfate (CdSO4)
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anhydrous (Thermo Fisher Scientific, Waltham, MA) were introduced to the plastic pots before
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transplant to achieve a concentration of 500 mg kg-1 CeO2NPs and/or 0.25 and 1.0 mg kg-1 Cd2+
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dry sand. Sand has the advantages of being a less complicated system than soil, due to less
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adsorption, maximizing the chemical lability of the added cadmium. Five plants for each
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treatment were grown at room temperature under fluorescent bulbs providing 250 µmol m2 s-1
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photosynthetic photon flux density (16 h light – 8 h dark photoperiod). Plants were fert-irrigated
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(Phyto Technology Lab, Shawnee Mission, KS). 80 mL of freshly
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with full strength Hoagland for 30 days. Plants grown in Hoagland without the addition of
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CdSO4 and CeO2NPs were used as controls.
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Growth analyses and biomass partitioning
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After the 30-day growth period, five plants per treatment were carefully removed from
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the growth media and rinsed with deionized water, divided into roots and leaves and then, tapped
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dry with a paper towel. The root and leaf tissues from each replicates were dried in an oven at 70
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ºC for 7 days to determine the dry biomass (DW), and concentrations of sodium and cerium in
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these tissues. Fresh root samples from each plant were collected and stored at 4 ºC in pure
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methanol for root anatomical analysis. Detailed descriptions of the analytical parameters and
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procedures are provided below.
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Cadmium and Cerium contents analyses
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A 0.5 g subsample of dry plant tissues was digested using a DigiPREP MS hot block
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digester (SCP Science, Clark Graham, Canada), following the EPA method 3050b 35. Dry leaves
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and roots of three replicates were ground and mixed with 4 mL of 70% (v/v) nitric acid. The
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mixture was maintained at room temperature overnight for predigestion, and then was digested in
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the hot block at 95 °C for 4 hours. After cooling to room temperature, 2 mL of 30% (w/v) H2O2
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was added to the mixture, further heated in the hot block at 95 °C for 2 hours. Finally, the Ce and
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Cd in the digestate was quantified by an inductively coupled plasma mass spectrometry (ICP-
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MS, Perkin Elmer mod. DRCII, Waltham, MA).
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Chlorophyll fluorescence
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Leaf chlorophyll was analyzed each week using a continuous excitation chlorophyll
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fluorescence analyzer (OS1p, Opti-Sciences, Hudson, NH). Leaves were acclimated to the dark
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using lightweight leaf clips for at least 30 min before measurements were taken 36. Baseline (F0)
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and maximum (Fm) fluorescence were measured and variable (Fv = Fm – F0) fluorescence, and
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the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) ratio were calculated.
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Chlorophyll analysis
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At the end of the experiment, 30 mg of leaf lamina were collected using a paper puncher,
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avoiding major veins. Leaf portions were placed in dimethyl formamide (DMF), and chlorophyll
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was extracted using the method described by Moran 37. The absorbance was read at 664 and 647
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nm using a UV-Vis spectrophotometer (model Lamba 35; PerkinElmer, Waltham, MA) and used
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to calculate leaf chlorophyll concentrations.
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Root Anatomical observations
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Series of hand sections of roots from each plant were prepared by cutting radial sections
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of fresh roots. For suberin visualization in fluorescence microscopy, the protocol developed by
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Lux, et al.
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freshly prepared solution of Fluorol Yellow 088 (Santa Cruz Biotechnology, Dallas, TX) (0.01%
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w/v, in lactic acid) at 70°C for 30 min and then rinsed in DI water (three baths of 5 min each).
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was adopted. Briefly, free hand sections from each treatment were incubated in a
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The roots were counter-stained with aniline blue (0.5% w/v, in water) at room temperature for 30
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min in darkness and then washed in DI water for 30 min. Samples were then mounted on slides
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using glycerol 50% and observed under a Zeiss Axiophot epi-fluorescence microscope
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(Oberkochen, Germany) and documented by a CoolSNAP cf CCD Camera (Photometrics,
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Tuscon, AZ). All anatomical observations were performed at the Microscopy and Imaging
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Center, Texas A&M University, College Station, TX.
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Image and statistical analyses.
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The length of the root barriers was measured on the fluorescent microscopic images using
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ImageJ (NIH, Bethesda, MD). Data were subjected to analysis of variance using a completely
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randomized experimental design. Since the experiment was set up as a 2 × 2 factorial
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experiment, where the first factor was CeO2NPs and second was Cd, a two-way ANOVA
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analysis was also conducted to understand if there was an interaction between the two
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independent variables: CeO2NPs and Cd. In addition, one-way ANOVA was performed and
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means separation between treatments was obtained by the Tukey’s test. Data were analyzed
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using the Minitab 17 Statistical Software (Minitab Inc., State College, PA).
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Results
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Growth analysis
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CeO2NPs alone were not associated with significant changes in the dry weight (DW)
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when compared with the control. However, leaf, root, and total biomass all were significantly
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lower in the presence of 1.0 mg kg-1 Cd and root biomass was significantly lower in the presence 10 ACS Paragon Plus Environment
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of 0.25 mg kg-1 Cd. (Fig. 1). Cadmium treated plants decreased their total biomass (Fig. 1A) by
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approximately 11% (Cd 0.25) and 31% (Cd 1.0) respectively. CeO2NPs did not aggravate the Cd
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toxicity (p CeO2NPs × Cd = 0.576). In fact, the total dry weight of soybeans treated with
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CeO2NPs + Cd 0.25 was slightly higher than plants exposed to Cd 0.25 alone. No significant
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differences was observed between plants treated by CeO2NPs + Cd 1.0 and Cd 1.0 for all plant
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tissues. Plants treated with CeO2NPs alone showed a 18% reduction in their root biomass and
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plants treated with Cd 0.25 and Cd 1.0 showed a decrease by 30 and 34%, respectively (mostly
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due to the effect of Cd alone, p Cd
