CuO nanoparticle dissolution and toxicity to wheat (Triticum aestivum

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CuO nanoparticle dissolution and toxicity to wheat (Triticum aestivum) in rhizosphere soil Xiaoyu Gao, Astrid Avellan, Stephanie N Laughton, Rucha Vaidya, Sónia Morais Rodrigues, Elizabeth A. Casman, and Gregory V. Lowry Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05816 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Environmental Science & Technology

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CuO nanoparticle dissolution and toxicity to wheat (Triticum aestivum) in rhizosphere soil

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Xiaoyu Gao†, §, Astrid Avellan †, §, Stephanie Laughton †, §, Rucha Vaidya †, §, Sónia M. Rodrigues‡, Elizabeth A. Casman§, #, and Gregory V. Lowry†, §, *.

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†

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§

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States

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‡

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#

Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States Center for Environmental Implications of NanoTechnology (CEINT), Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United

Centre for Environmental and Marine Studies (CESAM), Department of Chemistry, Universidade de Aveiro, 3810-193 Aveiro, Portugal Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States

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*Address correspondence to [email protected]

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Abstract:

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It has been suggested, but not previously measured, that dissolution kinetics of soluble nanoparticles such

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as CuO NPs in soil affect their phytotoxicity. An added complexity is that such dissolution is also

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affected by the presence of plant roots. Here, we measured the rate of dissolution of CuO NPs in bulk soil,

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and in soil in which wheat plants (Triticum aestivum) were grown under two soil NP dosing conditions:

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(a) freshly added CuO NPs (500 mg Cu/kg soil), and (b) CuO NPs aged for 28d before planting. At the

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end of the plant growth period (14 days), available Cu was measured in three different soil compartments:

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bulk (not associated with roots), loosely attached to roots, and rhizosphere (soil firmly attached to roots).

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The labile Cu fraction increased from 17mg/kg to 223mg/kg in fresh treatments and from 283 mg/kg to

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305mg/kg in aged treatments over the growth period due to dissolution. Aging CuO NPs increased the

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toxicity to Triticum aestivum (reduction in root maximal length). The presence of roots in the soil had

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opposite and somewhat compensatory effects on NP dissolution, as measured in rhizosphere soil. pH

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increased 0.4 pH units for fresh NP treatments and 0.6 pH units for aged NPs. This lowered CuO NP

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dissolution in rhizosphere soil. Exudates from T. aestivum roots also increased soluble Cu in porewater.

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CaCl2 extractable Cu concentrations in bulk vs. rhizosphere soil increased from 1.8mg/kg to 6.2mg/kg

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(fresh treatment), and from 3.4mg/kg to 5.4mg/kg (aged treatments). Our study correlated CuO NP

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dissolution and the resulting Cu ion exposure profile to phytotoxicity, and showed that plant-induced

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changes in rhizosphere conditions should be considered when measuring the dissolution of CuO NP near

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roots.

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TOC art:

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Introduction

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The anticipated benefits of nano-enabled agrochemicals include slow and controlled release of

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micronutrients, plant tissue-specific targeted release of micronutrients or pesticides, reduced amounts of

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agrochemicals being required, and generally lower toxicity compared to more soluble products1,2. Copper-

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based nanoparticles (NPs) are already on the agrochemical market3,4. Copper is an essential crop

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micronutrient. Deficiency may lead to reduced disease resistance5 and decreased crop yields6. However,

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at high concentrations, Cu can also be toxic to plants,7 the surrounding microbial communities,8 and soil

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invertebrates9. Due to its relatively slow dissolution, CuO NPs have been studied as a potential candidate

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for agrochemical use. It behaves differently from dissolved Cu2+ in soil, potentially affecting copper

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bioavailability, the release of Cu ions over time, and potential associated risks10–12. However, the

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connection between NP dissolution, the resulting dose of Cu ions and its toxicity to terrestrial plants, and

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the role of root exudates on this process have not been well elucidated due to a lack of appropriate

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characterization of the dissolution of the NPs in soil. Ideally, application rates of these novel materials

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should be based on their fate and effects in the terrestrial environment, their bioavailability and potential

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toxicity to plants. The toxic effect of Cu species is reflected in physiological changes in plant roots and

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shoots, such as decreased root length, increased root compactness, change in root color, shorter leaf

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length and decreased shoot biomass13–15. Hyperspectral imaging has been used to visualize NPs in plants

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and to confirm macroscopic evidence of NP toxicity16,17.

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Previous studies of the toxicity of CuO NPs to terrestrial plants assumed, but did not measure,

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dissolution behavior of CuO NP in soil. This has led to conflicting conclusions on the toxicity of CuO

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NPs. While some studies attributed the toxic effect of CuO NP to released ionic Cu15,18,19, others

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concluded the opposite20. For example, Servin et al. chose a Cu ion control concentration based on the

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assumption that only 10% of the CuO NP would dissolve in soil, the same fraction that dissolved in pure

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sand, rather than measuring CuO dissolution in soil. They concluded that dissolution of CuO NPs could

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not fully explain the plant toxicity because the plant responses differed from their Cu ion control .20 Much

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more than 10% CuO NP could have dissolved in soil because soil organic matter (SOM) acts as a Cu 2 ACS Paragon Plus Environment

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sink, increasing the amount of CuO NP that can be dissolved11. This weakens their conclusions about a

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NP-specific effect. Similar problems occurred in other studies 15,18,21–23. Breaking with this trend, Dimkpa

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et al. (2013) evaluated the total CuO NP dissolved in soil using a water-extraction method. 24

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Unfortunately, the water-extraction does not extract Cu bound to the soil solid matrix which accounts for

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most of the dissolved Cu in soils25–27. Thus, their assertion of a CuO NP-specific toxicity in soil is

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confounded by the potential that more Cu had dissolved than was assumed or measured. Recently Qiu et

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al. found that the toxicity of CuO NP, CuO bulk particles and soluble Cu (Cu(AC)2) depends on their

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solubility in soil, and that the distinction in solubility diminished after a 90-day aging period. However,

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the actual dissolution during the incubation periods (one day vs. 90 day) was not quantified. They

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successfully correlated the toxicity of NP to roots of Hordeum vulgare L. (5-day root elongation

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experiment) with ‘free Cu ions’ in soil pore water measured at a single time point before seeding;19

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though convincing, it should be noted that the dissolution during the 5-day toxicity test was not

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considered. While the relatively slow dissolution of CuO NP may result in unobservable impacts on

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toxicity during a relatively short 5-day toxicity test, dissolution at this rate would probably affect toxicity

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of NPs in longer tests.

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The dissolution of CuO NPs is a dissolution rate-limited process. Experimental approaches, such

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as extraction with CaCl2 or with diethylenetriaminepentaacetic acid (DTPA), have been used to predict

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the bioavailability or toxicity of metals in soil.28–31 CaCl2 extracts the Cu ions in soil pore water that are

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considered ‘readily available,’ while DTPA extracts the “labile” fraction including dissolved Cu in soil

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pore water (free Cu2+and Cu2+ complexed with soluble ligands such as dissolved organic matter (DOM)),

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but also the Cu2+ associated with soil solid phases, such as soil organic matter (SOM), clay particles, and

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iron oxide minerals.29–31 Whereas CaCl2 extracts metals that are ‘readily available’ to plants29, DTPA

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extracts this pool as well as the pool that may eventually become bioavailable in soil, the so called

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‘potentially available’ fraction32. One problem with using these extraction methods to predict the

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bioavailability of Cu based nanomaterials is that a single time point extraction does not capture the

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temporal dynamics of the CuO NP dissolution process. Our recent study used extraction methods at

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different times to monitor the kinetics of release of Cu ions from CuO NP in soil. In that study, the

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increase in DTPA extractable Cu over 30 days in soils was used to estimate the dissolved pool of Cu in

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soil.11 The availability of Cu ions increased with time over a 30d period, which may explain why previous

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efforts to correlate the extractable metals in metal-based NP-amended soils with their bioavailability or

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toxicity have generally failed33–35.

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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 3 ACS Paragon Plus Environment


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metal-based ENMs or the dissolution of ENMs in soil did not typically consider the impact of roots on Cu

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availability.11,12,33–35 Plant roots exude organic acids 36–38 that may affect the pH in rhizosphere. 39,40

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Although soil pH and organic carbon are known to be important factors influencing the dissolution

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behavior of CuO NPs in soil11,12, and previous studies have proposed that exudates from plant roots may

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affect the dissolution of CuO NP in the rhizosphere41, no studies have quantified this. Given that the

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rhizosphere is where plants interact with soil for nutrient uptake,42,43 a better understanding of how the

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roots impact NP dissolution and metal availability in the rhizosphere is needed to design nano-enabled

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agrichemicals with optimal properties for delivering nutrients.

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The objectives of this study are to quantify the influence of time and near-root chemical

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conditions on dissolution and lability of CuO NPs in rhizosphere soil, and to determine the influence of

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this dissolution on the toxicity of CuO NPs to Triticum aestivum during a 14-day plant growth period in

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soil. Wheat (Triticum aestivum) was used in this study because it is the 2nd most cultivated plant in the

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world, and it is sensitive to Cu deficiency44 or excess45. To evaluate the toxicity of CuO NP to plants, we

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measured the dissolution behavior of CuO NPs in soil in the presence of plants with emphasis on the soil-

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plant interface (rhizosphere) where roots interact with soil. The toxicity of Cu was evaluated by

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physiological changes in plant roots and shoots.

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Methods and materials

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Chemicals. Calcium chloride (≥99.0%, ACS grade) and hydrogen peroxide (30%, certified ACS) were

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purchased from Fisher Scientific. DTPA (>99%) and triethanolamine (TEA, ≥99.0% (GC)) were

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purchased from Sigma-Aldrich. Trace metal grade nitric acid (65%-70%) was purchased from VWR.

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Triticum aestivum seeds (Pembroke 2014) were bred by Dr. David Van Sanford (Department of Plant and

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Soil Sciences, University of Kentucky).

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Nanoparticles and Characterization of Nanoparticle properties. CuO NPs (~40 nm primary particle

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size), were purchased from Sigma-Aldrich. The primary size of particles, zeta potential, isoelectric point

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and hydrodynamic diameter have been characterized and reported in our previous study11. The details of

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characterization methods can be found in SI.

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Soils and Characterization of Soil Properties. Standard Lufa 2.2 soil (loamy sand) was purchased from

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Lufa Speyer, Germany. Lufa 2.2 soil contains 1.6 wt. % soil organic matter, and little total and available

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Cu (see supporting information SI, Table S1 and Table S2, control treatment). Using a well-characterized

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standard soil allows comparisons between studies. The high carbon organic content (about 1.6%) of Lufa

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2.2 makes this soil good for agricultural studies. Soil was air dried and sieved < 2mm before shipping.

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The soil was further air-dried for at least 24 hours before all experiments. Soil pH in different treatments 4 ACS Paragon Plus Environment

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was determined by the CaCl2 extraction method (see ‘Extraction methods’ section). Soil moisture content

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(1% for the air dried soil) was determined gravimetrically after oven-drying the soil at 105 ºC for 24 h46.

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Soil field moisture capacity (21%) was determined using a Haines apparatus with 0.1 bar pressure

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difference between the wet soil and the atmosphere.

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Soil amendment. The CuO NP suspension (containing Na2SO4), CuSO4 solution, or Na2SO4 solution

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(control treatment) were mixed with soil and brought to a moisture content of 21.7% (corresponding to

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~50% of the water holding capacity). The soil was mixed with wooden sticks in a beaker for 20min. The

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homogeneity was confirmed with the low standard deviation for the total Cu content measured by soil

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digestion data (SI, table S1). To test if CuO NP and CuSO4 treatments resulted in different Cu

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bioavailability and toxicity, the Cu ion concentration had to be high enough to ensure some CuO NPs

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remained in the soil during the study period. We chose 500mg/kg (as Cu) for the CuO NP treatment, and

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300 mg/kg (as Cu) for the CuSO4 treatment based on a preliminary study to assess the solubility of the

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CuO NPs in the Lufa 2.2 soil (SI, Figure S1). The results showed that the solubility of CuO NPs in Lufa

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2.2 soil was ~300mg/kg. Therefore, the selected concentrations provided a similar concentration of added

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Cu ion in both treatments after one month.

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Germination and plant growth. The seeds of Triticum aestivum were surface sterilized by submerging

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them in 10% sodium hypochlorite solution for 10 minutes, and then washed with DI water three times.

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The seeds were then kept immersed in DI water overnight on an end-to-end rotator. The following day,

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the seeds were transferred to a petri-dish containing moist tissue paper. The petri-dishes were covered

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with aluminum foil and incubated in the growth chamber for 7 days, until 90% germination was achieved.

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Germinated seeds were transplanted into syringes containing 120g of amended soils either immediately

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after adding the Cu (fresh treatment) or 28 days after the Cu was added (aged treatment).The plants were

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incubated in a growth chamber with constant moisture content and 16h-light/8h-dark cycle (25 °C for

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daytime and 21 °C for night time). A diluted Cu-free Hoagland solution (quarter strength) was added

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(1ml/day) to each syringe to maintain the moisture content of the soil as well as provide nutrients to

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plants. The concentration of Cu in soil and plant tissue was determined using a standard digestion method

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(EPA Method 3050b47) and ICP-MS analysis of Cu in the digestate. See SI. Adding moisture content did

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not induce any vertical transport of Cu, as suggested by Figure S4.

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Sampling of soil and plant tissue. Prior to transplanting the germinated seeds in soils, subsamples of

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each soil were collected from all treatments for DTPA extraction (2g of soil per extraction) to measure the

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labile metal fraction. After 14d of growth, rhizosphere soil, "loosely attached soil," and bulk soil (Figure

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S2) were collected for DTPA and CaCl2 extraction to determine the total dissolved metal and readily

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available metal, respectively, as described below. After the plants and roots were removed from the 5 ACS Paragon Plus Environment


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syringe, the soil remaining inside the syringe was defined as bulk soil, presumably minimally affected

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by the plant roots. The bottom 5mm of bulk soil was also collected to determine if there was significant

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vertical transport of Cu. No vertical transport of Cu was observed (SI Figure S4). The roots were

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separated from shoots. Both roots and shoots were photographed with a scale bar for determination of

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length. For each treatment, one plant root replicate was washed with 1mM KCl three times for Cytoviva

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analysis (described below). The remaining roots were shaken by hand in a 50 ml centrifuge tube, and the

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soil that detached during shaking was defined as loosely attached soil48 (Figure S2). After shaking, the

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roots were placed on aluminum foil and air dried in a fume hood for 24 hours. The roots were then shaken

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again in a 50 ml centrifuge tube, and the soil that detached during the air-drying process and the second

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shaking process was defined as rhizosphere soil49. Due to the small amount of rhizosphere soil collected

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per treatment, not all replicates were suitable for DTPA and CaCl2 extraction. The details of which

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samples were analyzed can be found in SI (Table S3 and Table S4). For CuSO4 treatments, the roots were

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highly compacted, precluding the collection of rhizosphere soil. The shoots and roots were oven-dried at

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105 ºC for 24 h. The mass of the dried roots and shoots was recorded before digestion for total Cu

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analysis (details in SI).

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Soil extraction. Two standard extraction fluids were used in this study. DTPA extractant was composed

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of 0.01M CaCl2, 0.005M DTPA and 0.1M triethanolamine (TEA) (pH=7.6). CaCl2 extractant was 0.01M

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CaCl2 without pH adjustment. All extractions were done using a reciprocal shaker at 180 rpm for 2 hours.

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It is important to note that the centrifuge tubes were laid horizontally in the shaker rather than vertically to

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provide the best extraction efficiency. For soil samples collected before the plant growth experiments, 2g

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of soil were extracted with 4ml DTPA extractant. For bulk soil samples, loosely attached soil samples and

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rhizosphere soil samples, 0.4g of soil was extracted with 0.8ml DTPA extractant, while 0.35g of soil was

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extracted with 3.5ml CaCl2 extractant. After extraction, all samples were centrifuged at 3000 rpm for 10

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min, and the supernatants were filtered using a 0.45 µm PTFE filter. The pH of the CaCl2 extracts for

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each soil fraction was measured (Figure 5). All samples were acidified with 20% HNO3 (final HNO3

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concentration, 2%) and Milli-Q-water before being analyzed by ICP-MS. The method for ICP-MS is

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provided in detail in the SI.

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Cytoviva analysis. The interaction between roots and NPs were visualized in fresh roots after a rinsing step

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in 10-3 M KCl, using a darkfield-based hyperspectral imaging (DF-HSI) system (CytoViva Inc., USA). See

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SI for additional details.

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Results

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Nanoparticle characterization. The properties of the CuO NPs have been previously described11.

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Briefly, the primary particle size was determined by TEM to be 38nm ± 1.7nm (278 particles counted,

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95% CI). The hydrodynamic diameter of an 80mg/kg CuO NP in an aqueous 5mM NaHCO3 suspension

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(pH=7) was measured to be 560nm±103nm (3 replicates, 95% CI, intensity averaged), and the zeta

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potential was -16.1mV±1.7mV (3 replicates, 95%CI) in the same suspension. The pH of the isoelectric

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point (pHiep) of the CuO NPs in a 5mM NaNO3 solution was 8.8, while the pHiep was 5.8 in the 5mM

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NaHCO3 solution11. The hydrodynamic diameter and zeta potential likely change after they are added to

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the soils due to interactions with soil components such as natural organic matter and calcium50,51.

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Change in extractability of Cu in bulk soil during the plant growth experiment. The DTPA

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extractable Cu in the bulk soil for CuO NP and CuSO4 amended soils are shown in Fig.1. The DTPA

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extractable Cu represents the Cu that was released from the CuO NPs during the treatment. The

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extractable Cu vs. time is shown for the 14d growth period for both the freshly added CuO NPs, and for

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the aged CuO NPs, where plants were added after the CuO NPs had aged for 28d prior to planting the

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germinated seeds. The total Cu concentration in the two treatments and in the control (unamended) soil is

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provided in the SI (Table S1).

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For the CuO NP treatments, the DTPA extractable Cu from bulk soil increased over time (Figure

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1a) (ANOVA test, P0.05). The DTPA extractable Cu in the

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aged CuO NP treatment was statistically significantly higher than both fresh and aged CuSO4 treatment

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(ANOVA test followed by Fisher’s LSD test for multiple comparison, P≤0.05) (Figure 1c). The CaCl2

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extractable Cu revealed a different order, with fresh CuSO4 treatment having the highest CaCl2

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extractable Cu, followed by the aged CuO NP treatment and the aged CuSO4 treatment, with the fresh

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CuO NP treatment having the lowest amount of CaCl2 extractable Cu. The CaCl2 extractable copper

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represents the “readily available” Cu in the porewater. 7 ACS Paragon Plus Environment


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Figure 1. Change in DTPA extractable Cu over time for each treatment: a) CuO NP treatment, b) CuSO4

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treatment, and comparison of mean of extractable Cu for each Cu treatments at the end of the plant

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growth period: c) DTPA extraction, d) CaCl2 extraction. Error bars show ± 1 standard deviation. In a and

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b, capital letters indicate significant differences between DTPA extractable Cu at four time points for

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CuO NP treatments (a) and CuSO4 treatments (b). In c and d, capital letters indicate significant

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differences in DTPA extractable Cu (c) and CaCl2 extractable Cu (d) among soils collected after plant

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harvesting (ANOVA test followed by Fisher’s LSD test for multiple comparisons, P≤0.05).

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Toxicity of CuSO4 and CuO NP. Root maximal length, root compactness (root mass/root maximal

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length), leaf lengths, shoot mass (Figure 2) and root morphology (SI Figure S5 and Figure S6) were used

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to evaluate the toxic effect of CuSO4 and CuO NPs.

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Root maximal length and root compactness indicated no visual toxic effect from the fresh CuO

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NP treatment. For other treatments, significant decreases in root maximal length (a decrease of 6.6cm,

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8.2cm, and 6.8cm compared to the control treatment for aged CuO NPs, fresh CuSO4, and aged CuSO4,

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respectively) were observed. Increased root compactness was observed for the aged CuO NP treatment

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(an increase of 4.0 mg/cm compared to the control) and for the fresh CuSO4 treatment (an increase of 5.1

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mg/cm compared to the control). Examples of shortened roots and compactness of roots are shown in the

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SI (Figure S5). Evidence of Cu toxicity was also observed in Cytoviva images. In comparison to the roots 8 ACS Paragon Plus Environment

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exposed to CuSO4 (SI, Figure S6), the roots exposed to CuO NP (fresh or aged) did not present the same

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damaged physiology. Roots exposed to CuSO4 (both fresh and aged) showed a brown damaged (necrotic)

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zone that was not found on any of the CuO NP exposed roots. No effects of Cu on the shoots (leaf length,

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biomass) were observed for the CuO NP treatments. Both the freshly amended and aged CuSO4

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treatments resulted in shorter third leaves (shortened by 5.4cm and 4.0 cm compared to the control for

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fresh and aged CuSO4 treatments, respectively). The freshly amended CuSO4 treatment also had less total

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shoot biomass compared to the control treatment.

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Some indication of toxicity was evident in all treatments except for the fresh CuO NP treatment.

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Aging of CuSO4 decreased its toxicity to Triticum aestivum, while aging of CuO NP increased its toxicity.

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Overall, the two CuSO4 treatments showed more toxic effects to Triticum aestivum compared with the

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two CuO NP treatments, even though the CuSO4 was added at a significantly lower Cu concentration

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(300mg/kg for CuSO4 treatments vs. 500mg/kg for CuO NP treatment).

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Figure 2. a) Root compactness and b) leaf length (leaf growth stage is noted with number, from 1 being

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the youngest to 3 the oldest) of wheat seedlings grown in freshly amended and aged CuO NP, CuSO4-

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amended soil, and control treatments. Error bars show ±1SD, * indicates P≤0.05; ** indicates P≤0.01.

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(ANOVA test followed by Fisher’s LSD test for multipal comparisions) compared to the control

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treatment.

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Cu root association and Cu uptake. The presence of CuO NPs associated with the roots after 2 weeks of

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plant growth in both fresh and aged CuO NP amended soils was investigated using enhanced dark-field

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hyperspectral imaging (DF-HSI) as shown in Figure 3. The pixels containing CuO NP have been

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highlighted in red. In both fresh and aged CuO NP amended soils (Figure 3), CuO NPs were found 9 ACS Paragon Plus Environment


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associated to specific locations on the roots, either to the root tip mucilage (Figure3 a, b, f, g), or to soil

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aggregates attached to the root hairs or root tips (Figure 3 a, c-i). For the concentration of Cu in roots, all

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Cu treatments were significantly higher than the control treatment. The Cu concentration in roots

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(577mg/kg, s.d.=46mg/kg, 6 replicates) was statistically significantly higher in the freshly amended CuO

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NP treatment than in the aged CuO NP treatment (400mg/kg, s.d.=60mg/kg, 6 replicates) or either ionic

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treatment (278mg/kg, s.d.=51mg/kg, 6 replicates for fresh CuSO4 and 442mg/kg, s.d.=67mg/kg, 6

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replicates for aged CuSO4) (Figure S7, a). For the shoot concentrations, no statistically significant

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differences were found for all Cu treatments (53mg/kg-88mg/kg) (Figure S7, b).

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Figure 3. Hyperspectral imaging of plant roots grown in soil with freshly amended CuO NPs (a-e) or

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after aging (f-i). The b, c and g views are magnified views from a and f. Pixels containing the reflectance

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spectra specific to CuO NPs are highlighted in red. CuO NPs and their aggregates were found associated

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to mucilage, root tissues and root hairs (red arrow), and to soil aggregates attached to those locations

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(yellow arrows). Scale bars: 25µm

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Effect of near-root environment on Cu availability from CuO NP treatment. Figure 4 shows the

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differences in extractable Cu in rhizosphere soil, loosely attached soil and bulk soil for fresh and aged

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CuO NP treatments. For the CaCl2 extraction in both fresh and aged CuO NP treatments (Figure 4a, b),

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the extractability of Cu in the rhizosphere soil was significantly higher than the extractability of Cu in the

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loosely attached soil or bulk soil (ANOVA test, P≤0.05).

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There were no statistically significant differences among DTPA extractable Cu measurements

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from rhizosphere soil, loosely attached soil and bulk soil in the freshly amended CuO NP treatment

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(Figure 4c). However, the DTPA extractable Cu in the rhizosphere soil in the aged CuO treatment was

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significantly lower than the DTPA extractable Cu in bulk soil, but similar to that measured in loosely

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attached soil (Figure 4d). In control experiments (Na2SO4), the CaCl2 extractable Cu was below the

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detection limit (0.08mg/kg in soil, 4ug/L for the diluted samples) in all soil samples.

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Aging increased the concentration of CaCl2 extractable Cu and DTPA extractable Cu in loosely

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attached soil and bulk soil, and increased the concentration of DTPA extractable Cu in rhizosphere soil (t

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test, P0.05).



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Figure 4. CaCl2 and DTPA extractable Cu in fresh (left side) and aged (right side) CuO NP amended

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rhizosphere soil, loosely attached soil and bulk soils. Error bars show ± 1 SD. Capital letters indicate

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significant differences between groups (One way ANOVA test followed by Fisher’s LSD test for multiple

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comparison, P≤0.05).

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Soil pH in bulk soil, rhizosphere soil and loosely attached soil. For all CuO NP treatments and the

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control treatment (no addition), the pH of the rhizosphere soils was significantly higher than the pH of the

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loosely attached and bulk soils (Figure 5a, b and c). In freshly amended CuO NP treatments and control

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treatments, the pH of the loosely attached soils were not statistically significantly different than the pH in

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the bulk soils. However, in aged CuO NP treatments, the pH of the loosely attached soil was statistically

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significantly higher than the pH in bulk soil. In bulk soil, the pH was the highest in freshly amended CuO


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NP treatments, followed by aged CuO NP treatment, followed by the control treatment, followed by the

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aged CuSO4 treatment, with freshly amended CuSO4 treatment having the lowest soil pH (Figure 5d).

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313 314 315

Figure 5. Mean ± SD of soil pH (measured using CaCl2 extraction) in rhizosphere soil, loosely attached

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soil and bulk soil in a) soil freshly amended with CuO NP, b) aged CuO NP treatment c) control soil, and;

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d) Comparison of pH of bulk soil among all treatments. Capital letters indicate significant differences

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(ANOVA test followed by Fisher’s LSD test for multiple comparison, P≤0.05).

319 320

Discussion

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CuO NP dissolution is linked to toxicity. Compared to Cu ions, the dynamic dissolution process of CuO

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NP in soil led to a very different Cu exposure profile for plants. At the end of the two growth periods, the

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DTPA-extracted Cu concentrations in CuO NP treatments were similar or even higher than in the CuSO4 13 ACS Paragon Plus Environment


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treatment. However, a decreasing trend in DTPA extractable Cu on CuSO4 treatments during the two

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plant growth periods was observed. This decrease can be attributed to the soil-organic matter interactions,

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solid-state diffusion of Cu ions into iron minerals or metal (co)precipitates52–54. Conversely, an increase in

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DTPA-extractable Cu over time was shown in CuO NP treatments (fresh treatment and aged treatment)

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during the two plant growth periods and the aging period. This can be attributed to the dissolution of CuO

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NP11,12. Thus, the plants in the freshly amended CuO NP soil were exposed to lower amounts of labile Cu

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compared to either the two CuSO4 treatments or the aged CuO NP treatment. These findings suggest that

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when evaluating the chemical availability or toxic effect of metal-based NPs in soil, single-time point

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chemical extractions at the end of the plant growth period cannot capture the dissolution process of NPs

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in soil, and thus may fail to predict the toxicity or bioavailability of NPs11,12,55. Considering that it is not

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feasible to uniformly dose Cu ions into soil over time to precisely mimic the dosing rate from NP

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dissolution, toxicity studies with soluble NPs should measure the dissolution rate in soil and monitor the

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behavior of soluble ions in soil, and interpret their results in light of the different dosing conditions that

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manifest.

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A significantly higher toxicity in CuSO4 treatments compared to the fresh CuO NP treatment is

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explained by the higher exposure of roots to labile Cu species, even though the CuO NP treatment had a

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higher total Cu concentration. Also, dissolution of CuO NPs over time gradually increased the available

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Cu in soil, leading to higher toxicity in the aged treatment. The opposite has been observed with CuSO4,

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where the available Cu in soil decreased over time, leading to lower toxicity to the plants in the aged

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treatment. The effects of time on toxicity of CuO NP and CuSO4 have already been observed19. The

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authors attributed this to the dissolution behavior of CuO NP, although without quantification. Here, we

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clearly showed that in order to correlate the chemical availability of CuO NPs with toxicity, the

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dissolution kinetics, i.e. predicting the total Cu released to soil during the growth period, should be

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considered. The dissolution kinetics can be modeled as first-order dissolution, with the rate constant fit to

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the extractable Cu over time11, and the total amount of Cu ion released from CuO NPs can be estimated

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by integrating the expression relating the change of extractable Cu over time. This observation is relevant

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to NP formulations of fungicides and micronutrients, so the release rate of the active ingredients can be

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better timed to the plant’s needs.

352 353

CaCl2 extractable Cu correlates with toxicity of CuO NP to wheat. Although DTPA extractable Cu

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gives a better indication of CuO NP dissolution because it extracts most of the Cu species in soil, CaCl2

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extractable Cu is better for correlating toxicity, since it measures dissolved Cu in pore water that can

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directly interact with plant roots. DTPA extraction would predict the toxicity of the aged CuO NP to be

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higher than the CuSO4 treatment (Figure 1 a, b). However, this was not the case. The aged CuO NP had 14 ACS Paragon Plus Environment

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lower toxicity compared to both the aged and the fresh CuSO4 treatment, indicating that the DTPA

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extraction cannot accurately predict toxicity for the CuO NPs. The CaCl2 extraction ranked them correctly

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(Figure 1d). Considering that the extractable Cu in CuO NP amended soil increased over time while the

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extractable Cu in CuSO4 amended soil decreased over time in Lufa 2.2 soil (SI, Figure S1), the wheat

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plants were exposed to a lower overall ‘readily available’ Cu (i.e. Concentration x time) in CuO NP

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treatments compared to the CuSO4 treatments. The lower CaCl2 extractable Cu in aged CuO NP treatment

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is a result of higher soil pH in aged CuO NP treatment compared to the fresh and aged CuSO4 treatment.

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Higher soil pH has been previously shown to lower Cu concentration in soil pore water 27,56.

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Root-associated CuO NP modulates toxicity. In the freshly amended CuO NP soil, although being

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exposed to a lower concentration of labile Cu, the roots of Triticum aestivum were actually exposed to

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higher total Cu concentration (Figure S7) than the other treatments. This is mainly due to CuO NPs'

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association with plant roots (Figure 3b). This exposure to CuO NPs did not lead to any detected toxic

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effects, indicating a low or de minimis level of toxic effects from the particle itself over the 14d growth

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period.

373 374

Root exudates affect CuO NP dissolution and availability. The increase in the pH of rhizosphere soil

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compared to bulk soil in our study indicates that the rhizosphere region was indeed influenced by the

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plant roots. Excretion of organic acid (dissociated ions) by plant roots , nitrogen uptake and ionic

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exchanges by plant roots may explain the higher pH of the rhizosphere soil compared to the pH of bulk

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soi39,40,57,58. The observed pH change in rhizosphere soil was not likely a result from the presence of CuO

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NP, as similar pH changes occurred with both the fresh CuO NP treatment and the negative control

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treatment (0.4pH unit, ANOVA test, P>0.05). However, in the aged CuO NP treatment, the pH increase

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was higher (0.6pH unit, ANOVA test, P

CuO nanoparticle dissolution and toxicity to wheat (Triticum aestivum

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