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Time and nanoparticle concentration affect the extractability of Cu from CuO NP amended soil Xiaoyu Gao, Eleanor Spielman-Sun, Sónia Morais Rodrigues, Elizabeth A. Casman, and Gregory V. Lowry Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04705 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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Environmental Science & Technology

Time and nanoparticle concentration affect the extractability of Cu from CuO NP amended soil

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Xiaoyu Gao†, §, Eleanor Spielman-Sun†, §, Sónia M. Rodrigues‡, Elizabeth A. Casman§, #, and Gregory V. Lowry†, §, *.

†

Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States

7

‡

Centre for Environmental and Marine Studies (CESAM), Department of Chemistry, Universidade de Aveiro, 3810-193 Aveiro, Portugal

8

§

Center for Environmental Implications of NanoTechnology (CEINT), Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United

9

States

10

#

11

*Address correspondence to [email protected]

12 13

Abstract

14

We assess the effect of CuO nanoparticle (NP) concentration and soil aging time on the

15

extractability of Cu from a standard sandy soil (Lufa 2.1). The soil was dosed with CuO NP or

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Cu(NO3)2 at 10 mg Cu kg-1 soil (mg/kg) or 100 mg/kg total copper, then extracted using either

17

0.01M CaCl2 or 0.005M DTPA (pH 7.6) extraction fluids at selected times over 31 days. For 100

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mg/kg CuO NP, the amount of DTPA-extractable Cu in soil increased from 3 wt% immediately

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after mixing to 38 wt% after 31 days. In contrast, the extractability of Cu(NO3)2 was highest

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initially, decreasing with time. The increase in extractability was attributed to CuO NP

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dissolution in soil. This was confirmed with synchrotron X-ray absorption near edge structure

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(XANES) measurements. The CuO NP dissolution kinetics were modeled by a first-order

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dissolution model. Our findings indicate that dissolution, concentration, and aging time are

24

important factors influencing Cu extractability in CuO NP-amended soil, and suggest that a time

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dependent series of extractions could be developed as a functional assay to determine the

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dissolution rate constant.

Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States

27 28

Introduction

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Copper based nanoparticles (NP) including metallic copper (Cu NP), copper oxides (Cu2O NP

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and CuO NP), and copper hydroxides (Cu(OH)2 NP) are manufactured nanomaterials that have

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been used as pesticides and fungicides because of their antimicrobial properties1, 2 .They can also

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be used as fertilizers to deliver micronutrient-Cu to plants, which can improve fertilizer

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ACS Paragon Plus Environment


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efficiency and crop yield3,4. Copper salt (mainly as Cu(NO3)2 or CuSO4) based micronutrients

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and pesticides have historically been widely used. Excessive use of Cu containing fertilizers and

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pesticides may lead to negative impacts on ecosystems, soil microorganisms, microbial

36

processes5, plants6 and soil invertebrates7.

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In the U.S., Cu containing fertilizers and pesticides are regulated, with the maximum

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application rate of 75 kg/ha/year (USEPA, 1993). However, these regulations were determined

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using highly soluble Cu salts (e.g. Cu(NO3)2 and CuSO4) in soil. Dynamic processes including

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aggregation, oxidation, and dissolution will likely make the available pool of Cu derived from

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Cu based NP time-dependent8, 9. While the importance of time on the fate and bioavailability of

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Cu salts is documented10-12, aging effects for Cu based NP has not been elucidated. In order to

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assess the impact of Cu based NP to agroecosystems, it is important to determine the factors

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controlling their bioavailability in soils.

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Chemical extraction methods are used to predict the bioavailability of metal in soil13.

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Several single extraction methods, originally developed to determine the fraction of metals in

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soil involved in geochemical equilibrium processes including sorption and precipitation, can

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predict the leaching of soil metals to groundwater, their impact on ecosystems, and their

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bioavailability for soil organisms or plants11-22. Two extraction methods, 0.01M CaCl2 extraction

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and 0.005M diethylenetriaminepentaacetic acid (DTPA) extraction (pH 7.3~7.6) are commonly

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used for predicting the bioavailability or lability of metals such as Cu, Zn and Cd, in soil13-20.

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CaCl2 extraction (0.01M) predicts metal bioavailability by mimicking the chemistry of soil pore

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water and targets the exchangeable metal ions in soil pore water which are ‘readily available’ to

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plants in soils14, 20, 21. DTPA is a strong chelating agent that mimics the chelating effect of root

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exudates to enhance the nutrient availability from soil for subsequent uptake 15. The DTPA

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extraction not only targets the free ions in soil pore water, but also the carbonate-bound and the

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organic-bound fractions of metal in soil, which could be ‘potentially available’ to plants14, 22.

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While these extraction methods for assessing the lability of Cu in Cu salt (CuSO4 and Cu(NO3)2)

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amended soil or for metal contaminated soils are well-developed, there are only a few reports

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using such methods with Cu-based NP or other metal/metal oxide nanoparticles in soil23-25.

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Recently, a few studies have used single time point CaCl2 extraction and DTPA

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extractions to predict the lability of metal/metal oxide nanoparticles in soil. Judy et al.25 used

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CaCl2 and DTPA extractions to estimate the bioavailability of ZnO-NP, TiO2-NP and Ag-NP in

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soil and concluded that these extraction methods could not predict their bioavailability to plants

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(Medicago truncatula) in Woburn sandy soil. Pradas del Real et al.23 used DTPA and CaCl2

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extractions to assess the labile pool of Ag in Ag NP amended soil, and concluded that the low

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extractability of Ag in soil was consistent with the low bioavailability of Ag to plants (wheat and

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rape) in a loamy soil. Xu et al.24 used CaCl2, EDTA and DTPA extractions to estimate the

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bioavailability of CuO NP and TiO2 NP to soil microbes and their community structures in a

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typical paddy soil. They observed that DTPA and EDTA extractable Cu in CuO NP amended

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soil correlated well with microbial activity (microbial biomass, soil enzyme activity, and total

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phospholipid fatty acids) in a CuO NP amended soil. So far, results from studies on the use of

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chemical extraction methods to predict the bioavailability of metals from nanoparticles in soils

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are contradictory and often inconclusive. One reason for this may be the fact that these studies

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did not assess the rates of transformations of NP in those soils and the corresponding effect on

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metal extractability. We hypothesize that aging time and concentration will be important factors

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influencing these particles’ transformation and bioavailability in soil, which may explain the

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absence of a correlation between extractability and bioavailability using a single time-point

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extractions23-25.

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The dissolution and transformation of some metal and metal oxide NP in soil have been

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determined. The dissolution of copper oxide nanoparticles over time in three soils was reported

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by McShane et al26. In their study, they measured an increase in free Cu2+ activity in soil pore

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water over time and concluded that CuO NPs were dissolving. However, the rate of dissolution

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of CuO NP was not modeled or reported. The present study extends this work by McShane et al.

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by measuring pore water and SOM-associated Cu species using well-established extraction

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methods designed to assess bioavailable fractions of Cu, by synchrotron X-ray analysis to

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confirm changes in copper speciation, and by determining the effect of NP concentration on the

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dissolution behavior. The transformations of metal and metal oxide nanoparticles in soil have

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been monitored using synchrotron X-ray absorption spectroscopy (XAS)27-32 to measure changes

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in metal speciation over time. Recently, Sekine et al.27 used XAS to monitor the change of

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speciation of Ag-NP, AgCl-NP and Ag2S-NP in soil over time. They observed that an increase in

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S-bound Ag species, including Ag2S-NP, Ag-cysteine and Ag-cysteine, correlates with the

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decrease in labile Ag determined using diffusive gradients thin films. However, the Ag NP

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transformation kinetics were not studied.

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The dissolution of a number of metal and metal oxide nanoparticles in water has been

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reported33-39. Most studies use empirical first-order dissolution models to describe their

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dissolution 35-38, and evidence suggests that the measured dissolution rate constants are

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concentration dependent36. However, the dissolution rate of metal and metal oxide nanoparticles

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in soils, where water content and SOM can greatly affect the dissolution, is less well-understood.

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These rates are needed to understand the dynamic nature of nanoparticulate metals relative to

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soluble metals added to soils and to parameterize fate and transport models for engineered

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nanomaterials. 40

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The objectives of the present study are to (a) compare the extractability of CuO NP with

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the extractability of Cu(NO3)2 in soil, (b) quantify the extractability of CuO NP as a function of

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time and nanoparticle concentration in a sandy (Lufa 2.1) soil (c) determine the fate processes

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influencing the extractability of CuO NP in soil and (d) to model the dissolution kinetics of CuO

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NP in soil from extraction experiments. We used 0.01 M CaCl2 and 0.005M DTPA (pH=7.6)

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extraction methods to study the extractability of Cu(NO3)2 and CuO NP in aerated soils over a

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one-month period at two different total added Cu concentrations (10 and 100 mg Cu kg-1 (mg/kg)

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dried soil). Changes in speciation of Cu in soil were monitored using XAS to infer the

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dissolution of CuO NP.

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Method and Materials

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Chemicals. CuO NP (50 nm), DTPA, (>99% (titration)) and triethanolamine (TEA, ≥99.0%

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(GC)) were purchased from Sigma-Aldrich. Cu(NO3)2 (>98% ACS grade), calcium chloride

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(≥99.0%, (ACS grade)) and sodium bicarbonate (≥99.7%, (ACS grade)) were purchased from

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Fisher Scientific.

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Nanoparticle Characterization. Primary particle size distribution of the CuO NP was

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characterized by transmission electron microscopy (TEM, Hitachi H-9000 TEM microscope

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operating at 300 kV). The hydrodynamic diameter and zeta potential of CuO NP in suspension

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(80 mg/kg as Cu in 5mM pH=7 NaHCO3 buffer) were determined by dynamic light scattering

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(Zetasizer Nano, Malvern). The isoelectric points of 80mM CuO NP in 5mM NaHCO3 buffer

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and in 5mM NaNO3 were calculated from measurements of the zeta potential of the particles in

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suspension over a range of pH. The crystal structure of CuO NP was determined by X-ray

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powder diffraction (XRD, Panalytical X’Pert Pro MPD X-Ray Diffractometer).

126 127

Soils and Characterization of Soil Properties. Standard soil (2.1-sandy soil) was purchased

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from Lufa, Germany. The standard soil (Lufa 2.1) was used because it is commonly used in

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bioavailability studies and therefore can enhance comparison from different studies. Lufa 2.1 soil

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also contains very little extractable Cu and total Cu (as discussed later in ‘Soil and nanoparticle

131

characterization’ section), making background interference minimal. Lufa soil was air dried and

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sieved < 2mm before shipping. The soil was further air-dried for 12 hours before all experiments.

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Soil pH was determined according to the standard procedures recommended by the USDA41.

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Specifically, 5 g of air-dried soil was mixed by hand for 10s with 5ml of deionized water. The

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pH of the solution was measured after allowing the mixture to settle for 10 minutes. To

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determine the soil moisture content, 2 g of the air dried soil were dried in an oven at 105 ºC for

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24 h 42. The moisture content was then determined gravimetrically. Soil field moisture capacity

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was determined using a modified cylinder method in which air-dried soil was added to a 15ml-

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graduated cylinder. Deionized water was then added into the cylinder to wet the top 2 cm of soil.

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After 24h, the wetting front in the soil moved downward. After removing the top 2 cm of soil,

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the moisture content of soil above the wetting front (which was assumed to be at soil’s field

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capacity) was determined.

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Soil amendment and incubation. Two doses of CuO NP and Cu(NO3)2 were used in our study:

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10mg Cu/kg dry soil for the low dose amendment, and 100 Cu mg/kg dry soil for the high dose

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amendment. These two doses were selected to investigate the influence of concentration on

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extractability of CuO NP in soil. While the low does is more realistic, the high dose provided

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sufficient Cu concentration for XAS study. Soils were amended with CuO NPs or Cu(NO3)2. All

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amended soil samples were incubated in 50ml centrifuge tubes under aerobic conditions between

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0 and 31 days before being extracted and digested. Additional details of the amendment

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procedure can be found in SI.

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Total Metal Concentration. Soil total metal concentration was determined using acid digestion

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according to USEPA Method 3050B (1996). According to the procedure, 1g of air-dried soil was

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digested with concentrated nitric acid and 30% hydrogen peroxide at 95 ºC using a hot plate.

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After digestion, the samples were centrifuged at 3000 rpm for 10 min, followed by filtration

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using 0.45um filter to remove fine particles in the supernatant. The filtered supernatant was

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diluted with Milli-Q water and acidified with 20% HNO3 (final HNO3 concentration was 2%) for

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analysis by ICP-MS (Agilent 7700x). The instrument was calibrated with a mixed calibration

160

standard (purchased from Agilent Technologies) every time before measurement. The calibration

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ranges used for different samples can be found in table S1.

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Extractions to assess the labile Cu in soil samples. After different incubation periods, 2.0 g of

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air-dried soils or 2.3 g of wet soils were extracted with two standard extractants: The first one

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(termed DTPA) uses a 4 mL mixture of 0.01M CaCl2, 0.005M DTPA and 0.1M triethanolamine

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(TEA) (pH=7.6). The second one (termed CaCl2) (pH=5) uses 20 mL of 0.01M CaCl2. All

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extractions were done using a reciprocal shaker at 180 rpm for 2 hours. Sample bottles were laid

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horizontally in the shaker. Both wet soil and air dried soil were used to study the effect of air

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drying. After extraction, all samples were centrifuged at 3000 rpm for 10 min, and the

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supernatants were filtered with using a 0.2 um PTFE filter. In order to monitor the impact of

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CuO NP suspension or Cu(NO3)2 solution on pH of soil, the pH of CaCl2 extracts for air-dried

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amended soil and a unamended soil (no nanoparticle or Cu(NO3)2 added) were also measured to

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estimate the soil pore water pH. The samples collected were further filtered with a 3kda filter to

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separate the dissolved and nanoparticulate fraction of Cu in extracts. All samples were acidified

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with 20% HNO3 (final HNO3 concentration was 2%) and Milli-Q-water and analyzed by ICP-

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MS. Due to the large difference between Cu concentrations from CaCl2 extracts and Cu

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concentration from DTPA extracts, different calibration ranges were used. The different

178

calibration ranges used for different samples can be found in Table S1 in supporting information.

179 180

Determination of Cu speciation in soils. Cu speciation in soils (Lufa 2.1) on 1, 4, 7 and 19 days

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after amendment was analyzed by Cu K-edge

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XAS at the Stanford Synchrotron Radiation Lightsource (SSRL) on Beamline 11-2. Spectra for

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both 100mg/kg and 10mg/kg amended soils were collected. However, the signal-to-noise ratio

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for the 10mg/kg amended soils was too poor for adequate speciation. Specifically, samples were

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lyophilized, ground with a mortar and pestle to achieve uniformity, pressed into pellets, and

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placed between Kapton tape. A double crystal Si (220) monochromator was calibrated by setting

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the first inflection of the K-edge of a metallic Cu foil to 8979 eV. Harmonic rejection was

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achieved by detuning the monochromator crystal by 25%. Spectra of soil samples were recorded

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in fluorescence mode at room temperature using a 100-element germanium detector. The scans

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were averaged, energy corrected using a metallic Cu foil standard, deadtime-corrected,

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background subtracted with E0 defined at 8988 eV, and de-glitched using SIXPack data analysis

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software43. Spectra were analyzed by linear combination fitting (LCF) using the following

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reference spectra: CuO NP, metallic Cu, CuSO4, Cu(NO3)2, CuPO4, Cu-cysteine, Cu2S

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(chalcocite mineral sample) ,CuS (covellite mineral sample), Cu- iron oxide, Cu+ sorbed to

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humic acid (Cu(I)-HA) and Cu2+ sorbed to humic acid (Cu(II)-HA). Inclusion of a reference

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spectrum into the combination fit required at least a 10% decrease in the R-value, indicating a

197

significant change to the quality of the fit.

198 199

Dissolution kinetics. For both extraction methods, the extractable Cu (either in pore water

200

(CaCl2), or pore water plus soil bound Cu (DTPA)) is assumed to increase proportionally as the

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CuO NPs dissolve.

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The increase in the extractability of Cu over time is modeled using equation 1,

203 ௗா

204

ௗ௧

= ݇൫‫ܧ‬௙௜௡௔௟ − ‫ܧ‬൯

(1)

205 206

where E is the concentration of extractable Cu at time t, k is the empirical 1st order extraction rate

207

constant, and Efinal is the concentration of extractable Cu at the end of experiment. If the

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dissolution of the CuO NP is the rate limiting step, i.e. the Cu-soil organic matter interaction is

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much faster than the dissolution of CuO NP in soil, then the measured extraction rate constants

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from both extractions should be similar, and equal to the CuO NP dissolution rate constant.

211 212 213

Results and Discussion

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Soil and nanoparticle characterization. Lufa 2.1 soil is a sandy soil, containing 3 wt% clay, 11

215

wt% silt and 86 wt% sand (as provided by Lufa). It has low organic matter content (organic

216

carbon content is 0.7 wt% as provided by Lufa). After air-drying, Lufa soil had 1.2 wt% moisture

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content. The soil pH was 5.6 and the field capacity was 16 wt%. The total Cu concentration of

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the unamended soil was 2.95±0.11mg/kg. Total Cu concentration measured in each of the

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amended soils is presented in Table S2. The DTPA extractable Cu in unamended soils ranged

220

from 0.37 to 0.53 mg/kg dried soil while the CaCl2 extractable Cu in unamended soils ranged

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from 0.005 to 0.024mg/kg (Table S3).

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The primary particle size of CuO NP (measured from TEM) was 38nm (s.d. =14nm, 278

223

particles were counted). The hydrodynamic diameter and zeta potential of 80mg/kg CuO NP in

224

pH=7, 5mM NaHCO3 buffer were 557nm (s.d. =56nm, 3 replicates, polydispersity index 10 d (p > 0.05, Kolmogorov-Smirnov test). This suggests that

277

the Cu may be fully dissolved and “aging” similarly to the Cu(NO3)2. However, the slight

9



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downward trend in extractability for t > 10d is not statistically significant (P > 0.05, one-way

279

ANOVA test). Although extraction procedures generally used air dried soils21,46 , we used both the air

280

dried soils (after incubation) and wet soils for extractions to investigate the influence of air

282

drying on extractability of CuO NP in soil. Our results indicated that air drying has no significant

283

effect (P>0.05, Kolmogorov-Smirnov test) on extractability of Cu in both CuO NP amended and

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Cu(NO3)2 amended soils. Thus, only the results from air dried soil is shown in Figure 1 for

285

clarity. Additional discussion on the effect of air drying can be found in the supporting

286

information.

Cu(NO3)2

6 4 CuO NP

2 0

287 (c)

Extractable Cu (mg /kg dried soil)

8

0

Cu(NO 3)2

50 CuO NP

25 0 0

Cu(NO 3)2

0.1

CuO NP

0.0 0

100mg/kg DTPA extraction

75

10mg/kg, CaCl 2 extraction

0.2

10 20 30 40 Incubation time (days)

100


(b) 0.3

10mg/kg, DTPA extraction

(a) 10



100mg/kg, CaCl 2 extraction

(d) 12



281

Cu(NO 3)2

8 4

CuO NP 0 0


288 289

Figure 1. Extractable Cu and in CuO NP and Cu(NO3)2 amended soils as a function of time and

290

the first order dissolution fit for CuO NP in soil: (a) DTPA extraction for 10 mg/kg amendment,

291

(b) CaCl2 extraction for 10 mg/kg amendment, (c) DTPA extraction for 100 mg/kg amendment

292

and (d) CaCl2 extraction for 100 mg/kg amendment. Error bars indicate ± 1 standard error.

293

Dashed lines indicate model fits using equation 1. For the low dose amendment, because CuO

294

NPs were fully dissolved after the 7-day sampling time, we modeled only the first 7 days.

295

represents extractable Cu in CuO NP amended soils air dried after incubation and

296

extractable Cu in Cu(NO3)2 amended soils air dried after incubation.

10


represents

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298 299

Figure 2. Fraction of small particles and dissolved ions (those passing 3kDa filter) in (a) DTPA

300

extracts and (b) CaCl2 extracts. D1, D2, D31 stand for 1 day, 2 days and 31 days after dosing.

301

Error bars indicate ± 1 standard error.

302 303

Fractions of dissolved Cu and particulate Cu in extracts. Bioavailability of Cu depends on its

304

speciation, e.g. free ions, complexed ions and particulate species47. We used filtration (first a

305

0.2-micron filter followed with a 3kDa filter) to distinguish between dissolved and particulate

306

species of Cu in each of the extracts. Figure 2 shows the fraction of Cu that passes the 3 kDa

307

filter (considered dissolved) in CaCl2 and DTPA extracts. For DTPA extraction, nearly all

308

extractable Cu (from 90% to 100%) was dissolved. This is because most Cu in DTPA extracts

309

bound with the chelating agent (DTPA) and the Cu-DTPA complex can pass through the 3kDa

310

filter. In contrast, filtration of the CaCl2 extract indicated the presence of Cu-containing particles

311

compared to the DTPA extracts (P0.05, one way ANOVA test).

316 317

Effect of CuO NP concentration on its extractability in soil. The concentration of added Cu

318

influences the extraction behavior for CuO NP compared to Cu(NO3)2. For the low Cu dose, the

319

extractability of Cu in CuO NP amended soil was the same as for the Cu(NO3)2 amended soil

320

after ~10 days. No statistically significant difference (p>0.05, Kolmogorov-Smirnov test) is 11



321

found for extractable Cu for both CaCl2 extractions and DTPA extractions between CuO NP

322

amended soil and Cu(NO3)2 amended soil on day 13, 19 and 31, suggesting that the CuO NP

323

were fully dissolved before 13 days in soil at the lower dose. The behavior was quite different at

324

the high Cu dose. For the high dose of added Cu, extractable Cu in Cu(NO3)2 amended soils was

325

always higher than the extractable Cu in CuO NP amended soil. The extractability of Cu from

326

the CuO NP amended soil increased over the entire 31day period, suggesting that CuO NP was

327

dissolving over 31 days, but the dissolution of CuO NP in soil was not complete. One possible

328

explanation on the persistence of CuO NP and the slower dissolution rate after ~7 days in the

329

high dose soil (100mg/kg Cu) is that the free Cu2+ in soil pore water approached saturation with

330

respect to CuO(s). Conversely, the lower dose system (10 mg/kg) was not oversaturated with

331

respect to the CuO(s) phase. While CaCl2 extraction is a well-established method to assess the

332

pore water concentration of dissolved Cu, the potential for artefact during the extraction and

333

uncertainty in the complexation constants for Cu and the NOM in our system prevents an

334

accurate determination of the degree of saturation in the pore water. .

335 336

Dissolution rate of CuO NP in soil. For the high dose of CuO NP (100 mg/kg), the first-order

337

extraction model describes the change of extractable Cu over time well (R2>0.995) (dashed lines

338

in Figure 1). However, we should note that Cu2+ ions dissolved from CuO NP can become

339

irreversibly bound with soil organic matter, making it unextractable by DTPA, as indicated in

340

former sections. This irreversible interaction is about 20% for our soils, and has a minimal effect

341

on the calculated dissolution rate constant. This is in part because it is a small fraction of the

342

total, and in part because the time scale for partitioning into this irreversible fraction is short, i.e.

343

less than 1d compared to the dissolution processes being investigated, i.e. many days to weeks.

344

For CaCl2 extraction, the fraction of extractable ionic Cu was significantly less, with only 2% to

345

10% of the ionic Cu being extractable because it targeted only Cu in soil pore water. Despite the

346

differences in the extractable amount of Cu, the modeled dissolution rate constants for DTPA

347

extractable Cu and CaCl2 extractable Cu are similar (Table 1). This indicates that the extractable

348

amount of Cu by either the DTPA or CaCl2 extraction can be used to monitor the CuO NP

349

dissolution in the soils. This is a natural consequence of a first-order dissolution process, which

350

scale with the ratio of the final and initial concentration (C/Co) so any process that reduced C and

351

Co by the same constant fraction will not affect the calculated rate. Moreover, it suggests that

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Cu2+ binding to SOM is rapid enough, such that dissolution of the CuO NP is the rate-limiting

353

process controlling both DTPA extractable Cu and CaCl2 extractable Cu in soil.

354 355

Effect of aging on speciation of Cu in Cu(NO3)2 and CuO NP amended soil. Speciation of Cu

356

in the 100mg/kg CuO NP and 100mg/kg Cu(NO3)2 amended soils were determined at selected

357

time points using XANES (Figure 3). Details regarding the spectra for model compounds and

358

fitting result can be found in the supporting information (Figure S7 and Table S4). The

359

speciation of Cu in Cu(NO3)2-amended soils can be adequately modeled using only the Cu(II)-

360

HA model compound, indicating that the Cu has predominantly Cu-O character, i.e. associated

361

with humic acids or potentially (but less likely) with clay or metal oxide surfaces of the solids.

362

This is consistent with prior speciation studies indicating that the main species of Cu in soil is

363

Cu(II)-HA using experimental approachs48-49 and with results of equilibrium partitioning

364

modeling50. This also suggests that Lufa 2.1 soil has the capacity to sorb up to 100mg/kg of

365

added Cu, because our data showed that all Cu in the 100mg/kg Cu(NO3)2 amended soil was

366

Cu(II)-HA. In contrast, the Cu speciation in CuO NP amended soil required both Cu(II)-HA and

367

CuO NP model compounds. In the high dose CuO NP amended soil, linear combination fitting

368

indicates that the presence of CuO decreases over time, with a subsequent increase in the Cu(II)-

369

HA. This suggests that the CuO NPs were dissolving relatively fast in the first 7 days and then

370

more slowly after that as the pore water becomes saturated with respect to CuO(s). The rapid

371

dissolution in the first 7 days in consistent with the DTPA and CaCl2 extractability data, which

372

increased most rapidly in the first 7 days, followed by a slower increase. The dissolution of CuO

373

NP slowed down after 7 days even though the soil has not reached its capacity to adsorb Cu,

374

which confirms our former assumption that dissolution of CuO NP is the limiting factor

375

controlling the extractability of Cu from soil. Note that we also analyzed the 10mg/kg soils and

376

the unamended soil samples, but the signal-to-noise ratio was too poor for adequate speciation.

377

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378

Page 14 of 23

Table 1. Modeled first-order dissolution parameters for CuO NP amended soil. Extraction type

k (day-1)

95% confidence

Half-life

E0 a

Efinal

intervals for k (day-1)

(days)

(mg/kg)

(mg/kg)

R2

High dose amendment DTPA extraction (dry soil) CaCl2 extraction (dry soil)

0.15

0.11-0.19

4.6

3.35

37.4

0.995

0.13

0.12-0.18

5.2

0.05

1.0

0.998

Low dose amendment DTPA extraction (dry soil) CaCl2 extraction (dry soil)

0.16

0.06-0.25

4.5

0.36

6.71

0.936

0.11

0.07-0.14

6.6

0.03

0.16

0.975

379 380

a: E0= initial extractable Cu at day 0 (intercept at y axis)

381 382

383 384 385

Figure 3. Change of Cu speciation in amended soils as inferred by XANES: in (a) Cu(NO3)2

386

amended soil and (b) CuO NP amended soil dosed at 100 mg/kg total Cu. The red dash lines are

387

fitted data while the black lines are experimental data. Model compounds used for the fits are

388

below the experimental spectra. The pie charts represent linear combination fits of the various

389

model compounds.

14


Page 15 of 23


390 391

Environmental Implications

392

The extractability of Cu from CuO NP-amended soils is different from that in soils dosed with

393

Cu ions as Cu(NO3)2, suggesting that the lability of CuO NP may be different from the lability of

394

the highly soluble Cu salts used as pesticides in soils. CuO NP was much less labile than

395

Cu(NO3)2 in soil immediately after they were added to the soil, but its lability increased over

396

time. The differences in lability between CuO NP and Cu(NO3)2 became negligible at low Cu

397

doses (10 mg/kg) after about 7 days, but differences in lability remained over 31 days for the

398

high dose. The increase of the labile pool of CuO NP over time was a result of their slow

399

dissolution. Thus, our research shows that dissolution is an important process controlling the

400

extractability of CuO NP in soil, but the dissolution rate and CuO NP persistence will be

401

concentration dependent. Moreover, the aging time in soil must be considered when assessing

402

the lability or bioavailability of CuO NP in soils as was also previously suggested by Sekine et al

403

for Ag NP, Ag2S NP and AgCl NP and McShane et al. for CuO NP (dosed at 500 mg/kg)26, 27,

404

along with the total applied dose. If toxicity is purely the result of the release of copper ion, the

405

regulatory limit for applying nano CuO in agriculture could be adjusted to consider its “slow

406

release” behavior and concentration-dependent persistence. Because of the relatively slow

407

dissolution behavior of CuO NP, the regulatory limit for CuO NP could be higher than that set

408

for Cu salts. This is especially true if, with some additional surface modification, the dissolution

409

rate of Cu-based nanoparticles could be further reduced. Compared with a direct spray

410

application of Cu salt, a slow sustained release of ions from CuO NP may have lower

411

environmental impact to groundwater and rivers because particles have lower leachability and

412

mobility. On the other hand, if CuO NPs exhibit nanoparticle specific toxicity51,52, for higher

413

doses where CuO NPs persist, regulations will need to consider this persistence if CuO NPs

414

show greater toxicity than the Cu salts. Overall, the regulation of nano enhanced particles might

415

be better based on their dissolution rate at the applied dose, which could be easily determined

416

with the methods used in this study.

417

This work advances our understanding of the fate of CuO NP in several important ways.

418

First of all, we found CuO NP dissolution is the rate limiting step in controlling the increase of

419

CaCl2 extractable Cu and DTPA extractable Cu in CuO NP amended soil, indicating the

420

dissolution process of CuO NP in soil is much slower than the Cu-SOM interaction. Thus, we

15



421

can monitor the dissolution of CuO NP in soil from either the increase in dissolved Cu in soil

422

pore water (as indicated by CaCl2 extraction) or increase in extractable Cu by DTPA extraction

423

(dissolved Cu plus Cu bound to SOM and carbonates). While McShane et al.26 suggested that soil

424

pH is an important factor controlling the dissolution of CuO NP in soil, we also suggest that the

425

amount of SOM in soil may be as or more important because it provided the sink for the released

426

Cu in the soils used here. Secondly, our research indicates that the concentration of soluble

427

nanoparticles added to the soils can affect temporal changes in Cu speciation, which in turn can

428

affect the interpretation of exposure or toxicity testing. At a low dose (10 mg/kg dried soil),

429

CuO NPs became fully dissolved within 10 days. Thus, at low doses, exposures to nanoparticles

430

after ~10 days are not occurring and exposures and toxicity testing would be expected to be

431

consistent with a dissolved Cu species. Moreover, the Cu species present was similar to Cu(II)-

432

HA found in the natural soil so responses to CuO NP amended soils at these low doses would

433

likely be similar to exposures to native soils with the same Cu concentration. However, using a

434

higher CuO NP dose (100 mg/kg dried soil), about 40% of CuO NPs remained undissolved after

435

31 days, potentially because the dissolution was limited by the solubility with respect to CuO(s).

436

In experiments using this high concentration, exposures and effects may be a result of

437

interactions with CuO NP and therefore different than for added ions or native soils.

438

Our research suggests that a single time point extraction after dosing soil may not be

439

adequate for predicting bioavailability unless that extraction is made at the same time as the end

440

point of interest (e.g. plant uptake). Rather, a time series of extractions after dosing may be more

441

appropriate for predicting the bioavailability of metal/metal oxide nanoparticles in soil. The time

442

series of extractions used here could be developed as a functional assay for studying the

443

dissolution kinetics of metal/metal oxide nanoparticles in soil. The functional assay approach has

444

recently been proposed as a means to empirically predict nanomaterial behaviors in complex

445

media53. The method that we developed is simple, and highly reproducible among the three

446

replicates in our experiments. The dissolution rate constant could be used for nanomaterial risk

447

forecasting in soil system, as suggested by Hendren et al53. Further studies need to confirm this

448

method using different metal/metal oxide nanoparticles in different soil systems. For example,

449

several well-known limitations of soil extractions methods, e.g. dilution effects, and the presence

450

of an “irreversibly bound” fraction of metal, exist. In the current study, the irreversibly bound

451

fraction was relatively low (

Time and Nanoparticle Concentration Affect the ... - ACS Publications

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