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Extracting metallic nanoparticles from soils for quantitative analysis: Method development using engineered silver nanoparticles and SP-ICP-MS Dina M. Schwertfeger, Jessica R. Velicogna, Alexander H. Jesmer, Selin Saatcioglu, Heather A. O. McShane, Richard P. Scroggins, and Juliska I. Princz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04668 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Analytical Chemistry

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Title: Extracting metallic nanoparticles from soils for quantitative analysis:

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Method development using engineered silver nanoparticles and SP-ICP-MS

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Authors: D. M. Schwertfeger,† Jessica R. Velicogna,† Alexander H. Jesmer,† Selin Saatcioglu,‡

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Heather McShane,§ Richard P. Scroggins,† Juliska I. Princz†,*

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†

Biological Assessment and Standardization, Environment Canada, Ottawa, Canada

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‡

Department of Civil and Environmental Engineering, Carleton University, Ottawa, Canada

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§

Department of Natural Resource Sciences, McGill University, Montreal, Quebec, Canada

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*

To whom correspondence may be addressed ([email protected])

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ACS Paragon Plus Environment


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Abstract

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Hindering progress in assessing the fate and toxicity of metallic engineered nanomaterials in the

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soil environment is the lack of an efficient and standardized method to disperse soil particles and

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quantitatively subsample the nanoparticulate fraction for characterization analyses. This study

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investigated various soil extraction and extract preparation techniques for their ability to remove

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nano-particulate Ag from a field soil amended with biosolids contaminated with engineered

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silver nanoparticles (AgNP), while presenting a suitable suspension for quantitative SP-ICP-MS

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analysis. Extraction parameters investigated included reagent type (water, NaNO3, KNO3, TSPP,

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TMAH), soil-to-reagent ratio, homogenization techniques as well as procedures commonly used

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to separate nanoparticles from larger colloids prior to analysis (filtration, centrifugation and

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sedimentation). We assessed the efficacy of the extraction procedure by testing for the

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occurrence of potential procedural artifacts (dissolution, agglomeration) using a dissolved /

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particulate Ag mass ratio and by monitoring the amount of Ag mass in discrete particles. The

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optimal method employed 2.5 mM TSPP used in a 1:100 (m/v) soil-to-reagent ratio, with

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ultrasonication to enhance particle dispersion and sedimentation to settle out the micron-sized

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particles. A spiked-sample recovery analysis showed that 96 ± 2% of the total Ag mass added as

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engineered AgNP was recovered, which included recovery of 84.1% of the particles added, while

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particle recovery in a spiked method blank was ~100%, indicating that both the extraction and

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settling procedure have a minimal effect on driving transformation processes. A soil dilution

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experiment showed that the method extracted a consistent proportion of nano-particulate Ag (9.2

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± 1.4% of the total Ag) in samples containing 100, 50, 25 and 10% portions of the AgNP-

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contaminated test soil. The nano-particulate Ag extracted by this method represents the upper

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limit of the potentially dispersible nanoparticulate fraction, thus providing a benchmark with


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which to make quantitative comparisons, while presenting a suspension suitable for a myriad of

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other characterization analyses.

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Key words: soil extraction, biosolids, nanomaterials, complex matrix

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INTRODUCTION

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With the increasing use of metal-based engineered nanoparticles (ENP) in consumer and

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industrial products and their likely release into the environment1, the potential risks of

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nanomaterials on environmental systems are currently under investigation by government

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regulatory agencies that require quantitative data to assess and compare these substances’ risks2-

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3

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concentrations of a toxicant are compared against predicted toxicity thresholds4. However, risk

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assessments for engineered nanoparticles are particularly challenging because they do not always

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conform to the conventional tools of risk assessment. For example, toxicological behaviour of

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trace metals are often related to exposure to the quantity of the total bioavailable mass;5 however,

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for nanomaterials it is also relevant to consider exposure to particle number, particle size and

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active surface area3. While identifying, counting and characterizing engineered nanomaterials in

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aqueous environmental media is riddled with complexities6, the multi-phase and dynamic nature

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of soils poses further challenges in carrying out these assessments for the terrestrial environment.

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There has yet to be a standard method for quantitatively sampling the potentially dispersible

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nano-particulate fraction, which could then be used for further quantitative and qualitative

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characterization analyses.

. During the environmental risk assessment process, predicted environmental exposure

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Manufactured Ag nanoparticles (AgNP) are one of the most widely used, and thus studied

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nanomaterials6, with the soil environment being an important exposure route due to land

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application of processed sewage sludge (i.e., biosolids), which are considered a major sink for

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AgNP1 released from consumer products. Manufactured AgNP are primarily used for their anti-

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microbial properties, presumably arising from the slow release of Ag+ as particles oxidize;7

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however, a growing number of studies have documented AgNP toxicity to non-target


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organisms8. Toxicity may occur via nano-specific mechanisms, such those causing oxidative

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stress9-11 or those induced from the direct uptake of the particle into living cells, or toxicity may

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occur via mechanisms involving the dissolved free ion (Ag+) released from AgNP12. It is

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unknown how significant these nano-specific mechanisms are compared to those involving Ag+

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when exposure occurs in the soil environment. While a number of toxicity studies have been

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conducted using natural soil exposures,13-16 the utility of these studies for environmental risk

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assessment is limited by their lack of quantitative exposure characterization.

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One of the most reliable and efficient quantification tools already established in most

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environmental laboratories is the ICP-MS. Recent advances in ICP-MS technology and

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computation has allowed for the sizing and counting of metallic nanoparticles.17-18 The use of

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ICP-MS requires samples be in an aqueous form and free of large particles (generally considered

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> 1 μm), thus necessitating soil extraction and phase separation techniques (e.g., centrifugation

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and filtering). By definition, all soil analyses based on extraction techniques are operationally

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defined. This means that measured values are inextricably linked to the overall method used to

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acquire the data, that is, the series of procedures including soil preparation, extracting reagent

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and procedure and phase separation technique. The challenge with using such data, from a risk

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assessment perspective, is the lack of comparability for data obtained from different methods. It

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would therefore be beneficial to use a standardized method for soil extraction / phase separation

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that is suitable for subsequent nanoparticulate characterization analyses.

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While development of a comprehensive method for extracting metallic nanoparticles from

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contaminated soils and/or biosolids has yet to be presented in the scientific literature, a number

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of studies have employed various soil extraction and phase separation techniques, often without

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much justification or validation. De-ionized water has been used to extract metallic NPs in



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transformation, mobility and toxicity studies.19-20 In these studies, where a surrogate of the

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natural “pore water” is meant to be extracted, just enough water is added to saturate soils, or

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form a saturated paste, which is a method traditionally used to extract and assess soluble salts,

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that is, constituents in the soil that readily re-solubilize upon wetting.21 When soils are dried

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prior to storage and/or extraction, soil particles flocculate or agglomerate as they come into

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closer proximity to each other with the collapse of their hydration shells.22 This drives

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aggregation and precipitation reactions that would not necessarily occur to soils in situ and are

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thus considered artifacts of the soil drying procedure.23 While using freshly sampled soil would

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be suitable for this kind of saturated pore water extraction, it is simply not practical or feasible

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for most experimental designs. Unlike de-ionized water, that offers very little (chemically) to

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help disperse flocculated soil particles, it would seem advantageous to rehydrate and extract

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nanoparticles from dried soils using a reagent that could facilitate re-dispersion of both natural

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soil, and engineered, nanoparticles.

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Dilute salt reagents (e.g., 0.01 M NaCl2, 0.01 M KNO3, 0.01 M CaCl2) are generally used to

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mimic the electrolytic properties of in situ soil pore water and have been commonly used to

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extract fractions of bioavailable trace metals.24-25 KNO3 has traditionally been used to extract

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electrostatically bound trace metals (i.e., ionic metal forms) and a recent study demonstrated how

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0.01 M KNO3 could be used to extract and analyze both Ag+ and AgNP forms within the same

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extract.26 While this scenario was advantageous from a mass balance perspective, the extracts

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showed only moderate soil dispersion and resulted in large dissolved-to-particulate Ag ratios,

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which is not ideal for SP-ICP-MS analysis. Sodium in an extracting reagent has the advantage of

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promoting the dispersion of soil particles. The relatively small ratio of charge-to-hydrated radius

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of the Na+ ion forms a large electrostatic double layer around particles driving electrostatic


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repulsion between particles.22 It is this electrostatic repulsion that is the major factor controlling

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the dispersion of engineered NP’s from soil particles.27 This would be particularly useful in cases

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where aluminosilicate clay is a major component of nanoparticle heteroaggregates.28 The

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literature shows that NaCl has been used to extract naturally occurring Fe- and Al-(hydr)oxide

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nanoparticles29 as well as engineered Ag nanoparticles.30

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The usefulness of the more alkaline extracting reagents, such as NaOH, tetrasodium

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pyrophosphate (TSPP) and tetramethylammonium hydroxide (TMAH), lies in their ability to

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disperse/dissolve soil organic matter (SOM), which has been found to be a major binding agent

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in the formation of heteroaggregates involving metallic nanoparticles in soils.28 TSPP is

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commonly used to disperse soil particles prior to particle size analysis.31 Regelink et al.29 found

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that 2 mM TSPP (adjusted to pH 7.7 to 8.8) extracted significantly more nano-sized Fe-

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(hydr)oxide than both 1 mM NaOH (pH 9.0) and 0.01 mM NaCl, moreover, the larger Fe-

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(hydr)oxide concentrations in the TSPP extract corresponded to larger concentrations of

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dissolved organic carbon. The strongly alkaline TMAH has been used to dissolve biological

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tissues in order to release and disperse Au and Ag nanoparticles for sizing and quantification via

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SP-ICP-MS.32 The use of TMAH to extract nanoparticles from soils has not yet been tested.

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A variety of techniques have been employed to separate out the nano-sized particles from

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larger particles contained in soil suspensions, a necessary step in preparing samples for ICP-MS

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analyses. In the Cornelis et al. study28 researchers used 0.45 µm cellulose-acetate centrifugal

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filters to extract nano-sized particulates from their saturated soil samples, while Unrine et al.20

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sequentially centrifuged (1000 x g for 5 min) and filtered samples using a glass microfiber filter

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and the commonly used 0.2 µm nylon syringe filter. In a study comparing the effect of pH on the

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speciation of naturally occurring Fe nanoparticles, Neubauer et al.33 centrifuged their soil



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suspensions at 3900 x g for 10 min followed by filtration through 0.45 um cellulose acetate filter.

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Studies investigating Ag recovery after passing AgNP suspensions through various filtering

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apparatus show recoveries as low as 12% for some brands of 0.45 µm centrifugal filters34 and

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73% for sequential 30 mm and 1 µm borosilicate glass syringe filtration.19 There has yet to be

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any consensus on how to prepare soil suspensions for analyses that require removal of micron-

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sized particles, such as SP-ICP-MS and field-flow fractionation (FFF) techniques.

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In this study, we investigated different extraction methods for their efficacy in removing

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nano-particulate Ag from soils amended with biosolids which had been spiked with engineered

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AgNP. The subsequent SP-ICP-MS analysis allowed us to count Ag-containing nanoparticles

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and obtain the mass of Ag within each particle, while at the same time measuring the

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concentration of dissolved Ag. Our approach to designing an optimal soil extraction procedure

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was to liberate the most Ag nanoparticles from the soil matrix, while minimizing nanoparticle

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dissolution and agglomeration processes, which would incur procedural artifacts.

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MATERIALS AND METHODS

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Nanomaterials. Silver nanoparticles with nominal diameters of 25 nm and 40 nm were

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purchased from nanoComposix (San Diego, CA) as PVP-coated (88%), dispersible, silver nano-

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spheres (i.e., powder form) and are hereto referred to by their nominal diameters. The bulk of the

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work (i.e., soil extraction experiments, centrifugation and sedimentation studies) was carried out

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with the 40 nm particles, while the sample filtration study included the 25 nm particles.

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Nanomaterial characterization (independent of the supplier’s) was carried out using transmission

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electron microscopy (TEM) (Tecnai G2 F20 TEM) with energy-dispersive X-ray spectroscopy

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(EDS), dynamic light scattering (DLS) (MalvernTM Nano ZS Zetasizer) and SP-ICP-MS (Perkin

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Elmer NexION 300D). Details of the characterization methods are presented elsewhere.35 Upon


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receipt of the nanomaterials, stock suspensions of the 25 and 40 nm AgNP were prepared to the

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nominal Ag concentrations of 20 mg L-1 and 15 g L-1, respectively, using NanopureTM water

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(Barnstead 2-cartridge water filtration system, > 18.3 MΩ·cm, TOC < 10 ppb). TEM images of

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these stock suspensions showed that both nanomaterials were well dispersed in water (Fig. S1)

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with most particles falling within the suppliers’ size specifications of 23.1 ± 6.9 nm for the 25

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nm particles and 38.6 ± 9.8 nm for the 40 nm particles. Further characterization results are

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presented and discussed in the Supporting Information.

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Soil and biosolids. Test soils (Batch A and B) were prepared by mixing AgNP-spiked

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biosolids with uncontaminated field soil. The soil used was an agricultural sandy loam (5.8

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pHCaCl2, 12.5 g kg-1 total organic carbon and 9% clay, 0.11 μg g-1 total Ag) which was air-dried

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and sieved (< 2 mm). Fresh biosolids (~ 20% solids w/w, 8.0 pH, 7.3 μg g-1) were obtained from

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a municipal wastewater treatment plant located in Southern Quebec. Biosolids were stored

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frozen and thawed when needed by mixing with NanopureTM water to form a slurry, then aged in

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sealed containers for 48 h at room temperature. The average redox potential of the aged slurry

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was -180 mV based on triplicate measurements using a Thermo-Scientific Redox/ORP electrode.

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An aliquot of the 15 g L-1 AgNP (40 nm) dispersion was added to the slurry, homogenized using

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a vortex mixer, aged a further 48 h at room temperature and subsequently added to the soil at a

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rate of 10 g biosolids (dry weight) per kg of soil, in order to achieve a nominal Ag concentration

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of 833 µg g-1 soil and a soil moisture content of 24%. Soil Batches A and B were prepared at

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different times and Batch A was aged two weeks prior to sub-sampling and air-drying, while

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Batch B was sub-sampled immediately and air-dried. Air-dried samples were stored in sealed

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plastic bags at ~ 6°C. A test soil contaminated with AgNO3 (Batch C, nominal Ag concentration



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= 833 μg g-1) was prepared in the same manner as Batch A, but AgNO3 was added to biosolids

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instead of AgNP.

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Soil extraction experiments. Three soil extraction experiments were carried out to test

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various extraction parameters. Details of the various treatments are provided in Table 1. In

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Extraction Experiment #1, test soil from Batch A was used to compare four extracting reagents:

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sodium nitrate (0.01 M NaNO3); tetrasodium pyrophosphate (TSPP) (0.25 mM Na4P2O7),

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potassium nitrate (0.01 M KNO3), and NanopureTM water. In addition to type of reagent,

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ultrasonication homogenization techniques were tested which included use of an ultrasonic wand

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(60 watts, OMNI International Sonic Ruptor 250 ultrasonic homogenizer, micro-titanium tip, 20

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kHz) and an ultrasonic bath (Branson 3510, 40 kHz, 130 watts). The extraction technique was

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carried out as follows: 8 g (air dry) test soil was weighed into a 50-mL polypropylene centrifuge

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tube and mixed with 40 mL of extracting solution which gave a soil-to-reagent ratio of 1:5

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(g:mL). Samples were mixed on a rotary mixer (30 rpm) for 30 min followed by one of three

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homogenization treatments: ultrasonic wand (2 min), ultrasonic bath (20 min) or no

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ultrasonication but samples were further mixed on the rotary mixer for an additional 20 min.

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Separation of nano- from micron-sized particles was conducted by centrifuging samples at 6000

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x g for 20 min followed by passing supernatants through a tandem 0.45 µm/ 0.22 μm nylon

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syringe filter (Millex® Millipore). Filtrates were then stored at 6°C in the dark overnight, prior to

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SP-ICP-MS analysis the next day. All samples were extracted in triplicate.

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Extraction Experiment #2 investigated the effects of increasing the concentration of TSPP

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(0.25 mM, 2.5 mM and 25 mM), as well as decreasing the soil-to-reagent ratio from 1:10 to 1:50.

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Also included was a soil sample to which no manufactured AgNPs were added, but instead the


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soil was spiked with AgNO3 (i.e., Soil Batch C). This sample was extracted with 2.5 mM TSPP

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using a 1:10 soil:reagent ratio. Also under investigation was the use of the strongly alkaline

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reagent, tetramethylammonium hydroxide (25% TMAH). The extraction procedure for samples

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involving the TSPP reagent was the same as that in Experiment 1, except for the sample

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treatment with soil-to-reagent ratio of 1:50, for which 50-mL of reagent was mixed with 1 g soil.

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After mixing on the rotary mixer for 30 min, all samples were homogenized using the ultrasonic

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wand for 2 min. Instead of removing micron-sized particles with centrifugation and filtration

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procedures, sample suspensions were left to settle overnight (~ 18 h) at room T, in the dark,

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allowing large particles to sediment out. To avoid sampling particles > 1 µm, sub-samples were

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taken between 0.5 and 1.0 cm depth from the sample surface, as calculated using Stoke’s law,36

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assuming ideal spherical particles. The extraction procedure for samples with the TMAH

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extraction was as follows: 0.5 g of soil was weighed into a 30-mL polypropylene tube, 10 mL of

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25% TMAH was added and the mixture was homogenized in the ultrasonic bath, at room T, for 1

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h. Sedimentation was carried out same as the TSPP samples. All samples were extracted in

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duplicate.

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A fresh batch of AgNP-contaminated test soils (i.e., Batch B) was used for Extraction

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Experiment #3 and all samples were extracted with 2.5 mM TSPP reagent using soil:reagent

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ratios of 1:10 or 1:100, and were mixed for 30 min, except for one sample for which the mixing

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time of 24 h was investigated. In addition, a soil dilution series was prepared which contained

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mixtures of 50/50, 25/75, and 10/90 (w/w) of Batch B soil / uncontaminated soil, resulting in

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nominal total soil Ag concentrations of 424, 212 and 85 μg g-1. All samples were extracted in

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duplicate, homogenized with the ultrasonication wand for 1.5 min and sedimentation was used to

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facilitate nano/micron particle separation. Note: the time used for homogenization with the wand



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was reduced from 2 min to 1.5 min in order to minimize heating of the wand. Following

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Extraction Experiment #3, the optimal extraction procedure was identified and used to

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investigate AgNP recovery from spiked samples, which has been described in detail in the

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Supporting Information.

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Filtration and centrifuge experiments. To investigate potential AgNP and Ag+ loss from

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filtration procedures often used to separate micron-sized from nano-sized particles, four sets of

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samples were prepared: two sets were prepared with the 25 nm AgNP material and two sets with

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AgNO3. Sample solutions were prepared in either water (Nanopure™) or a filtered soil extract.

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The filtered soil extract was prepared by extracting the uncontaminated test soil with 0.01 M

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KNO3 using a soil-to-solution ratio of 1:5, mixing on a rotary mixer for 30 min followed by

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centrifugation at 6000 x g for 20 min, then filtering through a tandem 0.45/0.22 μm syringe filter.

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Each sample set consisted of 100-mL solutions prepared with low, medium and high Ag

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concentrations, ranging from 2 to 150 μg L-1. Three 10-mL sub-samples were passed through a

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tandem 0.45 μm/0.22 μm nylon syringe filter and total Ag was analyzed by ICP-MS (standard

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mode) before and after filtration. To investigate potential AgNP loss from centrifuge procedures,

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two sets of AgNP (40 nm) suspensions were prepared: one set prepared in a water (NanopureTM)

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matrix, the other in a filtered soil extract matrix that was prepared in the same manner as

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described above, except 0.25 mM TSPP was used to extract the uncontaminated soil instead of

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KNO3. For each matrix, samples were prepared with low and high Ag concentrations which

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ranged from 2 to 200 μg L-1. Triplicate 50-mL sub-samples were centrifuged in a ThermoFisher

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Scientific Legend X1R Centrifuge using three different procedures: 1000 x g for 15 min, 3000 x

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g for 15 min and 6000 x g for 20 min. Initial sample suspensions and resulting supernatants were

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analyzed for total Ag by ICP-MS (standard mode).


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ICP-MS and SP-ICP-MS Analysis. All ICP-MS analyses were conducted using a

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PerkinElmerTM NexION 300D ICP-MS. Total Ag in solutions/suspensions were determined on

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samples acidified to 1% HNO3 (TraceMetalTM Grade, Fisher Chemical) at least 24 h prior to

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analysis, using the ICP-MS in standard mode. Calibration solutions were prepared fresh daily

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from a silver standard (1000 mg L-1 in 2% HNO3, High-Purity Standards) and indium (1 µg L-1)

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was used as the internal standard. Samples were analyzed in duplicate, with relative standard

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deviations (RSD) ≤ 10% between the duplicate readings or samples were re-analyzed. A 1 µg L-1

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solution of Ag in 1% HNO3 was used for quality control (QC) purposes and analyzed after every

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ten samples. Ag recovery in the QC sample was between 90 to 110%, or the preceding set of

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samples was re-analyzed after re-calibration.

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SP-ICP-MS analysis was performed using the same ICP-MS, but operating in single particle

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mode using the SyngistixTM Nano-Application module (PerkinElmer Inc., Waltham, MA).

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Instrumental and data acquisition parameters of the analysis are provided in Table 2. Transport

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efficiency was determined by the particle size method37 using a 60 nm Au suspension (NIST

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Reference Material 8013) for reference. Dissolved Ag calibration was performed using solutions

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ranging from 0.1 to 10 µg L-1 prepared using the purchased Ag stock solution previously

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mentioned, while Ag particle calibration was performed using suspensions prepared with 30 and

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60 nm silver nano-sphere suspensions purchased from Ted Pella (Redding, CA). Particle sizes of

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the Ted Pella AgNP were verified by TEM and were found to fall within the supplier’s

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specifications. For SP-ICP-MS analysis, all particles detected were assumed to be spherical and

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pure Ag0 with a density of 10.49 g cm-3. While this assumption likely holds true for the AgNP in

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the calibration and original stock suspensions, particles detected in the soil extracts likely include

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a variety of transformation products formed by the interactions of the manufactured AgNP with



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constituents in the biosolids / soil and thus particle geometry and mass fraction of the Ag analyte

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are actually unknown. Thus for the analysis of particulate Ag in the soil extracts, the data was

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processed using a default spherical shape and mass fraction of 1 in order to calculate an

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equivalent mono-metal particle size, denoted with the subscript “eqv” (e.g., sizeeqv), indicative,

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not of an actual particle size, but a reference size to aid in the understanding of the Ag mass

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detected. Particle size distributions (PSD and PSDeqv) were fit to a log normal smoothing

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function from which mean and mode particle sizes (or sizeseqv) were derived. Further details of

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the SP-ICP-MS analysis are provided in Supporting Information.

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RESULTS AND DISCUSSION

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Soil extraction experiments. In Extraction Experiment #1, the 0.25 mM TSPP reagent

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extracted significantly (p < 0.01) more Ag particles than the other reagents, given the same

279

homogenization procedure (Fig. 1). Likewise, the use of the ultrasonic wand always extracted

280

significantly (p < 0.01) more particles compared to the other homogenization treatments. For a

281

given reagent, use of the ultrasonic wand extracted 2- to 5-fold more particles than the ultrasonic

282

bath and on average 12-fold more particles than using no ultrasonication. Furthermore, the use of

283

either ultrasonic technique did not appear to enhance the extractability of dissolved Ag to the

284

same extent, as indicated by the lower dissolved-to-particulate Ag mass ratios (Table 1). The

285

lower the ratio, the less likely the extraction procedure is causing particle dissolution, or

286

targeting the dissolved Ag fraction, and is thus considered an indication of greater particle

287

extraction efficiency. The combination of using the ultrasonication wand with the 0.25 mM

288

TSPP reagent resulted in extracting the greatest concentration of particles while showing the

289

lowest dissolved-to-particulate mass ratio (Table 1). This procedure extracted 1250 ± 180 x 106


Page 15 of 29

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15 290

Ag-containing nanoparticles per g soil which represents 0.05% of the total Ag in the soil. The

291

particles extracted by all reagents contained similar amounts of Ag mass, as indicated by

292

equivalent mono-metal particle sizes: mode sizeeqv ranged from 34.5 to 39.9 nm (Table 1), while

293

mean sizeeqv ranged from 34.7 to 44.6 nm. While this equivalent size was consistent with the 40

294

nm AgNP initially added to the soil, it is doubtful that the particles, or particle surfaces,

295

remained untransformed during the biosolids-aging and soil-aging procedures. It should be noted

296

that the SP-ICP-MS is not a speciation technique, thus secondary precipitates (e.g., Ag2S, AgCl)

297

may be included in this analysis, as well as Ag deposited onto organic or clay surfaces, if there is

298

enough Ag mass to be included in the particulate fraction.

299

In Extraction Experiment #2, we investigated if increasing the concentration of TSPP would

300

further enhance the efficacy of the extraction. Unlike the first experiment where all homogenized

301

extracts were centrifuged and filtered, in this experiment sedimentation was used to settle out

302

large particles (see subsequent section on filtration and centrifuge experiments). Increasing the

303

TSPP concentration 10-fold (to 2.5 mM) extracted 3.2-fold more particles (Fig. 2) and reduced

304

the dissolved-to-particulate mass ratio from 5.5 to 2.5 (Table 1). However, increasing the TSPP

305

concentration to 25 mM resulted in unstable SP-ICP-MS readings and repeated failure of the SP-

306

ICP-MS quality control, likely due to Na interference. Thus, in order to supply more active TSPP

307

per g of soil, the soil-to-reagent ratio was lowered from 1:5 to 1:50. This 10-fold decrease in the

308

extraction ratio liberated twice as many particles (Fig. 2). Interestingly, using the lower ratio did

309

not extract any more dissolved Ag, thus the dissolved-to-particle ratio was reduced by about half,

310

to 1.3. The use of the extracting reagent 25% TMAH extracted a similar particle concentration as

311

the 2.5 mM TSPP extraction; however, the TMAH extracted much more dissolved Ag resulting

312

in the larger dissolved-to-particulate mass ratio of 10.7 (Table 1). This was probably due to



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Page 16 of 29

16 313

more complete digestion of the SOM which would have released more organically-complexed

314

Ag, but did not seem to improve the dispersion/extraction of AgNPs. We found that the TMAH

315

extracts were darker and more viscous than the TSPP extracts, and as such, the 18 h

316

sedimentation time was not sufficient to settle out larger particles. Despite dilution, these extracts

317

proved a challenge to analyze by SP-ICP-MS, for which even replicate readings from the same

318

extract showed RSD > 50%.

319

The TSPP extraction of the AgNO3-contaminated soil resulted in the detection of particulate

320

Ag for which the modal sizeeqv was 31.2 nm (Table 1). These data suggest the formation of

321

detectable Ag precipitates in the biosolids/soil mixture which is consistent with observations by

322

other researchers. For example, Whitely et al.19 detected nanoparticles (< 80 nm, mean diameters

323

≤ 40 nm) containing Ag in the soil pore waters from soils amended with AgNO3 and sewage

324

sludge using an AF4/ICP-MS analysis. Based on the redox potential and pH of our incubated

325

biosolids, and the relatively large availability of S, the formation of Ag2S is highly likely38,

326

particularly in the AgNO3 where Ag+ is added directly to the biosolids; however, these are not

327

conditions that would thermodynamically favor complete AgNP dissolution and precipitation of

328

Ag2S within the AgNP treatments, and thus particulate Ag2S is likely mixed with intact AgNP,

329

although varying degrees of surface transformations of the AgNP are likely.39 Once extracted,

330

further particle analysis can be made with regards to elemental composition by analyzing

331

samples for other elements/isotopes by SP-ICP-MS or by the use of imaging techniques (i.e.,

332

TEM/EDS). In addition, samples may be analyzed for the presence of known solid-phase species

333

(e.g., using EXASF or dark-field hyperspectral imaging).

334

In Soil Extraction Experiment #3, the effect of lowering the soil-to-reagent ratio was

335

investigated again, but this time the ratios compared were 1:10 and 1:100. The 10-fold lowering


Page 17 of 29

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17 336

of the extraction ratio resulted in a 20-fold increase in the number of particles extracted, while

337

the dissolved-to-particulate mass ratio decreased from 2.1 to 1.2 (Table 1). Results of the soil

338

dilution test showed that the TSPP extraction procedure extracted Ag particles in a fairly

339

consistent and predictable manner (Table 3). The number of particles extracted from samples

340

containing 50, 25 and 10% portions of the AgNP-contaminated soil were 70, 27 and 16% of the

341

number of particles detected in the AgNP-contaminated soil, while no significant changes to

342

particle sizeeqv were observed (Table 1). The proportion of particulate Ag mass relative to the

343

total Ag remained fairly constant, ranging from 7.5 to 11%. These data suggest that the

344

extraction procedure does not elicit a dose-dependent AgNP transformations.

345

Using the finalized extraction method, the number of particles recovered in the spike-recovery

346

sample was 84.1% of those measured in the reference suspension. Similarly 84.8% of the

347

particulate Ag mass was recovered (Table S1, supporting Information). A portion of the

348

unrecovered particulate Ag mass was recovered in the dissolved fraction resulting in 96.8%

349

recovery of the total Ag added. While these results suggest some degree of particle dissolution

350

may have occurred, no significant changes in sizeeqv were detected in the recovered particles

351

(Fig. S4). Neither particle dissolution, nor agglomeration, were evident in the spiked method-

352

blank, which showed ~ 100% recovery for all three Ag parameters. Thus the apparent AgNP

353

dissolution in the spiked soil samples likely occurred from interactions of the freshly added

354

AgNP and soil constituents, and not from the AgNP interaction with the TSPP reagent.

355

Other researchers have found TSPP to be a highly effective reagent for extracting metallic

356

nanoparticles from soil. Regelink et al.29 found that 2 mM TSPP extracted more Fe and Al

357

(hydr)oxide nanoparticles than either 5 mM NaCl or 1 mM NaOH, and related the efficacy of the

358

TSPP extraction to the greater extractability of SOM they also observed in TSPP extracts, thus



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Page 18 of 29

18 359

reasoning greater disaggregation of organo-mineral aggregates. Pyrophosphate is a tetravalent

360

anion and is thus highly effective at replacing SOM adsorbed onto mineral nanoparticulate

361

surfaces leading to greater extraction of SOM and mineral particles associated with organo-

362

mineral heteroaggregates. Scavenging and binding di- and tri-valent elements (i.e., Ca2+, Al3+,

363

Fe2+) that are normally complexed to, and flocculate, soil humic and fulvic components, would

364

also act to further disperse agglomerated SOM.39 While the TSPP used in the Regelink et al.

365

study was adjusted to pH 9.0, to avoid dissolution of the Fe- and Al-(hydr)oxide nanoparticles

366

under study, we did not lower the pH in our study given that these nano-(hydr)oxides likely

367

interact with engineered AgNP to form heteroaggregates28 thus their dissolution should further

368

enhance AgNP extractability. However, it should be noted that if one was trying to extract

369

engineered metallic oxide nanoparticles (e.g., CuO, CeO2), it would be best to understand oxide

370

particle solubility in the extremely alkaline conditions (> pH 10) of the TSPP solution, and lower

371

the reagent pH as necessary.

372

Filtration and Centrifuge Experiments. Results of the filtration experiment showed that

373

despite the stated particle size cut-off being almost an order of magnitude larger than the AgNP

374

used the experiment (i.e., 25 nm), the filtration procedure resulted in significant retention of

375

particles. Filtration retained 11 to 39% of Ag in the AgNP dispersions prepared in the water

376

matrix, and 67 to 72% of Ag in the dispersions prepared in the soil matrix (Table 4). In contrast,

377

retention of dissolved Ag (i.e., Ag+) by the filters was negligible, regardless of matrix. Initial Ag

378

concentration of the solutions did not appear to influence the degree of Ag retention by the filter.

379

Soil particles likely contributed to the clogging of filter pores which exacerbated filter retention

380

of particulate Ag, but had little effect on the dissolved species.


Page 19 of 29

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19 381

Results from the centrifugation experiment also revealed significant loss of AgNP in the

382

supernatant, even at the lowest speed of 1000 x g, which consistently showed recovery rates of

383

80 to 87%, with the initial AgNP concentration and sample matrix having little effect (Fig. 3).

384

Ag recovery in samples worsened with increased centrifuge speed and significantly lower

385

recoveries were observed in samples with higher initial AgNP concentrations. It is highly likely

386

that extracts from Extraction Experiment #1 were impacted by the combination of centrifuge and

387

filtration procedures used. Based on the losses observed in the filtration/centrifuge experiments,

388

predicted that sample 1-1A would contain 70 to 86% fewer Ag particles than sample 2-1, which

389

was essentially a replicate of 1-1A, but for which sedimentation was used to separate micron-

390

sized from nano-sized Ag particles. This estimate was not far off from the actual value: sample

391

1-1A showed approximately 68% fewer particles than sample 2-1 and a larger dissolved-to-

392

particulate mass ratio (Table 1), as expected.

393

While filtering has often been used to separate out larger particles, our results clearly show

394

that filtering with tandem 0.45 / 0.22 μm nylon filters removed a significant portion of the 25 nm

395

particles. This is consistent with the findings of other researchers using similar and different

396

types of filters.19, 34, 41 The accumulation of evidence suggests that filtering is not an appropriate

397

procedure to remove large particles from environmental samples prior to SP-ICP-MS analysis.

398

And while we found that the centrifugation procedures we used also removed a significant

399

portion of nanoparticles, further experimentation could be performed to potentially optimize

400

centrifuge speeds and times. However, the study by Gimbert et al.41 showed that removal of

401

nanoparticles by centrifugation was variable depending on soil type, suggesting that it may be

402

difficult to develop a centrifuge procedure suitable for a wide variety of soil types. Similar to the

403

conclusions of the Gimbert et al. study, we found that sedimentation was the most useful



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Page 20 of 29

20 404

technique to settle out large particles. However, unlike the Gimbert et al. study, where they

405

observed re-aggregation of nanomaterials in their water extracts with prolonged sedimentation

406

times (> 6 h), the TSPP seemed to keep particles dispersed during the full sedimentation time of

407

16 to 18 h, as supported by the spiked sample and spiked blank AgNP recoveries.

408

CONCLUSIONS

409

Results from this study showed that the 2.5 mM TSPP reagent, used in a soil-to-reagent ratio of

410

1:100 (g mL-1), with the added agitation of a sonication wand, extracted the greatest number of

411

AgNP per g of soil and was associated with the smallest relative dissolved Ag fraction. While

412

increasing the mixing time from 30 min to 24 h may potentially extract even more particles, the

413

added time to this procedure should be weighed against the increase in extraction efficiency for a

414

particular test soil. The procedure was shown to extract AgNP in a consistent manner,

415

proportional to the total Ag concentration, and did not appear drive particle dissolution nor

416

agglomeration, but rather, it allowed for a “snap-shot” of the quantity (i.e., number) of the

417

potentially dispersible Ag-containing nanoparticles in a soil, as well as the quantity of Ag mass

418

contained within them, as indicated by the particle sizeeqv. This is particularly useful when

419

investigating the relative changes to, or effects of, nanoparticles within a set of soil samples as is

420

often the case with transformation and ecotoxicity studies. Sedimentation was the best procedure

421

to separate large particles from the nano-particulates in extracts prior to sub-sampling for SP-

422

ICP-MS analysis. Because filtration and centrifugation removed significant portions of AgNP,

423

these procedures should not be used for quantitative analyses. These results provide a rational

424

basis for a standardized soil extraction method that is relatively free of procedural artifacts and

425

results in a quantitative sub-sample of the mineral/metallic soil nanoparticulate fraction that is

426

compatible with SP-ICP-MS analysis and likely suitable for other characterization techniques.


Page 21 of 29

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21 427

ACKNOWLEDGMENTS

428

Funding was provided through the Chemicals Management Plan through Environment and

429

Climate Change Canada. We thank Dr. J. Wang for his contribution for TEM-EDS imaging and

430

analysis, Helene Lalande at McGill University for her expertise in the total Ag analysis in soils

431

and Dr. B. Örmeci in the Department of Civil and Environmental Engineering, Carleton

432

University for her cooperation in the project. Also, thanks to Equilibrium Environmental Inc.

433

(Greg Huber) for the collection of the test soil and the Saint-Hyacinthe wastewater treatment

434

facility for supplying the biosolids.

435

SUPPORTING INFORMATION

436 437

The Supporting Information includes: (1) Characterization of nanomaterials; (2) SP-ICP-MS analytical details; and (3) Spike-recovery analysis (experimental details and results).

438

REFERENCES

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

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with engineered silver nanoparticles. 2016. Environ. Chem. Accepted for publication Nov. 21.Need DOI. (27) Baalousha, M.; Stolpe, B.; Lead, J. R., Flow field-flow fractionation for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems: A critical review. J. Chrom. A 2011, 1218 (27), 4078-4103. (28) Cornelis, G.; Pang, L.; Doolette, C.; Kirby, J. K.; McLaughlin, M. J. Transport of silver nanoparticles in saturated columns of natural soils. Sci. Total Environ. 2013, 463–464 (0), 120130. (29) Regelink, I. C.; Weng, L.; Koopmans, G. F.; van Riemsdijk, W. H. Asymmetric flow field-flow fractionation as a new approach to analyse iron-(hydr)oxide nanoparticles in soil extracts. Geoderma 2013, 202–203 (0), 134-141. (30) Peyrot, C.; Wilkinson, K. J.; Desrosiers, M.; Sauvé, S. Effects of silver nanoparticles on soil enzyme activities with and without added organic matter. Environ. Toxicol. Chem. 2014, 33 (1), 115-125. (31) Kroetsch, D.; Wang, C. Particle Size Distribution. In Soil Sampling and Methods of Analysis, 2nd ed.; Carter, M. R.; Gregorich, E. G., Eds. CRC Press: 2007. (32) Gray, E. P.; Coleman, J. G.; Bednar, A. J.; Kennedy, A. J.; Ranville, J. F.; Higgins, C. P. Extraction and Analysis of Silver and Gold Nanoparticles from Biological Tissues Using Single Particle Inductively Coupled Plasma Mass Spectrometry. Environ. Sci. Technol. 2013, 47 (9), 14315-14323. (33) Neubauer, E.; Schenkeveld, W. D. C.; Plathe, K. L.; Rentenberger, C.; von der Kammer, F.; Kraemer, S. M.; Hofmann, T. The influence of pH on iron speciation in podzol extracts: Iron complexes with natural organic matter, and iron mineral nanoparticles. Sci. Total Environ. 2013, 461–462 (0), 108-116. (34) Cornelis, G.; Kirby, J. K.; Beak, D.; Chittleborough, D.; McLaughlin, M. J. A method for determination of retention of silver and cerium oxide manufactured nanoparticles in soils. Environ. Chem. 2010, 7 (3), 298-308. (35) Schwertfeger, D. M.; Velicogna, J.; Jesmer, A.; Scroggins, R. P.; Princz, J. I. SP-ICP-MS Analysis of Metallic Nanoparticles in Environmental Samples with Large Dissolved Analyte Fractions. Anal. Chem. 2016. DOI:10.1021/acs.analchem.6b02716 (36) Hillel, D. Texture, Particle Size Distribution and Specific Surface. In Introduction to Soil Physics, Academic Press Inc.: San Diego, California, 1982; pp 33-37. (37) Pace, H. E.; Rogers, N. J.; Jarolimek, C.; Coleman, V. A.; Higgins, C. P.; Ranville, J. F. Determining transport efficiency for the purpose of counting and sizing nanoparticles via single particle inductively coupled plasma mass spectrometry. Anal. Chem. 2011, 83 (24), 9361-9369. (38) Lindsay, W. L. Silver. In Chemical Equilibria in Soils; Blackburn Press: Caldwell, New Jersey, 1979; pp 300-304. (39) Kaegi, R.; Voegelin, A.; Ort, C.; Sinnet, B.; Thalmann, B.; Krismer, J.; Hagendorfer, H.; Elumelu, M.; Mueller, E. Fate and transformation of silver nanoparticles in urban wastewater systems. Water Res. 2013, 47 (12), 3866-3877. (40) Hall, G. E. M.; Vaive, J. E.; MacLaurin, A. I. Analytical aspects of the application of sodium pyrophosphate reagent in the specific extraction of the labile organic component of humus and soils. J. Geochem. Explor. 1996, 56, 23-36. (41) Gimbert, L. J.; Haygarth, P. M.; Beckett, R.; Worsfold, P. J. The Influence of Sample Preparation on Observed Particle Size Distributions for Contrasting Soil Suspensions using Flow Field-Flow Fractionation. Environ. Chem. 2006, 3 (3), 184-191.



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Tables and Figures Table 1. Treatment details and selected results for the soil extraction experimentsa sample ID

soil batch

ratio soil: extracting reagent reagent

Extraction Experiment #1 1-1A A 0.25 mM TSPP 1-1B A 0.25 mM TSPP 0.25 mM TSPP 1-1C A 1-2A A 0.01 mM NaNO3 1-2B A 0.01 mM NaNO3 0.01 mM NaNO3 1-2C A 1-3A A 0.01 mM KNO3 0.01 mM KNO3 1-3B A 1-3C A 0.01 mM KNO3 1-4A A water 1-4B A water 1-4C A water Extraction Experiment #2 2-1 A 0.25 mM TSPP 2-2 A 2.5 mM TSPP 2-3 A 25 mM TSPP 2-4 A 2.5 mM TSPP 2-5 A 25% TMAH 2-6 C-AgNO3 2.5 mM TSPP Extraction Experiment #3 3-1 3-2 3-3 3-4 3-5 3-6

558 559 560 561

B B B B (50%) B (25%) B (10%)

2.5 mM TSPP 2.5 mM TSPP 2.5 mM TSPP 2.5 mM TSPP 2.5 mM TSPP 2.5 mM TSPP

mix time (min)

nano/ ultrasonica micron size -tion separation

mean sizeeqv (nm)

log [part.] (g-1 soil)

ratio diss./ part.

1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5 1:5

30 30 50 30 30 50 30 30 50 30 30 50

wand bath none wand bath none wand bath none wand bath none

C/F C/F C/F C/F C/F C/F C/F C/F C/F C/F C/F C/F

37.3 37.0 35.3 38.6 39.1 NA 36.3 35.3 34.5 39.6 39.9 NA

9.1 8.6 8.0 8.4 7.8 NA 8.2 7.9 7.8 8.8 8.1 NA

6.4 8.1 44 17 34 NA 20 39 45 13 23 NA

1:5 1:5 1:5 1:50 1:50 1:10

30 30 30 30 30 30

wand wand wand wand bath wand

SED SED SED SED SED SED

37.3 39.8 NA 35.5 38.4 31.2

9.6 10.1 NA 10.4 10.0 9.1

5.5 2.5 NA 1.3 10.7 4.4

1:10 1:10 1:100 1:100 1:100 1:100

30 1440 30 30 30 30

wand wand wand wand wand wand

SED SED SED SED SED SED

33.4 37.7 35.5 35.8 37.4 36.0

9.6 9.9 10.9 10.8 10.4 10.1

2.1 1.3 1.2 1.3 1.4 1.3

a

C/F = centrifuge and filtration (0.45/0.2 µm); SED = sedimentation; part = particles containing Ag; ratio diss./part. is the mass of Ag detected in the dissolved fraction divided by the mass of particulate Ag based on SP-ICP-MS; mean sizeeqv is based on the diameter of a mono-metal (Ag0) spherical particle; NA = not analyzed.


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25 562 563

Table 2. SP-ICP-MS instrumental and analytical details parameter group ICP-MS component

parameter

parameter value

ICP-MS model

Spray chamber

Perkin Elmer NexION 300D Low pressure PFA nebulizer Cyclonic, glass

Neb gas flow

1.04 L min-1

Plasma RF power

1600 W

Sample flow rate

0.270 mL min-1

Analyte monitored

107

Sample introduction

Manual with 60 s rinse, 20 s read delay

Transport efficiency Instrument dwell time Sample analysis time Readings per sample

9.1 to 9.4%

Nebulizer

ICP-MS settings

Single Particle Parameter

564 565 566 567 568 569 570 571

572 573 574 575 576 577 578 579

Ag

50 µs 60 s (or more) ~ 1.2 x 106 (or more)

Table 3. SP-ICP-MS Results for Soil Dilution Series (Extraction Experiment #3)a sample ID

total Ag (µg g-1)

particle concentration (109 part. g-1)

part. Ag/ total Ag (%)

3-3 3-4 3-5 3-6

848 424 212 85

88 (1) 62 (7) 24 (1) 13.7 (0.4)

7.5 (0.9) 10 (2) 8.4 (0.5) 11 (2)

a

The results are the mean of duplicate extractions; standard error is provided in brackets; total Ag for sample 3-3 was measured by ICP-MS (standard mode) on HNO3-digest samples, whereas total Ag values for all other samples were calculated as 50%, 25% or 10% of the measured value.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

26 580 581 582 583

Table 4. Total Ag in samples measured before and after filtration through a tandem 0.45 μm/0.22 μm nylon syringe filtera total Ag (µg L-1) Ag form

584 585 586

matrix

conc. level

before

after

% mean loss (SE)

AgNP

water

low med high

2.2 15.0 93.0

2.0 5.0 56.7

11 (1 ) 67 (3) 39 (2)

AgNP

soil extract

AgNO3

water

AgNO3

soil extract

low med high low med high low med high

2.2 14.4 126.6 2.1 52.3 170 2.7 54.4 158

0.6 4.0 42.0 2.1 51.8 162 2.5 54.6 154

72 (3) 72 (2) 67 (3) 0 (0.05) 1 (0.1) 5 (0.1) 6 (0.2) 0 (0.1) 3 (0.1)

a

SE = standard error of three replicates.


Page 27 of 29

27 587 1500

wand bath none

106 part. g-1 soil

1200 900 600 300 0

588 589 590 591 592 593 594 595 596 597 598 599 600 601

TSPP

KNO3

NaNO3

water

Figure 1. Results from Extraction Experiment #1. Particle concentrations in test soils (106 particles g-1 soil) resulting from the use of various extracting reagents (0.25 mM TSPP, 0.01 M KNO3, 0.01 M NaNO3, water), and the use of various homogenization techniques (ultrasonication wand, ultrasonication bath and no ultrasonication). Bars represent 95% confidence limits (α = 0.05) derived for the mean of three replicates.

30 25

109 parts g-1 soil

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 15 10 5 0

602 603 604 605 606 607 608

0.25 mM 2.5 mM 2.5 mM 25% TMAH TSPP (1:5) TSPP (1:5) TSPP (1:50) (1:50)

Figure 2. Particle concentrations (109 particles g-1 soil) in extracts from Extraction Experiment #2. Bars represent 95% confidence limits (α = 0.05) derived for the mean of two replicates.



28 609 water - low water - high soil extract - low soil extract - high

100

75

% Recovery

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 29

50

25

610 611 612 613 614 615 616 617 618 619

1000 x g

3000 x g

6000 x g

Figure 3. Silver % recovery in supernatants after centrifuging AgNP (40 nm) suspensions at three different speeds. Suspensions contained either low (2 or 15 µg L-1) or high (200 µg L-1) concentrations of total Ag and were prepared in either a simple water matrix or in a filtered soil extract matrix (0.25 mM TSPP). Bars represent standard error calculated for triplicate samples.


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29 620

TOC Artwork:

621 622

623 624


Extracting Metallic Nanoparticles from Soils for ... - ACS Publications

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