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Extraction and Analysis of Silver and Gold Nanoparticles from Biological Tissues Using Single Particle Inductively Coupled Plasma Mass Spectrometry Evan P. Gray, Jessica Coleman, Anthony J Bednar, Alan James Kennedy, James F. Ranville, and Christopher P. Higgins Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 12 Nov 2013 Downloaded from http://pubs.acs.org on November 15, 2013

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Environmental Science & Technology

Extraction and Analysis of Silver and Gold Nanoparticles from Biological Tissues Using Single Particle Inductively Coupled Plasma Mass Spectrometry

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Evan P. Gray†, Jessica G. Coleman††, Anthony J. Bednar††, Alan J. Kennedy††, James F.

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Ranville§, Christopher P. Higgins†*

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†Colorado School of Mines, Department of Civil and Environmental Engineering, 1500 Illinois

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St., Golden, Colorado, USA 80401.

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††U.S. Army Engineer Research and Development Center, Environmental Laboratory, 3909

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Halls Ferry Road, Vicksburg, Mississippi, USA 39180

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§Colorado School of Mines, Department of Chemistry and Geochemistry, 1500 Illinois St.

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Golden, Colorado, USA 80401

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KEYWORDS

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TMAH, Bioaccumulation, D. magna, L. variegatus, nanoparticle

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ABSTRACT

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Expanded use of engineered nanoparticles (ENPs) in consumer products increases potential for

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environmental release and unintended biological exposures. As a result, measurement techniques

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are needed to accurately quantify ENP size, mass, and particle number distributions in biological

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matrices. This work combines single particle inductively-coupled plasma mass spectrometry

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(spICPMS) with tissue extraction to quantify and characterize metallic ENPs in environmentally

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relevant biological tissues for the first time. ENPs were extracted from tissues via alkaline

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digestion using tetramethylammonium hydroxide (TMAH). Method development was performed

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using ground beef and was verified in Daphnia magna and Lumbriculus variegatus. ENPs

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investigated include 100 and 60 nm Au and Ag stabilized by polyvynylpyrrolidone (PVP). Mass-

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and number-based recovery of spiked Au and Ag ENPs was high (83-121%) from all tissues tested.

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Additional experiments suggested ENP mixtures (60 and 100 nm Ag ENPs) could be extracted

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and quantitatively analyzed. Biological exposures were also conducted to verify the applicability

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of the method for aquatic organisms. Size distributions and particle number concentrations were

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determined for ENPs extracted from D. magna exposed to 98 µg/L 100 nm Au and 4.8 µg/L 100

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nm Ag ENPs The D. magna nanoparticulate body burden for Au uptake was 613 ± 230 µg/kgww,

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while the measured nanoparticulate body burden for D. magna exposed to Ag ENPs was 59 ± 52

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µg/kgww. Notably, the particle size distributions determined from D. magna tissues suggested

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minimal shifts in the size distributions of ENPs accumulated, as compared to the exposure media.

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Introduction

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The increased use of engineered nanoparticles (ENPs) in consumer products has raised concerns

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over the environmental fate, potential toxicity, and overall risk these materials pose to both

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environmental and human systems [1,2,3]. Release routes of ENPs into environmental systems

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include waste disposal via landfill or via sewage treatment plants (solids or effluent), in addition

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to direct environmental releases such as spills or weathering of ENP containing products (paints,

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etc.) [3]. When released to wastewater, probabilistic flow modeling indicates that ENPs will

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largely be removed to the sludge phase while a smaller fraction will be released in the effluent

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[4,5,6]. Subsequent disposal of ENP-bearing sewage treatment plant sludge (i.e., biosolids) as

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crop fertilizer is of concern as a potential ENP environmental release pathway [5].


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The effects associated with ENP exposure are currently the subject of much research. ENPs

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have the potential to elicit a toxic response from their elemental composition, their specific size

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[7], or their reactivity, especially through the formation of reactive oxygen species.

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early toxicity testing of ENPs has been plagued by the lack of clear characterization and

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quantification of ENPs during the exposures, including assessing the relative impacts of the

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nanoparticulate and dissolved fractions of metals [8]. This inability to differentiate the form of a

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measured body burden (dissolved metal or ENP) in addition to the lack of characterization

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techniques for determining the aggregation state of the ENPs in tissues has limited robust scientific

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conclusions. The need to measure ENPs in tissues, specifically, the size distribution and particle

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number concentration (in addition to mass concentration), has been identified as a pressing need

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[7,9]. Despite the lack of appropriate methodology, the generally accepted approach is to use

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concurrent ENP and dissolved exposures to help identify ENP-specific effects [10,11,12].

However,

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The most common ENP sizing technique used for aqueous ENP dispersions is dynamic

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light scattering (DLS) [13], though other techniques, such as nanotracking analysis (NTA) or

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electron microscopy (EM) are also used [14]. EM allows particle size distributions to be obtained,

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but sample preparation makes quantitative assessments of particle number and mass distributions

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difficult [13]. These techniques are limited by requiring high concentrations (mg/L) to effectively

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analyze unknown samples [13,17,18] and their non-specificity for the target ENP. As detection of

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particles in mixtures can be problematic, separation techniques such as field flow fractionation

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(FFF), capillary electrophoresis, and hydrodynamic chromatography [1,19,20] have been

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employed to analyze polydisperse samples. Separation techniques impart a dilution factor which

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can make environmental sample analysis challenging [21]. While sensitive detectors, such as

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inductively coupled plasma mass spectrometry (ICP-MS), can be employed to counteract dilution,


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detection limits are still above predicted environmental concentrations (greater than 5 µg/L) [21].

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At present, the only technique currently capable of determining particle size and number and mass

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concentration for ENP-containing samples at or below 10 µg/L is single particle ICP-MS

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(spICPMS) [22]. Initial development of spICPMS was performed by Deguelder et al. [23] and

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further method refinement has continued [24,25,26,27]. As spICPMS is based on introducing

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individual ENPs into the ICP-MS plasma, this technique requires dilute solutions, and is thus most

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applicable in the low to mid ng/L range, making it ideal for environmental ENP sample analysis

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[13,26]. The main drawback of spICPMS is the size limit of ENP identification, which is currently

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approximately 20 nm for Au and Ag ENPs, but depends on instrument sensitivity and the ionic

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background for the metal of interest [24,25].

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While detection of ENPs in environmental samples and test media (external dose) using

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spICPMS is possible, the extraction of ENPs from tissues can provide direct determination of

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organism internal dose, and is essential for exposures in matrices where ENP concentrations are

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difficult to determine [7]. Traditional digestion using strong acids likely leads to the dissolution

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of most ENPs. The medical community conducted initial tests on non-acidic digestion using

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enzymes or strong bases for extracting metallic and non-metallic colloids from tissues [28,29].

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Enzymatic digestions would only facilitate ENP extraction where enzyme function does not

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require low pH, such as pepsin [29]. For example, an enzymatic digestion was developed using

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Proteinase K for the extraction of Ag ENPs from chicken tissue [30]. This digestion had a recovery

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of around 80% using asymmetrical field flow fractionation (AF4) -ICP-MS, however this recovery

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was reduced to 68% when using spICPMS. Alkaline digestions focus on sodium hydroxide

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(NaOH), potassium hydroxide (KOH) and tetramethylammonium hydroxide (TMAH) [28,29,31]

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to liberate ENPs from tissues. TMAH was also shown to have high extraction efficiency for


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dissolved metals [31], while NaOH and KOH generally cause particle aggregation [28,29]. In a

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related study, Schmidt et al [32] used TMAH in the extraction of ENPs from liver tissue, but was

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unable to identify particle size or number concentration using FFF-ICP-MS analysis.

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The objective of this work was to combine the sensitivity of spICPMS with a tissue

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extraction procedure to quantify ENPs in environmentally-relevant biological tissues.

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Specifically, this study quantitatively describes the extraction of ENPs spiked into representative

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mammalian tissue (ground beef), as well as two types of biological tissues commonly employed

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in environmental toxicity testing, Daphnia magna (D. magna) and Lumbriculus variegatus (L.

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variegatus). Extraction efficiencies, size distributions, and tissue concentrations (number and

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mass) were assessed. The applicability of this technique to resolve both ENP mixtures and

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dissolved elements from ENPs was evaluated. Lastly, the validated method was applied to

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ENP=exposed D. magna to confirm the ability of the procedure to liberate and characterize

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bioaccumulated ENPs.

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Materials and Methods

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Materials

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Gold and silver ENPs used in spike recovery and uptake experiments were purchased from

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nanoComposix (San Diego CA, USA) with polyvinylpyrrolidone (PVP) coatings in sizes of 100

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nm for Au and 100 and 60 nm for Ag (NanoXact). Additionally, 100 nm Au particles with surface

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associated tannic acid were purchased from BBI (Cardiff, UK) to determine the daily ICP-MS

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transport efficiency. Dissolved Au and Ag standards (CertiPrep) were purchased from SPEX

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(Metuchen NJ, USA). Optima grade hydrochloric (HCl) and nitric acid (HNO3) for Au and Ag

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standard preparation was purchased from Fisher Scientific (Fairlawn NJ, USA).

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extractions were performed using TMAH (electronic grade, 25% w/w) purchased from Alfa Aesar

Alkaline


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(MA, USA).

ACS grade ethylenediaminetetraacetic acid (EDTA) was purchased from

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Mallinkrodt (Paris KY, USA). All solutions and dilutions in this work were made using Barnstead

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Nanopure water (18 MΩ, Barnstead Nanopure Diamond, ultrapure water system).

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Single Particle ICP-MS

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Analysis of biological extracts and aqueous samples using spICPMS followed the method

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described by Pace et al. [25,26]. All samples were analyzed using a Perkin Elmer NexION 300q

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ICP-MS (Waltham MA, USA) equipped with a Meinhard Type (Type A, glass) nebulizer (Golden

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CO, USA) and a Perkin Elmer cyclonic spray chamber (Waltham MA, USA).

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calibration was achieved using a blank and at least four Au and Ag standards ranging from 0.1 to

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2 µg/L. All data were collected in single particle mode, with signals averaged for the entire

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analysis period (200 s). ICP-MS nebulization efficiency was calculated daily using the particle

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size method [26] using a 100 nm Au ENP (BBI) and ranged from 4-6% for all experiments

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described herein. The dwell time for all spICPMS measurements was 10 ms based on literature

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reported optimization [33] and 20,000 readings were collected for each sample resulting in a total

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analysis time per sample of 200s. Standard deviations for recovery, total mass determination and

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body burdens were calculated from triplicate samples with the exception of ENP-exposed D.

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magna body burdens, which were calculated from four replicate samples. Background signal

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corresponding to dissolved elements was removed from size distribution plots by identifying the

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minima between the resolved background and the ENP distribution. For visual data representation

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of ENP size distributions, readings from analytical replicates were combined, with the resultant

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figures representing 60,000 data points (with the exception of ENP=exposed D. magna body

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burden size distributions, which include 80,000 data points).

Instrument

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Sample Extraction Procedure

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All ENP tissue extraction method development was conducted using 93% lean ground beef

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purchased from a local market. Extraction of Au and Ag ENPs was optimized for ENP number

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and mass recovery by varying sample digestion time, TMAH digestion concentration, and sample

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cleanup procedures. Recovery was calculated by comparing the observed ENP number and mass

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distributions of an extracted sample to the same ENP analyzed in DI water. Total metal analysis

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was performed to confirm that aqueous standards represented total metals extraction (described

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below). Particle number distributions were determined directly by counting all ENPs observed in

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a sample, while ENP mass distributions were determined by integrating the observed counts.

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These counts were then converted to mass following the procedure outlined by Pace et al. [25,26].

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Initial sample digestion times were varied between 12 and 24 hours, while TMAH

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digestion concentrations included 5, 10, 15, 20 and 25% w/w (only data for 20% TMAH shown).

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Low gravity centrifugation (100 relative centrifugal force Fisher Scientific Model 228) and coarse

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filtration (1 µm Whatman nylon) and simple dilution were all tested as sample clean-up steps. All

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tissue samples were bath sonicated (Fisher Scientific FS140D, 135 W) for one hour at the start of

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each digestion to aid in breaking down tissue and preventing particle aggregation. Sample

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digestion mass was held constant at 0.5 g tissue with 10 ml of TMAH solution for beef and L.

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variegatus (20:1 solvent to tissue), while D. magna digestion mass was much lower with 5 D.

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magna (approx. 1.75 mg) per digestion. Digested samples were diluted a minimum of 1:20 to

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produce a final maximum TMAH concentration of 1%, which was maintained in cases where

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additional dilutions were performed.

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Method Validation Spike Recovery Experiments

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Initial method development indicated that 24-hour digestion with a 20% TMAH solution at room

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temperature and only sample dilution cleanup yielded the highest recoveries. Specifically, 20%

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TMAH was chosen after optimization testing for both Au and Ag ENPs using a variety of TMAH

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concentrations. Recovery determinations between filtration (1 µm nylon), centrifugation, and

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simple dilution were made qualitatively by comparing the observed number of ENPs for each

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cleanup step. Significant losses of ENPs were observed using filtration and centrifugation, similar

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to previous work [30,34]. Simple dilution provided the highest relative recovery and was thus used

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for further experiments. Spike recovery experiments were conducted at 98 µg/kg wet weight (ww)

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for Au ENPs and 19 µg/kgww for Ag ENPs by diluting stocks (concentrations verified by traditional

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ICP-MS analysis) prior to pipetting ENP solutions onto beef samples in 15 ml polypropylene tubes.

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Tissue spiking concentrations were purposefully selected to be environmentally relevant while

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simultaneously still allowing spICPMS analysis to be performed. TMAH was added to beef

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samples immediately after each ENP spike. Au and Ag ENPs in extracts were diluted at least 1 to

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20 prior to spICPMS analysis. As biological samples from ENP-exposed organisms are unlikely

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to undergo immediate extraction and analysis, the issue of sample preservation was evaluated by

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freezing spiked beef samples for seven days and comparing “fresh” vs. “aged” samples. For spike

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recovery experiments involving L. variegatus and D. magna tissues, spiked tissue concentrations

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in D. magna were 2.2 mg/kgww for Au ENPs and 420 µg/kgww for Ag ENPs. L. variegatus spiked

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tissue concentrations were lower at 98 µg/kgww for Au ENPs and 19 µg/kgww for Ag ENPs.

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Statistical differences (p < 0.05) between tissue types were determined using an ANOVA followed

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by a post hoc Tukey Test (Origin, V9.0, OriginLab, Northampton, MA, USA).

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Complex Sample Analysis

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TMAH extraction coupled to spICPMS was tested using samples spiked with 60 and 100 nm ENPs

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and samples spiked with Ag+ and Ag ENPs. Mixture experiments were conducted using 60 and

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100 nm Ag ENPs spiked into tissue samples at equivalent nominal particle number concentrations

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(1.8 x 1010 particles/kgww). Dissolved Ag was spiked into tissues at a concentration of 40 µg/kgww.

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Two types of dissolved Ag experiments were conducted, one in which only dissolved Ag was

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added to the tissue (40 µg/kgww) and one in which both dissolved and 100 nm Ag ENPs were added

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(40 µg/kgww Ag+ and 19 µg/kgww Ag ENPs). All experiments were conducted in triplicate.

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Biological Uptake Experiments

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As spike recovery experiments do not reflect the actual conditions following ENP exposure,

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additional experiments with exposed organisms were conducted with D. magna. Briefly, D.

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magna uptake experiments were conducted following EPA 2021.0 [35], with slight modifications.

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D. magna adults were used instead of neonates to provide sufficient tissue mass for digestion and

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analysis. All adult D. magna were obtained from Aquatic Bio Systems (Fort Collins, CO, USA).

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Test beakers contained 25 mL of EPA moderately hard water [35] fortified with the appropriate

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mass of ENPs and five D. magna. Four replicate beakers were used per treatment. Uptake of Au

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ENPs was conducted at 98 µg/L (9.7 x 109 particles L-1), while uptake of Ag ENPs was conducted

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at 4.8 µg/L (8.7 x 108 particles L-1). Test duration was 48 hours with a 16:8 hour light:dark

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photoperiod at 20°C. D. magna were briefly washed in 20mM EDTA post exposure to remove

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any surface associated metal ions prior to TMAH digestion, but were not depurated post exposure.

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Water samples were taken at 0, 12, 24, 36 and 48 hours from each exposure chamber to monitor

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potential changes in ENP exposure during testing. Water samples were frozen at -80°C upon


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collection, but were thawed to room temperature immediately prior to dilution in water and

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spICPMS analysis.

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Total metals determination

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To enable comparison between this technique and a total metals digestion, microwave digestion

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was performed using a MARS 6 microwave digestion system (CEM Corporation, Matthews NC).

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The digestion procedure was a modified EPA 200.2 [35] to accommodate high pressures. Ramp

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time was 15 min followed by a hold time of 30 min at 175 oC. Tissue mass (0.5 g) and spike

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concentrations (98 µg/kgww Au and 19 µg/kgww Ag) were identical to TMAH spike recovery

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experiments. Concentrated HNO3 (9 ml) and concentrated HCl (3 ml) was added to Au ENP

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containing samples, while only 12 ml of HNO3 was added to Ag ENP containing samples to avoid

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AgCl formation. Samples were predigested overnight prior to microwave digestion.

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Bioaccumulation metrics

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While biodynamic models of ENP uptake are likely to provide the most accurate estimates of ENP

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bioaccumulation [36,37], to enable comparisons of Au and Ag ENP bioaccumulation from

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aqueous exposures, the bioconcentration factor (BCF) was calculated:

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𝐵𝐶𝐹 =

𝐶𝑜𝑟𝑔𝑎𝑛𝑖𝑠𝑚 ⁄𝐶 𝑤𝑎𝑡𝑒𝑟

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with Corganism expressed as µg/kgww and Cwater as µg/L, resulting in a BCF with units of L/kgww.

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Measured (as opposed to nominal) Cwater values were employed. This approach assumes steady

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state, which is not necessarily a valid assumption [7,38], but at present, may be a useful metric for

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comparing bioaccumulation across several studies [39].

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Results and Discussion

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Method Performance in Model Mammalian Tissues

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The TMAH tissue extraction method coupled to spICPMS analysis was effective at quantitatively

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identifying 100 nm Au and Ag ENPs spiked into ground beef spiked at 98 µg/kgww Au and 19

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µg/kgww Ag (Table 1). When compared to ENPs analyzed in water, recovery of Au ENPs on a

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particle number basis was 94 ± 3% (± standard deviation), while recovery based on the total mass

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was 89 ± 3% (Table 1). Similarly, the number-based recovery of Ag ENPs extracted from beef

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was 95 ± 2%, while the total mass-based recovery was 104 ± 2% (Table 1). The minor

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discrepancies between the two metrics may be a result of slight changes in ENP size of either the

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extracted ENP or its reference standard (prepared in DI water). Despites this, the TMAH extraction

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process did not lead to observable alterations (< 5 nm) in the size distributions of either Au or Ag

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ENPs when compared to ENPs analyzed in water (Figure 1). No consistent bias in recovery based

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on ENP number as compared to ENP mass was observed between tissue types (Tables S2, S3).

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Particle mass-based recoveries do appear to be higher for Ag ENPs as compared to Au ENPs for

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all tissue types tested. Ag ENP recoveries on a mass basis are all over 100%, which was likely

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caused by a change in the Ag ENP reference standard (prepared in DI water). Figure S3 shows

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that for all tissue types tested, less Ag mass (~ 3.5%; 2 nm smaller diameter) was detected for the

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ENP standard as compared to the tissue-extracted ENPs. This unintended dissolution of Ag ENP

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reference standards illustrates the need to ensure reference standards are stable with respect to

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dissolution. Stabilization can be achieved through the addition of surfactants and/or other

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alterations of solution chemistry that prevent dissolution [40].

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Table 1. Particle number and particle mass based recoveries of 100 nm Au and Ag ENPs extracted from different biological tissues using TMAH. Beef and L. variegatus were spiked at 98 µg/kgww Au while Ag was spiked at 19 µg/kg ww. D. magna were spiked at 2.2 mg/kgww Au and 0.42 mg/kg ww Ag. Tissue Matrix Ground Beef 7 Day Frozen Beef D. magna L. variegatus Ground Beef 7 Day Frozen Beef D. magna L. variegatus

Particle Number Recovery % ±SD Au 94 ± 3 90 ±4 95 ±2 95 ±3 Ag 94 ±2 106 ±2 83 ±5 95 ±3

Particle Mass Recovery % ±SD 89 ±3 88 ±2 92 ±2 95 ±3 104 ±2 121 ±4 105 ±8 107 ±7

Figure 1. Au ENPs analyzed in H2O and TMAH extracted from beef (A). Ag ENPs analyzed in H2O and TMAH extracted from beef (A). Tissue concentration were 98 µg/kgww for Au ENPs and 19 µg/kgww for Ag ENPs. Percent readings and peak mode (Readings, % , Mode, nm) are (8.5, 100), (7.9, 98), (9.9,87), (9.4, 92) for Au H2O, Au Beef, Ag H2O, Ag Beef. 247

Matrix spike experiments in beef indicate that the 20% TMAH digestion maintaining a

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ratio of at least 20:1 (solution to tissue mass) is a highly efficient extraction procedure when

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coupled to spICPMS. Recoveries observed in all spiking experiments were all within the 75-125%

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range for the development of extractions for which there is no historical record of recovery values

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[41]. Recovery was reported on a traditional mass basis, and also on a particle number basis, as

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this metric (in addition to size distribution) are likely just as (if not more) important for


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understanding ENP biological interactions [39]. In an enzymatic digestion procedure, Loeschner

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et al [30] reported AF4–ICP-MS recovery of around 80%, however, spICPMS recovery was 68

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±13% as compared to AF4–ICP-MS recovery.

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(comparison to a total metal digestion), and as such total recoverable Ag may be well below 80%

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using enzymatic digestion.

No total Ag recovery values are reported

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Extraction of ENPs from tissues is likely dependent on ENP tissue concentration in

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exposed organisms. Spike recovery tests in this work were performed at 98 to 19 µg/kgww and are

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three orders of magnitude lower than the tissue concentration of 197 mg/kgww used in enzymatic

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digestion by Loeschner et al [30]. Both techniques diluted samples to ng/L concentrations after

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aqueous based digestion prior to analysis. Digestion at 197 mg/kgww is well above predicted

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environmental concentrations, but was intended to reflect possible food contamination rather than

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environmental exposure. In contrast, the TMAH digestion procedure described in this work is

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effective at extracting ENPs spiked into tissues at lower, more environmentally relevant

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concentrations (i.e., down to 19 µg/kgww). Despite differences in digestion tissue concentration,

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neither digestion procedure altered the primary size distribution of the ENPs.

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Complex Samples and Matrices

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Alkaline tissue digestion for Au ENPs was previously attempted in the literature, with extracts

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being analyzed using FFF- ICP-MS [32]. FFF-ICP-MS was capable of baseline separation of a 60

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and 100 nm ENPs in a 25% TMAH solution with bovine serum albumin present. However, no

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separation of 60 and 100 nm ENPs was observed when analyzing rat livers using the same

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extraction and analysis procedure. In this work, resolution of a mixture of 60 and 100 nm Ag ENPs

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extracted from tissues using TMAH was nearly baseline and observed peak shapes were nearly

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identical to the 60 and 100 nm Ag ENPs run individually in water (Figure 2). These results suggest


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Figure 2. Overlay of 60 and 100 nm Ag ENPs (PVP) analyzed in water (A) compared to 60 and 100 nm Ag ENPs extracted simultaneously from ground beef (B).

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that spICPMS is a better detection technique for ENP analysis as compared to FFF-ICP-MS for

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TMAH-based extraction of complex tissues if metal core particle size is the desired endpoint,

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provided the ENPs extracted are within the spICPMS analytical range. AF4 separation coupled to

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ICP-MS can easily separate mixtures, even after tissue digestion [30].

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hydrodynamic diameter of the particles, which can provide useful information on transformation

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or accumulation of surface coatings on the metal particle core. Non-ideal particle-membrane

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interactions during AF4 separations, may prevent accurate sizing of unknown samples using

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particle standards or FFF theory, however.

AF4 reports the

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In addition to providing good ENP resolution in extracted tissue samples, TMAH digestion

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coupled to spICPMS is capable of separating dissolved and ENP elements present in tissue

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samples. Figure 3 shows two digested beef samples, both of which were spiked with 40 µg/kgww

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Ag+. The results presented in Panel B in Figure 3 are from samples that were also spiked with 100

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nm Ag ENPs to 19 µg/kgww ENP Ag. A clear ENP peak was observed above the dissolved Ag

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background that was present in both panels (Figure 3). The presence of the dissolved Ag does not

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shift the observed ENP size distribution (Figure 3A inlay) from the particles analyzed in water


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(Figure 1B, H2O). In previous work, TMAH was chosen specifically to liberate Cd+ from mouse

Figure 3. Extraction of dissolved and ENP Ag (100 nm) from beef (A) compared to extraction of dissolved Ag only from beef (B), with converted size distribution in inlay.

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liver samples while not causing dissolution of quantum dots also present in the sample [31]. This

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ability of TMAH to dissolve tissues and preserve dissolved and ENPs is consistent with the results

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presented herein.

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Total Metal Digestion

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The total mass recovered using microwave-assisted digestion was comparable to ENP mass

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recovered using TMAH digestion coupled to spICPMS analysis.

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distribution, when converted to mass distributions and integrated, enabled the calculation of the

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total TMAH extractable ENP mass in each sample. The recovered mass was then compared to the

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total acid extractable ENP (metal) mass. Comparing the TMAH extracted mass to total metals

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analysis indicated that TMAH extraction liberated 90-96% of the total mass depending on the

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specific particle type analyzed (Table 2). These results indicate that TMAH extraction is capable

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of liberating all ENPs from tissues, similar to a more traditional acid digestion. Further, the

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recovery of Au and Ag ENPs using TMAH extraction was between 75-125%, indicating that this

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extraction procedure is valid for different ENP and tissue types (Table 1, Table S1).

Measured spICPMS size


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Table 2. Total acid extractable Au and Ag from beef spiked at 98 µg/kg ww Au and 19 µg/kgww Ag both as ENPs. Recovery is shown as a comparison of TMAH and total metals extraction. Element Au Ag

Acid Digested (µg) (±SD) 4.9 (± 0.2) x 10-02 9.4 (± 0.9) x 10-03

TMAH Extracted (µg) (±SD) 4.4 (± 0.06) x 10-2 9.0 (± 0.3) x 10-3

% Recovery 90 96

308 309

Method Performance in Environmentally Relevant Tissues

310

Matrix spike experiments to validate TMAH extraction in the toxicologically relevant

311

organisms D. magna and L. variegatus showed high recovery for both Au and Ag ENPs. D. magna

312

particle number based recovery ranged from was 83% to 105% while L. variegatus recovery varied

313

from 95 to 105% (Table 1). Excellent recovery values (between 80-120%) from both mass and

314

particle number based approaches indicated complete recovery of ENPs. Further, as with the beef

315

tissue, no size shift was observed in Au ENPs spiked into either D. magna or L. variegatus as

316

compared to ENP standards. Mass and particle number based recoveries were similar between

317

beef, D. magna, and L. variegatus.

318

Extracted tissue concentrations in spike recovery tests cover two orders of magnitude, from

319

2.2 mg/kgww in D. magna to 19 µg/kgww in L variegatus. Extracted size distribution, particle

320

number, and mass concentrations were clearly quantified for all samples tested in this tissue

321

concentration range. Importantly, the concentrations of the tissue spike experiments were below

322

the ENP (presumably not dissolved metal) body burdens observed for ENP uptake studies in the

323

literature. For example, Coleman et al. [42] observed a body burdens ranging from 1.7 to 4.4

324

mg/kgww for L. variegatus exposed to 4.6 mg/L Ag ENPs.

325

Method Validation with Exposed D. magna

326

Extracted size distributions of Au and Ag ENPs from exposed D. magna (Figure 4) were consistent

327

with the extracted size distributions in matrix spike experiments (Figure 1A and 1B).


16

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328


Figure 4. Uptake of 100 nm Au (A) and Ag (B) ENPs into D. magna exposed at 98 µg/L Au ENPs and 4.8 µg/L Ag ENPs, as compared to reference Au and Ag ENPs in water. Percent readings and peak mode (Readings, %, Mode, nm) are (9.0, 102), (2.4, 98), (6.6, 86), (1.2, 94) for Au ENPs in water, Au ENPs in D. magna, Ag ENPs in water, and Ag ENPs in D. magna, respectively. Histograms are normalized to the largest bin for each experiment.

329

The particle number concentrations extracted from D. magna ranged from 6.2 (± 2.0) x 1010

330

particles/kgww for Au exposure to 1.3 (± 1.2) x 1010 particles/kgww for Ag exposure (Table 3). The

331

average mass-based body burden in extracted D. magna was 613 ± 230 µg/kgww for Au ENPs and

332

73 ± 64 µg/kgww for Ag ENPs (Table 3). When size distributions of ENPs extracted from exposed

333

D. magna are compared to the reference ENPs, there is no observable shift in ENP size

334

distributions (< 5 nm), at least for this short exposure period. For Ag uptake, the bioaccumulated

335

ENPs exhibited the same shift to a larger size that was observed in Ag spike recovery experiments.

336

This is likely a result of ENP standard dissolution as discussed previously. No significant dissolved

337

signal was observed in extracted daphnia for either Au or Ag exposures. Measurement of D.

338

magna exposure media indicated the Au ENP exposure concentration ranged from 91 to 94 µg/L

339

throughout the test. Nanoparticulate Ag levels were below the initial exposure concentration of 4.8

340

µg/L at the start of the experiment and showed a decreasing trend over the 48-hour test (Figure

341

S2). As the exposure mass and particle number varied across the 48-hour exposure, a range of

342

BCFs were determined for Ag uptake experiments (Table 3).


17


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Table 3. Tissue concentrations, both particle number and mass based for exposed D. magna. Measured dose is included and BCFs (L/kgww) are shown.

Sample analyzed

Element (100 nm ENP)

Tissue concentration (µg/kgww) (±SD)

D. magna

Au

613 (±230)

D. magna (media at T=0)

Ag

D. magna (media at T=48 hours)

74 (±64)

Ag

Tissue concentration (particles kgww-1) (±SD) 6.2 (±2.0) x 1010 1.3 (±1.2) x 10

Measured Exposure concentration (µg/L)

Measured Exposure BCF (Mass based)

Nominal Exposure BCF (Mass based)

94 (±7.2)

6.6

6.3

3.4 (±0.56)

22

10

15 2.3 (±0.57)

31

343 344

Comparisons of ENP Bioaccumulation

345

The primary objective of this study was to develop and validate a method for quantifying metal-

346

based ENPs in environmentally relevant biological tissues. To validate the approach,

347

bioaccumulation was measured in short-term aquatic only exposures using D. magna. The D.

348

magna readily accumulated Au and Ag ENPs in the 48 hour exposure, reflecting the fact that D.

349

magna are filter feeders which can be exposed to ENPs through their high filter rate [39]. When

350

compared to the range of BCFs reported for Ag ENPs in D. magna (after converting to dry weight

351

(dw) tissue concentrations using a dry-to-wet conversion factor of 0.08, [43]) the log BCFs

352

(L/kgdw) measured in this study (1.9 for Au ENPs, 2.3 to 2.5 for Ag ENPs) are significantly lower

353

than those previously reported (range log BCF of 3.16 to 4.66, mean of 4.04 ± 0.39 [39]). BCF

354

values for D. magna in this work resulted from a short term, low exposure (19 µg/L Ag) test where

355

organisms were not fed. Chronic (21 day) uptake studies have indicated that D. magna ENP

356

ingestion is heavily influenced by the presence of food and the exposure concentration, with

357

ingestion rate increasing disproportionately with increasing exposure concentration [38] The


18

Page 19 of 24


358

literature reported BCF values were determined from chronic D. magna exposures where

359

organisms were fed thought the exposure, and where test concentrations were greater than this

360

work [38,44]. Given the differences between exposures and potential uptake kinetics for Ag ENPs,

361

these differences likely explain the observed variability between BCFs. Further, the consistency

362

between the TMAH spICPMS procedure and total metal analysis (as measured in spike-recovery

363

experiments, Table 2), suggests these differences are likely real, and may have arisen due to the

364

fact that the bioaccumulated nanoparticulate mass reported herein is directly measured as ENPs.

365

Previous studies measuring total Ag mass in tissue likely reflect both dissolved and nanoparticulate

366

mass.

367

Implications

368

This work is the first to show that ENPs can be extracted from a number of different tissues

369

to directly determine uptake of ENP number, size and mass distributions using TMAH digestion

370

coupled to spICPMS. High mass and number-based recoveries indicate that this extraction

371

procedure is efficient at liberating particles from organism tissues. The applicability of the

372

extraction procedure in beef, D. magna, and L. variegatus suggest that this procedure may be

373

applicable to a wide range of tissues.

374

discriminating dissolved Ag signal from Ag ENPs, and was capable of resolving a mixture of 60

375

and 100 nm ENPs. Both mixtures and dissolved elements are likely to be encountered in

376

environmental samples. The concentration detection limit was not experimentally determined in

377

this study, but the relative ease of detection and characterization at 19 µg/kgww suggests that this

378

number could be lower without significant further optimization.

Further, the extraction procedure was capable of

379

Despite the size detection limit of approximately 15-20 nm for spICPMS based on current

380

instrumental sensitivities [24,25,30], this work indicates that metal-based ENPs can be extracted


19


Page 20 of 24

381

from biological tissues and quantitatively analyzed at environmentally relevant concentrations.

382

Importantly, tissues with high lipid content, such as fish, or with different cellular makeup, such

383

as plants, were not tested and method validation would be necessary in these tissues prior to

384

determination of uptake for these matrices. Finally, changes in the primary particle size due to

385

biological coatings would not be detectable using this method, and TMAH extraction would likely

386

lead to disaggregation of any ENPs which have aggregated in vivo..

387

Though limitations exist, this extraction procedure coupled to the highly sensitive

388

analytical technique spICPMS helps fill a necessary metrology gap by allowing the direct

389

determination of organism dose in exposed organisms, specifically yielding ENP number and size

390

information for the primary particle in addition to total mass uptake.

391

ACKNOWLEDGMENTS

392

We would like to thank the US Army Corps of Engineers for funding this research (Grant

393

W912HZ-09-0163). We would also like to thank the Ranville research group for helping in

394

spICPMS troubleshooting and method development.

395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411

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