Multi-method assessment of PVP-coated silver nanoparticles and artificial sweat mixtures

Abstract

Research presented here utilizes silver nanoparticles (AgNPs) as a case study for how the immediate local environment alters the physical and chemical properties of nanomaterials. Dermal exposure is a primary route for exposure to many of the consumer products containing AgNPs. Interactions between AgNPs and human sweat/perspiration are critical for understanding how changes in Ag speciation will impact exposure. Previous studies have examined silver release from AgNP-containing products after exposure to artificial sweat (AS), however there is no basic assessment of how mixtures of AgNPs and AS alter the physical and chemical properties of AgNPs. The current research evaluated changes in size, aggregation, chemical composition, and silver speciation of four different sizes of AgNPs exposed to four different formulations of AS. The AS formulations were from standardized methods with different chemical compositions, ionic strengths, and pH. Samples were collected at four-time intervals for analysis using dynamic light scattering, UV–Vis spectroscopy, and single-particle inductively coupled plasma-mass spectrometry. Each mixture was also prepared for speciation analysis using X-ray absorption spectroscopy and scanning electron microscopy coupled to energy-dispersive X-ray analysis. The equivalent diameter measurements from the three techniques followed the order of DLS > UV–Vis > spICP-MS. Speciation analyses indicate significant changes for the smaller NPs, while the largest (100 nm) NPs had less measurable differences. This study shows the need to fully understand what specific information an analytical technique might provide and to use those techniques properly in tandem to give the fullest answer to a given research question.

1. Introduction

Research regarding exposure and toxicity of nanomaterials (NMs) to humans has been widely studied over the past two decades with much of the research focusing on the question of whether engineered NMs produce adverse outcomes or responses when exposed to various organisms, tissues, and cells (Baek and An, 2011; Zhang et al., 2011; Wang et al., 2016; Notter et al., 2014). Silver is an especially attractive material for use in consumer products because of its antibacterial properties (Kim et al., 2007; Ahmadi et al., 2013; Le Ouay and Stellacci, 2015). AgNPs have been shown to be toxic to zebrafish, algae, and earthworms (Asharani et al., 2008; Navarro et al., 2008; Shoults-Wilson et al., 2011), ionic silver to plankton and fish (Kennedy et al., 2010), and silver chloride (AgCl) to fungi and bacteria (Paulkumar et al., 2013; Trinh et al., 2015), each of which are very common species of silver in AgNPs-containing consumer products (Rogers et al., 2018; Tulve et al., 2015; Benn et al., 2010). These species become important for describing their activity and reactivity in various bodily systems, such as in stomach fluids for ingestible products (Rogers et al., 2012; Mwilu et al., 2013) or against the skin for products designed for wear or disinfection (Larese et al., 2009; Davis et al., 2012; Bianco et al., 2015; Wasukan et al., 2015). The use of AgNPs in textiles for odor control and cleanliness has led to a particularly robust research area on these specific consumer products (Benn and Westerhoff, 2008; Geranio et al., 2009; Stefaniak et al., 2014; Ding et al., 2016). Research on textiles often uses artificial sweat or perspiration to simulate the conditions in which these products would be exposed during use (Kulthong et al., 2010; Yan et al., 2012; Hedberg et al., 2014; Kim et al., 2017; Rovira et al., 2017; Gagnon et al., 2019; von Goetz et al., 2013; Wagener et al., 2016), but there is seemingly no basic assessment of the interaction between AgNPs and different formulations of AS.

Although a comprehensive AS has been designed and tested by Harvey et al. (2010), Midander et al. showed that the release of several heavy metals in simple AS was likely sufficient in determining the bioaccessible portion of the metals when compared with the release in comprehensive AS (Midander et al., 2016). Since human sweat can vary by pH and composition depending on personal and environmental factors (Sato et al., 1989; Shirreffs and Maughan, 1997; Patterson et al., 2000; Baker et al., 2018), the formulations chosen here were ones defined in national and international standards that spanned an appropriate range of pH. Undertaking this study serves to help justify the usage of artificial sweat formulations in research pertaining to silver nanoparticles and comparatively to evaluate the ability of different analytical techniques to quantify the size and speciation of pristine and chemically-altered silver nanoparticles. Further, identifying similarities and differences between methods enables researchers, industry, and/or regulators to determine if a more complex artificial sweat formulations is critical for understanding the changes in NM speciation using Ag as a case study. Demonstrating clear distinctions between national and international methods provides a better format for developing harmonized testing guidelines.

The object of the current study was two-fold. The first objective was to determine how different formulations of AS alter the particle size, aggregation, and chemical speciation for a range of AgNPs of different sizes. The second objective was to compare the data provided by three different techniques for quantifying particle size and two different techniques for determining changes in particle chemistry. In the current study, we utilized four different standardized formulations of AS: the U.S. Consumer Product Safety Commission (CPSC), the International Organization for Standardization: Acidic and Basic Sweat Formulations (ISOa and ISOb, respectively), and the American Association of Textile Chemists and Colorists Acidic Sweat (AATCC). These AS solutions were combined with an ionic silver solution and four different sizes of dispersed polyvinylpyrrolidine (PVP)-coated AgNPs, which were chosen because of their steric stabilization mechanism and the difficulty of isolating environmentally-relevant altered NPs (Gagnon et al., 2019; El Badawy et al., 2010; Tejamaya et al., 2012; Levard et al., 2012; Huynh and Chen, 2011). Samples were pulled from these mixtures at four time intervals for analysis using DLS, UV–Vis spectroscopy, spICP-MS, XAS, and SEM/EDX, to determine how the size and chemical speciation of the AgNPs changes when exposed to sweat. The techniques for quantifying particle size were solution-based techniques which require only dilution of the sample for measurement (DLS, UV–Vis spectroscopy, and spICP-MS). For the solid phase analysis, the solutions were dried under vacuum or nitrogen in an effort to concentrate particles prior to analysis. These techniques have been well studied using AgNPs in various capacities (Mwilu et al., 2013; Paramelle et al., 2014; Yang et al., 2016; Mitrano et al., 2012a; Lombi et al., 2013; Scheckel et al., 2010; Aznar et al., 2017; Amendola et al., 2010), yet this consolidation of techniques has not yet been performed to the authors’ knowledge for AgNPs and this gap in the research is an important one to be processed and filled.

2. Materials and methods

2.1. Sample preparation

PVP-coated silver nanoparticles were purchased as aqueous dispersions from nanoComposix (San Diego, CA) with nominal sizes 20, 50, and 100 nm (AgNPs-20, −50, and −100) at a nominal concentration of 20 mg/L Ag from the NanoXact line. Additional PVP-coated silver nanoparticles were purchased as lyophilized powders from the National Institute for Standards and Technology (NIST; Gaithersburg, MD) with nominal size 75 nm (AgNPs-75). The 75 nm particles were resuspended in 100 mL of ultrapure water (ASTM Type 1) from a Super-Q Water Purification System from MilliporeSigma (Burlington, MA) to a nominal concentration of 20 mg/L Ag. An ionic silver solution (AgIon) of nominal concentration 20 mg/L Ag was prepared by dissolving 3.1 mg of silver nitrate (AgNO₃) from Thermo Fisher Scientific (Waltham, MA) in 100 mL of ultrapure water with 2 drops of concentrated trace metal grade nitric acid (HNO₃) from Thermo Fisher added for stability. All silver solutions/dispersions were stored in their stock bottles or high-density polyethylene (HDPE) bottles wrapped in aluminum foil at 4 ± 2 °C and used within nine months of purchase or dispersion.

AS mixtures were selected and prepared according to standard guidelines using sodium chloride (NaCl), sodium hydrogen phosphate, anhydrous (Na₂HPO₄, 99%), sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O, 99%), l-histidine monohydrochloride monohydrate (l-histidine HCl·H₂O, 98%), dl-lactic acid (85%), and sodium hydroxide (NaOH) from Thermo Fisher (Table 1).

Table 1.

Chemical composition and pH specifications for utilized artificial sweats.

AS	CPSC	ISOa	ISOb	AATCC
NaCl	0.9%	0.5%	0.5%	1.0%
L-histine HCL·H2O	-	0.05%	0.05%	0.025%
NaH2PO4·2H2O	-	0.22%	0.25%	-
Na2HPO4	-	-	-	0.1%
DL-lactic acid	-	-	-	0.1%
pH	5.8 ± 0.3a	5.5 ± 0.2	8.0 ± 0.2	4.3 ± 0.2

^aNot specified; value is the average of measurements from 10 solutions.

AS(CPSC) was prepared according to “Experimental Methodology for the Collection and Analysis of Surrogate and Hand Wipes on CCA-Treated Wood” from the Consumer Product Safety Commission (CPSC). AS(ISOa) was prepared according to the acidic formulation in “ISO 105-E04:2013 Textiles – Tests for Colour Fastness – Part E04: Colour Fastness to Perspiration” from the International Organization for Standardization (ISO). AS(ISOb) was prepared according to the basic formulation in “ISO 105-E04:2013 Textiles – Tests for Colour Fastness – Part E04: Colour Fastness to Perspiration” from the ISO. AS (AATCC) was prepared according to “AATCC 15–2013 Colorfastness to Perspiration” from the American Association of Textile Chemists and Colorists (AATCC). All AS solutions were stored in HDPE bottles at room temperature (RT; 19–22 °C) and were prepared fresh daily.

1:1 mixtures from a minimum 1.0 mL of silver and 1.0 mL of AS were prepared for each possible silver solution/dispersion (AgIon, AgNPs-20, AgNPs-50, AgNPs-75, AgNPs-100) with water and artificial sweat (AS(CPSC), AS(ISOa), AS(ISOb), AS (AATCC)) in polypropylene (PP) centrifuge tubes wrapped in aluminum foil to minimize air and light exposure. These ratios were chosen to have a high enough concentration of silver to run all analytical techniques while still retaining relevancy for the ratio of silver and sweat that can be experienced by an individual when accounting for the amount of silver present in consumer products (Benn et al., 2010; Gagnon et al., 2019; Wagener et al., 2016; Quadros et al., 2013) and sweat rates in humans (Kilding et al., 2009; Mehnert et al., 2002; Shirreffs et al., 2005). The mixtures were placed on a Model E6010 fixed speed reciprocal shaker from Eberbach Corporation (Ann Arbor, MI) and agitated for 15 min at RT with a speed of 180 osc/min. After shaking, a minimum of 0.5 mL of each mixture was removed by adjustable pipette from each tube and transferred to polystyrene (PS) round-bottom tubes and diluted 1:10 with ultrapure water. The silver and sweat mixtures were stored at RT in a laboratory cabinet and volume was pulled from each additionally after 1, 4, and 7 days.

2.2. pH, DLS, zeta potential, and UV–Vis measurements

The pH of the 1:10 dilutions was measured using an accumet XL25 Dual Channel pH/Ion Meter with an accumet Ag/AgCl pH combination electrode from Thermo Fisher, which was calibrated daily using reference standards of pH 4, 7, and 10 also from Thermo Fisher. pH was recorded when the value had stabilized for at least 3 s.

After measuring the pH, each dilution was transferred into a PS cuvette for dynamic light scattering (DLS) measurement using the Zetasizer Nano ZS from Malvern Panalytical (Malvern, UK) equipped with a 50 mW at 532 nm laser and a backscatter collection angle of 173°. Reported values are the average of five replicates obtained from a minimum of 10 scans of 10 s each. Quality control was assessed using the DTS1235 Zeta potential transfer standard (ZPTS) from Malvern with a hydrodynamic diameter (HDD) of 360 ± 36 nm (based on repeated measurements).

After measuring the HDD, each dilution was then transferred to a tapered quartz cuvette for UV–Vis absorption measurement using the UV-2700 UV–Vis Spectrophotometer from Shimadzu Scientific Instruments (Columbia, MD). Measurements were background corrected with a water-filled second quartz cuvette over a range of 200.00–900.00 nm (light source changeover at 323.0 nm) using a fast scan speed, sampling interval of 1.0 nm, slit width of 5.0 nm, and accumulation time of 0.1 s. Quality control was assessed using the ZPTS with a maximum absorbance wavelength of 206 nm (based on repeated measurements). Plots and statistical analyses were prepared or performed using OriginPro, Version 2019 from OriginLab Corporation (Northampton, MA).

2.3. spICP-MS measurements

Each 1:10 dilution of the mixtures was diluted further 1:10,000 with ultrapure water to a minimum of 5 mL for spICP-MS measurement using the Single Nanoparticle Application Module with the Agilent 7900 ICP-MS and ASX-500 Series Autosampler from Agilent Technologies. (Santa Clara, CA). A 100-ppm silver standard in 2% HNO₃ from Elemental Scientific (Omaha, NE) was diluted to 1 ppb with ultrapure water for calibration of the silver ionic response and a secondary source silver standard was prepared in the same way for calibration verification. 1% HNO₃ dilutions were also prepared but were found to be less consistent between primary and secondary solutions and thus were not used. A 1000-ppm gold standard in 2% hydrochloric acid (HCl) from Elemental Scientific was diluted to 1 ppb with ultrapure water for calibration of the gold ionic response. 1% HCl dilutions were prepared using trace metal grade HCl from Fisher but were found to be less consistent between batches and thus were not used. Standard reference material (SRM) 8013 gold nanoparticles with certified size of 56.0 ± 0.5 nm (by transmission electron microscopy, TEM) from NIST were used for determination of the nebulization efficiency based on size at a nominal concentration of 50 ppt in ultrapure water. Quality control was assessed using 1:20,000 dilutions in ultrapure water of the AgNPs-50 stock dispersion, with a manufacturer reported size of 47 ± 4 nm by TEM, and the AgNPs-75 stock dispersion, with a certified size of 74.6 ± 3.8 nm by TEM and a reported size of 69.2 ± 0.9 nm by spICP-MS. All solutions/dispersions were prepared fresh daily.

The instrument was tuned daily using a 1 ppb solution of Ce, Co, Li, Mg, Tl, and Y in 2% HNO₃ from Agilent; no internal standard was used. The glassware used consisted of 0.4 mL/min flow concentric glass MicroMist nebulizer, quartz Scott-style spray chamber, straight quartz connector, and quartz torch with 2.5 mm injector from Glass Expansion (Pocasset, MA). Instrument settings consisted of 1500 W RF power, 15.00 L/min plasma gas flow rate, 1.1 L/min carrier gas flow rate, 0.9 L/min auxiliary gas flow rate, and 2 °C spray chamber temperature. Initial sample flow rates ranged from 0.331 to 0.362 mL/min measured with a TruFlo Sample Monitor from Glass Expansion. Sample data was collected for 30 s with an acquisition time of 100 μs and peak integration to determine the mass in each particle signal. Consecutive rinses of 5% HNO₃/5% HCl, 5% HNO₃, and ultrapure water were run for 60 s each following every sample/QC measurement; additionally, blank washes of ultrapure water were run between samples, to ensure there was no significant carryover of ionic or nanoparticulate silver. The background equivalent diameter for all samples was typically between 5.5 and 8.5 nm with an ionic cutoff between 19 and 21 nm where overlap did not occur.

2.4. XAS measurements

Quarter-inch diameter holes were drilled in a piece of quarter-inch thick polyvinyl chloride (PVC) sheet. One 25-nm pore size mixed cellulose esters membrane filter from MilliporeSigma was taped over each hole using Kapton polyimide tape from Thermo Fisher wide enough to secure the entire filter to the PVC sheet. The PVC sheet was then flipped over to present filter paper wells. 1:1 mixtures of 0.5 mL of each silver solution/dispersion and 0.5 mL of water and each AS were prepared by adjustable pipette in PP centrifuge tubes wrapped in aluminum foil. The mixtures were placed on a Model E6010 fixed speed reciprocal shaker and agitated for 15 min at RT with a speed of 180 osc/min. After shaking, 0.25 mL of each mixture was removed by adjustable pipette from each tube and dropped into one of the empty wells. The loaded PVC sheet was then loosely covered with a laboratory surface protector sheet and put into a glovebox antechamber. The antechamber was evacuated until the samples had completely dried onto the filters. After removing the PVC sheet from the antechamber, each filter was removed and covered with a second piece of Kapton tape to seal it within tape. The silver and sweat mixtures were stored at RT in a laboratory cabinet and an additional 0.25 mL was pulled from each tube and dried onto filters again after 1 and 7 days. Samples were kept protected from light as much as possible during preparation and before analysis.

The taped samples provided a localized area of dried material that were sectioned into three pieces and overlaid on top of each other before sealing within another two pieces of Kapton tape. These samples were measured for Ag K-edge XAS at the Materials Research Collaborative Access Team (MRCAT) 10-ID beamline at the Advanced Photon Source (APS) operated by the U.S. Department of Energy (DOE) at Argonne National Laboratory (Lemont, IL) Segre et al., 2000. The energy of the incident X-rays was scanned using a Si (111) monochromator consisting of a cryogenically-cooled first crystal and a 250-mm long second crystal. A Pt-coated flat harmonic rejection mirror was moved into the beam and a beam size of 400 μm by 400 μm was used. Incident beam energy was calibrated to the first derivative inflection point of the absorption edge (25514 eV) of a silver foil reference standard. 5 step scans of each sample were collected from −200 eV to 10 k relative to the Ag K-edge. Spectra for the Ag foil, along with silver oxide (Ag₂O), silver chloride (AgCl), silver phosphate (Ag₃PO₄), and silver sulfide (Ag₂S) from Thermo Fisher, were measured for comparison; all standards except the foil were diluted with PVP from Thermo Fisher, then pressed into 13-mm diameter pellets and sealed in Kapton tape. Spectra were collected in both transmission mode, using an aluminum spectroscopy ion chamber, and fluorescence mode, using a Lytle-type fluorescence detector. Merging of the measured spectra were first performed using LARCH Newville, 2013, followed by background removal, normalization, and rebinning using ATHENA Ravel and Newville, 2005. Linear combination fitting (LCF) was performed with the Ag standards on the normalized spectra of the samples using ATHENA with a fitting range of −30 to 100 eV relative to the absorption edge. Principal component analysis (PCA) was performed to indicate that at most three standards were required, so best fits from combinatorics were selected by comparison of the resulting R-factor and reduced chi-square, with priority given to results using two standards instead of three.). Linear combination fitting (LCF) was performed with the Ag standards on the normalized spectra of the samples using ATHENA with a fitting range of −30 to 100 eV relative to the absorption edge. Principal component analysis (PCA) was performed to indicate that at most three standards were required, so best fits from combinatorics were selected by comparison of the resulting R-factor and reduced chi-square, with priority given to results using two standards instead of three.

3. Results and discussion

3.1. Pristine materials

The sizing results for the pristine AgNPs dispersed in water, for the three different techniques, are presented in Table 2 and Fig. 1. Although SEM was conducted for imaging purposes the instrument was optimized for the collection of EDX spectra and was not used for measuring particle or aggregate sizes.

Table 2.

Equivalent diameters from DLS with polydispersity index (PDI), UV–Vis spectroscopy, and spICP-MS of nanoparticles in mixtures of water and nanoparticulate silver

H2O		Day 0	Day 1	Day 4	Day 7
AgNps-20	deq (nm, DLS)	180 ± 12	161 ± 11	147 ± 36	157 ± 1
	PDI (DLS)	0.21 ± 0.03	0.42 ± 0.06	0.42 ± 0.07	0.24 ± 0.01
	deq (nm, Uv-Vis)	8	18	20	15
	deq (nm, spICP-MS)	34a	34a	34a	24b
AgNPs-50	deq (nm, DLS)	60 ± 1	62 ± 1	63 ± 1	62 ± 1
	PDI (DLS)	0.15 ± 0.02	0.13 ± 0.01	0.12 ± 0.02	0.14 ± 0.01
	deq (nm, Uv-Vis)	60	58	59	57
	deq (nm, spICP-MS)	42	41	39	39
AgNPs-75	deq (nm, DLS)	103 ± 1	100 ± 1	101 ± 1	104 ± 1
	PDI (DLS)	0.05 ± 0.02	0.06 ± 0.02	0.05 ± 0.02	0.05 ± 0.01
	deq (nm, Uv-Vis)	77	77	77	74
	deq (nm, spICP-MS)	54	44	34	40
AgNPs-100	deq (nm, DLS)	101 ± 1	105 ± 1	105 ± 1	109 ± 1
	PDI (DLS)	0.07 ± 0.01	0.07 ± 0.01	0.04 ± 0.01	0.06 ± 0.02
	deq (nm, Uv-Vis)	89	90	92	90
	deq (nm, spICP-MS)	71	75	67	77

^aParticle peak signals overlap with the ionic peak signals.

^bFew particle signals were present.

Fig. 1.

Change in equivalent diameter from DLS, UV–Vis spectroscopy, and spICP-MS over 7 days for mixtures of water and nanoparticulate silver. Standard deviation bars for DLS measurements are omitted for clarity.

DLS measurements show that all but the AgNPs-20 were stable over the 7-day period. The DLS results for the 20 nm particles indicated a mean particle diameter between 180 and 140 nm over the 7 days indicating aggregation of the particles. The DLS results were unable to distinguish a difference in the size between the 75 and 100 nm AgNPs with an average measured value of 102 and 105 nm for the 75 and 100 nm particles, respectively. Sizing results based on surface plasmon resonance (SPR, calculated using the sizes and simulated peak maxima for spherical citrate-capped AgNPs by Paramelle et al. (2014)) were able to distinguish the four different particle sizes and the measured values were very close to the reported values (Table 2). As with the DLS data, particle size for the 50, 75, and 100 nm particles were stable over the 7-day period. Notably the AgNPs-20 diameters were much smaller than those from DLS, with all calculated results much closer to the nominal values of the nanoparticles. Nanoparticle aggregates would expect to have a red-shifted spectrum (Blakey et al., 2013), but since no higher wavelength peak was measured in the full 200–900 nm range, it could be possible that a few larger aggregates in the AgNPs-20 samples dominate scattering in the DLS and a smaller fraction of individual particles is solely responsible for the UV–Vis absorbance. Single particle ICP-MS measurements were unable to clearly and consistently differentiate the particulate signals from the ionic signals for the AgNPs-20 samples. The values that were calculated are based on the extraction of the particle data from an overlapping ionic signal that will result in an over estimation of the equivalent particle diameter. The lack of an appreciable large mass signal in the 20 nm particles provides additional evidence suggesting the DLS results are due to small number of larger aggregates dominating the scattering signal. The diameters obtained for the AgNPs-75 samples were smaller than expected and inconsistent over the 7 days, leading to the mean having no statistically significant difference (Tukey’s means comparison; probability different, 0.87) from the AgNPs-50 samples. Only the 100 nm AgNPs had a clearly discernible equivalent diameter that differed from the three other AgNPs (Fig. 1).

The LCF results for the speciation of the AgNPs dispersed in water are presented in Fig. S1 and Table S1. The XANES data showed evidence of Ag⁰ and AgCl immediately after suspending the NPs in ultrapure water. For the 50, 75, and 100 nm particles, Ag⁰ was the dominant phase present. However, for the 20 nm particles the LCF results indicated that approximately 80% of the Ag was in the form of AgCl. Results from the SEM/EDX analysis also indicated that S was present in the 20 and 50 nm particles, that Cl and S were present in the 75 nm, and Cl was present in the 100 nm particles (Figs. S2–S9). The EDX results agree reasonably well with the XANES data, but there was no evidence of a significant Ag₂S phase present in any of the NPs. The SEM images do not show evidence of individual particles for the 20 nm (Fig. S2), but individual particles are observable for the other three particle sizes (Figures S4, S6, and S8).

3.2. Artificial sweats

Two major differences separate the artificial sweats from each other, chemical composition and pH, as described in Table 1. In terms of chemical composition, it was expected that interaction with the silver would be entirely driven by NaCl or the sodium phosphate species, since AgCl and Ag₃PO₄ are thermodynamically favored compounds and the anions are present in high concentrations (900:1 to 1900:1 chloride to silver ratio, 75:1 to 170:1 phosphate to silver ratio). The organic compounds were not expected to significantly influence any reactions because of their lower concentrations in the solutions.

Box plots showing the pH for each of the solutions with and without AgNPs over 7 days are provided in Fig. 2. Analysis of variance within and between AS indicated there was no statistical difference in pH over the 7-day period for each AS. There were statistical differences between groups due to the initial adjustment of pH (ISOb and AATCC differed significantly from each other and the others). Minor trends were observable for pH of the AS when no silver is present, which shifted the pH to more neutral values. The addition of AgNPs stabilized the pH over the course of 7 days. Numerical values for the pH measurements are provided in the Supporting Information (Table S2).

Fig. 2.

Half box plots of pH change over 7 days for mixtures of water or AS with nanoparticulate silver. Colors are black for water or AS with no silver, purple for AgNPs-20, blue for AgNPs-50, green for AgNPs-75, and yellow for AgNPs-100.

3.3. Artificial sweat nanoparticle mixtures

DLS measurements taken over 7 days for all the AS + AgNPs mixtures are shown in Fig. 3 and a table containing the numerical values, where available, for the equivalent diameters obtained using DLS, UV–Vis spectroscopy, and spICP-MS is provided in the Supporting Information (Table S3). Several general trends from the DLS data were apparent. First, the hydrodynamic diameter (HDD) for the AgNPs increased with time for the acidic AS formulations, and second, the largest increases in HDD occurred for the AS formulations with the highest concentrations of chloride (CPSC and AATCC). Finally, the 75 nm particles were the most stable AgNPs over the 7 days for the 4 different AS formulations.

Fig. 3.

Change in equivalent diameter from DLS, UV–Vis, and spICP-MS over 7 days for mixtures of water or AS with nanoparticulate silver. “NR” indicates AgNP suspension with either no absorbance peak or a peak that was <390 or >500 nm. Green and purple circles surrounding the spICP-MS data indicate that few particle signals were present (green) or particle and ionic signal peaks were overlapping (purple).

UV–Vis spectroscopy measurements taken over 7 days for all of the AS + AgNPs mixtures are also presented in Fig. 3. Many of the samples did not exhibit a detectable plasmon resonance peak that would be associated with AgNPs. The peak maxima and spectra obtained for all mixtures are provided in the Supporting Information (Table S4, Figs. S10–S14). The UV–Vis data provide a much different picture than DLS results, but it is important to remember that the SPR absorbance is dependent on the presence of Ag metal at the surface and particles less than ~100 nm (Zook et al., 2011). The SPR peak was absent from the 20, 50 and 100 nm AgNPs and all but one of the acidic AS for the 75 nm (AATCC). The loss of the SPR peak in the acidic AS at the initial time zero indicates an immediate interaction with the AgNP surfaces to produce some form of silver salt species that does not occur under more basic conditions. For the AS (AATCC)+AgNPs-75 mixture, there were minimal shifts in the maximum peak intensity (Fig. S14) indicating no change in the aggregation of the particles over the 7 days, in contrast to the results from the DLS data.

SpICP-MS measurements taken over 7 days for all the AS + AgNPs mixtures are shown in Fig. 3 with the equivalent diameters determined using the bulk density of silver. Table S3 additionally provides diameters calculated using the bulk density and mass fraction of silver chloride, which are significantly larger than those calculated with silver, but considering the UV–Vis and DLS results and the speciation presented below, the results in Fig. 3 are likely undervalued but indeterminable by how much with using spICP-MS as a standalone analysis. The total particle counts and accompanying concentration, as well as the signal distribution and particle size distribution charts, for each mixture are provided in the Supporting Information (Table S5, Figs. S15–S39). The results obtained using spICP-MS had many of the same groupings that were identified with UV–Vis spectroscopy and DLS, with some limitations identified for the technique. Particle size values obtained for mixtures containing AgNPs-20 had issues with overlapping particle and ionic peak signals, actual particle diameters in these mixtures are likely smaller than reported here. The mixtures with AgNPs-100 had low particle counts in most of the samples because when the particles begin to react and aggregate as described in previous techniques, there are fewer numbers of particles contributing to the distribution and a reliable and robust equivalent diameter cannot be calculated. For the AgNPs-50 mixtures, only the AS(ISOb) had sufficient particle counts over all 7 days for analysis. By the end of the 7 days, there were low count rates within the acidic AS, while the AgNPs-50 for AS (AATCC) exhibited low count rates with the first measurement. Since the other techniques indicate possible aggregation, larger particles might have been present, though it is unclear what the upper bound for particle size is for spICP-MS measurements besides the physical limitations of 140 μm for the nebulizer inner diameter, 30 μm for the primary aerosol, and 10–20 μm for the tertiary aerosol in the sample introduction process (Todolí et al., 2000; Mora et al., 1997; Todolí and Mermet, 2006). All of the AgNPs-75 mixtures were very consistent between each other and over the 7 days and did not experience size change or a detrimental decrease in particle counts. Research using spICP-MS for the analysis of altered or environmental samples has been increasingly geared towards processing samples off- or online with spICP-MS, but further progress is still needed in these areas (Yang et al., 2016; António et al., 2015; Mitrano et al., 2012b, 2014; Tuoriniemi et al., 2012; Bolea-Fernandez et al., 2019; Clark et al., 2019; Correia et al., 2018).

3.4. Speciation and elemental analysis

The Demeter, ATHENA Software Package (Ravel and Newville, 2005) was used to conduct a principal component analysis (PCA) to determine the minimum number of components required to interpret the sample data spectra which included each NP size, time point, and artificial sweat formulation. Results from the PCA indicated three components were required to describe 99.99% of the variance between spectra. For speciation and elemental analysis, five species were considered for the mixtures being studied: elemental Ag, Ag₂O (from oxidized material through light and/or air), AgCl and Ag₃PO₄ (from reaction with the anions in the artificial sweats), and Ag₂S (most thermodynamically stable silver product) (Levard et al., 2012). Target transforms of Ag reference compounds indicated the three most probable species present in the sample were Ag⁰, AgCl, and Ag₂S. The three reference compounds were used to successfully fit all of the sample spectra.

Fig. 4 presents a summary plot of the results, and a table of the full LCF results along with the fits are in the Supporting Information (Table S7; Figs. S40–S43). After 7 days the quality of the XANES spectra collected was significantly reduced for the AS in comparison to day 0 and 1 and was more pronounced for the smaller particles (Figs. S40–S43). The reduced quality of the spectra likely indicates a reduction in the number of particles that were retained on filters due to the potential dissolution, transformation, and settling of AgNPs over time. The impact of time and AS formulation on Ag speciation was determined by comparing the results of the LCF analysis of the AgNPs suspended in water to those suspended in the AS, and across the 7-day time period. A change in the relative abundance of a specific Ag species by greater than 10% for at least one of the three species over 7 days or from the species distribution in water was considered significant (error bars in Fig. 4 represent a ±10% error). A difference in 10% was chosen as a conservative estimate of the uncertainty in the XANES LCF analysis. Differences that exceed 10% are identified by the presence of a green dot in Fig. 4. The ability of the different AS formulations to alter the speciation of Ag may be assessed by comparing the speciation of Ag suspended in water and the different AS a function of size and time. The size of the AgNPs was related to the degree of chemical transformation with the 20 nm particles undergoing the greatest change in speciation and the 100 nm particles the least when comparing the speciation in AS and water. After less than a day, regardless of the matrix, Ag₂S was identified as a component with at least a 10% relative abundance. The appearance of the Ag₂S was accompanied most often by a greater decrease in the amount of Ag⁰ compared to AgCl present. There were minimal changes in the relative of species in the 100 nm particles over the 7-day time period; there is an increase in the amount of AgCl present in the AATCC, but the difference is less than 10% when compared to the AgNPs suspended in water. The formation of Ag₂S was also greatest for the 20 nm particles. For the 50 and 75 nm particles, the ISOa formulation exhibited an Ag species distribution most similar to water. The other three AS formulations resulted in the formation of Ag₂S or significant differences in the ratio of Ag⁰ to AgCl present. The AS (AATCC) formulation resulted in the greatest change in Ag speciation over time and compared with the water suspension. The presence of AgCl in water dispersed samples makes it challenging to assess if one specific AS formulation was more aggressive than the others. Focusing only on changes in the abundance of Ag⁰ in the AS formulations and comparing that with the abundance of Ag⁰ in the water dispersion and over time, changes in the abundance of Ag⁰ occurred most often the AS(CPSC) and AS (AATCC) formulations for the 20, 50, and 75 nm particles, indicating that the most important factor governing changes in Ag⁰ is the Cl concentration.

Fig. 4.

Relative abundance of different Ag species for mixtures of water and AS over a 7-day period. The error bars represent a 10% error in the relative abundance of each species. A green circle in the plot indicates at least a 10% difference in the relative abundance of one of the Ag species over 7 days.

The EDX results for thechemically-altered AgNPs exposed to AS for 1 day were compared to the results from the AgNPs dispersed in water to determine if there was an enrichment in the amount of Cl or S present. An increase in the value of the ratio for the AgNPs suspended in AS would indicate an enrichment with respect to either Cl or S (Table 3). The results did not correlate well with the XANES data. The 20 and 50 nm particles showed an enrichment in the amount of Cl present in agreement with the chemical transformation of Ag⁰ to AgCl. There was no indication of S enrichment for any of the particles and there was a decrease in the amount of Cl present in the 75 and 100 nm particles. The lack of good agreement between the two techniques for characterizing changes in chemical speciation/composition of the solid phase is related to the particle specific analysis of EDX. The XANES data is a survey of the bulk composition of all particles present, while the EDX data is representative only of the particles where a spectrum was collected.

Table 3.

Elemental ratios of Cl and S to Ag from EDX spectra obtained from SEM analysis.

AgNP	Matrix	Day	Cl:Ag	S:Ag
20 nm	H2O	0	0.000	0.048
20 nm	CPSC	1	0.017	0.003
50 nm	H2O	0	0.002	0.001
50nm	ISOa	1	0.050	0.000
75nm	H2O	0	0.012	0.021
75nm	AATCC	1	0.000	0.000
100nm	H2O	0	0.005	0.000
100nm	ISOb	1	0.004	0.000

3.5. Transformation of AgNPs

The extent of particle transformation for the 4 different particles sizes was greatest for the 20 and 50 nm particles. For the 20 nm particles, there was an increase in the DLS hydrodynamic diameter, a low number of particles identified with spICP-MS and a significant increase in the presence and abundance of Ag₂S in all of the AS matrices. The reduced quality of the XANES data after 7 days, increased abundance of Ag salts, and a low number of particles for spICP-MS indicate that there was significant dissolution, transformation, and settling of the particles and that the elevated pH of the AS(ISOb) formulation inhibited formation of Ag₂S (Figs. 2, 3, and S40–S43; Tables S3, S5, and S7). The extent of transformation for the 50 nm particles was not as extensive as for the 20 nm. Increases in the hydrodynamic diameter and indications of few particles or overlapping peaks from the spICP-MS are well correlated significant reductions in the abundance of Ag⁰ in the CPSC and AATCC (Fig. 3, Fig. 4). The data for the 75 nm particle size was stable throughout the 7 days even though the XANES data indicated significant changes in the Ag speciation for three of the AS formulations (Fig. 4).

AgNPs exposed to the AS(CPSC) and AS (AATCC) formulations experienced the greatest change in measured particle size based on hydrodynamic diameter and a reduction in the total number of particles counted over the seven days (Fig. 3 and Table S5). The greatest chemical transformation in the AgNPs was associated with CPSC, ISOb, and AATCC formulations based on the LCF results. As previously noted, the CPSC and AATCC have a significantly higher concentration of NaCl compared with the ISO formulations (Table 1) indicating that increased Cl is related to increased transformation of AgNPs.

4. Environmental impact

The results from the water mixtures displayed great invariability in the equivalent diameters using all techniques over the course of a seven-day timespan. Since the solutions were protected from light and air between sampling days, this is unsurprising and gave a good comparison for the results with the artificial sweats. Only the AgNPs-20, which display large aggregates in SEM and DLS, but which are detected below or merged with the ionic signal peak in spICP-MS, are not particularly useful to this analysis and would need further study to validate the results noticed with the larger particles.

The basicity of the mixtures made with AS(ISOb) provided results that were similar to those in water. Besides for a decrease in particle count and equivalent size for the AgNPs-100 mixture using spICP-MS, all other measurements were consistent over seven days for the larger set of particles. From the XAS and SEM/EDX results, as well as the presence of peaks in the UV–Vis spectra, the composition of these nanoparticles is determined to be primarily elemental silver. This AS helps display that pH plays a very strategic role in the transformation of AgNPs, more so than chemical composition, and that those individuals with basic perspiration could experience a different exposure than people with more acidic sweat. The presence and greater quantity of nanoparticles in basic AS is supported by previous results seen by von Goetz et al. and Wagener et al. (von Goetz et al., 2013; Wagener et al., 2016). The results from the acidic sweat mixtures of AS(CPSC), AS(ISOa), and AS (AATCC) were very similar to each other. For the AgNPs-20, −50, and −100 mixtures, the DLS equivalent diameters increased over seven days, the particle count and peak signals went away quickly in the spICP-MS measurements, and no peaks that could be correlated to AgNPs were present in the UV–Vis spectra. The presence of chlorine in the available EDX spectra and LCF fittings for the XAS spectra that indicate primarily elemental Ag, along with the lack of peaks in the UV–Vis spectra, support the conclusion that the acidity promotes the oxidation of Ag (0) to Ag(I) and formation of a silver chloride shell around these nanoparticles. The only size-related differences occurred in these mixtures because of how the AgNPs-75 mixtures did not follow the trends observed with the other nanoparticles. The AgNPs-75 solutions saw no change in the DLS and spICP-MS measurements over seven days, and the AS(CPSC) and AS(ISOa) mixtures had no UV–Vis peaks, though the AS (AATCC) mixtures did. Rather than an effect related to the size of these particles, since the AgNPs-50 and −100 samples behaved similarly, our conclusion is that there is something different in the preparation process or surface chemistry of the AgNPs-75 that is not fully represented in this work. Lyophilization and resuspension in water of the other nanoparticle stock solutions would be a way of narrowing down the possible reasons for the difference in reactivity.

Ultimately, the research presented here shows the great necessity of using different techniques to characterize the change in nanoparticle systems, since one method cannot provide all of the information. DLS is a useful broad-spectrum technique but lacks any element specificity and can be complicated by adsorbed species in environmental samples. UV–Vis spectroscopy is not useable with silver salts except to support an argument of shell formation. SpICP-MS makes assumptions that are very limiting in the results, and although it can process rather dilute samples, it also takes the longest time to process a set of samples. EDX is limited to elemental compositions unless assumptions are made to correlate to actual speciation and XAS cannot fully differentiate core-shell possibilities and struggles with samples of lower concentration. By using even two or three of these techniques in conjunction with one another, a better picture about the fate and transformation of nanoparticles can be determined and is necessary even with the relatively simple systems presented herein.

Abbreviations

AgNPs

silver nanoparticles

PVP

Polyvinylpyrrolidone

AS

artificial sweat

DLS

dynamic light scattering

HDD

hydrodynamic diameter

PDI

polydispersity index

SPR

surface plasmon resonance

spICP-MS

single particle inductively coupled plasma-mass spectrometry

XAS

X-ray absorption spectroscopy

XANES

X-ray absorption near edge spectroscopy

LCF

linear combination fitting

PCA

principal components analysis

SEM

scanning electron microscopy

EDX

energy dispersive X-ray analysis

NMs

nanomaterials

CPSC

Consumer Product Safety Commission

ISO

International Organization for Standardization

AATCC

American Association of Textile Chemists and Colorists