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Our knowledge of uptake, toxicity and detoxification mechanisms as related to nanoparticles' (NPs') characteristics remains incomplete. Here we combine the analytical power of three advanced techniques to study the cellular binding and uptake and the intracellular transformation of silver nanoparticles (AgNPs): single-particle inductively coupled mass spectrometry, mass cytometry and synchrotron X-ray absorption spectrometry. Our results show that although intracellular and extracellularly bound AgNPs undergo major transformation depending on their primary size and surface coating, intracellular Ag in 24 h AgNP-exposed human lymphocytes exists in nanoparticulate form. Biotransformation of AgNPs is dominated by sulfidation, which can be viewed as one of the cellular detoxification pathways for Ag. These results also show that the toxicity of AgNPs is primarily driven by internalized Ag. In fact, when toxicity thresholds are expressed as the intracellular mass of Ag per cell, differences in toxicity between NPs of different coatings and sizes are minimized. The analytical approach developed here has broad applicability in different systems where the aim is to understand and quantify cell-NP interactions and biotransformation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Material flow analysis shows that soil is a key repository for silver (Ag) from (nano)silver-functionalized consumer products, but the potential effects of Ag toxicity, via Ag + release, on soil microbial communities and their ecosystem services remains largely unknown. We examined the responses of multiple microbial biomarkers to increasing Ag + doses (nine concentrations, 0-2000 mg kg -1 ) in nine different soils representing a wide range of soil properties. Analyses included substrate-induced microbial respiration, nine different soil enzyme activities, and quantification of bacterial 16S-rRNA (SSU) and fungal intergenic spacer (ITS) copies. The resulting half-maximal effective concentrations (EC 50 ) for Ag ranged from ∼1 to >500 mg kg -1 and showed soil-specific responses, including some hormesis-type responses. Carbon cycle-associated enzyme activities (e.g., cellobiohydrolase, xylosidase, and α/β-glucosidase) responded similarly to Ag. Sulfatase and leucine-aminopeptidase activities (linked to the sulfur and nitrogen cycles) were the most sensitive to Ag. Total organic carbon, and to a lesser extent pH, were identified as potentially useful response predictors, but only for some biomarkers; this reflects the complexity of soil Ag chemistry. Our results show Ag toxicity is highly dependent on soil characteristics and the specific microbial parameter under investigation, but end point redundancies also indicated that representative parameters for key microbial functions can be identified for risk assessment purposes. Sulfatase activity may be an important Ag toxicity biomarker; its response was highly sensitive and not correlated with that of other biomarkers.

With advances in nanotechnology, particles with various size, shape, surface chemistry, and composition can be easily produced. Nano- and microparticles have been extensively explored in many industrial and clinical applications. Ensuring that the particles themselves are not possessing toxic effects to the biological system is of paramount importance. This paper describes a proof of concept method, in which a microfluidic system is used in conjunction with a cell microarray technique aiming to streamline the analysis of particle-cell interaction in a high throughput manner. Polymeric microparticles, with different particle surface functionalities, were first used to investigate the efficiency of particle-cell adhesion under dynamic flow. Silver nanoparticles (AgNPs, 10 nm in diameter) perfused at different concentrations (0 to 20 μg/mL) in parallel streams over the cell microarray exhibited a higher toxicity compared to the static culture in the 96-well-plate format. This developed microfluidic system can be easily scaled up to accommodate a larger number of microchannels for high throughput analysis of the potential toxicity of a wide range of particles in a single experiment.

Quantification of cell-associated nanoparticles (NPs) is a paramount question in both nanomedicine and nanotoxicology. Inductively coupled plasma mass spectrometry is a well-established method to resolve cell-associated (metal) NPs in bulk cell populations, however, such analysis at single cell level remains a challenge. Here we used mass cytometry, a technique that combines single cell analysis and time-of-flight mass spectrometry, to quantitatively analyze extra- and intracellular silver (Ag) in individual Ag NP exposed human T-lymphocytes. The results revealed significant population heterogeneity: for example, in lymphocytes exposed to 3 μg of 30 nm branched polyethylene imine coated Ag NPs/mL the extracellularly bound Ag varied from 79 to 560 fg and cellular uptake from 17 to 121 fg. Similar amplitude of heterogeneity was observed in cells exposed to various doses of Ag NPs with other sizes and surface coatings, demonstrating the importance of single cell analysis when studying NP-cell interactions. Although mass cytometry has some shortcomings such as inability to analyze potential transformation or dissolution of NPs in cells, we consider this method as the most promising for quantitative assessment of cell-NP interaction at single cell level.

While the importance of nanoparticle (NP) characterization under relevant test conditions is widely recognized in nanotoxicology, few studies monitor NPs behavior in the presence of exposed organisms. Here we studied the behavior of nine types of silver nanoparticles (AgNPs) during the 48 h algal toxicity test. In particular, we investigated NP aggregation and dissolution by time-resolved inductively coupled plasma mass spectrometry and ultrafiltration and performed mass balance measurements to study the distribution of Ag in the test system. We also determined the amount of extra- and intracellular Ag by chemically etching AgNPs on the surface of algal cells and used dark field microscopy for their imaging. We observed that positively charged branched polyethilenimine (bPEI)-coated AgNPs tend to aggregate in the presence of algae and interact with test vessels and algal cells, while citrate-coated AgNPs have a tendency to dissolve. On the other hand, with large variation of half-maximum effective concentration (EC50) across tested NPs (5.4 to 300 ngAg mL -1 ), Ag internalized by the algal cells at EC50 was similar (0.8 to 3.6 ngAg mL -1 ) for all AgNP types. These data show that while sorption to the vessels, dissolution, and aggregation impact on the distribution of AgNPs in the test system and on interactions with algal cells, AgNP toxicity is strongly correlated with the NP-cell surface interaction and internalization of Ag.

The widespread use of silver nanoparticles (Ag-NPs) results in their movement into wastewater treatment facilities and subsequently to agricultural soils via application of contaminated sludge. On-route, the chemical properties of Ag may change, and further alterations are possible upon entry to soil. In the present study, we examined the long-term stability and (bio)availability of Ag along the "wastewater-sludge-soil" pathway. Synchrotron-based X-ray absorption spectroscopy (XAS) revealed that ca. 99% of Ag added to the sludge reactors as either Ag-NPs or AgNO3 was retained in sludge, with ≥79% of this being transformed to Ag2S, with the majority (≥87%) remaining in this form even after introduction to soils at various pH values and Cl concentrations for up to 400 days. Diffusive gradients in thin films (DGT), chemical extraction, and plant uptake experiments indicated that the potential (bio)availability of Ag in soil was low but increased markedly in soils with elevated Cl, likely due to the formation of soluble AgClx complexes in the soil solution. Although high Cl concentrations increased the bioavailability of Ag markedly, plant growth was not reduced in any treatment. Our results indicate that Ag-NPs entering soils through the wastewater-sludge-soil pathway pose low risk to plants due to their conversion to Ag2S in the wastewater treatment process, although bioavailability may increase in saline soils or when irrigated with high-Cl water.

The scientific understanding of nanoparticle (NP) release and transformations they undergo during the product life cycle is hampered by the narrow scope of many research endeavors in terms of both breadth of variables and completeness of analytical characterization. We conducted a comprehensive suite of studies to reveal overarching mechanisms and parameters for nanosilver transformations either still adhered to the fabric or when released after washing. Laboratory prepared nanoenhanced fabrics were investigated: three Ag variants and one Au used as an unreactive reference to separate mechanical from chemical releases. Sequential combinations of sunlight irradiation and/or washing in seven different detergent formulations was followed by NP characterization divided into two groups: (1) dissolved and particulate matter in the wash solutions and (2) the fraction that remained on the fabric. Analytical techniques included spICP-MS, XANES, TEM, SEM, and total metals analysis of fabric digests and wash water filtrates. Sunlight irradiation stabilizes metallic Ag upon washing. Detergents containing oxidizing agents assisted with Ag particle release but not Au NPs, inferring additional chemical mechanisms. While particle size played some role, the NP capping agent/fabric binder combination was a key factor in release. When particles were released, little alteration in size was observed. The use of well-controlled fabrics, unreactive reference materials, and a life-cycle based experimental regime are paramount to understanding changes in Ag speciation and release upon use of nanoenhanced textiles.

Here, we evaluate the extent of sorption of silver nanoparticles (AgNPs) with different primary sizes (30 and 70 nm) and surface properties (branched polyethylene imine, "bPEI" and citrate coating) to laboratory plastic during (eco)toxicological testing. Under conditions of algal growth inhibition assay, up to 97% of the added AgNPs were sorbed onto the test vessels whereas under conditions of in vitro toxicological assay with mammalian cells, the maximum loss of AgNPs was 15%. We propose that the high concentration of proteins and biomolecules in the in vitro toxicological assay originating from serum-containing cell culture medium prevented NP sorption due to steric stabilisation. The sorption of AgNPs to test vessels was clearly concentration dependent. In the conditions of algal growth inhibition assay at 10 ng AgNPs/mL, up to 97% of AgNPs were lost from the test while at higher concentrations (1000 ng AgNPs/mL), the loss of AgNPs was remarkably smaller, up to 64%. Sorption of positively charged bPEI-coated AgNPs was more extensive than the sorption of negatively charged citrate-coated AgNPs and, when calculated on a mass basis, more 70 nm-sized Ag than 30 nm Ag sorbed to plastic surfaces. In summary, this study demonstrates that the loss of AgNPs during (eco)toxicological tests due to sorption on test vessel surfaces is significant, especially in diluted media (e.g. in algal growth medium) and at low NP concentrations. Thus, to ensure the accurate interpretation of (eco)toxicological results, the loss of AgNPs due to adsorption to test vessels should not be overlooked and considered for each specific case.

Silver-induced selective pressure is becoming increasingly important due to the growing use of silver (Ag) as an antimicrobial agent in biomedical and commercial products. With demonstrated links between environmental resistomes and clinical pathogens, it is important to identify microbial profiles related to silver tolerance/resistance. We investigated the effects of ionic Ag stress on soil bacterial communities and identified resistant/persistent bacterial populations. Silver treatments of 50-400 mg Ag kg(-1) soil were established in five soils. Chemical lability measurements using diffusive gradients in thin-film devices confirmed that significant (albeit decreasing) labile Ag concentrations were present throughout the 9-month incubation period. Synchrotron X-ray absorption near edge structure spectroscopy demonstrated that this decreasing lability was due to changes in the Ag speciation to less soluble forms such as Ag(0) and Ag2S. Real-time PCR and Illumina MiSeq screening of 16S rRNA bacterial genes showed β-diversity changes, increasing α-diversity in response to Ag pressure, and immediate and significant reductions in 16S rRNA gene counts with varying degrees of recovery. These effects were more strongly influenced by exposure time than by Ag dose at these rates. Ag-selected dominant OTUs principally resided in known persister taxa (mainly Gram positive), including metal-tolerant bacteria and slow-growing Mycobacteria., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Increasing commercial use of nanosilver has focussed attention on the fate of silver (Ag) in the wastewater release pathway. This paper reports the speciation and lability of Ag in archived, stockpiled, and contemporary biosolids from the UK, USA and Australia, and indicates that biosolids Ag concentrations have decreased significantly over recent decades. XANES revealed the importance of reduced-sulfur binding environments for Ag speciation in materials ranging from freshly produced sludge to biosolids weathered under ambient environmental conditions for more than 50 years. Isotopic dilution with (110 m)Ag showed that Ag was predominantly non-labile in both fresh and aged biosolids (13.7% mean lability), with E-values ranging from 0.3 to 60 mg/kg and 5 mM CaNO3 extractable Ag from 1.2 to 609 μg/kg (0.002-3.4% of the total Ag). This study indicates that at the time of soil application, biosolids Ag will be predominantly Ag-sulfides and characterised by low isotopic lability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Engineered zinc oxide (ZnO) and silver (Ag) nanoparticles (NPs) used in consumer products are largely released into the environment through the wastewater stream. Limited information is available regarding the transformations they undergo during their transit through sewerage systems before reaching wastewater treatment plants. To address this knowledge gap, laboratory-scale systems fed with raw wastewater were used to evaluate the transformation of ZnO- and Ag-NPs within sewerage transfer networks. Two experimental systems were established and spiked with either Ag- and ZnO-NPs or with their dissolved salts, and the wastewater influent and effluent samples from both systems were thoroughly characterised. X-ray absorption spectroscopy (XAS) was used to assess the extent of the chemical transformation of both forms of Zn and Ag during transport through the model systems. The results indicated that both ZnO- and Ag-NPs underwent significant transformation during their transport through the sewerage network. Reduced sulphur species represented the most important endpoint for these NPs in the sewer with slight differences in terms of speciation; ZnO converted largely to Zn sulfide, while Ag was also sorbed to cysteine and histidine. Importantly, both ionic Ag and Ag-NPs formed secondary Ag sulfide nanoparticles in the sewerage network as revealed by TEM analysis. Ag-cysteine was also shown to be a major species in biofilms. These results were verified in the field using recently developed nanoparticle in situ deployment devices (nIDDs) which were exposed directly to sewerage network conditions by immersing them into a municipal wastewater network trunk sewer and then retrieving them for XAS analysis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Long-term speciation and lability of silver (Ag-), silver chloride (AgCl-), and silver sulfide nanoparticles (Ag2S-NPs) in soil were studied by X-ray absorption spectroscopy (XAS), and newly developed “nano” Diffusive Gradients in Thin Films (DGT) devices. These nano-DGT devices were designed specifically to avoid confounding effects when measuring element lability in the presence of nanoparticles. The aging profile and stabilities of the three nanoparticles and AgNO3 (ionic Ag) in soil were examined at three different soil pH values over a period of up to 7 months. Transformation of ionic Ag, Ag-NP and AgCl-NPs were dependent on pH. AgCl formation and persistence was observed under acidic conditions, whereas sulfur-bound forms of Ag dominated in neutral to alkaline soils. Ag2S-NPs were found to be very stable under all conditions tested and remained sulfur bound after 7 months of incubation. Ag lability was characteristically low in soils containing Ag2S-NPs. Other forms of Ag were linked to higher DGT-determined lability, and this varied as a function of aging and related speciation changes as determined by XAS. These results clearly indicate that Ag2S-NPs, which are the most environmentally relevant form of Ag that enter soils, are chemically stable and have profoundly low Ag lability over extended periods. This may minimize the long-term risks of Ag toxicity in the soil environment.

In order to accurately assess the potential environmental risk posed by silver nanoparticles (Ag-NPs), their transformation and fate must be investigated in natural systems. This has proven to be very challenging due to the difficulties encountered in retrieving/analyzing NPs dispersed in complex and heterogeneous environmental matrices at relevant (i.e., low) concentrations. In this study, we overcame this challenge by immobilizing functionalized Ag-NPs onto plasma polymerized solid substrates to form "nano in situ deployment devices" (nIDDs). This method allowed us to retrieve and analyze the Ag-NPs after 48 h of direct exposure in freshwater-sediment and saltwater-sediment environments. The type and extent of Ag-NPs transformation was expected to vary along the water-sediment continuum as sediments typically contain steep gradients in solute concentrations and redox potential. To trace the distribution of redox sensitive elements (e.g., Fe, Mn), Diffusive Equilibration in Thin-films (DET) devices were inserted into the sediments alongside the nIDDs. Chemical transformation of the immobilized Ag-NPs across the water-sediment continuum was investigated after retrieval by synchrotron radiation X-ray Absorption Spectroscopy. Linear combination fitting of Ag K-edge X-ray absorption spectra indicated that the chemical transformations of Ag-NPs in both freshwater and saltwater sediments were strongly affected by the redox conditions over the investigated range. Silver bound to reduced sulfur was the principal product of Ag-NP transformations but different extents of transformation were observed for Ag-NPs exposed to different depths in the sediment. These field results add important insights about the transformation of Ag-NPs in heterogeneous environments.

Silver nanoparticles (Ag-NPs) are used in a wide variety of products, prompting concerns regarding their potential environmental impacts. To accurately determine the toxicity of Ag-NPs it is necessary to differentiate between the toxicity of the nanoparticles themselves and the toxicity of ionic silver (Ag) released from them. This is not a trivial task given the reactive nature of Ag in solution, and its propensity for both adsorption and photoreduction. In the experiments reported here, we quantified the loss of silver from test solutions during standard ecotoxicity testing conducted using a variety of different test container materials and geometries. This sensitive (110m)Ag isotope tracing method revealed a substantial underestimation of the toxicity of dissolved Ag to the green algae Pseudokirchneriella subcapitata when calculated only on the basis of the initial test concentrations. Furthermore, experiments with surface-functionalized Ag-NPs under standard algal growth inhibition test conditions also demonstrated extensive losses of Ag-NPs from the solution due to adsorption to the container walls, and the extent of loss was dependent on Ag-NP surface-functionality. These results hold important messages for researchers engaged in both environmental and human nanotoxicology testing, not only for Ag-NPs but also for other NPs with various tailored surface chemistries, where these phenomena are recognized but are also frequently disregarded in the experimental design and reporting.

The use of nanoscale Ag in textiles is one the most often mentioned uses of nano-Ag. It has previously been shown that significant amounts of the Ag in the textiles are released upon washing. However, the form of Ag present in the textiles remains largely unknown as product labelling is insufficient. The aim of this study was therefore to investigate the solid phase speciation of Ag in original and washed silver textiles using XANES. The original Ag speciation in the textiles was found to vary greatly between different materials with Ag(0), AgCl, Ag2S, Ag-phosphate, ionic Ag and other species identified. Furthermore, within the same textile a number of different species were found to coexist. This is likely due to a combination of factors such as the synthesis processes at industrial scale and the possible reaction of Ag with atmospheric gases. Washing with two different detergents resulted in marked changes in Ag-speciation. For some textiles the two detergents induced similar transformation, in other textiles they resulted in very different Ag species. This study demonstrates that in functional Ag textiles a variety of different Ag species coexist before and after washing. These results have important implications for the risk assessment of Ag textiles because they show that the metallic Ag is only one of the many silver species that need to be considered., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The fate and lability of added soluble Ag in soils over time was examined by measurement of labile metal (E-value) by isotopic dilution using the (110m)Ag radioactive isotope and the solid-phase speciation of Ag by X-ray absorption near edge structure (XANES) spectroscopy. After two weeks of ageing the E-values for Ag decreased by 20-90% with a further decrease of 10-40% after six months. The overall decrease in labile Ag for all soils after the 6 month ageing period was 50-100%. The ageing was more rapid and pronounced in the alkaline soils. XANES results for Ag in soils indicated that for the majority of soils the added Ag(+) was reduced to metallic Ag over time, and associations with Fe-oxohydroxides and reduced S groups in organic matter also decreased Ag lability. Strong positive correlations were found between metallic Ag and non-labile Ag and between organic carbon and Ag bonded with S species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The transformation and environmental fate of engineered nanomaterials (ENMs) is the focus of intense research due to concerns about their potential impacts in the environment as a result of their uniquely engineered properties. Many approaches are being applied to investigate the complex interactions and transformation processes ENMs may undergo in aqueous and terrestrial environments. However, major challenges remain due to the difficulties in detecting, separating, and analyzing ENMs from environmental matrices. In this work, a novel technique capable of in situ study of ENMs is presented. By exploiting the functional interactions between surface modified silver nanoparticles (AgNPs) and plasma-deposited polymer films, AgNPs were immobilized on to solid supports that can be deployed in the field and retrieved for analysis. Either negatively charged citrate or polyethylene glycol, or positively charged polyethyleneimine were used to cap the AgNPs, which were deployed in two field sites (lake and marina), two standard ecotoxicity media, and in primary sewage sludge for a period of up to 48 h. The chemical and physical transformations of AgNPs after exposure to different environments were analyzed by a combination of XAS and SEM/EDX, taken directly from the substrates. Cystine- or glutathione-bound Ag were found to be the dominant forms of Ag in transformed ENMs, but different extents of transformation were observed across different exposure conditions and surface charges. These results successfully demonstrate the feasibility of using immobilized ENMs to examine their likely transformations in situ in real environments and provide further insight into the short-term fate of AgNPs in the environment. Both the advantages and the limitations of this approach are discussed.