Ruth C. Merrifield

Stability of Citrate, PVP, and PEG Coated Silver Nanoparticles in Ecotoxicology Media

Silver nanoparticles (AgNPs) are present in the environment and a number of ecotoxicology studies have shown that AgNPs might be highly toxic. Nevertheless, there are little data on their stability in toxicology media. This is an important issue as such dynamic changes affect exposure dose and the nature of the toxicant studied and have a direct impact on all (eco)toxicology data. In this study, monodisperse citrate, PVP, and PEG coated AgNPs with a core size of approximately 10 nm were synthesized and characterized; their behavior was examined in standard OECD media used for Daphnia sp. acute and chronic tests (in the absence of Daphnia). Surface plasmon resonance, size, aggregation, and shape were monitored over 21 days, comparable to a chronic exposure period. Charge stabilized particles (citrate) were more unstable than sterically stabilized particles. Replacement of chloride in the media (due to concerns over chloride-silver interactions) with either nitrate or sulfate resulted in increased shape and dissolution changes. PVP-stabilized NPs in a 10-fold diluted OECD media (chloride present) were found to be the most stable, with only small losses in total concentration over 21 days, and no shape, aggregation, or dissolution changes observed and are recommended for exposure studies.

Molecular toxicity of cerium oxide nanoparticles to the freshwater alga Chlamydomonas reinhardtii is associated with supra-environmental exposure concentrations

Abstract Ceria nanoparticles (NPs) are widely used as fuel catalysts and consequently are likely to enter the environment. Their potential impacts on. biota at environmentally relevant concentrations, including uptake and toxicity, remain to be elucidated and quantitative data on which to assess risk are sparse. Therefore, a definitive assessment of the molecular and phenotypic effects of ceria NPs was undertaken, using well-characterised mono-dispersed NPs as their toxicity is likely to be higher, enabling a conservative hazard assessment. Unbiased transcriptomics and metabolomics approaches were used to investigate the potential toxicity of tightly constrained 4–5u2009nm ceria NPs to the unicellular green alga, Chlamydomonas reinhardtii, a sentinel freshwater species. A wide range of exposure concentrations were investigated from predicted environmental levels, to support hazard assessment, to supra-environmental levels to provide insight into molecular toxicity pathways. Ceria NPs were internalised into intracellular vesicles within C. reinhardtii, yet caused no significant effect on algal growth at any exposure concentration. Molecular perturbations were only detected at supra-environmental ceria NP-concentrations, primarily down-regulation of photosynthesis and carbon fixation with associated effects on energy metabolism. For acute exposures to small mono-dispersed particles, it can be concluded there should be little concern regarding their dispersal into the environment for this trophic level.

Synthesis and Characterization of Polyvinylpyrrolidone Coated Cerium Oxide Nanoparticles

There is a pressing need for the development of standard and reference nanomaterials for environmental nanoscience and nanotoxicology. To that aim, suspensions of polyvinylpyrrolidone (PVP)-coated ceria nanoparticles (NPs) were produced. Four differently sized monodispersed samples were produced by using different PVP chain lengths. The chemical and physical properties of these NPs were characterized as prepared and in different ecotoxicology exposure media. Dynamic light scattering analysis showed that the samples were monodispersed, with an unchanged size when suspended in the different media over a 72 h period. Electron microscopy confirmed this and revealed that the larger (ca. 20 nm) particles were aggregates composed of the smaller individual particles (4-5 nm). Electron energy loss spectroscopy (EELS) showed that the smallest and largest samples were composed almost entirely of cerium(III) oxide, with only small amounts of cerium(IV) present in the largest sample. Dissolved cerium concentrations in media were low and constant, showing that the NPs did not dissolve over time. The simple synthesis of the these NPs and their physical and chemical stability in different environmental conditions make them potentially suitable for use as reference materials for (eco)toxicology and surface water environmental studies.

The critical importance of defined media conditions in Daphnia magna nanotoxicity studies

Due to the widespread use of silver nanoparticles (AgNPs), the likelihood of them entering the environment has increased and they are known to be potentially toxic. Currently, there is little information on the dynamic changes of AgNPs in ecotoxicity exposure media and how this may affect toxicity. Here, the colloidal stability of three different sizes of citrate-stabilized AgNPs was assessed in standard strength OECD ISO exposure media, and in 2-fold (media2) and 10-fold (media10) dilutions by transmission electron microscopy (TEM) and atomic force microscopy (AFM) and these characteristics were related to their toxicity towards Daphnia magna. Aggregation in undiluted media (media1) was rapid, and after diluting the medium by a factor of 2 or 10, aggregation was reduced, with minimal aggregation over 24h occurring in media10. Acute toxicity measurements were performed using 7nm diameter particles in media1 and media10. In media10 the EC50 of the 7nm particles for D. magna neonates was calculated to be 7.46μgL(-1) with upper and lower 95% confidence intervals of 6.84μgL(-1) and 8.13μgL(-1) respectively. For media1, an EC50 could not be calculated, the lowest observed adverse effect concentration (LOAEC) of 11.25μgL(-1) indicating a significant reduction in toxicity compared to that in media10. The data suggest the increased dispersion of nanoparticles leads to enhanced toxicity, emphasising the importance of appropriate media composition to fully assess nanoparticle toxicity in aquatic ecotoxicity tests.

The concentration-dependent behaviour of nanoparticles

Environmental context Studies of manufactured nanoparticles (NPs) in the environment have been performed almost exclusively at high NP concentrations. These data lead to misunderstandings related to NP fate and effects at relevant environmental concentrations, which are expected to be low. A better understanding of the concentration-dependent behaviour of NPs will improve our understanding of their fate and effects under environmentally realistic conditions. Abstract This rapid communication highlights the importance of nanoparticle concentration in determining their environmental fate and behaviour. Notably, two fate processes have been considered: dissolution and aggregation. The decrease in nanoparticle concentration results in increased dissolution and decreased aggregate sizes, inferring higher potential for environmental transport of nanoparticles.

The concentration-dependent aggregation of Ag NPs induced by cystine

Cystine is widely used in cell culture media. Cysteine, the reduced form of cystine, is widely used to scavenge dissolved Ag in eco-toxicological studies to differentiate dissolved vs. nanoparticle uptake and toxicity. However, little is known about the impact of cysteine and cystine on the aggregation behavior of Ag NPs, in particular as a function of Ag NP concentration. Herein, we investigate how cystine (0-300μM) affects the stability of citrate-, polyvinylpyrrolidone-, and polyethylene glycol-coated silver nanoparticles (cit-Ag NPs, PVP-Ag NPs and PEG-Ag NPs, respectively) with and without Suwannee River fulvic acid (SRFA) as a function of Ag NPs concentration using UV-vis spectroscopy at environmentally and ecotoxicologically relevant Ag NP concentrations (ca. 125-1000μgL(-1)). The results demonstrate, for the first time, the concentration-dependent aggregation of cit-Ag NPs in the presence of cystine with a shift in the critical coagulation concentration (CCC) to lower cystine concentrations at lower cit-Ag NP concentrations. At the highest cit-Ag NP concentration (1000μgL(-1)), reaction limited aggregation was only observed and no CCC was measured. SRFA slowed the aggregation of cit-Ag NPs by cystine and aggregation occurred in reaction limited aggregation (RLA) regime only. No CCC value was measured in the presence of SRFA. Cystine replaces citrate, PVP and PEG coatings, resulting in aggregation of both electrostatically and sterically stabilized Ag NPs. These findings are important in understanding the factors determining the behavior of Ag NPs in cell culture media. Also due to the similarity between cystine and cysteine, these results are important in understanding the uptake and toxicity of Ag NPs vs. Ag ions, and suggest that the reduction of the toxicity of Ag NPs in the presence of cysteine could be due to a combined effect of scavenging Ag(+) ions and Ag NP aggregation in the presence of cysteine.

High Resolution STEM-EELS Study of Silver Nanoparticles Exposed to Light and Humic Substances.

Nanoparticles (NPs) are defined as particles with at least one dimension between 1 and 100 nm or with properties that differ from their bulk material, which possess unique properties. The extensive use of NPs means that discharge to the environment is likely increasing, but fate, behavior, and effects under environmentally relevant conditions are insufficiently studied. This paper focuses on the transformations of silver nanoparticles (AgNPs) under simulated but realistic environmental conditions. High resolution aberration-corrected scanning transmission electron microscopy (HAADF STEM) coupled with electron energy loss spectroscopy (EELS) and UV-vis were used within a multimethod approach to study morphology, surface chemistry transformations, and corona formation. Although loss, most likely by dissolution, was observed, there was no direct evidence of oxidation from the STEM-EELS. However, in the presence of fulvic acid (FA), a 1.3 nm oxygen-containing corona was observed around the AgNPs in water; modeled data based on the HAADF signal at near atomic resolution suggest this was an FA corona was formed and was not silver oxide, which was coherent (i.e., fully coated in FA), where observed. The corona further colloidally stabilized the NPs for periods of weeks to months, dependent on the solution conditions.

Single-particle inductively coupled plasma mass spectroscopy analysis of size and number concentration in mixtures of monometallic and bimetallic (core-shell) nanoparticles

It is challenging to separate and measure the physical and chemical properties of monometallic and bimetallic engineered nanoparticles (NPs), especially when mixtures are similar in size and at low concentration. We report that single particle inductively coupled mass spectroscopy (SP-ICP-MS), alongside field flow fractionation (FFF), has allowed for the accurate measurement of size and particle number concentrations of mixed metallic nanoparticles (NPs) containing monometallic NPs of gold (Au) and silver (Ag) and a bimetallic core-shell structured NP (Au@Ag) of equivalent size. Two sets of these NPs were measured. The first contained only 60nm particles, where the Au@Ag NP had a 30nm core and 15nm shell to make a total diameter of 60nm. The second contained only 80nm particles (Au@Ag NP core particle of 50nm with a 15nm shell). FFF separation was used here as a sizing technique rather than a separation technique. It was used to confirm that suspensions containing either individual or mixtures of the Au 60nm, Ag 60nm and AuAg 60nm suspensions eluted together and were of the same size. Similarly, FFF was used to show that suspensions containing individual or mixtures of the equivalent 80nm, eluted together and were of the same size. Although the 60nm and 80nm suspensions did not elute at the same time they were not run together. SP-ICP-MS is then used to identify the size and concentration of the particles within the suspension. Successful separation of the NPs was effected and the limits of the instrument were obtained.

Determining the Concentration Dependent Transformations of Ag Nanoparticles in Complex Media: Using SP-ICP-MS and Au@Ag Core–Shell Nanoparticles as Tracers

The fate, behavior, and impact of engineered nanoparticles (NPs) in toxicological and environmental media are driven by complex processes which are difficult to quantify. A key limitation is the ability to perform measurements at low and environmentally relevant concentrations, since concentration may be a key factor determining fate and effects. Here, we use single particle inductively coupled mass spectroscopy (SP-ICP-MS) to measure directly NP diameter and particle number concentration of suspensions containing gold-silver core-shell (Au@Ag) NPs in EPA moderately hard water (MHW) and MHW containing 2.5 mg L-1 Suwannee River fulvic acid. The Au core of the Au@Ag NPs acts as an internal standard, and aids in the analysis of the complex Ag transformations. The high sensitivity of SP-ICP-MS, along with the Au@Ag NPs, enabled us to track the NP transformations in the range 0.01 and 50 μg L-1, without further sample preparation. On the basis of the analysis of both Au and Ag parameters (size, size distribution, and particle number), concentration was shown to be a key factor in NP behavior. At higher concentration, NPs were in an aggregation-dominated regime, while at the lower and environmentally representative concentrations, dissolution of Ag was dominant and aggregation was negligible. In addition, further formation of ionic silver as Ag NPs in the form of AgS or AgCl was shown to occur. Between 1 and 10 μg L-1, both aggregation and dissolution were important. The results suggest that, under realistic conditions, the role of NP homoaggregation may be minimal. In addition, the complexity of exposure and dose in dose-response relationships is highlighted.

Citrate-Coated Silver Nanoparticles Growth-Independently Inhibit Aflatoxin Synthesis in Aspergillus parasiticus

Manufactured silver nanoparticles (Ag NPs) have long been used as antimicrobials. However, little is known about how these NPs affect fungal cell functions. While multiple previous studies reveal that Ag NPs inhibit secondary metabolite syntheses in several mycotoxin producing filamentous fungi, these effects are associated with growth repression and hence need sublethal to lethal NP doses, which besides stopping fungal growth, can potentially accumulate in the environment. Here we demonstrate that citrate-coated Ag NPs of size 20 nm, when applied at a selected nonlethal dose, can result in a >2 fold inhibition of biosynthesis of the carcinogenic mycotoxin and secondary metabolite, aflatoxin B1 in the filamentous fungus and an important plant pathogen, Aspergillus parasiticus, without inhibiting fungal growth. We also show that the observed inhibition was not due to Ag ions, but was specifically associated with the mycelial uptake of Ag NPs. The NP exposure resulted in a significant decrease in transcript levels of five aflatoxin genes and at least two key global regulators of secondary metabolism, laeA and veA, with a concomitant reduction of total reactive oxygen species (ROS). Finally, the depletion of Ag NPs in the growth medium allowed the fungus to regain completely its ability of aflatoxin biosynthesis. Our results therefore demonstrate the feasibility of Ag NPs to inhibit fungal secondary metabolism at nonlethal concentrations, hence providing a novel starting point for discovery of custom designed engineered nanoparticles that can efficiently prevent mycotoxins with minimal risk to health and environment.