Alan J. Kennedy

Fractionating Nanosilver: Importance for Determining Toxicity to Aquatic Test Organisms

This investigation applied novel techniques for characterizing and fractionating nanosilver particles and aggregates and relating these measurements to toxicological endpoints. The acute toxicity of eight nanosilver suspensions of varying primary particle sizes (10-80 nm) and coatings (citrate, polyvinylpyrrolidone, EDTA, proprietary) was assessed using three aquatic test organisms (Daphnia magna, Pimephales promelas, Pseudokirchneriella subcapitata). When 48-h lethal median concentrations (LC50) were expressed as total silver, both D. magna and P. promelas were significantly more sensitive to ionic silver (Ag(+)) as AgNO(3) (mean LC50 = 1.2 and 6.3 μg/L, respectively) relative to a wide range in LC50 values determined for the nanosilver suspensions (2 -126 μg/L). However, when LC50 values for nanosilver suspensions were expressed as fractionated nanosilver (Ag(+) and/or <4 nm particles), determined by ultracentrifugation of particles and confirmed field-flow-fractograms, the LC50 values (0.3-5.6 μg/L) were comparable to the values obtained for ionic Ag(+) as AgNO(3). These results suggest that dissolved Ag(+) plays a critical role in acute toxicity and underscores the importance of characterizing dissolved fractions in nanometal suspensions.

Factors influencing the partitioning and toxicity of nanotubes in the aquatic environment

Carbon nanotubes (NTs) may be among the most useful engineered nanomaterials for structural applications but could be difficult to study in ecotoxicological evaluations using existing tools relative to nanomaterials with a lower aspect ratio. Whereas the hydrophobicity and van der Waals interactions of NTs may suggest aggregation and sedimentation in aquatic systems, consideration regarding how engineered surface modifications influence their environmental fate and toxicology is needed. Surface modifications (e.g., functional groups and coatings) are intended to create conditions to make NTs dispersible in aqueous suspension, as required for some applications. In the present study, column stability and settling experiments indicated that raw, multiwalled NTs (MWNTs) settled more rapidly than carbon black and activated carbon particles, suggesting sediment as the ultimate repository. The presence of functional groups, however, slowed the settling of MWNTs (increasing order of stability: hydroxyl > carboxyl > raw), especially in combination with natural organic matter (NOM). Stabilized MWNTs in high concentrations of NOM provided relevance for water transport and toxicity studies. Aqueous exposures to raw MWNTs decreased Ceriodaphnia dubia viability, but such effects were not observed during exposure to functionalized MWNTs (> 80 mg/L). Sediment exposures of the amphipods Leptocheirus plumulosus and Hyalella azteca to different sizes of sediment-borne carbon particles at high concentration indicated mortality increased as particle size decreased, although raw MWNTs induced lower mortality (median lethal concentration [LC50], 50 to >264 g/kg) than carbon black (LC50, 18-40 g/kg) and activated carbon (LC50, 12-29 g/kg). Our findings stress that it may be inappropriate to classify all NTs into one category in terms of their environmental regulation.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

Characterization of silver nanoparticles using flow-field flow fractionation interfaced to inductively coupled plasma mass spectrometry

The ability to detect and identify the physiochemical form of contaminants in the environment is important for degradation, fate and transport, and toxicity studies. This is particularly true of nanomaterials that exist as discrete particles rather than dissolved or sorbed contaminant molecules in the environment. Nanoparticles will tend to agglomerate or dissolve, based on solution chemistry, which will drastically affect their environmental properties. The current study investigates the use of field flow fractionation (FFF) interfaced to inductively coupled plasma-mass spectrometry (ICP-MS) as a sensitive and selective method for detection and characterization of silver nanoparticles. Transmission electron microscopy (TEM) is used to verify the morphology and primary particle size and size distribution of precisely engineered silver nanoparticles. Subsequently, the hydrodynamic size measurements by FFF are compared to dynamic light scattering (DLS) to verify the accuracy of the size determination. Additionally, the sensitivity of the ICP-MS detector is demonstrated by fractionation of μg/L concentrations of mixed silver nanoparticle standards. The technique has been applied to nanoparticle suspensions prior to use in toxicity studies, and post-exposure biological tissue analysis. Silver nanoparticles extracted from tissues of the sediment-dwelling, freshwater oligochaete Lumbriculus variegatus increased in size from approximately 31-46nm, indicating a significant change in the nanoparticle characteristics during exposure.

Potential for Occupational Exposure to Engineered Carbon-Based Nanomaterials in Environmental Laboratory Studies

Background The potential exists for laboratory personnel to be exposed to engineered carbon-based nanomaterials (CNMs) in studies aimed at producing conditions similar to those found in natural surface waters [e.g., presence of natural organic matter (NOM)]. Objective The goal of this preliminary investigation was to assess the release of CNMs into the laboratory atmosphere during handling and sonication into environmentally relevant matrices. Methods We measured fullerenes (C60), underivatized multiwalled carbon nanotubes (raw MWCNT), hydroxylated MWCNT (MWCNT-OH), and carbon black (CB) in air as the nanomaterials were weighed, transferred to beakers filled with reconstituted freshwater, and sonicated in deionized water and reconstituted freshwater with and without NOM. Airborne nanomaterials emitted during processing were quantified using two hand-held particle counters that measure total particle number concentration per volume of air within the nanometer range (10–1,000 nm) and six specific size ranges (300–10,000 nm). Particle size and morphology were determined by transmission electron microscopy of air sample filters. Discussion After correcting for background particle number concentrations, it was evident that increases in airborne particle number concentrations occurred for each nanomaterial except CB during weighing, with airborne particle number concentrations inversely related to particle size. Sonicating nanomaterial-spiked water resulted in increased airborne nanomaterials, most notably for MWCNT-OH in water with NOM and for CB. Conclusion Engineered nanomaterials can become airborne when mixed in solution by sonication, especially when nanomaterials are functionalized or in water containing NOM. This finding indicates that laboratory workers may be at increased risk of exposure to engineered nanomaterials.

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

Expanded use of engineered nanoparticles (ENPs) in consumer products increases the potential for environmental release and unintended biological exposures. As a result, measurement techniques are needed to accurately quantify ENP size, mass, and particle number distributions in biological matrices. This work combines single particle inductively coupled plasma mass spectrometry (spICPMS) with tissue extraction to quantify and characterize metallic ENPs in environmentally relevant biological tissues for the first time. ENPs were extracted from tissues via alkaline digestion using tetramethylammonium hydroxide (TMAH). Method development was performed using ground beef and was verified in Daphnia magna and Lumbriculus variegatus . ENPs investigated include 100 and 60 nm Au and Ag stabilized by polyvynylpyrrolidone (PVP). Mass- and number-based recovery of spiked Au and Ag ENPs was high (83-121%) from all tissues tested. Additional experiments suggested ENP mixtures (60 and 100 nm Ag ENPs) could be extracted and quantitatively analyzed. Biological exposures were also conducted to verify the applicability of the method for aquatic organisms. Size distributions and particle number concentrations were determined for ENPs extracted from D. magna exposed to 98 μg/L 100 nm Au and 4.8 μg/L 100 nm Ag ENPs. The D. magna nanoparticulate body burden for Au ENP uptake was 613 ± 230 μg/kgww, while the measured nanoparticulate body burden for D. magna exposed to Ag ENPs was 59 ± 52 μg/kgww. Notably, the particle size distributions determined from D. magna tissues suggested minimal shifts in the size distributions of ENPs accumulated, as compared to the exposure media.

Surfactive stabilization of multi-walled carbon nanotube dispersions with dissolved humic substances

Soil humic substances (HS) stabilize carbon nanotube (CNT) dispersions, a mechanism we hypothesized arose from the surfactive nature of HS. Experiments dispersing multi-walled CNT in solutions of dissolved Aldrich humic acid (HA) or water-extractable Catlin soil HS demonstrated enhanced stability at 150 and 300 mg L(-1) added Aldrich HA and Catlin HS, respectively, corresponding with decreased CNT mean particle diameter (MPD) and polydispersivity (PD) of 250 nm and 0.3 for Aldrich HA and 450 nm and 0.35 for Catlin HS. Analogous trends in MPD and PD were observed with addition of the surfactants Brij 35, Triton X-405, and SDS, corresponding to surfactant sorption maximum. NEXAFS characterization showed that Aldrich HA contained highly surfactive domains while Catlin soil possessed a mostly carbohydrate-based structure. This work demonstrates that the chemical structure of humic materials in natural waters is directly linked to their surfactive ability to disperse CNT released into the environment.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

Effects of hardness, chloride, and acclimation on the acute toxicity of sulfate to freshwater invertebrates

The acute toxicity of sulfate to Ceriodaphnia dubia, Chironomus tentans, Hyalella azteca, and Sphaerium simile was assessed to support potential updates of Illinois (USA) sulfate criteria for the protection of aquatic life. The mean lethal concentrations to 50% of a sample population (LC50s), expressed as mg S04(-2)/L, in moderately hard reconstituted water (MHRW) were as follows: 512 mg/L for H. azteca, 2,050 mg/L for C. dubia, 2,078 mg/L for S. simile, and 14,134 mg/L for C. tentans. At constant sulfate (approximately 2,800 mg/L) and hardness (106 mg/L), survival of H. azteca was positively correlated with chloride concentration. Hardness also was found to ameliorate sodium sulfate toxicity to C. dubia and H. azteca, with LC50s for C. dubia increasing from 2,050 mg SO4(-2)/L at hardness = 90 mg/L to 3,516 mg SO4(-2)/L at hardness = 484 mg/L. Using a reformulated MHRW with a similar hardness but higher chloride concentration and different calcium to magnesium ratio than that in standard MHRW, the mean LC50 for H. azteca increased to 2,855 mg/L, and the LC50 for C. dubia increased to 2,526 mg/L. Acclimation of C. dubia to 500 and 1,000 mg SO4(-2)/L for several generations nominally increased mean LC50 values compared with those cultured in standard MHRW.

Impact of Organic Carbon on the Stability and Toxicity of Fresh and Stored Silver Nanoparticles

Studies investigating the impact of particle size and capping agents on nanosilver toxicity in pristine laboratory conditions are becoming available. However, the relative importance of known environmental mitigating factors for dissolved silver remains poorly characterized for nanosilver in context with existing predictive toxicity models. This study investigated the implications of freshly prepared versus stored 20 and 100 nm nanosilver stocks to freshwater zooplankton (Ceriodaphnia dubia) in presence and absence of dissolved organic carbon (DOC). Results indicated that while the acute toxicity of nanosilver decreased significantly with larger size and higher DOC, storage resulted in significant increases in toxicity and ion release. The most dramatic decrease in toxicity due to DOC was observed for the 20 nm particle (2.5-6.7 fold decrease), with more modest toxicity reductions observed for the 100 nm particle (2.0-2.4 fold) and dissolved silver (2.7-3.1 fold). While a surface area dosimetry presented an improvement over mass when DOC was absent, the presence of DOC confounded its efficacy. The fraction of dissolved silver in the nanosilver suspensions was most predictive of acute toxicity regardless of system complexity. Biotic Ligand Model (BLM) predictions based on the dissolved fraction in nanosilver suspensions were comparable to observed toxicity.