NTA: Environmental Impact of Nanoparticles

The need to characterize different properties of nanomaterials continues to grow rapidly. Since the commercialization of the technique in 2004, Nanoparticle tracking Analysis (NTA) has become increasingly prevalent in a wide variety of different research fields and industrial applications. In this forth chapter of the Nanoparticle Tracking Analysis (NTA) application and usage review, we discuss the reports of the use of NTA in developing methods to study the impact of nanoparticles and analysis of environmental samples. 

NTA in the Development of test methods for nanoparticle impact studies

Despite being a relatively new technique, NTA has been recognized as showing promise as an analytical method which could furnish not just information about nanoparticle size and, equally importantly, concentration but that it could do so in complex sample types of high polydispersity (Montes-Burgos et al., 2010; Lynch 2008, Montes-Burgos et al., 2007; Gornati et al., 2009) and that methods such as NTA could be considered as one of a number of means by which the environmental impact and potential cellular toxicity of nanoparticles could be studied in the future (Borm et al., 2006; Tenuta 2008, Tran and Anton, 2009, Kuhlbusch et al., 2010; Hassellöv and Kaegi 2009, Stolpe et al., 2011) and methods to study their effects on other biological barriers have been addressed (Linn et al., 2010). Tran et al. (2011) also developed a hypothetical model for predicting the toxicity of high aspect ratio nanoparticles while Karlsson (2010) compared the comet assay in nanotoxicology research to results obtained by NTA.

In another such study, NTA was considered to share many features in common with conventional flow cytometry but unique in the deeply sub-micron size range. NTA was considered “a direct and fast technique by which nanoparticles in their natural solvated state in a liquid can be rapidly detected, sized and concentration measured. The technique can be used to complement existing techniques for the sizing of nanoparticles (e.g., DLS, PCS) allowing data obtained from these methods to be validated by direct microscopical observation of the sample”.(Bendre et al., 2011)

In an interesting and thought-provoking extension of assessing possible sources of nanoparticles Chen et al. (2012) undertook a TEM and NTA-assisted study in the characterization and preliminary toxicity assay of nano-titanium dioxide additive in sugar-coated chewing gum. They described a facile and highly reliable separation method of TiO2 particles from food products (focusing on sugar-coated chewing gum) claiming their work to be the first comprehensive characterization study on food nanoparticles by multiple qualitative and quantitative methods. Surprisingly, their results showed that the number of food products containing nano-TiO2 (<200 nm) is much larger than known and consumers have already often been exposed to engineered nanoparticles in daily life. Over 93% of TiO2 in gum is nano-TiO2, and it is unexpectedly easy to come out and be swallowed by a person who chews gum.

Noël also used NTA amongst other techniques for analysis of nanoparticle aggregates in his work on generating nano-aerosols from TiO2 (5 nm) nanoparticles showing different agglomeration states as applied to toxicological studies (Noël et al., 2012) and Cabot et al. (2012) used NTA to measure changes in tobacco smoke particle size over a series of different time points providing an input into residence time estimates, thus aiding dose calculations to the lower airways.

More recently Ling et al. (2013) have reviewed a range of detection and identification instruments and their calibration for the analysis of air/liquid/surface-borne nanoscale particles suggesting that microscopy analysis for particle morphology can be performed by depositing air-borne or liquid-borne nanoparticles on surfaces. Detection limit and measurement resolution of the liquid-borne nanoparticles could be enhanced by aerosolizing them and taking advantage of the well-developed air-borne particle analyzers. NTA was tested on TiO2 aggregate particles

Similarly, Asimakopoulou et al. (2013) discussed the development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles. Their proposed technology was validated with various types of nanoparticles (Diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labelled nanoparticles and delivery of aerosolized nanoparticles was demonstrated by NTA visualization of the nanoparticle fluorescence in the cell cultures following exposure.

Wang et al. (2013) addressed the need for suitable methods for high throughput screening of physicochemical properties of nanomaterials (NM) and their immediate environments to allow better understanding of NM bioactivities, prioritization of NMs for further testing, and the building of computational models to predict NM toxicity.

Recognizing that nanosilver, due to its small particle size and enormous specific surface area, facilitates more rapid dissolution of ions than the equivalent bulk material; potentially leading to increased toxicity of nanosilver, Reidy et al. (2013) critically reviewed current knowledge and recommendations relating to mechanisms of silver nanoparticle release, transformation and toxicity:

In anticipation of increasing regulatory measurements requirements for nanomaterials and their toxicity, a number of studies on metrology have been undertaken. Thus, Brown et al. (2013) have reviewed nano-object count metrology and a best practice framework. Emphasizing that harmonized methods for identifying nanomaterials by size and count for many real world samples do not currently exist and that, while particle size remains the sole discriminating factor for classifying a material as ‘nano’, inconsistencies in size metrology will continue to confound policy and decision-making they suggested that substantial scientific scrutiny is needed in the area of nanomaterial metrology to establish best practices and to develop suitable methods before implementation of definitions based solely on number percent nano­object content for regulatory purposes. Strong cooperation between industry, academia and research institutions will be required to fully develop and implement detailed frameworks for nanomaterial identification with respect to emerging concentration measurement­based metrics. When discussing NTA specifically, they pointed out that NTA may have difficulty with agglomeration since the agglomerate will appear as a single scattering centre. In these cases, the sample dispersion may have to be altered through further dilution or sonication. Dispersing aids may be used but should not index match the particles. Larger particles in a population may be removed through filtration, centrifugation or settling prior to analysis.

Similarly, Pettitt and Lead (2013) have outlined characterization requirements for nanomaterial regulation. They proposed some minimum physicochemical parameters required to adequately describe NMs for regulatory purposes and discussed the most appropriate mechanisms to obtain those data in terms of the overarching delivery mechanism. Guiding principles for particle characterization during the hazard testing required to comply with regulations were examined.

Finally, Vincent (2013) has briefly reviewed NTA for the characterization of nanomaterials for toxicological assessment.

Analysis of Environmental Samples

An early appreciation was published on the role that NTA could play in the analysis of nanoparticles contained within or extracted from complex environmental samples. Thus, Borm et al. (2006) and Tenuta (2008) cited NTA as a possible technology for the development of systematic research strategies concerning their analysis and specific examples were described relating to TiO2 nanoparticles in natural aquatic media (Holmberg et al., 2008) and the oxidation of organic pollutants in aqueous solutions by nanosized copper oxide catalysts (Ben-Moshe et al., 2008)

More recent studies have shown that the complexity of interactions between NEPs and environmental matrices is extremely complex represents a significant challenge in both their quantification and modelling but in which NTA may play a role (Gornati et al., 2009; Hartmann 2011, Njuguna et al., 2011; Tran et al., 2011; Kuhlbusch et al,. 2010; Sentein et al., 2011)

Quik and his co-workers have also studied the role that natural colloids play in the sedimentation of CeO2 nanoparticles using NTA to analyze natural river waters from two major European rivers in which they showed that heteroaggregation of the metal oxide with or deposition onto the solid fraction of natural colloids was the main mechanism causing sedimentation in relation to homoaggregation (Quik et al., 2012). MacCuspie et al. (2011) had earlier studied the potential hazards of gold species in a variety of cellular and aqueous systems.

Schwyzer has studied both the colloidal stability (Schwyzer et al., 2011 and 2012) and solubilization (Schwyzer et al., 2010) of carbon nanotubes under natural conditions and Reed et al. (2012), in their study of the detection of single walled carbon nanotubes (CNT) by monitoring embedded metals (intercalated in the CNT structure), found that during analysis of split samples by both single particle inductively coupled plasma mass spectrometry (spICPMS) and NTA, the quantification of particle number concentration by spICPMS was several orders of magnitude worse than by NTA. They postulated that this was a consequence of metal content and/or size, caused by the presence of many CNTs that do not contain enough metal to be above the instrument detection limit, resulting in undercounting CNTs by spICPMS, though spICPMS is still a more sensitive technique for detecting the presence of CNTs in environmental, materials, or biological applications.

Domingos and his co-workers carried out a typical such study involving analysis of nanoparticle suspensions using several state-of-the-art analytical techniques (transmission electron microscopy; atomic force microscopy; dynamic light scattering; fluorescence correlation spectroscopy; nanoparticle tracking analysis; flow field flow fractionation). Theoretical and analytical considerations were evaluated, results were compared, and the advantages and limitations of the techniques were discussed. No “ideal” technique was found for characterizing manufactured nanoparticles in an environmental context as each technique had its own advantages and limitations (Domingos et al., 2009).

NTA has also been used amongst other techniques to study and compare three Silica nanotracer nanoparticulates and their transport in soils (Vitorge et al., 2010) and the nano-sized Fe2O3 waste powder adsorption with arsenite (As3+) in the steel industry has also been studied (Prasad et al., 2011).

Given the increasing prevalence of nanoparticles in consumer products and processes, their release and appearance in wastewaters have attracted increasing attention. Thus the release of nanosized biocides from wood coatings have been studied with NTA (Künniger et al., 2010) as have nanoscale components of toothpastes. Peetsch and Epple (2011) and Farkas et al. (2011) have also reported the characterization of the effluent from a nanosilver producing washing machine using NTA to follow silver movement. Rezić (2011) had also described the determination of engineered nanoparticles on textiles and in textile waste-waters.

Similarly, the widespread use of TiO2 as a major sunscreen component and the associated fate, behavior and environmental risks in the UK has been studied (Johnson et al., 2011).

The use of NTA as a new tool in the study of nanoparticles in environmental samples and for toxicological studies has been reviewed recently. In a study of stability of CeO2 in de-ionized water and electrolyte-containing fish medium the dispersions were monitored using various techniques, for a period of 3 days. NTA was found to provide useful data which was complementary to zeta potential, particle size via DLS, fluorescence and UV–Vis spectroscopy and SEM and specifically was shown to provide useful, quantitative information on the concentration of nanoparticles in suspension although limited in its ability to accurately track the motion of large agglomerates found in the fish medium (Tantra et al., 2011).

Vähä-Nissi et al. (2011) have described the use of NTA in their study on the safe production and use of nanomaterials with special reference to aqueous dispersions from biodegradable and/or renewable polymers while Wilkinson et al. (2011) suggested solution-engineered palladium nanoparticles as a model for health effect studies of automotive particulate pollution.

In his assessment of the need for standardized methods and environmental monitoring programs for anthropogenic nanoparticles, Paterson reviewed the available techniques emphasizing the critical need for methods capable of qualitatively and quantitatively measuring such pollutants. He issued a challenge to national and international regulatory and research agencies to help develop standard methods, quality assurance tools, and implement environmental monitoring programs for this class of pollutants citing NTA as being one such technique that could supply important information.(Paterson et al., 2011). von der Kammer et al. (2011) reiterated this point in their recent discussion on the general considerations associated with the isolation of engineered nanoparticles from highly complex environmental samples (von der Kammer et al., 2011)

In other work related to the development of test methods for Health and Safety risk management, Dolez and her co-workers used NTA to measure the penetration of nanoparticles through protective gloves in conditions simulating occupational use. Involving nanoparticles applied as powder and colloidal solutions to different materials subject to various types of static and dynamic mechanical deformations simultaneously with nanoparticle exposure. In determining that the development of the test method also involved the identification of appropriate nanoparticle detection techniques, Dolez concluded that while methanol-based sampling solutions could be centrifuged on grids or mica substrates for analysis by microscopy techniques, NTA and ICP-MS could also be used to directly detect nanoparticles in water-based sampling solutions (Dolez et al., 2011).

In his assessment of new single particle methods for detection and characterization of nanoparticles in environmental samples, Tuoriniemi (2013) evaluated NTA for the measurement of number concentration and size distributions. The technique was considered suitable for monitoring and measuring exposure at “relatively high” (> 106 particles mL-1) concentrations but NTA was considered relatively unspecific in the sense that it is difficult to distinguish particles of different materials. To increase sensitivity and specificity, single particle inductively coupled plasma mass spectrometry (spICPMS) was assessed for element specific characterization of particles in liquid samples. Also, recognizing that variable pressure or environmental scanning electron microscopes (ESEM) could be applied on a vast range of sample types with “no or very little sample preparation”, backscattered electron (BSE) imaging in such an instrument was chosen as a base for developing a method for quantification of particles in solid samples. The technique was applied for quantifying particles in toxicity tests involving soil biota and was considered to be sensitive enough to cover the concentration range that is typically of interest in such tests. It was concluded that due to the information obtained on a single particle basis, electron microscopy is a suitable complementing technique for spICPMS measurements, which otherwise give little information about the structure of the particles. It should be noted however, that this study did not apparently consider the high capital and running costs of these techniques nor the sample analysis time which might significantly curtail sample throughput which was a particular limitation.

Multiple complimentary techniques were used to characterize bare and polymer-coated nTiO2 and nZnO particles under a range of environmentally relevant conditions: dynamic light scattering, nanoparticle tracking analysis, scanning electron microscopy and transmission electron microscopy. Percolation of suspensions of such materials through angular sand columns showed uncoated (bare) NPs demonstrated high retention within the water saturated granular matrix and both bare nTiO2 and nZnO deposition onto sand was found to be time-dependent. In contrast to bare particles, polymer-coated NPs were highly stable in suspension and exhibited significant transport potential (Petosa et al., 2011). Petosa et al. (2013) have more recently extended this work to the study of the mobility of nanosized cerium dioxide and polymeric capsules in quartz and loamy sands saturated with model and natural groundwaters. Laboratory-scale columns were used to examine the mobility of polyacrylic acid (PAA)-coated cerium dioxide nanoparticles (nCeO2) and an analogous nanosized polymeric capsule (nCAP) in water saturated quartz sand or loamy sand. ENP suspensions were characterized using dynamic light scattering and NTA to establish aggregate size. Enhanced particle retention was also observed in loamy sand in comparison to the quartz sand, emphasizing the need to consider the nature of the aqueous matrix and granular medium in evaluating contamination risks associated with the release of ENPs in natural and engineered aquatic environments.

Raychoudhury et al. (2011) similarly investigated the straining of polyelectrolyte-stabilized (coated with carboxymethyl cellulose) nanoscale zero valent iron particles (CMC-NZVI)) during transport through granular porous media using NTA to demonstrate that CMC-NZVI particles, despite of their small size (NTA determined hydrodynamic diameters of 167–185 nm and transmission electron microscopy imaged diameters of approximately 85 nm), may be removed by straining during transport, especially through fine granular subsurface media. A tailing effect observed in the particle breakthrough curves, was attributed to detachment of deposited particles.

Mallampati et al. (2012) has used NTA to assess enhanced heavy metal immobilization in soil by grinding with addition of nanometallic Ca/CaO dispersion mixtures concluding that it might be due to adsorption and entrapment of heavy metals into newly formed aggregates, thereby prompting aggregation of soil particles and enclosure/binding with Ca/CaO-associated immobile salts. Shang et al. (2012) reported a study of transport and retention of engineered nanoporous silicate particles (ENSPs) that are designed for treatment and remediation of contaminants such as uranium in groundwater and sediments using NTA and DLS to periodically monitor the quality of the ENSP dispersion. NTA has also been used amongst other techniques to study nanoparticulates transport in soil organisms as diverse as earthworm (Hooper et al., 2011) and the influence of humic acid on TiO2 nanoparticles in test media (Mullinger et al., 2011)

Tourinho et al. (2012) have recently reviewed the literature dealing with the fate and effects of metal-based NPs in soil. In the environment, the characteristics of NPs (e.g., size, shape, surface charge) and soil (e.g., pH, ionic strength, organic matter, and clay content) will affect physical and chemical processes, resulting in NP dissolution, agglomeration, and aggregation. They point to the lack of standards existing for toxicity tests with NPs and, more importantly, that the reporting of associated characterization data is sparse, or missing, making it impossible to interpret and explain observed differences in results among studies. NTA, with its ability to generate higher resolution particle size distribution information and number frequency distributions, is advantageous in this respect.

Ramirez-Garcia et al. (2011) reported on a highly successful and original protocol for the dispersion of titania nanoparticles in biocompatible fluids for in vitro and in vivo studies of the nanoparticle–biology interaction. Using stabilizers to obtain dispersions of 45 and 55nm diameters at concentrations up to 10mg/ml and the sizing techniques of Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Differential Centrifuge Sedimentation (DCS) were used to characterize the different suspensions and the suitability of each was compared while Tenuta et al. (2011) have described the elution of labile fluorescent dye from nanoparticles during biological use.

In a comprehensive review of the use of flow field-flow fractionation (FlFFF) for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems, Baalousha et al. (2011) compared numerous detection techniques applied to FlFFF (including inductively coupled plasma-mass spectroscopy, light scattering, NTA, UV-absorbance, fluorescence, transmission electron microscopy, and atomic force microscopy), demonstrating that FlFFF provides a wealth of information on particle properties including, size, shape, structural parameters, chemical composition and particle-contaminant association.

Exploiting the ability of NTA to more accurately measure the particle size distribution profile of polydisperse systems than other techniques, Raychoudhury et al. (2011) demonstrated that aggregation resulted in a change in the particle size distribution (PSD) of carboxymethyl cellulose (CMC)-modified nanoparticles of zero-valent iron (NZVI)of with time when were investigated in laboratory-scale sand packed columns and that the change in PSD over time was influenced by the CMC-NZVI concentration in suspension. They showed that changes in particle sizes over time led to corresponding changes in single-collector contact efficiencies, resulting in altered particle deposition rates over time using a coupled aggregation-colloid transport model to demonstrate how changes in PSD can enhance or reduce the transport of CMC-NZVI in column experiments.

In studying the toxicity of ZnO nanoparticles to Folsomia candida, Waalewijn-Kool et al. (2012) showed that differences in methods of spiking exposure media to test dispersion size characteristics made little difference to the reproductive capacity of the organism, NTA and TEM both showing the toxicity of the ZnO was not related to particle size. Yu (2011) studied colloid transport in surface runoff through dense vegetation.

Hadioui et al. (2012) described a multimethod quantification of Ag+ release from nanosilver, suggesting part or all of the toxicity attributed to silver nanoparticles (nAg) may be due to the release of free silver (Ag+). Using NTA to determine nAg size and number prior to employing ion-exchange technique (IET) centrifugal ultrafiltration and single particle inductively coupled plasma mass spectrometry (SP ICP-MS) to determine very low concentrations of free or dissolved Ag in commercial suspensions of nAg.

Following earlier work in which Gallego-Urrea et al. (2011) considered the applications of NTA to the determination of size distributions and concentrations of nanoparticles in environmental, biological and food samples, Luo et al. (2013) have compared NTA to the use of atmospheric scanning electron microscopy (ASEM) in the visualization and characterization of engineered nanoparticles in complex environmental and food matrices such as supernatant of natural sediment, test medium used in ecotoxicology studies, bovine serum albumin and tomato soup, concluding that ASEM analysis was found to be a complementary technique to existing methods that is able to visualize ENPs in complex liquid matrices and to provide ENP size information without extensive sample preparation.

While Saleh (2013) showed aggregation behavior of nanomaterials under biological exposure conditions, Wilkinson (2013), in an attempt to gain a fuller insight into the health effects from PM, suggested it could only be achieved through practical investigation of the mode of toxicity from distinct types of particles and required techniques for their identification, monitoring, and the production of model fractions for health studies. Accordingly he undertook a comprehensive collection and chemical analysis of particulates at the origin of emission in order to provide clearer insight into the nature of the particulates at exposure and add detail to aid risk assessment. Taking the approach of in vitro cytotoxicity testing, nanoparticles of types typical to automotive emissions, were synthesized and extensively characterized using SEM-EDS, X-ray diffraction (XRD), transmission electron microscopy (TEM),dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). The produced model magnetite and palladium nanoparticles were found to induce toxicity in human pulmonary epithelial cells (A549 and PBEC) as well as impact severely on immunological and renal cells (221 B- and 293T-cells) in a dose-dependent manner.

Finally, Park et al. (2013) have posed the question of whether the results of regulatory ecotoxicity testing of engineered nanoparticles are the relevant to the natural environment. Proposing that many studies have explored the toxicity of ENPs to aquatic organisms but these studies have usually been performed with little understanding of ENPs behavior in the test media and the relationship between behavior in the media to behavior in natural waters, their study evaluated and compared the aggregation behavior of four model gold nanoparticle types (coated with neutral, negative, positive and amphoteric cappings) in standard ecotoxicity test media and natural waters. In standard media, positive and neutral nanoparticles (NPs) were stable whereas amphoteric and negative NPs generally showed substantial aggregation. In natural waters, amphoteric NPs were generally found to be stable, neutral and positive NPs showed substantial aggregation while negative NPs were stable in some waters and unstable in others. Humic acid addition stabilized the amphoteric NPs, destabilized the positive NPs and had no effect on stability of negative NPs. Given the dramatically different behaviors of ENPs in various standard media and natural waters, they suggest current regulatory testing may either under- or over-estimate the toxicity of nanomaterials to aquatic organisms and that, therefore, there is a pressing need to employ ecotoxicity media which better represent the behavior of ENPs in natural system. Prior to testing, all model study particles were characterized by TEM and NTA. Reed et al. (2013) and Reed (2013) used using single particle-inductively coupled plasma-mass spectrometry (spICPMS) to detect single walled carbon nanotubes by monitoring embedded metals using trace catalytic metals intercalated in the CNT structure as proxies for the nanotubes. Interestingly, analysis of split samples by both spICPMS and NTA showed the quantification of particle number concentration by spICPMS to be several orders of magnitude lower than by NTA. They postulated that this was a consequence of metal content and/or size, caused by the presence of many CNTs that do not contain enough metal to be above the instrument detection limit, resulting in undercounting CNTs by spICPMS. However, they claimed that since the detection of CNTs at low ng L_1 concentrations is not possible by other techniques, spICPMS was still a more sensitive technique for detecting the presence of CNTs in environmental, materials, or biological applications.


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