Measuring environmental impact of nanomaterial wastes and contaminants using Nanoparticle Tracking Analysis

The increasing number of nanomaterial based consumer products raises concerns about their possible impact on the environment. Suitable methods for their analysis are a particular problem in this regard. This white paper reviews the use of Nanoparticle Tracking Analysis (NTA) in addressing this need.


The increasing number of nanomaterial based consumer products raises concerns about their possible impact on the environment. Suitable methods for their analysis are a particular problem in this regard. This white paper reviews the use of Nanoparticle Tracking Analysis (NTA) in addressing this need.

Nanoparticle Tracking Analysis (NTA) Overview

NTA utilizes the properties of both light scattering and Brownian motion in order to obtain the particle size distribution of samples in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via a 20x magnification microscope onto which is mounted a camera. The camera, which operates at approximately 30 frames per second (fps), captures a video file of the particles moving under Brownian motion within the field of view of approximately 100 μm x 80 μm x 10 μm (Figure 1).

Figure 1: Schematic of the optical configuration used in NTA.

The movement of the particles is captured on a frame-by-frame basis. The proprietary NTA software simultaneously identifies and tracks the center of each of the observed particles, and determines the average distance moved by each particle in the x and y planes. This value allows the particle diffusion coefficient (Dt) to be determined from which, if the sample temperature T and solvent viscosity η are known, the sphere-equivalent hydrodynamic diameter, d, of the particles can be identified using the Stokes-Einstein equation (Equation 1).

Equation 1

where KB is Boltzmann’s constant.

NTA is not an ensemble technique interrogating a very large number of particles, but rather each particle is sized individually, irrespective of the others. An example of the size distribution profile generated by NTA is shown in Figure 2.

Figure 2: An example of the size distribution profile generated by NTA. The modal size for this sample is found to be approximately 70 nm, with larger sized particles also present.

In addition, the particles’ movement is measured within a fixed field of view (approximately 100 μm by 80 μm) illuminated by a beam approximately 10 μm in depth. These figures allow a scattering volume of the sample to be estimated; by measuring concentration of the particles within this field of view and extrapolating to a larger volume it is possible to achieve a concentration estimation in terms of particles per mL for any given size class or an overall total.

Monitoring Nanoparticle Exposure

As discussed by Paterson et al. (2011) there is an urgent need for standardized methods and environmental monitoring programs for anthropogenic nanoparticles in order to appropriately assess the risks to biological species due to potential nanoparticle exposure. In doing so they 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 thereby generating baseline data that could facilitate the environmental risk assessment evaluations that are currently virtually absent.

NTA has recently become an ASTM method for the analysis of particle size distribution of nanomaterials in suspension being one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region, The ASTM (2012) guide describes the NTA technique for direct visualization and measurement of Brownian motion, generally applicable in the particle size range from several nanometers until the onset of sedimentation in the sample and is acknowledged as being capable of being routinely applied in industry and academia as both a research and development tool and as a QC method for the characterization of sub-micron systems.

Given the recognized importance of the subject of nanoparticles and their analysis and the fact that nanoparticles are already used in several consumer products including food, food packaging and cosmetics, and their detection and measurement in food represent a particularly difficult challenge, the European Commission published in October 2011 its recommendation on the definition of ‘nanomaterial’. This will have an impact in many different areas of legislation, such as the European Cosmetic Products Regulation, where the current definitions of nanomaterial will come under discussion regarding how they should be adapted in light of this new definition. This new definition calls for the measurement of the number-based particle size distribution in the 1–100 nm size range of all the primary particles present in the sample independently of whether they are in a free, unbound state or as part of an aggregate/agglomerate. Recently, Linsinger et al. (2012) have analyzed the requirements on measurements for the implementation of the European Commission definition of the term 'nanomaterial’.

Calzolai et al. (2012) have subsequently reviewed methods for measuring nanoparticles size distribution in food and consumer products. They gave an overview of the current state of the art, focusing particularly on the suitability of the most used techniques for the size measurement of nanoparticles when addressing this new definition of nanomaterials illustrating the problems to be overcome in measuring nanoparticles in food and consumer products with some practical examples. In assessing NTA and in comparison the other such techniques, they acknowledged that NTA was effective in overcoming the inherent weaknesses of the DLS and static light scattering methods when confronted with mixtures of relatively similarly sized particles and had a number of important advantages including relatively low instrument cost and high sensitivity which can detect nanoparticles at concentrations as low as 106 particle/cm3. They did point out however, the inherent limitation of the technique in not being able to detect nanoparticles below 10-20 nm meant it did not meet the full requirements of the EU definition and was, furthermore, a technique which required expertise on the part of the operator. In analyzing foodstuffs, Famelart et al. (2013) recently used NTA to determine heat-induced effects on the particle size distribution of casein micelles through the formation of disulphide bonds formed during acid gelation of preheated milk in the presence and absence of N-ethylmaleimide (NEM), a thiol-blocking agent.

Treatment of Wastes and Contamination

As nanoparticles become more widely spread throughout industry and consumer products, release from, and exposure to, such nanoparticle-containing materials becomes of increasing concern and the subject of intense study. Here we describe specific examples of the use of NTA for sizing and concentration measurement of nanoparticles in development of monitoring protocols as might be applied to industrial products and manufacturing processes.

Thus, Sachse et al. (2012) have studied the effect of nanoclay on dust generation during drilling of polymer nanocomposites, using NTA to follow particle size distribution and quantity. While there is currently a lack of information available in the literature on the nano and ultrafine particle emission rates from these, it was shown that the influence of nanoclay on mechanical drilling of PA6 composites, in terms of dust generation, has been reported with more particles in the size range between 175 and 350 nm being generated during drilling of the nanocomposites, these particles deposit in a shorter time. In a similar type of application, Njuguna et al. (2011) have investigated the nanoparticles generated from nanofiller reinforced polymer nanocomposites during structural testing.

Künniger et al. (2010) investigated the consequences for functionality and the aquatic environment of the release of conventional and nano-sized biocides from coated wooden façades during weathering. Extending these studies to show that Ag-NPs are likely transformed to silver complexes, which are considerably less toxic than ionic silver, Künniger et al. (2013), in her comparative study of metallic silver nanoparticles (Ag-NP), most recently compared conventional organic biocides used as transparent, hydrophobic coatings of wooden outdoor façades.

Cabot et al. (2012) have used NTA to monitor changes in tobacco smoke particle size when measured over a series of different time points. The health effects of automotive particulate pollution, specifically related to engineered Pd-nanoparticles, were studied by Wilkinson et al. (2011) using NTA and DLS to track particle aggregation in cell growth media. The measurement of soot-in-oil agglomerates from automotive engines was recently carried out by NTA and compared to TEM (La Rocca et al., 2013). Diluting used sump oil in heptane, both techniques showed that soot-in-oil exists as agglomerates with average size of 120 nm but that NTA was able to measure particles in polydisperse solutions and report the size and volume distribution of soot-in-oil aggregates with the advantage of being fast and relatively low cost compared with TEM.

A new SAE Standard (equivalent to MIL-L-21260) covering military engine oils suitable for preservation, break-in, and lubrication of reciprocating internal combustion engines in equipment used in combat/tactical service has recently been proposed in which NTA was used to establish protocols for measuring soot agglomerates size distribution in used automotive lubricant oils (SAE Standard (2013).

Peetsch and Epple (2011) employed DLS, NTA, SEM, energy-dispersive X-ray spectroscopy (EDX), X-ray powder diffraction (XRD), atomic absorption spectroscopy (AAS), thermogravimetric analysis (TG), and elemental analysis in their characterization of the solid components of three desensitizing toothpastes and a mouth wash.

Having established that, to March 2011, there existed over 100 food and food-related nanoproducts, Chen et al. (2012) investigated and developed a simple test for the characterization and preliminary toxicity assay of nano-titanium dioxide additive in sugar-coated chewing gum. Using NTA their results surprisingly 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 and that 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. Similarly, van Landuyt et al. (2013) showed by NTA that nanoscale particles exist in dental abrasives (up to 60vol %) and that dental personnel (and patients) may inhale nano-sized dust particles (<100 nm) during abrasive procedures to shape, finish or remove restorations.

Recognizing that no standard test method is currently available for evaluating the efficiency of personal protective equipment against nanoparticles, in particular in the case of gloves, Dolez et al. (2011) used NTA and other techniques to determine the rate of nanoparticle penetration through protective gloves in conditions simulating glove-nanoparticle occupational interaction. They reported on commercial 15 nm TiO2 nanoparticles-powder and colloidal solutions in 1,2-propanediol, ethylene glycol and water and for four types of protective gloves (disposable nitrile and latex as well as unsupported neoprene and butyl rubber gloves) they showed that mechanical deformations and contact with colloidal solution liquid carriers may affect glove materials. Preliminary results obtained with TiO2 powder indicated a possible penetration of nanoparticles through gloves following mechanical deformations.

Textile materials with engineered nanoparticles (ENPs) have excellent properties as they are antibacterial, antimicrobial, water resistant and protective. The textile industry has recognized the importance and the advantages of ENPs, so they now comprise one of the fastest developing branches of processing and are the subject of significant patent activity, some of which employs NTA analysis in the description (Corona et al., 2013). The most important sources of ENPs released to the environment from textiles are textile-industry wastewaters and waters from large hospital or hotel laundries. Rezić (2011) has reviewed analytical techniques for the characterization of ENPs on textiles. In this context, the increasing number of nanomaterial-based consumer products raises concerns about their possible impact on the environment. In assessing the effluent from a commercially available silver nanowashing machine, Farkas et al. (2011) used inductive coupled mass spectrometry (ICP-MS) and TEM to confirm the presence of an average of 10 nm silver nanoparticles but employed NTA to determine that 60–100 nm particles were also present. The effluent was shown to have negative effects on a natural bacterial community as its abundance was clearly reduced when exposed to the nanowash water and they suggested that if washing machines capable of producing AgNPs become a common feature of households in the future, wastewater will contain significant loadings of AgNPs which might be released into the environment. Ling and Pui (2013) also used NTA to characterize nanoparticles from abrasive waterjet machining (AWM) and electrical discharge machining processes showing a peak size of 100-200 nm and that while the filtration systems of the cleaning systems were found to remove 70 and 90 % the nanoparticles present, the particle concentration of the filtered water from the AWM was still four times higher than that found in regular tap water.

Nanoparticle-containing matrices are being increasingly investigated for the removal of environmental pollutants from industrial process wastewaters. NTA was employed by Prasad et al. (2012) in their study of the adsorption of arsenite (As3+) on nano-sized Fe2O3 waste powder from the steel industry while Savu et al. (2010) earlier assessed the generation of airborne nanoparticulates during pulsed laser welding processes and considered methods for their removal.

Mallampati et al. (2012) demonstrated, in part by employing NTA, the enhanced heavy metal immobilization in soil by grinding with addition of a nanometallic Ca/CaO dispersion mixture. Raychoudhury et al. (2011) assessed the transport of two polyelectrolyte-stabilized zerovalent iron nanoparticles in porous media for the remediation of contaminated subsurface environments. Using DLS, NTA and laser Doppler velocimetry, they measured the aggregate size and surface charge of bare and carboxymethylcellulose-coated nZVI particles.

Similarly, Cheng et al. (2012) have recently described the synthesis of carbon-coated, porous and water-dispersive Fe3O4 nanocapsules with a diameter of about 120 nm (as determined by NTA) and their excellent performance for heavy metal removal applications. The heavy metals removal test they employed demonstrated the excellent affinity of nanocapsules, the high efficiency for different metals (>90%), 79 mg g−1 adsorption capacity for Pb2+ and ultrafast removal process (Pb2+, 99.57%) within 1 minute).

In developing a simple and rapid room-temperature aerosol deposition method to fabricate TiO2 films for photokilling/photodegradation applications, Park et al. (2012) used NTA to demonstrate a mean size of approximately 1µm on fracturing following impacting a glass substrate to form a functional thin film, a process known as aerosol deposition.

Investigating new techniques for enhanced oil recovery (EOR) Hendraningrat et al. (2012a) have undertaken a glass micromodel experimental study of hydrophilic nanoparticles retention for EOR, in which NTA was used to enumerate particles in both the influent and effluent in a glass micromodel. Further work reported an evaluation of oil recovery using nanofluid injection onto several water-wet Berea sandstone core plugs (Hendraningrat et al. (2012b). Hendraningrat and his colleagues have subsequently carried out and reported numerous further studies in this area in which NTA was used to determine the size, size distribution and concentration of nanoscale particles used in the field of EOR. Li et al. (2013) showed that a hydrophilic silica nanoparticles suspension enabled improved oil recovery by a 2-phase flow system. Hendraningrat also reported a coreflood investigation of nanofluid enhanced oil recovery again in low-medium permeability Berea sandstone (Hendraningrat et al., 2013a and 2013b) as well as comparing the effect of some parameters influencing enhanced oil recovery process using these silica nanoparticles (Hendraningrat et al., 2013c). The latest data regarding these studies has been reported recently: the retention of nanoparticles during flooding experiment in several water-wet Berea cores was investigated in 3 different ways involving continuously increasing pressure during single-phase coreflood experiment with microscopic visualization under SEM integrated with Energy Dispersive X-Ray Spectroscopy (EDX) to distinguish nanoparticles with other elements and NTA particle measurement between influent and effluent (Hendraningrat et al., 2013d).


The ability of the NTA technique to generate high resolution particle size distribution data as well as nanoparticle concentration data makes the technique ideally suited to the testing of filters and filtration processes.

Ling et al. (2011) have used NTA to measure particle (50–500 nm) concentration upstream and downstream of the filter to determine the filtration efficiency of a model membrane filter, the Nucleopore® filter, for application in the purification and disinfection of drinking water as well as removal of NPs in highly pure chemicals used in the industries. NTA measurements were found reliable within a certain concentration limit (about 108–1010 particles/cm3) and they stated that experimental results are comparable with previously published data obtained using an aerosolization method, thus validating the capability of the NTA technique.

Co-workers Boulestreau and Schulz have carried out extensive studies of filtration using NTA as the primary method for testing filter efficiency and performance. Thus, in describing the online analysis of the nanoparticles to prevent membrane fouling by a secondary effluent, Boulestreau et al. (2011a and 2011b) tested NTA in terms of reliability and reproducibility of the device as well as the impact of the prefiltration on the measurements made. They showed that NTA was able to measure the particle size distribution and the absolute particle concentration of particles between 100 and 1000 nm in secondary effluent. Their results showed clearly a relationship between the amount of nanoparticles below 200 nm and the filtration behavior. Further such work by Schultz et al. (2011) on improving understanding and prevention of membrane fouling in treated domestic wastewater used NTA to demonstrate that a combination of ozonation/coagulation showed synergistic effects and which led to an additional decrease of submicron particle content and further improvement of the filtration performance.

More recently Boulestreau and co-workers have described the on-line use of NTA in which it was used to optimize the ozonation and the coagulation conditions in a filter system. They stated that the fact that the absolute size and concentration of the nanoparticles can be observed within a few minutes thus allowing users to detect the effect of ozonation and coagulation on the nanoparticles and that the NTA instrument is “a highly capable device to analyze the nanoparticles” (Boulestreau et al., 2012).

Schulz (2012) described his work on sub-micron particle analysis to characterize fouling in tertiary membrane filtration in which he tested a combination of pre-ozonation, coagulation and subsequent low-pressure membrane filtration as an option for tertiary wastewater treatment. He showed that “by Nanoparticle Tracking Analysis (NTA) a reliable and reproducible detection of the colloid content in treated domestic wastewaters is possible. The effects of the pre-treatments on sub-micron particle size distribution and on the absolute concentration can be detected”. The results of his work demonstrated that ozonation and coagulation were found to reduce the content of small colloids < 200 nm by forming larger agglomerates, resulting in a better filterability of the water. A combination of both treatments shows synergetic effects and a further reduction of the particle content as well as of the total fouling resistance was observed. More recently, Boulestreau and Miehe (2013) have published guidelines for the use of online fouling monitoring in tertiary treatment; work carried out under a Project entitled OXERAM 2. In order to improve performance of both polymeric membrane and a microsieve pilot scale process, on-line monitoring was implemented. After a literature review and extensive laboratory testing at the Technical University of Berlin, two instruments were selected as being ideal for this purpose. The first was on-line NTA which was used to give “reliable and reproducible information about the concentration and size distributions of the colloidal fractions in the tested treated domestic wastewater”. The other instrument was a simple turbidometer but which was found to be less informative than NTA. As part of the same project, Godehardt et al. (2013) also used NTA in their study on the role of organic substances in tertiary treatment via oxidation and membrane filtration.

In an unrelated filtration problem, that of fractionation of nanocellulose by a foam filter, NTA was used in an attempt to measure bacterial nanocellulose in a sample of enzymatically pretreated nano-fibrillated cellulose from softwood. The length of nanofibres (many 10s of microns) often precluded the analysis of such material though sub-micron nanocrystalline cellulose was accessible to NTA (Tanaka et al., 2012).

Luechinger et al. (2010) earlier described a facile, broadly applicable method to prepare nanoporous silver films between 0.5−5 μm and 30−300 nm using soluble salt nanoparticles as pore templates testing them with filtration of aqueous dispersions of carbon nanoparticles (20 nm primary particle size) at a filtration efficiency of >99.6%.

In a study of the significance of electrostatic protein-polysaccharide interactions using bovine serum albumin (BSA) and sodium alginate (Na-Alginate) to specifically illustrate the contribution of this form of non-covalent network to membrane fouling, NTA was used to help demonstrate that soluble complex formation is governed by lowering zeta-potential sufficiently to enable positively charged micro-regions on the protein to bridge between negatively charged carboxyl groups on the alginate (Neemann et al. (2013)).

In his development of a recirculating aquaculture system in which accumulation of fine suspended solids and colloids can be avoided by integrating a membrane filtration unit into the system, Holan et al. (2013a) used NTA to identify how the feeding regime affected membrane performance and fouling phenomena caused by dissolved and submicron colloidal particles in the system and how the membrane impacted general water quality and particle characterization. He further reported on this system in his extended work on the Intensive rearing of cod larvae, Gadus morhua. Holan et al. (2013b) thus showed there is a great potential of implementing a membrane filtration system in aquaculture recycling systems.


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