Chady Stephan

The National Nanotechnology Initiative defines engineered nanomaterials (ENM) as those with dimensions of 1–100 nm, where their unique characteristics enable novel applications to be carried out. ENMs often possess different properties than their bulk counterparts of the same composition, making them of great interest for a broad spectrum of industrial, commercial, and healthcare uses. However, the widespread application of ENMs will inevitably lead to their release into the environment, which raises concerns about their potential adverse effects on the ecosystems and their impact on human health.

	This installment of “Atomic Perspectives” discusses the theory of single-particle inductively coupled plasma–mass spectrometry (SP-ICP-MS) and its use in characterizing and sizing metal-based nanoparticles. The single-particle analytical technique can quantitate the difference between ionic and particulate signals, measure the particle concentration (particles per milliliter), and determine the particle size and size distribution. In addition, it enables users to investigate particle agglomeration and dissolution. As a result, SP-ICP-MS is a key analytical technique in assessing the fate, behavior, and distribution of ENMs in various sample matrices. Besides discussing its fundamental principles, the real-world applicability of SP-ICP-MS will be exemplified by evaluating its capability in various sample matrices (1).

	The unique properties of engineered nanoparticles (ENPs) have made them an essential component of many consumer, industrial, and healthcare products and as a result created intense interest in their environmental behavior (2). For this reason, it is not only important to know the type, size, and distribution of nanoparticles in soils, potable waters, and wastewaters, but it is also crucial to understand their environmental cycle. Therefore, to ensure the future development of nanotechnology products, there is clearly a need to evaluate the risks posed by these ENPs that will require proper tools to fully understand their impact on the ecosystem. Current approaches to assess exposure levels include predictions based on computer modeling, together with direct measurement techniques. Predictions through modeling are based on knowledge of how they are emitted into the environment and by their behavior in the samples being studied. Although the life cycles of ENPs are now starting to be understood, very little is known about their environmental behavior. Prediction through life-cycle assessment modeling requires validation through measurement at environmentally significant concentrations. For ENPs that are being released into the environment, extremely sensitive methods are required to ensure that direct observations are representative in time and space. ENPs differ from most conventional ‘‘dissolved’’ chemicals in terms of their heterogeneous distributions in size, shape, surface charge, composition, degree of dispersion, and so on. For this reason, it is not only important to determine their concentrations, but also these other important metrics.

	The measurement and characterization of nanoparticles is therefore critical to all aspects of nanotechnology. In the field of environmental health, it has become clear that complete characterization of nanomaterials is important for interpreting the results of toxicological and human health studies. Metal-containing ENPs are a particularly significant class because their use in consumer products and industrial applications makes them the fastest growing category of nanoparticles (3).

	Many analytical techniques are available for nanometrology, only some of which can be successfully applied to environmental health studies. Methods for assessing particle size distributions include electron microscopy, chromatography, laser-light scattering, ultrafiltration, and field-flow fractionation. However, the lack of specificity of these techniques is problematic for complex environmental matrices that may contain natural nanoparticles having polydisperse size distributions and heterogeneous compositions. For this reason, sensitive detection techniques are needed if specific information about the elemental composition and concentration of the nanoparticles is required. Unfortunately, difficulties can also arise with some detection techniques due to a lack of sensitivity for characterizing and quantifying particles at environmentally relevant concentrations.

	One technique that is proving invaluable for detecting and sizing metallic nanoparticles is SP-ICP-MS (4). Its combination of elemental specificity, sizing resolution, and unmatched sensitivity makes it extremely applicable for the characterization of ENPs containing elements such as Ag, Au, Ti (TiO

), to name a few, which have been integrated into larger products such as consumer goods, foods, pharmaceuticals, and personal care products.

	Much of the early work has focused on the use of ICP-MS with particle separation techniques, such as field-flow fractionation and chromatography (5). However, more recently, SP-ICP-MS is showing a great deal of promise in several application areas, including the determination of silver nanoparticle concentrations in complex samples (5). This technique is suited to differentiate between the analyte in solution and existing as a nanoparticle without any prior separation techniques, simplifying nanoparticle analysis while eliminating complex sample preparation steps (6). This ability allows SP-ICP-MS to provide information on the size and size distribution of nanoparticles, as well as the dissolved concentration of the analyte.

	SP-ICP-MS involves introducing nanoparticle-containing samples at environmentally significant concentrations into the ICP-MS system and collecting time-resolved data. Because of the very low elemental concentrations and the transient nature of ionized nanoparticles, high sensitivity and very short measurement times are necessary to ensure the detection of individual particles as ion pulses (7,8). The number of observed pulses at the detector is related to the nanoparticle concentration by the nebulization efficiency and the total number of nanoparticles in the sample, while the size of the nanoparticle is related to the pulse intensity. Figure 1 represents the full cycle process of characterizing the size of nanoparticles that have been released into the environment from a manufactured product using SP-ICP-MS.

	In this example, nano-Ag bactericide imbedded in athletic socks is released during simulated wash cycles (step 1, Figure 1). By analyzing a simple aqueous solution with the ICP-MS system (step 2) and collecting data using the SP-ICP-MS technique (step 3), the size, size distribution, concentration, and associated dissolved material can be quantified. The dissolved Ag content is at low signal intensity, with nanoparticles creating pulses above this background where the height of the pulse relates to the mass of analyte and the number of pulses correlates to the concentration of nanoparticle in the samples. The size distribution of particles in the sample can be calculated using well-understood SP-ICP-MS theory (step 4) (9). A histogram of nanoparticle diameter versus number of events (nanoparticle number) can then be created to visualize the nanoparticle distribution in the sample, in addition to calculating the concentration of both nanoparticle and dissolved fractions released from the products (step 5). Let’s take a closer look at the individual steps in this process.

	Effectively detecting and measuring single, individual nanoparticles with ICP-MS requires operating the instrumentation in a different manner than when analyzing dissolved samples. Figure 2 shows traces from both dissolved- and single-nanoparticle analyses. In Figure 2a, a steady-state signal results from measuring dissolved elements; the output when detecting single particles is quite different, as illustrated for 60-nm silver particles in Figure 2b. Each spike in Figure 2b represents a particle. The difference in the way these data are generated plays a key role in understanding single particle analysis. The easiest way to understand this is to compare the processes involved when both dissolved elements and particles are measured.

	When dissolved elements are measured, aerosols enter the plasma where the droplets are desolvated and ionized. The resulting ions enter the quadrupole to be sorted by their mass-to-charge ratios (

). The quadrupole spends a certain amount of time at each mass before moving to the next mass; the time spent analyzing each mass is called the “dwell time.” After each dwell time measurement, a certain amount of time is spent for the electronics to stabilize before the next measurement is performed. This stabilization time is called “settling time”-that is, overhead and processing time. When analyzing dissolved elements, the resulting signal is essentially a steady-state signal. However, considering the dwell and settling times, a significant amount of the signal is not measured because of the settling time of the electronics, a critical aspect when analyzing nanoparticles, as shown in Figure 3. It is important to point out that with dissolved ions, the part of the signal that is missed is not critical because the elements are dissolved and produce a continuous signal.

	Particles present in an aqueous solution are introduced to the plasma the same way as dissolved solutions. As the droplets are desolvated in the plasma, the resulting particles are ionized producing a burst of ions (one ion cloud per particle). The ions then pass into the quadrupole. However, using conventional ICP-MS data collection, alternating between dwell time and settling time, ion clouds are not always detected. If, for example, the ion cloud happens to fall within the dwell time window, it will be detected. Otherwise, if it passes into the quadrupole or reaches the detector during the settling time, it will not be detected, leading to an inaccurate counting efficiency. Figure 4 shows that an ion cloud from a single particle can be missed if it falls outside of the dwell time window, as demonstrated by the first and third dwell time windows. However, when the ion cloud from a single particle falls within the dwell time window, it is detected, as represented by the peak falling within the second dwell time window. When multiple particles are detected in rapid succession, the resulting signal is a series of peaks, each one originating from a particle, as shown in Figure 5.

	Figure 6 is a representation of the timing parameters involved in ICP-MS analysis. The three axes represent signal intensity, mass (

), and time. With conventional analyses of dissolved ions, the mass and intensity axes are the most important: The resulting spectra are plots of m/z versus intensity. The time axis is important when analyzing a transient signal. The quadrupole scan speed is important when measuring multiple elements in a transient signal, such as for laser ablation or multielement speciation analyses.

	However, when measuring transient signals for a single mass, the time axis (transient data acquisition speed in Figure 6) becomes important, since enough data points must be acquired to define the peak. For example, with high performance liquid chromatography (HPLC)–ICP-MS, usually 4–10 points per second are enough to define a peak. Comparing HPLC peaks to single-particle signals, the ion packets from each particle are typically 1000 times narrower than peaks produced by HPLC. Therefore, data must be acquired significantly faster for single-particle analysis. Since only a single mass is typically being measured for single particle analysis, the quadrupole scan speed is not important, and the time axis becomes the “transient data acquisition speed,” which encompasses both the dwell and settling times. For that reason, the faster the transient data acquisition speed, the better suited the system is for single-particle analysis.

	In SP-ICP-MS, transient data acquisition speed consists of two parameters: dwell time (reading time) and settling time (overhead and processing time). It is very important that the ICP-MS is able to acquire signals at a dwell time that is shorter than the particle transient time, therefore avoiding false signals generated from partial particle integration, coincidence and agglomerates or aggregates. The shorter the settling time, the less chance there is of missing a particle. Figure 7 demonstrates the importance of settling time using a constant 100-μs dwell time and a constant time window. In Figure 7a, there are only two 100-μs windows to detect particles; the rest of the time is overhead, where data cannot be acquired. In this case, there are only about 100 measurements made in one second. Therefore, most of the time is wasted by scanning and settling. Figure 7b is the same time scale, but with a settling time of 100 μs. Therefore, more time is spent measuring and looking for nanoparticles-about 5000 measurements in one second. However, still half of the time is wasted. Figure 7c represents the ideal situation with no settling time. This allows for 10,000 measurements per second, with no wasted time being spent scanning and settling-the ideal situation for SP-ICP-MS.

	Another benefit of fast continuous data acquisition is that multiple points can be measured from a single particle, thus eliminating the chances that particles are missed or that only partial ion clouds from particles are detected. Figure 8 shows how this can be accomplished. In Figure 8a, the signal from a single particle is measured multiple times. The signal from each time slice is plotted, which defines the peak. When multiple particles are detected, the resulting peaks are a series of time slices, as shown in Figure 8b.

	Figure 9 shows how typical single-particle responses can be converted into peaks that define a single particle. In Figure 9a, data was collected in fast continuous mode (no settling time) with a dwell time of 100 μs. When the intensities from the first 1.6 s are plotted, it is seen that six points define a peak. In Figure 9b, the dwell time was reduced to 50 μs, which leads to twice as many data points being acquired. As a result, the peak shape is defined by 12 points, leading to a different peak shape. These examples demonstrate the benefit of sampling multiple data points per particle.

	Converting Nanoparticle Pulse Counts to Nanoparticle Diameter

	It is important to emphasize that ICP-MS is a mass-based technique where particle size is determined by relating the pulse intensity to an elemental mass. With traditional ICP-MS analysis, the first step in this process is to create a calibration curve using dissolved standards. This curve connects the signal intensity from the instrument to the concentration of the analyte entering the plasma. The next step is to relate the concentration of the dissolved analyte to a total analyte mass that enters the plasma during each reading. This relationship between analyte concentration and the mass observed per event is called the “mass flux,” which is highly dependent on the transport efficiency of the sampling process. This transport efficiency must be calculated for each instrument and under the given run conditions for the mass flux to be accurate. In this way, the resulting calibration curve relates signal intensity (counts/event) to a total mass transported into the plasma per event.

	By using well-understood SP-ICP-MS principles, the intensity of each individual pulse (counts/event) can then be transformed using the mass flux calibration curve to determine the particle mass, which can then easily be converted to particle diameter, by knowing the density and assuming the geometry (shape) of the particle is spherical (10). This process is exemplified in Figure 10, which shows a signal of multiple silver nanoparticles over time (Figure 10a), with an individual pulse at the bottom left (Figure 10b) and the dissolved ionic calibration curve on the top (Figure 10c). On the top right is shown the mass of the particle (in micrograms) per dwell time after nebulizer efficiency and flow rate have been applied (Figure 10d). The mass of the nanoparticle is then converted to a diameter, which is based on the mass fraction and the density, which is based on the shape of the particle (Figure 10e).

	Let’s now take a look at the real-world applicability of SP-ICP-MS by evaluating its capability and suitability for characterizing nanoparticles in some common sample matrices, including surface waters and human blood.

	Assessing the Fate of Silver Nanoparticles in Surface Water Using SP-ICP-MS

	Most analytical techniques are not suitable for environmental matrices, because nanoparticle concentrations are typically very low (11). Historically, particle size has been measured by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM), while dissolved content has been measured by ultrafiltration. These common techniques have known limitations for measuring low concentrations in the presence of colloidal species in complex waters.

	On the other hand, SP-ICP-MS has been found to be a promising technique for detecting and characterizing metal nanoparticles at very low concentrations. Silver nanoparticles are one of the most frequently studied types of nanoparticles, because they are among the most common nanomaterials found in consumer products such as laundry detergents and personal hygiene products and tend to release free Ag ions, especially at low concentrations. The aim of this study was to investigate the use of SP-ICP-MS for the detection and characterization of metal nanoparticles in environmental waters where they can be involved in various physicochemical processes.

	It is important to point out these types of dissolved species can also be determined by ultrafiltration followed by total metal quantification using ICP-MS or atomic absorption spectrometry. However, this procedure is time consuming since it requires the preequilibration of the filtration membrane for at least three cycles of centrifugation, which is typically 20 min each (12). Additionally, aggregates and remaining stable silver nanoparticles can be counted and measured by other commonly used techniques (DLS, TEM) but SP-ICP-MS is the only practical method that can distinguish between silver nanoparticles and other colloids in surface waters in a timely manner.

	The ICP-MS system (NexION 350X, PerkinElmer Inc.,) instrumental parameters used for this analysis are given in Table I. SP-ICP-MS data acquisition was carried out using the instrument’s Syngistix Nano Application software module (13). The sample introduction system consisted of a quartz cyclonic spray chamber, a type C0.5 concentric glass nebulizer, and a 2-mm-bore quartz injector.

	Commercially available suspensions of gold and silver nanoparticles were used in this work. A reference material (NIST 8013) consisting of a suspension of gold nanoparticles (60 nm nominal diameter, 50 mg/L total mass concentration and stabilized in a citrate buffer) was used to determine the nebulization efficiency. Suspensions of silver nanoparticles were used, including citrate-coated (40 and 80 nm nominal diameter) and bare (80 nm nominal diameter) nanosilver suspensions (Ted Pella Inc.).

	The surface water was sampled in Rivière des Prairies (Montreal, Canada) and filtered with 0.2-μm filter paper before spiking with silver nanoparticles. Silver nanoparticle suspensions were added to water samples with concentrations ranging from 2.5 to 33.1 μg Ag/L and left to equilibrate under continuous and gentle shaking. Before SP-ICP-MS analysis, small aliquots of the samples were diluted to below 0.2 μg Ag/L.

	Data acquisition was performed in triplicate for each sample, and deionized (DI) water was analyzed between replicates to check memory effects. As shown in Figure 11, the Syngistix Nano Application Module generates plots of frequency versus size (nanometers) plots with an interactive focusing window, eliminating the need for any subsequent data processing using theoretical equations described earlier.

	As previously described, SP-ICP-MS data processing is based on distinguishing between the signal of dissolved metal and that of nanoparticles, counting the pulses (or events) corresponding to individual nanoparticles and converting their intensities to particle size. The frequency of the events (pulses) provides particle number concentration, and the intensity of each pulse is proportional to the mass of analyte. The latter was converted to volume and then into size knowing the density and the geometry of the particle using the Syngistix Nano Application module for fully automated data acquisition; subsequent manual data processing is not required.

	Even after filtration of surface waters at 0.2 μm, an NTA system (LM14, NanoSight Ltd.) with a green laser at 532 nm showed the presence of colloidal particles with an average diameter of approximately 110 nm. Therefore, the addition of metal nanoparticles to this complex matrix will make their detection and characterization very difficult, if not impossible, with commonly used techniques such as DLS and TEM because they lack specificity to elemental composition.

	Furthermore, even the determination of the dissolved fraction that is usually performed by ultrafiltration may be inadequate because silver ions could adsorb onto the surface of the colloids and, therefore, be retained by the filtration membrane. As a result, the proportion of dissolved metal will be underestimated. SP-ICP-MS measurements were found to be more effective and to have fewer limitations than other techniques. Additionally, the presence of other insoluble particles does not interfere with the analysis of silver nanoparticles, as the Ag signal is recorded independently of the other constituent elements of the colloids.

	The study carried out further investigations to better understand the evolution of the average particle diameter and the percentage of dissolved metal over time in both pure and river water (14). The individual data will not be presented here, but in all cases, the average particle size of the persistent nanoparticles remains substantially constant. For suspensions of particles with a nominal diameter greater than 40 nm, between 50 and 80% of the particles persist for at least five days of equilibration in pure and surface water. Under the experimental conditions of this work, the coating appears to have no significant effect on the dissolution of nanoparticles over time-both citrate-coated and bare silver nanoparticle (80 nm) suspensions showed a slight decrease of particulate silver by approximately 20% during five days. Additionally, for the same size and equilibration time, the proportion of dissolved silver was found higher in the case of citrate-coated silver nanoparticles. This does not necessarily mean that bare nanosilver is more stable than citrate-coated silver nanoparticles. It could be that the release of silver ions may be due to oxidation to residual Ag ions adsorbed on the surface of silver nanoparticles or bonded to the coating. We believe that the stability and behavior of nanoparticles in any medium will depend on the synthesis procedure. This is supported by the fact that smaller particles with a nominal diameter below 40 nm tend to dissolve at a faster rate.

	Determination of Gold and Silver Nanoparticles in Blood Using SP-ICP-MS

	The small size of nanoparticles and their potential for enhanced reactivity resulting from larger surface area per volume has raised concerns about their adverse effects on human health. Although these properties may enhance their desired application benefits, they may also introduce new, unwanted toxic effects (15). Two metal nanoparticles, gold nanoparticles and silver nanoparticles, have been widely studied. Gold nanoparticles are particularly interesting due to their desired intrinsic properties such as high chemical stability, well-controlled size and surface functionalization, while silver nanoparticles, due to their antibacterial properties, are often applied in wound disinfection and in coatings of medical devices and prostheses, and also in commercial textiles, cosmetics and household goods (16). As a result, concerns have been raised about the migration of silver nanoparticles from bandages or medical devices into open wounds and thus, the blood stream. These concerns emerge from recent publications showing that nanoparticles can directly be taken up by the exposed organs and are able to translocate using the blood stream to secondary organs, such as the central nervous system, potentially affecting the growth characteristics of embryonic neural precursor cells (17). Therefore, the need exists for researchers to detect and measure nanoparticles in blood. The work described here explores the ability of SP-ICP-MS to detect and measure gold and silver nanoparticles in whole blood (18).

Experimental Samples, and Sample Preparation

	A blood Standard Reference Material (Seronorm Trace Elements in Whole Blood, Level I) was diluted 20 times with tetramethylammonium hydroxide (TMAH) plus 0.1% Triton-X. Gold and silver nanoparticles (gold-30 and 60 nm, NIST 8012, 8013; silver-40 and 60 nm, Ted Pella Inc.) were added to each blood sample at various concentrations. To break up any agglomerated particles, the stock solutions were sonicated for 5 min before spiking in the blood. The blood samples were manually shaken before analysis.

	The ICP-MS system (NexION 350X, PerkinElmer, Inc.) instrumental parameters used for this analysis are given in Table II. SP-ICP-MS data acquisition was carried out using the instrument’s Syngistix Nano Application software module. Calibrations were carried out with both dissolved and particulate gold or silver standards, which are shown in Table III.

	Two rinses were used between samples: 1% HCl plus 0.1% Triton-X was aspirated to dissolve and remove any residual gold particles, followed by deionized water to remove traces of the hydrochloric acid. A solution of 1% HNO

 plus 0.1% Triton-X was used as a rinse solution for silver particles. This two-solution rinse approach was found to be essential because residual acid could dissolve particles in the sample. Each rinse solution was aspirated for 1 min.

	Initial tests were performed with gold nanoparticles. Figure 12 shows a blood sample spiked with a mixture of 30- and 60-nm Au nanoparticles (each approximately 100,000 particles/mL). There are clearly two size distributions, indicating that both particle sizes are seen. This sample was analyzed three times consecutively, with the measured particle sizes shown in Table IV. These results demonstrate both accuracy and repeatability of the methodology.

	Next, 40- and 60-nm Ag nanoparticles were added to the blood samples so that the final, total particle concentration was about 200,000 particles/mL. Figure 13 displays the detected particle distribution, and Table V shows results for three replicates of the spiked blood sample. It’s worth pointing out that the software module first converts the peak area intensity into mass and the resulting mass into diameter. This is then displayed as frequency of events versus nanoparticle diameter, as described earlier.

	To see if lower particle concentrations could be detected in blood, only 40-nm Ag nanoparticles were spiked into the blood samples at a nominal concentration of 50,000 particles per milliliter, half the concentration of the previous analysis. The data shown in Table VI demonstrate that even at low concentrations, Ag nanoparticles can be accurately and reproducibly measured in blood.

	As has been shown, measuring single particles with ICP-MS is quite different than measuring dissolved species. The most important factor when measuring single particles is the speed at which data can be acquired: since particle ionization events are on the order of microseconds, rapid data acquisition and elimination of the settling time between measurements are crucial. Continuous measurement allows multiple readings per particle ionization event, which results in more-accurate size determinations. For SP-ICP-MS analysis, continuous data acquisition at a dwell time shorter than or equal to 100 μs is the most important instrumental requirement for precise nanoparticle counting and sizing.

	The investigation has also shown that when using automated data acquisition software optimized for SP-ICP-MS measurements, it is possible to study the behavior of silver nanoparticles in surface water without using any subsequent manual data processing. The technique has allowed the effective and selective measurement of changing particle size, aggregation and dissolution over time at low concentrations. Based on these data, SP-ICP-MS is uniquely suited to provide such information on the fate of metal nanoparticles at very low concentrations in environmental surface waters. This work has also demonstrated the ability of SP-ICP-MS to rapidly and accurately detect and measure gold and silver nanoparticles in whole blood, both at low concentrations and in mixtures. Although this study has just focused on gold and silver nanoparticles in surface waters and blood samples, there is no doubt that it is applicable to other types of metal and metal oxide nanoparticles in a variety of complex matrices including wastewater, effluents, culture media, as well as biological tissue and fluids (19,20).

		White Paper: Weighing the Benefits and Risks of Nanotechnology, Single Particle ICP-MS Compendium of Applications, PerkinElmer Nanomaterial Reference Library, 

https://www.perkinelmer.com/lab-solutions/resources/docs/Nanomaterials_SP-ICP-MS_Compendium(012982_01).pdf

		Nanotech-Enabled Consumer Products Continue to Rise: The Project on Emerging Nanotechnologies, 2017, 

http://www.nanotechproject.org/news/archive/9231/

		D.M. Mitrano, A. Barber, A. Bednar, P. Westerhoff, C.P. Higgins, and J.F. Ranville,

		D.M. Mitrano, E.K. Leshner, A. Bednar, J. Monserud, C.P. Higgins, and J.F. Ranville, 

Articles

The National Nanotechnology Initiative defines engineered nanomaterials (ENM) as those with dimensions of 1–100 nm, where their unique characteristics enable novel applications to be carried out. ENMs often possess different properties than their bulk counterparts of the same composition, making them of great interest for a broad spectrum of industrial, commercial, and health care uses. However, the widespread application of ENMs will inevitably lead to their release into the environment, which raises concerns about their potential adverse effects on the ecosystems and their impact on human health.

Single-Particle ICP-MS: A Key Analytical Technique for Characterizing Nanoparticles

Single-particle inductively coupled plasma–mass spectrometry (SP-ICP-MS) is an exciting new technique for detecting and characterizing metal nanoparticles at very low concentrations. It is fast and can provide significantly more information than other traditional techniques, including particle number concentration, particle size, and size distribution, in addition to the concentration of dissolved metals in solution. The added benefit of using ICP-MS is that it can distinguish between particles of different elemental compositions. The study will investigate the use of SP-ICP-MS to track the release of engineered nanoparticles (ENPs) into the environment and to better understand their fate and behavior specifically in drinking, surface, and wastewater samples. 

	
	The unique properties of engineered nanoparticles (ENPs) have created intense awareness in their environmental behavior. Because of the increased use of nanotechnology in consumer products, industrial applications, and healthcare technology, nanoparticles are more likely to enter the environment. For this reason, it is not only important to know the type, size, and distribution of nanoparticles in soils, potable waters, and wastewaters, but it is also crucial to understand their impact on the growing mechanism of crops used for human consumption. Therefore, to ensure the future development of nanotechnology products, there is clearly a need to evaluate the risks posed by these ENPs, which will require proper tools to fully understand their toxicological impact on human health. Current approaches to assess exposure levels include predictions based on computer modeling, together with direct measurement techniques. Predictions through modeling are based on knowledge of how they are emitted into the environment and by their behavior in the samples being studied. Although the life cycles of ENPs are now starting to be understood, very little is known about their environmental behavior. Prediction through life-cycle assessment modeling requires validation through measurement at environmentally significant concentrations. For ENPs that are being released into the environment, extremely sensitive methods are required to ensure that direct observations are representative in time and space. ENPs differ from most conventional ‘‘dissolved’’ chemicals in terms of their heterogeneous distributions in size, shape, surface charge, composition, and degree of dispersion. For this reason, it is not only important to determine their concentrations, but also these other important metrics, particularly when they are discharged and interact with their real-world surroundings.
	
	

Impact of Nanoparticles Released into the Environment


	
	When nanoparticles enter the environment, they can undergo a number of potential transformations that depend not only on the properties of the nanoparticles but also on the medium they are being released into. These changes typically involve chemical and physical processes, but they can also involve biodegradation of surface coatings used to stabilize many nanomaterials. For example, the toxicity impact of nanomaterials on algae involves adsorption onto cell surfaces, which has the potential to disrupt transportation through the cell membrane. Additionally, larger organisms can directly ingest nanoparticles, and then enter the food chain when they are consumed by other aquatic and terrestrial forms of life. These processes are further complicated by aggregation of nanoparticles with other natural minerals and natural colloids, which will dramatically change their fate and potential toxicity in the environment.
	
	Nanomaterials from domestic, medical, and industrial sources may also undergo significant changes during wastewater cleaning processes, such as the conversion of silver nanoparticles to silver sulfide in treatment plants. In addition, aggregation of the nanomaterials with other minerals and organic matter in the wastewater often results in the nanomaterial combining with other solids in the effluent, rather than remaining as a dispersed nanoparticle suspension. So, apart from atmospheric deposition or accidental spillage of nanomaterials directly into rivers, lakes, and the surrounding land, wastewater treatment remains by far the largest source of nanomaterial contamination either from the runoff into groundwater sources and drinking water supplies or from the raw sewage sludge that is often spread onto the soil as a fertilizer (1).
	
	
	


	
	Therefore, risk assessment for nanomaterial released into the environment is still evolving, and reliable measurements of environmentally significant concentrations remain challenging. Predicted environmental concentrations based on current usage are low, but they are expected to increase as the use of nanomaterials increases. At this early stage, comparisons of estimated exposure data with known toxicity data indicate that the predicted environmental concentrations are orders of magnitude below those known to have environmental effects on living plant and animal biota. As more toxicity data are generated under environmentally relevant conditions, risk assessments for nanomaterials will improve to produce accurate assessments that ensure environmental safety.
	
	Many analytical techniques applied in materials science and other scientific disciplines could also be applied to ENP analysis. However, environmental and biological studies may require that methods be adapted to work at extremely low concentrations in complex matrices. The most pressing research needs are the development of techniques that introduce minimal artifacts to optimize sensitivity, specificity, and throughput, as well as differentiating between naturally occurring particles and manufactured nanoparticles, together with a better understanding of their dissolution properties.


	
	The measurement and characterization of nanoparticles is therefore critical to all aspects of nanotechnology. In the field of environmental health, it has become clear that complete characterization of nanomaterials is important for interpreting the results of toxicological and human health studies. Metal-containing ENPs are a particularly significant class because their use in consumer products and industrial applications makes them the fastest growing category of nanoparticles (2).
	
	Although many analytical techniques are available for nanometrology, only some can be successfully applied to environmental and health studies. Methods for assessing particle size distributions include electron microscopy, chromatography, laser light scattering, ultrafiltration, and field flow fractionation. However, the lack of specificity of these techniques is problematic for complex environmental matrices that may contain natural nanoparticles having polydisperse size distributions and heterogeneous compositions. For this reason, extremely sensitive detection techniques are needed if specific information about the elemental composition and concentration of the nanoparticles is required. Unfortunately, difficulties can also arise with some detection techniques because of a lack of sensitivity for characterizing and quantifying particles at environmentally relevant concentrations.
	
	


	
	One technique that is proving invaluable for detecting and sizing metallic nanoparticles is single-particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) (3). Its elemental specificity, sizing resolution, and unmatched sensitivity make it extremely applicable for the characterization of ENPs containing elements such as Ag and Au and compounds such as TiO

that have been integrated into larger products such as consumer goods, foods, pharmaceuticals, and personal care products.
	
	Much of the early work has focused on the use of ICP-MS with particle separation techniques, such as field flow fractionation and chromatography (4). However, more recently, SP-ICP-MS has shown a great deal of promise in several applications areas, including the determination of concentrations of silver nanoparticles in complex samples (5). This technique is suited to differentiate between the analyte in solution and existing as a nanoparticle without any prior separation techniques, simplifying nanoparticle analysis while eliminating complex sample preparation steps (6). This ability allows SP-ICP-MS to provide information on the size and size distribution of many varied and different metal-based nanoparticles, as well as the dissolved concentration of the analytes under study. Let’s take a closer look at the fundamental principle of SP-ICP-MS.

	SP-ICP-MS is based on the measurement of the signal intensity produced by a single particle. Nanoparticle suspensions are sufficiently diluted to minimize the chances that more than one particle reaches the plasma at a time. The particle is atomized and ionized, producing a signal of relatively high intensity, which is measured as a pulse. This process is exemplified in Figure 1, which shows particles (P) and dissolved analyte in the sample aerosol entering the plasma and being ionized. The ions then pass through the interface region into the ion optics where they are eventually separated in the mass spectrometer. In this manner, particles are detected as individual pulses, whereas the dissolved analyte contributes to a continuous background signal. The frequency of pulses (events) provides the particle number concentration, whereas the intensity of each pulse is proportional to the mass of the nanoparticle. Because of the short transient nature of the pulse, very short integration times are necessary to maximize the detection of individual particles as pulses of ions after they are ionized by the plasma.

	This process is exemplified in Figure 2, which shows both metal nanoparticles and metal ions in solution being ionized by an ICP-MS system. The signal from the dissolved ions is represented by the continuous signal below the dashed line, while the ionized pulses of nanoparticles are represented by the individual spikes. For this approach to work effectively, the speed of data acquisition and the response time of the detector must be fast enough to capture the time-resolved nanoparticle pulses, which are typically 300–400 µs (3). If the electronics are not fast enough, multiple or many pulses can easily pass through in a single integration window leading to inaccurate particle counting and sizing. For this application, the ICP-MS should be capable of using dwell times shorter than the particle transient signal time, thus avoiding false signals generated from clusters of particles. In practice, for an instrument that is optimized for nanoparticle characterization, this means using dwell times of 100 µs or less and a settling time of zero, so that the pulse can be fully characterized and precisely integrated using a peak area integration algorithm (7).

	It is important to emphasize that ICP-MS is a mass-based technique, so in SP-ICP-MS, the particle size is determined by relating the pulse intensity to an elemental mass. With traditional ICP-MS analysis, the first step in this process is to create a calibration curve using dissolved standards. This curve connects the signal intensity from the instrument to the concentration of the analyte entering the plasma. The next step is to relate the concentration of the dissolved analyte to a total analyte mass that enters the plasma during each reading. This relationship between analyte concentration and the mass observed per event is called the 

, which is highly dependent on the transport efficiency of the sampling process and the instrument ion optics. This transport efficiency must be calculated for each instrument and under the given run conditions for the mass flux to be accurate. In this way, the resulting calibration curve relates signal intensity (counts/event) to a total mass transported into the plasma per event. So by using well-understood SP-ICP-MS principles, the intensity of each individual pulse (counts/event) can then be transformed using the mass flux calibration curve to determine the particle mass, which is easily converted to particle diameter, by knowing the density and assuming that the geometry (shape) of the particle is spherical (8,9). This is exemplified in Figure 3, which shows a signal of multiple gold nanoparticles over time (Figure 3a), with an individual pulse on the left-hand-side at the bottom (Figure 3b) and the dissolved ionic calibration curve on the top (Figure 3c). The top right-hand-side of Figure 3d shows the mass of the particle (in micrograms) per dwell time after nebulizer efficiency and flow rate have been applied. The mass of the nanoparticle is then converted to a diameter, based on the mass fraction and the density, based on the shape of the particle (Figure 3e) (10). Note: If the nanoparticle is rod-shaped (for example) a different calculation is used.

	Now, let’s take a look at the real-world applicability of SP-ICP-MS by evaluating its capability and suitability for characterizing nanoparticles in environmental samples.

Real-World Applicability of SP-ICP-MS for Water Samples

	Silver (Ag) nanoparticles are among the most commonly used nanoparticles in consumer products, such as fabrics, deodorants, and detergents because of their antimicrobial properties. Therefore, it is expected that Ag ENPs will find their way into the environment, necessitating ways to accurately and rapidly detect and characterize them in a variety of environmental matrices. Work has already been performed demonstrating the ability to successfully characterize Ag ENPs and their dissolution characteristics in a variety of drinking water (11), surface water (12), and wastewater samples (13). Let’s take a closer look at these three very different application requirements for SP-ICP-MS. Note: All the data presented in the following section were generated on a NexION 350D ICP-MS system using the Syngistix Nano Application Software Module (PerkinElmer Inc.) (14). 

	SP-ICP-MS is an ideal tool for assessing the efficiency of drinking water treatment systems in removing silver, gold, and titanium dioxide (TiO

) nanoparticles without using any other analytical technique. To evaluate the effectiveness of the water treatment process for these types of nanoparticles, Donovan and colleagues (11) collected water samples both pre- and post-treatment at three water treatment plants in Missouri. None of the six waters contained measurable amounts of Ag or Au, either as particles or dissolved species. However, all source water samples contained TiO

, as shown in Table I. Plants 1 and 2 effectively removed both dissolved Ti and TiO

 particles, as evidenced by the lower amounts present in the post-treatment waters than the pretreatment samples. The results from Plant 3 differed from the first two plants in that Ti-containing particles could still be detected after treatment, although significantly less than the pretreatment sample. However, all the dissolved Ti was removed. 

	To check the accuracy of this method, spike recovery tests were performed for all metals, both as dissolved (Diss.) and particle (Part.) concentrations. Three samples were spiked with 2 μg/L of the dissolved metals and 100-nm nanoparticles at a concentration equal to 1 × 10

 particles/mL. Table II shows the results of these spike recovery studies, which indicate accurate recoveries, except for the concentration of TiO

 nanoparticles, which recovered below 10% for each sample. Low recovery for TiO

 particles was most likely because of aggregation in the standards and in the water matrix. Without the addition of a surfactant, these uncapped TiO

 particles tended to aggregate, which resulted in particle loss between dilutions and in the water matrix where they had an opportunity to react and form new species or aggregate further and fall out of solution. The highest recovery obtained for these particles in ultra-pure water and in a surface water matrix were 24% and 9%, respectively.

	For this work, Hadioui and colleagues (12) investigated the efficiency of SP-ICP-MS for the detection and characterization of metal nanoparticles in fresh surface waters where they can be involved in various physicochemical processes as shown in Figure 4. In this example, dissolved silver (A), including released free Ag ions (C, D) and soluble complexes (E) can easily and instantly be measured by SP-ICP-MS. These dissolved species could also be determined by ultrafiltration followed by total metal quantification using ICP-MS or atomic absorption spectroscopy (AAS), but this procedure is time consuming since it requires the pre-equilibration of the membrane for at least three cycles of centrifugation, generally 20 min each (15). Aggregates (B) and remaining stable silver nanoparticles (G) can be counted and measured by other commonly used techniques such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and transmission electron microscopy (TEM), but SP-ICP-MS is the only method that can distinguish between nano-Ag (nAg) and other metal-based colloids in surface waters.

	The surface water was sampled from a river in Montreal, Canada, and filtered with 0.2-μm filter paper before spiking with silver nanoparticles. Nano-Ag suspensions were added to water samples with concentrations ranging from 2.5 to 33.1 μg Ag/L and left to equilibrate under continuous and gentle shaking. Commercially available suspensions of gold and silver nanoparticles were used in this work. A National Institute of Standards and Technology (NIST) reference material (RM 8013) consisting of a suspension of gold nanoparticles (60 nm nominal diameter, 50 mg/L total mass concentration, and stabilized in a citrate buffer) was used to determine the nebulization efficiency, while two types of silver nanoparticle suspensions were used for the dissolution studies-a citrate-coated version (40 nm and 80 nm nominal diameter) and a noncoated version (80 nm nominal diameter). In addition, 60-nm nominal diameter Ag nanoparticles were used for quality control (QC) purposes.

	An interactive view of data acquisition of intensity as a function of time for the 60-nm nominal diameter silver nanoparticles is shown in Figure 5. In this example, the concentration of the analyte was 208 ng/L and was converted to volume and then into size knowing the density and the geometry of the particle using the Syngistix Nano Application Module, with no further need for manual data processing.

	It is also important to emphasize that even after filtration of surface waters at 0.2 μm, the NTA method, which is commonly used for this type of analysis, showed the presence of quite significant amounts of colloidal particles with an average diameter of ~110 nm. Therefore, the addition of metal nanoparticles to this complex matrix would make their detection and characterization very difficult, if not impossible, with the commonly used techniques previously mentioned, such as DLS and TEM. Furthermore, even the determination of the dissolved fraction that is usually performed by ultrafiltration may be inadequate because silver ions may adsorb on the surface of the colloids and, therefore, be retained by the filtration membrane. Consequently, the proportion of dissolved metal will be underestimated. SP-ICP-MS measurements were found to be more effective and to have fewer limitations than other techniques. Indeed, the presence of other insoluble particles did not interfere with the analysis of silver nanoparticles, as the signal of Ag is recorded independently of the other constituent elements of the colloids.

	The same group of researchers found that the SP-ICP-MS technique allowed the effective and selective measurement of changing particle size, aggregation, and dissolution over time at low concentrations. In fact, they concluded that SP-ICP-MS is probably the only suitable technique that can provide such information on the fate of metal nanoparticles at very low concentrations typically found in environmental waters (15).
	
	

	Another common, more complex matrix that must be evaluated for the determination of Ag ENPs is wastewater samples from wastewater treatment plants. The complexity and variety of the wastewater matrices can make the analysis of ENPs extremely challenging. Figure 6 is a representation of a wastewater treatment plant that shows the possible pathways that could introduce nanomaterials into the environment and the potential impact they might have on the surrounding land and water supplies (1).

	A recently published study by Azodi and colleagues (13) evaluated the ability of SP-ICP-MS to characterize Ag ENPs in three common wastewater matrices: mixed liquor, effluent, and alginate solutions. The wastewater samples were collected from a wastewater treatment plant near Montreal, Quebec, Canada. The effluent wastewater was collected after the secondary settling tank, while the mixed liquor was collected after the secondary aeration tank. The effluent wastewater is the final treated wastewater that is discharged to the river from this treatment plant, while the mixed liquor is the wastewater that leaves the aeration tank after biological treatment to settle the suspended solids in the settling tank. As a result, the mixed liquor has much higher levels of suspended solids and a relatively higher dissolved carbon content compared to the effluent wastewater.

	Alginate, more commonly known as alginic acid, is an anionic polysaccharide distributed widely in the cell walls of brown algae, which is present at parts-per-million levels in wastewaters and comprises the dissolved organic carbon fraction of wastewaters. The alginate solution was used as a known control-surrogate for comparison with the wastewater samples. All solutions were prepared using the alginic acid sodium salt from brown algae (at 6 ppm) in deionized water by shaking end-over-end for 1 h. Ag ENPs capped with polyvinylpyrrolidone (PVP) with a mean diameter of 67.8 ± 7.6 nm (as determined with TEM) were spiked into 10 mL of all samples at a concentration of 10 ppb (5 million particles/mL). The samples were then diluted 10–1000× with deionized water and sonicated for 5 min before analysis. All samples were prepared in triplicate. To determine the accuracy of SP-ICP-MS, the Ag ENPs were added to deionized water at a concentration of 0.1 ppb (50,000 particles/mL). The SP-ICP-MS measurement determined the mean size of the Ag ENPs to be 63.2 nm (which agrees with the TEM measurements), and the concentration to be 53,758 particles/mL, thus validating the accuracy of the measurements.

	Next, the Ag ENPs were measured in a 6 ppm alginate solution. Once the accuracy of the technique was ensured, it was followed by the measurement of the effluent wastewater and mixed liquor samples. First, the total Ag concentration was measured in both wastewater samples and was found to range from 25 to 40 ppt, levels that should not inhibit the determination of Ag ENPs. Figure 7a shows the Ag particle size distribution for 0.1 ppb (50,000 particles/mL) in the alginate sample, which corresponds to a mean particle size of 66.1 nm, with a concentration of 52,302 particles/mL. The agreement between the measured and TEM-determined particle sizes indicates that the alginate matrix does not affect the measurement accuracy as exemplified in Figures 7b and 7c, which show the measured particle size distributions for the effluent and mixed liquor respectively. Table III shows the measured particle sizes of both samples that agree with the certificate value, together with particle concentrations, which are close to the calculated value, indicating that neither of the wastewater matrices impact the measurement and strongly suggesting that Ag ENPs can be successfully measured in wastewater samples.

	The detection limits for both the Ag particle size and concentrations in the wastewaters were also determined. For determining the particle size detection limits, the diluted samples were analyzed without any Ag ENPs being added. The detection limit was determined by running the unspiked wastewater matrices and observing the particle size, which corresponded to the smallest recorded peak. For the effluent, the detection limit was about 18 nm, while for the mixed liquor, it was about 12 nm. To determine the lowest concentration of Ag ENPs that could be detected, Ag ENPs were spiked into deionized water and diluted multiple times (Note: Since the wastewater matrices were shown not to affect the results, the detection limit was only measured in deionized water). The measured particle concentration was then recorded for each concentration. The particle concentration detection limit was determined to be the spiked particle concentration where the measured concentration did not change when the sample was diluted. In this work, the Ag ENP particle concentration detection limit was determined to be 25 ppt (12,500 particles/mL).

	SP-ICP-MS demonstrates very exciting potential for the characterization of nanoparticles in many varied types of environmental samples. It is a very effective tool to assess the efficiency of drinking water treatment systems in removing certain nanoparticles, without the need for any additional techniques. It has also allowed the effective and selective measurement of changing particle size, aggregation, and dissolution over time at low concentrations in natural surface waters. Additionally, the technique has shown the versatility to measure and analyze nanoparticles in various matrices found in a wastewater treatment plant. There is no question that compared to other traditional analytical methods, SP-ICP-MS offers unique capabilities in tracking the fate and behavior of metal nanoparticles at extremely low levels in the environment. Although this study has focused on the effectiveness of the technique for the characterization of nanoparticles likely to be present in environmental water samples, it is also applicable to other types of metal and metal oxide nanoparticles in a variety of complex environmentally significant matrices including plant material (16) and biological tissue (17).

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 is the manager of global applications of nanotechnology at PerkinElmer, Inc., in Woodbridge, Ontario, Canada. 

 is the principal of his own freelance writing and scientific consulting company, Scientific Solutions, based in Gaithersburg, Maryland. Direct correspondence to: 
	

SP-ICP-MS demonstrates excellent potential for characterizing nanoparticles in varied types of environmental samples.

Chady Stephan

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