Analytical chemistry of engineered nanomaterials: Part 2. analysis in complex samples (IUPAC Technical Report)

2.1.1 Sample preparation from liquid samples

One of the most common methods used for the pre-fractionation and pre-concentration of nanomaterials prior to characterization is membrane filtration which will remove particles larger than the membrane pore size [13]. However, when nanomaterial size is near the pore size of the membrane, only a fraction of the nanomaterial will be filtered. This partial filtration is caused by the variability of the membrane pore size, the non-spherical shape of the nanomaterials, particularly those present in environmental samples, and possible interactions such as electrostatic repulsion. Centrifugation and particularly ultracentrifugation is a second common approach that is used to separate or pre-concentrate nanomaterials in suspensions according to particle size and density.

Cloud-point extraction (CPE) has also been employed for pre-concentrating nanomaterials. Surfactant is added to an aqueous suspension at a concentration above the critical micelle concentration, and the sample is heated to the cloud point temperature [13]. Above this temperature, the surfactant separates from the liquid phase, and the nanomaterial is concentrated in the surfactant phase which can be separated by centrifugation. This method has been used in a number of studies as a key tool to enrich and extract nanoparticles from complex environmental matrices, prior to analysis and to preserve the colloidal status of unstable environmental samples [14]. Indeed, many different CPE protocols have been applied to the extraction and/or pre-concentration of metal ions, nanoparticles, or organic substances, and the speciation of metals and some other elements. Remarkably, the opportunity to combine state-of-the-art multi-element analyses using ICP-MS with CPE is still largely unexploited. Furthermore, the specificity of CPE and a detailed comparison with filtration and centrifugation methods have yet to be established. The dispersion of samples containing nanomaterials frequently uses sonication to break up particle agglomerates. The addition of energy to a colloidal system can result in unexpected or unwanted effects; therefore, sonication parameters (intensity, duration, temperature) must be optimized.

Extraction is a general technique often used for separation/preconcentration and speciation analysis. Various types of extraction in connection with atomic absorption spectrometry (AAS), ICP-OES, and ICP-MS have been applied for metallic nanoparticles, particularly in environmental water samples (see Tables in Section 3). A solid-phase extraction (SPE) can be coupled with sequential elution. The magnetic solid-phase extraction (MSPE) procedures have shown excellent performance with a low limit of detection (LOD), high extraction factors (EF), high recoveries, and acceptable precision [15], [16], [17]. It is important to ensure that the extraction process does not have an impact on the particle size distribution or agglomeration state, which underlines the importance of well-planned particle extraction procedures. To isolate nanoparticles from biological matrices, tissue degradation under acid or basic conditions is used. However, digestions are likely to lead to particle dissolution or aggregation, or nanoparticle surface alterations, which will provide false information about the physical state of the particles [18]. Therefore, except for resistant nanoparticles such as TiO₂, acid solubilization of biological tissues is generally not recommended. Alkali and enzymatic solubilizations have been used with success, although published results indicate that numerous factors may affect the efficiency of these methods.

2.1.2 Sample preparation from solid matrices

Direct solid sampling and slurry sampling have helped significantly to decrease the number of preliminary operations in the analytical process for the handling of solid samples. Slurries are solid dispersions in a liquid phase that can be transported as solutions, enabling the direct determination of an analyte, reducing the time required for analysis, and minimizing the risks of contamination by circumventing sample decomposition with wet or dry oxidation procedures. Some examples of the slurry sampling technique have provided interesting results. Direct slurry sampling with graphite furnace AAS (GFAAS) was used for the detection and sizing of metal nanoparticles in chicken meat matrix reference material [19]. Slurry sampling was also used for the determination of TiO₂ nanoparticles in river water by ICP-MS and ICP-OES [20], assessment of composition of surface-modified TiO₂ nanoparticle catalyst by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) [21], chemical analysis of size-tailored magnetic colloids of nickel and cobalt nanoferrites by ICP-OES [22], accurate sizing of CeO₂ nanoparticles by the analysis of the optical forward-scattered field [23] and for many other purposes. Nonetheless, several problems including calibration, weighing errors, sample inhomogeneity, etc., may become important, unless experiments are carefully optimized [24].

The selection of sample preparation technique for analysis of nanoparticles is determined by the sample matrix and objective of the analysis. Nanoparticles in liquid samples are usually subjected to preconcentration with varying degree of success using membrane filtration and centrifugation. On the other hand, the potential of cloud point extraction seems to be still largely unexploited and future research focus could bring this technique into forefront. The extraction of nanoparticles from solid samples is still dominated by sample/tissue degradation with alkali and enzymatic dissolution being the most used, however, direct analysis solid and slurry samples is on the rise.

2.3.1 Microscopy and spectroscopy

Electron microscopy (EM) is able to visualize nanoparticles at the single-particle level, and hence provides information about their physical size, shape, or aggregation state. As with other particle counting methods, it is essential to analyze a large number of particles to ensure counting statistics that are representative of the original sample. The number of particles that must be counted is larger for ENMs with high polydispersity. Except for three-dimensional tomographic methods, EM provides a two-dimensional representation of the particles. Most EM techniques function under a vacuum, potentially affecting the aggregation state and observed size of the particles. Environmental scanning electron microscopes (ESEM) have an inferior resolution (tens of nanometers), but they can be used under less strict vacuum conditions and even in liquid, which is useful for reducing artefacts due to agglomeration. Despite this advantage, the lower resolution is problematic for small particles sizes. Cryogenic preparation techniques or the embedding of samples in hydrophilic resins have also been used to conserve colloidal samples; however, the techniques are still not widespread for use with nanoparticles. As an example, ESEM has been employed to study nanoparticle uptake and translocation in plants [39].

The power of microscopy for chemical analysis is in its combination with spectroscopic tools to provide information on elemental composition when coupled with energy dispersive X-ray spectroscopy (EDS) and chemical structure when coupled with electron energy-loss spectroscopy (EELS) [18]. The techniques of EDS and EELS are complementary, covering measurements of both light and heavy elements.

Transmission electron microscopy (TEM) is generally the most useful EM technique for characterizing nanoparticles in complex matrices, as well as some nanoparticle coatings [33]. It is able to provide a spatial resolution below 1 nm, which can be matched by scanning electron microscopes (SEM) fitted with a field-emission electron gun. For instance, nanoparticles could be well characterized through the use of high-resolution field-emission gun scanning electron microscopy in combination with X-ray analysis (FEG-SEM/EDS) [40]. In this case, the chemical composition of nanoparticles was identified in earthworm tissue after exposure to contaminated soil.

High-resolution TEM is able to provide images with a subatomic level resolution which are useful for obtaining information on the crystal structure of nanomaterials. Selected-area electron diffraction (SAED) and convergent-beam electron diffraction (CBED) are complementary in that they can be used in combination with conventional TEM to further identify the crystal structures. These microscopy spectroscopies could be applied to any characterization scenario; however, both matrix complexity and concentration levels can practically limit their application [29, 33]. High-resolution scanning transmission electron microscopy (HRSTEM) has a unique ability to image local cellular environments adjacent to a nanoparticle at near atomic resolution and can be used in combination with EDS and EELS. These tools can be used to analyze particle location, translocation and potential reformation, ion dispersion, and the in vivo synthesis of second-generation nanoparticles. Such analyses can provide an in-depth understanding of tissue–particle interactions and insight into the effects that are caused by the nanoparticles [41].

Although optical microscopy has a much lower resolution than EM and often requires covalently attached fluorophore on nanoparticles, the recent combination of enhanced dark-field microscopy with hyperspectral imaging (EDF-HSI) has been successfully used to analyze ENMs in complex matrices. The analysis is performed under atmospheric pressure in the original sample matrix, and the results provide both spectral and spatial information. This technique enables fast and quantitative insights into the interactions between nanomaterials and organisms at the cellular or invertebrate level [42]. The utilization of EDF-HSI for the analysis of ENMs in biological matrices has been reviewed [43, 44].

2.3.2 Continuous separation techniques

The use of hyphenated techniques, such as continuous separation techniques coupled to elemental, light scattering, or spectroscopy detectors, is increasing due to their ability to simultaneously obtain particle size and multi-elemental information. These techniques were initially developed to reduce polydispersity and complexity of the sample prior to analysis. Field flow fractionation (FFF) is a separation technique that fractionates ENMs in a laminar flow in a narrow channel under the application of an external perpendicular field [45], [46], [47]. This method provides size information over a range of 1 nm to 100 mm, depending on the separation mode, allows one to maintain the native conditions of the dispersed sample and is compatible with coupling to a range of online detectors (e.g., ICP-OES, ICP-MS, UV–Vis, dynamic light scattering) and off-line collection of fractions for microscopy or other analyses.

While commonly-used FFF detectors, including UV, light-scattering, and fluorescence, do not provide nanoparticle compositional information and do not respond directly to the mass concentration of nanoparticles, ICP-OES and ICP-MS coupled to FFF can possess both nanoparticle sizing and compositional analysis at the μg L⁻¹ level with a detection level at least 10 to 100-fold lower than DLS or UV-FFF techniques [48]. Numerous variations of the FFF technique are continuously being developed including asymmetric flow FFF (AF4), sedimentation, thermal, magnetic, and electrical FFF. Excellent recoveries of nearly 90 % have been observed for Ag nanoparticles when the various flows were properly optimized [49]. Thus, FFF combined with elemental and sizing detectors has emerged as a tool with great potential for analysis of samples in complex matrices, including environmental and biological samples and consumer products [50].

The chromatographic separation of nanoparticles can be performed using hydrodynamic chromatography (HDC) and size exclusion chromatography (SEC). The main disadvantage of these techniques is the long analysis time per sample (around 30 min) and/or low sample injection volume (generally 20 μL), resulting in particle number concentration for environmentally relevant nanoparticles that fall below instrumental detection limits [18]. Whereas SEC has a limited size separation range, HDC has a low separation power with respect to either SEC or FFF [49]. Gel electrophoresis (GE) and capillary electrophoresis (CE) with UV–Vis or fluorescence detection are the most used techniques for the separation and characterization of nanoparticles based on their size, shape, and surface functionalization. The advantage of CE is the high resolution that is attainable in addition to its capacity to analyze both ionic species and nanoparticles. Although the potential of these two techniques has been examined, especially with respect to the gels (GE) and buffers (CE), they have rarely been applied to real world-samples. In both GE and CE, the addition of ionic surfactants plays a critical role in the separation of nanoparticles. The CE separations of bioconjugated quantum dots serve as examples [51]. The separation techniques have also been extensively used in the detection of nanoparticles in complex matrices and thus these techniques are discussed further in Section 3.

2.3.3 Atomic spectrometric techniques

Conventional atomic spectrometric techniques like ETAAS and ICP-OES can provide total elemental concentrations for a variety of samples. However, they are only able to provide information about the physicochemical form of the element (whether present as dissolved species or particles), when used together with size fractionation techniques (e.g., filtration, centrifugation) or when used online as an element-specific detector for a size fractionation method (e.g., FFF, HDC, SEC, EC).

Among different versions of ICP-MS available on the market, mass analyzers such as quadrupole (ICP-Q-MS), scanning sector field (SF-ICP-MS), static sector field with multi-collector detector (MCICP-MS), and time of flight (ICP-TOF-MS) are the most commonly used, each having several advantages and disadvantages [18, 52]. The quadrupole ICP-MS instruments are most appropriate for the detection of mono-elemental ENMs only. Triple quadrupole ICP-MS (ICP-QQQ-MS) offers an improved detection, e.g., for SiO₂ and for TiO₂ nanoparticles in complex matrices. Analysis of nanocomposites, naturally formed or unknown ENMs requires the simultaneous detection of all of the elements present in the single particle, which can be achieved by ICP-TOF-MS [52]. The enhanced capability of ICP-TOF-MS is reflected by its significantly higher cost.

Today, the single-particle ICP-MS (spICP-MS) is widely used for the analysis of metal-containing nanoparticles [53], [54], [55], [56]. It is exploited to give:

1. Qualitative information about the presence of particulate and/or dissolved forms of specific elements.

2. Characterization of the mass of a given element per particle. This information can be converted into particle size as long as information about the composition, shape, and density of the particles is known or assumed.

3. Quantitative information about particle number concentrations and mass concentrations of the dissolved and/or particulate forms of the inorganic nanoparticles [57].

This technique utilizes a standard ICP-MS setup and makes use of ultrafast, time-resolved detection to probe nanoparticles that are introduced into diluted suspensions, (ideally) one by one. The technique can be used down to parts per trillion particle number concentration levels. Quantification can be performed using nanoparticle standards of the same elemental composition or dissolved standard solutions, after taking into account the nebulization efficiency, to obtain particle size and size distributions. A comprehensive and systematic study of the accuracy, precision, and robustness of spICP-MS, using a rigorously characterized reference material issued by the National Institute of Standards and Technology (NIST), has been performed as well as necessary advancement toward full validation and adoption of spICP-MS by the broader research community [58]. Signal drift correction and flicker correction have been solved as well [59, 60]. Limitations of spICP-MS for the determination of nanoparticle size distributions and dissolution have been presented, demonstrating an impact of the ICP-MS sample introduction system [61].

Recent instrumental developments of spICP-MS, from the sample introduction system to the detector to coupling of spICP-MS to separation and fractionation techniques have been reviewed, along with some of the remaining challenges [62]. Among the important challenges are the sample preparation, the attainment of high transport efficiency, and the optimization of experimental conditions to ensure that nanoparticles enter the ICP fully vaporized, atomized, and ionized, regardless of their elemental composition, size, and sample matrix. The attainment of mass resolutions that provide isotopic information, the simultaneous multielement detection of nanoparticle signals (of the order of hundreds of microseconds), and the lack of appropriate reference materials for calibration are also challenges. Innovations and advances in the field of spICP-MS are represented by the coupling of spICP-MS to separation techniques such as capillary electrophoresis (CE), electrospray-differential mobility analysis (ES-DMA), AF4, flow injection (FI), isotope dilution, laser desorption and ablation, and time-of-flight (TOF) [62, 63].

Recently, a multi-mode determination of nanoparticle metal mass fraction and number concentration has been achieved based on a dual inlet system consisting of a pneumatic nebulizer and a microdroplet generator [64]. This setup provided seamless and robust operation in a total of three analysis modes. Each analysis mode is based on a different calibration principle, thus constituting an independent way to determine metal mass fractions and nanoparticle number concentrations and improving the validation capabilities of spICP-MS for nanoparticle analysis [64].

X-ray absorption spectroscopy (XAS) at both the near-edge X-ray absorption spectroscopy (XANES) region and the extended X-ray absorption fine structure (EXAFS) region, have proven to be invaluable tools for in situ structural characterization of ENMs [33, 65, 66]. The XANES spectra provide information about the oxidation state, fractional d-electron density, and the electronic environment of the absorbing atom, while the EXAFS spectra yield information about the element coordination such as the number, type, and distance of the backscattering atoms surrounding the central absorbing atom. Due to limitations in sensitivity (mg kg⁻¹ range), synchrotrons are usually needed to acquire the XAS spectra, so this technique is neither routine nor readily available [30].

2.3.4 Electroanalytical techniques

Electrochemistry is also considered as an efficient and cost-effective method able to detect, characterize and quantify various nanomaterials. Concerning the analysis of ENMs, two electroanalytical techniques have been particularly well developed – voltammetry of immobilized particles (VIP) and particle collision coulometry (PCC) – complementary techniques which can be used in combination. The VIP technique involves the immobilization of the nanoparticles on the electrode surface, which means that nanoparticles are separated from the media in which they are suspended, typically using a drop and dry approach, electrostatic interactions, or chemical deposition [67]. Various electrode materials, including chemically modified ones, have been developed for this purpose. If the surface coverage of the electrode is low enough, the peak potential can be directly related to the diameter of the nanoparticle; the peak potential and the area under the voltammetric peak can then be used to get qualitative and quantitative information, respectively. A molar-ratio method was applied to the determination of the stoichiometry and apparent binding constant of gold nanoparticle-organic capping complexes using voltammetric data [68].

For PCC, a modification of the baseline of a chronoamperogram (current vs. time plot) is evaluated when a metal or metal oxide nanoparticles randomly impacts the surface of a microelectrode held at a fixed potential, due to the oxidation/reduction of the nanoparticle. The charge involved in the process is related to the mass of the electroactive species [69]. Sensitivity can be increased via electrocatalysis, mass transfer, or using magnetic fields. Using electrochemical impedance spectroscopy and electrochemical scanning microscopy silver species present in linear low-density polyethylene (LLDPE) films with different Ag(I)/Ag(0) ratios and silver nanoparticle features usable as food contact polymers were characterized [70].

There is a large number of techniques available for determination and characterization of nanoparticles, and the selection of the technique really depends on the objective of the analysis. No single technique can be used as a universal tool to answer all analytical challenges and thus combining the advantages of individual techniques is necessary. Microscopic and spectroscopic analysis provide superior spatial resolution; however, they are not techniques of choice for particle number concentration. Continuous separation techniques are great tools for analysis of nanoparticles in complex matrices, but lack resolution when samples containing particles of similar size are analyzed. Atomic spectrometric techniques currently dominate the field of nanoparticles analysis because they are able to provide information about (i) the presence of particulate and/or dissolved forms of the analyte, (ii) mass of the analyte per particle, and (iii) particle number concentration. However, majority of spectrometric instruments used have quadrupole detector, and thus can monitor only one element at a time. Electrochemical analysis and X-ray absorption spectroscopy are very useful in chemical speciation of nanoparticles.

3.1.1 Engineered nanomaterials (ENMs) in waters

Many environmental ENM analyses involve water samples and spiked waters (surface, river, lake, sea, waste ones as blank material with known quantity values of ENMs added), which are analyzed directly or after a suitable dilution. As thermodynamic equilibrium does not exist for nanomaterials in water, physicochemical and chemical properties of nanomaterials in water depend on the surrounding media and may change during any handling/preparation process. Moreover, several artifacts may occur during sample transport, storage, and preparation, such as nanomaterial loss due to attachment to vials, contamination, and transformation. Therefore, it is essential to test the effect of transport, storage, and preparation on the nanomaterial by conducting control experiments with known quantities of well-defined particles [13]. For instance, filtration is often used because it removes microbial contamination and suspended particles in addition to aggregates. A recent filtration study has compared six different membrane filters for Ag and CeO₂ nanoparticles in aqueous samples analyzed by the emerging technique of spICP-MS. The highest recoveries were obtained for Milli-Q, rainwater, and river water samples with filtration over a polypropylene membrane [81].

Aside from traditional techniques for the determination of metal-containing nanoparticles in environmental waters, less conventional approaches for sample pretreatment, preconcentration, on- and offline detection, size characterization and quantification of metal-containing nanoparticles in environmental samples have also been used. Among them, cloud-point and solid-phase extractions provide high preconcentration factors and recoveries, and electrothermal AAS provides possibilities for the still challenging distinction between dissolved ions and nanoparticles should be mentioned. The combination of separation and detection, such as FFF and HDC with ICP-MS, UV–Vis, light scattering, and fluorescence detectors, is proved useful for the analysis of metal and metal oxides nanoparticles in waters containing organic matter, natural waters, and municipal wastewaters. Some recent studies are indicated in Table 3.

The technique of spICP-MS represents today the routine technique for the analysis of nanoparticles in natural waters and spiked waters [57]. The explosive growth and improvement in spICP-MS including the development of various sample introduction systems helps to analyze and understand nanoparticle behavior in complex matrices. In combination with size separation techniques, such as HDC or electrospray-differential mobility analysis, it is highly promising for the assessment of the shape and structure-related information on nanoparticles and their aggregates [82]. In that case, HDC provides information about the hydrodynamic diameter and spICP-MS gives information on the nanoparticle core which can help to distinguish between spherical and non-spherical particles as well as between pristine and surface-modified nanoparticles [83]. In a comparison with several methods including AF4, DLS, and TEM, the combination of HDC and spICP-MS is recommended for particle size and concentrations determination in waters with high recoveries [84]. Particle coincidence and dissolved analyte interferences, matrix effects, data acquisition and processing, the influence of instrument components, and other factors should be considered [54, 56, 62, 85]. Detection and quantification of ENMs requires their distinction from natural nanoparticles with similar intrinsic properties such as composition, size, surface chemistry, and other. Application of multi-element analysis to individual particles using spICP-TOF-MS revealed that specific metal elements are often present in natural nanoparticles but no specific multi-element signatures were detected for ENMs [86].

Many nanoplastics are retained in wastewater even after the wastewater treatment plant and others end up in the sludge. Nanoplastics have not been adequately researched and are thus difficult to characterize [87]. Some but not all quantification techniques require complex sample pretreatment or total removal of the organic-rich matrix or biofilms present. Regarding characterization and quantification, together with the FTIR and Raman spectroscopy as the most common techniques for analysis of plastic functional groups, other destructive methods, such as pyrolysis gas chromatography mass spectrometry (Py-GC-MS) and thermal extraction desorption gas chromatography mass spectrometry (TED-GC-MS), may be desirable.

3.1.2 ENMs in soil and sediments

The introduction of ENMs from anthropogenic contributions to soils and sediments occurs through industrial activities, wastewater sludge, consumer, and agricultural products. The isolation and pre-concentration of metal nanoparticles is an important component in the preparation of soil samples. Conventional extraction methods involve the use of dialysis, centrifugation, filtration, liquid–liquid extraction, or liquid–solid extractions, which can affect the physicochemical properties of the nanoparticles [88]. The principal methods used to detect and characterize ENMs in soils and sediments include TEM, HDC, AF4, and DLS. Both HDC and AF4 are typically coupled to specific detectors, such as DLS, ICP-MS, or fluorescence. The approach based on the combination of in situ complementary and hyphenated analytical techniques can effectively be used to interrogate and quantify these complex interactions involving natural matrices [89]. As in water matrices, at low concentrations of ENMs, most conventional analytical techniques are not able to take advantage of inherent differences (e.g. in terms of composition, isotopic signatures, element ratios, structure, shape, or surface characteristics) to distinguish between naturally occurring nanoparticles and ENMs of similar composition [90]. However, spICP-TOF-MS has been shown to distinguish between such nanoparticles and ENMs in soil by simultaneously detecting multiple elements or isotopes on an individual particle level [91].

For the detection, speciation, and quantification of micro- and nanoplastics in alpine snow, new highly sensitive method based on thermal desorption–proton transfer reaction–mass spectrometry (TD-PTR-MS) could be of interest closing the methodological gap of chemical characterization and quantification at a nanogram scale [92].

3.1.3 Nanoparticles in air and aerosols

Aerosols are very complex in structure and can contain a great number of different chemical compounds even within a single particle. They should be analyzed for both size and chemical composition, in real-time or off-line applications. Characterization of aerosol nanoparticles is critical to the advancement of science and practical nanotechnologies. Key areas cover, among others, rapid aerosol nanoparticle measurements, detection, characterization, and behavior of low nanometer particles, nanoparticle size distribution measurements, and particle standards for size, concentration, morphology, and structure.

Single-particle aerosol mass spectroscopy (SPAMS) is employed for the determination of aerosol particles, thus including emissions and distribution patterns of atmospheric nanoparticles. In comparison to filter-based analytical techniques and traditional individual particle measurements, SPAMS is advantageous due to its continuous real-time detection of single particles [93].

Among challenges on the novel field of nanoplastics and other pollutants in environmental samples such as snow, natural and drinking water, and air there are their low concentrations, consequences of non-existent standards, losses during sample preparation, weathering and oxidation changes, as well as degradation. The amount of organic matter in the soil matrix, the discrimination and identification of large aggregates are also challenging, and can be solved by coupling size fractionation to molecular analysis [113].

3.5.1 Fate of engineered nanomaterials (ENMs) in environmental samples

The quantification and characterization of ENMs are crucial for the assessment of their environmental fate, transport behavior, and health risks. At the same time, ENMs are not static entities, and their characterization studies must also include information about the dynamics of the interfacial region between the nanomaterial surface and the surrounding medium. Thus, the identification and accurate characterization of engineered and naturally occurring nanoparticles is a difficult task. Biological interactions and environmental fate of engineered nanoparticles are affected by colloidal stability and aggregation. The fate of ENMs refers to both the chemical and physical changes (transformations) occurring to the particles while in natural systems, also to their mobility and localization in a specific environment. The physicochemical properties of the nanomaterial and the environmental conditions (e.g., pH, ionic strength, organic matter, and oxygen concentration) determine which transformation processes are relevant. The transformation processes may occur simultaneously or interfere with each other. Especially important is the fact that the pristine and transformed forms of nanoparticles may differ in uptake and toxicity (Fig. 4).

Fig. 4:

Schematic overview of the processes determining the forms/species in which nanomaterials may occur in the natural environment; adapted from [57].

For instance, Ag nanoparticles adsorb dissolved organic matter (DOM), preferentially short-chained DOM compared to long-chain and aromatic ones, thus inhibiting aggregation at higher Ag nanoparticles concentrations [200]. The surface composition of TiO₂ (5 nm) and α-Fe₂O₃ (2 nm) nanoparticles was shown to be controlled by the adsorption of biological components (proteins and amino acids), inorganic oxyanions (phosphates and carbonates), and environmental ligands (humic acid). The extent of surface adsorption depends on the solution phase composition and the affinity of different components to adsorb to the nanoparticle surface with a range of possible surface interactions, adsorption energetics, and adsorption modes including reversible adsorption, irreversible adsorption, and co-adsorption [201]. The Ag nanoparticles released into the environment are mostly transformed into silver sulfide (Ag₂S) nanoparticles, and corresponding speciation analysis (Ag₂S, Ag, and AgCl nanoparticles in the presence of Ag⁺ ions) by selective extraction and sequential elution is of importance for understanding environmental processes [202]. Fates of ZnO and CeO₂ nanoparticles during the typical drinking water treatment process investigated by spICP-MS confirmed that particle concentrations were reduced by a minimum of 60 % and most were reduced by more than 95 % from source water to final drinking water [203].

The major transformations of ENMs include:

1. Dissolution. A transformation process is referred to as dissolution if it breaks the solid nanoscale (below 100 nm) form of the material down into ionic or molecular dispersions due to interactions with water or other solvents. Dissolution only affects inorganic nanomaterials (metal or metalloid). Organic and carbon-based inorganic nanomaterials, such as fullerenes and carbon nanotubes, do not dissolve, at least not in the same manner as metals or other chemical elements.

2. Attachment and aggregation with natural particles (heteroaggregation). Nanomaterials are prone to form agglomerates with the natural suspended matter, natural colloids, and aerosols or attach to the surfaces of larger natural particles such as soil and sediment grains.

3. Sorption of natural organic matter. Natural organic matter can be sorbed by particles in aqueous media to form a coating around the surface of the nanomaterial, also described as an eco-corona. The presence of a natural organic matter (NOM) coating is known to interfere with dissolution and heteroaggregation. Depending on the type of NOM, heteroaggregation is reduced by NOM that provides additional stability to nanomaterials by replacing the original coatings, resulting in electrostatic and steric repulsion between particles [204, 205].

The different properties of ENMs result in significant differences in the fate, transformation, antimicrobial activities, and effects on biological wastewater treatment [206]. Understanding the behavior of nanoparticles in the environment, such as their permeability into biological tissues, becomes key to understanding their toxicological effects. Analytical monitoring of the size distribution of the nanoparticles over time is a useful indication of their stability, which will influence other biological (including toxicity) and chemical reactions [56]. The fate of nanoparticles in the environment can be monitored by numerous analytical techniques mentioned above (Section 2). Most techniques used for nanoparticle aggregation analysis are limited to ensemble measurements or require harsh sample preparation that may introduce artifacts. An ideal method would analyze aggregate size in situ with single nanoparticle resolution. The spICP-MS method was established as a high-throughput analytical technique to quantify nanoparticle size distributions and aggregation in situ [207]. In addition, the novel coupled methods (called hyphenation techniques which typically represent the combination of a separation technique with spectroscopic detection technology) like electrospray-differential mobility analysis with single-particle inductively coupled plasma mass spectrometry (ES-DMA-spICP-MS) should have the capacity for real-time size, mass, and concentration measurement of nanoparticles on a particle-to-particle basis. The feasibility of this technique was validated through both concentration and mass calibration using NIST gold nanoparticle reference materials. The independent and simultaneous quantification of nanoparticle size and mass can provide detailed information on nanoparticle aggregation states, differentiating aggregated nanoparticles and nonaggregated states based on the “apparent density” derived from both DMA size and spICP-MS mass [208]. Using a combination of X-ray based spectroscopic image analysis with near edge X-ray absorption fine spectra (NEXAFS) possessed necessary analytical chemical information on nanoplastics particles [209].

Material flow models indicate that a variety of ENMs may accumulate in waste streams, generating nanowaste following the disposal of end-of-life ENMs and nano-enabled products. Challenges in nanowaste characterization and appropriate analytical techniques include: separation techniques coupled to spectrometry-based methods as promising tools to detect nanowaste and determine particle size distributions in liquid waste samples; standardized leaching protocols that can be applied to generate soluble fractions stemming from solid wastes, while microfiltration and ultrafiltration can be used to enrich nanoparticulate species; imaging techniques combined with X-ray-based methods as powerful tools for determining particle size, morphology, and screening elemental composition. Nonetheless, the quantification of nanowaste is currently hampered due mainly to difficulties in differentiating engineered from naturally occurring nanoparticles. A promising approach to deal with these challenges might be the application of nanotracers with unique optical properties, or elemental or isotopic fingerprints [210]. For analytical monitoring of fate of nanoplastics in municipal waste water treatment plants, the use of nanoparticles labeled with a rare metal like Pd or In is proposed [211].

3.5.2 Fate of engineered nanomaterials (ENMs) in biological systems and biodistribution

The biotransformation of nanoparticles involves different processes, including aggregation/agglomeration, and reactions with biomolecules that affect metabolism and are reflected in their toxicity [212]. Exposure of biological systems to ENMs can happen via different routes including ingestion of food or medicines, intravenous injection, inhalation, and skin contact (e.g., by body care products or textiles). Moreover, some nanoparticles are studied and used as drug carriers, radiosensitizers, and imaging agents. Their biodistribution is essential for evaluating their efficacy and safety [170]. The biodistribution of ENMs is affected by their chemical/elemental composition, size and size distribution, shape, coatings, surface properties, and their stability. For instance, cellular uptake of Ag nanoparticles is size-, dose- and coating-dependent [213]. The results of biodistribution studies give qualitative and often quantitative insights into the organs and tissues in which ENMs accumulate, and consequently, allow the determination of the actual dosage of ENMs at the site of toxicity as well as the organs that are at risk for suffering adverse effects from the ENMs [214]. Biodistribution studies are often carried out as in vivo studies for which test animals (often rats and mice) are commonly used, or, in some dedicated cases, humans [215]. Commonly, different parameters of interest are varied and tested, including ENM concentration, size, and shape as well as the duration of exposure, which is often up to 90 days.

From an analytical point of view, the reliable detection and quantification of the tissue distribution of ENMs is not trivial and requires well-developed methods and multi-instrumental approaches. All steps of the analysis must be properly addressed, including sample collection, sub-sampling, sample storage, sample preparation, and final instrumental analysis. For the dissolution of tissues and particle-containing body fluids, acid digestion in open or closed systems (generally with nitric acid in combination with hydrochloric acid, hydrofluoric acid, and hydrogen peroxide), enzymatic digestion or soft dissolutions and extractions are often used [214]. A variety of imaging and analytical methods have been developed to study nanoparticles in cells with benefits and limitations associated with each method. Here, HRSTEM should be mentioned with its unique ability to image the local environment of a nanoparticle at near-atomic resolution and apply analytical tools to these environments such as EDS and EELS [216]. The combination of ICP-MS and EM has been shown to be a valuable tool to identify which organs or tissues contain the most ENMs. Various types of ICP-MS and spICP-MS and related combined techniques together with XANES spectroscopy were used in the development of methylmercury detoxification mechanism via in vivo formation of HgSe nanoparticles [11].

The spICP-MS method was used to observe a size decrease for CeO₂ nanoparticles in lung and liver tissue over time [217], to detect Au nanoparticles in biological fluids [218], and to monitor the absorption and distribution of Ag nanoparticles into deeper skin layers in the ionized form [219]. In combination with DLS and UV–Vis, spICP-MS was also employed to evaluate changes in size, aggregation, chemical composition, and silver speciation of four different sizes of Ag nanoparticles exposed to four different formulations of artificial sweat [220]. To identify single nanoparticles of metals and metal oxides in biological tissues spICP-MS combined with FFF was used [214]. Complementary techniques (spICP-MS, TEM, and HPLC-ICP-MS) revealed the degradation of 40 nm citrate-stabilized Au nanoparticles in rat liver to smaller particles and low-molecular weight Au species [221]. Inductively coupled plasma mass spectrometry, X-ray fluorescence, and X-ray absorption spectroscopy helped to demonstrate the whole-body biodistribution of administrated SiO₂-Fe₃O₄ core–shell nanoparticles [222]. The application of μ-XANES showed the biotransformation of 25 nm CuO and CuSO₄ into Cu₃(PO₄)₂ (acute assays), whereas 40 and 80 nm CuO remained as CuO (chronic assays) [223]. Mass cytometry techniques have been developed for single-cell analyses and used to quantify the cellular association of inorganic nanoparticles [171], and the heterogeneous interaction between nanoparticles and human immune cells at a single-cell level [172].

Analytical methods for the determination of nanoliposomes and their associated fate in vivo include fluorescence labeling, radiolabeling, magnetic resonance imaging (MRI), MS, and computed tomography, each with its applicability and limitations [224]. Nanoplastics are potentially more hazardous than microplastics because of their small size, allowing them to penetrate cell membranes, induce toxicological impact on their environment, and cause possible risk to humans [225]. The mutual interrelationship between vascular plants and micro- and nanoplastics began to attract attention considering both the effect of vascular plants on the fate of these plastics and the effect of plastics on vascular plants [226]. Fluorescence confocal microscopy (LSCM) and SEM have been used to observe the internalization and specific locations of nanoplastics after absorption on plants [227].