Atomic Spectroscopy

Atomic spectroscopy is the most used method for the determination and quanti fi-cation of trace elements in environmental samples. In this method, high heat is used to decompose the sample in atoms and ions (a process called atomization) that are measured by specific detectors. Atoms are detected and quantified based on the emission (optical emission spectrometry, OES) or absorption (atomic absorption spectrometry, AES) of light, while ions are separated based on mass-to-charge ratios (mass spectrometry, MS) (Skoog et al.1998).

Currently, atomization for OES is produced by a type of discharge called plasma, which is supported by argon and usually called “inductively coupled plasma (ICP)” (Boss and Fredeen2004). According to Skoog et al. (1998)“plasma is an electrical conducting gaseous mixture containing a significant concentration of cations and electrons, at similar concentrations.” Argon ions in the mixture can absorb a big amount of energy from an external source to maintain highly elevated

temperatures (as high as 10,000 K). The basic principle of ICP consists in the introduction of a liquid sample into the argon plasma in the form of an aerosol produced by a nebulizer. The droplets forming the aerosol are carried along with some vapor that undergoes atomization to produce free atoms and ions that can be measured and quantified. Two main techniques, optical emission spectrometry (OES) and mass spectrometry (MS), coupled to the ICP (ICP-OES/MS), are among the most used for uptake determination and quantification of different elements.

ICP-OES provides lower cost analysis when compared to ICP-MS; thus, it is widely used as quantification tool. However, this technique can only be used to determine the elemental composition or dissolution of the NMs (Elzey 2010) and metallic elements of the NMs taken up by plants (Larue et al.2012a). Moreover, with this technique, it is not possible to discriminate between the amounts of NMs adsorbed/absorbed by plant roots (Larue et al.2012a).

On the other hand, ICP-MS is widely used to measure the number of single charged ions in a sample and separate them according to the mass-to-charge ratio (Boss and Fredeen 2004). According to Thomas (2013), ICP-MS is the “fastest growing trace element analysis technique” currently available. ICP-MS offers high sensitivity for metals/metalloids with detection limits ranging from the parts per million (ppm) to the parts per trillion (ppt) levels (Schaumann et al.2015; Arruda et al.2015). The ICP-MS alone can only be used to determine concentration and composition. However, in the last decade, ICP-MS has been coupled with other analytical techniques, allowing the measurement of other variables, including par-ticle size and distribution, thus increasing its capabilities for NMs determination in environmental samples. Examples of ICP-MS coupled techniques used to measure the uptake of ENMs by plants include single particle analysis (SP-ICP-MS),field flow fractionation (FFF-ICP-MS), and laser ablation (LA-ICP-MS) (Fig.3.1).

3.3.1.1 Single Particle (SP-ICP-MS)

Single particle analysis is a technique that“quantifies the number of particles in a volume offluid” (Degueldre and Favarger2003). It was developed to detect indi-vidual particles in aqueous suspensions. In this technique, the analyte is spatially concentrated, allowing the introduction of only one particle into the ICP, where atoms or ions are detected as a single pulse. The number of counts is related to the number of atoms, and the frequency of the pulses is proportional to the concen-tration of particles (Laborda et al. 2011). ICP-MS operated in the single particle mode allows the possibility of analyzing individual NPs, thanks to the reading of thousands of signals within a very short dwell time (*10 ms) (Mitrano et al.2012).

The dwell time is a key element in this analytical technique; normally, as the dwell time decreases, the resolution is higher.

Few references describe the use of this technique to determine the size and size distribution of NPs in a sample. Advantages of this technique include the following:

(1) It allows working with dilute solutions, reduces or eliminates sample preparation, avoids NP agglomeration, and it is faster than microscopy techniques; (2) it has

relatively high sensitivity because it is able to discriminate particles of 10 nm diameter. However, SP-ICP-MS requires a lot of statistical work and multiple run-ning (Arruda et al.2015), and many diluted solutions can represent a challenge when working with complex matrices. After several improvements, this technique has shown capabilities for the detection and quantification of MNMs in biological tis-sues. Dan et al. (2015) quantified the uptake and translocation of Au NPs in tomato (Solanum lycopersicum L.) plants by SP-ICP-MS. These researchers exposed polyvinylpyrrolidone (PVP)-coated Au NPs (40 nm) to hydroponically grown tomato plants for four days. Then, they digested the samples with Macerozyme R-10, a multicomponent enzyme mixture that contains cellulose (0.1 unit/mg), hemicellulose (0.25 unit/mg), and pectinase (0.5 unit/mg). Dan et al. (2015) ana-lyzed the digests by using SP-ICP-MS and were able to determine “the size, size distribution, particle concentration, and dissolved Au concentration.” The authors reported that 20 nm was the required size for quantification of the Au NPs with the SP-ICP-MS, while the concentration detection limit was 1000 NPs/mL.

3.3.1.2 Laser Ablation (LA-ICP-MS)

This hyphenated technique allows the analysis of solids without the need of chemical dissolution. It provides less contamination risk in small samples, reduced time for sample preparation, and increased sample throughput with less spectral interferences (Mokgalaka and Gardea-Torresdey2006; Koelmel et al.2013). One of thefirst reports about the use of LA-ICP-MS for the determination of the uptake of NPs by plants was performed in tobacco (Nicotiana tabacum) exposed to Au NPs of different sizes (Judy et al. 2011). Using this technique, the researchers deter-mined the presence of Au in the mesophyll of tobacco leaves harvested from plants treated with Au NPs of 5, 10, and 15 nm. They used a“LSX-213 laser ablation system that removed 400× 400 μm²craters, the depth of which ranged from 8 to 10μm as measured using a Nikon Eclipse 90i light microscope.” According to the authors, “gold concentration reported as log counts per second (CPS) of m/z 197 (Au) normalized by CPS for m/z 66 (Zn) to account for the mass of tissue removed from each laser burst.” However, this study only proved the presence of Au within tissues, with no mention of the Au form. The technique was improved by Koelmel et al. (2013) that used a culture system with no presence of ionic Au. They fed rice plants with surface modified Au NPs proven to be stable. The use of LA-ICP-MS allowed these researches to show the uptake and spatial distribution of Au NPs in shoots and roots of rice plants. Koelmel et al. (2013) were able to quantify the concentration of Au NPs in different tissues, separated by particle surface change.

This study reported that negatively charged Au NPs were more abundant in rice shoots, compared to neutral or positive charged Au particles. However, the authors concluded that the technique can only be used to determine the uptake of insoluble nanoparticles.

3.3.1.3 Field Flow Fractionation (FFF-ICP-MS)

Fieldflow fractionation is a group of techniques that allows separation and sizing of molecules through the application of different fields and modes of operation (Mitrano et al.2012). The basis of FFF is, thus, physical separation of the particles.

The analytes are passed through a channel that does not involve the use of a stationary phase. The channel with laminar flow is subjected to a certain field (sedimentation,flow, electrical, or thermal) that allows the separation of particles based on size or mass. The advantages of this technique include a high resolution for size fractionation, which varies from 1µm up to 1 nm (Dubascoux et al.2010), and its capability for analyzing nanoparticles in complex matrices when coupled to a high-resolution detector, such as ICP-MS (Artiaga et al.2015). A variant of FFF is asymmetrical flow field flow fractionation (AF4), a widely used technique for environmental analysis of both natural and manufactured NPs that allows the possibility of performing “multi-element analysis when coupled to MS” (Mitrano et al. 2012). Palomo-Siguero et al. (2015) used AF4-ICP-MS and TEM to detect chitosan-modified selenium NPs (CS-Se NPs) in radish (Raphanus sativus) plants.

The CS-Se NPs were extracted from the root by using 0.1 % chitosan, 0.034 M ascorbic acid, and 0.24 M acetic acid as an extracting solution. The extracts were centrifuged at 10,000 rpm for 10 min, and the supernatant was injected into the AF4-UV-ICP-MS that showed CS-Se NPs extracted from lateral roots. TEM images show the presence of spherical Se NPs with an estimated particle diameter of 25± 8 nm, mainly interconnected, assembled or aggregated.