Overview of analytical techniques discussed in this review with examples of application.
\r\n\t
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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"60829",title:"Detection Methods of Nanoparticles in Plant Tissues",doi:"10.5772/intechopen.74101",slug:"detection-methods-of-nanoparticles-in-plant-tissues",body:'\nOver the past decades, nanotechnology has been widely applied on commercial products on the market, including biosensor, catalysts to optics, antimicrobial activity, computer transistors, electrometers, and wireless electronic logic and memory schemes. In the agriculture sector, nanoparticles are often incorporated into nano-formulated pesticides, fertilizers, and nanobiosensors for crop protection [1]. Therefore, the application of engineered nanomaterials worldwide inevitably caused the release and accumulation of nanoparticles in the environment [2].
\nAs the basic components of the ecosystem, plants are sessile and their roots absorb nutrients and water as well as contaminants from their environment. Accumulating evidence demonstrated that engineered nanoparticles could be released from some commercial products, further be taken in and accumulated in plant tissues. As plants may serve as a potential pathway for the transportation of nanoparticles through the food chain [3], the increasing applications of engineered nanomaterials in the world have raised a growing concern about their potential adverse impacts on ecosystems, food safety and human health [2, 4]. Therefore, to evaluate potential environmental risks imposed by nanoparticle application, it is important to understand the interaction between nanoparticles and plants, as well as NP’s behavior and toxicity in plants. However, the behavior of NPs in plants and phytotoxicity mechanism are so complicated that contradictory results regarding the effects of nanoparticles on plants were obtained from various studies during the past decade [5, 6, 7, 8, 9]. These conflicting results indicate that impacts of nanoparticles on plants largely depend on the type and concentration of nanoparticles, plants species, tissue exposed, and the experimental conditions [6, 10, 11].
\nThere are various engineered nanoparticles with different size, morphology, and properties. Engineered nanoparticles also exhibit distinct physical and chemical properties with different environmental behaviors and toxicity in comparison with their bulk counterparts, which could be attributed to the small size at nanoscale (1–100 nm) and high surface-to-volume ratios of nanoparticles [12]. Upon nanoparticle exposure, the directly contact between nanoparticles and roots leads to the uptake of nanoparticles by roots and translocation of nanoparticles in plants [10, 13]. Different types of nanoparticles exhibit distinct behaviors and translocation characteristics. During interaction with biological environments, nanoparticles can also be transformed by plants, which in turn alter environmental fate and toxic properties of nanoparticles [14, 15]. Therefore, the toxic effect and behavior of nanoparticles are determined by not only the initial properties (such as particle size, shape, structure, charge, elemental composition, mass concentration, and state of aggregation etc.), but also by the physicochemical evolution [16, 17, 18]. Hence, in order to accurately assess the phytotoxicity of nanoparticles, it is necessary to determine the original characteristics of NPs before treatment, uptake and translocation, cellular internalization and intracellular biotransformation during interaction with plants.
\nApproaches to detect and characterize NPs during plant-NP interaction are thus becoming crucial in our studies. Nowadays, a variety of analytical techniques have been developed to provide the necessary information regarding plant-NP interaction, including microscopy imaging, chromatography, spectrometry-based techniques, and so on. In this review, we describe the advantages and limitations of a selection of current most frequently-used methods in the study of uptake, distribution, translocation and biotransformation of NPs in plants. We also exemplify the usage of these analytical techniques with instances from recent studies.
\nTransmission electron microscopy (TEM) and scanning electron microscopy (SEM) are considered to be the most popular techniques for the analysis of nanomaterials. Electron microscopy straightforwardly captures projected area of the particles, providing visualization of true particle size dimensions.
\nIn TEM process, a focused electron beam is transmitted through a specimen, an image is formed from the interaction of the electrons with the sample. The image is then magnified and focused onto an imaging device. TEM is capable of imaging at a significantly higher resolution (down to the sub-nanometer) than light microscopes, it can visualize as small as a single column of atoms, which is thousands of times smaller than a resolvable object seen in a light microscope.
\nIn SEM process, a focused beam of electrons scans the surface of the sample; interaction between electrons and atoms in the surface of sample produces various signals that contain information about the sample’s surface topography and composition. Then an image is formed upon focusing of scattered electrons. SEM can achieve resolution better than 1 nm [19]. It can be used to image intact sample as well as sectioned sample [15].
\nThrough visualization of nanoparticle position within a cell or tissue, TEM/SEM can provide the precise nanoparticle information about their size, structure, shape, morphology, dispersion or aggregation state, which is informative for assessing in vitro nanoparticle uptake and localization. Nanoparticle sizes are calculated and expressed as a sphere diameter having a similar projected area as the projected image of the nanoparticle. Particle size analysis is carried out by manually using a marking device to move along the nanoparticles. A mean linear dimensional measure of the nanoparticles is obtained by dividing the total length of the nanoparticles by the total number of nanoparticles counted [20]. In addition, when combined with spectroscopic methods, characterization of the composition of the internalized nanoparticles became possible [21]. Owing to the high lateral resolution of TEM, it could also be used to trace the dynamics of individual NPs in a living cell or plant tissues.
\nScanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). A typical STEM is a conventional TEM equipped with additional scanning coils, detectors and necessary circuitry. Like a conventional TEM, images are formed by electrons transmitting through a thin specimen. The difference is that in STEM the electron beam is focused to a fine spot (with spot size 0.05–0.2 nm), then it scans over the sample in a raster. The rastering of the beam across the sample makes STEM suitable for combination with analytical techniques such as annular dark-field imaging and spectroscopic mapping, to obtain information on the structure of nanoparticles with sub-nanometer resolution and their chemical composition [22, 23].
\nDark-field microscopy with a STEM, such as high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), can be used to distinguish elements with high atomic number (Ag, Au, etc.) from the major elements in organisms (C, N, O, etc.) with a high spatial resolution (down to 1 nm) [24].
\nDynamic light scattering (DLS) technique is the most commonly employed high-throughput technique to measure nanoparticle size and determine aggregation state of nanoparticles in aqueous suspensions. In DLS analysis, the Brownian movements of the NPs in aqueous suspensions cause constructive and destructive interference, which results in time-dependent fluctuations in scattering intensity. Then the average particle size can be calculated from these time-dependent fluctuations in scattering intensity by application of the autocorrelation function and subsequent calculation of the exponential decay [25]. Meanwhile, the zeta potential, a key indicator of the stability of colloidal dispersions, is measured rapidly using DLS. DLS is able to analyze samples containing very broad distributions of species; it can also detect very small amounts of the higher mass species [25].
\nEnergy-dispersive X-ray spectroscopy (EDS) is used as an analytical technique to analyze a sample’s elemental composition or chemical characterization. EDS applies a high-energy electron beam which focuses into the sample of interest to excite in an inner shell an electron and to eject it from the shell, thus generating an electron hole. Then an electron from the outer shell with higher-energy fills the hole, which releases the energy in difference in between the outer shell and the inner shell in the form of X-ray [26]. As each element has a unique set of peaks on electromagnetic emission spectrum determined by its unique atomic structure, through measuring the number and energy of the X-rays emitted from the sample by an EDS instrument, the information about elemental composition of the sample is obtained [26].
\nThere are two main types of X-ray spectroscopy-based techniques that can be used to analyze speciation and localization of NPs within the plant tissue: X-ray fluorescence (XRF) spectrometry and X-ray absorption (XAS) spectrometry. Both of them are based on measuring the spectra of emission or absorption of X-radiation. The absorption of X-ray photons by element is controlled by the photo-electric effect.
\nWhen sample is subjected to X-ray radiation, incident X-rays (photons) of a definite energy shine on the samples. If the energy of incident X-rays that reaches the sample is lower than the binding energy (E0) of the core electrons of the element, the atoms of this element do not participate in the absorption process. While with increasing energy of the incident X-ray photons, a point will be reached where their energy is approximately equal to the binding energy of the core electrons. At this point a sharp increase in absorption of the X-ray photons occurs [27]. The energy absorbed by the core electron elevates it into a higher energy state or electron orbital, which is unoccupied. This excited core electron is referred to as a photoelectron. At the binding energy (E0), the photoelectron is ejected from the atom into the continuum [28]. As a result, a vacancy in the shell of the core electron is created in a core orbital. In order for the atom to return to the ground state, an electron from a higher energy orbital (e.g. L or M) fills the vacancy consequently, emitting X-ray photons in the form of fluorescence with characteristic energy corresponding to the difference between the two electronic levels’ binding energies. These X-ray photons have characteristic energies for each element in the periodic table, confers element-specificity to the absorption and fluorescence spectroscopy.
\nDuring XRF process, the emitted fluorescence signal can be recorded at each position and used to generate XRF elemental maps. XRF is a nondestructive technique, it can be used to identify and determine the concentrations of elements present in biological samples, as well as providing information of in situ localization of elements in the samples.
\nDuring XAS process, the energy of the incident X-ray beam is progressively increased beyond the binding energy, thus the emission of fluorescence and absorption of the incident X-ray progressively increases, generating a characteristic X-ray absorption spectrum by detecting and recording the absorption or fluorescence at each energy point. The main feature of the XAS spectrum is a sharp, step-like curve called the absorption edge [28, 29].
\nThe XAS spectrum is conventionally divided into two parts according to the energy region. The region comprising the pre-edge, the edge-jump and post-edge covering the energy range from approximately −50 to +50 eV of the absorption edge is defined as X-ray absorption near edge structure (XANES). The region from +50 to +1000 eV above the edge is defined as extended X-ray absorption fine structure (EXAFS) [28]. XANES is particularly sensitive to the oxidation states of elements and the electronegativity of the ligands, and it provides electronic structural information about the oxidation state and local geometry of the absorbing metal atom. EXAFS can provide information about the element coordination such as the identity and number of the coordinating atoms, and the interatomic distance between the central absorbing atom and its next nearest neighbors.
\nXAS is an element specific spectroscopic technique that provides specific qualitative information about chemical species at very high (subatomic) spatial resolution and is able to analyze almost any type of samples including amorphous (non-crystalline) materials in situ, requiring minor or no sample preparation prone to modify the chemical species. XAS experiments require an intense and polychromatic X-ray source. Synchrotron radiation is a very intense, collimated and polarized X-ray source, with a continuous band of wavelengths from around the μm (infrared) to the pm (hard X-ray) range [30]. Nowadays, owing to the development of synchrotron radiation facilities, the combination of synchrotron radiation with XAS is proved to be a powerful technique for speciation analysis of chemical elements.
\nXAS spectroscopy can be performed as bulk analyses to assess the overall speciation of the chemical of interest in the sample (usually homogenized). For bulk-XAS experiment, a beam in the size range of a few hundred μm2 to a few mm2 is used to illuminate sample, the XAS spectra obtained are generally representative of the average speciation of the chemical in the sample [28, 31]. Whereas, the signal of minor species in the sample, which accounts for less than 5–10% of the total analyte, is insufficient to be resolved and quantified from the bulk spectra. In this case, bulk-XAS analysis is insufficient for us to obtain specific information from a complex and heterogeneous mixture of biological sample. This limitation can be overcome by decreasing the beam size to the range of tens of nm to a few um, and using thin section of sample. This kind of laterally-resolved XAS analysis is referred to as μ-XAS.
\nFrom μ-XAS spectra, the information attained at each point of analysis is only representative of the spot probed and not of the overall speciation in the sample. Therefore, a trade-off exists between detecting minor species and obtaining the overall speciation of the analyte in the whole sample. A strategy to solve the problem is coupling bulk XAS analyses with laterally resolved techniques such as μ-XRF, μ-XAS and μ-XRD. In a typical work flow, laterally resolved μ-XRF elemental maps are first collected to identify spots of interest, which are then further probed by μ-XAS analysis [28].
\nRecently, a new approach termed XANES imaging has been developed with the capacity to analyze element speciation and full lateral distribution over large areas of sample. In a XANES imaging process, an elemental map of the sample will be firstly generated to identify interesting areas, then the μ-X-ray fluorescence signals from the interesting areas are collected repeatedly over progressively increased incident X-ray energies and scan across the characteristic absorption edge of the target element. These resulting maps can be aligned and stacked, then the XANES spectra can be extracted from individual pixels or groups of pixels over regions of interest, eventually, the spatial distribution of both major and minor species within the sample will be obtained [28]. A detailed information regarding comparison among bulk-XANES, μ-XANES and XANES imaging is provided in a review by Gräfe et al. [28].
\nX-ray diffraction (XRD) is a nondestructive technique for characterizing crystallographic structure or elemental composition of crystalline materials. It can reveal information about the crystal structure, crystalline phase, preferred crystal orientation (texture), average crystallite size and strain of materials. The constructive interference of a monochromatic beam of X-rays diffracted at specific angles from each set of lattice planes in the crystalline sample will produce X-ray diffraction peaks, intensities of which are determined by the distribution of atoms within the lattice, therefore, an X-ray diffraction pattern will be generated which reflects the periodic atomic arrangements in the sample.
\nFor synchrotron-based X-ray diffraction (SR-XRD) technique, the high intensity and well-defined wavelength of the incident synchrotron radiation will generate a better resolution of diffraction peaks and make SR-XRD capable in detecting minor constituents in a sample [27]. In addition, XRD is capable of 20 μm lateral resolution with minimal sample preparation requirements, can be used as a valuable complementary or alternative methods to XAS analysis. A limitation of this method is that it’s not applicable for amorphous materials; it can only characterize crystalline samples.
\nX-ray computed microtomography (μCT) uses X-ray to create cross-sections of a sample that can be used to produce three-dimensional digital images of the sample’s internal structure at a micron level spatial resolution without destroying the original sample [32].
\nIn an absorption-edge synchrotron radiation-based μCT process, a high flux, monochromatic X-ray beam passes through the sample, a scintillator converts the transmitted X-rays into visible light and the resulting absorption projection is captured by a photodetector to produce 2D radiographs. The sample is then rotated (or the X-ray source and detector are rotated about the object) by a small angle, a series of 2D X-ray absorption images is captured successively between 0° and 180°. Using mathematical principles of tomography, this series of images is then reconstructed to produce a 3D image, thus a 3D distribution of the element of interest within the sample is obtained [27, 32].
\nScanning transmission X-ray microscopy (STXM) is a type of X-ray microscopy that allows in situ mapping of elements at high lateral resolution within a specimen. STXM uses a Fresnel zone plate to focuses synchrotron soft X-ray absorption beamline into a small spot, the sample is placed at the focus of the zone plate and scanned by X-ray, then a film or charged coupled device detector is used to detecting the transmitted X-rays intensity that pass through the specimen [33].
\nSTXM-XAS, a technique that in-situ conditions of a XAS experiment with a STXM microscope, is capable of determining chemical speciation with a spatial resolution of 10–30 nm [34]. STXM-XAS can handle samples with thicknesses up to 20 micron at 1.5 keV, which makes it possible to study a wider and more flexible range of materials, including various plant tissues [33].
\nSecondary ion mass spectrometry (SIMS) uses an energetic ion beam to bombard a sample, particles from the top few atomic layers of the sample surface are then removed, resulting in the consequent liberation of ions, known as secondary ions. These secondary ions are then sorted on the basis of their energy in the instrument’s electrostatic sector and later dispersed in a mass spectrometer to produce a map giving information about the elemental or molecular distribution within the sample [29, 35].
\nNano secondary ion mass spectrometry (NanoSIMS) is a nanoscopic scale resolution chemical imaging mass spectrometer based on SIMS [35]. The main advantage of NanoSIMS over other SIMS is the ability to operate at high mass resolution, while maintaining both excellent signal transmission and high lateral resolution (down to 50 nm) with a low detection limit (mg/kg range). It is capable of measuring most elements in the periodic table, from hydrogen to uranium, as well as their different isotopes. These advantages of NanoSIMS make it one of the most powerful tools to quantitatively investigate elemental distribution in organisms at the cellular level [36, 37]. It is reported that Nano-SIMS has been used for the analyses of NPs in biological samples including plant tissue [36].
\nA limitation of NanoSIMS technique is that it is difficult to analyze elements with poor secondary ion yield, such as Zn, Cd, and Mn [37]. In addition, it is a destructive technique, which can be a disadvantage for some samples. This problem can be overcome by using high-pressure freezing followed by freeze substitution to preserve cellular and subcellular structures as well as elemental distributions of plant cells [29].
\nInductively coupled plasma (ICP) based analytical techniques can provide quantitative elemental composition of a wide variety of sample types, including solids, liquids, and suspensions. Inductively coupled plasma-optical emission Spectrometry (ICP-OES) can be used to measure nanoparticle number concentration and elemental composition within a sample. As ICP-based techniques involve the use of liquid phases, suspensions could be analyzed directly, but solid samples have to be pretreated for the digestion of the matrix [21]. Generally, solid samples are dissolved or digested using acid in a microwave to get volatile analytic species. The sample solution is then nebulized into the core of inductively coupled argon plasma, where a flame temperature in a range from 6000 to 10,000 K vaporizes the nebulized solution, thus the analytic species are atomized, ionized and thermally excited. The excited atoms and ions return to low energy position, emitting electromagnetic radiation at wavelengths characteristic of a particular element, then the analytic species can be detected and quantified with an optical emission spectrometer (OES) through measuring the intensity of radiation, which is converted to elemental concentration by comparison with calibration standards.
\nInductively coupled plasma-mass spectrometry (ICP-MS) is an inorganic elemental analysis technique based on atomic mass spectrometry. ICP-MS consists of an ion source, a sampling interface, ion lens, a mass spectrophotometer and a detector system [18]. ICP sources are mainly used for metal analysis. It is an ideal ionization source for mass spectrometry, and can ionize over 90% of many elements. Mass spectrophotometer (e.g. ion trap, quadrupole or time-of-flight) covers different mass-to-charge ranges; differ in mass accuracy and achievable resolution.
\nDuring ICP-MS process, the ICP source is used to decompose, atomize and ionize a sample of interest. The ions generated in the high temperature argon plasma core are subsequently sorted by mass with the mass spectrophotometer and subjected to further elemental and isotopic analysis. The identities of the ions are determined by their mass-to-charge ratio using a mass analyzer, while the ions intensity is measured at ppt to ppm levels using the ion detector, then the intensity measurements are converted to elemental concentration by comparison with calibration standards. With the high sensitivity and specificity, ICP-MS has been widely used for the detection, characterization, and quantification of nanoparticles [38].
\nICP-MS can be used in single particle mode to characterize individual particles, termed single particle inductively coupled plasma-mass spectrometry (SP-ICP-MS). During SP-ICP-MS process, the sample is first suspended in a nebulized liquid and subsequently carried to argon plasma, where the sample is sequentially desolvated and atomized and ionized, creating a plume of ions. The ions pass through the mass spectrometer where they are separated by mass-to-charge ratio and detected using a time resolved analysis acquisition. The sample solution needs to be diluted sufficiently to ensure low concentrations (ppt to ppb) that no more than one particle will enter the plasma at a time. By using sufficiently short integration (dwell) time which is a duration for the instrument to take a reading, thousands of fast and individual readings are generated to capture nanoparticle event as a discrete signal pulse, each pulse is assumed to correlate to one nanoparticle event [39, 40, 41]. Based on ionic calibration standard, the particle mass can be determined by the intensity of the ICP-MS response. If the density of the elemental constituents of the particle is known, the theoretical size of the particle can be determined. If the transport efficiency from the nebulizer to the plasma is known, then the particle number concentration can be further calculated [38, 42].
\nSP-ICP-MS has been widely applied to measure particle size, size distribution, number concentration and elemental composition of nanoparticles in biological samples, demonstrating it as a powerful tool in quantifying NPs. To deal with biological tissues, a strong acid extraction procedure is required to release the NPs from the matrix. This introduces the possible dissolution of metal NPs which challenges the accuracy of the final analytical data. To solve this problem, Dan et al. studied recoveries of gold NPs when using such a special macerating enzyme that appeared to release the NPs from plant tissue without changing the size distribution of the NPs [43]. With the aid of enzymatic digestion, we have applied SP-ICP-MS analysis to characterize Ag NPs internalized by Arabidopsis, thus having established a new technique and opened up new research domain in our lab [44]. Overview of these analytical techniques including advantages and limitations with examples of application in plant-NP interaction studies is provided in Table 1.
\nTechnique | \nInformation provided | \nSpatial resolution | \nDetection limit | \nAdvantages | \nLimitations | \nExamples of application | \n
---|---|---|---|---|---|---|
TEM | \nSize, size distribution, shape, distribution, aggregation state, structure | \n>0.1 nm | \nmg/kg | \nHigh resolution, in vivo | \nDestructive, sample preparation, high vacuum condition, insensitive to light elements | \nCucumber [54] | \n
SEM | \nSize, size distribution, shape, distribution, aggregation state, structure | \n1 nm to 1 μm | \nmg/kg | \nHigh resolution, in vivo | \nHigh vacuum, sample preparation, insensitive to light elements | \nEichhornia crassipes [50] | \n
STEM | \nSize, size distribution, shape, distribution, aggregation state, structure | \n<0.1 nm | \nmg/kg | \nAtomic resolution, analysis of low concentrations (ppm), in vivo | \nSample preparation, insensitive to light elements | \nChlamydomonas reinhardtii [36] | \n
DLS | \nSize distribution, zeta potential, hydrodynamic diameter | \n3 nm-μm | \n\n | In situ and real-time measurement, rapid and simple analysis | \nDifficult to interpret heterogeneous size distributions, aggregates, dust particles can ruin the measurements on nanoparticles, multiple scattering and particle interactions in high concentrations, limited capability on polydisperse samples | \nCapsicum annuum L. [47] Romaine lettuce [48] | \n
EDS | \nElemental composition, distribution | \n\n | \n | Nonquantitative analysis | \n\n | \n |
XAS | \nOxidation state, elemental composition, structure | \nppm | \nmg/kg | \nIn vivo, minor species can be investigated | \nCause beam damage artifacts | \nCucumber [54] | \n
XRF | \nSolid state speciation, quantitative bulk analysis, isotope ratios, morphology | \n\n | mg/kg | \nNondestructive, in vivo | \n\n | Landoltia punctata [51] | \n
XRD | \nStructure, size | \n1–3 wt% | \n\n | Nondestructive | \n\n | \n |
μ CT | \nDistribution | \n\n | \n | In vivo, nondestructive, 3D visualization | \n\n | Wheat [52] | \n
STXM | \nSize, shape, visualization | \n30 nm | \n\n | In vivo, no sample preparation, nondestructive, liquid conditions | \n\n | Cucumber [59] | \n
NanoSIMS | \nDistribution, elemental composition, surface properties | \n50 nm | \n\n | In vivo, high mass resolution, high lateral resolution | \nDestructive, difficult to analyze some elements with poor secondary ion yield, such as Zn, Cd, and Mn | \nChlamydomonas reinhardtii [36] | \n
ICP-OES | \nElemental composition, concentration | \nNot available | \nμg/l | \nQuantitative analysis | \nDo not provide information on particle shape or diameter | \nRice [53] | \n
ICP-MS | \nBulk elemental composition, number concentration, mass concentration | \nNot available | \nng/l | \nQuantitative analysis, high sensitivity, low background signal, rapid and simple analysis | \nDo not provide information on particle shape or diameter, minimum particle size is limited by ICP-MS sensitivity, background and dissolved element content, narrow optimum range of particle number concentrations | \nArabidopsis thaliana [49] | \n
SP-ICP-MS | \nConcentration, number concentration, mass concentration | \nNot available | \nng/l | \nQuantitative analysis, high sensitivity, low background signal, rapid and simple analysis | \nDo not provide information on particle shape or diameter, minimum particle size is limited by ICP-MS sensitivity, background and dissolved element content, narrow optimum range of particle number concentrations | \nSoybean and rice [55] Arabidopsis thaliana [44] | \n
Overview of analytical techniques discussed in this review with examples of application.
Although a range of techniques are available to detect and characterize uptake, translocation and biotransformation of NPs in plant tissue, no single technique can provide all information regarding plant-NP interaction. Sufficient information is often obtained by the combination of these analytical techniques, which could provide complementary information mutually. Here in this section literature examples from recent studies are used to demonstrate the application of different techniques in the study of plant-NP interaction.
\nCareful characterization of NPs is critical for accurately assessing the impacts of nanoparticles on plants and understanding their behavior. When initiate an experiment, NPs will either diffuse or aggregate within certain biological media due to different characteristics of the media (i.e. pH, ionic strength, concentration and redox conditions) [45, 46], the aggregation state of NPs will result in quite distinct properties from original NPs. Therefore, the characterization of original NPs is often the first step before NPs application [45, 46, 47]. Zhang et al. used TEM images to observe the shape and size of nCeO2 before applied to romaine lettuce. XRD spectrum confirmed the cubic fluorite structure of nCeO2; then ICP-MS was used to confirm the purity of nCeO2. Measuring zeta potential and hydrodynamic size of nCeO2 by DLS analysis indicated a significant aggregation of particles after mixing nCeO2 with nutrient solution. After nCeO2 application, μ-XRF analysis showed that Ce mostly distributed outside the roots. TEM images confirmed that large amount of nCeO2 aggregates distributed on the root surface [48]. Yang et al. used TEM to measure averaged size of CeO2-NPs suspended in deionized water, and XRD was employed to detect average primary particle size of CeO2-NPs in dry powder samples, as well as to confirm the crystal structures of CeO2-NPs [49]. Vinković et al. used DLS, TEM and ICP-MS to characterize AgNPs in ultrapure water (UPW) and sterilized tap water used for the plant watering (TWW) [47]. By measuring hydrodynamic diameter, zeta potential and polydispersity index (PdI) of citrate-coated AgNPs, DLS results showed that the volume size distribution in UPW was bimodal with 90% of smaller particles (12.9 ± 9.1 nm) and only 9% of bigger particles (87.6 ± 41.7 nm). The zeta potential value equal to −16.9 ± 0.6 mV indicated electrostatic stabilization of AgNPs in UPW. While after suspension in TWW, AgNPs aggregation occurred due to higher ionic strength of TWW. Further TEM analysis confirmed the presence of flocculated and aggregated AgNPs in TWW. ICP-MS was used to estimate the stability of AgNPs upon dissolution, results showed that total Ag was lower than 0.5% in TWW, which implies that Ag+ release was not occurred in TWW [47].
\nIn order to understand the uptake mechanism of NPs and their translocation pathway, imaging techniques are often employed to visualize the distribution and morphology of NPs upon exposure, EDS can provide information on their chemical composition, while ICP-based techniques are used to measure particle number concentration, size distribution and mass concentration. Zhao et al. used SEM imaging to find that the root tip of Eichhornia crassipes after CuO NP exposure was thinner than unexposed root. EDS analysis of the aggregates attached on epidermis showed the presence of 37.6% (w/w) of Cu, confirmed that CuO NPs presented on the surface of root tips. Further through TEM imaging, dark aggregates with high electron density were detected in the intercellular spaces of cortical tissues in roots, and EDS analysis confirmed the presence of Cu on these aggregates, indicating that CuO NPs were taken up by roots and located in intercellular spaces [50]. Yang et al. used ICP-MS to find that CeO2-NPs were taken up from root and subsequently translocated to shoot tissues in Arabidopsis thaliana, Ce accumulation was much higher in CeO2-NP treatments than those in CeO2-bulk and ionic Ce treatments, indicated that the toxicity resulted from the CeO2-NPs per se rather than from the dissolved Ce ions. TEM images showed the presence of a large number of needle-like particle aggregations in the intercellular regions and the cytoplasm of leaf cells [49]. Stegemeier et al. used synchrotron-based μ-XRF to visualize silver distribution in duckweed roots exposed to Ag0 NPs or Ag2S NPs, or to AgNO3. The silver Kα XRF maps showed clear differences in the distribution of Ag for each type of Ag used. The silver was distributed throughout the root tip and showed highest concentrations near the apical meristem after exposure to AgNO3. A similar distribution of Ag in root tip was shown after exposure to Ag0-NPs. While after exposure to Ag2S-NPs, a hotspot of silver located at the end of the root cap, suggested that silver was not readily internalized in this case [51]. Pradas del Real et al. firstly used μ-CT to create 3D reconstructed image of wheat root after Ag NPs exposure for in situ 3D visualization, then μ-XRF was used to provide 2D elemental distribution [52]. Combination of μ-CT and μ-XRF showed the presence of localized Ag accumulation regions with a size of 1–4 μm adhering on the epidermis. Nano-CT technique capable of higher spatial resolution revealed that these AgNPs accumulated preferentially in discontinuities between root epidermal cells. In addition, many AgNPs were fixed on root hairs. With the methods to study Ag2S-NPs treatment, μ-XRF showed that Ag is mainly colocalized with S, μ-CT and nano-CT showed that these Ag accumulation regions with a size from 3 to 8 μm presented mostly on the root surface. Through ICP-MS analysis, a higher Ag content in root and shoot was observed after exposure to AgNPs compared to AgNO3 exposure, suggesting a nano-specific accumulation mechanism [52]. Peng et al. used ICP-OES to measure Cu content in root of rice after adding CuO NPs to the soil. The results showed that Cu content in roots was significantly increased, with a much higher content than aboveground parts. μ-XRF analysis indicated that Cu accumulated in the aleuronic layer of rice, but not the polished rice [53]. In another study, In order to study whether CeO2 NPs can move from the roots to shoots in cucumber after the root was exposed to CeO2 NPs, TEM and EDS analyses were performed and the presence of Ce particles in the xylem sap was confirmed, suggested that Ce-containing species could be transported throughout the whole plant by vascular system. ICP-MS analysis also confirmed the uptake of CeO2 NPs from root to shoot [54]. Li et al. used the macerozyme R-10 tissue extraction method followed by SP-ICP-MS to study the uptake and size distribution of AgNPs in soybean and rice. Both SP-ICP-MS and TEM measurements indicated that the size of Ag-containing NPs were 2–3 times larger than the originally dosed AgNPs after exposure to AgNPs, indicating the AgNPs biotransformation processes were involved [55].
\nBiotransformation is defined as biochemical modification by living organisms [16, 56]. Biotransformation of NPs by plant may modify the toxicity, behavior, and fate of NPs in the plant tissue. Biotransformation process may involve redox, dissolution, sulfidation, aggregation, and adsorption of macromolecules and ion [10, 57]. XAS and STXM are the most frequently used techniques to characterize the speciation of NPs during cellular internalization and intracellular biotransformation. Zhao et al. used EDS technique to find that S present on aggregates in the intercellular spaces of cortical tissues in Eichhornia crassipes (water hyacinth) roots after CuO NP exposure, indicating that CuO NPs (or other Cu species) interacted with S-containing compounds such as cysteine. XANES was employed to identify Cu species in roots and leaves after CuO NPs internalization. XANES analysis revealed that CuO NPs in roots mainly kept the original pattern (65.7% of CuO). Other Cu species included Cu-Ac (14.2%), Cu2(OH)PO4 (8.7%) and 7.6% of Cu2S. XRD spectrum of original CuO NPs showed that all peaks belonged to CuO, and no peak on Cu2S was detected, indicating that the observed Cu2S in roots were formed after incubation with CuO NPs [50]. Zhang et al. used Bulk-XANES technique to study transformation of nCeO2. XANES spectra of root and shoot showed similar feature as the initial nCeO2; Results showed that Ce in lettuce mostly presented as CeO2, with a small fraction of CePO4 in roots (4.3%) and Ce carboxylates (3.5%) in leaves, suggested that nCeO2 can release small amount of Ce3+ with the assistance of organic acids and reducing substances in root exudates [48, 58]. Stegemeier et al. used EXAFS spectra to determine Ag speciation in duckweed (Landoltia punctata) roots after exposure to Ag0 NPs or Ag2S NPs, or AgNO3, revealed that more photo-reducible Ag species were generated after exposure to ionic Ag [51]. In contrast, a higher prevalence of sulfur associated Ag species (as a mixture of Ag2S (64%) and Ag-thiol (53%) were produced after exposure to Ag0 NPs or Ag2S NPs treatment. Bulk EXAFS analysis of Ag2S-NP treatment indicated that plant is unable to dissolve or transform a significant amount of the Ag2S-NPs after 24 h exposure [51]. In another study, μ-XANES spectroscopy was employed to determine speciation of Ag at root after Ag NPs exposure. μ-XANES revealed that Ag was mostly present as metallic Ag in the epidermis, but inside the roots Ag was homogeneously distributed in the cell walls of the cortex as a mixture of Ag-thiol species and other ionic Ag species, suggested the biotransformation of Ag occurred. Moreover, no Ag(0) was observed inside roots, implied that Ag-NPs were completely dissolved and complexed by organic ligands [52]. Peng et al. used Bulk-XANES to analyze translocation and transformation of CuO NPs in rice, the results revealed that Cu element mainly existed in the form of copper citrate, only a small portion of Cu kept original CuO form in roots, stems, and leaves of rice after CuO NP treatment. During CuO NPs internalization in rice, one-third of Cu(II) was transformed to Cu(I) which was mainly associated with cysteine. CuO, copper citrate, and copper (I) acetate all accounted for nearly 30% of the total Cu in the chaff [53]. Ma et al. combined μ-XRF and μ-XANES to detect CeO2 NPs or its transformation species in the xylem sap, shoots and roots of cucumber after exposure to CeO2 NPs, revealed that about 15% of Ce was reduced from Ce(IV) to Ce(III) in the roots after treatment, and Ce was transported as a mixture of Ce(IV) and Ce(III) from roots to shoots through xylem, while was transported almost only in the form of CeO2 from shoots back to roots through phloem [54]. Peng et al. used bulk-XANES to analyze Cu speciation in the tissues of rice plants after exposure to CuP NPs, indicated that Cu was combined with cysteine, citrate, and phosphate ligands, and some of the Cu (II) was transformed to Cu (I) during CuO NP uptake, and confirmed that CuO NPs were transported from the roots to the leaves. In order to further study Cu biotransformation in cellular level, they firstly used μ-XRF to map Cu element distribution in the root; the results revealed that Cu was mainly localized in the root epidermis and exodermis. Then μ-XANES was employed to determine speciation of Cu element at selected spots in μ-XRF map. In addition, combination of STXM with Cu L3-edge XANES spectroscopy was used to map the in situ elemental composition of Cu in the root cells, the results confirmed that speciation of Cu in the root cells and the intercellular space existed in the form of Cu-citrate and CuO NPs, respectively [10]. Zhang et al. used TEM to detect the uptake and localization of nano-Yb2O3 in cucumber roots after exposure, found that a lot of high electron-dense dark deposits looked like fine needle-shaped nanoclusters in the intercellular spaces and middle lamellas in the cross sections of cucumber roots, later EDS analysis confirmed the presence of Yb in these dark deposits. In order to identify the chemical species of Yb in these dark deposits, the chemical distribution was mapped by STXM, and NEXAFS spectra were extracted, results indicated that this compound was inferred to be YbPO4, suggesting that Yb2O3 particles and YbCl3 were all transformed to YbPO4 in the intercellular regions of the roots, and indicating that biotransformation and internalization of Yb2O3 nanoparticle took place in plant cell, which conferred phytotoxicity to plant [59]. In another study, Zhang et al. used the same methodology to investigate the biotransformation of CeO2 NPs in cucumber. TEM images showed the presence of needle-like clusters on the epidermis and in the intercellular spaces of cucumber roots after CeO2 NPs exposure. STXM imaging indicated that the chemical composition of needle-like clusters is CePO4. Further XANES analysis showed that Ce presented in the roots as CeO2 and CePO4 while in the shoots as CeO2 and cerium carboxylates, confirming biotransformation of CeO2 NPs in plant cells [16]. In order to determine the toxicity and fate of nanoparticles upon exposure to plants, Wang et al. combined a variety of techniques to investigate the cellular internalization and intracellular biotransformation of silver nanoparticles in Chlamydomonas reinhardtii. NanoSIMS was firstly applied to analyze the distributions of Ag in algal cells, silver was observed to accumulate predominantly on the cell walls and in the cytoplasm of the algae after exposure to AgNPs. Then HAADF-STEM was performed to examine the accurate localization and morphology of Ag, HAADF image showed that a set of bright spots located mainly in the periplasmic space and cytoplasm, TEM was further used to observe morphology of these bright spots. EDS analysis showed that these bright spots were Ag-containing substances; moreover, Ag and S always occurred concomitantly. EDS-mapping confirmed that Ag was almost exclusively co-localized with S in the cytoplasm of algae but not in the periplasmic space. Later, Synchrotron based Ag K-edge XAS was performed to further identify Ag speciation after exposure. It was found that Ag glutathione complexes and Ag2S represented the main speciation, suggested that Silver was also found to coexist with sulfur inside the cytoplasm in the form of Ag-GSH and Ag2S [36]. A regular work flow of NPs characterization during plant-NP interaction with the application of the most-frequently used techniques is shown in Figure 1.
\nSchematic diagram represent a regular work flow of NPs characterization in plant. A selection of analytical techniques is shown. Red dots indicate NPs at the moment of application. Yellow and blue dots indicate different elemental species of NPs after biotransformation in plants. The images of SP-ICP-MS, EDS, TEM, STXM, μ-XRF and μ-XANES are adapted from [16, 18, 44, 60].
Although the combination of these techniques described in this review is capable of taking over most of the task on the characterization of NPs during plant-NP interaction, considerable limitations of these techniques still remain to overcome. Many techniques are destructive, such as TEM, SEM and nanoSIMS, which means the same sample cannot be analyzed twice or by another method for validation. Analytical artifacts are sometimes inevitable during some sample preparation procedures. Because biological samples is usually hetero-dispersed and multicomponent, with diverse elemental compositions and sometimes contain multiple types of NPs, the analysis of NPs in these samples is thus quite complicated and a variety of methodology is utilized to provide complementary information, while the results measured by these different methods are not always comparable, which may partially due to different sample preparation procedures in different techniques. Further, instrument operation procedures and statistical analyses are likely to contribute to the complexity and uncertainty. Another challenge arises when analyze samples with low concentrations of the analyte. In non-hyperaccumulating plant species, visualizing the spatial distribution of NPs and detecting reliable in situ information about the chemical speciation of trace elements will be very difficult. In this case, analytical techniques with high sensitivity are desired to measure low concentrations of NPs. An ideal analytical technique should be able to simultaneously determine all parameters regarding plant-NP interaction, such as article size, morphology, structure, size distribution, mass concentration, translocation, elemental speciation and etc. It should be sensitive and accurate enough for in situ detection and characterization of trace element in complex biological samples in a non-destructive way. Although none of the existing techniques are able to solely provide all the information desired, we believe that a promising evolution of analytical methodology is taking place and will be capable of fulfilling requirements as much as desired to provide sufficient information about plant-NP interaction.
\nAuthors thank the NIE AcRF grant (RI 8/16 CZ).
\nAuthors declare no conflict of interest.
DLS | dynamic light scattering |
EDS | energy-dispersive X-ray spectroscopy |
EXAFS | extended X-ray absorption fine structure |
ICP-MS | inductively coupled plasma-mass spectrometry |
ICP-OES | inductively coupled plasma-optical emission spectrometry |
NanoSIMS | nano secondary ion mass spectrometry |
NPs | nanoparticles |
SEM | scanning electron microscope |
SP-ICP-MS | single particle-inductively coupled plasma-mass spectrometry |
STEM | scanning transmission electron microscope |
STXM | scanning transmission X-ray microscopy |
TEM | transmission electron microscope |
XANES | X-ray absorption near edge structure |
XAS | X-ray absorption spectrometry |
XRD | X-ray diffraction |
XRF | X-ray fluorescence spectrometry |
μCT | X-ray computed microtomography |
In the last decades, with the machinery modernization and the consolidation of the sector of modern inputs, agriculture has been growing at a fast pace, with pesticides being one of the main instruments that drive the agricultural sector in productivity gains [1]. However, the indiscriminate use of these substances has easily reached non-target organisms and their effects on the environment are varied, ranging from the reduction in the availability and quality of water to the compromise of air and food quality, harming human health. Also, it can directly affect cellular structures of aquatic or terrestrial organisms resulting in damage to biodiversity [2].
In the early 1960s, society began to worry about the adverse effects and potential risk that these pesticides posed to human health and the environment. In several countries, production, marketing, and use of many of these compounds, in particular those considered persistent organic pollutants (POPs), such as organochlorines, were banned [3]. With the ban on most organochlorine compounds (less toxic, but with greater bioaccumulation in the environment), after the Second World War, carbamate and organophosphate pesticides had their use intensified. Also, it became the most used pesticides worldwide, being widely used in developing countries with a predominantly agricultural economy [4].
These toxic substances have the potential to cause various biochemical and genetic injuries to non-target organisms. Carbamates and organophosphates, for example, are potent inhibitors of the acetylcholinesterase enzyme, which damages the nervous system of an exposed organism [5, 6]. This enzyme acts in the hydrolysis of the acetylcholine neurotransmitter in cholinergic synapses. Its inhibition can lead the individual to death due to cholinergic hyperstimulation. Pesticides are also known for their mutagenic and carcinogenic effects. They react with nucleic acids causing adverse reactions in the body. Thus, monitoring and controlling the presence of these substances in the environment are necessary, since these compounds have become a human health and environmental problems [7].
Pesticides or agrochemicals are defined as:
“Products and agents of physical, chemical or biological processes, intended for use in the sectors of production, in the storage and processing of agricultural products, in pastures, in the protection of forests, native or implanted, and of other ecosystems and also of urban environments, whose purpose is to change the composition of flora or fauna, in order to preserve them from the adverse action of living beings considered harmful” [8].
According to the harmful species that intend to eliminate, these compounds are classified as insecticides, fungicides, herbicides, acaricides, rodenticides, molluscicides, among others. Herbicides represent 48% of the total pesticides, which is followed by insecticides (25%) and fungicides (22%) [9]. Depending on the chemical class, they can be grouped into pyrethroids, organochlorines, organophosphates, carbamates, benzoylureas, neonicotinoids, among others [10].
Pesticides arrive in the environment carried by runoff and leaching of rainwater, irrigation, and drainage or by spraying, as shown in Figure 1. Among these processes, runoff and leaching can contaminate reservoirs, lakes, and rivers. Also, they expose aquatic organisms at levels of pesticides that can be toxic to many species. Once present in the aquatic environment, these compounds can penetrate the organisms orally - through the ingestion of contaminated food, respiratory - through the gills, and dermal - through the surface of the body. In most cases, these organisms tend to suffer bioaccumulation [1, 13]. Pesticide exposure can cause numerous physiological changes by direct influence on certain cellular structures, for example, on the lysosomal membrane, which can be degraded or can react with nucleic acids, resulting in several genetic injuries that cause adverse reactions in the body [2].
Pesticide paths to the aquatic environments. Source: [11, 12].
Currently, there is a growing concern about the exacerbated use of pesticides since, in recent decades, aquatic ecosystems have received alarmingly large amounts of these compounds, which are released by urban communities, rural properties, and industries. Thus, society started to worry about the adverse effects of these substances and their potential risk [14]. According to Silva et al. [4], carbamates and organophosphates are the most used pesticides worldwide. They together account for more than 50% of what is marketed.
Organophosphate pesticides (OPs) comprise a large number of substances classified chemically as esters, amides, or derivatives of pentavalent phosphoric acids. Carbamates (CBs) are esters, or N-substituted derivatives of carbamic acid (carbamic acid monoamide) (Figure 2). Both have low water solubility and are, in general, easily hydrolyzable in alkaline environments [10, 15, 16]. In general, OPs need biotransformation to become toxicologically active, unlike CBs that are already bioactive.
Organophosphate pesticides: (a) methyl-paration (triesters of phosphoric acid), (b) carbamate carbofuran (esters derived from carbamic acid).
Carbamates and organophosphates affect the nervous system of organisms. They inhibit the activity of the enzyme acetylcholinesterase (AChE), as demonstrated by Wang et al. [17]. In their study, AChE inhibition in carp (Cyprinus carpio) exposed to various concentrations of organophosphates, malathion, and triazophos, as well as carbamates fenobucarb and carbosulfan, was evaluated. In equitoxic mixtures, the authors noted that AChE activity was inhibited by the combination of triazophos and malathion, as well as triazophos and carbosulfan, with synergism occurring. The effects of organophosphates on the behavior and activity of the AChE of zebrafish larvae have also been studied, through exposure to chlorpyrifos and malathion, and changes in swimming speed (hypoactivity and hyperactivity), rest and tigmotatism have been found [18].
Recently, benzoylurea, a class of pesticide that in the past was not considered an acetylcholinesterase inhibitor, since its main mode of action is the inhibition of chitin biosynthesis in insects (which interrupts the incorporation of N-acetylglycosamine monomers), demonstrated anticholinesterase potential [19]. In 2011, this class of pesticides represented 3.6% of the world’s pesticide market. Since then, its commercial importance has grown over the years [20].
The intensive use of pesticides in agricultural cultivation has been one of the main problems responsible for the contamination of aquatic ecosystems. It is due to both the deposition and consequent accumulation of these contaminants in the environment and the sensitivity of the organisms. Currently, there is an increasing number of studies in which fish, for example, are used as indicators of pesticides in the aquatic ecosystem, since these substances, even in low concentrations, can affect their physiology and survival capacity [17, 21, 22].
These organisms are sources of biologically active molecules. When their functioning is altered, compromise the organism’s physiological functions, which culminates in genetic, biochemical, morphological, ecological, or behavioral changes [23]. These biomolecules are considered as biomarkers, and their measurement has been used in biomonitoring programs to detect exposure to toxic substances in the aquatic environment [24]. This early detection allows identifying the presence of the contaminant, even before it causes significant changes in the health of the exposed individuals.
Among exposure biomarkers, recent studies showed great interest in enzyme biomarkers as an alternative for monitoring impacted aquatic environments due to their high specificity and speed in responding to changes from target substances [4, 6, 17, 21, 25, 26]. The use of enzymes as biomarkers is based on inhibitory or inductive interference caused by contaminants in their catalytic activity. Most of these toxic compounds have a high affinity for electron pairs found in the amino acids that form the enzymes, such as the sulfhydryl - SH groups and other functional groups from the catalytic site [5, 27]. Among the main enzymes used extensively for this purpose, cholinesterase enzymes stand out (ChEs; EC 3.1.1.x).
Two distinct cholinesterases are found in vertebrate and invertebrate aquatic organisms, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). AChE is a hydrolase that predominates mainly in erythrocytes, neurons, ganglia of the autonomic nervous system, and terminal motor plates. Its main function is to promote the hydrolysis of the neurotransmitter acetylcholine. It releases acetate and choline in the cholinergic synapses. Due to its key function in the control of synaptic transmission, this enzyme becomes one of the most vulnerable molecular targets to the action of neurotoxic agents. For this reason, it has been widely studied in aquatic organisms and proposed for use in monitoring programs, given its sensitivity [5, 6]. On the other hand, BChE predominates in plasma, liver, neuroglia, pancreas, and digestive tract walls. It has not fully clarified its function, and the absence of its activity has been reported in the brains of several fish species [4, 28].
These enzymes are widely used in biomonitoring of aquatic ecosystems. They are used as biomarkers of the presence of two specific classes of pesticides: carbamates and organophosphates, which generally have low environmental persistence, especially when compared to organochlorines, but with greater toxicity. These substances act by inhibiting enzymatic activity. It interacts with the steratic site by phosphorylation (organophosphates) or carbamoylation (carbamates) (Figure 3) [12, 29].
Steps of inhibition by organophosphorus (A) and carbamates (B): I – Approaching of the organophosphorus (OP) or carbamate (CB) pesticide into the bottom of the catalytic cavity attracted by the choline binding sub-site (for OP only) and transition state in the interaction between enzyme and the pesticide. In particular, the bonds involved; II - Scheme representing the two occurrence possibilities during the existence of the enzyme-OP complex: spontaneous reactivation (left) or aging (right – only OP); III – Free enzyme; IV - Before undergoing aging (only OP), R2 was attracting electrons from the phosphorus atom. After the removal of R2, these electrons are shared with “O”-Serine, strengthening the binding, which cannot be hydrolyzed.
Inhibition, once initiated, tends to generate acute or chronic intoxication. Depending on the degree of exposure to the toxic substance, the individual may die, due to over-stimulation of his nervous system, since with AChE inhibition, acetylcholine accumulates in neuromuscular junctions and cholinergic synapses [22, 30]. The signs and symptoms of carbamate poisoning are similar to those of organophosphates. They differ only in the duration and intensity of toxicity. The moderate effects of carbamates compared to organophosphates are due to the fact that they reversibly inhibit acetylcholinesterase (hydrolysis with enzyme regeneration) and are rapidly metabolized in vivo [31].
The anticholinesterase action of these pesticides, simultaneously, causes AChE inhibition of central and peripheral nervous tissue. Also, they inhibit erythrocyte AChE and plasma BChE [29]. According to the data from the Food and Agriculture Organization (FAO, 2007) [32], inhibition of cholinesterase activity from 20% characterizes the action of anticholinesterase agents. After 50% inhibition, clinical signs are visualized, and after 90% inhibition, the organism dies.
The in vitro study of acetylcholinesterase activity in various fish species, such as arapaima (Arapaima gigas), peacock bass (Cichla ocellaris), tambaqui (Colossoma macropomum), zebrafish (Danio rerio), jaguar cichlid (Parachromis managuensis), streaked prochilod (Prochilodus lineatus), cobia (Rachycentron canadum), and tilapia (Oreochromis niloticus), have been proposed to be used in the detection of harmful physiological effects of pesticides to these aquatic organisms [4, 6, 33, 34, 35]. In addition to these, we can mention the works of GHAZALA et al. [26], who tested the effect of three sublethal concentrations of the profenofos and carbofuran pesticides on the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the brain, gills, muscle, kidney, liver, and blood of the species Labeo rohita (Indian carp). These authors found that exposure to both pesticides affected the functions of these organs, including metabolism and neurotransmission. Araújo et al. [6] also reported in vitro inhibition of acetylcholinesterase by carbamate and organophosphate pesticides in the brain of the Jaguar cichlid, showing high degree of toxicity.
These studies have confirmed fish as a practical and economically viable source of acetylcholinesterase, which is capable of making water resource biomonitoring procedures routine.
Pesticides, in general, are known to have genotoxic, mutagenic, and carcinogenic action, since they interact chemically with the genetic material, promoting changes in the DNA molecule. These alterations in the organisms’ DNA can cause serious consequences, since, at the individual level, they damage cells and organs and can even affect their reproductive function [36]. Among the most used methodologies for assessing DNA damage in aquatic organisms, the micronucleus (MN) test stands out, which allows the observation of macrolesions in the genome quickly, simply, and minimally invasive [37, 38, 39]. This test consists of a blood smear on a slide (Figure 4A) and is commonly applied to fish erythrocytes, oysters hemocytes, and crabs as an alternative in the detection of genotoxic agents, such as pesticides, in environmental biomonitoring programs [36, 40, 41, 42].
Micronucleus test (A) and Comet assay (B) to evaluate DNA da mages in aquatic organisms.
Currently, it is one of the most used cytogenetic tests in the field of toxicological genetics since it is a sensitive test for detecting structural either-or numerical chromosomal changes [43, 44]. In addition to the micronucleus test, nuclear morphological changes (NMC) can also be analyzed. Several studies describe the presence of these changes in fish cells as a result of exposure to genotoxic substances [7, 44, 45].
In addition to these tests, the Comet Assay (single cell gel electrophoresis assay) is also one of the most used in the evaluation of genomic damage caused by pesticides. It presents high sensitivity in detecting pre-mutagenic lesions in individual cells. It is a technique capable of detecting microlesions in DNA, which are genomic lesions that can be repaired [46]. In this technique, cells that have damages in their DNA, form different fragments which tend to migrate at different speeds during the electrophoretic run, forming a comet under fluorescence microscopy (Figure 4B).
Among the studies that demonstrate the action of pesticides in aquatic organisms, we can mention the study by Silva et al. [7] that evaluated the genotoxic potential of the herbicide trifluralin (one of the herbicides most used in weed control) on Colossoma macropomum (tambaqui). The mutagenic and genotoxic effects of different concentrations of trifluralin (0.25, 0.5, 0.75, 1.0 mg L−1) in peripheral erythrocytes of C. macropomum, were investigated using the micronucleus test (MN), assay comet, and apoptosis. After an exposure period of 96 h, the results showed a significant rate of micronuclei and nuclear abnormalities in erythrocytes from C. macropomum exposed to 0.5, 0.75, 1.0 mg L−1 of trifluralin compared to the group control, thus confirming the genotoxicity of the herbicide trifluralin in the investigated species.
In the search for high productivity, the use of pesticides has been intensified in agricultural crops. Also, the indiscriminate use of these substances has reached non-target organisms, causing deleterious effects on biodiversity, especially in the aquatic ecosystem.
To mitigate these impacts, several methodologies have been used to detect exposure to these toxic substances in aquatic environments. Among them, the methodologies that aim to evaluate the exposed organism at the biochemical and genetic level, as described in this review, show efficiency. It allows the early identification of the presence of the contaminant even before it causes significant changes in the health of the exposed individual, as well as before higher levels of biological organization are reached. It is worth mentioning that the pesticides present in aquatic ecosystems can accumulate in high concentrations in the organisms throughout the trophic level reaching the human being.
Monitoring and controlling the presence of these substances in the environment is necessary since these compounds have become a human and environmental health problem. Allied to this, there is a need for more incentives for the adoption of sustainable agroecological practices, as well as the prohibition of harmful active ingredients to the environment, added to the strict inspection by competent environmental agencies.
The authors would like to thank the Laboratory of Comparative Physiology and Animal Behavior (LabFCCA), the Laboratory of Evolutionary and Environmental Genomics (LAGEA) and the Directorate of Environmental Management (DGA) of the Infrastructure Superintendence of the Federal University of Pernambuco/UFPE for their support and collaboration in the execution of this work.
IntechOpen aims to guarantee that original material is published while at the same time giving significant freedom to our Authors. We uphold a flexible Copyright Policy, guaranteeing that there is no transfer of copyright to the publisher and Authors retain exclusive copyright to their Work.
',metaTitle:"Publication Agreement - Monograph",metaDescription:"IntechOpen aims to guarantee that original material is published while at the same time giving significant freedom to our authors. For that matter, we uphold a flexible copyright policy meaning that there is no transfer of copyright to the publisher and authors retain exclusive copyright to their work.",metaKeywords:null,canonicalURL:"/page/publication-agreement-monograph",contentRaw:'[{"type":"htmlEditorComponent","content":"When submitting a manuscript, the Author is required to accept the Terms and Conditions set out in our Publication Agreement – Monographs/Compacts as follows:
\\n\\nCORRESPONDING AUTHOR'S GRANT OF RIGHTS
\\n\\nSubject to the following Article, the Author grants to IntechOpen, during the full term of copyright, and any extensions or renewals of that term, the following:
\\n\\nThe foregoing licenses shall survive the expiry or termination of this Publication Agreement for any reason.
\\n\\nThe Author, on his or her own behalf and on behalf of any of the Co-Authors, reserves the following rights in the Work but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Work as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\\n\\nThe Author, and any Co-Author, confirms that they are, and will remain, a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\\n\\nSubject to the license granted above, copyright in the Work and all versions of it created during IntechOpen's editing process, including all published versions, is retained by the Author and any Co-Authors.
\\n\\nSubject to the license granted above, the Author and Co-Authors retain patent, trademark and other intellectual property rights to the Work.
\\n\\nAll rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the specific approval of the Author or Co-Authors.
\\n\\nThe Author, on his/her own behalf and on behalf of the Co-Authors, will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Work as a consequence of IntechOpen's changes to the Work arising from the translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits as determined by IntechOpen.
\\n\\nAUTHOR'S DUTIES
\\n\\nWhen distributing or re-publishing the Work, the Author agrees to credit the Monograph/Compacts as the source of first publication, as well as IntechOpen. The Author guarantees that Co-Authors will also credit the Monograph/Compacts as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Work.
\\n\\nThe Author agrees to:
\\n\\nThe Author will be held responsible for the payment of the agreed Open Access Publishing Fee before the completion of the project (Monograph/Compacts publication).
\\n\\nAll payments shall be due 30 days from the date of issue of the invoice. The Author or whoever is paying on behalf of the Author and Co-Authors will bear all banking and similar charges incurred.
\\n\\nThe Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Work worldwide for the full term of the above licenses, and shall provide to IntechOpen, at its request, the original copies of such consents for inspection or the photocopies of such consents.
\\n\\nThe Author shall obtain written informed consent for publication from those who might recognize themselves or be identified by others, for example from case reports or photographs.
\\n\\nThe Author shall respect confidentiality during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Author and Co-Authors are confidential and are intended only for the recipients. The contents of any communication may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\\n\\nAUTHOR'S WARRANTY
\\n\\nThe Author and Co-Authors confirm and warrant that the Work does not and will not breach any applicable law or the rights of any third party and, specifically, that the Work contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy.
\\n\\nThe Author and Co-Authors confirm that: (i) the Work is their original work and is not copied wholly or substantially from any other work or material or any other source; (ii) the Work has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) Authors and any applicable Co-Authors are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) Authors and any applicable Co-Authors have not assigned, and will not during the term of this Publication Agreement purport to assign, any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\\n\\nThe Author and Co-Authors also confirm and warrant that: (i) he/she has the power to enter into this Publication Agreement on his or her own behalf and on behalf of each Co-Author; and (ii) has the necessary rights and/or title in and to the Work to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licences in this Publication Agreement. If the Work was prepared jointly by the Author and Co-Authors, the Author confirms that: (i) all Co-Authors agree to the submission, license and publication of the Work on the terms of this Publication Agreement; and (ii) the Author has the authority to enter into this biding Publication Agreement on behalf of each Co-Author. The Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each Co-Author.
\\n\\nThe Author agrees to indemnify IntechOpen harmless against all liabilities, costs, expenses, damages and losses, as well as all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of, or in connection with, any breach of the agreed confirmations and warranties. This indemnity shall not apply in a situation in which a claim results from IntechOpen's negligence or willful misconduct.
\\n\\nNothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\\n\\nTERMINATION
\\n\\nIntechOpen has the right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Author and/or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Author and/or any Co-Author (being a private individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Author and/or any Co-Author (as a corporate entity) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for, or enters into, any compromise or arrangement with any of its creditors.
\\n\\nIn the event of termination, IntechOpen will notify the Author of the decision in writing.
\\n\\nIntechOpen’s DUTIES AND RIGHTS
\\n\\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen, at its discretion, agrees to publish the Work attributing it to the Author and Co-Authors.
\\n\\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen agrees to provide publishing services which include: managing editing (editorial and publishing process coordination, Author assistance); publishing software technology; language copyediting; typesetting; online publishing; hosting and web management; and abstracting and indexing services.
\\n\\nIntechOpen agrees to offer free online access to readers and use reasonable efforts to promote the Publication to relevant audiences.
\\n\\nIntechOpen is granted the authority to enforce the rights from this Publication Agreement on behalf of the Author and Co-Authors against third parties, for example in cases of plagiarism or copyright infringements. In respect of any such infringement or suspected infringement of the copyright in the Work, IntechOpen shall have absolute discretion in addressing any such infringement that is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\\n\\nIntechOpen has the right to include/use the Author and Co-Authors names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Work and has the right to contact the Author and Co-Authors until the Work is publicly available on any platform owned and/or operated by IntechOpen.
\\n\\nMISCELLANEOUS
\\n\\nFurther Assurance: The Author shall ensure that any relevant third party, including any Co-Author, shall execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\\n\\nThird Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\\n\\nEntire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by, or on behalf of, the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (known as the "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of any fraudulent pre-contract misrepresentation or concealment.
\\n\\nWaiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\\n\\nVariation: No variation of this Publication Agreement shall have effect unless it is in writing and signed by the parties, or their duly authorized representatives.
\\n\\nSeverance: If any provision, or part-provision, of this Publication Agreement is, or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted. Any modification to, or deletion of, a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\\n\\nNo partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Author or any Co-Author, nor authorize any party to make or enter into any commitments for, or on behalf of, any other party.
\\n\\nGoverning law: This Publication Agreement and any dispute or claim, including non-contractual disputes or claims arising out of, or in connection with it, or its subject matter or formation, shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of, or in connection with, this Publication Agreement, including any non-contractual disputes or claims.
\\n\\nPolicy last updated: 2018-09-11
\\n"}]'},components:[{type:"htmlEditorComponent",content:'When submitting a manuscript, the Author is required to accept the Terms and Conditions set out in our Publication Agreement – Monographs/Compacts as follows:
\n\nCORRESPONDING AUTHOR'S GRANT OF RIGHTS
\n\nSubject to the following Article, the Author grants to IntechOpen, during the full term of copyright, and any extensions or renewals of that term, the following:
\n\nThe foregoing licenses shall survive the expiry or termination of this Publication Agreement for any reason.
\n\nThe Author, on his or her own behalf and on behalf of any of the Co-Authors, reserves the following rights in the Work but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Work as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Author, and any Co-Author, confirms that they are, and will remain, a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Work and all versions of it created during IntechOpen's editing process, including all published versions, is retained by the Author and any Co-Authors.
\n\nSubject to the license granted above, the Author and Co-Authors retain patent, trademark and other intellectual property rights to the Work.
\n\nAll rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the specific approval of the Author or Co-Authors.
\n\nThe Author, on his/her own behalf and on behalf of the Co-Authors, will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Work as a consequence of IntechOpen's changes to the Work arising from the translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits as determined by IntechOpen.
\n\nAUTHOR'S DUTIES
\n\nWhen distributing or re-publishing the Work, the Author agrees to credit the Monograph/Compacts as the source of first publication, as well as IntechOpen. The Author guarantees that Co-Authors will also credit the Monograph/Compacts as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Work.
\n\nThe Author agrees to:
\n\nThe Author will be held responsible for the payment of the agreed Open Access Publishing Fee before the completion of the project (Monograph/Compacts publication).
\n\nAll payments shall be due 30 days from the date of issue of the invoice. The Author or whoever is paying on behalf of the Author and Co-Authors will bear all banking and similar charges incurred.
\n\nThe Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Work worldwide for the full term of the above licenses, and shall provide to IntechOpen, at its request, the original copies of such consents for inspection or the photocopies of such consents.
\n\nThe Author shall obtain written informed consent for publication from those who might recognize themselves or be identified by others, for example from case reports or photographs.
\n\nThe Author shall respect confidentiality during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Author and Co-Authors are confidential and are intended only for the recipients. The contents of any communication may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\nAUTHOR'S WARRANTY
\n\nThe Author and Co-Authors confirm and warrant that the Work does not and will not breach any applicable law or the rights of any third party and, specifically, that the Work contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy.
\n\nThe Author and Co-Authors confirm that: (i) the Work is their original work and is not copied wholly or substantially from any other work or material or any other source; (ii) the Work has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) Authors and any applicable Co-Authors are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) Authors and any applicable Co-Authors have not assigned, and will not during the term of this Publication Agreement purport to assign, any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Author and Co-Authors also confirm and warrant that: (i) he/she has the power to enter into this Publication Agreement on his or her own behalf and on behalf of each Co-Author; and (ii) has the necessary rights and/or title in and to the Work to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licences in this Publication Agreement. If the Work was prepared jointly by the Author and Co-Authors, the Author confirms that: (i) all Co-Authors agree to the submission, license and publication of the Work on the terms of this Publication Agreement; and (ii) the Author has the authority to enter into this biding Publication Agreement on behalf of each Co-Author. The Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each Co-Author.
\n\nThe Author agrees to indemnify IntechOpen harmless against all liabilities, costs, expenses, damages and losses, as well as all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of, or in connection with, any breach of the agreed confirmations and warranties. This indemnity shall not apply in a situation in which a claim results from IntechOpen's negligence or willful misconduct.
\n\nNothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\nTERMINATION
\n\nIntechOpen has the right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Author and/or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Author and/or any Co-Author (being a private individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Author and/or any Co-Author (as a corporate entity) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for, or enters into, any compromise or arrangement with any of its creditors.
\n\nIn the event of termination, IntechOpen will notify the Author of the decision in writing.
\n\nIntechOpen’s DUTIES AND RIGHTS
\n\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen, at its discretion, agrees to publish the Work attributing it to the Author and Co-Authors.
\n\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen agrees to provide publishing services which include: managing editing (editorial and publishing process coordination, Author assistance); publishing software technology; language copyediting; typesetting; online publishing; hosting and web management; and abstracting and indexing services.
\n\nIntechOpen agrees to offer free online access to readers and use reasonable efforts to promote the Publication to relevant audiences.
\n\nIntechOpen is granted the authority to enforce the rights from this Publication Agreement on behalf of the Author and Co-Authors against third parties, for example in cases of plagiarism or copyright infringements. In respect of any such infringement or suspected infringement of the copyright in the Work, IntechOpen shall have absolute discretion in addressing any such infringement that is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\nIntechOpen has the right to include/use the Author and Co-Authors names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Work and has the right to contact the Author and Co-Authors until the Work is publicly available on any platform owned and/or operated by IntechOpen.
\n\nMISCELLANEOUS
\n\nFurther Assurance: The Author shall ensure that any relevant third party, including any Co-Author, shall execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\nThird Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\nEntire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by, or on behalf of, the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (known as the "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of any fraudulent pre-contract misrepresentation or concealment.
\n\nWaiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\nVariation: No variation of this Publication Agreement shall have effect unless it is in writing and signed by the parties, or their duly authorized representatives.
\n\nSeverance: If any provision, or part-provision, of this Publication Agreement is, or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted. Any modification to, or deletion of, a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\nNo partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Author or any Co-Author, nor authorize any party to make or enter into any commitments for, or on behalf of, any other party.
\n\nGoverning law: This Publication Agreement and any dispute or claim, including non-contractual disputes or claims arising out of, or in connection with it, or its subject matter or formation, shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of, or in connection with, this Publication Agreement, including any non-contractual disputes or claims.
\n\nPolicy last updated: 2018-09-11
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. 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