Dr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\\n\\n
Seeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\\n\\n
Over these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\\n\\n
We are excited about the present, and we look forward to sharing many more successes in the future.
\\n\\n
Thank you all for being part of the journey. 5,000 times thank you!
\\n\\n
Now with 5,000 titles available Open Access, which one will you read next?
Preparation of Space Experiments edited by international leading expert Dr. Vladimir Pletser, Director of Space Training Operations at Blue Abyss is the 5,000th Open Access book published by IntechOpen and our milestone publication!
\n\n
"This book presents some of the current trends in space microgravity research. The eleven chapters introduce various facets of space research in physical sciences, human physiology and technology developed using the microgravity environment not only to improve our fundamental understanding in these domains but also to adapt this new knowledge for application on earth." says the editor. Listen what else Dr. Pletser has to say...
\n\n\n\n
Dr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\n\n
Seeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\n\n
Over these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\n\n
We are excited about the present, and we look forward to sharing many more successes in the future.
\n\n
Thank you all for being part of the journey. 5,000 times thank you!
\n\n
Now with 5,000 titles available Open Access, which one will you read next?
\n'}],latestNews:[{slug:"intechopen-partners-with-ehs-for-digital-advertising-representation-20210416",title:"IntechOpen Partners with EHS for Digital Advertising Representation"},{slug:"intechopen-signs-new-contract-with-cepiec-china-for-distribution-of-open-access-books-20210319",title:"IntechOpen Signs New Contract with CEPIEC, China for Distribution of Open Access Books"},{slug:"150-million-downloads-and-counting-20210316",title:"150 Million Downloads and Counting"},{slug:"intechopen-secures-indefinite-content-preservation-with-clockss-20210309",title:"IntechOpen Secures Indefinite Content Preservation with CLOCKSS"},{slug:"intechopen-expands-to-all-global-amazon-channels-with-full-catalog-of-books-20210308",title:"IntechOpen Expands to All Global Amazon Channels with Full Catalog of Books"},{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"}]},book:{item:{type:"book",id:"1970",leadTitle:null,fullTitle:"Virtual Reality and Environments",title:"Virtual Reality and Environments",subtitle:null,reviewType:"peer-reviewed",abstract:"Virtual Reality is clearly interdisciplinary research. 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She became a Teacher of Mathematics at the Berzsenyi Dániel Teacher Training College in 1988. Dr. Lányi obtained the Dr. Univ. degree at the University of Veszprém, Hungary in Physical-chemistry (1993), and the Ph.D. degree at the University of Veszprém, Hungary in Computer Science (2000). She has worked as a software engineer and as an associate professor for program languages at the University of Pannonia.\nCurrently, she is focused on virtual reality and its application, user interface design, computer graphics for informatics engineering students and using multimedia in the education for teacher training courses. Ph.D. and Masters’ supervision has an emphasis on multimedia/ virtual reality for the rehabilitation of children with disabilities and patients with mental health issues. She has supervised altogether 180 BSc and MSc thesis works from 1997. Her students received numerous awards.\nDr. Lányi received several awards, the most important ones are: “Master teacher” award of the Hungarian Ministry of Education (2001), the \\"Kalmar\\" award from the John von Neumann Computer Society (2016), the “Hungarian Higher Education Plague” of the Ministry of Human Capacities (2016), the “Diamond-Award from the Association for the Advancement of Assistive Technology in Europe (2015), which is a personal recognition, granted for outstanding work in advancing assistive technology in Europe and the “King Salman Award for Disability Research” of the King Salman Center for Disability Research (2018).\nShe was the secretariat manager of EDeAN in 2009 and the representative of Hungary in IFIP Technical Committee 13: Human-Computer Interaction (TC13) in the period of 2008-2018. 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1. Introduction
Exposure of humans to nanoparticles (NPs) can occur accidentally by environmental particles (e.g., air pollution) and intentionally because a variety of consumer products, cosmetics, and medical products contain NPs. Release of NPs during the manufacturing process may result in exposure of workers by the dermal, oral, and inhalation route.
Exposure to air pollution, such as ultrafine particles, is known to cause inflammatory airway diseases and cardiovascular problems in humans [1]. Pope et al. [2] concluded that even low levels of ambient particle exposure have a significant effect on mortality. The log-linear dose–response relationship between particles and mortality, which was found in their study, highlighted the risk upon exposure to relatively low ambient levels.
Less information is available on health risk of inhaled NPs at the workplace. Reported exposure levels in manufactories for printed electronics, nanoscale metal oxides, and ceramics were low and below the allowed limits [3-6]. On the other hand, exposure to metal NPs in precious metal refinery was higher than recommended limits, and mitigating measures were suggested [7]. Adverse effects of respiratory exposure to occupational NPs included allergic symptoms of the respiratory tract and accumulation in the lung and peritoneum [8-10].
The lung is also one of the suggested routes for noninvasive medication because, compared to dermal and oral exposure, inhalation provides higher bioavailability of active pharmaceutical ingredients. The thinness of the air–blood barrier allowing fast entry to the systemic blood circulation combined with the large absorption area of the lung and the low degree of inactivation through enzymatic degradation by enterocyte and liver enzymes are the main reasons for high systemic levels of inhaled drugs. Several NP-based products, such as nano-salbutamol and nano-fluticasone [11, 12] for chronic obstructive pulmonary disease and asthma and nano-atropine sulfate against organophosphorus poisoning [13], successfully finished testing in healthy volunteers. Furthermore, the safety of heavy chains of Fab fragments of antibodies produced by Nanobody® technology for respiratory syncytial virus infections has been demonstrated in a recent phase I trial [14]. Technosphere® (bis-3,6(4-fumarylaminobutyl)-2,5-diketopiperazine that adsorbs active pharmaceutical ingredients) insulin has passed clinical phase II and III trials [15] and is currently being reviewed by the FDA as inhaled mealtime insulin for managing hyperglycemia in patients with type 1 and type 2 diabetes mellitus. All applications are intended for longer time periods and raise the issue of potential adverse effects on the respiratory epithelium upon chronic exposure.
2. Epithelial barriers of the lung
Airborne particles pass the conducting airways (trachea and large bronchi) and the small bronchi and bronchioli and finally reach the alveoli, where gas exchange takes place. The surface area of the lung is estimated to be 80–140 m2 large [16]. This large surface is covered by a small amount of lining fluid, approximately 10–20 ml in total, and separates the environment from the systemic blood circulation by only a thin air–blood barrier (0.1–0.2 µm thick).
2.1. Airway lining fluid
The mucus layer covering the trachea and main bronchi is much thinner than the mucus layer in the oro-gastrointestinal tract. Indications of mucus thickness vary according to the different techniques that were used for the measurement (Fig. 1). The layer appears to possess a minimum thickness of 5–10 µm in the large airways, while a thickness of 6.9 µm has been measured for segmental bronchi [17, 18]. Other data report a constant thickness of 7 µm of the periciliary layer of all large airways that possess cilia covered by a mucus gel layer with maximum thickness of 5 µm in the trachea and to 0.5 µm in the bronchi [19]. There is a gradual decrease in thickness of this layer, and 1.8 µm of lining fluid has been measured for human bronchioles. It is likely that the mucus gel layer is not continuous and exposure to particles might differ between cells [20]. Mucus and surfactant have different compositions; mucus contains up to 95 % of water, followed by 2–3 % glycoproteins, 0.1–0.5 % proteoglycans, and 0.3–0.5 % lipids. Surfactant, on the other hand, consists of up to 90 % of lipids and 10 % of proteins and therefore resembles more the composition of the human plasma membranes, which on average contain 50 % lipid and 50 % protein [21]. The surfactant is not only reducing the surface tension of the alveoli and preventing their collapse but presents also an effective barrier against dehydration and invasion of pathogens.
2.2. Airway epithelium
The epithelium of the airways representing the main barrier for molecules and particles in aerosols becomes thinner from the conducting (large) airways toward the alveoli, where gas exchange takes place. In the large airways, the epithelium consists of columnar bronchial epithelial cells with microvilli at their apical surface and goblet cells (Fig. 1). Smaller bronchi, bronchioles, have a cubic epithelium and secretory Clara cells, while alveoli are coated with a squamous epithelium consisting of alveolar epithelial cell types I and II. Alveolar macrophages migrate on the surface of this epithelium.
Figure 1.
Barriers for particle uptake by the respiratory system. The mucus layer of the large bronchi can be divided in the periciliary (PL) surrounding the cilia of the bronchial epithelial cells and the gel layer (GL). The epithelial layer of the large (conducting) airways consists of columnar bronchial epithelial cells with cilia (BE) and mucus-producing goblet cells (GC). In the bronchioli, cuboidal bronchial epithelial cells (BE) and mucus-producing Clara cells (C) are found. All epithelial cells reside on a basement membrane (BM). The air–blood barrier at the alveolus consists of alveolar epithelial cell type I (AT-I) and surfactant-producing AT-II cells. Alveolar macrophages (M) migrate on top of the alveolar epithelial cell layer. On the other side of the basement membrane, endothelial cells (EC) of capillaries are located.
Cytotoxicity testing is one of the first steps in the evaluation of toxicants and preclinical testing of drugs. Cytotoxicity of conventional compounds is routinely studied after 4–48 h of exposure because conventional toxicants either acutely damage cells or are degraded by the cell. In contrast to many conventional compounds, NPs have the ability to accumulate in cells and can cause cellular effects also after longer times, for instance, by interference with organelle function. Both extent and effect of cellular accumulation of NPs are currently largely unknown. Most likely these effects are both particle dependent (e.g., content of metal ions, solubility/biodegradability) and cell dependent (expression of uptake routes, proliferation rate, intracellular location). The thickness of the mucus layer on top of intestinal cells can reach up to 900 µm, while respiratory cells are covered only by a mucus layer of 12 µm. Permeability of the epidermis is low due to the formation of the stratum corneum. In addition to the lack of a protective acellular layer, the turnover time of lung cells is slow. Cells of colon crypts are completely renewed after 3–4 days, and cells of the epidermis need 39 days. In contrast to that, after one year, only 7 % of alveolar cells are renewed [22-24]. This combination results in a higher accumulation and a lower regenerative capacity. Accumulation of NPs upon chronic inhalation has been shown, for instance, for titanium dioxide NPs applied to rats over 28 days and three months [25].
In vitro studies could help to estimate the extent and the consequences of cellular accumulation and classify NPs according to their potential for chronic effects. Such information could decrease the amount of labor- and cost-intensive and ethically problematic animal studies. As some defense mechanisms, particularly mucociliary clearance and clearance by the systemic circulation, are lacking in cellular models, such studies might be a tool to identify NPs with a low potential of accumulation and cytotoxicity and probably a lower need for chronic in vivo testing.
3. Components of a representative chronic cytotoxicity model
In order to assess the effects of NP accumulation in a relevant way, the in vitro model has to fulfill several requirements. The important biological parameters are 1) selection of the appropriate cells, 2) physiologically relevant culture system, 3) appropriate NP exposure, 4) demonstration of cellular uptake and accumulation, and 5) information on cell damage/cytotoxicity (Fig. 2). For interpretation of the findings, changes in physicochemical properties of particles have to be taken into account.
Figure 2.
Key parameters for physiologically relevant testing of chronic exposure of airway cells. One potential realization of the principle (a) is presented (b).
3.1. Selection of the appropriate cells
Cellular studies are performed with primary cells or with cell lines. Primary cells have to be isolated from fresh tissues prior to the experiments. They retain many of the cell-specific properties but have a limited life span in culture. Due to the reduced availability of fresh human tissue, mainly rat or murine primary cells are used. In addition to potential inter-species differences, quality (cell viability) and purity (contamination with other cells) of the preparation may vary leading to low reproducibility of the experiments. In contrast to primary cells, immortalized cells, cell lines, provide more reproducible results. Cell lines show a much slower senescence rate and are termed “immortal.” Cell lines have different origins; they may arise during transformation of tumors, due to the presence of a viral gene that partially deregulates the cell cycle, by introduction of telomerase and by fusion with cancer (myeloma) cells. The negative side of immortalization is that cells lose several cell-specific functions. Telomerase immortalized cells are currently believed to be the best way of immortalization because relatively many cell-specific functions are still present but only few TERT-immortalized bronchial epithelial cell lines are available by commercial suppliers, mainly by the American Type Culture Collection (ATCC). As a general rule, the selection of one or the other types of cell is based on the scientific question that should be addressed. Studies on cell-specific functions are performed better in primary cells. Basic cellular processes, such as proliferation, necrosis, and apoptosis, are generally studied in cell lines. Several cell lines have been described as representative models for respiratory cells [26]. They differ in their ability to form a tight intercellular barrier, expression of specific transporters, and secretion products typical for the respective cells. A549 cells and NCI-H441 cells are derived from alveolar epithelial cells. Although they do not form a monolayer with sufficient tightness, A549 cells show surfactant production and have been used in numerous toxicity studies [27, 28]. NCI-H441 cells are the preferred line for transport studies since they form polarized tight monolayers and express organic cation transporters and P-glycoprotein. Cell lines derived from bronchial epithelium comprise BEAS-2B, NuLi-1, 16HBE14o-, and Calu-3 cells. BEAS-2B cells lack tight junctions and mucus production but express organic cation transporters. NuLi-1 cells have been characterized to a lower extent and are rarely used although they form a tight monolayer. 16HBE14o- cells have the ability to form tight junctions and express a variety of transporters. Their main disadvantage is that they do not show the desired phenotype when cultured at an the air–liquid interface, the most physiologically relevant type of culture for cells of the respiratory system.
Calu-3 cells have been described as the suitable model for pulmonary permeability studies [29] with good correlation of permeability values with drug absorption from the rat lung in vivo [30]. Calu-3 cells are derived from bronchial submucosal glands, which explains their ability to produce airway surface liquid, mucins, and other immunological active substances [31]. When cultured on membranes at an air–liquid interface, they form cellular monolayers with tightness and expression of membrane transporters similar to the situation in vivo. Calu-3 cells show mucin-containing granules at the apical pole of the cell (e.g., [32]), but a mucus layer covering the cells cannot be discerned. This problem is linked to the high solubility of mucus in water. After fixation of the tissue and staining either with anti-mucin antibodies or with alcian blue, no mucus layer can be discerned neither in native bronchial tissue nor in Calu-3 cells [33].
3.2. Culture conditions for respiratory cells
Culture at an air–liquid interface is required to provide the most physiologically relevant exposure conditions. This culture method is characterized by supply of the cells with nutrients only from the basal side. This is usually obtained by culturing the cells on membranes in polystyrene housings. These inserts vary in membrane material and pore size and provide the cells with enough support and nutrient supply to form a stable monolayer. Air–liquid interface culture, where the apical part of the cell is exposed to air and not to medium, compared to conventional culture is illustrated in Fig. 3. Respiratory cells cultured at an air–liquid interface show increased differentiation resulting in production of mucus in Calu-3 cells and surfactant in A549 cells [34, 35].
Figure 3.
Different ways of culture: Cell grown at the bottom of well and exposed to NPs suspended in medium (a). In culture at an air–liquid interface, cells are grown on membranes and supplied with nutrients from the basal side. In this culture, respiratory cells secrete pulmonary surfactant (blue line). Cells are exposed to NPs applied as aerosols (b). AC: alveolar cell.
For the testing of particle effects after prolonged exposure, a constant cell population is desired. Routine cell culture systems are less ideal because cells on plastic surfaces proliferate faster than cells in the human body, and intracellularly located NPs are diluted to a greater extent than in vivo. In addition, the high proliferation rate makes compensation of minimal damage possible. Fast proliferation in the culture system has another disadvantage in the evaluation by physiology-based assays used for cytotoxicity testing. All results are normalized to the data of the untreated control cells. This normalization poses the problem that if the untreated cells show physiological growth inhibition due to high cell densities, cell damage by NP could be overlooked.
In order to prevent nonphysiologically high proliferation rates, physiologically relevant culture systems have been developed. Culture in a polar environment, such as growth on microspheres and on membranes, slows down proliferation. Three-dimensional systems such as hanging drops and seeding in hydrogels, on microspheres, and on other scaffolds have been developed. These systems are suitable for several types of cells but do not provide adequate conditions for respiratory cells because cells are cultured submerged. An adaptation of the hanging drop method, however, has been developed and validated for evaluation of gaseous toxicants up to 20 days [36]. Cell culture on membrane inserts, which has been developed to study active and passive transport mechanisms of compounds across cell monolayers, appears suitable. A microfluidic system has been adapted in a way that culture on transwells at an air–liquid interface is possible and A549 cells cultured that way maintained differentiation and viability for 14 days [37]. It is, however, not clear whether under these culture conditions a stable monolayer of cells is obtained since some research groups report multilayer formation of A549 cells in air–liquid interface culture already after three days, while other studies observed A549 monolayers for up to four weeks of air–liquid interface culture [38]. Also 16HBE14o- cells start the formation of multilayers after 17 days in this culture. One hypothesis for the formation of multilayers was absence of cell removal from the apical side by medium changes. Calu-3 bronchial epithelial cells in air-liquid interface culture appear to be suitable because transepithelial electrical resistance (TEER) values remain constant for 17 days [39], while the most often used Caco-2 cells maintain constant values for a maximum of seven days.
3.3. Exposure with particles
Several methods have been used to expose cells to aerosols either as dry powders or by nebulization of solutions. Deposition by aerosols results in a heterogeneous distribution pattern (e.g., [40]) not unlike the in vivo situation where also different densities of particle deposition have been measured [41]. Several groups assessed the effects of environmental NPs (diesel exhaust, smoke) in vitro using either diffusion chambers or more advanced devices in static or dynamic exposure. Setups usually used exposures over 15–60 min, where the aerosol is generated and cells are exposed in a humid atmosphere at physiological temperature (37 °C). Particle deposition in most of the systems is driven by sedimentation and diffusion. Only few established systems, including electrostatic aerosol in vitro exposure system (EAVES) and CULTEX® radial flow system, employ electrostatic precipitation. The Voisin chamber [42, 43], Minucell system [44, 45], Nano Aerosol Chamber In Vitro Toxicity [46, 47], biological aerosol trigger [48], air–liquid interface cell exposure (ALICE) system [49-51], and electrostatic aerosol in vitro exposure system [52, 53] were developed by specific researcher groups. Other systems, such as CULTEX® [54, 55], CULTEX® RFS, and VITROCELL® [56], are commercially available. CULTEX® and VITROCELL® systems have been used in the testing of volatile organic compounds, copper NPs, carbon NPs, zinc oxide NPs, gold NPs, polystyrene NPs, cerium oxide NPs, and laser printer emission particles [44, 57-60]. ALICE system [49-51] and VITROCELL/PARI BOY [61] have been used for aerosolization of NP-containing liquid aerosols. The deposition rates by these devices showed considerable variations related to the aerosolization technique. Related to the total aerosolized amount in nebulizers particle deposition ranged between 0.037 % for aerosolized polystyrene particles/well in the VITROCELL/PARI BOY system, 0.157 % in the ALICE system [51], and 2.8 % in the optimized ALICE CLOUD system [62]. These systems may also lead to particle-dependent differences in deposition [61].
To avoid differences in delivery due to material properties, the use of manual devices, like the ones developed for animal exposures, might be advantageous. Delivery by these devices is easy to perform, less expensive, and technically less demanding and results in a higher relative deposition (amount of particles on cells related to total amount aerosolized) than by the more physiological setups mentioned before. Frequently used syringe-based devices are available from Penn Century Ltd and deliver aerosols generated from dry powder or from suspensions. MicroSprayer® IA-1C aerosolizer for liquid aerosols and DP-4 Dry Powder Insufflator™ for powder aerosols have been used for intratracheal delivery of nanoparticles and occupational and environmental toxicants to mice and rats [63-66]. Both devices have also been used in cellular studies [35, 61, 67-74]. Particle sizes produced by DP-4 Dry Powder Insufflator™ were linked to the respective sizes of the formulated or of the not-formulated powders [75] and are roughly similar before and after aerosolization [76]. The high amount of material that has to be applied is problematic for in vitro studies. The minimal amount is linked to the accuracy of the analytical balance and can be 1 mg as the minimum. An additional problem represents the potential cell damage due to application. When using different distances between tip of the device and cell surface, an optimum distance, when no cell damage occurred, can be identified. With aerosolization devices from suspensions, such as the MicroSprayer® IA-1C aerosolizer, also small amounts of NPs can be applied and the system, therefore, is less dependent from the material. These systems show less electrostatic interaction between particles and devices, aggregation of particles, and hygroscopy that decrease reproducibility of the experiments. The main disadvantage of the MicroSprayer® IA-1C aerosolizer is the generation of droplet in sizes of 20 µm that under normal conditions cannot reach the deep lung.
4. Readout parameters
In order to correlate cytotoxicity and accumulation, particle parameters and cellular uptake of the NPs have also to be determined.
4.1. Particle stability
Stability of particles, including chemical stability as well as colloidal stability may change over the incubation time. Chemical stability is changed by degradation and dissolution of the particles, while colloidal stability is influenced by pH, ions, and macromolecules in the biological fluid. Biodegradability is given for therapeutic aerosols, not for NP inhaled as air pollution and at the workplace. Detection of degradation products is particle specific and usually detected by mass spectrometry. Particle characterization includes changes in bulk composition, in size and structure, and in surface composition, surface charge, electrophoretic properties, surface hydrophobicity, and aggregation [77]. Changes in bulk composition are measured by energy dispersive X-ray spectroscopy (EDS), inductively coupled plasma mass spectrometry (ICP-MS), and combustion analysis (CA) but are less likely to show incubation-dependent changes in biological systems. Particle imaging by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy can identify changes in size and structure. X-ray photoelectron spectroscopy (XPS), Brunauer–Emmett–Teller (BET) method, atomic force microscopy (AFM), and laser-Doppler velocimetry (LDV) are common techniques to characterize surface properties of NPs. Agglomeration can be followed by static light scattering (SLS), photon correlation spectroscopy (PCS)/dynamic light scattering (DLS), small-angle neutron scattering (SANS), and nanoparticle tracking analysis (NTS) [78].
The most important parameter that has to be monitored during prolonged incubation is colloidal stability. Colloidal stability of electrostatically stabilized particles is relatively low because the surface charges are neutralized by ions in the solution and particles prone to agglomeration. Binding of macromolecules may cause complex changes in surface charge, composition, and hydrophobicity and often results in particle agglomeration. Coating of the NPs with hydrophilic bulky molecules, such as polyethylene glycol (PEG), prevents binding of proteins and sterically hinders agglomeration. For instance, gold NPs with PEG coating are stable in physiological solution for 2–15 days [79]. Capping with biocompatible bulky organic molecules, such as serum albumin and starch, also stabilized silver NPs better than electrostatic repulsion by citrate [80]. This coating is absent for unintentionally inhaled NPs from the workplace and environment, which therefore shows lower colloidal stability. Depending on the particle properties with longer incubation in cell culture medium, either the average size of the agglomerates or the fraction of large aggregates increased [81].
4.2. Cellular uptake/accumulation
Colorimetric reading might lack sensitivity but in principle is useful for quantification. Prussian blue staining, for instance, is an established technique for demonstration of cellular uptake of iron oxide particles [82]. Uptake can either be performed on the single-cell level or in the entire population of exposed cells with fluorometric reading and flow cytometry. Also inductively coupled plasma mass spectrometry (ICP-MS) is used for this purpose [83]. Microscopy-based techniques, such as laser scan microscopy and transmission electron microscopy, may be suitable to confirm intracellular localization but are too laborious for quantification. Software programs, e.g., provided as ImageJ macro, could be used to analyze data from more cells [84]. Systems based on transmission electron microscopy scanning, rapid scanning X-ray fluorescence microscopy, and NMR relaxometric techniques are more specialized techniques for quantification of intracellular NPs [85-87].
4.3. Cytotoxicity
The assessment of cytotoxicity commonly uses changes in physiology such as cell number, membrane integrity, amount of DNA or protein, and metabolic activity as readout and indicated as changes in viability. Signals that are obtained after treatment with samples are referred to untreated or solvent-treated cells. Screening assays are usually followed by other tests because decrease of viability can be the result of a variety of influences: depletion of nutrients, interference with organelle function, changes in pH, disruption of membrane integrity, induction of apoptosis, inhibition of proliferation, etc. When cells are cultured on membranes and not in plates, also measurements of TEER values can be used to indicate cell damage. A breakdown in TEER values can be caused by disruption of intercellular junctions or by induction of cell death. Since no indicators are used in these measurements, TEER measurements belong to the label-free detection techniques. A variety of other label-free assays are used for cytotoxicity testing of NPs. They have the advantage that no dye can interfere with cell metabolism or with the particles. NPs have been reported to interfere with conventional cell-based assays in various ways and cause false-positive and false-negative results [88, 89]. Interference of NPs with colorimetric, fluorometric, and luminescent readout is usually identified by inclusion of additional controls and performance of more than one assay. The majority of the aforementioned methods in acute cytotoxicity screening can also be used for the assessment of long-term exposure.
5. Own exposure system
In order to fulfill the requirements for organ-typical testing of NP accumulation, an exposure system based on Calu-3 cells has been developed, which is described in the following.
5.1. Particles accumulation in Calu-3
5.1.1. Methodology
Calu-3 cells were cultured on polyethylene terephthalate membranes with pore size of 3 µm at an air–liquid interface and showed constant TEER values from the 10th day until the end of the observation period, which was chosen at the 28th day (Fig. 4a). During the establishment of the model, cultures with different amounts of liquid in the basal compartment were characterized. When 1,500 µl was applied to the basal compartment, a pseudostratified columnar but not completely stable epithelium was formed. It appeared that the cells did not form a continuous monolayer but that at some locations also multilayers were formed. The epithelium was not stable because, as has been already hypothesized by Lehmann et al., removal of the medium detached cells of the upper layer [38]. When only 500 µl was applied to the basal chamber, the Calu-3 cells formed a stable simple columnar epithelium. The different morphology appears to be caused by different volumes that reached the apical compartment resulting from hydrodynamic pressure of the fluid in the basal chamber. When 1,500 µl was applied to the basal compartment, the apical chamber contained about 250 µl of fluid. At volumes of 500 µl in the basal chamber, only 50 µl liquid was collected from the apical compartment.
Accumulation of 1.8 µg/cm2 and 9 µg/cm2 fluorescently labeled 20 nm carboxyl-functionalized polystyrene particles was studied. Addition as suspension in 10 µl cell culture medium was preferred to application as aerosol by MicroSprayer® IA-1C aerosolizer because data from pilot experiments did not show differences between these application forms. Furthermore, data were more reproducible upon application in small volume than with MicroSprayer® IA-1C aerosolizer. Particles in cell culture medium measured 42 nm with zeta potential of -41 mV. Cells were incubated with the particles for seven days with medium changes in the basal compartment every other day. After seven days, supernatants of cells were collected, cells were rinsed in cell culture medium, TEER measured, and cells incubated with the same amount of NPs for another seven days. This procedure was repeated up to a whole observation time of 28 days. Membranes with cells at each time point (7, 14, 21, 28 days) were subjected to quantification and visualization of uptake. For quantification of cellular uptake, cells were removed from the membrane by trypsin treatment and counted by electrochemical sensing and cellular uptake determined by fluorescence plate reader. Cellular uptake was determined using a standard curve produced from particle solutions diluted with cell culture medium containing cells. This dilution medium was chosen to correct for interference like quenching or autofluorescence by cells. Confocal images obtained by laser scan microscopy allowed visualization of stratification of the Calu-3 cell layer as well as verification of intracellular uptake. In addition, immunocytochemical detection of mucin 5AC was used to identify mucus production of the cells.
5.1.2. Results
During the entire exposure time, no decrease in TEER values was noted, suggesting absence of cell damage (Fig. 4a). Uptake of particles showed intercellular and regional differences (Fig. 4b). Over the entire observation time, Calu-3 cells formed a continuous monolayer, but not all cells showed obvious staining with anti-mucin 5AC antibody as indication for mucus production. This situation resembles the in-vivo condition, where also a non continuous mucus layer in the bronchi has been reported [20]. Cellular uptake of NPs was higher in cells with no mucus production (Fig. 4c). For both concentrations of polystyrene particles, linear cellular accumulation was noted (Fig. 4d).
Figure 4.
Accumulation of 20 nm fluorescently labeled carboxyl-functionalized polystyrene particles (red) in Calu-3 cells over 28 days. TEER values remained constant over the entire exposure time (a). Differences in accumulation of NPs between cells are seen (b). In the volume-rendered 3-D reconstruction performed on the z-series of images at d28, accumulation of NPs mainly in cells showing no prominent anti-mucus production (anti-mucin 5AC staining; green) is seen. Arrow indicates the membrane on which the Calu-3 cell monolayers were grown (c). Quantification of cellular uptake by fluorometric reading shows a linear accumulation of the particles over time (d). Doses of 1.8 µg/cm2 and 9 µg/cm2 were applied and theoretical uptake (Theory) is extrapolated based on the uptake after seven days. Scale bar: 20 µm.
5.2. Discussion of the presented model
Calu-3 cells were cultured on polyethylene terephthalate membranes with pore size of 3 µm. These pores should allow the passage of the particles, but pilot experiments using naked membranes showed that only a fraction of the carboxyl-functionalized polystyrene particles (52 %) was detected on the other side of the membrane. Permeation of polystyrene particles across 3 µm polycarbonate membranes has been reported differently; while one study recovered almost 100 % of 50 nm carboxyl-functionalized and plain polystyrene particles after 6 h [90], another group reported passage of about 25 % of 37 nm carboxyl-functionalized polystyrene particles [91]. Low translocation rates after 24 h of incubation across the same membranes are also reported for 50 nm silica NPs; more than 90 % of the applied particles were trapped in the membranes [92]. These studies suggest that particle transport across cellular monolayers may give erroneous data on cellular transport of polystyrene particles.
Growth of Calu-3 cells in air–liquid interface culture with different volume of medium in the basal chamber could help in identifying differences between monolayered and multilayered epithelium. However, the multilayers that were obtained when using a greater volume in the basal chamber were less stable, and morphological variations and less constant TEER values were obtained.
The currently used system is based on fluorescence labeling of the particles. This labeling may cause changes in surface properties or size. For polystyrene particles, changes in surface properties by fluorescence labeling can be excluded because the particles are synthesized and subsequently swollen in organic fluid containing the dye. This is followed by gradient evaporation of the solvent and repeated washing of the particles to remove dye from the surface [93]. Any fluorescent labeling mode, however, bears also the problem of stability because loss of the label may reduce the measured accumulation values when freshly prepared solutions are used for preparation of the standard curve. The advantage of the polystyrene particles that were used in this study is that they showed stable fluorescence over the entire incubation time of 28 days. Preparation of the standard curves using stock solutions, which are kept under the same conditions as for the cellular exposures, may be more appropriate. It is, however, not excluded that the dilution influences stability since fluorescently labeled particles are more stable in concentrated than in diluted solution (https://www.bdbiosciences.com/documents/BD_Accuri_SPHERO_RainbowCalibration_Particles_ProdInfoSheet.pdf). In addition, stability may be different for intracellular- and extracellular-located particles. Degradation of the label by intracellular enzymes might be expected to reduce the stability of the fluorescence of intracellularly located particles.
The estimate of NP uptake in this study was based on particle uptake after seven days; this uptake could be the result of an inhibited uptake already at that time. Sequential exposure to different NPs and measurement at shorter time points than seven days could be used to answer the question whether the presence of intracellular particles affects the uptake of new particles.
The type of intracellular localization/accumulation may influence cellular effects. Most NPs are taken up by endocytotic mechanisms that deliver their content to lysosomes. Studies on correlation of intralysosomal and extralysosomal accumulation to cell damage may provide further information.
In the concentration range tested, no obvious inhibition of particle uptake was seen, suggesting that cells can handle these particle concentrations without obvious damage. For the identification of potential threshold values and mechanistic studies, exposure to higher concentrations of NPs has to be performed. In addition to that, studies on other particle sizes may provide further insight into cellular effect of inhaled NPs.
In addition to cytotoxicity, exposure to NPs may induce inflammation and including cytokine release as readout parameter may identify this effect. Secretion of the cytokines interleukin-6 and interleukin-8 has been used as an indication for inflammatory response in Calu-3 cells [94].
Co-culture of alveolar epithelial cells with macrophages could provide more insight into the interplay of different cell types at the alveolar barrier. It is questionable that both cell types can be observed over longer periods because of the different medium requirements and proliferation rates.
6. Conclusion
Chronic cytotoxicity testing of respiratory cells is linked to specific problems. Only few cells tolerate air–liquid interface culture for a prolonged time and without change in morphology. Exposure to aerosol may present particle-dependent delivery rates. The presented exposure system fulfilled all requirements for physiologically relevant testing of prolonged contact to NPs by respiratory exposure. Doses that were applied in the model are in the same order of magnitude as therapeutic aerosols [95]. The data showed relatively low inhibition of particle uptake by intracellularly accumulated NPs. Study of particle transport is limited by the fact that insert membrane do not allow unimpeded passage of the particles.
\n',keywords:"Nanoparticles, Chronic effects, Particle uptake, Particle accumulation, Cytotoxicity, Respiratory system",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/48546.pdf",chapterXML:"https://mts.intechopen.com/source/xml/48546.xml",downloadPdfUrl:"/chapter/pdf-download/48546",previewPdfUrl:"/chapter/pdf-preview/48546",totalDownloads:1558,totalViews:362,totalCrossrefCites:2,totalDimensionsCites:6,hasAltmetrics:0,dateSubmitted:"October 26th 2014",dateReviewed:"April 24th 2015",datePrePublished:null,datePublished:"July 15th 2015",dateFinished:"June 14th 2015",readingETA:"0",abstract:"Nanoparticles (NPs) are included in a variety of consumer products including cosmetics, food, and food packaging. They are also used in medical products for dermal and oral application and for inhalation. The thinness of the air–blood barrier, the large absorption area of the lung, and the relatively low inactivation by enzymes provide fast entry to the systemic blood circulation at high drug concentrations. In addition to intended uptake, exposure to airborne particles from the environment and to NPs released during the manufacturing process may occur. Cytotoxicity is routinely studied for 4–48 h of exposure, but NPs may accumulate in cells and can cause cellular effects after longer times. Both extent and consequences of cellular NP accumulation are currently largely unknown.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/48546",risUrl:"/chapter/ris/48546",book:{slug:"nanomaterials-toxicity-and-risk-assessment"},signatures:"Eleonore Fröhlich and Claudia Meindl",authors:[{id:"174043",title:"Prof.",name:"Eleonore",middleName:null,surname:"Fröhlich",fullName:"Eleonore Fröhlich",slug:"eleonore-frohlich",email:"eleonore.froehlich@medunigraz.at",position:null,institution:{name:"Medical University of Graz",institutionURL:null,country:{name:"Austria"}}},{id:"174089",title:"BSc.",name:"Claudia",middleName:null,surname:"Meindl",fullName:"Claudia Meindl",slug:"claudia-meindl",email:"claudia.meindl@klinikum-graz.at",position:null,institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Epithelial barriers of the lung",level:"1"},{id:"sec_2_2",title:"2.1. Airway lining fluid",level:"2"},{id:"sec_3_2",title:"2.2. Airway epithelium",level:"2"},{id:"sec_5",title:"3. Components of a representative chronic cytotoxicity model",level:"1"},{id:"sec_5_2",title:"3.1. Selection of the appropriate cells",level:"2"},{id:"sec_6_2",title:"3.2. Culture conditions for respiratory cells",level:"2"},{id:"sec_7_2",title:"3.3. Exposure with particles",level:"2"},{id:"sec_9",title:"4. Readout parameters",level:"1"},{id:"sec_9_2",title:"4.1. Particle stability",level:"2"},{id:"sec_10_2",title:"4.2. Cellular uptake/accumulation",level:"2"},{id:"sec_11_2",title:"4.3. Cytotoxicity",level:"2"},{id:"sec_13",title:"5. Own exposure system",level:"1"},{id:"sec_13_2",title:"5.1. Particles accumulation in Calu-3",level:"2"},{id:"sec_13_3",title:"5.1.1. Methodology",level:"3"},{id:"sec_14_3",title:"5.1.2. Results",level:"3"},{id:"sec_16_2",title:"5.2. Discussion of the presented model",level:"2"},{id:"sec_18",title:"6. Conclusion",level:"1"}],chapterReferences:[{id:"B1",body:'Van Hee VC, Kaufman JD, Budinger GR, Mutlu GM. 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Drug transport across pulmonary epithelial cell monolayers: effects of particle size, apical liquid volume, and deposition technique. Journal of Aerosol Medicine and Pulmonary Drug Delivery 2010; 23(3) 119-27.'},{id:"B74",body:'Diab R, Brillault J, Bardy A, Gontijo AV, Olivier JC. Formulation and in vitro characterization of inhalable polyvinyl alcohol-free rifampicin-loaded PLGA microspheres prepared with sucrose palmitate as stabilizer: efficiency for ex vivo alveolar macrophage targeting. International Journal of Pharmaceutics 2012; 436(1-2) 833-9.'},{id:"B75",body:'Hoppentocht M, Hoste C, Hagedoorn P, Frijlink HW, De Boer AH. In vitro evaluation of the DP-4M PennCentury insufflator. European Journal of Pharmaceutics and Biopharmaceutics 2014; 88(1) 153-9.'},{id:"B76",body:'Duret C, Wauthoz N, Merlos R, Goole J, Maris C, Roland I, Sebti T, Vanderbist F, Amighi K. 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Cellular targets and mechanisms in the cytotoxic action of non-biodegradable engineered nanoparticles. Current drug metabolism 2013; 14(9) 976-88.'},{id:"B89",body:'Meindl C, Absenger M, Roblegg E, Fröhlich E. Suitability of cell-based label-free detection for cytotoxicity screening of carbon nanotubes. BioMed Research International 2013; 2013 564804.'},{id:"B90",body:'Schulze C, Schäfer U, Voetz M, Wohlleben W, Venzago C, Lehr C. Transport of metal oxide nanoparticles across Calu-3 monolayers modelling the air-blood barrier. EURO-NanoTox-Letters 2011; 1 0001-0011.'},{id:"B91",body:'Cartwright L, Poulsen MS, Nielsen HM, Pojana G, Knudsen LE, Saunders M, Rytting E. In vitro placental model optimization for nanoparticle transport studies. International Journal of Nanomedicine 2012; 7 497-510.'},{id:"B92",body:'George I, Vranic S, Boland S, Borot MC, Marano F, Baeza-Squiban A. Translocation of SiO2-NPs across in vitro human bronchial epithelial monolayer. Journal of Physics: Conference Series 2013; 429 012022.'},{id:"B93",body:'Zhang Q, Han Y, Wang W, Zhang L, Chang J. Preparation of fluorescent polystyrene microspheres by gradual solvent evaporation method. European Polymer Journal 2009; 45 550-6.'},{id:"B94",body:'Zhu Y, Chidekel A, Shaffer TH. Cultured human airway epithelial cells (calu-3): a model of human respiratory function, structure, and inflammatory responses. Critical Care Research and Practice 2010; 2010.'},{id:"B95",body:'Angelo R, Rousseau K, Grant M, Leone-Bay A, Richardson P. Technosphere® Insulin: Defining the Role of Technosphere Particles at the Cellular Level. Journal of Diabetes Science and Technology 3, 545- 54.'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Eleonore Fröhlich",address:"eleonore.froehlich@medunigraz.at",affiliation:'
Center for Medical Research, Medical University of Graz, Graz, Austria
Center for Medical Research, Medical University of Graz, Graz, Austria
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York and William Neal Reynolds",authors:[{id:"13418",title:"Prof.",name:"David",middleName:null,surname:"Jordan",fullName:"David Jordan",slug:"david-jordan"}]},{id:"13135",title:"Computational Biology, Protein Engineering, and Biosensor Technology: a Close Cooperation for Herbicides Monitoring",slug:"computational-biology-protein-engineering-and-biosensor-technology-a-close-cooperation-for-herbicide",signatures:"Giuseppina Rea, Fabio Polticelli, Amina Antonacci, Maya Lambreva, Sandro Pastorelli, Viviana Scognamiglio, Veranika Zobnina and Maria Teresa Giardi",authors:[{id:"13746",title:"Dr.",name:"Giuseppina",middleName:null,surname:"Rea",fullName:"Giuseppina Rea",slug:"giuseppina-rea"},{id:"15034",title:"Dr.",name:"Fabio",middleName:null,surname:"Polticelli",fullName:"Fabio Polticelli",slug:"fabio-polticelli"},{id:"15038",title:"Dr.",name:"Amina",middleName:null,surname:"Antonacci",fullName:"Amina Antonacci",slug:"amina-antonacci"},{id:"15039",title:"Dr.",name:"Sandro",middleName:null,surname:"Pastorelli",fullName:"Sandro Pastorelli",slug:"sandro-pastorelli"},{id:"15040",title:"Dr.",name:"Maya",middleName:null,surname:"Lambreva",fullName:"Maya Lambreva",slug:"maya-lambreva"},{id:"15041",title:"Dr.",name:"Maria Teresa",middleName:null,surname:"Giardi",fullName:"Maria Teresa Giardi",slug:"maria-teresa-giardi"},{id:"15178",title:"Dr.",name:"Veranika",middleName:null,surname:"Zobnina",fullName:"Veranika Zobnina",slug:"veranika-zobnina"},{id:"22620",title:"Dr.",name:"Viviana",middleName:null,surname:"Scognamiglio",fullName:"Viviana Scognamiglio",slug:"viviana-scognamiglio"}]},{id:"13136",title:"Statistical Based Real-Time Selective Herbicide Weed Classifier",slug:"statistical-based-real-time-selective-herbicide-weed-classifier",signatures:"Irshad Ahmad and Abdul Muhamin Naeem",authors:[{id:"13787",title:"Prof.",name:"Irshad",middleName:null,surname:"Ahmad",fullName:"Irshad Ahmad",slug:"irshad-ahmad"},{id:"23691",title:"Prof.",name:"Abdul Muhamin",middleName:null,surname:"Naeem",fullName:"Abdul Muhamin Naeem",slug:"abdul-muhamin-naeem"}]},{id:"13137",title:"Variable Rate Herbicide Application Using GPS and Generating a Digital Management Map",slug:"variable-rate-herbicide-application-using-gps-and-generating-a-digital-management-map",signatures:"Majid Rashidi and Davood Mohammadzamani",authors:[{id:"14094",title:"Prof.",name:"Majid",middleName:null,surname:"Rashidi",fullName:"Majid Rashidi",slug:"majid-rashidi"}]},{id:"13138",title:"Soil Electrical Conductivity as One Possible Tool for Predicting of Cirsium Arvense Infestation Occurrence",slug:"soil-electrical-conductivity-as-one-possible-tool-for-predicting-of-cirsium-arvense-infestation-occu",signatures:"Milan Kroulik, Atonin Slejska, Dana Kokoskova and Veronika Venclova",authors:[{id:"13477",title:"Dr.",name:"Milan",middleName:null,surname:"Kroulik",fullName:"Milan Kroulik",slug:"milan-kroulik"},{id:"14742",title:"Prof.",name:"Antonin",middleName:null,surname:"Slejska",fullName:"Antonin Slejska",slug:"antonin-slejska"},{id:"14743",title:"Dr.",name:"Dana",middleName:null,surname:"Kokoskova",fullName:"Dana Kokoskova",slug:"dana-kokoskova"},{id:"14744",title:"Dr.",name:"Veronika",middleName:null,surname:"Venclova",fullName:"Veronika Venclova",slug:"veronika-venclova"}]},{id:"13139",title:"Herbicides in the Soil Environment: Linkage between Bioavailability and Microbial Ecology",slug:"herbicides-in-the-soil-environment-linkage-between-bioavailability-and-microbial-ecology",signatures:"M. Celina Zabaloy, Graciela P. Zanini, Virginia Bianchinotti, Marisa A. Gomez and Jay L. Garland",authors:[{id:"13386",title:"Dr.",name:"Maria Celina",middleName:null,surname:"Zabaloy",fullName:"Maria Celina Zabaloy",slug:"maria-celina-zabaloy"},{id:"14735",title:"Dr.",name:"Graciela P.",middleName:null,surname:"Zanini",fullName:"Graciela P. Zanini",slug:"graciela-p.-zanini"},{id:"14736",title:"Dr.",name:"Virginia",middleName:null,surname:"Bianchinotti",fullName:"Virginia Bianchinotti",slug:"virginia-bianchinotti"},{id:"14737",title:"Dr.",name:"Marisa",middleName:null,surname:"Gomez",fullName:"Marisa Gomez",slug:"marisa-gomez"},{id:"23759",title:"Dr.",name:"Jay",middleName:null,surname:"Garland",fullName:"Jay Garland",slug:"jay-garland"}]},{id:"13140",title:"Application of Mutated Acetolactate Synthase Genes for Herbicide Resistance to Crop Improvement",slug:"application-of-mutated-acetolactate-synthase-genes-for-herbicide-resistance-to-crop-improvement",signatures:"Masanori Shimizu, Kiyoshi Kawai, Koichiro Kaku, Tsutomu Shimizu and Hirokazu Kobayashi",authors:[{id:"14759",title:"Dr.",name:"Hirokazu",middleName:null,surname:"Kobayashi",fullName:"Hirokazu Kobayashi",slug:"hirokazu-kobayashi"}]},{id:"13141",title:"Transgenic Tall Fescue and Maize with Resistance to ALS-Inhibiting Herbicides",slug:"transgenic-tall-fescue-and-maize-with-resistance-to-als-inhibiting-herbicides",signatures:"Hiroko Sato, Tadashi Takamizo, Junko Horita, Kiyoshi Kawai, Koichiro Kaku and Tsutomu Shimizu",authors:[{id:"13817",title:"Dr.",name:"Hiroko",middleName:null,surname:"Sato",fullName:"Hiroko Sato",slug:"hiroko-sato"},{id:"13865",title:"Dr.",name:"Tadashi",middleName:null,surname:"Takamizo",fullName:"Tadashi Takamizo",slug:"tadashi-takamizo"},{id:"23766",title:"Dr.",name:"Junko",middleName:null,surname:"Horita",fullName:"Junko Horita",slug:"junko-horita"},{id:"23767",title:"Dr.",name:"Kiyoshi",middleName:null,surname:"Kawai",fullName:"Kiyoshi Kawai",slug:"kiyoshi-kawai"},{id:"23768",title:"Dr.",name:"Koichiro",middleName:null,surname:"Kaku",fullName:"Koichiro Kaku",slug:"koichiro-kaku"},{id:"23769",title:"Dr.",name:"Tsutomu",middleName:null,surname:"Shimizu",fullName:"Tsutomu Shimizu",slug:"tsutomu-shimizu"}]},{id:"13142",title:"Pollen Mediated Gene Flow in GM Crops: the Use of Herbicides as Markers for Detection. the Case of Wheat",slug:"pollen-mediated-gene-flow-in-gm-crops-the-use-of-herbicides-as-markers-for-detection-the-case-of-whe",signatures:"Iñigo Loureiro, Concepción Escorial, Inés Santín and Cristina Chueca",authors:[{id:"13724",title:"Dr.",name:"Maria-Cristina",middleName:null,surname:"Chueca",fullName:"Maria-Cristina Chueca",slug:"maria-cristina-chueca"},{id:"13725",title:"Dr.",name:"Ines",middleName:null,surname:"Santin-Montanya",fullName:"Ines Santin-Montanya",slug:"ines-santin-montanya"},{id:"13726",title:"Dr.",name:"Iñigo",middleName:null,surname:"Loureiro",fullName:"Iñigo Loureiro",slug:"inigo-loureiro"},{id:"13727",title:"Dr.",name:"Maria-Concepcion",middleName:null,surname:"Escorial",fullName:"Maria-Concepcion Escorial",slug:"maria-concepcion-escorial"}]},{id:"13143",title:"Overview of Analytical Techniques for Herbicides in Foods",slug:"overview-of-analytical-techniques-for-herbicides-in-foods",signatures:"Hua Kuang, Libing Wang and Chuanlai Xu",authors:[{id:"14502",title:"Prof.",name:"Chuanlai",middleName:null,surname:"Xu",fullName:"Chuanlai Xu",slug:"chuanlai-xu"},{id:"14802",title:"Dr.",name:"Hua",middleName:null,surname:"Kuang",fullName:"Hua Kuang",slug:"hua-kuang"}]},{id:"13144",title:"Enantioseparation and Enantioselective Analysis of Chiral Herbicides",slug:"enantioseparation-and-enantioselective-analysis-of-chiral-herbicides",signatures:"Lixia Jin, Weiliang Gao, Ling Li, Jing Ye, Chunmian Lin and Weiping Liu",authors:[{id:"15144",title:"Dr.",name:"Chunmian",middleName:null,surname:"Lin",fullName:"Chunmian Lin",slug:"chunmian-lin"}]},{id:"13145",title:"Residual Herbicide Dissipation in Vegetable Production",slug:"residual-herbicide-dissipation-in-vegetable-production",signatures:"Timothy Grey and William Vencill",authors:[{id:"13361",title:"Dr.",name:"William",middleName:null,surname:"Vencill",fullName:"William Vencill",slug:"william-vencill"},{id:"13772",title:"Dr.",name:"Timothy",middleName:null,surname:"Grey",fullName:"Timothy Grey",slug:"timothy-grey"}]},{id:"13146",title:"Solid-Phase Extraction for Enrichment and Separation of Herbicides",slug:"solid-phase-extraction-for-enrichment-and-separation-of-herbicides",signatures:"Pyrzynska Krystyna",authors:[{id:"13414",title:"Prof.",name:"Krystyna",middleName:null,surname:"Pyrzynska",fullName:"Krystyna Pyrzynska",slug:"krystyna-pyrzynska"}]},{id:"13147",title:"Chemometric Strategies for the Extraction and Analysis Optimization of Herbicide Residues in Soil Samples",slug:"chemometric-strategies-for-the-extraction-and-analysis-optimization-of-herbicide-residues-in-soil-sa",signatures:"Cristina Díez, Enrique Barrado and José Antonio Rodríguez",authors:[{id:"13645",title:"Dr.",name:"Cristina",middleName:null,surname:"Diez",fullName:"Cristina Diez",slug:"cristina-diez"},{id:"15104",title:"Dr.",name:"Enrique",middleName:null,surname:"Barrado",fullName:"Enrique Barrado",slug:"enrique-barrado"}]},{id:"13148",title:"Membrane Treatment of Potable Water for Pesticides Removal",slug:"membrane-treatment-of-potable-water-for-pesticides-removal",signatures:"Anastasios Karabelas and Konstantinos Plakas",authors:[{id:"14176",title:"Dr.",name:"Anastasios",middleName:null,surname:"Karabelas",fullName:"Anastasios Karabelas",slug:"anastasios-karabelas"},{id:"15404",title:"Dr.",name:"Konstantinos",middleName:"Vasileios",surname:"Plakas",fullName:"Konstantinos Plakas",slug:"konstantinos-plakas"}]},{id:"13149",title:"Eletrochemical Oxidation of Herbicides",slug:"eletrochemical-oxidation-of-herbicides",signatures:"Sidney Aquino Neto and Adalgisa Rodrigues De Andrade",authors:[{id:"13473",title:"Prof.",name:"Adalgisa",middleName:"Rodrigues",surname:"De Andrade",fullName:"Adalgisa De Andrade",slug:"adalgisa-de-andrade"},{id:"13476",title:"Prof.",name:"Sidney",middleName:null,surname:"Aquino Neto",fullName:"Sidney Aquino Neto",slug:"sidney-aquino-neto"}]},{id:"13150",title:"The Bioassay Technique in the Study of the Herbicide Effects",slug:"the-bioassay-technique-in-the-study-of-the-herbicide-effects",signatures:"Pilar Sandín-España, Iñigo Loureiro, Concepción Escorial, Cristina Chueca and Inés Santín-Montanya",authors:[{id:"13724",title:"Dr.",name:"Maria-Cristina",middleName:null,surname:"Chueca",fullName:"Maria-Cristina Chueca",slug:"maria-cristina-chueca"},{id:"13725",title:"Dr.",name:"Ines",middleName:null,surname:"Santin-Montanya",fullName:"Ines Santin-Montanya",slug:"ines-santin-montanya"},{id:"13726",title:"Dr.",name:"Iñigo",middleName:null,surname:"Loureiro",fullName:"Iñigo Loureiro",slug:"inigo-loureiro"},{id:"13727",title:"Dr.",name:"Maria-Concepcion",middleName:null,surname:"Escorial",fullName:"Maria-Concepcion Escorial",slug:"maria-concepcion-escorial"},{id:"23961",title:"Pilar",name:"Sandin",middleName:null,surname:"España",fullName:"Sandin España",slug:"sandin-espana"}]},{id:"13151",title:"Plasmodesmata: Symplastic Transport of Herbicides Within the Plant",slug:"plasmodesmata-symplastic-transport-of-herbicides-within-the-plant",signatures:"Germani Concenco and Leandro Galon",authors:[{id:"13555",title:"Dr.",name:"Germani",middleName:null,surname:"Concenco",fullName:"Germani Concenco",slug:"germani-concenco"},{id:"15476",title:"Prof.",name:"Leandro",middleName:null,surname:"Galon",fullName:"Leandro Galon",slug:"leandro-galon"}]},{id:"13152",title:"7-Keto-8-Aminopelagonic Acid Synthase as a Potential Herbicide Target",slug:"7-keto-8-aminopelagonic-acid-synthase-as-a-potential-herbicide-target",signatures:"In-Taek Hwang, Dong-Hee Lee and No-Joong Park",authors:[{id:"14070",title:"Dr.",name:"In-Taek",middleName:null,surname:"Hwang",fullName:"In-Taek Hwang",slug:"in-taek-hwang"}]},{id:"13153",title:"Possibilities of Applying Soil Herbicides in Fruit Nurseries – Phytotoxicity and Selectivity",slug:"possibilities-of-applying-soil-herbicides-in-fruit-nurseries-phytotoxicity-and-selectivity",signatures:"Zarya Rankova",authors:[{id:"13412",title:"Dr.",name:"Zarya",middleName:null,surname:"Rankova",fullName:"Zarya Rankova",slug:"zarya-rankova"}]},{id:"13217",title:"Herbicide Sulcotrione",slug:"herbicide-sulcotrione",signatures:"Nanxiang Wu",authors:[{id:"14277",title:"Dr.",name:"Nanxiang",middleName:null,surname:"Wu",fullName:"Nanxiang Wu",slug:"nanxiang-wu"},{id:"14804",title:"Senior Experimentalist",name:"Feng",middleName:null,surname:"Jin",fullName:"Feng Jin",slug:"feng-jin"},{id:"14805",title:"assistant researchers",name:"Yong",middleName:null,surname:"Jin",fullName:"Yong Jin",slug:"yong-jin"}]},{id:"13154",title:"The Hemodynamic Effects of the Formulation of Glyphosate-Surfactant Herbicides",slug:"the-hemodynamic-effects-of-the-formulation-of-glyphosate-surfactant-herbicides",signatures:"Hsin-Ling Lee and How-Ran Guo",authors:[{id:"14877",title:"Dr.",name:"How-Ran",middleName:null,surname:"Guo",fullName:"How-Ran Guo",slug:"how-ran-guo"},{id:"14881",title:"Dr.",name:"Hsin-Ling",middleName:null,surname:"Lee",fullName:"Hsin-Ling Lee",slug:"hsin-ling-lee"}]},{id:"13155",title:"Herbicides and Protozoan Parasite Growth Control: Implications for New Drug Development",slug:"herbicides-and-protozoan-parasite-growth-control-implications-for-new-drug-development",signatures:"Ricardo B. Leite, Ricardo Afonso and M. Leonor Cancela",authors:[{id:"14471",title:"Dr.",name:"M. Leonor",middleName:null,surname:"Cancela",fullName:"M. Leonor Cancela",slug:"m.-leonor-cancela"},{id:"14472",title:"MSc.",name:"Ricardo",middleName:null,surname:"B. Leite",fullName:"Ricardo B. Leite",slug:"ricardo-b.-leite"},{id:"14473",title:"Msc",name:"Ricardo",middleName:null,surname:"Afonso",fullName:"Ricardo Afonso",slug:"ricardo-afonso"}]},{id:"13156",title:"Synthesis and Evaluation of Pyrazine Derivatives with Herbicidal Activity",slug:"synthesis-and-evaluation-of-pyrazine-derivatives-with-herbicidal-activity",signatures:"Martin Doležal and Katarína Kráľova",authors:[{id:"13650",title:"Prof.",name:"Martin",middleName:null,surname:"Dolezal",fullName:"Martin Dolezal",slug:"martin-dolezal"},{id:"14876",title:"Dr.",name:"Katarina",middleName:null,surname:"Kralova",fullName:"Katarina Kralova",slug:"katarina-kralova"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"69157",title:"Harmonic Resonance Analysis for Wind Integrated Power System and Optimized Filter Design",doi:"10.5772/intechopen.89167",slug:"harmonic-resonance-analysis-for-wind-integrated-power-system-and-optimized-filter-design",body:'\n
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1. Introduction
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Voltage source converter (VSC) is widely used in industrial, commercial and renewable power generation applications. Out of many possible configurations, the three-phase and three-wire is widely used. During the last decade, the penetration of renewable energy sources has increased around the world. This is due to the increased concern worldwide about the carbon emission [1, 2]. The utilisation of energy from uncertain and variable sources has become possible with the use of these converters only.
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For the purpose of this study, the analysis of VSCs is carried out assuming the ideal operating conditions of grid. But VSCs are never operating under such conditions in practical. In a relatively weak system, VSCs are subject to various power quality disturbances, such as unbalance voltage, voltage swell and swag, notches, etc. [3]. The occurrence of such disturbances causes various problems like ripple in torque of generator and motor, increased losses, abnormal tripping of protective devices, malfunction of sophisticated control system, reduction in the expected lifetime of equipment, etc. [4].
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There are two types of harmonics generated by VSCs. One is characteristics harmonics, which are related with the switching operations of the IGBTs inside the VSC. And second is non-characteristics harmonics. The voltage ripple on DC side of VSC generates harmonics on its AC side current. According to [5], the non-characteristics harmonics are generated by the unbalanced voltage in the AC side. However the quantification of magnitude of such harmonics is not simple and cannot be done with deterministic method. The non-characteristics harmonics are considered as the steady-state low-frequency components which would not appear if the grid voltage is balanced. The unbalance grid voltage has fundamental frequency negative sequence component and third-order positive sequence component.
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Though it is possible to eliminate zero sequence third harmonic component using transformer of proper vector group, the non-characteristics third-order positive sequence harmonics cannot eliminate transformers with delta-connected winding.
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In [6], the author has proposed DC voltage control to eliminate DC oscillating voltage when AC side is unbalanced. To achieve this, VSC has to operate with constant AC power control. However, the effect of control on AC current is not discussed. It is important to analyse the distorted and unbalanced AC side current, when such control is implemented. In this case, the currents of AC side of converter contain non-characteristics low-frequency component such as fundamental negative sequence and third harmonic components of positive and negative sequence.
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\n
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2. Modelling of the voltage source converter
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The total DC current Idc is flowing to the DC side of VSC. Vdc is the instantaneous voltage across the capacitor, and id is the instantaneous DC current. The quality factor of DC capacitor is assumed to be high, so the series resistance is neglected. The instantaneous power is supplied by the renewable energy source, i.e. wind turbine. The instantaneous current Is is supplied by the external source. It is equal to zero when VSC operates as a reactive power compensator.
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The block diagram of VSC control structure is depicted in Figure 1. The control system consists of (i) voltage control, (ii) a phase lock loop (PLL), (iii) current reference calculation block and (iv) current control. The real power reference is calculated by the PI controller, which considers the DC voltage and desired active power through the VSC to the grid. The instantaneous reactive power is calculated by the separate loop, which may consider either the desired power factor or the reference voltage. The dq frame is synchronised with the positive sequence fundamental voltage of the grid at PCC with the help of PLL. It converts three phase voltages Va, Vb and Vc into Vd+ and Vq+, which are converted in the alpha-beta reference frame voltages Vα and Vβ. Then current reference is obtained by the α-β to abc transformation. These reference currents are compared with actual current, and modulating signals md and mq are generated. These signals are finally transformed into ma, mb and mc to generate pulse width modulated (PWM) signal.
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Figure 1.
Control structure of grid-connected wind converters.
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Both switching frequency and grid voltage distortion can cause poor power quality. A filter design is a subject that requires trade-off between filter performance and the control bandwidth. Filters are required to meet power quality standard, avoid parallel resonance and improve power quality.
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Inverter for grid interfacing will need to incorporate interface filters to attenuate the injection of current harmonics.
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3. Impedance-based stability analysis
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This method was proposed in [7]. The system impedance is partitioned into source and grid impedance. The source impedance is either represented by Thevenin’s equivalent circuit or Norton equivalent circuit (Figures 2 and 3). Thevenin’s equivalent circuit consists of ideal voltage source in series with the series impedance (Zs), whereas the load impedance is modelled by series impedance (Zl). Since the converter circuit is non-linear, it is represented by the small signal circuit. This linear representation of circuit is valid only for the small perturbation of signal. With this assumption, the current (I) flowing from source to load is given by
System stability analysis is based on the assumption that the source voltage and load impedance remain stable. So, V(s) and 1/Z(s) are stable. So, the stability depends on the extreme right-hand side of Eq. (2). It is given by
The close observation of Eq. (3) reveals the important characteristics. It is a transfer function with unity gain and feedback equal to Zs(s)/Zl(s). According to the linear control theory, the H(s) is stable, if and if only, when ration Zs(s)/Zl(s) meets the requirement of Nyquist stability criterion [7].
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In the above analysis for stability, it is assumed that the source is ideal voltage source and it remains stable under unloaded condition. However, the grid-connected inverters are usually current controlled. Hence above analysis is not much useful. So, the source should be represented as current source. To arrive at the equivalent current source, the same small signal voltage source is modified. The voltage across load is given by
Similar to the above analysis, the current source is assumed to be stable under unloaded condition. The load is stable when connected to ideal current source. Under this condition, I(s) and 1/Yl(s) are stable. Under this condition, stability of V(s) depends on stability of second term of Eq. (5). This again resembles the closed-loop transfer function with negative feedback. The gain is unity and the feedback factor is Ys(s)/Yl(s). Therefore the system is stable, if, along with above conditions, it meets the Nyquist criterion. In Eq. (5) admittances are used instead of impedance, though the analysis still can be carried out in terms of the impedances. In this case, Eq. (5) becomes
It is important to note the requirement of stability. In the voltage source model, the output impedance of source should be as low as possible (ideally zero); whereas in the current source model, the output impedance should be as high as possible (ideally infinite).
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4. Grid-connected inverters
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The modelling of impedance of grid-connected VSC has very important use in analysis of stability and resonance phenomena when converter is integrated into the grid [5]. The grid-connected converter used in renewable sources is modelled as a current source in parallel with an impedance, i.e. Norton’s equivalent circuit [6]. The stability of grid-connected inverter can be determined by Nyquist criterion [8]. The control structure of most of the VSCs is developed in the rotating dq reference frame [7]. The phase lock loop is used to synchronise converter with grid [9]. The use of complex multiple-loop control structure introduces non-linearities. These are generally overlooked in the simplified low-order modelling [10]. On the flip side, the detailed model introduces complexity and cross-couplings between various terms, which makes the determination of output impedance cumbersome. The trade-off way suggested in some literatures is to linearise the model by small signal analysis technique.
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The impedance of converter-interconnected generator is affected by various factors such as control parameters, PLL, switching delays and converter harmonic filters. The converter is basically controlled by output current signal. In Figure 4, i1 is the converter current and i2 is the grid current. The converter is controlled either by i1 or i2. If the grid current is the control variable, then the current control loop is
where Y21 is the forward trans-admittance of the filter, \n\n\nG\nPI\n\n\ns\n\n\n is the proportional-integral-type current controller and \n\n\nG\nD\n\n\ns\n\n\n is the switching delay. Here, the converter output voltage is considered as pure sinusoidal; if there is a noise in the voltage, then it needs to be considered as a disturbance signal. If, the converter is controlled by taking converter current i1, then Y21 is replaced by output conductance Y11. The forward transconductance Y21 is given by
Higher-order controller also can be used for current control, but here simple PI controller is considered for the sake of simplicity. The delay, estimated by Pade’s approximation technique, is given by
The frequency response of output impedance is plotted here with the line impedance. The phase at the intersection of two curves gives the phase margin (PM).
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5. Phase lock loop
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\n\n\nG\nPLL\n\n\ns\n\n=\nf\n\ns\nw\nk\n\n\nE11
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The phase lock loop is required for PI-based controller. So, the structure of PLL should be thoroughly assessed for stability. Proper structure of PLL and control scheme helps to mitigate adverse impact on the system. The purpose of PLL is to synchronise converter with grid. Harmonics in the grid may penetrate to the converter through the PI controller. If control parameters are not properly chosen, this may produce harmonics through circular effect. Figure 5 shows the block diagram of PLL. The detail block diagram is given in Figure 6.
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Figure 5.
Phase lock loop.
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Figure 6.
Block diagram of simple phase lock loop.
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Figure 6 is the traditional second-order generalised integrator–quadrature signal generator (SOGI-QSG) PLL. It can filter out higher-order harmonics, where ui is the input signal; ui′ and qui′ are two output signals, which are in quadrature; k is the damping coefficient; and ω′ is the output angular frequency of PLL. Eqs. (14) and (15) show the transfer function of PLL.
In traditional SOGI-QSG PLL, the DC component in the input signal is not suppressed by the PLL. To overcome this problem, a minor modification is made in above PLL. The modified PLL is shown in Figure 7.
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Figure 7.
Block diagram of modified phase lock loop.
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Transfer functions of modified PLL are given in Eqs. (16)–(18). This structure reduces the tracking error of PLL.
The bode plot of one of the PLL used in [12] is given here. The bandwidth of the PLL is 33 Hz. It attenuates harmonics of 1 kHz to −30 dB. However, the effect of PLL with other controllers and output filter needs to be investigated for crucial stability analysis (Figure 8).
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Figure 8.
Frequency response of simple phase lock loop.
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6. Damped passive filter topologies
\n
Different types of passive filters are used in converter-based renewable generation sources. The effectiveness of filter, particularly passive type, primarily depends on the grid strength and variation of grid impedance with time. LCL is the common type of filter used widely. The variation in grid impedance affects the performance of LCL filter. Hence, the design of LCL filter is a trade-off between robustness and damping of resonance. The effective impedance with simple PI controller is explained here with RL filter topology.
Equation (25) shows that there are two zeros and one pole. First, the output impedance decreases at −20 dB per decade up to first zero at α. At α, the impedance response becomes flat, and at β the impedance starts increasing at 20 dB per decade (Figure 9). So, the response of integrated filter becomes similar to that of series resonance filter. The selection of α and β depends on the parameter selection of Lfc, Rfc, kP and ki. Bode plot for RL filter without and with PI current controller is given in Figures 10 and 11, respectively.
\n
Figure 9.
Grid-connected inverter with filter.
\n
Figure 10.
Frequency response of impedance with RL filter without PI controller.
\n
Figure 11.
Frequency response of impedance with RL filter with PI controller.
\n
\n
\n
6.2 L-C filter
\n
LC filter is used in converters for industrial applications like variable frequency drive (VFD) and uninterrupted power supply (UPS). It is simple in construction and relatively less costly. The analysis of LC filter with PI controller is given here (Figure 12).
\n
Figure 12.
L-C filter.
\n
As per the standard practice, the value of L is selected such that its impedance at fundamental frequency should not drop by more than 3% of rated voltage. The capacitive reactance offers 1/5th of the fundamental inductive reactance at switching frequency of converter (around 3–4 kHz) to absorb harmonics effectively. Based on these criteria, LC filter is widely designed. The frequency response of LC filter with and without PI controller is given in Figures 13 and 14, respectively. It is clear from the difference in bode plot that PI controller changes the frequency response.
\n
Figure 13.
Frequency response of LC filter without PI controller.
\n
Figure 14.
Frequency response of LC filter with PI controller.
\n
\n
\n
6.3 L-C-L filter
\n
Initially LC filter was used for converter applications, but grid-connected inverter has unique requirements that LC filter may not provide. Properly designed LCL filter may overcome the drawbacks of LC filter (Figure 15).
Figure 16 shows the frequency response of LCL filter together with PI controller. It is clear from Eq. (29) and from Figure 16 that the PI controller increases the order of filter, so the frequency response of passive filter gets changed by the controller action.
\n
Figure 16.
Frequency response of LCL filter with PI controller.
\n
\n
\n
\n
7. Optimised filter design
\n
The design criterion for filter design should comply with the regulatory requirement. As per IEEE 519-1992 standard, the current harmonics for weak grid condition (Isc/IL) should be less than 0.3%. The ripple is caused by pulse width modulated signal. Output voltage varies from zero level to DC voltage level (Vdc). The modulated wave causes ripple in the current, which can be reduced by proper selection of output filter parameters. Typical L-C-L filter is used in most of the inverter. The L1-C-L2 filter has three unknowns. The selection of these parameters depends on various factors. Grid condition is one of them. The strength of the grid decides the effectiveness of filters. Typical grid impedance varies from 5 to 8% with X/R ratio in the range of 7–10 [13].
\n
The switching frequency decides the ripple level and ripple frequency. Generally, inverter switching frequency remains in the range of 3–5 kHz. The ripple current is reduced either by increasing switching frequency or by using passive filter at the inverter/converter output. Higher switching frequency is selected to reduce the ripple current at the generation point, but it adversely affects the converter (IGBT) losses [14]. The second option is to use large inductor at the output of converter, but this not only incurs high cost but also increases the core losses. Typically 20% ripple current is expected in the output current. Keeping this in consideration, the inductor L1 is given by [7]
The capacitor rating is selected such that the reactive power of capacitor is neither too high nor too low. Higher reactive power demands more power from converter, which causes more loss in reactor L1 and also more loss in converter switches. A lower value of capacitor will increase the inductor size. So, the capacitor is selected so that the reactive power should be in the range of 15–20% of the rated power.
In LCL filter, the damping can be achieved by simply adding series resistance in series with capacitor C. It is obvious that large value of damping resistance (Rd) gives large damping. But damping is effective around the resonance point only [15]. Above the resonance point, damping weakens. Also, the large value of resistance causes higher losses (Figure 17).
\n
Figure 17.
Type I LCL filter.
\n
Impedance of Type I LCL filter with damping resistance is given by
Frequency response of LCL filter with damping resistor is given in Figure 18. The grid parameters are Lg = 0.212 mH and Rg = 0.0095 Ohm, whereas the filter parameters are L1 = 0.450 mH, L2 = 0.300 mH and C = 270 uF. The damping resistance is varied from 0.05 to 0.8 Ohm. It is clear from the plot that the response of impedance is similar to inductive impedance. The notch is observed at the resonant frequency, which can be dampened by resistor in series with capacitor. The gain margin is −40 dB and phase margin is around 120°. So, as per the Nyquist criterion, the filter response is very stable.
\n
Figure 18.
Nyquist plot of Type I LCL filter.
\n
\n
\n
7.1.2 Type II filter
\n
In Type II LCL filter (Figure 19), the damping can be achieved by simply adding a parallel combination of resistor and inductor in series with capacitor C. The effect of inductor is investigated using Nyquist plot. The value of Rf = 10 Ohm and Lf is varied from 50 to 500 μH. The effect of increase in Lf observed using Nyquist plot is given in Figure 20.
\n
Figure 19.
Type II LCL filter.
\n
Figure 20.
Nyquist plot of Type II LCL filter.
\n
Impedance of Type II LCL filter with damping inductor and resistance is given by
From the bode graph, it is observed that the gain margin is 70–100 dB for various values of Lf. Similarly, the phase margin is 270°. As the value of Lf is increased, there will be reduction in the gain margin.
\n
\n
\n
7.1.3 Type III filter
\n
In Type III LCL filter, the damping can be achieved by simply adding a parallel combination of resistor and inductor in series with capacitor C. The effect of inductor is investigated using Nyquist plot (Figure 21).
\n
Figure 21.
Type III LCL filter.
\n
Impedance of Type III LCL filter with damping inductor and resistance is given by
Impedance shows two resonance points: first is parallel resonance and second is series resonance (Figure 22). At first resonance point, the output impedance increases, and at series resistance it is at the minimum value. The phase margin of filter is around 210°. This ensures the stability of filter. The resonant frequency will shift with change in grid resistance, so the damping effect is difficult to predict.
\n
Figure 22.
Nyquist plot of Type III LCL filter.
\n
\n
\n
7.1.4 Type IV filter
\n
In Type IV LCL filter, the damping can be achieved by simply adding a parallel combination of resistor and capacitor Cf. The effect of resistor value is investigated using Nyquist plot (Figure 23).
\n
Figure 23.
Type IV LCL filter.
\n
Impedance of Type IV LCL filter with damping resistor Rf in series with capacitor Cf is given by
The response of Type IV filter is similar to the tuned filter. The notch in frequency response of impedance is observed at the resonant frequency (Figure 24). This notch can be damped by putting higher value of resistor in series with the capacitor Cf. The phase of impedance sharply changes from −90 to 90°, which means the nature of impedance turns from capacitive to inductive. The ratio of Cf/C decides the damping effectiveness. The larger the Cf/C ratio, the larger will be the damping. Also, with increasing value of Cf/C ratio, the loss in resistor Rf is also increases as more and more current tends to flow in it [16, 17, 18].
\n
Figure 24.
Nyquist plot of Type IV LCL filter.
\n
\n
\n
7.1.5 Type V filter
\n
In Type V LCL filter, the damping can be achieved by simply adding a parallel combination of resistor and inductor with capacitor C. The effect of inductor is investigated using Nyquist plot (Figure 25).
Type V filter response shows two resonance frequency (Figure 26). The first resonance creates high voltage distortion, while the second resonance point gives rise to current distortion. If converter generates harmonics equal to this resonance, then there will be high voltage distortion and current distortion. However, with lower value of resistance, damping can be achieved. An additional inductor Lf can reduce the resistive loss.
\n
Figure 26.
Nyquist plot of Type V LCL filter.
\n
\n
\n
\n
7.2 Active damping of filter
\n
Grid-connected converters may not function stably under the harmonic resonance condition. Harmonic resonance occurs when the converter impedance and grid impedance becomes equal in magnitude and 180° out of phase. The converter is generally connected to grid through filters, which is mostly LCL type. So, the parameters of LCL filters play an important role in keeping the successful functioning of converter. Also, the control structure of the converter shapes its output impedance. So, the control parameter should be selected such that it keeps the converter in the safe zone at all frequency. This is explained further here with analysis.
\n
The inverter output characteristics depend on several factors like parameter of LCL filter, grid impedance, type of controller and control parameters. By selecting the appropriate parameter, the inverter can be operated with resistive output impedance, inductive output impedance and capacitive output impedance. Most of the inverters are operated with inductive (L-type) output impedance. This inverter is known as the L-type inverter. Here it is explained how an inverter impedance can be made capacitive (C-type inverter) with selection of virtual current control loop.
\n
The output impedance of inverter is controlled by the virtual impedance. The single-line diagram of inverter with control loop is shown in Figures 27 and 28. The control is implemented with current loop and voltage loop, which gives good tracking behaviour and good output voltage. The feedback of current is taken from branch between the two capacitors. The first capacitor has a value of βC, and the second capacitor has a value of (1-β)C. Thus, the overall value of capacitor is C. The output impedance of system after adding virtual branch GV is given by
With the above value of GV, the output impedance becomes capacitive. So, by selection of proper control loop and control parameter, the shaping of inverter output impedance can be effectively done. Also, the desired value of damping can be achieved. This method is known as active damping method. Like this, the inverter can be made either L-type or R-type.
\n
\n
\n
\n
8. Conclusion
\n
The characteristics and response of impedance of grid-connected inverter depend on various factors like selection of output filter and its parameter, controller type and its parameters, structure of PLL, the delay in switching of converter, etc. In this chapter, using first-order PI controller and different controller types, it is explained in a simple way. The complexity of impedance increases with the order of the controller and its structure. Also, the reshaping of inverter impedance can be done by selection of suitable control structure. By adding virtual impedance, the output impedance can be made inductive, capacitive or reactive without adding any additional hardware. In this work different variants of passive filter are explained, and how their output impedance behaves at different frequency is also explained with the use of Nyquist plot. The active damping method is explained, whereby damping is achieved without using resistor in the filter, which is considered as a very energy-efficient method of achieving damping. The output impedance can be reshaped either by external hardware in the form of filter or by adding virtual control loop in the controller. The stability of converter depends on how converter output impedance interacts with the grid impedance. Numerous efforts have been made and can be found in literatures to achieve the stability only by reshaping the impedance. However, the stability region can be expanded by reshaping of impedance up to a certain extent only. Only reshaping of impedance does not guarantee the converter stability with changing grid condition. Also, the converter is more likely to fall in an unstable region with weak grid condition, but reshaping of impedance helps to prevent converter-based generation from going unstable. By analysing the output impedance in the frequency domain, the controller parameters can be adjusted to reshape the output impedance. By increasing the proportional gain of the PI controller, the magnitude of the output impedance can be increased and can enhance the ability of harmonic rejection. By increasing the integral gain of the PI controller, the phase of the output impedance can be increased to improve the stability of the system [19, 20] (Table 1).
\n
\n
\n
\n\n
\n
Switching frequency
\n
4 kHz
\n
\n
\n
Switching delay
\n
20 μS
\n
\n
\n
Lc
\n
4 mH
\n
\n
\n
Lg
\n
0.4 mH
\n
\n
\n
Cf
\n
5 μF
\n
\n
\n
kp
\n
1.2
\n
\n
\n
ki
\n
11.6
\n
\n\n
Table 1.
Converter parameter.
\n
\n\n',keywords:"voltage source converter (VSC), pulse width modulation (PWM), grid-connected converter, filter, harmonic resonance, doubly fed induction generator (DFIG), Nyquist criteria, damped filter",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/69157.pdf",chapterXML:"https://mts.intechopen.com/source/xml/69157.xml",downloadPdfUrl:"/chapter/pdf-download/69157",previewPdfUrl:"/chapter/pdf-preview/69157",totalDownloads:239,totalViews:0,totalCrossrefCites:0,dateSubmitted:"May 8th 2019",dateReviewed:"August 13th 2019",datePrePublished:"November 27th 2019",datePublished:"February 26th 2020",dateFinished:"September 20th 2019",readingETA:"0",abstract:"As the contribution of renewable energy sources is increasing year over year, the effect of harmonics on power system becomes important, and it requires special attention. In conventional power sources, the harmonics is not generated at the source side; only load side is contributing in the harmonics. But renewable energy sources, particularly wind and solar, are based on power electronic devices, so it generates harmonics. This harmonics may have an adverse effect on the system. Harmonic resonance is one of the phenomena, due to which the harmonics are amplified and give rise to several trivial issues. Various methods are used to control the harmonics in the system. Harmonic filter is one of the simple ways to absorb the harmonics generated at load and generation side. Various filter designs have been found in literature as well as in the field. The filters are classified according to their design, construction and operation method. There are two main categories, active filters and passive filters. The passive filters are widely used due to its simplicity and lesser cost. However, to achieve the better performance, it is also used with active filters, and this combination is known as hybrid filter. The response of filters is modified as per the system requirement using various techniques. In this work, the impedance characteristics of various filters are discussed and analysed. Also, how the control structure of power electronic devices affects or modifies the output impedance of converter is also discussed.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/69157",risUrl:"/chapter/ris/69157",signatures:"Jignesh Pravinbhai Patel and Satish Kantilal Joshi",book:{id:"7636",title:"Wind Solar Hybrid Renewable Energy System",subtitle:null,fullTitle:"Wind Solar Hybrid Renewable Energy System",slug:"wind-solar-hybrid-renewable-energy-system",publishedDate:"February 26th 2020",bookSignature:"Kenneth Eloghene Okedu, Ahmed Tahour and Abdel Ghani Aissaou",coverURL:"https://cdn.intechopen.com/books/images_new/7636.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",isbn:"978-1-78984-591-4",printIsbn:"978-1-78984-590-7",pdfIsbn:"978-1-83880-372-8",editors:[{id:"172580",title:"Dr.",name:"Kenneth Eloghene",middleName:null,surname:"Okedu",slug:"kenneth-eloghene-okedu",fullName:"Kenneth Eloghene Okedu"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:[{id:"304472",title:"Mr.",name:"Jignesh Pravinbhai",middleName:null,surname:"Patel",fullName:"Jignesh Pravinbhai Patel",slug:"jignesh-pravinbhai-patel",email:"jignesh.pravinbhai@gmail.com",position:null,institution:null},{id:"309509",title:"Dr.",name:"Satish Kantilal",middleName:null,surname:"Joshi",fullName:"Satish Kantilal Joshi",slug:"satish-kantilal-joshi",email:"skjoshi@ieee.org",position:null,institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Modelling of the voltage source converter",level:"1"},{id:"sec_3",title:"3. Impedance-based stability analysis",level:"1"},{id:"sec_4",title:"4. Grid-connected inverters",level:"1"},{id:"sec_5",title:"5. Phase lock loop",level:"1"},{id:"sec_6",title:"6. Damped passive filter topologies",level:"1"},{id:"sec_6_2",title:"6.1 R-L filter",level:"2"},{id:"sec_7_2",title:"6.2 L-C filter",level:"2"},{id:"sec_8_2",title:"6.3 L-C-L filter",level:"2"},{id:"sec_10",title:"7. Optimised filter design",level:"1"},{id:"sec_10_2",title:"7.1 Passive damping of filter",level:"2"},{id:"sec_10_3",title:"7.1.1 Type I filter",level:"3"},{id:"sec_11_3",title:"7.1.2 Type II filter",level:"3"},{id:"sec_12_3",title:"7.1.3 Type III filter",level:"3"},{id:"sec_13_3",title:"7.1.4 Type IV filter",level:"3"},{id:"sec_14_3",title:"7.1.5 Type V filter",level:"3"},{id:"sec_16_2",title:"7.2 Active damping of filter",level:"2"},{id:"sec_18",title:"8. Conclusion",level:"1"}],chapterReferences:[{id:"B1",body:'Blaabjerg F, Zhe C, Kjaer SB. Power electronics as efficient interface in dispersed power generation systems. IEEE Transactions on Power Electronics. 2004;19(5):1184-1194\n'},{id:"B2",body:'Ali MH, Wu B, Dougal RA. An overview of SMES applications in power and energy systems. IEEE Transactions on Sustainable Energy. 2010;1(1):38-47\n'},{id:"B3",body:'Flourentzou N, Agelidis VG, Demetriades GD. VSC-based HVDC power transmission systems: An overview. IEEE Transactions on Power Electronics. 2009;24(3):592-602\n'},{id:"B4",body:'Xia J, Fang X, Chow JH, Edris A, Uzunovic E, Parisi M, et al. A novel approach for modeling voltage-sourced converter-based FACTS controllers. IEEE Transactions on Power Delivery. 2008;23(4):2591-2598\n'},{id:"B5",body:'He J, Li YW. Generalized closed-loop control schemes with embedded virtual impedances for voltage source converters with LC or LCL filters. IEEE Transactions on Power Electronics. 2012;27(4):1850-1861\n'},{id:"B6",body:'Sun J. Impedance-based stability criterion for grid-connected inverters. IEEE Transactions on Power Electronics. 2011;26(11):3075-3078\n'},{id:"B7",body:'Blaabjerg F, Teodorescu R, Liserre M, Timbus AV. Overview of control and grid synchronization for distributed power generation systems. IEEE Transactions on Industrial Electronics. 2006;53(5):1398-1409\n'},{id:"B8",body:'Middlebrook RD. Input filter considerations in design and application of switching regulators. In: Proceedings of the IEEE-IAS Annual Meeting; 1976. pp. 366-382\n'},{id:"B9",body:'Freijedo FD, Yepes AG, Lopez O, Vidal A, Doval-Gandoy J. Three-phase PLLs with fast postfault retracking and steady-state rejection of voltage unbalance and harmonics by means of lead compensation. IEEE Transactions on Power Electronics. 2011;26(1):85-97\n'},{id:"B10",body:'Wang F, Duarte JL, Hendrix MAM, Ribeiro PF. Modeling and analysis of grid harmonic distortion impact of aggregated DG inverters. IEEE Transactions on Power Electronics. 2011;26(3):786-797\n'},{id:"B11",body:'Beres RN, Wang X, Blaabjerg F, Liserre M, Bak CL. Optimal design of high-order passive-damped filters for grid-connected applications. IEEE Transactions on Power Electronics. 2016. DOI: 10.1109/TPEL.2015.2441299\n'},{id:"B12",body:'Sun J. Small-signal methods for AC distributed power systems—A review. IEEE Transactions on Power Electronics. 2009;24(11):2545-2554\n'},{id:"B13",body:'Li X-Q, Wu X-J, Geng Y-W, Zhang Q. Stability analysis of grid-connected inverters with an LCL filter considering grid impedance. Journal of Power Electronics. 2013;13(5)\n'},{id:"B14",body:'Jiao J, Nelms RM. Regulating output impedance using a PI controller to improve the stability of a single phase inverter under weak grid. In: 2016 IEEE 16th International Conference on Environment and Electrical Engineering (EEEIC); 2016\n'},{id:"B15",body:'Yang W, Wang M. Impedance modeling and output impedance coupling analysis of three-phase grid-connected inverters. In: 2018 IEEE International Power Electronics and Application Conference and Exposition (PEAC); 2018\n'},{id:"B16",body:'Zhong Q-C, Zeng Y. Control of inverters via a virtual capacitor to achieve capacitive output impedance. IEEE Transactions on Power Electronics;29(10):5568-5578. DOI: 10.1109/tpel.2013.2294425\n'},{id:"B17",body:'He Y, Lai C-t, Chung S-h, Zhang X, Wu W. Use of series negative impedance to cancel the effect of equivalent grid impedance on the grid connected inverter in the DPGS. In: 2018 IEEE Applied Power Electronics Conference and Exposition (APEC); 2018\n'},{id:"B18",body:'Zhong Q-C, Zeng Y. Universal droop control of inverters with different types of output impedance. IEEE Open Access. 2016. DOI: 10.1109/ACCESS.2016.2526616\n'},{id:"B19",body:'Messo T, Luhtala R, Aapro A, Roinila T. Accurate impedance model of grid connected inverter for small signal stability assessment in high-impedance grid. In: 2018 International Power Electronics Conference (IPEC-Niigata 2018 -ECCE Asia); 2018\n'},{id:"B20",body:'Ling P, Peng Z, Chao G. An impedance reshaping control strategy to enhance adaptability to grid for grid-connected inverters. In: 2018 China International Conference on Electricity Distribution (CICED); 2018\n'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Jignesh Pravinbhai Patel",address:null,affiliation:'
Department of Electrical Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India
Department of Electrical Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India
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XML Typesetting and pagination - web (PDF, HTML) and print files preparation
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For Authors who are still unable to obtain funding from their institutions or research funding bodies for individual projects, IntechOpen does offer the possibility of applying for a Waiver to offset some or all processing feed. Details regarding our Waiver Policy can be found here.
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Indexing and listing across major repositories, see details ...
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Dissemination and Promotion
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Benefits of Publishing with IntechOpen
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Proven world leader in Open Access book publishing with over 10 years experience
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The Open Access Publishing Fee (OAPF) is payable only after your full chapter, monograph or Compacts monograph is accepted for publication.
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OAPF Publishing Options
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1,400 GBP Chapter - Edited Volume
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4,000 GBP Compacts Monograph - Short Form
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*These prices do not include Value-Added Tax (VAT). Residents of European Union countries need to add VAT based on the specific rate in their country of residence. Institutions and companies registered as VAT taxable entities in their own EU member state will not pay VAT as long as provision of the VAT registration number is made during the application process. This is made possible by the EU reverse charge method.
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Services included are:
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An online manuscript tracking system to facilitate your work
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XML Typesetting and pagination - web (PDF, HTML) and print files preparation
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Permanent and unrestricted online access to your work
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Exceeds 20 pages (for chapters in Edited Volumes), an additional fee of 40 GBP per page will be required
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If a manuscript requires Heavy Editing or Language Polishing, this will incur additional fees.
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Your Author Service Manager will inform you of any items not covered by the OAPF and provide exact information regarding those additional costs before proceeding.
\n\n
Open Access Funding
\n\n
To explore funding opportunities and learn more about how you can finance your IntechOpen publication, go to our Open Access Funding page. IntechOpen offers expert assistance to all of its Authors. We can support you in approaching funding bodies and institutions in relation to publishing fees by providing information about compliance with the Open Access policies of your funder or institution. We can also assist with communicating the benefits of Open Access in order to support and strengthen your funding request and provide personal guidance through your application process. You can contact us at oapf@intechopen.com for further details or assistance.
\n\n
For Authors who are still unable to obtain funding from their institutions or research funding bodies for individual projects, IntechOpen does offer the possibility of applying for a Waiver to offset some or all processing feed. Details regarding our Waiver Policy can be found here.
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Added Value of Publishing with IntechOpen
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Choosing to publish with IntechOpen ensures the following benefits:
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Indexing and listing across major repositories, see details ...
\n\t
Long-term archiving
\n\t
Visibility on the world's strongest OA platform
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Live Performance Metrics to track readership and the impact of your chapter
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Dissemination and Promotion
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Benefits of Publishing with IntechOpen
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Proven world leader in Open Access book publishing with over 10 years experience
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+5,200 OA books published
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Most competitive prices in the market
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Fully compliant with OA funding requirements
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Optimized processes, enabling publication between 8 and 12 months
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Personal support during every step of the publication process
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+146,150 citations in Web of Science databases
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Currently strongest OA platform with over 150 million downloads
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