**About the Editor**

### **Jos ´e Mar´ıa Salvador-Gonz´alez**

Jose Mar ´ ´ıa Salvador-Gonzalez is a Professor at the Complutense University of Madrid. His ´ publications are in Mariology and religious studies. He holds twenty degrees from Rome, Paris, Louvain, Caracas and Madrid universities. He has five PhDs: in Aesthetics and Art Sciences, Universite Panth ´ eon-Sorbonne, Paris I (1981); in Social Sciences, Universidad Central de Venezuela ´ (2002); in Art History (2007); in Sciences of Religions (2018); and in Philosophy (2022). The last three were obtained from the Complutense University of Madrid. He is the Director of the journal De Medio Aevo and the CAPIRE Research Group.


**4.2 Planning HCW management**

There are basically two types of HCW management; a national level HCW management and a local level (individual healthcare institute or service provider) HCW management. In some cases, regional (provincial, prefectural) level HCW management is set between the national and local levels.

*Technical Cooperation for Enhancing Infectious Healthcare Waste Management DOI: http://dx.doi.org/10.5772/intechopen.102604*

#### *4.2.1 National/regional HCW management plan and strategy*

The purpose of planning a national/regional HCW management is to improve HCW management at the national and regional (e.g., provincial) levels, where strong political commitment is required. In the planning process, it is required to involve relevant ministries and professional organizations including academics in the HCW management field.

The goals of the national/regional HCW management plan are: to declare the government's intentions to improve HCW management, to define overall national/ regional strategies and plan for improving HCW management, to specify activities and timeline for implementation, and to define the roles and responsibilities of authorities concerned & other stakeholders.

#### 1.Assessment study

The first step for formulating a national HCW management plan is to conduct a national assessment study on HCW management, where the following four points have to be clarified: (i) an inventory of existing healthcare institutes (waste generators) and HCW treatment facilities; (ii) analysis of existing legislation, regulations, and rules; (iii) existing HCW stream and its management practices if any; and (iv) implementation agency and human resource on HCW management.

The inventory of HCW generation sources and GIS (geographical information system) map are crucial for planning HCW management, which covers all healthcare institutes including hospitals, clinics, and primary healthcare (PHC) institutions. A regression model will be applied to estimate the amount of HCW generated by them using the outpatient, inpatient, and bed numbers.

#### 2.Planning

Specific objectives toward developing a national HCW management plan should include the following five key objectives: (1) to understand the present situation and setting the purpose of the plan, (2) to develop the legal and regulatory framework, (3) to develop financial investments and resources for HCW management, (4) to develop capacity building program, and (5) to set up a monitoring plan. The expected general contents of the national HCW management plan are as shown in **Table 2**.

#### *4.2.2 Local HCW management plan*

When planning a local HCW management, the first thing that must be done is to clarify the executing agency based on the legal system, and that agency will make the plan. The local HCW management plan is created by each healthcare institute and/or service provider based on the above-mentioned national/regional plan. It is required to be specific and practical depending on given local conditions.

Specific objectives toward developing a local HCW management plan needs to include the following six key components based on the direction of the national/ regional HCW management plan: (1) designing HCW management system, (2) segregation of HCW at source, (3) HCW handling, storage, and transport, (4) treatment technologies, (5) waste disposal, (6) staff training, and (7) monitoring:

#### **Table 2.**

*General contents of the national HCW management plan.*

#### 1.Designing HCW management system

Regarding the treatment and/or sterilization of infectious HCW, there are basically two types of HCW management systems; distributed (on-site) and centralized (offsite) systems. The distributed one is a system in which a healthcare institute has its own (relatively small-scale) treatment facility and processes infectious HCW by itself. On the other hand, the centralized system is a system in which a private or public service provider collects infectious HCW and transports it from each healthcare institute based on a contract and centrally processes it in the service provider-owned treatment facility.

The advantages of the distributed system are: complete control of infectious HCW by the generator, mitigating the risk of exposure during waste collection & transportation, and reducing unknown risks in the treatment by a service provider. However the distributed system has the following disadvantages: the healthcare institute has to get a relatively high financial burden for a treatment facility, and also become responsible for meeting all regulatory requirements on infectious HCW treatment, which needs additional resources.

#### *Technical Cooperation for Enhancing Infectious Healthcare Waste Management DOI: http://dx.doi.org/10.5772/intechopen.102604*

On the other hand, the advantages of a centralized system are minimization of cost and responsibility for the treatment of infectious HCW. Each healthcare institute can concentrate only on source separation and appropriate waste discharging. The HCW generators, in particular small-scale healthcare institutes, benefit from the quality of service and the economies of scale. The disadvantages are indirect control of infectious HCW management due to outsourcing.

In the case of a centralized system, two management plans, on-site and off-site plans, are required, and coordination and cooperation between the two actors are indispensable.

These distributed and centralized systems are often combined in a country/region to act as a hybrid system for actual HCW management (**Figure 7**). As shown in the **Figure 7**, the generated HCW is first separated into non-infectious HCW and infectious HCW (sharps and infectious) at the source. Non-infectious HCW is treated in the MSW management flow, while infectious HCW is sent for on-site or off-site treatment. If there is no treatment facility or in the case of the treatment capacity is insufficient, an emergency controlled cell is installed at the landfill site for direct disposal as an emergency measure.

2.Segregation at source

Segregation at source (source separation) is one of the most important steps to successfully manage HCW. As shown in **Figure 1**, only about 15% of the HCW is hazardous, treatment and disposal costs could be greatly reduced if proper segregation were performed. Segregating hazardous from nonhazardous waste reduces also greatly the risks of infecting SWM workers.

Segregation consists in separating the different waste streams based on the hazardous properties of the waste, the type of treatment and disposal practices that are applied. A recommended way of identifying HCW categories is by sorting the waste into color-coded and well-labeled bags or containers.

#### **Figure 7.**

*Outline and options for HCW management in developing countries. Dashed parts are emergency measures under limited conditions, which is an example in Palestine.*

#### 3.Handling, storage, and transport

HCW workers have the greatest occupational risk (the Risk A in **Figure 3**), where the hazard is from direct contact with sharps and infectious waste. Sharps can cause puncture wounds, scratches, and scrapes, where infectious agents can penetrate the skin. The use of special containers for sharps is absolutely necessary. In the HCW handling process, there is also potential for exposure through inhalation of pathogencontaining aerosols or particulates [20].

The best way to minimize the risk of exposure is to ensure that the infectious waste is properly isolated. Some basic principles [20] are: packaging the infectious HCW properly; avoiding physical contact with the infectious HCW; using personal protective equipment (PPE); and handling the infectious HCW as little as possible.

Another factor to be considered is public health including informal waste pickers if the hazardous infectious waste is directly disposed without any treatment as an emergent measure under limited conditions. In that case, no one has easy access to discarded needles and syringes, so that waste sharps' containers need the following features: puncture resistance, impermeability, rigidity, tamper resistance, and proper marking [31].

#### 4.Treatment technologies

The purpose of treatment is to change the biological character of infectious HCW to eliminate, or at least to significantly reduce, its potential for causing negative impacts. The three most common techniques used to treat infectious HCW are incineration (various types are available), steam sterilization (autoclaving), and microwaving (**Table 3**). Other currently available techniques include irradiation, chemical disinfection, and so on.

#### 5.Disposal

When infectious HCW waste has been properly treated, the waste is no longer infectious. The treated HCW can be handled in the same way as normal municipal waste. However there are two exceptional cases [18]: for sharps and pathological wastes, additional processing before disposal is necessary; if other hazardous substances such as pharmaceutical, chemical, or radioactive waste are contained, there must be additional treatment before disposal.

In the case of sharps waste treated by steam sterilization, intact sharps are possibly sent to a landfill where workers are at risk for injury; therefore, they should be shredded or destroyed when be treated. Pathological waste treated by steam sterilization also requires additional processing since body parts or organs can be recognizable, which should not be directly disposed in the landfill.

In developing countries, sometimes no suitable HCW treatment facility is available and the only option is direct landfilling. In such a case, it is necessary to avoid using an open dumpsite and dispose a landfill having a shielded structure. It is necessary to immediately cover the soil at the time of waste disposal to prevent the dissipation of HCW and protect the environment.

#### 6.Staff training

HCW management training is an effective intervention for preventing infections and improving the occupational safety of the HCWs through building awareness,

**Table 3.** *Comparison of most common HCW treatment technologies and landfilling [14, 20, 32–35].*


**3. Histopathology**

Ovarian tumors are classified according to the World Health Organization (WHO) proposal for gynecological tumors. Ovarian cancer has high cellular heterogeneity, and most of the primary ovarian tumors can be integrated into three major groups, namely epithelial, sex cord and ovarian stroma, and germ cell tumors [2].

Although the ovarian epithelial surface represents only a small fraction of all ovarian cell types, EOC is the most common, corresponding to almost 60% of all ovarian tumors [19, 20]. According to the criteria proposed by the WHO in 2014, EOC can be divided into seven histological subcategories, namely serous, mucinous, endometrioid, clear cell, Brenner, seromucinous, and undifferentiated [2]. All the mentioned histological subtypes, except for the undifferentiated type, are further subdivided into benign, borderline, and malignant neoplasia, depending on the optical microscopy characteristics.

Sex cord and stroma tumors arise from the ovarian connective tissue, often responsible for hormone secretion. These tumors encompass a vast group of tumors, for which the subgroup of "pure" ovarian stromal tumor is the most frequent (9% of all OC), usually with benign behavior. Also in this group of tumors, granulosa cell tumors are associated with aggressive behavior and represent 1% of all OC. Regarding the germ subgroup, a mature cystic teratoma is very common (32% of all OC), although the remaining germ cell tumors, both benign and malignant, are rare, representing 3–5% of all OC cases [2, 21].

is still discussed, it has an impact in early disease stages not only to define FIGO staging but also to establish the need for adjuvant treatment, with a significant impact in survival [27, 28]. Blood dissemination is less frequent and usually occurs in advanced disease stages [23, 24].

Ovarian Cancer Overview: Molecular Biology and Its Potential Clinical Application

http://dx.doi.org/10.5772/intechopen.73863

61

A considerable number of clinical-pathological factors have been implicated in OC prognosis. Disease stage, tumor size, histological subtype, differentiation degree, and residual tumor after surgery are considered as the classic prognostic factors. More specifically, the extent of residual disease after surgery is regarded as a major prognostic factor, shown to influence the chemotherapy response and survival [29–33]. Inclusively, a recent meta-analysis has shown that residual tumor is a more powerful prognostic determinant than FIGO stage [31]. The correct histological classification of EOC is also crucial, since it is an independent prognostic factor and provides a guideline for therapeutic management [8, 27]. Performance status (PS) and age are also important factors having an impact on the prognosis and, ultimately, in the

Numerous studies have been conducted to assess the clinical significance of molecular alterations in OC. However, so far, the obtained results do not allow a prognostic biomarker to be universally accepted, although the determination of *BRCA* germline mutations has been recently approved as a predictive biomarker for OC. Recently, the development and the application of new genomic technologies have allowed the description of molecular signatures integrated into prognostic and predictive models. In particular, the Cancer Genome Atlas Project (TCGA) has been critical in adding to our knowledge, as it has been used to confirm the importance of *BRCA* genes to serous OC patients survival, as well as being able to help to describe a transcriptional signature with prognostic relevance [34] (this will be investigated further in a separate chapter).

The therapeutic strategy for EOC is based on cytoreductive surgery and staging, followed by adjuvant chemotherapy with the duplet platinum/taxane [8, 20]. As mentioned above, the extent of surgery is a determinant for survival and response to chemotherapy, since these parameters vary significantly depending on the success (optimal or suboptimal) of the surgical procedure [33]. Systemic therapy with cytotoxic agents plays a fundamental role in the treatment of this neoplasia. Chemotherapy is generally recommended in the EOC, including early stages with histopathological criteria of poor prognosis (FIGOIA/IB G3, FIGO IC, FIGO II, or clear cell histology at any stage). However, stage IA or IB G1 or G2 tumor patients, if adequately staged (i.e., with peritoneal washings, assessment of the contralateral ovary and fallopian tube, pelvic and para-aortic node assessment and omentectomy), have a better prognosis and can be treated with surgery alone without the need for adjuvant chemotherapy [35–37] (**Figure 1**).

**5. Prognostic factors**

decision of medical treatment [27].

**6. Treatment**

**6.1. First-line treatment**


 There are many risks — both threats and opportunities — which may impact on a company's resources, projects and profitability. Risk means different things to different businesses and organizations. Undoubtedly, the risk represents both a potential threat and potential opportunity for businesses.

Every business and decision involves a certain amount of risk. Risk might cause a loss to a company. This does not mean, however, that businesses cannot take risks. As disengagement and risk aversion may result in missed business opportunities, which will lead to slower growth and reduced prosperity of a company. In today's increasingly complex and diverse environment, it is crucial to find the right balance between **risk aversion and risk taking**. To do this it is essential to understand the complex, out of the whole range of economic, technical, operational, environmental and social risks associated with the company's activities. However, risk management is about much more than merely avoiding or successfully deriving benefit from opportunities. Risk management is the identification, assessment, and prioritization of risks. Lastly, risk management helps a company to handle the risks associated with a rapidly changing business environment. When risk management does receive attention, it is often in response to unforeseen (and usually negative) events.

The impact of the global economic crisis has varied from one country to another: not all countries, sectors and organizations were affected in the same hard way by it. Even, the impact of the financial crisis is varying widely across companies within the same sector. In today's post-crisis economy effective risk management is a critical component of any successful management strategy. In complex and rapidly changing situations, as today's supply chains and partnership arrangements tend to be, management needs to consider all risks within the enlarged business connections.

Understanding of the risk management is vital for both practitioners and researchers. The emergence of new insights into approaches and models can help address multifaceted risk management issues.

There are five parts in this book of 23 chapters. The papers are organized according to theoretical, methodological and practical issues and areas of risk management: Part 1

#### XIV Preface

provides new insights into theoretical approaches and models for risk management, Part 2 deals with risk and supply chain management, Part 3 focuses on specific aspects of enterprise risk management, Part 4 examines risk management practice across different projects and industries, and Part 5 discusses emerging issues related to climate change and climate risk management. The authors touched on a wide range of risk management issues. Consequently, in the context of the thematic classification scheme, some papers fall into more than one category.

I consider it an honour and privilege to have had the opportunity to edit this book. I am particularly grateful to all the authors for their outstanding contributions, and to Mirna Cvijic, the publishing process manager at InTech, for her kind assistance in publishing this book.

#### **Dr. Nerija Banaitiene**

Department of Construction Economics and Property Management, Faculty of Civil Engineering, Vilnius Gediminas Technical University, Lithuania

**Approaches and Models for Risk Management**

**Chapter 1**

© 2012 Hürlimann, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Hürlimann, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Biometric Solvency Risk for Portfolios of General**

The main theoretical goal of the present exposé is to extend the results presented in Hürlimann [1] to the Markov chain model of life insurance, which enables modeling all single life/multiple life traditional contracts subject to biometric risk with multiple causes of decrement. In particular, a complete risk modeling of single-life insurance products with mortality and disability risks requires the specification of a Markov model with three states. As novel illustration we offer to the interested practitioner an in-depth treatment of

The present investigation is restricted to biometric risks encountered in traditional insurance contracts within a discrete time Markov chain model. The current standard requirements for the Solvency II life risk module have been specified in QIS5 [2], pp.147-163. QIS5 prescribes a solvency capital requirement (SCR), which only depends on the time of valuation (=time at which solvency is ascertained) but not on the portfolio size (=number of policies). It accounts explicitly for the uncertainty in both trends (=systematic risk) and parameters (=parameter risk) but not for the random fluctuations around frequency and severity of claims (=process risk). In fact, the process risk has been disregarded as not significant enough, and, in order to simplify the standard formula, it has been included in the systematic/parameter risk component. For the purpose of internal models and improved risk management, it appears important to capture separately or simultaneously all risk components of biometric risks. A

As starting point, we recall in Section 2 the general solvency rule for the prospective liability risk derived in [1], Section 2, which has resulted in two simple liability VaR & CVaR target capital requirements. In both stochastic models, the target capital can be decomposed into a solvency capital component (liability risk of the current period) and a risk margin component (liability risk of future periods), where the latter must be included (besides the

**Life Contracts (II) The Markov Chain Approach**

Werner Hürlimann

**1. Introduction**

http://dx.doi.org/10.5772/48374

Additional information is available at the end of the chapter

endowment contracts with waiver of premium by disability.

more detailed account of our contribution follows.


**1. Introduction**

The main goal of this article is to consider the development and operation of a deterministic control unit (DCU) for an arbitrary technological process in real time (RT). Modern onboard equipment and networks increase in their complexity from year to year, with the number of tasks undertook by such systems growing significantly. Therefore, the industry needs to ensure a high level of reliability during the operation of such systems. For this purpose, aircraft onboard networks' developers need effective mechanisms for ensuring deterministic data delivery. DCU is a deterministic real-time technological complex, which controls a technological process (TP). Its operation depends on external influencing factors, which move DCU from an internal state to a uniquely defined (deterministic) new state in a specific predetermined time interval. DCU is based on a distributed information computing environment (RIVS) [1] and built of the computing modules (CM) of the same type. A network should connect these models. There are several elements, which are the typical

**Citation:** Kosyanchuk, V.; Selvesyuk, N.; Zybin, E.; Novikov, V.; Olenev, V.; Solovyov, A.; Semyonov, M. Application of a Deterministic Optical Network Model for the Implementation of an Expert System Knowledge Base for Information Transmission Failure Management. *Eng. Proc.* **2023**, *33*, 51. https:// doi.org/10.3390/engproc2023033051

Academic Editors: Askhat Diveev, Ivan Zelinka, Arutun Avetisyan and Alexander Ilin

Published: 19 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

RIVS modules: N computing modules, K control loops for technological systems, L data concentrators. All of these modules have access to a local information system (LIS). Most TP problems are solved using RIVS, but the time for solving the task should not exceed a certain maximum task duration time. The time for each task from the control loop is determined from the permitted operating time of a separate system control loop and the entire DCU. The key DCU functioning process is the exchange of the information between its subsystems. Transmitted or received data determine the correct operation of functional software applications (FSP) and the whole TP.

#### **2. Local Information System Requirements**

Several DCU LIS features distinguish it from other real-time and information networks:

Such control signals are time markers used for the synchronization of the system, and control and notification signals [5–10].

Typical LISs face problems of data transmission delays, which makes the operation of the system unpredictable and leads to packet collisions. Of course, it is possible to significantly increase the data speed or use specialized type of quality of service (QoS). This QoS is scheduling, which ensures data transmission in accordance with a schedule specified for the whole network during the configuration phase. However, unfortunately, during the operation of the DCU, unexpected delays and collisions occur. This produces an effect on the work of the scheduler, so existing networks cannot provide complete determinism, which is very important for onboard networks. Regarding this, the main sources of collisions should be excluded from the network architecture to ensure complete determinism. Most such delays are caused by switches and routing mechanisms.

The construction of LIS DCU should be based on the following principles: the "pointto-point" organization of the data exchange between the FSP of different CMs; using broadcast mode for sending packets as the main mode; using of distributed memory for data exchange between FSPs. Such LIS principles could be implemented in deterministic real-time optical networks (DOS RV). Such networks transmit the packets without switching and use distributed memory principles for data exchange between end systems. All-optical networks are a type of networks where the switching, multiplexing, and data transmission are organized by purely optical technologies, rather than by electronic technologies. DOS RVs are built as an information transmission system with two types of topologies: a star and a ring. They contain *N* VMs with the FSPs function and a spectral SDA (SSDA). Figure 1 shows a block diagram of the DOS RV.

Some key principles should be taken into account to build an effective DOS RV [11,12]. The optical technology of wavelength division multiplexing (WDM) should be used as a transport architecture. WDM consists in the transmission of many different data flows over the single optical fiber, but in separate optical *λ*-channels. Each channel has its own optical frequency called the *λ*-channel. Information exchange is based on the principle of distributed shared memory (DSM). Memory access is performed via the simple "read" and "write" commands. Each FSP has a particular memory area, which is used to exchange data with external subscribers of the fiber optic medium. It is performed via the *λ*-channel specially allocated for this FSP, and this frequency is fixed for the data flow in terms of the whole network.

**Figure 1.** DOS RV structure.

There are a few important steps to organize the work of DOS RV:

#### **3. The DOS Model in the Automation Design and Development System for Information Exchanges of Real-Time Systems**

To design communication protocols that implement information exchange in the DOS RV networks, modern methods should be used. Such a methods should cover all the system development stages (see Figure 2).

**Figure 2.** Methodology for development of communication protocols for real-time systems.

The design process begins with the gathering of technical requirements for the future protocol and their analysis. Technical requirements are divided into requirements for the internal mechanisms of the protocol and requirements for the operation of devices based on this protocol. Regarding this, the first version of the specification of the future protocol was developed. A simulation was used to test and verify the developed protocol.

The modeling process creates layered and network models. The network model is created after at least one successful version of the layered model has been created. Each of these types of models solves its own problems and checks the operation of different mechanisms of the protocol. After creating the models, it is necessary to configure them, and then carry out detailed modeling. If an error is found at any of the parallel stages, it is necessary to create a bug report, send it to the expert group for review, and stop work in the parallel branch until the specification is checked or clarified. After analyzing the error, the specification is refined and remodeled. When both simulation teams report that no more errors have been found, the simulation results are compared to the original technical requirements. If the requirements are not met, the specification is sent back for revision, otherwise the document is finalized and can be sent to further stages of the project, for example, hardware implementation.

The gathering of technical requirements is carried out by an expert group, which includes representative customers, developers, researchers who have the necessary knowledge and information on the further use of the protocol. The completeness of the specifications is an important issue, as the requirements will be the basis for deciding whether there are errors in the protocol.

Next, the specification of the designed communication protocol is written. Such a document usually includes a general description of the protocol stack (or a single protocol), data transfer formats, protocol mechanisms and algorithms in accordance with the layered architecture, interlayer interfaces, and examples of the application of the above mechanisms or the protocol as a whole. Once this task is completed and the initial draft of the specification is ready, the document is submitted for modeling. Modeling is carried out in order to check the underlying algorithms and can be carried out already in the process of writing the specification. To do this, an intermediate document is already given for

modeling; it also passes through various checks, but is returned back to the revision stage if such a specification does not meet the technical requirements.

Further modeling should be carried in two parallel branches: layered protocol modeling (light gray blocks in Figure 2) and the system of devices modeling (dark gray blocks in Figure 2). When modeling a layered protocol stack, the model layers fully correspond to the protocol structure, and the interfaces between these layers correspond to service access points. Thus, to carry out the simulation, it is sufficient to fully describe the operation of the protocol in relation to one device. To obtain a result, it is necessary to organize data transfer between two devices only, since, for the task of analyzing the protocol, network interaction and the mechanisms of operation as part of a larger system do not matter. When writing a device interaction model, the network operation and the exchange of various information packets are modeled. With this approach, the interaction of the constituent elements of the processes inside the device is not considered. In order to effectively use the communication protocol modeling, both of these approaches should be used. This is the only way to detect most of the errors in the specification.

The development of a layered model can be carried out with software and formal models can be created using well-known mathematical tools. For example, the Petri nets theory, the automata theory, queuing systems, etc. can be used. Programming languages based on formal methods could be used, for example, SDL, GPSS, etc. Setting up such a model consists of setting protocol parameters, and other properties that should be described in the specification.

For modeling a system of devices, programming languages are chosen that can build large systems with the use of multiple modules, ensuring the parallel operation of these modules. One of the most efficient languages for these purposes is SystemC. It covers a wide range of protocol development areas and can be used for architecture design, hardware and software simulation, system behavior description, and functional verification. Finally, SystemC supports hardware modeling and design. Using the SystemC network model, you can set various configuration parameters, such as the data rate, number of nodes and switches, time delays, routing tables, number of ports in switches, etc. Also, on network models, it is possible to automate the process of designing a network topology, the tracking of routes, and setting various network properties.

During the tuning of the model, some errors in the model itself can be detected and, as a result, a request is sent to correct the model. During the model development phase and the modeling phase, errors in the designed specification can be found. In this case, a detailed description of the error is generated. The team developing another model in a parallel methodology branch is notified that the modeling process should be temporarily suspended. Next, an expert evaluates what changes are needed in the specification, and then corrects it. When changes are made to the models, the specification is resubmitted for modeling.

Once the simulation process is completed successfully and no more specification errors are found, the simulation results are compared to the original specifications. In case of discrepancy, the specification is sent for correction. Otherwise, the project team creates the final version of the specification and submits it for implementation [13].

Since modeling is such an important part of the communication protocol development process, it is necessary to have an automated system that allows one to set up a software model and run it for execution. Such CAD systems are becoming an integral part of the technology, since they can significantly speed up the development and reduce the cost of the process of creating new technological solutions. Let us consider what the process of creating such a CAD system for DOS RV networks could look like.

Modern onboard networks consist of a large number of elements with different functionality. All these elements are interconnected through the infrastructure of the onboard network. Such networks are easy to operate as long as they are small enough, and as long as they have predictable behavior and are used for a simple set of information flows without strict requirements and restrictions. However, as the network grows larger, information flows become denser, requirements and constraints become more stringent, and design becomes a challenge. In such cases, there is a need to create a software packages for computer-aided design and network modeling.

For DOS RV networks, there is a need to create an automated design and simulation system that will cover the full cycle of development and simulation of the onboard network, starting with the design of the network topology and ending with obtaining network simulation results and statistical data (see Figure 3).

**Figure 3.** Architecture of CAD for DOS RV.

The designed CAD DOS RV will use a graphical interface that allows for an optical onboard network to be assembled from a variety of network nodes and a spectral multiplexer (in the future, several multiplexers) corresponding to the physical implementations that are available for building networks. A configuration interface will be provided for setting parameters, configuring protocols, and test scenarios.

Software modules (SM) will perform functions corresponding to the various stages of the onboard network design. SM-1 is responsible for designing the network topology and the evaluation of the physical characteristics, such as the mass of cables and devices, power consumption, and the evaluation of the required number of data exchange channels in the network. SM-2 is responsible for complete network configuration, including functions for configuring administration protocols. SM-3 is used to adjust the "Scheduling" quality of service implemented in the DOS RV protocols. At SM-3, the design work is completed, and at SM-4, the entire network is launched for simulation. The simulation proceeds in accordance with the specified work scenarios, which imply turning off devices, introducing errors into the network, changing settings, etc. Upon completion of the simulation, the user has a complete set of diagnostic information on the operation of the DOS RV and graphical information on statistics.

To implement the simulation functions, it is necessary to create an effective simulator of the DOS RV operation. This model can be used to solve the problem of modeling protocol stack specifications, testing mechanisms, and testing prototypes of future real networks on the model. The developed model allows one to work with the simulation environment in a graphical user interface, perform simulations of various scenarios, and generate detailed diagnostic information. As the first step in the implementation of the DOS RV protocols, a point-to-point network model was created. It consists of two nodes and a data transmission channel. Using the developed model, a detailed study of the mechanisms of the transport layer, the link layer, and the administration layer was carried out and the efficiency of the developed mechanisms was shown. To do this, we tested the transmission of various packets with different qualities of service, interrupts, time-codes, commands for working with distributed memory, and requests for the administration level [14].

This model will form the basis of the extended version, which will be able to set flexible work scenarios and use the full DOS RV network infrastructure. The model is implemented in the SystemC language, which is used to model parallel systems. In the future, we plan to use this model as part of an expert system for preparing and making decisions in the event of an exchange failure in DOS RV.

#### **4. The Concept of Applying the DON Model to Form the Knowledge Base of the Expert System for Preparation and Decision-Making**

Currently, an urgent problem is that of designing real-time onboard intelligent systems. Such systems are based on the integration of models capable of adapting, modifying, learning, representing, and operating knowledge, focused on the specifics of the problem area and the corresponding type of uncertainty, which reflects their ability to develop and change their state.

When implementing a real-time decision support and decision-making (RTDSDM), it is necessary to take into account the specifics of such systems. The system should obtain a solution under the time constraints determined by the real controlled process. It should take into account the time factor when describing a problem situation and in the process of finding a solution. Also, it is impossible to obtain all the objective information necessary for the decision, and, in connection with this, the use of subjective, expert information. There is a need to have a multivariate search, and the need to apply methods of plausible inference and active participation in the process of the search and intellectual inference. Case-based decision search methods can be used in many RTDSDM blocks (analyzer, decision search blocks, explanations, modeling, and forecasting) and can improve the efficiency of decision-making in various problem situations.

Precedent-based intelligent inference is an approach that allows a new problem to be solved by using or adapting a solution to an already known problem, i.e., using the already accumulated experience in solving similar problems.

The disadvantages of case-based reasoning include the following:

The main purpose of using the apparatus of precedents within the framework of RTDSDM is to issue a ready-made solution to the operator for the current situation based on precedents that have already taken place in the past when managing a given or similar object (system).

It is obvious that in order to structure information and quickly access it from the RTDSDM core, it is necessary to form a specialized knowledge base (KB) containing a set of precedents, each of which describes an emergency situation and certain methods for solving it. One such solution, which allows for parrying the failures that have arisen, is the dynamic reconfiguration of the DOS RV. In addition, information about an emergency situation can be taken both from the real experience of a qualified pilot, and formed using simulation modeling implemented in CAD based on the DOS RV model. The DOS RV model can also be used in RTDSDM to analyze the current state of the network and predict failure situations. This function is implemented by the method of the continuous comparison of the functioning of the network and the DOS RV model implemented in the core of the RTDSDM.

The intelligent inference function is produced by a specialized expert system (SES), which is part of the RTDSDM. To increase flexibility and expand the ability to solve a wider range of problems, SES should rely on both classical search algorithms using standard calculators and specialized parallel algorithms implemented on the basis of coprocessors. One of these coprocessors is a neural network computer or a neurocontroller (NC). On the basis of an NC, it is possible to deploy artificial neural networks with different architectures and different specifics of functioning, which will allow hidden connections and nonlinear patterns to be identified in the data stored in the knowledge base, which, in turn, will significantly increase the flexibility and depth of intellectual inference carried out in the SES. The generalized structure of the RTDSDM is shown in Figure 4a.

**Figure 4.** (**a**) The structure of the RTDSDM based on the DOS RV model; (**b**) NC architecture.

As can be seen from Figure 4a, the RTDSDM includes a tera-network calculator or neurocontroller. This element can be realized using a microprocessor, on the basis of FPGA, and in the form of a special module representing a system-on-chip (SoC). Depending on the variant of realization, the pro-cems in the NC can be sequential (in the case of a microprocessor), parallel (in the case of an FPGA), and combined (in the case of a SoC). The NC architecture is shown in Figure 4b.

As can be seen from Figure 4b, a NC consists of neurons *Nj*, whose inputs are multiplied by weight coefficients *ai j*; each neuron transforms the weighted sum of inputs using the activation function *Fj* and the output is formed *dj*, where *i* = *j* = 1, *n*. Taking into account the peculiarity of neuron architecture, namely, the presence of many inputs and only one output, the NC can be described using the neuron interaction matrix *Mnxn* (NIM), whose elements outside the diagonal are the weights of the inputs of the neuron with the number corresponding to the row number, and the diagonal element is the output of this neuron.

In addition to the NIM, it is also necessary to describe the set of activation functions used in the NC with reference to each neuron. For this purpose, we introduce the vector of activation functions *F<sup>n</sup>* (VAF), whose elements *fi* indicate the number of the activation function implemented in the neuron *Ni*. Odds *aij* in the NIM affect the slope of the activation function of each neuron; however, to increase the flexibility of the NC, it is also necessary to introduce bias coefficients *bi*, which allow one to move the activation function of each neuron to the left or right. To this end, we introduce the vector of bias coefficients *Bn*, containing *bi*. Thus, the NC is mathematically uniquely described by the set

$$\{M^{\text{nxn}}, B^n, F^n\},\tag{1}$$

where *n* is the number of neurons in the NC. The NC dynamics can be described by the following transformation:

$$d\_l = (M \times d\_{t-1} + b) \tag{2}$$

Due to the fact that the NIM is used as part of the SES, it is possible to significantly increase the flexibility of decision-making and increase the speed of some (e.g., nonformalizable) computations. This is possible due to the fact that on the NIM, it is possible to implement a large number of different classes of neural networks (multilayer perceptrons, Hopfield network, Boltzmann machine, etc.), both direct propagation and recurrent, without changing the NC architecture. It should also be noted that, in the case of using a parallel implementation of NIM (on FPGA), the response time of the NC is determined by the FPGA production technology and currently reaches units of nanoseconds.

#### **5. Conclusions**

The use of a commutation-free optical environment in onboard systems and the construction of a distributed system of support and decision-making on its basis allows one to significantly increase the flexibility and scalability of the onboard equipment complex, simplify and accelerate the process of its reconfiguration (for example, in the case of dynamic reconfiguration), and to achieve high determinism of states of all onboard processes and systems. It should also be noted that the use of the information network model, which is formed and used in the CAD environment during the development of DOS RV, allows one to obtain a dual-purpose model (both at the design stage and at the stage of intelligent control of the information network environment), to create a single, continuous process of development, and the application of DOS RV. In general, the efficiency of work is significantly increased and financial costs are optimized. The considered approach is methodically applicable to the development of other components of real-time technological systems.

**Author Contributions:** Funding acquisition, V.K.; conceptualization, N.S.; writing—review & editing, E.Z.; writing—original draft preparation, V.N.; software, V.O.; methodology, A.S.; validation, M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was financially supported by the Ministry of Science and Higher Education of the Russian Federation under agreement No. 075-15-2022-1024.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.


# **New Advances in Oil, Gas and Geothermal Reservoirs**

Edited by Daoyi Zhu Printed Edition of the Special Issue Published in *Energies*

www.mdpi.com/journal/energies

## **New Advances in Oil, Gas and Geothermal Reservoirs**


**Conflict of interest**

The authors declare no conflict of interest.

*The Influence of Microbial Metabolites in the Gastrointestinal Microenvironment on Anticancer… DOI: http://dx.doi.org/10.5772/intechopen.88137*


**The Role of the Statistical Method of Motor Unit Number Estimation (MUNE) to Assess the Potential Therapeutic Benefits of Riluzole on Patients with Pre-symptomatic Familial Amyotrophic Lateral Sclerosis**

Arun Aggarwal

[91] Kim, D.S., et al., Highly Pure and Expandable PSA-NCAM-Positive Neural Precur‐ sors from Human ESC and iPSC-Derived Neural Rosettes. PloS one, 2012. 7(7): p.

[92] Qiang, L., et al., Directed conversion of Alzheimer's disease patient skin fibroblasts

[93] Israel, M.A., et al., Probing sporadic and familial Alzheimer's disease using induced

[94] Mou, X., et al., Generation of disease-specific induced pluripotent stem cells from pa‐ tients with different karyotypes of Down syndrome. Stem cell research & therapy,

[95] Marchetto, M.C., et al., A model for neural development and treatment of Rett syn‐ drome using human induced pluripotent stem cells. Cell, 2010. 143(4): p. 527-39.

into functional neurons. Cell, 2011. 146(3): p. 359-71.

pluripotent stem cells. Nature, 2012. 482(7384): p. 216-20.

e39715.

182 Current Advances in Amyotrophic Lateral Sclerosis

2012. 3(2): p. 14.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56559


*<IP_ADDRESS>. Crayfish production*

*<IP_ADDRESS>. Shrimp/prawn production*

20 Aquaculture - Plants and Invertebrates

*<IP_ADDRESS>. Crab production*

Shrimps are grown in brackish water, whereas prawns grow in freshwater. The shrimp production is done with the fields of holding salty water located in west coast and deltaic areas of eastern India where the salt-resistant rice varieties are grown. Due to limited availability of salt water, only one crop (integrated rice-shrimp farming) is grown mostly in the months of July to September every year. The channels are arranged to control the water supply into the field or pond during harvesting of rice, and the shrimp is protected in the ponds/trenches. At this stage, rice field consists of substantial number of shrimp larvae having less water. The shrimp harvesting in this case is usually done after 3 months of rice harvest. To clearly say the method followed to culture rice and shrimp is through alternative system (semi-intensive monoculture) with few modifications and balanced diet supplied during the culture. The average production rate of shrimp per hectare in this method is 1440 kg. *Penaeus monodon* is the most common species grown in this type of culturing. *Penaeus merguiensis, Penaeus indicus* and

The most popular species of freshwater prawn grown in the integrated rice-prawn culture is *Macrobrachium rosenbergii* (*M*. *rosenbergii*). The monoculture of *M. rosenbergii* in the rice fields is in practice in North Kuttanad, where almost 248 hectares are cultivated integrated riceprawn culture in a year [34]. Like rice-shrimp farming, the alternative system of culturing is adapted for rice-prawn culture (semi-intensive monoculture). The productivity of prawn monoculture with rice is around 700 kg/hectare. On the other hand, the polyculture of prawn (*M. rosenbergii*) along with fish (*Catla catla*, *Labeo rohita* and *Ctenopharyngodon idella*) in the rice fields is also in practice, where the productivity of prawn is 285 kg/hectare, which has almost two times less production of monoculture of prawn in the integrated system. The yield of

*Penaeus monodon* with mixed cultures in the rice fields is about 2135 kg/hectare [35].

More than two decades ago, the role of indigenous technology in the production of rice-crab culture is well described [36]. The crabs *Oziotelphusa senex senex* or *Parathelphusa hydrodromus* are commonly called as 'freshwater rice field crabs' since their occurrence is naturally in the rice fields. Besides this *Eriocheir sinensis* (Chinese mitten crab) and *Scylla serrata* (mud crab) are the major crab species used for rice-crab integrated farming. Mitten crab is a famous cultural species of China, whereas the mud crab is the most economic crab identified and grows throughout the world. Both these species grow in saline water and are cultured along with salt-resistant rice species in the case of integrated culture. *Scylla serrata* is popular in Asian continent. This crab culture is picking up in the Indian states such as Kerala, Tamil Nadu, Karnataka, Andhra Pradesh, West Bengal, Maharashtra, Odisha, Gujarat and Union territories. However, the crab culture is influenced by many factors, including environmental,

Crabs depend on naturally available food in the rice fields. Anyhow, supplementation of external feed is needed during fast-growing periods. The management of rice-crab culture is much easier than other RAFs. But the only management aspect that needs to be addressed is regulation of pesticide usage. Since the usage of pesticides is unavoidable for rice yield, it can be managed by selecting less toxic pesticides with a method of spray on rice stems and

*Metapenaeus ensis* are alternative species to grow in this type of system.

biological, physical as well as socio-economic factors [37, 38].

Similar to crabs, crayfishes are the strongest crustaceans that tolerate unfavorable conditions than other species. The most common species used in rice-crayfish integrated system are *Procambarus clarkii* and *Cherax quadricarinatus*. The rice-crayfish culture is common in Southern United States (*Procambarus clarkii*) and in Australia (*Cherax quadricarinatus*). The adult crayfish is used as broodstock to get seed in the ponds. The broodstock is released into field after 1 week of rice seedling implantation. On the other hand, the crayfish seed is also obtained from hatcheries. Besides this the seed also obtained from the rice-crayfish integrated system after harvesting, where the adult crayfish produce juveniles which are used as seed for immediate culture. Unlike other crustaceans rice-crayfish culture is also done with sequential farming. In this system before 2 weeks of harvesting the rice, the water is drained to facilitate crayfish to make burrows [44]. Watering the field after harvesting leads to growth of ratoon crop (occurrence of regrowth from rice stubbles) and facilitates the growth of insects, zooplankton, worms and molluscs, which promotes the direct growth of flushed out crayfish from the burrows [45]. The optimum weight for harvesting crayfish is about 15–60 g. The average yield of crayfish in this system is about 1960 kg/hectare [46].

Many other aquatic species can be integrated with rice culture. The typical integration of aquaculture with rice system followed in Asian countries is presented in **Figure 6**.


**1. Introduction**

Metals present numerous benefits for everyday life. They have contributed to the development of civilization, to the modernization and development of industries. In some cases, they are essential for maintaining the metabolism of the human or animal body, or they are indispensable in the growth of plants as microelements [1, 2]. However, if metals exceed certain concentration levels (even trace amounts), they can contribute to environmental pollution and can lead to devastating effects on living organisms [3, 4]. **Figure 1** shows the main sources of metal pollution and their transport through an environment. Water is the most responsible environmental factor, with the help of which the heavy metals present at improper concentrations can reach living organisms. Furthermore, heavy metals have the property of bioaccumulation since they could not be biodegradable, leading to critical health issues [5–7]. Therefore, water pollution with heavy metals represents a global concern and the World Health Organization (WHO) established the maximum admitted level of heavy metal concentrations in drinking water [8].

**Figure 1.** *Sources and transport of heavy metals through an environment.*

The removal of heavy metals from water sources or wastewater before discharge is an important problem that must be carried out to minimize pollution, reduce their interference with beneficial uses, and eliminate their negative effects on the environment. In some cases, in addition to eliminating the toxic effect of the metals present in waters upon the environment, these water treatment methods also have the purpose of recovering and revaluing the metals, especially if we are talking about rare and precious metals (such as gold, silver, platinum, palladium, rhodium, ruthenium, iridium, and osmium). The present chapter summarizes the treatment methods employed for heavy metals removal, describing new advanced developments, and highlighting the advantages and disadvantages of each in terms of efficiency, accuracy, feasibility, and kinetic.

### **2. Chemical precipitation**

Chemical precipitation (**Figure 2**) is the process of transforming soluble metal ions into insoluble metal compounds using various precipitating agents, such as hydroxides, carbonates, sulfides, sulfates, phosphates, chlorides, and sodium borohydride, usually followed by a separation step (sedimentation, filtration, settling, and centrifugation) [9–11].

The most frequently used method in industry is precipitation under hydroxide form. The method is based on the low solubility of metal hydroxides (reaction 1) at alkaline values of the mass reaction pH [1, 3].

$$\rm{M^{n+}\\_aq} + n(\rm{OH})^{-}\,\_{\rm{aq}} = \rm{M(OH)}\_{\rm{n\,\,pp}} \tag{1}$$

The solubility of hydroxides is dependent on pH. For each metal, there are solubility diagrams that represent the graphical representation of the solubility function of *Heavy Metals Removal from Water and Wastewater DOI: http://dx.doi.org/10.5772/intechopen.110228*

**Figure 2.** *Scheme of the precipitation system.*

pH, which present importance in the establishment of the pH value at which the desired metal ions present the lowest solubility [12]. Different metals have different values of the optimal precipitation pH, so maximizing the removal efficiency of a certain metal can lead to a significant decrease in the removal degree of another metal.

Sometimes for this reason it is necessary to use two stages of treatment to obtain a high removal degree of all the metals present in the wastewater. On the other hand, the fact that metals present in water precipitate at different pH values can lead to a selective separation of them. The main advantages of the method are: (1) easy of operation, could be applied without requiring a pretreatment; (2) could remove several parameters at once; (3) could be performed at ambient temperature; (4) could be automated; and (5) presents low costs if lime is used as a precipitating agent. At the same time, the method presents also some disadvantages, such as requires a high amount of precipitating agent, consequently generating a large amount of sludge (especially if lime is used as precipitating agent); the pH must be strictly controlled; does not remove complex metals; the efficiency of the separation step influence the treatment performance [13]. Among the metals that can be removed using this method, could be mentioned: iron, copper, zinc, cadmium, beryllium, cobalt, mercury, manganese, and aluminum. Chromium can only be removed if it is found in the trivalent form.

Besides pH, other parameters and factors must be considered (such as temperature, ionic strength, formation of complexes, formation of other solid phases, alkalinity, and formation of buffer solutions) because they affect the solubility of metal hydroxides and consequently the efficiency of metal ions removal through precipitation. The metal ions'solubility increases with the temperature increasing, with ionic strength increasing, or with the formation of complexes. Around the optimum pH value of metal ions precipitation under hydroxide form takes place in the formation of another solid phase, such as carbonates form. Certain anions present in wastewater from metal processing (chlorines, sulfates) can also precipitate the metals present. The carbonates and sulfates present in the water, as well as some organic anions can precipitate with the calcium ions added in the form of lime to adjust the pH. The metal ions'solubility decreases as the formatted precipitate is subject to aging and is transformed into ideal crystals [12].

Instead of hydroxide precipitation or together with it is used precipitation under carbonates forms (reaction 2).

$$\rm{M^{n+}\,\_{aq}} + n(CO\_3)^{2-}\,\_{aq} = M\_2(CO\_3)\_{n\text{ pp}}\tag{2}$$

In contrast to metal hydroxides, metal carbonates are formed at lower values of the reaction mass pH, and present higher density, which leads to the improvement of the removal process performance. Lower metal solubilities can be obtained by increasing the carbonate dose at pH values lower than the optimal pH for the precipitation of hydroxides. The main disadvantages of this method are represented by its reduced kinetic and by the possible foaming of the reaction mass due to CO2 release [14]. This method is frequently used for lead removal due to the high solubility of lead hydroxide [15, 16].

Metal sulfides are compounds that also present a much lower solubility than metal hydroxides. Thus, metal ions can be removed from aqueous solutions by precipitating them in the form of sulfides according to reactions 3–6 [1, 11, 17, 18].

$$\text{H}\_2\text{S}=\text{HS}^- + \text{H}^+\tag{3}$$

$$\mathbf{H}\mathbf{S}^- = \mathbf{S}^{2-} + \mathbf{H}^+ \tag{4}$$

$$\mathbf{M}^{2+} + \mathbf{S}^{2-} = \mathbf{M} \mathbf{S}\_{\text{PP}} \tag{5}$$

$$\text{M}^{2+} + \text{HS}^{-} = \text{MS}\_{\text{pp}} + \text{H}^{+} \tag{6}$$

For the precipitation of metals in the form of sulfides, soluble sulfides can be used, for example, sodium sulfide (Na2S), calcium polysulfide (CaS), or sodium hydrosulfide (NaHS), or insoluble sulfides, such as ferrous sulfide (FeS). The partially soluble ferrous sulfide is added as a suspension. Most metals have lower solubility than FeS, so heavy metals precipitate as sulfides, while FeS is solubilized (reaction 7). The reaction takes place around pH = 8, and Fe precipitates in the form of hydroxides [1, 11, 17].

$$\mathbf{M}^{2+} + \mathbf{FeS} = \mathbf{MS} + \mathbf{Fe}^{2+} \tag{7}$$

Due to its reducing character, an important advantage of this method is the fact that FeS can be used directly in the removal process of Cr(VI) from water (reaction 8). The sulfur is oxidized to its basic state, and chromium precipitates in the form of hydroxide [19–21].

$$\text{H}\_2\text{CrO}\_4 + \text{FeS} + 2\text{H}\_2\text{O} = \text{Cr(OH)}\_3 + \text{Fe(OH)}\_3 + \text{S} \tag{8}$$

Another advantage of the precipitation under sulfide ions is the ability to direct the precipitation of complex metals. In this case, it does not require any pretreatment steps or multiple stages to remove different metals. Unfortunately, the sulfide precipitates are in the form of small particles that present poor sedimentation properties, sometimes requiring the use of coagulation agents. The main disadvantage of this method consists of the strict conditions for operation and handling of the resulting sludge due to the possibility of toxic hydrogen sulfide formation. The advantages and disadvantages of the main precipitation agents used for the removal of metal ions from water and wastewater are summarized in **Table 1**.

*Heavy Metals Removal from Water and Wastewater DOI: http://dx.doi.org/10.5772/intechopen.110228*

**Table 1.**

*The advantages and disadvantages of the main precipitation agents used for the removal of metal ions from water and wastewater.*

Precipitation in the form of sulfates is generally applied to remove barium from aqueous solutions [22–24]. Precipitation in the form of phosphates is generally applied for the elimination of trivalent metals (iron, aluminum, and chromium). The precipitation in the form of chlorides is particularly applied in combination with the oxidation of cyanides for the removal and recovery of silver [25]. Sodium borohydride is an effective reducing agent and is used to remove Pb, Hg, Ni, Cd, Co, Cu, and some precious metals [26, 27].

All these mentioned factors and variables that influence the precipitation raise difficulties in the evaluation of the treatment process. So that predicting the efficiency of the process requires the evaluation of a theoretical and experimental study for each individual case. For example, Serrano and his coworkers studied the removal of Fe (III), Cd(II), and Zn(II) using hydroxides precipitation combined with flotation. The precipitation efficiency was determined function of pH, metal ions initial concentration, treatment time, and dosage of the precipitating agent. The simultaneous removal of 99% for all three studied metal ions was obtained using a pH value of 10.3, an initial concentration of metal ions between 1 mol/L to 15 mol/L, and a treatment time of 15 minutes [9]. Zhang and Duan studied the removal of heavy metals by precipitation using magnesium hydroxy carbonate as a precipitating agent. They obtained a residual concentration under the maximum admitted value using a dosage of the precipitation agent of 0.3 g/50 mL of residual solution at a pH value of 7.1 [12]. Sadeghi et al. studied the removal of lead through precipitation using sodium sulfide and sodium carbonate as precipitation agents. In each case was obtained a removal efficiency

>95% at pH = 11 [15]. The lead ions removal was also studied by precipitation with sodium carbonate by Hu et al. In this case, the researchers used a ball milling process to increase the reaction between lead salts and the precipitation agent, achieving a removal degree of 99% [16]. Regarding precipitation in the form of sulfides, studies have been focused on minimizing the production of toxic hydrogen sulfide. In this regard, Phol studied the metal ions precipitation using other sulfur-containing precipitation agents, such as potassium/sodium thiocarbonate (STC), 2,4,6 trimercaptotiazine (TMT), sodium dimethyldithiocarbamate (SDTC), 1,3 benzenediamidoethanethiol (BDETH 2), 2,6-pyridinediamidoethanethiol (PyDET), or pyridine-based thiol ligand (DTPY) [17]. In this way, the metal ions are binding by the precipitation agent and form metal complexes and are avoided the H2S formation but to obtain an efficient removal of metal ions is necessary to use a higher dose of precipitation agents. Prokkola et al. studied the metal ions precipitation from acidic mine drainage (AMD) by using HS, resulting from sulfate reduction reaction. The resulted H2S gas and ionic HS� during anaerobic treatment were recycled in the precipitation process. The optimum pH value of the precipitation process was 5.5, when is achieved a residual concentration of metal ions <30 μg/L [18]. It is observed that regardless of the precipitation agent used, the degree of metal ions removal from aqueous solutions is influenced by the pH value, treatment time, precipitation agent dose, and initial concentration of metal ions in the solution. By adjusting these influencing factors, the removal capacity of metal ions from wastewater can be significantly improved, which provides the theoretical basis for the practical application of these technologies from the laboratory scale, updated to pilot scale, and further to the industrial level.


**6.3 PAPP-A**

*Prediction of Maternal and Fetal Syndrome of Preeclampsia*

**26**

**6.2 sFlt-1/PlGF ratio**

*endothelial dysfunction.*

**Figure 2.**

of preeclampsia [55].

ing and monitoring PE [56].

Researches show that sFlt-1 is an antiangiogenic molecule and therefore seems to be involved importantly in the pathogenesis of preeclampsia. High levels of circulatings sFlt-1 in early pregnancy are associated with the later commencement of preeclampsia. An in vitro research shows that sFlt-1 inhibits tube formation of endothelial cells from human umbilical vein. In essential cytotrophoblast cell culture, sFlt-1 production and mRNA expression are related inversely to oxygen saturation. A twofold elevation in the level of sFlt-1 was also observed when normal villous explants were exposed to a hypoxic state (1% oxygen), compared with physiologic exposure to 5% oxygen. Therefore, it is reasonable that the hypoxic placenta releases an excess of sFlt-1 into the maternal circulation, which induces maternal endothelial dysfunction and clinical symptoms of preeclampsia. There is also a tendency that an excess of sFlt-1 production can trigger events in the pathogenesis

*An excessive production of sFlt-1 is known in patients with preeclampsia. As an antagonist, it binds with high affinity to free PIGF in maternal serum. Thereby, proangiogenic effects by binding of PIGF to membranous sFlt-1 receptors (a vascular endothelial growth factor) are inhibited and are thought to be responsible for*

Especially, the sFlt-1/PlGF ratio connects to the clinical condition of the disease,

differentiates between healthy and preeclamptic pregnancies, and gives a shortterm prediction of disease development. Consequently, the estimation of sFlt-1 and PlGF was measured in clinical routine as a reliable and meaningful tool in examin-

Research on antiangiogenesis factors such as sFlt-1 failed to convince as the exclusive marker for the prediction of preeclampsia in the first trimester [51]. Verlohren et al. showed that the combination of angiogenesis and antiangiogenesis factors, at least in the second and third trimesters, may offer the possibility of a risk classification by an sFlt-1/PlGF ratio. It was found that patients with preeclampsia

Pregnancy-associated plasma protein A (PAPP-A), an insulin-like growth factorbinding protein protease, is secreted by the syncytiotrophoblast. As part of the first-trimester screening, it has long been used in risk calculation for chromosomal abnormalities. We could show that patients with decreased levels of PAPP-A in maternal blood during the first trimester develop preeclampsia [54], especially an early-onset preeclampsia as revealed also by others [34, 59, 60, 74].


*2.2. Experimental Design*

In order to assess the ultrastructural abnormalities induced by the Phorate and Dimethoate in the fish renal cells, the fish for each insecticide were placed in four experimental conditions: a control and three different exposure concentrations. Based on the selected doses of each insecticide and the time of exposure. All groups had an equivalent no. of fishes, i.e., 5 fish per group in a glass aquarium. After completion of the exposure period of 72 h, the fish were euthanized, and the kidneys were removed to assess ultrastructural abnormalities using transmission electron microscopy (TEM). It was briefly washed in 0.09% normal saline soon after biopsy of the animal in order to remove the associated mucus or blood from it. The kidney was cut into small pieces of 1 × 1 mm. Then, these tissues were fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer for 8–12 h at 4 ◦C. Then, the tissues were washed in buffer three times, each for one hour duration at 4 ◦C. The tissues were post fixed in 1% osmium tetroxide for 1 h at 4 ◦C. The tissues were then dehydrated through an acetone series (30%, 50%, 70%, 90% and 100%) for 15 min each. The dehydrated tissues were then cleared off the acetone by propylene oxide for 30 min and then infiltrated and embedded in a liquid resin epoxy. After embedding, the resin blocks were then thin-sectioned and these ultrathin sections (70 nm) were placed on metal grids and stained with electron dense stains, with uranyl acetate and lead citrate. These samples were then examined and photographed for ultrastructural changes by TEM (TECNAI 200 kV, FEI/Phillips, Hillsboro, OR, USA) at AIIMS, New Delhi for observations and photography.

## **3. Results**

Kidney samples from the control fish showed normal structural features. The nucleus was oval in shape with heterochromatin evenly distributed throughout the nucleoplasm and in a binucleate condition. The heterochromatin was uniformly distributed (Figure 1b). The mitochondria were tubular with distinct cristae (Figure 1a); stacks of rough endoplasmic reticulum were easily seen around the nucleus and mitochondria (Figure 1d) lysosomes were also seen. The basement membrane also had normal nuclei. Additionally, the pedicels which arise from the specialised epithelial cells (podocytes) were easily seen forming narrow infiltration slits in the glomerulus (Figure 1c).

**Figure 1.** Transmission electron micrographs of control renal cells with intact normal architecture of cellular organelles: (**a**) normal mitochondria with distinct cristae (white filled arrow) (scale 0.2 μm); (**b**) nucleus, though binucleate (white filled arrow) but with uniformly distributed chromatin (white arrow head) (scale 1 μm); (**c**) pedicel with infiltration slits (white filled arrow) (scale 0.5 μm); (**d**) stacks of endoplasmic reticulum (boxed), mitochondria (arrow) and lysosomes (arrow head) (scale 1 μm).

In both the Phorate- and Dimethoate-treated fish, the ultrastructural changes were evident (Figures 2–4). Marked changes were observed in the shape of nuclei in which the nuclear envelope showed shrinkage and, hence, an irregular outline. The mitochondria displayed degenerative changes with condensed and rounded appearance, having disorganized lamellar cristae. The endoplasmic reticulum was also disorganized and fragmented and less in number. Vacuolization was also very much evident in the cytoplasm of the treated fish.

In the glomerulus of Phorate-treated fish, most of the podocytes exhibited swollen, fused and short pedicel with a significant increase in filtration slit width. These degenerative changes became more and more evident with the increase in the concentration of the Phorate for the exposure time period of 72 h. With the increase in dose, the number of mitochondria increased with distinct degenerative changes. The degenerative changes became more pronounced with the increase in the concentration of the genotoxins, as was revealed by severe deformation and nuclear blebbing of the nucleus and other structures. This increase in the dose led to an increase in the apoptotic stimuli, which was confirmed by the apoptotic body near the nucleus (Figure 4a), and nuclear membrane shrinkage was observed in all the treated fish renal cells.

**Figure 2.**

**Figure 3.**

**Figure 4.**

## **4. Discussion**

Ultra-morphological analyses through electron microscopy are the best tool to detect various cell intoxication symptoms. Transmission electron microscopy (TEM) produces a two-dimensional image of the cell. It provides the minute details of surface alterations which cannot be observed by the simple light microscope such as an increase or decrease in the number of organelles, nuclear degeneration, loss of cytoplasmic integrity, etc. However, one of the main drawbacks of electron microscopy is that this technique cannot be used to quantify the apoptotic cells [32]. Microscopy is therefore used in the interpretation of the various parameters used to measure the extent of toxicity and contributes to the establishment of measures that aim to prevent environmental contamination, a risk not only to the ecosystem but also to human health [33]. It will support follow-up studies to further elucidate the underlying cellular mechanisms of pesticide toxicity in wildlife. It is pertinent to mention here that the number of fish used in the present study was limited, therefore more work needs to be carried out on true replicates so that there is no room of doubt for the confirmation of the results.

The results of the present study on the ultrastructural changes observed in the kidney cells of common carp, *Cyprinus carpio communis,* exposed to two organophosphate insecticides viz. Phorate and Dimethoate have been compared with the previous findings of other researchers, who observed the toxicological effects of other toxicants in different species. Nandita [34] conducted TEM studies on the kidney of *Ctenopharyngodon iddellus* exposed to an organophosphate monocrotophos (36% *w*/*w* for 15, 30 and 45 days of duration). In the treated fish, several ultrastructural abnormalities were observed in the kidney. The prominent changes included degenerative changes in the nucleus and mitochondria, and cytoplasm in the proximal convoluted tubule showed vacuolization. The number of mitochondria were found to be tremendously increased upon exposure. The degenerative changes were found to be directly linked to the dose concentration of the toxicant as the cellular structures were not clearly visible with a high dosage of the toxicant. Shrinkage of glomerulus, enlargement of Bowman's capsule, dissociation of epithelial lining and vacuolization of the cytoplasm were also observed in the kidney of fish, *Cirrhinus mrigala*, exposed to metal, lead acetate (chronic toxicity study of 30 days with sub lethal concentrations of 14.1 and 28.2 ppm) [35]. Pesticides cause physiological and biochemical changes in the fish species and greatly influence their behavioural activities. The tubular degeneration was also observed in the kidney of zebrafish, *Danio rerio,* when exposed to a combination of various pesticides (sublethal concentration of 8.4 and 4.2 μg/L of Chloropyrifos 50% + Cypermethrin 5% EC for 7, 14, 21 and 28th day) [36]. Kidney of Asian stinging catfish, *Heteropneutes. fossilis* treated with chlorpyrifos (1/50 of LC50 and 1/10 of LC50 for a duration of 30 days) also revealed tubular degeneration, vacuolization and loss of glomerular [37]. Similar effects were observed in the kidneys of the brown trout, *Salmo trutta m. fario* when exposed to cadmium and zinc poisoning (4.4 mg/kg and 11 mg/kg, respectively, for a 46-week exposure period [38]. Aluminium also showed its toxic effects on the kidney of Tilapia, *Tilapia zillii*, by causing glomerular shrinkage. This freshwater fish was exposed to three levels of aluminium (25, 50, 100 μg/L) for 96 h [39]. TEM of Almix herbicide at sublethal concentrations of 66.67 mg/L for 30 days on freshwater teleost kidney was found to induce necrosis in the nucleus, severe vacuolization, whorl pattern of ER, and also mitochondrial degeneration [40].

Glomerular podocytes, on the other hand, are highly specialized cells and their most unique features are interdigitated foot processes with filtration slits in between. They are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier [41]. Swollen and fused podocytes and increased infiltration slits observed through TEM in the glomerulus is likely to cause glomerular disease, resulting in failure of the filtration barrier [41]. The ultrastructural examination of the kidney of rainbow trout, *Oncorhynchus mykiss,* exposed to synthetic dyes revealed melanomarcophages increased in number and melanin was more abundant in the cells of treated fish [42].

The kidney thus serves as a major route of excretion of metabolites of xenobiotics [40] and receives the largest proportion of postbranchial blood. It is more likely to undergo histopathological alterations under pesticide stress [14] and, hence, deserves to be examined at cellular level, which is possible only through transmission electron microscopy. The cellular degeneration which occurred in the kidney due to the Phorate and Dimethoate toxicity might have also affected the osmoregulatory function of the fish as the kidney is not only the excretory organ, but also functions as osmoregulatory organ of the fish. The teleost kidney is one of the first organs to be affected by pollutants in water [41]. Thus, the overall ultrastructural changes observed in the kidney of common carp in the present study proved that these organophosphorus insecticides viz. Phorate and Dimethoate are potent genotoxicants which showed their deleterious effects on the osmoregulatory organ of the fish. Previous findings have also proved Phorate to be genotoxic on various other parameters, including the activities of carbohydrate metabolic enzyme, succinate dehydrogenase in common carp [14] and also altered protein content in various tissues of common carp [8]. Phorate also induced pronounced pathological toxicity in the brain of common carp, *Cyprinus carpio* and the extent of damage and degeneration were progressive with the exposure time period and the concentration of Phorate [16]. Phorate also has deleterious effects on humans as it causes cellular damage, as well as changes in DNA structure [7].

The degree of damage to the gill morphology of the Dimethoate-treated fish showed direct correlation to the exposure period, (24, 48 and 96 h when exposed to sub lethal concentration of 0.96 mg/L), while the oxygen consumption and ventilation rate showed a significant decrease during the exposure period to *Cyprinus carpio* [24]. Studies have also shown that Dimethoate can adversely affect the reproductive fitness of male common carps, even at sublethal concentration, both in short-term and long-term exposure, 96 h and 36 days, respectively, when exposed to 0.96 mg/L and 0.48 mg/L [25]. The carp fingerlings exhibit a number of abnormalities in their behaviour when exposed to Dimethoate such as gulping of surface water, uncoordinated movements, tremors and convulsions, excessive mucus secretion, and imbalanced swimming, ending in a collapse to the bottom of the aquarium with wide mouth open [10–12]. Histopathological examination of liver of carp exposed to a sublethal concentration of Dimethoate revealed disorganization of hepatocytes, rupture of blood vessels, vacuolization of cytoplasm, pyknotic nuclei and necrosis of the hepatic tissue [23]. *Oncorhynchus mykiss* fish showed remarkable behavioural abnormalities such as loss of balance, erratic swimming, and convulsion and a decrease in protein concentration with the increase in exposure to Dimethoate [19]. Shrinkage of glomerulus, dilation of lumen, vacuolization and tubular degeneration were histopathological changes observed in the kidney of common carp exposed to Dimethoate [21]. Dimethoate-exposed Labeo showed a decrease in protein content [22]. Nortan [27] studied ultrastructural effects of diethylnitrosamine (DENA) and trichloro ethylene (TCE) on the kidney of Japanese medaka, *Oryzias latipes.* Tissues of control and DENA and TCE exposed medaka for 32 weeks were observed by TEM. The kidney of the treated group showed cytotoxic effects along the length of proximal tubule, which were more pronounced in region II, distented proximal tubule, cellular necrosis, membrane fragments, pyknotic nuclei, nuclear membrane disintegration. Marlasca et al., [42] reported degranulation and dilation of endoplasmic reticulum of hepatocytes and renal cells with packed melanosomes in all the developmental stages of rainbow trout exposed to two synthetic dyes (for 30 days) by TEM. Spazier et al., [43] reported severe cellular disintegration of spleen of Anguilla fish exposed to contaminated waters. The cells revealed necrotic changes marked by swelling of the mitochondria. Godar et al., [44] observed apoptosis by dissociation of ribosomes, ER dilation, degeneration of nucleus and chromatin condensation through TEM on inducing the photo toxicity of ultraviolet radiations (UV) of different wave band regions on L5178Y-R murine lymphoma (LYR) cells. Anglade et al., [45] observed apoptotic changes such as loss of contact with surrounding tissues, shrinkage and chromatin condensation in myelinated neurons of substantia nigra in four normal aged humans. Hagar et al., [46] reported ultrastructural anomalies in β-cells of rat pancreas on exposure to dimethoate, as there was a tremendous increase in the number of vacuoles and dilated ERs. The treated group received dimethoate orally via gavage (21 mg/kg) daily for two months. Brajuskovic et al., [47] reported ultrastructural changes such as a reduction in the size of mitochondria of B-chronic lymphocytic leukaemia (B-CLL). The proliferation of smooth

endoplasmic reticulum and the presence of several cytosolic electron dense granules were reported in the liver hepatocytes of two the fish species exposed to contaminated waters [48]. The ultrathin sections of the kidney of a malathion-exposed (dose of 27 mg/kg b.wt/day for one month) rat revealed positive signs of degeneration including narrowing of capillary space and deposition of electron dense material in the podocytes and vacuolation of podocyte cytoplasm with fusion of its foot processes [49]. In a nutshell, these examples clearly reveal that our findings are in line with previous studies that suggest that, like other toxicants, these two insecticides, i.e., Phorate and dimethoate, also induce abnormal degenerative changes in cells.


*13.2. Aggrecan Isolation Procedures*

To isolate aggrecan from cartilage for analysis, it must be dissociated from its ternary complex formation with HA and link protein. This is achieved by using chaotropic agents such as guanidinium hydrochloride (GuHCl), which disrupts the water structure of the tissue, opens up the dense collagenous structure allowing dissociation of the aggrecan–HA–link protein complexes and release of aggrecan monomer which diffuses out of the tissue and is recovered in the extraction solution [228]. Cartilage is initially diced into small pieces to reduce the diffusive pathways out of the tissue for effective extraction; broad spectrum protease inhibitors covering all four mechanistic classes of proteases are included in the extraction solution to protect the aggrecan from proteolysis. Homogenisation procedures, which are commonly used in the extraction of proteins from other soft connective tissues, cannot be used for the isolation of aggrecan in an intact form since their high shear forces fragment the aggrecan. The cartilage extract can then be subjected to anion exchange chromatography on support matrices derivatised with anionic ligands such as diethylaminoethyl (DEAE) or sulphopropyl [229], dissociative size exclusion chromatography in 4-M GuHCl containing buffers using open pore gel chromatographic media such as Sephacryl or Sepharose CL2B [230], or density gradient equilibrium isopycnic ultracentrifugation in high concentrations of CsCl [231]. This latter procedure relies on aggrecan's high buoyant density in CsCl gradients of ≥1.55 g/mL to isolate aggrecan; while extracted proteins typically have buoyant densities of 1.3–1.35 g/mL, HA has a buoyant density of 1.4–1.45 g/mL. Density gradient ultracentrifugation can be conducted in the presence of 4M GuHCl to ensure aggrecan is isolated free of other interactive components also present in the cartilage extract [231,232].


**Morphological, Chemical, and Genetic Characteristics of Korean Native Thyme Bak-Ri-Hyang (***Thymus quinquecostatus* **Celak.)**

#### **Minju Kim 1, Jun-Cheol Moon 2, Songmun Kim 1,\* and Kandhasamy Sowndhararajan 3,\***

**\*** Correspondence<EMAIL_ADDRESS>(S.K<EMAIL_ADDRESS>(K.S.); Tel.:+82-33-250-6447(S.K.);+91-422-2642095(K.S.)

Received: 27 April 2020; Accepted: 25 May 2020; Published: 28 May 2020

**Abstract:** Bak-ri-hyang (*Thymus quinquecostatus* Celak.) is an important medicinal and aromatic plant in Korea. *T. quinquecostatus* population and is always mixed with other thyme cultivars during cultivation and marketing. Hence, this study aimed to determine the genetic variability and the essential oil composition of three Korean native thyme, *T. quinquecostatus* cultivars collected from the Wolchul, Jiri, and Odae mountains, in comparison with six commercial thyme cultivars (*T. vulgaris*), to distinguish Bak-ri-hyang from other thyme cultivars. The composition of essential oils obtained from nine individuals was analyzed by gas chromatography–mass spectrometry (GC–MS). The random amplified polymorphic DNA (RAPD) analysis was accomplished using 16 di fferent primers. The GC–MS analysis revealed that Wolchul, creeping, golden, and orange cultivars belong to the geraniol chemotype. Whereas the Odae, lemon, and silver cultivars belong to the thymol chemotype. Further, linalool was the most abundant component in carpet and Jiri cultivars. The RAPD analysis demonstrated that all thyme cultivars showed characteristic RAPD patterns that allowed their identification. In total, 133 bands were obtained using 16 primers, and 124 bands were polymorphic, corresponding to 93.2% polymorphism. Cluster analysis of RAPD markers established the presence of clear separation from nine thyme cultivars. The highest dissimilarity and similarity coe fficient of the RAPD markers were 0.58 and 0.98, respectively. According to the RAPD patterns, the nine thyme cultivars could be divided into two major clusters. Among three Korean cultivars, the Wolchul and Odae cultivars were placed into the same cluster, but they did not show identical clustering with their essential oil compositions. The findings of the present study suggest that RAPD analysis can be a useful tool for marker-assisted identification of *T. quinquecostatus* from other *Thymus* species.

**Keywords:** essential oil; genetic; RAPD; thyme; *Thymus quinquecostatus*; *Thymus vulgaris*