*4.2. Age and Nationality of Women in the Food-Handling Sector*

The data analyses reveal that, in the food-handling sector, over 90% of workers are women. Most of them are Spanish and married (Table 2). The results obtained from the partial surveys do not evince significant differences among the data by area.

**Table 2.** Horticultural areas studied.

Source: Compiled by the authors.

The distribution is even across age groups, with the exception of women under 20. This is evidence that most women have been working in the sector for years.


*5.4.2 Fuel*

Neem became threatened due to its useful oils produced; it usually burnt in lamps throughout many countries. Its wood has long been in the practice of burning for food as well. Furthermore, husk produced by the seeds, mainly employed as fuel. Because of the tree's good growth and valued firewood, it has become the most vital plantation species in northern Nigeria. It is also grown for fuel around large towns. Charcoal made from this neem wood stands excellent quality, with a rich value only somewhat below that of coal from Nigeria's some of the eastern parts. Neem is very common, especially in towns and villages, in the northern regions despite been threatened [6].

## *5.4.3 Medicinal values of neem*

Although masses in India insisted on the effectiveness of neem actions in treating many ailments, the pharmacological properties have hardly been threatened to severe trials with controls. It has been proclaimed that, neem trees have been in practice in curing many diseases most especially the oil extracted from it, but many findings contradict with the claim to be wise enough in curing children's at the tender age [22]. Neem being a Meliaceae family, a famous plant with medicinal attributes since time immemorial. *Azadirachta indica* have a numerous medicinal attributes. Notably, leaf and bark were used in the treatment of gingivitis, periodontitis, sores, boils, enlarged spleen and malarial fever during childbirth, measles, smallpox, head scald, as well as cutaneous affections [23]. Moreover, seed oil from neem and essential oils of both leaves and bark possessed the potentialities of curing many ailments notably; *Mycobacterium* and *Plasmodium* [24]. Neem oil was used as contraceptive (Vaginal infections) and mosquito repellent due to the fact that, nimbin, nimbinin were found and nimbidin inclusive [23].

#### **5.5 Neem needed a tougher law of protection**

Neem deterioration is increasingly appeared in large areas of Nigerian states most especially in neighboring countries like; Niger, Cameroun, Chad and Mali as a *Noteworthy Threatened Plant Species in the Sahel Region, Nigeria DOI: http://dx.doi.org/10.5772/intechopen.93975*

result of inaction to the problem occurred in the areas by the government and nongovernmental agencies that can curve the menace. Certain measures can lead to the solution to the neem declination which will definitely enhance the socioeconomic effects of neem deteriorations [25]. Neem tree served as an insecticide by possessing some pesticidal components, it attacks so many as widely practiced in West Africa. But in some parts of the world, such as India, Far East Asia, it defoliates and also kills the tree. Reports have been documented that an oriental yellow scale damaged several neem trees in across West African countries including North eastern Nigeria and Eastern Niger, which lead to an eminence drought in the Sahel, which turned many neem trees weak and sickly [26].

## **6. Conclusion**

The biodiversity has been lost at a higher rate in most parts of Nigeria especially in Sahel region, where people solely depend on plants to carry out their life activities. Many factors have contributed a lot most importantly human related, such as: medicinal purposes, industrialization, technological advancements and settlements for urbanization. Moreover, direct causes of biodiversity loss in Nigeria made up of; poor law enforcement and weak laws, much demand from forest products, cultural practices which contributed tremendously in cutting down of vegetation and lead to loss in biodiversity as depicted, many plants would likely to be in extinction if care is not much taken. Proper awareness on the sustainable use of these mostly utilized species within the Sahel region should be forcefully and continuously communicated to the communities living of the resource so that they could one day be the custodian and guardian of their forests and natural resources. We believed that effective conservation and sustainable used of natural resources got to be community based, coupled with national and international law to safeguard their livelihood.

### **Acknowledgements**

We really appreciates the Yobe State University and Universiti Putra Malaysia for funding and facilities rendered for the preparation of this publication. We are thankful to the traditional medicine practitioners, herbs gatherers and sellers for their cooperation in sharing their knowledge of plants.

#### **Conflict of interest**

The present book chapter contribution has no conflict of interest declared.

*Endangered Plants*


**Author details**

Carol A. Cummings1 \* and Rosemary Reilly-Chammat2

1 Rhode Island College, Providence, RI, United States

2 Rhode Island Department of Elementary and Secondary Education, Providence, RI, United States

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is 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.

*Perspective Chapter: Strengthening and Empowering Professional Health Educators' Capacity… DOI: http://dx.doi.org/10.5772/intechopen.108980*


**2. Electrodialysis bipolar membrane: principles and definitions**

Industrial wastewater differs in industrial pollutant components depending on the types of industries in which they are formed. This difference plays a major role in the selection of wastewater treatment technologies.

Treatment technologies applied for wastewater recycling; Secondary Treatment (IA), Nutrient Removal, Filtration, Surface Filtration, Microfiltration (MF), Ultrafiltration (UF), Flotation, Nanofiltration (NF), Reverse Osmosis (TO), Electrodialysis (ED), Carbon Adsorption, Ion Exchange, Advanced Oxidation and Disinfection [1]. **Figure 1** shows the corresponding pore sizes of the pressureoperated membranes and their ability to hold specific wastewater components.

Membrane Processes (mainly reverse osmosis (RO) systems) and desalination plants are increasing day by day. In last two decades, over 10.000 membrane treatment plants have been established in most countries [3]. Daily treatment capacity of these plants may access 100 million m3/day in 2020.

With the introduction of low cost membrane modules in the 1960s and 1970s, membranes were widely used in industrial areas [4]. RO process (pore diameter < 0.0001 μm) can remove dissolved solids, bacteria, viruses and other microorganisms present in water [5]. By operating RO systems, which is one of the wastewater recycling processes, both high quality process water (filtrate flow) production is provided, and concentrate flow with high pollution load but low flow (silk) is formed. In RO systems with flow and concentrate modifications, approximately almost 90% filtrate and 10% concentrate can be formed from the inlet flow at high pressure [3].

The disposal of the concentrate from Membrane systems is still the main focus of most scientific research. This is an important issue for most country for the protection of water bodies and soil. As well known, discharged wastewater streams are still being used for irrigation. High salt content in concentrate streams means desertification of most valuable agricultural areas. The concentrate originating from membrane processes should be disposed or treated with feasible system.

Bipolar membranes are a type of ion-selective membranes first introduced in the 1950s (**Figure 2**). Bipolar membranes are composite membranes consisting of a Cation exchange membrane and an Anion exchange membrane [7]. Cation exchange membrane and anion exchange membranes, which are among ion exchange membranes, are heterogeneous, while bipolar membranes are homogeneous. Homogeneous bipolar membranes can be prepared from many different

**119**

lyte solution.

processes [7].

the functional groups or chemicals [7]:

*A New Approach for Membrane Process Concentrate Management: Electrodialysis Bipolar…*

materials. The properties of bipolar membranes formed from different materials are

Earlier studies on electrodialysis began before the World War II in Germany. Industrial and pilot scale applications have been developed since 1950. The first applications were about obtaining drinking water from sea water. Later, in applications in the food industry, it was tried to produce demineralized sugarcane sugar. If we consider the electrodialysis system as a black box, as a result of natural brine feeding, acid and base are formed at the output of the system, this picture is shown

Electrodialysis processes is one of the membrane process that the driving force is an electrical field. EDBM system consist of anionic, cationic and bipolar membranes [9]. EDBM systems are widely used in chemical industry, in food industry,

Using bipolar membranes together with ion exchange membranes in electrodialysis processes, high quality process water production can be possible and EDBM may become a more viable method for industrial wastewater treatment applications [10]. In the EDBM process, direct current (DC) is supplied to the electrodes to electrolyse the water molecules. Electrical potential between anode and cathode works as a driving force for the movement of electrons in electrolyte solution [8]. In EDBM process, bipolar membranes realizes the acid and base production in electro-

Organic acids such as lactic acid, ascorbic acid, salicylic acid, amino acid and inorganic acids such as hydrofluoric (HF) acid, sulfuric acid (H2SO4), hydrochloric acid (HCl) can be produced using EDBM systems. Alkali bases potassium hydroxide (KOH), Sodium Methoxide (CH3NaO) can also be produced in this systems [11].

In electrodialysis systems that separate water with bipolar membranes, an acid - base is formed from a very efficient energy-related salt concentration due to the accumulation of hundreds of cell units between 2 electrodes, such as conventional electrodialysis

Some catalytic reactions take place in electrodialysis systems using bipolar membranes. Reactions between water molecules and functional chemical groups occur as Eq. (1), (2), (3), (4) [12]. The catalytic mechanism is underlined by chemical reaction model of water dissociation, that is, the water splitting could be considered as some type of proton-transfer reaction between water molecules and

biochemistry industry and environmental protection technologies [9].

*DOI: http://dx.doi.org/10.5772/intechopen.93985*

given in the table below (**Table 1**).

*Water splitting function of a bipolar membrane [6].*

**Figure 2.**

in the picture below [8] (**Figure 3**).

#### **Figure 1.**

*Pressure operated membrane technology [2].*

*A New Approach for Membrane Process Concentrate Management: Electrodialysis Bipolar… DOI: http://dx.doi.org/10.5772/intechopen.93985*

#### **Figure 2.**

*Electrodialysis*

**2. Electrodialysis bipolar membrane: principles and definitions**

(IA), Nutrient Removal, Filtration, Surface Filtration, Microfiltration (MF), Ultrafiltration (UF), Flotation, Nanofiltration (NF), Reverse Osmosis (TO), Electrodialysis (ED), Carbon Adsorption, Ion Exchange, Advanced Oxidation and Disinfection [1]. **Figure 1** shows the corresponding pore sizes of the pressureoperated membranes and their ability to hold specific wastewater components. Membrane Processes (mainly reverse osmosis (RO) systems) and desalination plants are increasing day by day. In last two decades, over 10.000 membrane treatment plants have been established in most countries [3]. Daily treatment capacity of

With the introduction of low cost membrane modules in the 1960s and 1970s,

membranes were widely used in industrial areas [4]. RO process (pore diameter < 0.0001 μm) can remove dissolved solids, bacteria, viruses and other microorganisms present in water [5]. By operating RO systems, which is one of the wastewater recycling processes, both high quality process water (filtrate flow) production is provided, and concentrate flow with high pollution load but low flow (silk) is formed. In RO systems with flow and concentrate modifications, approximately almost 90% filtrate and 10% concentrate can be formed from the inlet flow at high pressure [3]. The disposal of the concentrate from Membrane systems is still the main focus of most scientific research. This is an important issue for most country for the protection of water bodies and soil. As well known, discharged wastewater streams are still being used for irrigation. High salt content in concentrate streams means desertification of most valuable agricultural areas. The concentrate originating from membrane processes should be disposed or treated with feasible system. Bipolar membranes are a type of ion-selective membranes first introduced in the 1950s (**Figure 2**). Bipolar membranes are composite membranes consisting of a Cation exchange membrane and an Anion exchange membrane [7]. Cation exchange membrane and anion exchange membranes, which are among ion exchange membranes, are heterogeneous, while bipolar membranes are homogeneous. Homogeneous bipolar membranes can be prepared from many different

in the selection of wastewater treatment technologies.

these plants may access 100 million m3/day in 2020.

Industrial wastewater differs in industrial pollutant components depending on the types of industries in which they are formed. This difference plays a major role

Treatment technologies applied for wastewater recycling; Secondary Treatment

**118**

**Figure 1.**

*Pressure operated membrane technology [2].*

*Water splitting function of a bipolar membrane [6].*

materials. The properties of bipolar membranes formed from different materials are given in the table below (**Table 1**).

Earlier studies on electrodialysis began before the World War II in Germany. Industrial and pilot scale applications have been developed since 1950. The first applications were about obtaining drinking water from sea water. Later, in applications in the food industry, it was tried to produce demineralized sugarcane sugar. If we consider the electrodialysis system as a black box, as a result of natural brine feeding, acid and base are formed at the output of the system, this picture is shown in the picture below [8] (**Figure 3**).

Electrodialysis processes is one of the membrane process that the driving force is an electrical field. EDBM system consist of anionic, cationic and bipolar membranes [9]. EDBM systems are widely used in chemical industry, in food industry, biochemistry industry and environmental protection technologies [9].

Using bipolar membranes together with ion exchange membranes in electrodialysis processes, high quality process water production can be possible and EDBM may become a more viable method for industrial wastewater treatment applications [10].

In the EDBM process, direct current (DC) is supplied to the electrodes to electrolyse the water molecules. Electrical potential between anode and cathode works as a driving force for the movement of electrons in electrolyte solution [8]. In EDBM process, bipolar membranes realizes the acid and base production in electrolyte solution.

Organic acids such as lactic acid, ascorbic acid, salicylic acid, amino acid and inorganic acids such as hydrofluoric (HF) acid, sulfuric acid (H2SO4), hydrochloric acid (HCl) can be produced using EDBM systems. Alkali bases potassium hydroxide (KOH), Sodium Methoxide (CH3NaO) can also be produced in this systems [11].

In electrodialysis systems that separate water with bipolar membranes, an acid - base is formed from a very efficient energy-related salt concentration due to the accumulation of hundreds of cell units between 2 electrodes, such as conventional electrodialysis processes [7].

Some catalytic reactions take place in electrodialysis systems using bipolar membranes. Reactions between water molecules and functional chemical groups occur as Eq. (1), (2), (3), (4) [12]. The catalytic mechanism is underlined by chemical reaction model of water dissociation, that is, the water splitting could be considered as some type of proton-transfer reaction between water molecules and the functional groups or chemicals [7]:

#### **Table 1.**

*Ion exchange polymers of bipolar membrane layers [8].*

$$\text{B} + \text{H}\_2\text{O} \leftrightarrow \text{BH}^\* \dots \text{OH}^- \leftrightarrow \text{BH}^\* + \text{OH}^- \tag{1}$$

$$\text{B} \text{H}^\* + \text{H}\_2\text{O} \leftrightarrow \text{B} \dots \text{H}\_3\text{O} \leftrightarrow \text{B} + \text{H}\_3\text{O} \tag{2}$$

$$\text{A}^- + \text{H}\_2\text{O} \leftrightarrow \text{AH} \dots \text{OH}^- \leftrightarrow \text{AH} + \text{OH}^- \tag{3}$$

$$\text{AH} + \text{H}\_2\text{O} \leftrightarrow \text{A}^- \dots \text{H}\_3\text{O}^\* \leftrightarrow \text{A}^- + \text{H}\_3\text{O} \tag{4}$$

**121**

**Figure 4.**

**Figure 3.**

**3. Usage areas of EDBM process**

*membrane; M +, cation; X- anion; H +, hydrogen ion; R, OH−*

The bipolar membrane electrodialysis process is used in the latest technological way as an integrated process for the supply of potable water and industrial salt water and recovery of industrial effluent. On the other hand, both in chemical processes and environmental protection processes, they have been successfully applying in recent years. Another field of use of bipolar membrane is the chemical industry, where new products such as H2SO4 and NaOH are produced from a salt such as Na2SO4. Indeed, this method has become widespread recently and is used successfully. Especially, the production of acid and base without producing

*Bipolar membrane and EDBM: BP, bipolar membrane; A, anion selective membrane; C, cation selective*

*functions; (b) acid and base production; (c) acid production; (d) base production [12].*

 *or CH3O. (a) Bipolar membrane and its*

*A New Approach for Membrane Process Concentrate Management: Electrodialysis Bipolar…*

*DOI: http://dx.doi.org/10.5772/intechopen.93985*

*Schematic illustration of EDBM process as a black-box [8].*

where BH+ and A− refer to the catalytic centers. The catalytic sites provide an alternative path with low effective activation energy for water splitting into hydrogen and hydroxyl ions [7]. EDBM configurations including acid base production schematic diagrams are given in **Figure 4** [12].

*A New Approach for Membrane Process Concentrate Management: Electrodialysis Bipolar… DOI: http://dx.doi.org/10.5772/intechopen.93985*

#### **Figure 3.**

*Electrodialysis*

*Anion exchange layer*

Poly-vinylidene fluoride blend with poly-vinyl benzyl chloride

Poly-divinylbenzene-codimethylamino

Poly-methyl methacylate-coglycidyl

Grafted perfluorinated polymer

Poly-butadiene-co-styrene Sulfonic acid

Poly-sulfone Aminated

*Ion exchange polymers of bipolar membrane layers [8].*

propyl methacrylamide

*Cation exchange layer*

methacrylate

membranes

*Separate contact layer* Poly-vinyl amine

**Table 1.**

 EDBM configurations including acid base production

+ − +− +↔…↔ + (1)

− −− + ↔… ↔ + (3)

− +− + ↔… ↔ + (4)

refer to the catalytic centers. The catalytic sites provide an

+ + ↔… ↔ + (2)

*Schematic illustration of EDBM process as a black-box [8].*

#### **Figure 4.**

*Bipolar membrane and EDBM: BP, bipolar membrane; A, anion selective membrane; C, cation selective membrane; M +, cation; X- anion; H +, hydrogen ion; R, OH− or CH3O. (a) Bipolar membrane and its functions; (b) acid and base production; (c) acid production; (d) base production [12].*

#### **3. Usage areas of EDBM process**

The bipolar membrane electrodialysis process is used in the latest technological way as an integrated process for the supply of potable water and industrial salt water and recovery of industrial effluent. On the other hand, both in chemical processes and environmental protection processes, they have been successfully applying in recent years. Another field of use of bipolar membrane is the chemical industry, where new products such as H2SO4 and NaOH are produced from a salt such as Na2SO4. Indeed, this method has become widespread recently and is used successfully. Especially, the production of acid and base without producing

waste and the production of organic - inorganic acids increased the interest in this method. Many researchers have worked on this subject. It is possible to find many studies especially on acetic acid, propionic acid, gluconic acid, citric acid and lactic acid. In fact, some model studies have started to be carried out recently. Biotechnological research is ongoing to reduce costs in the electrodialysis process in order to reduce the cost in order to ensure acid recovery.

When ED and EDBM processes are compared with other treatment methods, it is an important advantage that it provides recovery from pollutants in wastewater and salt water. Studies show that it is possible to recover pollutants from solutions with one or more contaminants. In addition to this, the process of making acid and base production possible with EDBM process takes another step forward [13].


**1. Introduction**

The interindividual variability in the metabolism of xenobiotics and drug response is extensive and many factors are involved with this variation including genetic composition, gender, age, co-administration of medication, individual physiology, pathophysiology and presence of other environmental factors (alcohol consumption, smoking, eating habits).

To produce their therapeutic effects, the drug must be present in appropriate concentrations at its site of action. Although the therapeutic concentrations are dependent on the given dose, they will also depend on the magnitude and rate of absorption, distribution, biotransforma‐ tion, and excretion. Pharmacokinetics studies the course and distribution of drug and its metabolites in different tissues, covering the mechanisms of absorption, transport, metabolism and excretion. In addition, pharmacodynamics concentrates on the biochemical and physio‐ logical effects of drugs and their mechanism of action. Proteins involved in drug effects are defined as target molecules and include not only (direct) receptors, but also proteins associated with mechanism of action such as e.g. signal transducer proteins [1].

After its administration, a drug is absorbed and then distributed throughout the body, requiring the coordinated functioning of various proteins, including metabolic enzymes, trafficking proteins, receptor proteins, and others. Medication can enter the body as ei‐ ther active drugs or as inactive prodrugs. Most drugs are metabolized in the liver to make them more soluble for subsequent elimination through the kidneys or intestines. Prodrugs require metabolic conversion, also called biotransformation, to liberate the ac‐ tive compound. Complete biotransformation of any one drug typically requires several different enzymes. [2]. Genetic variability has been described to have effect on drug ab‐ sorption and metabolism and its interactions with the receptors. This forms the basis for

slow and rapid drug absorption, poor, efficient or ultrarapid drug metabolism and poor or efficient receptor interactions [3]. The consequences of such variations can lead to ad‐ verse drug reaction and/or terapeutic failure.

INH-induced adverse reactions include fever, nausea, vomiting, hepatotoxicity, skin reactions, gastrointestinal and neurological disorders. Only in the early 1970s, the occurrence of severe liver injury as a side effect of this drug was recognized, resulting in the death of some patients [8]. Among the first-line anti-TB drugs, INH is the main associated with drug-induced hepatotocixity with a frequency ranging from 1 to 30% in different populations [9]. Other drugs causing liver injury are mainly reported in combination with INH [10, 11]. Drug-induced hepatotoxicity is defined as a serum alanine aminotransferase (ALT) level three times greater than the upper limit of normal (ULN) with clinical symptoms or five times the ULN without symptoms. In both cases treatment should be interrupted and, generally, a modified or alternative regimen is introduced [9]. Because these adverse reactions do not only affect morbidity and mortality rate but also lead to treatment interruptions, failure and relapse, adverse reactions contribute to the spread of the disease and the emergence of multidrug

Tuberculosis Pharmacogenetics: State of The Art

http://dx.doi.org/10.5772/54984

107

Adverse Drug Reactions (ADRs) are common causes of hospitalization and lead to large costs to society. There are two main financial burdens due to illnesses caused by ADRs: that of treating and that of avoiding them [12]. The occurrence of serious and fatal ADRs has been extensively studied in hospitalized patients and a meta-analysis of prospective studies in approximately forty hospitals in the United States of America (USA) suggests that 6-7% of hospitalized patients suffer from serious ADRs and 0.32% of patients develop fatal ADRs [13]. This results in approximately 100,000 deaths annually in the U.S. and an annual cost of over a hundred billion dollars to the society due to prolonged hospitalization and reduced produc‐ tivity [3, 13]. Furthermore, it has been estimated that ADRs are responsible for up to 7% of all admissions in hospitals in the United Kindown (UK) and 13% in medical clinics in Sweden [3], which shows the magnitude of this problem in the context of chemotherapy and drug development. Additionally, in France, a 10-year study in the Liver Unit of Hôpital Beaujon in Paris showed that among all patients hospitalized with acute hepatitis, 10% were due to adverse reaction to drugs and the prevalence of drug hepatotoxicity in patients older than fifty years exceeded 40%. In Japan and other Eastern countries, drugs are responsible for about

Liver injury is the most common ADR and the main complication during chemotherapy since liver is the central organ for the biotransformation and excretion of most drugs and xenobiotics [14-17]. There are basically six mechanisms involving primarily the hepatocyte injury. The reactions of mono-oxygenase cytochrome P450 (CYP450) with certain drugs generate toxic metabolites that bind to intracellular proteins, leading to calcium homeostasis pump dysfunc‐ tion with consequent disruption of actin fibers and cell lysis. Some drugs affect transport proteins in the cell membrane interrupting the flow of bile and then causing cholestasis. Several reactions involving CYP P450 can promote binding of the drug to the enzyme, with consequent exposure of this complex on the cell surface for recognition by T cells and antibody production as part of the autoimmune response. Finally, certain drugs may promote hepatic injury mediated by programmed cell death (apoptosis) or being capable of inhibiting respiration and/

resistence (MDR).

10-20% of cases of fulminant hepatitis [14].

or mitochondrial beta-oxidation [17].

In this context, pharmacogenetics is the study of genetic variations associated with individual variability in drug response, including differences in efficacy, drug-drug interactions, and the relative risk of an adverse response to drugs. It includes the study of genetic polymorphisms that could affect the expression or activity of drug transporters, drug metabolizing enzymes and drug receptors [2-4].

It's estimated that 99.9% of the human genome sequence between individuals is identical and genetic differences in polulations are called mutations if they are present in less than 1% and polymorphisms when present in at least 1% of a population. A single-nucleotide polymor‐ phism (SNP) involves a replacement of one nucleotide base with any one of the other three and occuring at approximately one out of every 1,000 bases in the human genome [5].

A mutation or polymorphism in genes that encode metabolic enzymes, carriers or receptors can affect the drug pharmacokinetics and pharmacodinamics leading to undesired therapeutic effects. The identification of these genetic markers which predicted if a person responds well or not to a specific drug could help to select the right medication in right dosage, maximizing the eficacy and preventing or reducing the adverse drug reactions.


**3. Results**

Based on the re-picking phase and distribution of available BMKG stations, we determined the location of the hypocenter using the NonLinLoc Global mode. The estimated mean error in x, y, and z directions using the covariances values are 10,198 km, 14,081 km, and 19,035 km, respectively. The distribution frequency estimated error location in the x, y, and z directions is shown in Figure S9. The distribution between the number of events and the azimuthal gap is shown in Figure S10, and the epicentral distance distribution is shown in Figure S11. We limit the value of travel time residuals in the range of −10 ≤ initial travel time residuals (itr) ≤ 10 to control the quality of the input data for the tomographic inversion process. The bad picks can be traced in unit 20 of simulps output file, as shown in Figure S12. Before the inversion, the initial travel time residual values varied in the range of −9.993 s to 9.858 s, with a mean value of −1.111 s. After inversion, the final travel time residuals varied in the range of −7.148 to 6.197 s, with a mean value of 0.156 s, forming a Gaussian distribution centered around 0 s. Tomographic inversion achieved the convergence after the 15th iteration with the weighted Root Mean Square (RMS) residual reduced by 47%, initially from 0.82312 s down to 0.43871 s (Figure S13). The RMS residual before the inversion ranged from 0.06 to 1.26 s with an average value of 0.54 s. After inversion, the range of values was down to 0.04 to 0.83 s with a mean value of 0.36 s (Figure S14) or reduced by about 33%.

The Interpretation of the tomogram becomes valid and has a high confidence level by performing a resolution test of the seismic tomography model. Therefore, we present the CRT results shown in Figure 5 for Vp and Vp/Vs of profiles A-A , B-B , C-C , and D-D and Figure S15 horizontal section for Vp and Vp/Vs with the recovered area is delineated by dashed purple lines. The CRT positive recovery for A-A is beneath the Gorontalo Basin, Sulawesi's north arm, and the Celebes Sea up to a depth of 210 km. The CRT positive recovery for the B-B profile is beneath the Halmahera Volcanic Arc up to a depth of 240 km; for profile C-C the CRT positive recovery is shown beneath the Celebes and Molucca Seas, and the Morotai Basin up to a depth of 210 km. For profile D-D , the CRT positive recovery is beneath Sulawesi's north arm, the Molucca Sea, and Halmahera Island up to a depth of 300 km. These results are consistent with the density distribution of P- and S-rays as revealed by the DWS, RHC, and DRE results (Shown in Figures S6–S8); the highest Vp and Vp/Vs values are listed in Table S1. The primary resolution test we used to interpret the tomographic results was the CRT, supported by the DWS, RHC, and DRE results. Hence, this research focused on the travel time tomography results in four areas in the subduction zone beneath Sulawesi's north arm, the subduction zone beneath the Halmahera Volcano Arc, and the double subduction zone of the Molucca Sea.

**Figure 5.** *Cont.*

**Figure 5.** The vertical cross-section of CRT profiles. Profile A-A (south–north), B-B (south–north), C-C (west–east), and D-D (west–east) are vertical cross-sections of the CRT recovery model from the inversion process. The purple dashed line is the positive CRT recovery area for tomographic interpretation. The first column is Vp perturbation, the second column is Vp/Vs perturbation, and the red triangles signify volcanoes.

Tomographic inversion results of cross-section A-A , as shown in Figure 6 as follows:

**Figure 6.** Vertical cross-section of A-A (south–north) of the Molucca Sea Plate, crossing the Minahasa Peninsula, Sulawesi's north arm, and the Celebes Sea. The red color indicates low velocity and low Vp/Vs ratio, and the blue color indicates high velocity and high Vp/Vs ratio. The A1 and A2 are areas for the interpretation of tomographic inversion results. The black dots signify earthquakes within a radius of 30 km ≤ A-A ≤ 30 km, and the yellow stars are significant earthquakes. The green line and the green three-dimensional surface plot are the slab2 models of the Molucca Sea Plate. The yellow line is the slab2 model of Sulawesi [45]. NST is the North Sulawesi Trench. The red triangles signify volcanoes. The blurred area is an area that is not well-resolved.

Tomographic inversion results of cross-section B-B', as shown in Figure 7 as follows:

**Figure 7.** Vertical cross-section of B-B' (south–north) crossing beneath the Halmahera Volcanic Arc. The red color indicates low velocity and low Vp/Vs ratio. The blue color indicates high velocity and high Vp/Vs ratio. The B1, B2, and B3 are areas for the interpretation of tomographic inversion results. The green line and the green three-dimensional surface plot are the slab2 models of the Molucca Sea Plate [45]. The black dots signify earthquakes within a radius of 30 km ≤ B-B ≤ 30 km. SeT: Seram Trench; HT: Halmahera Trench; Bi: Bibinoi Volcano; Bs: Batusibela Volcano; Am: Amasing Volcano; Br: Buku-Rica Volcano; Rr: Rogi-Rogi Volcano; Ja: Jailolo Volcano; To: Todoko-Ranu Volcano; Ga: Gamkonora Volcano; Ib: Ibu Volcano; Tb: Tobaru Volcano. The blurred area is an area that is not well-resolved.

Tomographic inversion results of cross-section C-C , as shown in Figure 8 as follows:

**Figure 8.** Vertical cross-section of C-C (west–east) crossing the Celebes Sea, Sangihe Island, Molucca Sea, and Morotai Basin. The red color indicates low velocity and low Vp/Vs ratio. The blue color indicates high velocity and high Vp/Vs ratio. The C1, C2, and C3 are areas for the interpretation of tomographic inversion results. The black dots signify earthquakes within a radius of 30 km ≤ C-C ≤ 30 km, and the yellow star is a significant earthquake. The green line and the green three-dimensional surface plot are the slab2 models of the Molucca Sea Plate, and the brown line is the slab2 model of the Philippines [45]. ST: Sangihe Trench; HT: Halmahera Trench; the blurred area is an area that is not well-resolved.

Tomographic inversion results of cross-section D-D , as shown in Figure 9 as follows:

**Figure 9.** Vertical cross-section of D-D (west–east) crossing Sulawesi's north arm, the Molucca Sea, and Halmahera Island. The red color indicates low-velocity areas and low Vp/Vs ratio, and the blue color indicates high-velocity areas and high Vp/Vs ratio. The D1, D2, D3, D4, and D5 are areas for the interpretation of tomographic inversion results. The black dots signify earthquakes within a radius of 30 km ≤ D-D ≤ 30 km, and the yellow star is the magnitude of a significant earthquake. The green line and the green three-dimensional surface plot are the slab2 models of the Molucca Sea Plate; the yellow line is the slab2 model of Sulawesi [45]. Thus, So: Soputan Volcano; ST: Sangihe Trench; the reserve arrow is Tifore island; HT: Halmahera Trench; Ja: Jailolo Volcano; To: Todoko-Ranu Volcano; Wa: Wato-Wato Volcano; the blurred area is an area that is not well-resolved.

Three-dimensional tomographic inversion results combine cross-section profile A-A and profile D-D , as shown in Figure 10 as follows:

**Figure 10.** The 3D visualization of the Molucca Sea Plate, combining vertical cross-section profile A-A (south–north) and profile D-D' (west–east) from the P-wave tomography inversion results. The earthquake events follow the high Vp anomaly pattern. The red color indicates low-velocity areas and low Vp/Vs ratio, and the blue color indicates high velocity and high Vp/Vs ratio. The white dots signify earthquakes; the yellow stars signify earthquakes with significant magnitudes. The red triangle is a volcanic arc located in Sangihe and Halmahera.


**3.3.1 Zona pellucida binding assay**

The assessment of the ability of sperm cells to bind the homologous zona pellucida (ZP) is the useful test for prediction of spermatozoal fertilizing ability (Hermansson et al., 2006). It is assumed that it is reliable test to detect sperm damage at a molecular level, which is not visible by microscopic analysis, because binding is receptor-ligand mediated reaction. The test may be done in two ways: by using intact homologous oocytes (ZP-binding assay, ZBA) and by using bisected hemizonae (hemizona binding assay, HZA) (Kawakami et al., 1998; Rijsselaere et al., 2005). In ZBA spermatozoa are coincubated with oocytes obtained from sliced ovaries. The number of spermatozoa that bound to ZP is counted with contrast-phase microscopy. The disadvantage of ZBA is the fact that the attachment of sperm cells to zona depends on the oocyte. This feature was partly overcome in HZA. Bisected by micromanipulation two parts of ZP are coincubated with spermatozoa. As a result the direct comparison of sperm cells from two origins may be done (Ivanova et al., 1999; Mayenco-Aguirre & Pérez Cortés, 1998).

A sublethal damage that occurres during cryopreservation leads to loss of sperm surface proteins, segregation of membrane proteins, inactivation of membrane-bound enzymes and decreased lateral protein diffusion within the membrane (Watson, 1995). Kadirvel et al. (2011) observed significant reduction of the zona binding ability of cryocapacitated buffalo bulls spermatozoa, and further reduction of binding ability of frozen-thawed spermatozoa, after incubation, in either capacitating, or non capacitating medium. Similar results have been obtained in bulls (Fazeli et al., 1997) and humans (Amann et al., 1999) spermatozoa, with significantly reduced binding ability to the zona pellucida after freezing and thawing.

Methods of Assessment of Cryopreserved Semen 565

enabled researchers to gain accurate information about the morpho-functional status of

This work was supported by the Polish National Science Centre, grant no N N311 530040.

Agarwal, A.; Ozturk E. & Loughlin, K.R. (1992). Comparison of semen analysis between the

Agarwal, A.; Prabakaran, S.A. & Said T.M. (2005). Prevention of oxidative stress injury to

Aitken, R.J. (1995). Free radicals, lipid peroxidation and sperm function. *Reprod Fertil Dev* 7,

Aitken, R.J.; Harikss, D. & Buckingham, DW. (1993). Analysis of lipid peroxidation

Aitken, R.J.; Wingate, J.K.; De Iuliis, G.N. & McLaughlin, E.A. (2007). Analysis of lipid

Almeida, J. & Ball, B.A. (2005). Effect of α-tocopherol and tocopherol succinate on lipid

Álvarez, M.; García-Macías, V.; Martínez-Pastor, F.; Martínez, F.; Borragán, S.; Mata, M.;

Amann, R.P. (1999). Cryopreservation of sperm. In: *Encyclopedia of Reproduction*, vol. 1.

Amann, R.P.; Shabanowitz, R.B.; Huszar, G. & Broder, S.J. (1999). In vitro sperm-binding

Arruda, R.P.; Ball, B.A.; Gravance, C.G.; Garcia, A.R. & Liu IKM. (2002). Effects of extender

Bailey, J.L. & Buhr, M.M. (1994). Cryopreservation alters the Ca2+ flux of bovine

Bailey, J.L., Bilodeau J.F. & Cormier, N. (2000). Semen cryopreservation in domestic animals:

Ballachey, B.E.; Evenson, D.P. & Saacke, R.G. (1988). The sperm chromatin structure assay.

Benoff, S. (1997). Carbohydrates and fertilization: an overview. *Mol Hum Reprod* 3, 599–637. Bilodeau, J-F., Chatterjee, S.; Sirard, M-A. & Gagnon, C. (2000). Levels of antioxidant

Blesbois, E.; Grasseau, I.; Seigneurin, F.; Mignon-Grasteau, S.; Saint Jalme, M. & Mialon-

peroxidation in human spermatozoa using BODIPY C11. *Mol Hum Reprod* 13, 203-

Garde, J.; Anel, L. & De Paz, P. (2008). Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos)

assay to distinguish differences in populations of human sperm or damage to

and cryoprotectants on stallion sperm head morphometry. *Theriogenology* 58, 253–6.

Relationship with alternate tests of semen quality and heterospermic performance

defences are decreased in bovine spermatozoa after a cycle of freezing and

Richard, M.M. (2008). Predictors of success of semen cryopreservation in chickens.

two Hamilton-Thorn semen analysers. *Andrologia* 24, 327–329.

mechanisms in human spermatozoa. *Mol Reprod Dev* 35, 302-315.

peroxidation in equine spermatozoa. *Anim Reprod Sci* 87, 321-337.

sperm resulting from cryopreservation. *J Androl* 20, 648–654.

a damaging and capacitating phenomenon. *J Androl* 21, 1-7.

spermatozoa. *Theriogenology* 70, 1498-1506.

spermatozoa. *Can J Anim Sci* 74, 45–52.

thawing. *Mol Reprod Dev* 55, 282-288.

of bulls. *J Androl* 9, 109-115.

*Theriogenology* 69, 252-261.

Academic Press, Burlington, MA, USA, 773-783.

spermatozoa and mechanisms of sperm cryoinjury.

sperm. *J Androl* 26, 654-60.

**5. Acknowledgments**

659-668.

211.

**6. References**

The reduced binding ability of the frozen-thawed spermatozoa might be due to the higher proportion of acrosome reacted and damaged spermatozoa, after cryopreservation and thawing. Moreover, impaired receptor-ligand interaction in frozen-thawed spermatozoa could be caused by cryoelution of an "essential ligand" from the sperm surface that has been described in human (Amann et al., 1999).