*Article* **Ecological Synthesis of CuO Nanoparticles Using** *Punica granatum* **L. Peel Extract for the Retention of Methyl Green**

**Mongi ben Mosbah 1,2, Abdulmohsen Khalaf Dhahi Alsukaibi 3 , Lassaad Mechi 3 , Fathi Alimi 3 and Younes Moussaoui 2,4,\***

**Abstract:** The aqueous extract from the bark of *Punica granatum* L. was invested to generate CuO nanoparticles from CuSO4 using a green, economical, ecological, and clean method. The synthesized nanoparticles were characterized and were successfully used as adsorbents for methyl green retention of an absorptive capacity amounting to 28.7 mg g−1 . Methyl green equilibrium adsorption data were correlated to the Langmuir model following the pseudo-second order kinetics model. This study clearly corroborates that copper nanoparticles exhibit a high potential for use in wastewater treatment.

**Keywords:** CuO nanoparticles; green synthesis; *Punica granatum* L.; biosorption; methyl green

**Citation:** ben Mosbah, M.; Alsukaibi, A.K.D.; Mechi, L.; Alimi, F.; Moussaoui, Y. Ecological Synthesis of CuO Nanoparticles Using *Punica granatum* L. Peel Extract for the Retention of Methyl Green. *Water* **2022**, *14*, 1509. https://doi.org/ 10.3390/w14091509

Academic Editors: Martin Wagner and Sonja Bauer

Received: 19 March 2022 Accepted: 4 May 2022 Published: 8 May 2022

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**Copyright:** © 2022 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/).

#### **1. Introduction**

Currently, nanotechnology is a very interesting field of research due to the successful application of nanomaterials in numerous areas, namely medicine, electronics, environment, biology, and optics [1–4]. Nanoparticles are known for their small size, their large area surface, and their dimensions ranging from 1 to 100 nm [5,6]. The physico-chemical parameters of these materials make them good candidates for a variety of applications such as the removal of pollutants from contaminated waters, biological activities, energy storage systems, etc., [1,7–13]. Numerous techniques have been used to synthesize nanomaterials in the form of particles, colloids, powders, rods, aggregates, tubes, wires, and thin films. These techniques are classified into physical, biological, and chemical methods. Physical methods involve evaporation and mechanical treatments [14,15], whereas chemical methods involve sonochemistry, microemulsion, and electrochemistry [4,14,16]. The latter have the merit of synthesis at a temperature below 350 ◦C [14,17]. Biological methods rely on the preparation of nanoparticles by such microorganisms as plant extracts, bacteria, yeasts, and fungi [18–20]. These methods have the benefits of easy scaling, non-toxicity, respect for the environment, and reproducibility of manufacturing [19,21]. Thus, the orientation towards natural products gained the widest attention owing to worldwide development, favoring good health, and mitigating the risk of disease. In this respect, the valorization of plant biomass is of paramount importance relating to the protection of the environment [22,23]. Renewable natural resources perform a key role in economic activities [24,25]. In this context, multiple natural resources have been investigated as sources of bioproducts. Components of biomass extracts, including phenols, flavonoids, tannins and other bioproducts, proved to be responsible for reducing metal salts to nanoparticles [13,23,26,27].

CuO nanoparticles are known as a p-type semiconductor [28,29] used as materials for Li batteries, sensors, solar energy, magnetic storage media, semiconductors, catalysis, and adsorption [29–31]. CuO nanoparticles can be synthesized by chemical reduction-based

processes including sol-gel, precipitation, bio-template, microwave heating, electrospinning, and ion exchange [28]. For example, Kumar et al. [32] synthesized CuO nanoparticles from copper nitrate through a hydrothermal reaction and used them for the adsorption of methyl orange from aqueous solution. They found that this synthetic method provided CuO nanoparticles with good adsorption performance. In another study, Darwish et al. [33] prepared CuO nanoparticles through the microwave heating method and the obtained materials were used for the retention of methyl orange. The obtained results indicate that experimental kinetics data correlated with the second-order model and the adsorption was endothermic and spontaneous and governed by a physical process [33]. Moreover, CuO nanoparticles biosynthesized using *Tamarindus indica* extracts exhibit excellent photostability and significant antibacterial activity against bacterial strains [34]. Likewise, Anand et al. [35] showed that CuO nanoparticles synthesized by the microwave combustion method can be used to cure urinary tract infection.

Within this framework, the basic objective of this work lies in using an ecological method for the elaboration of a nanomaterial for the adsorption of aqueous pollutants. In this case, it is a question of examining the possibility of using *Punica granatum* L. bark extract for the preparation of CuO nanoparticles. Indeed, Tunisia is a country with a Mediterranean climate that has several types of plants, among which *Punica granatum* is a drought tolerant species, and it benefits from great capacities to adapt to environmental conditions characterized by marked climatic aridity. The fruits of this plant are rich in secondary metabolites [36–38] which can be used as binding agents to generate nanoscale materials.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Copper sulfate (CuSO4 ≥ 99%), hydrochloric acid (HCl, 37%), sodium hydroxide (NaOH ≥ 98%), and methyl green (C23H25N3Cl2.ZnCl2) were purchased from Sigma-Aldrich (Tunis; Tunisia). All chemicals were invested as received without any additional purification.

#### *2.2. Extract Preparation*

perature of 4 °C.

of 5%.

The bark extract of *Punica granatum* L. was prepared according to the steps illustrated in Figure 1. *Water* **2022**, *14*, x FOR PEER REVIEW 3 of 17

**Figure 1.** Schematic representation of the extract preparation. **Figure 1.** Schematic representation of the extract preparation.

*Punica granatum* bark was washed and dried in the shade in a dry and aerated environment at room temperature (25 ± 2 °C). After drying, the material was cut into small pieces. To prepare the extracts, 200 g of obtained material were mixed with 800 mL of *Punica granatum* bark was washed and dried in the shade in a dry and aerated environment at room temperature (25 ± 2 ◦C). After drying, the material was cut into small pieces.To prepare the extracts, 200 g of obtained material were mixed with 800 mL of distilled

distilled water. The obtained mixture underwent mechanical stirring at a temperature between 50 and 60 °C, until we obtained a pasty solution. Subsequently, the mixture was filtered using Whatman filter paper No.01, before it was recovered and stored at the tem-

Chemical composition of *Punica granatum* bark was specified according to standardized methods. The ash content was estimated according to the T211 om-07 method. A given mass (m0) of the material was calcined in an oven at 600 °C for 6 h to provide a mass

TAPPI methods, namely, T212 om-07, T207 cm-08, and T 204-cm-07 were invested to determine the extractives in 1% NaOH, for cold and hot water, and for ethanol-toluene, respectively. Then, the α-cellulose, Klason lignin and holocellulose were quantified using T203 cm-99, T222 om-06, and Wise et al. [39] methods, respectively. All the experiments were duplicated and the difference between the values was within and experimental error

A total of 100 mL of 0.1 mol L−1 copper sulphate solution (24.96 g of CuSO4.5H2O in 100 mL distilled water) was incorporated with 100 mL of the freshly resulting extract of the *Punica granatum* bark. The solution was agitated at 70 ± 2 °C for 3 h. The solution turned to a very dark green color. Then, after filtration, the residue was recovered and

dried for 24 h at 50 °C. The resulting solid was eventually crushed (Figure 2).

m1 m0

× 100 (1)

Ash content =

*2.3. Chemical Composition of Punica granatum Bark*

(m1). The ash content is provided by Equation (1):

*2.4. CuO Nanoparticles Biosynthesis*

water. The obtained mixture underwent mechanical stirring at a temperature between 50 and 60 ◦C, until we obtained a pasty solution. Subsequently, the mixture was filtered using Whatman filter paper No.01, before it was recovered and stored at the temperature of 4 ◦C.

#### *2.3. Chemical Composition of Punica granatum Bark*

Chemical composition of *Punica granatum* bark was specified according to standardized methods. The ash content was estimated according to the T211 om-07 method. A given mass (m0) of the material was calcined in an oven at 600 ◦C for 6 h to provide a mass (m1). The ash content is provided by Equation (1):

$$\text{Ash content} = \frac{\text{m}\_1}{\text{m}\_0} \times 100\tag{1}$$

TAPPI methods, namely, T212 om-07, T207 cm-08, and T 204-cm-07 were invested to determine the extractives in 1% NaOH, for cold and hot water, and for ethanol-toluene, respectively. Then, the α-cellulose, Klason lignin and holocellulose were quantified using T203 cm-99, T222 om-06, and Wise et al. [39] methods, respectively. All the experiments were duplicated and the difference between the values was within and experimental error of 5%.

#### *2.4. CuO Nanoparticles Biosynthesis*

A total of 100 mL of 0.1 mol L−1 copper sulphate solution (24.96 g of CuSO4.5H2O in 100 mL distilled water) was incorporated with 100 mL of the freshly resulting extract of the *Punica granatum* bark. The solution was agitated at 70 ± 2 ◦C for 3 h. The solution turned to a very dark green color. Then, after filtration, the residue was recovered and dried for 24 h at 50 ◦C. The resulting solid was eventually crushed (Figure 2). *Water* **2022**, *14*, x FOR PEER REVIEW 4 of 17

**Figure 2.** Graphical representation for CuO nanoparticles formation using extract of *Punica granatum* bark. **Figure 2.** Graphical representation for CuO nanoparticles formation using extract of *Punica granatum* bark.

#### *2.5. Methyl Green Adsorption on CuO Nanoparticles 2.5. Methyl Green Adsorption on CuO Nanoparticles*

The adsorption tests were carried out with a solution of methyl green. To improve the adsorbent capacity of the prepared CuO nanoparticles, we opted for the optimization of the following conditions: pH of the solution, mass of the adsorbent, initial concentration, and contact time. The adsorption tests were carried out with a solution of methyl green. To improve the adsorbent capacity of the prepared CuO nanoparticles, we opted for the optimization of the following conditions: pH of the solution, mass of the adsorbent, initial concentration, and contact time.

using a Beckman DU 800 spectrophotometer. The pH study was indicated in the form of

The impact of the adsorbent dose was traced through varying the adsorbent mass from 2 to 500 mg in 10 mL of 50 mg L−1 methyl green solution at natural pH solution (pH = 6.5). Next, the solutions were agitated for 12 h, then centrifuged, filtered and the residual concentration was estimated. The study of the adsorbent dose corresponds to a graphical

To ensure good adsorption, we examined the impact of contact time on the adsorption of methyl green on CuO nanoparticles. Likewise, the previously reported steps were followed. Adsorbent (20 mg) was added to 10 mL of methyl green solution (50 mg L−1, pH = 6.5). The solutions were stirred for a time of 20 to 420 min. The impact of contact time was obtained through a graphical representation of the quantity adsorbed versus time.

The kinetics study was carried out by following the same steps as those described previously with a solution of methyl green (50 mg L−1). The mixtures were stirred for a

For adsorption isotherm, experiments were conducted by varying the initial concentration of methyl green from 5 to 120 mg g−1 (pH = 6.5, amount of adsorbent = 20 mg, time = 60 min). The models of Langmuir [40], Freundlich [41], Temkin [42], and Dubinin–Ra-

dushkevich [43] (Table 1) were invested to model the experimental results.

The pH effect was explored through adjusting the pH in the range 2−10 by integrating a solution of sodium hydroxide or hydrochloric acid (0.1 mol L−1). A total of 20 mg of CuO nanoparticles was added to 10 mL of methyl green solutions (50 mg L−1). The solutions

a graphical representation of the adsorbed amount versus pH.

representation of the adsorbed amount versus the adsorbent dose.

time ranging from 20 to 420 min.

The pH effect was explored through adjusting the pH in the range 2−10 by integrating a solution of sodium hydroxide or hydrochloric acid (0.1 mol L−1 ). A total of 20 mg of CuO nanoparticles was added to 10 mL of methyl green solutions (50 mg L−1 ). The solutions were agitated at 120 rpm for 12 h. The specimens were then centrifuged and the methyl green concentration was specified by UV-Visible spectroscopy at a wavelength of 630 nm using a Beckman DU 800 spectrophotometer. The pH study was indicated in the form of a graphical representation of the adsorbed amount versus pH.

The impact of the adsorbent dose was traced through varying the adsorbent mass from 2 to 500 mg in 10 mL of 50 mg L−1 methyl green solution at natural pH solution (pH = 6.5). Next, the solutions were agitated for 12 h, then centrifuged, filtered and the residual concentration was estimated. The study of the adsorbent dose corresponds to a graphical representation of the adsorbed amount versus the adsorbent dose.

To ensure good adsorption, we examined the impact of contact time on the adsorption of methyl green on CuO nanoparticles. Likewise, the previously reported steps were followed. Adsorbent (20 mg) was added to 10 mL of methyl green solution (50 mg L−1 , pH = 6.5). The solutions were stirred for a time of 20 to 420 min. The impact of contact time was obtained through a graphical representation of the quantity adsorbed versus time.

The kinetics study was carried out by following the same steps as those described previously with a solution of methyl green (50 mg L−1 ). The mixtures were stirred for a time ranging from 20 to 420 min.

For adsorption isotherm, experiments were conducted by varying the initial concentration of methyl green from 5 to 120 mg g−1 (pH = 6.5, amount of adsorbent = 20 mg, time = 60 min). The models of Langmuir [40], Freundlich [41], Temkin [42], and Dubinin– Radushkevich [43] (Table 1) were invested to model the experimental results.

**Table 1.** Theoretical models.

qads and qe correspond to the adsorbed amount and the adsorbed amount at equilibrium (mg g−1 ), respectively; Ce refers to the concentration of the solution at equilibrium (mg L−1 ); KL and KF indicate Langmuir and Freundlich constants, respectively; (1/n) denotes Freundlich coefficient; B represents Temkin constant relating to heat of adsorption; AT signifies Temkin constant relating to adsorption potential; R expresses universal gas constant (kJ kg−1 mol−1 K−1 ); T refers to temperature (K); β corresponds to constant related to the mean free energy of adsorption; ε stands for Polanyi potential.

For desorption experiments, the recuperated adsorbent after adsorption was recovered in 50 mL of a solution of 1.0 mol L−1 hydrochloric acid, for 3 h. Then the sample was shaken and centrifuged and washed with distilled water. Finally, the material was dried and reused for another adsorption cycle.

#### *2.6. Characterization Techniques*

Scanning electron microscopy was performed out by a HITACHI S-3400N (Paris, France) device (voltage: 0.5–30 KW, resolution: 4.5 nm).

Energy dispersive X-ray spectrometry (EDS) was carried out using a Perkin Elmer DIFFRAX (Paris, France).

The thermograms were obtained by testing the samples under nitrogen flow for the temperature ranges from 30 to 950 ◦C for the raw material and from 30 to 600 ◦C for the synthesized nanoparticles (heating rate: 10 ◦C min–1). The used device was the Analyzer Pyris 6 Perkin Elmer thermogravimetric analyzer (Paris, France).

The infrared analysis was conducted with a Nicolet iS 50 FT-IR Spectrometer Thermo Scientific (Paris, France). The pellets were obtained with anhydrous KBr 1% (*wt*/*wt*) of product.

#### **3. Results and Discussion**

*3.1. Characterization of Punica granatum Bark*

By applying standard methods, we determined the chemical composition of the bark of *Punica granatum* (Table 2).

**Table 2.** Chemical composition (content in%) of the bark of *Punica granatum* and some plant biomass collected from literature.

n.a.—not available; Holocell.—holocellulose content; 1% NaOH—content in 1% sodium hydroxide; ET—content in ethanol/toluene; H. water—hot water content; C. water—cold water content.

The bark of *Punica granatum* has an approximately high rate of extractives in cold and hot water (28%, 37.6%). These rates were higher than those of annual and perennial plants, non-wood and wood. However, extractives in a 1% NaOH solution (32.4%) were lower than those of some annual and perennial plants as well as vine stems but was higher than those of non-wood depicted in Table 2. Indeed, the bark of *Punica granatum* is rich in ester of hydrolysable ellagitannins, mainly punicalagin. Ellagitannins are high molecular weight, water-soluble polyphenols which contain several hydroxyl functions [55,56].

The extractables in the ethanol/toluene mixture displayed a rate of 46.5% which was higher than that of the materials reported in Table 2. Furthermore, the lignin content (22%) proved to be lower than that of wood plants such as date palm (27%) [44], and vine stems (28.1%) [48]. Nevertheless, it remained higher than that of olive wood (15.6%) [45], and higher than some non-wood and annual plants such as prickly pear (4.8%) [44], *Astragalus armatus* (16.8%) [53], and *Stipagrostis pungens* (12%) [52]. The holocellulose content (16.2%) and α-cellulose (7.2%) were lower than those of non-wood, annual, and wood plants exhibited in Table 2. On the other side, the bark of *Punica granatum* presented ash content comparable to that encountered for the sources outlined in Table 2.

The infrared spectrum displayed broad stretching vibrations around 3200–3600 cm−1 of OH groups and a band around 1360 cm−1 reflecting alcoholic, phenolic, and acidic groups. In addition, bands of punicalagin alkyl groups appeared at 2800–2970 cm−1 . The band at 1740 cm−1 was assigned to carbonyl groups. Moreover, the emergence of two bands **Mongi ben Mosbah1,2**

**Younes Moussaoui 2,4,\***

**Green**

*Article*

at 1620 cm−1 and 1430 cm−1 was attributed to the stretching of the C=C aromatic rings and the band at 1052 cm−1 was ascribed to the vibration d stretch CO. Moreover, the bands at 1229 and 637 cm−1 were associated respectively with the aromatic C-H bending and the C-O bond. **, Abdulmohsen Khalaf Dhahi Alsukaibi3 , Lassaad Mechi3 , Fathi Alimi3and**

The atomic composition of *Punica granatum* bark powder was specified using EDX analysis. The spectrum (Figure 3) suggested the presence of carbon, oxygen, iron, sodium, potassium, and calcium. *Water* **2022**, *14*, x FOR PEER REVIEW 7 of 17

**Figure 3.** EDX analysis of *Punica granatum* bark.**Figure 3.** EDX analysis of *Punica granatum* bark. **Figure 3.** EDX analysis of *Punica granatum* bark.

**Ecological Synthesis of CuO Nanoparticles Using**

The thermogram of the *Punica granatum* bark (Figure 4) revealed four stages. The first stage presented a mass loss of 5% from 30 ◦C to 200 ◦C. This loss was due to material dehydration. The second stage presented the largest mass loss (90%) occurring between 200 and 450 ◦C, during which the components of the lignocellulosic fibers and part of the lignin chain were degraded [57]. The third stage ranged between 450 and 600 ◦C. This region characterizes the thermal degradation of the remaining lignin and the complete decomposition of the carbon chains of the *Punica granatum* bark. Above 600 ◦C (the fourth stage) ash appeared and a constant mass was obtained. The thermogram of the *Punica granatum* bark (Figure 4) revealed four stages. The first stage presented a mass loss of 5% from 30 °C to 200 °C. This loss was due to material dehydration. The second stage presented the largest mass loss (90%) occurring between 200 and 450 °C, during which the components of the lignocellulosic fibers and part of the lignin chain were degraded [57]. The third stage ranged between 450 and 600 °C. This region characterizes the thermal degradation of the remaining lignin and the complete decomposition of the carbon chains of the *Punica granatum* bark. Above 600 °C (the fourth stage) ash appeared and a constant mass was obtained.

**Figure 4.** TGA curve of *Punica granatum* bark. (Blue lines indicate the three stages of mass loss) **Figure 4.** TGA curve of *Punica granatum* bark. (Blue lines indicate the three stages of mass loss).

The *Punica granatum* bark is an intrinsic source of biomolecules, involving condensed

act as ligating agents that are responsible for stabilizing and styling the copper nanoparticles. Indeed, the hydroxyl groups of the biomolecules performthe role of complexing agents that fix the copper ions of the copper sulphate solution. Although the structure of the complex formed was not determined, it canbe the product of the coordination of copper ions with the functional groups of the molecules present in the *Punica granatum* bark

*3.2. Preparation and Characterization of CuO Nanoparticles*

#### *3.2. Preparation and Characterization of CuO Nanoparticles*

The *Punica granatum* bark is an intrinsic source of biomolecules, involving condensed tannins, ellagic acids, catechins, punicalin, and punicalagin [55,56]. These biomolecules act as ligating agents that are responsible for stabilizing and styling the copper nanoparticles. Indeed, the hydroxyl groups of the biomolecules performthe role of complexing agents that fix the copper ions of the copper sulphate solution. Although the structure of the complex formed was not determined, it canbe the product of the coordination of copper ions with the functional groups of the molecules present in the *Punica granatum* bark extract. The formed complexes underwent decomposition and led to the release of CuO nanoparticles.

The SEM images (Figure 5) of the synthesized CuO nanoparticles display a smooth surface with the arrangement of the layers on top of each other forming a multilayer structure connected with strong molecular interactions with a large variation in the distribution of the particle size and shape. This accounts for the grafting of copper ions on the organic chains of the *Punica granatum* extract. Indeed, we noticed a beam of white light reflected on the material surface referring to the response of the copper. *Water* **2022**, *14*, x FOR PEER REVIEW 2 of 2

**Figure 5.** SEM microphotographs of synthesized CuO nanoparticles. **Figure 5.** SEM microphotographs of synthesized CuO nanoparticles.

Cu Element Weight (%) Atomic (%) C K 25.93 53.44 O K 19.49 21.01 Cu K 54.58 25.55 The spectrum obtained throughX-ray microanalysis (Figure 6) is indicative of peaks with strong energy for the copper found between 1 and 8 KeV, which confirms the obtaining of CuO nanoparticles. Additionally, about 54.58% of copper was quantified in the synthesized material. In addition, it is noteworthy that oxygen (19.49%) and carbon (25.93%) were noticed as constituents of the capping molecules surrounding the copper nanoparticles.

Cu

keV

Cu

0 1 2 3 4 5 6 7 8 9 10

**Figure 6.** EDX analysis of CuO nanoparticles.

0.0

0.5

1.0

1.5

O

C

2.0

2.5

3.0

3.5

**Figure 5.** SEM microphotographs of synthesized CuO nanoparticles.

**Multilayers**

**Pores**

20.0 kV 4.4 mm × 600 SE 50.0 µm 20.0 kV 5.3 mm × 600 SE 50.0 µm

20.0 kV 4.4 mm × 100 SE 500 µm 20.0 kV 4.4 mm × 420 SE 100 µm

**Figure 6.** EDX analysis of CuO nanoparticles. **Figure 6.** EDX analysis of CuO nanoparticles. **Figure 6.** EDX analysis of CuO nanoparticles.

The thermogravimetric analysis of the synthesized CuO nanoparticles (Figure 7) revealed three main areas of mass loss: The first zone representing about 10% located between 30 and 120 ◦C corresponded to the release of adsorbed water. The second zone of mass loss (approximately 40%) lies between 120 and 500 ◦C, subdivided into two subregions of degradation: The first sub-region of mass loss (approximately 25%) observed between 120 and 300 ◦C, was attributed to the degradation and opening of the organic molecules constituting bio-capping [58], which stands for the organic support of the extract present in the prepared nanoparticles. The second sub-region (about 15%) ranging between 300 and 500 ◦C, was ascribed to the thermal resistance of copper which is evidenced through the slowing down of degradation processes followed by thermal decomposition of organic chain residues. The third zone located above 500 ◦C presented a mass stability that was recorded with a total mass loss of 51.45%. This proves that a percentage of 48.55% metallic copper was present in the prepared nanoparticles. This behavior is in good agreement with facts reported by EDX analysis. The thermogravimetric analysis of the synthesized CuO nanoparticles (Figure 7) revealed three main areas of mass loss: The first zone representing about 10% located between 30 and 120 °C corresponded to the release of adsorbed water. The second zone of mass loss (approximately 40%) lies between 120 and 500 °C, subdivided into two subregions of degradation: The first sub-region of mass loss (approximately 25%) observed between 120 and 300 °C, was attributed to the degradation and opening of the organic molecules constituting bio-capping [58], which stands for the organic support of the extract present in the prepared nanoparticles. The second sub-region (about 15%) ranging between 300 and 500 °C, was ascribed to the thermal resistance of copper which is evidenced through the slowing down of degradation processes followed by thermal decomposition of organic chain residues. The third zone located above 500 °C presented a mass stability that was recorded with a total mass loss of 51.45%. This proves that a percentage of 48.55% metallic copper was present in the prepared nanoparticles. This behavior isin good agreement with facts reported by EDX analysis.

**Figure 7.** TGA curve of CuO nanoparticles. (Blue lines for main areas of mass loss and black line for sub-region of mass loss) **Figure 7.** TGA curve of CuO nanoparticles. (Blue lines for main areas of mass loss and black line for sub-region of mass loss).

*3.3. Study of the Adsorption of Methyl Green on CuO Nanoparticles*

#### *3.3. Study of the Adsorption of Methyl Green on CuO Nanoparticles*

*Water* **2022**, *14*, x FOR PEER REVIEW 10 of 17

The synthesized CuO nanoparticles were used as an adsorbent for the retention of methyl green. Thus, various parameters were investigated such as methyl green concentration, contact time, adsorbent dose, and pH. The synthesized CuO nanoparticles were used as an adsorbent for the retention of methyl green. Thus, various parameters were investigated such as methyl green concentration, contact time, adsorbent dose, and pH.

3.3.1. Influence of pH, Adsorbent Dose and Contact Time on the Methyl Green Adsorption onto CuO Nanoparticles 3.3.1. Influence of pH, Adsorbent Dose and Contact Time on the Methyl Green Adsorption onto CuO Nanoparticles

The influence of pH is illustrated in Figure 8a. The adsorbed amount (qads (mg g−1 )) increased with the pH, increasing from 2 to 6. The maximum adsorbed amount was reached for a pH ranging from 6 to 8 such as those reported using loofah fiber [59], titanium dioxide [60], CoFe2O4/rGO nanocomposites [61], Graphene oxide [62], activated bentonite [63], and activated carbon [64]. Indeed, when the pH value increased, many of the anionic sites were available, contributing to the increased retention of methyl green. For low pH values, H+ ions were present on the surface of CuO nanoparticles, which entailed an electrostatic repulsion between the surface of the CuO nanoparticles and the molecules of methyl green, hampering the process of adsorption. Therefore, the natural pH (pH = 6.5) of the methyl green solution was used for the rest of the work. The influence of pH is illustrated in Figure 8a. The adsorbed amount (qads (mg g−1)) increased with the pH, increasing from 2 to 6. The maximum adsorbed amount was reached for a pH ranging from 6 to 8 such as those reported using loofah fiber [59], titanium dioxide [60], CoFe2O4/rGO nanocomposites [61], Graphene oxide [62], activated bentonite [63], and activated carbon [64]. Indeed, when the pH value increased, many of the anionic sites were available, contributing to the increased retention of methyl green. For low pH values, H+ ions were present on the surface of CuO nanoparticles, which entailed an electrostatic repulsion between the surface of the CuO nanoparticles and the molecules of methyl green, hampering the process of adsorption. Therefore, the natural pH (pH = 6.5) of the methyl green solution was used for the rest of the work.

**Figure 8.** Influence of pH (**a**), adsorbent amount (**b**), and contact time (**c**) on the methyl green adsorption onto CuO nanoparticles. (Blue line for adsorbed quantity and green line for removal) **Figure 8.** Influence of pH (**a**), adsorbent amount (**b**), and contact time (**c**) on the methyl green adsorption onto CuO nanoparticles. (Blue line for adsorbed quantity and green line for removal).

The adsorbed amount of methyl green decreased with an increase inadsorbent dose (Figure 8b). This is accounted for in terms of adsorption sites, which remained unsaturated throughout the adsorption process. Thus, a mass of 20 mg of adsorbent was The adsorbed amount of methyl green decreased with an increase inadsorbent dose (Figure 8b). This is accounted for in terms of adsorption sites, which remained unsaturated

throughout the adsorption process. Thus, a mass of 20 mg of adsorbent was considered sufficient to ensure good adsorption of the methyl green on the CuO nanoparticles with complete elimination of the methyl green (elimination rate = 100%). This adsorbent mass value was selected for the current work.

To determine the time required to ensure good elimination of the methyl green, a study of the influence of the contact time on the adsorbed amount was conducted (Figure 8c). The profile of the adsorption process was characterized by two different stages. The first stage was described throughout the first 60 min of which several adsorption sites were available on the CuO nanoparticles surface. In the second stage, the elimination of methyl green remained unchanged, reflecting the saturation of the active adsorption sites. Therefore, a contact time of 60 min was considered sufficient to ensure adsorption equilibrium.

#### 3.3.2. Kinetic Study

Both the pseudo-first order Equation (2) [65–67] and pseudo-second order Equation (3) [66–68] models were invested to characterize the kinetics of adsorption of methyl green on CuO nanoparticles.

$$\ln(\mathbf{q}\_{\mathbf{e}} - \mathbf{q}\_{\mathbf{t}}) = \ln \mathbf{q}\_{\mathbf{e}} - \mathbf{k}\_{\mathbf{l}} \mathbf{t} \tag{2}$$

$$\frac{\mathbf{t}}{\mathbf{q}\_{\rm t}} = \frac{1}{\mathbf{k}\_2 \mathbf{q}\_e^2} + \frac{\mathbf{t}}{\mathbf{q}\_{\rm t}} \tag{3}$$

where qe and qt (mg g−1 ) refer, respectively, to the adsorbed quantities at equilibrium and at time "t"; k1 and k2 represent the constants for pseudo-first and pseudo-second order model, respectively.

The value of the adsorbed amount of methyl green calculated (qcal = 25.6 mg g−1 ) using the pseudo-second order model was comparable to the experimental value (qexp = 24.9 mg g−1 ) (Table 3). Additionally, the regression coefficient value in the case of the pseudo-second order model (R2 = 0.999) was high in comparison with that recorded for the pseudo-first order model. This suggests that the adsorption of methyl green on CuO nanoparticles adheres to the pseudo-second order kinetics. Likewise, according to ∆q, RMSE and χ 2 , the pseudo-second order model displays lower errors and is in good consistency with the experimental data. Similar phenomena have been reported for the adsorption of methyl green onto various adsorbent such as loofah fiber [59], titanium dioxide [60], zeolite H-ZSM-5 [69], carbon nanotubes [70], and graphene sheets [71].

**Table 3.** Kinetics parameters of methyl green adsorption on CuO nanoparticles.

#### 3.3.3. Adsorption Isotherms

The Temkin, Langmuir, Freundlich, and Dubinin–Radushkevich isotherms were used to characterize the adsorption of methyl green on the prepared nanoparticles (Table 4).The

It seems that the Langmuir fit (R2 = 0.9892) fits the methyl green adsorption data better than the other three models. It turns out that the Langmuir model is the most adequate to characterize the adsorption of methyl green on CuO nanoparticles. This result was checked by the low value of the chi-square test χ 2 , and the standard deviation ∆q, reflecting monolayer adsorption. Moreover, the values of dimensionless equilibrium constant RL, (Equation (4)) were lower than 1, reflecting the favorable adsorption of methyl green on the CuO nanoparticles. This fact was confirmed by the value of the Freundlich coefficient (1/n = 0.2294), which was lower than 1 [67,72–74].

$$\mathbf{R\_L} = \frac{1}{1 + \mathbf{K\_L}\mathbf{C\_0}}\tag{4}$$

The maximum adsorbed amount of methyl green on CuO nanoparticles calculated from the Langmuir model, was 28.7 mg g−1 .

A comparison of the adsorption capacity of methyl green onto CuO nanoparticles with other adsorbents reported by other researchers was shown in Table 5. It can be seen that CuO nanoparticles showed comparable adsorption capacity with respect to zeolite H-ZSM-5 [69] and grapheme oxide [62]. The adsorption capacity of CuO nanoparticles was higher than those of loofah fiber (18.2 mg g−1 ) [59] and bamboo [75], and was lower than that of titanium dioxide (384.6 mg g−1 ) [60], activated carbon from *Brachychiton Populneus* fruit shell [64], activated bentonite [63], and graphene sheets [71]. Therefore, the CuO nanoparticles were suitable and promising for the removal of methyl green from aqueous solutions since they have a relatively higher adsorption capacity.

**Table 5.** Reported maximum adsorption capacities in the literature for methyl green adsorption.

3.3.4. Desorption and Adsorbent Reuse

To assess the feasibility and applicability of adsorbent reuse and therefore to increase the use effectiveness of the prepared nanoparticles, desorption of the adsorbed methyl green was performed. Departing from the study of the pH effect, it was demonstrated that adsorption of methyl green was defective in acidic medium. Thus, desorption was conducted by washing the recuperated adsorbent after adsorption with a hydrochloric acid and afterwards with distilled water. Next, the adsorbent was dried and reused for the adsorption of methyl green. The adsorption/desorption procedure was repeated five times and results were summarized in Table 6.

**Table 6.** Adsorption-desorption of methyl green.

The maximum adsorption capacity of CuO nanoparticles decreased from 24.9 mg g−1 to 17.1 mg g−1 after five cycles.The regeneration results revealed that the adsorption efficiency of CuO nanoparticles dropped of only about 8% after three cycles. The adsorption/desorption tests corroborated appropriate reuse characteristics of the adsorbent after three cycles with a better performance for methyl green sorption.

#### **4. Conclusions**

The green synthesis of metallic nanoparticles has accumulated interest over the past decade due to their distinctive properties that make them applicable in various fields. Metal nanoparticles synthesized using plants have been shown to be non-toxic and environmentally friendly. In this study, a very cheap and simple conventional method was used to obtain the CuO nanoparticles using the extract from the bark of *Punica granatum*. The nanoparticles' purity was confirmed through EDX, yielding the presence of elemental copper oxide containing 25.93, 19.49, and 54.58%, of carbon, oxygen, and copper, respectively. The synthesized CuO nanoparticles demonstrated a great performance in the adsorption of methyl green with an adsorption capacity of 28.7 mg g−1 under optimal conditions (60 min, contact time; 2 g L−1 , adsorbent dose; pH, 6.5; 25 ± 2 ◦C). The adsorption of methyl green on the CuO nanoparticles follows the pseudo-second order model and the adsorption isotherm is in good conformity with the Langmuir model. CuO nanoparticles can be reused with good recycle properties for three cycles. Regarding the good sorption performance of

the prepared CuO nanoparticles, the use of environmentally friendly *Punica granatum* bark can constitute an ecological, simple, and economical method for the elaboration of materials in order not only to purify contaminated waters but also to enact additional environmental applications. The nanoparticles obtained through plant extracts have the potential to be widely used in the medical field as dressings or therapeutic drugs. Thus, green chemistry provides a new innovative technique that does not use hazardous substances for the design and development of variable activity materials.

**Author Contributions:** Literature review, data collection, field work, M.b.M.; methodology and writing—original draft preparation, M.b.M. and Y.M.; designed and revised the manuscript, L.M. and F.A.; writing—review and editing, A.K.D.A.; visualization and supervision, Y.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by scientific research deanship at University of Ha'il—Saudi Arabia through project number RG-191251. The APC was funded by scientific research deanship at University of Ha'il—Saudi Arabia through project number RG-191251.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research has been funded by scientific research deanship at University of Ha'il—Saudi Arabia through project number RG-191251.

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

#### **References**


**Notes**

1 Initially "Yalu" was not a person's name but a title that means "old grandfather" or "old ancestor", used as "an honorific title for a prestigious leader". Due to the long use of honorific titles and the difference in Hmong dialects, the meaning of "Yalu" was gradually blurred until it was treated as a person's name (Wu 2012, pp. 34–37).


*3.1.1 Devices used in vibration therapy*

### *<IP_ADDRESS> Local vibration therapy*

The devices used in local vibration therapy are relatively simple and are small, and in general, are portable devices device that applies vibration directly over the muscle or the tendon [20]. Buttons are available for selecting the operational conditions of the intervention characteristics, which consider the frequency of mechanical vibration. Local vibration therapy is very important for several individuals that are in long-term immobilization, immobilization by a cast, or being in the early rehabilitation period after surgery [10, 20].

### *<IP_ADDRESS> Systemic vibration therapy*

The device used in Systemic vibration therapy is a vibrating platform. Systemic vibration therapy is a clinical intervention in which mechanical vibration generated on a vibrating platform is transmitted to the entire body of the person who is in contact with the base of the platform producing the whole-body vibration exercise in the individual [8, 12, 16].

There are two main types of vibrating platforms, which are shown in **Figure 6A** and **B**. **Figure 6A** represents a vibrating platform where the base performs a movement with vertical displacement as a result of the force applied. The oscillating of this base can be vertical or triplane way. **Figure 6B** has demonstrated the side alternating vibrating platform, where the base performs side alternating movements (up and down) [8, 12, 16].

In the side alternating vibrating platform, the base rests on a central fulcrum. When turned on, while the right side of the base goes up, the left side goes down, generating mechanical vibration, like a seesaw. The mechanical vibration is then transmitted to the individual who is in contact with the base, and this platform is called the side alternating platform [8, 12, 16].

In the vertical platform, the whole base of the platform goes up and down which is called the vertical displacement type. This vertical displacement may be due to a simple displacement of the base, lowering and raising. In this case, the vibrating

**Figure 6.** *Vibrating platforms and oscillation of their bases.*

platform is called a synchronous vertical type of platform. This vertical displacement can also be a result of the base displacement in three planes. In this case, the platform is named the triplane vertical platform [8, 12].

It is easy to verify that, associated with the mechanical vibration, a force is generated, which is responsible for the displacement of the base of the vibrating platform, and it is transmitted to the person's body. This force arises from the action of the acceleration imposed by the vibrating platform on the body, which has a certain mass [8, 12, 16].


**4. Detection of archaeological sites based on remote sensing techniques**

massol Castle found that for the majority of the time periods, the PM10 readings exceeded the

**Figure 10.** AOT levels in the Limassol area. High AOT levels are noted in the area near the Limassol Castle.

**0**

**200**

**400**

**600**

**800**

**1000**

**1200**

A similar approach was followed for the Paphos town using daily MODIS AOT data. The re‐ sults have shown that 54% of the measurements for air quality was above the threshold of AOT 300 (AOT 0.300) (see Figure 11). This analysis suggest that cultural heritage sites near the Pa‐ phos town (e.g. *Nea Paphos, Tombs of the Kings* etc) are exposed to air pollutants half the time.

**Paphos AOT values**

**Figure 11.** Paphos AOT values (sample = 109 measurements) in blue. In red circle is the threshold air quality limit of 300 (AOT 0.300). In the y-axis, AOT value is multiplied by 1000 (to match MODIS data) (Themistocleous et al., 2012a).

**Paphos AOT Air Quality Threshold**

limit value (50 μg/m3), indicating a high level of air pollution in the area.

72 Remote Sensing of Environment: Integrated Approaches

Several Neolithic settlements ("magoules") are located in the Thessalian plain in central Greece. These sites are typically found as low hills raised up to 5-10 m. Alexakis et al., (2009; 2011) has recently shown that the detection of several unknown sites is possible based on remote sensing and GIS analysis. The study aimed to combine several types of remote sens‐ ing data (e.g. Landsat TM/ETM+, ASTER, Hyperion, IKONOS) and DEM in order to im‐ prove the detection of these subsurface remains (Figure 12). The satellite data were statistically analyzed, together with other environmental parameters, to examine any kind of correlation between environmental, archaeological and satellite data. Moreover, different methods were compared for the detection of Neolithic settlements. The results of the study suggested that the complementary use of different imagery can provide more satisfactory results.

Further to the Alexakis study, Agapiou et al., (2012a) argued that the detection of the settle‐ ments is possible based on ground spectroradiometric measurements. Several spectroradio‐ metric measurements have indicated that each magoula has its own spectral characteristics related to its own morphological characteristics. The study has found that the highest peak of the magoula tends to give high NDVI and SR values (similar to the flat – healthy regions) while the slope of the magoula has lowest NDVI and SR values (and for the other indices as well). The extraction of each magoula requires further analysis and enhancement techniques in cases where the spatial resolution of the satellite image used is low. Local histogram en‐ hancements can identify magoules as a small difference of NDVI values at the same parcel (Figure 13).

**Figure 12.** Magoula *Neraida* using ASTER image (left). Magoula *Melissa 1* using IKONOS image (RGB - 321) (right).

Similar results were found following the application of the Tasselled Cap algorithm (Figure 14 to a series of Landsat TM/ETM+ multispectral images. The Tasselled Cap transformation is used to enhance spectral information for Landsat images, and it was specially developed for vegetation studies. The first three bands of the Tasseled Cap algorithm result are charac‐ terized as follow: band 1: brightness (measure of soil); band 2: greenness (measure of vegeta‐ tion); band 3: wetness (interrelationship of soil and canopy moisture).

Phenological studies of crops for the detection of buried archaeological remains were al‐ so evaluated (Agapiou et al., 2012b) It was found that the phenological cycle of crops for 'archaeological' and 'non archaeological areas' can be used as a "remote" approach in or‐ der to locate buried architecture remains. In Figure 15, the phenological cycle of an ar‐ chaeological site (*Almyros II*) and the phenological cycle of a healthy site (Site 3) are examined. A small NDVI difference is evident (Case A, Figure 15) which is associated with buried archaeological remains. This is due to the fact that soil over the archaeologi‐ cal remains seems to have a different moisture content compared to their surroundings. Therefore, although there exist similar climate characteristics and crop cultivation techni‐ ques, there is a difference in amplitude of the NDVI cycle of the archaeological and non-

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**Figure 15.** Phenological cycle of the Neolithic settlement (solid line) and the healthy site 3 (dashed line) (Agapiou et

Contemporary techniques and methods such as computer graphics, virtual reality, multime‐ dia technology, and information technology can be integrated in Web GIS technologies, in order to act as a uniform digital tool for documentation, protection and preservation of cul‐ tural heritage (Agapiou et al., 2010c; Hadjimitsis et al., 2006). In order to document and map known archaeological sites and monuments, several techniques may be used, including la‐ ser scanning, 3D modelling and GIS. In this section, applications from several monuments in

**5. Documentation of cultural heritage sites using remote sensing**

**techniques, GIS and laser scanning**

archaeological areas.

al., 2012b)

Cyprus are presented.

**Figure 13.** NDVI results for *Prodromos II* site (in green circle). (a) Raw satellite image without any radiometric enhancements, (b) satellite image with a linear max-min enhancement applied to all image, (c) max-min enhancement applied to the area around *Prodromos II* and (d) modified max-min enhancement applied to the area around *Prodromos II*. The magoula is indicated with the red arrow (Agapiou et al., 2012c).

**Figure 14.** Tasseled Cap results for *Nikaia 16* site (in red circle), (a) Brightness, (b) greenness, (c) wetness and (d) RGB of the first three components of the T-K algorithm (Agapiou et al., 2012c).

Phenological studies of crops for the detection of buried archaeological remains were al‐ so evaluated (Agapiou et al., 2012b) It was found that the phenological cycle of crops for 'archaeological' and 'non archaeological areas' can be used as a "remote" approach in or‐ der to locate buried architecture remains. In Figure 15, the phenological cycle of an ar‐ chaeological site (*Almyros II*) and the phenological cycle of a healthy site (Site 3) are examined. A small NDVI difference is evident (Case A, Figure 15) which is associated with buried archaeological remains. This is due to the fact that soil over the archaeologi‐ cal remains seems to have a different moisture content compared to their surroundings. Therefore, although there exist similar climate characteristics and crop cultivation techni‐ ques, there is a difference in amplitude of the NDVI cycle of the archaeological and nonarchaeological areas.

**Figure 13.** NDVI results for *Prodromos II* site (in green circle). (a) Raw satellite image without any radiometric enhancements, (b) satellite image with a linear max-min enhancement applied to all image, (c) max-min enhancement applied to the area around *Prodromos II* and (d) modified max-min enhancement applied to the area around *Prodromos II*. The

**Figure 14.** Tasseled Cap results for *Nikaia 16* site (in red circle), (a) Brightness, (b) greenness, (c) wetness and (d) RGB

magoula is indicated with the red arrow (Agapiou et al., 2012c).

74 Remote Sensing of Environment: Integrated Approaches

of the first three components of the T-K algorithm (Agapiou et al., 2012c).

**Figure 15.** Phenological cycle of the Neolithic settlement (solid line) and the healthy site 3 (dashed line) (Agapiou et al., 2012b)


**6.3. ISO 22157-2 bamboo: determination of physical and mechanical properties: part 2: laboratory manual**

The purpose of this standard, originally written as a technical report is to disseminate best practice test methods in order to make these globally available but also to outline the 'how to' for the tests specified in ISO 22157-1 [15, 16]. On a number of issues, this standard refers to existing national and supranational standards which should be followed, especially for bamboo application in temperate climates (e.g. W. Europe, Canada). This standard contains a number of examples, templates and backgrounds to the tests specified in [15].

be affected by the pull-out force evolution with time due to root growth (**Figure 4**). As shown before, the overall slope stability of a bioengineered slope can be assessed using existing slope stability analysis methods [34] taking into account both long-term (drained) and short-term

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Internal stability analysis consists of checking the mechanical capacity of the bamboo culms

The characteristic strength values should be obtained according to ISO 22157 [15, 16]. Suggested characteristic strength values for any bamboo species can be found in [35]. These values should be adapted to the bamboo moisture content by using the moisture content correction factor included in [35] which is based on NSR [36] and EN 384 [37]. Bamboo live pole (pegs) strength characteristic values have been shown in preceding epigraphs (see **Table 3**). The values for the material factor of safety are specified in ISO 22156 [30]. Recommended

In soil bioengineering works, bamboo culms work under Service Class 3 conditions (relative humidity >85%) and in-ground conditions. This situation can be reflected by means of the service class and load duration factor (Kmod) which can be determined from the existing

Other factors making allowance for other conditions (e.g. earthquakes, connection between

Bamboo structures' internal stability checks follow Eurocode 5: Design of timber structures. This design scheme is detailed in ISO 22156 [30] and in [38]. Because of the specific bamboo culm shape, the cross-sectional area to be used in the design calculation is the following:

where *A* is the net area of section; *De* is the bamboo culm outer diameter; *t* is the bamboo culm

By comparing the existing stress values with the element bending capacity, shear, axial ten-

Additionally, Eqs. (2) and (3) can also be useful for determining the minimum bamboo culm

The forces exerted on the bamboo culms can be determined by using traditional structural calculation theory (**Figure 5**). Typically, the moment, shear and axial stress diagrams should

\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Accordingly, the elastic section modulus (*Selastic*) for the bamboo case will be.

sion or axial compression, the internal stability check can be fulfilled.

be generated and their maximum values used for internal stability checks [29].

4 (*De*2 − (*De* − 2*t*)2) − ∑*area of any holes* (2)

32*De* (3)

which are fulfilling structural functions within the ground bioengineering work.

(undrained) conditions.

standards [30, 35].

average thickness.

*A* = \_\_

**7.2. Internal stability check**

values for this factor can also be found in [35].

elements of different rigidity, etc.) can also be found in [35].

Π

*Selastic* = Π(*De*4 − (*De* − 2*t*)4)

dimeter fulfilling the internal stability condition [29].

The above ISO standards provide the basis for design with bamboo culms. However, they do not include content relating to living bamboo (design and testing) and the use of a combination of living and inert bamboo in a structure. The existing standards need to be updated and expanded to reflect the growing research on test methods and material characterization of both living and inert bamboo, especially covering the durability and evolution of load transfer mechanisms with time. This update could be partially covered by an attempt to use the existing timber-based test methods for characterization and design [29] which could be beneficial in engaging engineers and architects [31] in the use of bamboo for bioengineering purposes.


**1. Introduction**

Sexual assault can be characterized as gender inequality, power imbalance, and hatred that generally results in violence against women. This fact does not exclude children, male, and LGBTI+ victims. Based on surveys on violence against woman performed in the USA, approximately 8 million of women and 3 million of men have experienced rape once or more in their lifetime [1, 2]. In most of the cases, the perpetrators are known to the victims, and false notations like "rape happens to young- and good-looking women" or that "it is committed by mentally problematic people" are incorrect. Likewise, rape does not frequently occur in places that are not properly illuminated or uninhabited remote areas as it is generally acknowledged, but mostly happen in very familiar setting like the home of the victim or the offender. Attractive sexy clothing is never considered a reason for rape [3].

There is a series of well-reported problems that survivors of sexual assaults suffer, in the short and long run following the rape. Medical impacts on genital organs because of physical traumas, pregnancy and unsafe abortion, and sexually transmitted diseases are complications seen in the short run, whereas anxiety, depression, post-traumatic stress disorder (PTSD), substance abuse, suicidal behavior, eating disorders, and even dental health problems affect the victims in the long run [4–7]. PTSD influences the sexual life of the sufferers in a long run too. Considering rape in war, genocide, prisons, and police stations, the magnitude of the problem increases tremendously [8]. Nevertheless, it is well reported that millions of women are affected all over the world regardless of their age, socioeconomic position, and economic or educational status [2, 9, 10].

In a detailed study that was undertaken in Turkey, while women constitute 35–40% of victims of sexual assault, the proportion of children is 50%, and the average age of these children is 11.5 years. The youngest child was a girl of less than one year of age and a 1.5-year-old boy. We see that 5% adult males, 5% mentally handicapped individuals, 1–3% elderly people, and 90-year-old senior ladies are also victims of this crime.  The reasoning for the attempts was reported as the victim was responsible for the attack, the aggressor was sexually aroused, and that this act was the result of sexual provocation. The way that a 1-year-old child provokes the aggressor has no valid explanation [11].

The study in mention revealed that the rate of realization of sexual attack in desolate and dark places is around 10% only. The riskiest environment seems to be the home of the attacker or the victim, with a rate of 60%. The rate of attack in a different house is around 20%. Another misconception is the belief that the victim of sexual assault must have suffered extensive physical injury. In practice, physical damage can be detected only in one-fourth of the cases. The reason for this is that physical violence is used in only half of the cases, and detectable damage occurs in half of them. Again, while the aggressor is mostly thought to be a stranger, victim screening studies show that 75% of the perpetrators against women and 90% against children are familiar people [11].

Resolving the case, identifying the perpetrator and desiring his/her punishment is an expected reaction of the victims, families, and the societies. Yet, conviction rates for sexually committed crimes are considerably low. There are various reasons that prevent victims to disclose rape cases, the main of them being the possibility of the shift of the blame toward themselves and the disbelief by the legal system. Sufferers think that both police and their families will disregard their claims and disbelieve them.

The negative attitude of the society, the fear that the claim will not be taken seriously, the thought that the aggressor will not be punished, care for the perpetrator himself (in case of close relatives or friends), and the perpetrator's possibility of retaliation can be counted as the reasons for such a low rate of reporting the event. However, the "fear of not being able to prove the incident" forms a serious obstacle for the victim to apply to the court.

It is necessary to discuss the reasons for refusal to notify in Turkey because the society approach is interesting. For many years, the concept of honor was related to sexuality for women and not for men. The veil of honor was present in legislation until 2005. Forced sexual intercourse was not considered a crime, and the abortion with the promise of marriage was legal. All these show that the laws perceived this crime as an act of sexuality only. In this case, the woman's dress and behavior have always been questioned, and the investigation of the victim's responsibility in the attack has been discussed on both social and legal grounds. The criminal law has been changed in 2005, and it has been stated that sexual assault is a crime motivated by domination and control, and it is not a sexually motivated action. However, the negative societal conception is still there. As a result, victims in Turkey are truly rejected from their families and

#### *Sexual Assault Crisis Center: The First Interdisciplinary Effort in Turkey DOI: http://dx.doi.org/10.5772/intechopen.104531*

their communities following a rape victimization, the sexual aggression remains undisclosed, and the rapist is rarely convicted [11, 12].

The "one-stop center" model for supporting survivors of sexual violence has gained attention in recent years. There are several centers functioning in this aspect primarily in the USA. We established the first full-scale sexual assault center, in which we provide confidential support to survivors with specially trained staff over a wide range of services. Apart from performing forensic medical examination, evidence collection, reporting to the police, providing medical care, making a police statement, and giving psychological support are within the scope of the establishment. Although it is not very easy to establish and operate, not surprisingly, there are many positive benefits for survivors. We hope that this will be a model for the Turkish legal system and that this effort will form the basis for a national understanding.


**2.3 Programming interfaces**

The actual handling of a message transfer, that is the execution of the respective communication protocols through the different networking layers, is much too complex and too hardware-oriented to be done at application level. Therefore, the application programmer is usually provided with appropriate communication libraries that hide the hardware-related part of message transfers and hence allow the development of platform-independent parallel applications.

1 for example by means of *process identifiers*

from advantages of specialized hardware (Gropp et al., 1999). Although MPI is, in contrast to the libraries mentioned in the last section, *not* a specific implementation but just an interface standard, the standardizing processes was accompanied by the development of a prototype

Hierarchy-Aware Message-Passing in the Upcoming Many-Core Era 157

Today, two different compatibility levels can be distinguished:3 Compatibility with MPI-1 means that an MPI implementation provides support for all features specified in the MPI standard Version 1.3. And compatibility with MPI-2 means as opposed to MPI-1 that the respective MPI implementation also provides support for the extensions specified up to the MPI standard Version 2.2. Altogether, these two levels incorporate a function set of about 280 MPI functions. However, many MPI applications just use a handful of them, mostly focusing on the actual message handling. To begin with the term of a message, the tuple (*address*, *count*, *datatype*) defines an MPI message buffer, in which *count* describes the amount of elements of *datatype* beginning at *address*. Thus, it is ensured that the receiving side obtains the same data even if it uses another data format than the sending side. To distinguish between several messages, a *tag* is introduced that represents the message type defined by the user application. Furthermore, MPI defines the concepts of the *context* and *groups* aggregated in a so-called *communicator*. Only messages with a valid context (that is in terms of a matching communicator) will be received and processes may be combined to logical groups by means of the communicator. In addition to these basic concepts, a wide range of further mechanism like non-blocking, buffered or synchronizing communication, as well as collective operations and a particular error handling is provided. Although most MPI applications are written according to the SPMD paradigm, MPI-2 also features process spawning and support for

The MCAPI, recently developed by the Multicore Association, resembles an interface for message-passing like MPI. However, in contrast to MPI and sockets which were primely designed for inter-computer communication, the MCAPI intends to facilitate lightweight inter-core communication between cores on one chip (Multicore Association, 2011). These may be even those which execute code from chip internal memory. Therefore the MCAPI tries to avoid the von Neumann bottleneck4 using as less memory as it is necessary to realize communication between the cores. According to this, the two main goals of this API are extremely high-performance and low memory footprint of its implementations. In order to achieve these principals, the specification sticks to the KISS5 principal. Only a small number of API calls are provided that allow efficient implementations on the one hand, and the opportunity to build other APIs that have more complex functionality on top of it, on the other hand. For an inter-core communications API, such as MCAPI, it is much easier to realize these goals because an implementation does not have to concern issues like reliability

2 Nowadays, two more popular and also freely available MPI implementations exist: Open MPI (Gabriel

3 Currently, the specifications of the upcoming MPI-3 standard are under active development by the

4 It describes the circumstance that program memory and data memory share the same bus and thus

and reference implementation: MPICH (Gropp et al., 1996).2

programs written according to the MPMD paradigm.

**2.3.4 The Multicore Communications API (MCAPI)**

et al., 2004) and MPICH2 (Gropp, 2002).

result in a shortage in terms of throughput.

5 Keep It Small and Simple

working groups of the Message-Passing Interface Forum.

#### **2.3.1 The Berkeley Socket API**

A very common communication interface is the Berkeley Socket API, also known as BSD Sockets. A *socket* is a communication termination endpoint that facilitates the access to various transport layer protocols, such as TCP and UDP (Winett, 1971). Although usable to communicate between processes on the same machine, their intention is to enable the inter-process communication over computer networks. This can be done either first establishing a connection via creating a stream socket for the TCP protocol, or connectionless using datagram sockets for the UDP protocol. The sockets are managed by the operating system which organizes the access to the underlying network. They are used in a Client-Server manner, what means that the connection establishment between a pair of processes must be triggered in an asymmetric way, starting from the client side. Afterwards, messages may be exchanged bidirectional via the socket by using simple send and receive functions (Stevens et al., 2006).

#### **2.3.2 Communication libraries for parallel environments**

Besides simple send and receive functions, communication libraries especially for parallel environments do not only offer simple Client-Server relations, but rather provide support for a session management covering all parallel processes, including process startup and an all-to-all connection establishment. Commonly such libraries also offer additional features, as for example, for conducting collective operations or for transparent data conversion. In the course of time, several of such communication libraries had been developed, usually driven by the demand for new libraries in connection with new hardware platforms. Examples are: *NX, NX/2* and *NX/M* that are libraries developed by Intel for a past generation of dedicated multi-computers (Pierce, 1988), *Zipcode* is a software system for message-passing developed by the California Institute of Technology (Skjellum & Leung, 1990), *P4: Portable Programs for Parallel Processors* is a socket-based communication library by Argonne National Laboratory (Butler & Lusk, 1994), *Chameleon* is no communication library by itself but rather a macro-based interface to several underlying communication libraries (Gropp & Smith, 1993), and *PVM: Parallel Virtual Machine* is still a very common communication library (Dongarra et al., 1993) that has also been extended by the ability to be runnable in Grid environments (Geist, 1998).

#### **2.3.3 The Message-Passing Interface (MPI)**

When looking at the diversity of communication libraries listed in the last section, it becomes obvious that writing portable parallel applications was hardly possible in those days. Hence, there was a strong demand for the creation of a unified interface standard for parallel communication libraries in the early 1990s. This demand for an easy portability of parallel applications to always new generations of parallel machines eventually led in 1993 to the definition of such a unified library interface by the so-called Message-Passing Interface Forum. The goal was to define a communication standard that is hardware and programming language independent but still meets the requirements of high-performance computing. The result was the Message-Passing Interface Standard (MPI), which is a specification of a library interface (Message Passing Interface Forum, 2009). The main objective is that users do not need to compromise among efficiency, portability and functionality without having to abstain 6 Will-be-set-by-IN-TECH

A very common communication interface is the Berkeley Socket API, also known as BSD Sockets. A *socket* is a communication termination endpoint that facilitates the access to various transport layer protocols, such as TCP and UDP (Winett, 1971). Although usable to communicate between processes on the same machine, their intention is to enable the inter-process communication over computer networks. This can be done either first establishing a connection via creating a stream socket for the TCP protocol, or connectionless using datagram sockets for the UDP protocol. The sockets are managed by the operating system which organizes the access to the underlying network. They are used in a Client-Server manner, what means that the connection establishment between a pair of processes must be triggered in an asymmetric way, starting from the client side. Afterwards, messages may be exchanged bidirectional via the socket by using simple send and receive functions (Stevens

Besides simple send and receive functions, communication libraries especially for parallel environments do not only offer simple Client-Server relations, but rather provide support for a session management covering all parallel processes, including process startup and an all-to-all connection establishment. Commonly such libraries also offer additional features, as for example, for conducting collective operations or for transparent data conversion. In the course of time, several of such communication libraries had been developed, usually driven by the demand for new libraries in connection with new hardware platforms. Examples are: *NX, NX/2* and *NX/M* that are libraries developed by Intel for a past generation of dedicated multi-computers (Pierce, 1988), *Zipcode* is a software system for message-passing developed by the California Institute of Technology (Skjellum & Leung, 1990), *P4: Portable Programs for Parallel Processors* is a socket-based communication library by Argonne National Laboratory (Butler & Lusk, 1994), *Chameleon* is no communication library by itself but rather a macro-based interface to several underlying communication libraries (Gropp & Smith, 1993), and *PVM: Parallel Virtual Machine* is still a very common communication library (Dongarra et al., 1993) that has also been extended by the ability to be runnable in Grid environments

When looking at the diversity of communication libraries listed in the last section, it becomes obvious that writing portable parallel applications was hardly possible in those days. Hence, there was a strong demand for the creation of a unified interface standard for parallel communication libraries in the early 1990s. This demand for an easy portability of parallel applications to always new generations of parallel machines eventually led in 1993 to the definition of such a unified library interface by the so-called Message-Passing Interface Forum. The goal was to define a communication standard that is hardware and programming language independent but still meets the requirements of high-performance computing. The result was the Message-Passing Interface Standard (MPI), which is a specification of a library interface (Message Passing Interface Forum, 2009). The main objective is that users do not need to compromise among efficiency, portability and functionality without having to abstain

**2.3.1 The Berkeley Socket API**

**2.3.2 Communication libraries for parallel environments**

et al., 2006).

(Geist, 1998).

**2.3.3 The Message-Passing Interface (MPI)**

from advantages of specialized hardware (Gropp et al., 1999). Although MPI is, in contrast to the libraries mentioned in the last section, *not* a specific implementation but just an interface standard, the standardizing processes was accompanied by the development of a prototype and reference implementation: MPICH (Gropp et al., 1996).2

Today, two different compatibility levels can be distinguished:3 Compatibility with MPI-1 means that an MPI implementation provides support for all features specified in the MPI standard Version 1.3. And compatibility with MPI-2 means as opposed to MPI-1 that the respective MPI implementation also provides support for the extensions specified up to the MPI standard Version 2.2. Altogether, these two levels incorporate a function set of about 280 MPI functions. However, many MPI applications just use a handful of them, mostly focusing on the actual message handling. To begin with the term of a message, the tuple (*address*, *count*, *datatype*) defines an MPI message buffer, in which *count* describes the amount of elements of *datatype* beginning at *address*. Thus, it is ensured that the receiving side obtains the same data even if it uses another data format than the sending side. To distinguish between several messages, a *tag* is introduced that represents the message type defined by the user application. Furthermore, MPI defines the concepts of the *context* and *groups* aggregated in a so-called *communicator*. Only messages with a valid context (that is in terms of a matching communicator) will be received and processes may be combined to logical groups by means of the communicator. In addition to these basic concepts, a wide range of further mechanism like non-blocking, buffered or synchronizing communication, as well as collective operations and a particular error handling is provided. Although most MPI applications are written according to the SPMD paradigm, MPI-2 also features process spawning and support for programs written according to the MPMD paradigm.


*3.3.3 Fe2O3 nanoparticles*

Iron oxide (III) is a very stable oxide, it crystallizes in hexagonal form and is found in nature as the mineral hematite α-Fe2O3. The nanostructures of this oxide take different forms as they are nanowires, nanotubes, nanospheres, etc. [47]. Although its synthesis has been widely studied, its possible antibacterial effect not. The Fe2O3 NPs bactericidal effect against *E. coli* and *S. aureus* has been reported, where an increase of this effect is observed, as the concentration of iron oxide NPs increases [48]. A bactericidal effect has also been seen on *P. aeruginosa* with a minimum inhibitory concentration of 0.06 mg/L [49]. Another study reports on the bactericidal activity of nanostructured hematite against a variety of Grampositive and Gram-negative bacteria; *P. aeruginosa*, *S. aureus*, *K. pneumoniae*, *Lysinibacillus sphaericus* and *Bacillus safensis* [50]; proposing some mechanisms of action depending on the activity observed in each stage of the growth of the bacteria in question. A bactericidal effect of NPs of Fe2O3 against *S. epidermidis* has even been determined [51].

From its properties, its possible application in the remediation of the environment and water, as well as in the biomedical area, has been proposed, due to the different studies of cytotoxicity that have been carried out [47].

#### *3.3.4 MgO nanoparticles*

Magnesium oxide is in nature as the mineral periclase [52]. The antibacterial activity of MgO against Gram-positive and Gram-negative bacteria has been reported. It has been proposed that MgO NPs can damage the cell membrane causing the loss of intracellular contents and causing the death of bacterial cells [53]. The generation of reactive oxygen species has been attributed to the surface alkalinity of the MgO NPs [54]. The antibacterial activity of NPs of MgO against Gram-negative bacteria has been evaluated; *E. coli* and *P. aeruginosa* (500 and 1000 μg/mL) and in a Gram-positive bacterium; *S. aureus* (1000 μg/mL) [55]. The MgO NPs potentiated lipid peroxidation induced by ultrasound in the liposomal membrane. In this case the mechanism of action could be associated to the presence of defects, or to the lack of oxygen on the surface of the NP, leading to lipid peroxidation and the generation of reactive oxygen species [55]. The antibacterial effect and mechanism of action of NPs of MgO against strains of *Campylobacter jejuni*, *E. coli* and *Salmonella enteritidis* has been studied [56]. In this case, it was observed that the permeability of the bacteria's membrane, after exposure to the MgO NPs, was compromised, finding the presence of hydrogen peroxide that would subsequently cause cell death. Studies of *P. aeruginosa* and *S. aureus* versus MgO NPs showed a greater zone of inhibition in *S. aureus* than in *P. aeruginosa* [56]. Based on previous work, the authors note that the bactericidal action of MgO NPs may be due to the binding of surface oxygen to bacteria. As the surface area of the particles increases, the concentration of oxygen ions on the surface increases, which results in a more effective destruction of the cytoplasmic membrane and the cell wall of the bacteria.

**21**

*In vitro Antimicrobial Activity Evaluation of Metal Oxide Nanoparticles*

**4.** *In vitro* **methods for antimicrobial evaluation of nanoparticles based**

Bacteria exposed to antimicrobials are under selective pressure to evolve and adapt, this natural process leads to antimicrobial resistance. Human kind is facing the growing threat of rapid evolution and dissemination of bacteria resistant to multiple antibiotics. There is, therefore, an urgent need to develop

Antimicrobial agents include disinfectants, antiseptics, and antibiotics. New agents must be exhaustively tested for efficacy and safety. Evidence-based selection of the microorganisms and the evaluation system is of paramount importance for adequate interpretation of the test results, and for extrapolating from *in vitro* to

The use of nanoparticles especially based in metal oxides emerge as new antimicrobial agents, therefore it is necessary to test the efficacy of nano-antimicrobials against representative bacterial species. One known limitation of the testing systems currently in use, is that formulations are often challenged *in vitro* with one

Regulatory agencies require adherence to well established evaluation systems. Regulatory tests applicable to disinfectants, antiseptics or therapeutic antimicrobials vary greatly; and could include to nanoparticles with potential use as

When evaluating chemical disinfectants, bacterial endospores are considered the microbial life-form hardest to kill, followed in descendent order by mycobacte-

In the United States, the Food and Drug Administration (FDA) regulates chemical sterilants and high level disinfectants (HLD) that are used to reprocess medical instruments [59]. The AOAC International sporicidal and tuberculocidal tests are the accepted methods for evaluation. For a liquid chemical sterilant, the FDA standard tolerates no failures in the AOAC sporicidal test 966.04, and accepts no survivors in simulated-use testing with a challenge inoculum of six logs of spores. The FDA defines HLD as sterilants used under the same contact conditions but for only the contact time needed to reduce *Mycobacterium bovis* in 6 log10 in the tuberculocidal test 965.12 [60]. Moreover, to be approved, the disinfectants should be subjected to worse case scenarios, such as the presence of organic or inorganic

In Europe, the CEN/TC 216 technical committee produces current and future disinfectant testing standards [61]. Standard EN-14885-2006 indicates test methods to be used to substantiate claims for products intended for instrument disinfection [62], including mycobacterial/tuberculocidal (EN-14348, EN 14563), bactericidal (EN-13727, EN-14561) and fungicidal (EN-13624, EN-14562) activity tests but the terms "sterility, sterile, sterilization, sterilant" fall outside the scope of CEN/TC 216. In the US, high, intermediate and low level disinfectants, are regulated by the Environment Protection Agency (except HLD intended to reprocess medical instruments, which fall under FDA's jurisdiction). Intermediate level

microbial species at the time, and rarely against multi-species biofilms.

*DOI: http://dx.doi.org/10.5772/intechopen.84369*

**metal oxide**

real-life scenarios.

**4.1 Regulatory testing**

antimicrobials.

*4.1.1 Disinfectants*

ria, bacteria in vegetative form, and viruses.

contamination, and under simulated use conditions.

new antimicrobials [57, 58].


*Edited by Julio Elito Jr.*

"Non-Tubal Ectopic Pregnancy" is a comprehensive book, written in an organized and concise format. The book offers an immersion into non-tubal ectopic pregnancy and the reader is invited, chapter after chapter, to visit the most important aspects of nontubal ectopic pregnancies. The book covers all aspects of non-tubal ectopic pregnancies including epidemiology, diagnosis, and management. Experts from all over the world have contributed to it, bringing the best from their research.The book presents the reader with the latest advances on non-tubal ectopic pregnancies.

ISBN 978-1-83880-454-1

Non-tubal Ectopic Pregnancy

Published in London, UK © 2020 IntechOpen © curtoicurto / iStock


**1. Introduction**

Type 1 diabetes (T1DM) is a complex autoimmune disorder resulting from the destruction of pancreatic insulin producing β-cells due to the loss of self-tolerance. There has been a rapid increase in disease incidence worldwide and T1DM is increasingly being diagnosed in even younger individuals and in those from diverse cultural backgrounds. Insulin replacement therapy remains the only viable option for individuals with T1DM. Ensuring any new therapies are meeting a very high safety bar and are superior to insulin replacement therapy remains a challenge. Indeed, treatment with global immunosuppression, while somewhat effective, has significant side effects and is not a practical solution to prevent T1DM which commonly develops in early life. Also, despite insulin's excellent safety profile, management of insulin replacement therapy involves multiple daily injections, pumps and continuous blood glucose monitoring or closed loop systems which are costly and complex to maintain, particularly in younger children. These therapies also bring increased risk for life-threatening high (hyperglycemia) or low (hypoglycemia) blood glucose concentrations as well as increased risk for chronic complications and shortened lifespan. Despite significant progress made in the development of therapeutics for T1DM and some remarkable results in the pre-clinical models, a successful translation into clinic is yet to occur. Due to the T1DM's multifactorial nature, the necessity to find links between genetic predisposition, immune system abnormalities and environmental triggers is becoming increasingly apparent. Therefore, exploring relationships between these contributing factors is necessary for better understanding of the disease progression and for design of the best therapeutic approaches. More recently, multiple studies have focused on the complex biology and involvement of the receptor for advanced glycation end products (RAGE) and its ligands in inflammation, autoimmunity, diabetes complications, apoptosis, and endoplasmic reticulum stress including the exploration of various ways to alleviate these. This presents an exciting new avenue for the development of targeted RAGE-related therapeutics and their translational potential from pre-clinical models to humans.


Laparoscopic Liver Resection for Hepatocellular Carcinoma

*Melina Vlami, Nikolaos Arkadopoulos and Ioannis Hatzaras*

### **Abstract**

Hepatocellular carcinoma (HCC), remains one of the most common causes of cancer-related death globally. HCC typically arises in the setting of chronic liver disease and cirrhosis and as such, treatment must be balanced between the biology of the tumor, underlying liver function and performance status of the patient. Hepatic resection is the procedure of choice in patients with high-performance status who harbor a solitary mass (regardless of size). Before the first laparoscopic hepatectomy (LH) was described as early as 1991, open hepatectomy (OH) was the only choice for surgical treatment of liver tumors. LH indications were initially based solely on tumor location, size, and type and was only used for partial resection of the anterolateral segments. Since then, LH has been shown to share the benefits of other laparoscopic procedures, such as earlier recovery and discharge, and reduced postoperative pain; these are obtained with no differences in oncologic outcomes compared to open resection. Specific to liver resection, LH can limit the volume of intraoperative blood loss, shorten portal clamp time and decrease overall and liver-specific complications. This chapter will offer an overview of standard steps are in pursuing laparoscopic liver resection, be it for a minor segmentectomy or a lobectomy.

**Keywords:** hepatocellular carcinoma, resection, laparoscopic, technique

#### **1. Introduction**

Despite advances in medical, surgical and locoregional therapies, hepatocellular carcinoma (HCC), the most common primary liver cancer, remains one of the most common causes of cancer-related death globally. Hepatocellular carcinoma is the fifth most common frequently occurring cancer in men, the ninth in women and is the second leading cause of death from cancer worldwide. It is estimated that by 2025 more than 1 million individuals will be affected by liver cancer annually.

HCC typically arises in the setting of chronic liver disease and cirrhosis. In fact, the rate of disease occurrence depends upon the complex interplay between the host, disease and environmental factors. This type of liver cancer contributes to up to 40% of all patient deaths in cirrhosis, making it the single most common cause of death in this patient population. The most prominent and well researched risk factors for HCC are Hepatitis B and C infections, accounting for 50% of all cases. Furthermore, there is a clear geographical distribution in the epidemiology of hepatocellular carcinoma, with the highest incidence seen in developing countries with high rates of chronic hepatitis B and aflatoxin exposure. In contrast the lowest incidence rates are seen in some European countries that also have a lower incidence of the before mentioned risk factors. Interestingly, increasing Hepatitis B vaccination, effective Hepatitis C treatment, reducing levels of aflatoxin exposure are now shifting the global epidemiology of HCC. Metabolic disorders, including Non-Alcoholic Steatohepatitis (NASH) and diabetes mellitus, along with obesity and insulin resistance, are now emerging as direct causative factors of HCC, particularly in the West. These evolving patterns of demographic and epidemiologic characteristics bear interesting implications in the diagnosis and management of patients with HCC [1–4].

#### **2. Management**

Cirrhosis patients should be followed within surveillance programs, that aim for early detection of suspicious nodules and effective treatment. Diagnosis of HCC is achieved with imaging and corroborated with an increased tumor marker alpha-fetoprotein blood (AFP) testing. Percutaneous biopsy is seldomly required for diagnosis.

HCC treatment in the setting of liver cirrhosis must be balanced between the biology of the tumor, and host characteristics such as the underlying liver function, presence or not of portal hypertension and ECOG status of the patient. When evaluating a patient for resection, the functional liver remnant must be carefully assessed and its adequate vascular inflow and outflow ascertained, along with biliary drainage. In the event of marginal functional liver remnant, portal vein embolization should be entertained to decrease the possibility of post-operative liver failure.

The most common staging systems for HCC include the Barcelona Clinic Liver Cancer (BCLC), Cancer of the Liver Italian Program (CLIP), and pathologic tumornode-metastasis (pTNM). In clinical practice, there is no ideal system that can be applicable to every patient in predicting survival [5].

#### **3. The BCLC system**

The Barcelona Clinic Liver Cancer (BCLC) staging system is widely used since its inception and remains the most validated and reliable system for prognostication, due to its treatment recommendations based on stage and its ability to offer predictions on patient survival. The BCLC staging system uses variables addressing tumor stage, liver functional status, physical status and cancer-related symptoms. Subsequently, the BCLC staging system can link the stages described with a treatment algorithm.

The initial authors of the BCLC staging system created a position of safety algorithm that proposes:

The combination of tumor specific staging criteria along with host specific information regarding severity of cirrhosis and symptoms have gained the BCLC *Laparoscopic Liver Resection for Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.102981*

staging system wide adoption by clinicians around the world. Criticisms of the BCLC staging system focus on the outdated studies the guidelines were based on and the available surgical and intensive care techniques that were available at the time these were first reported.

In fact, using modern approaches to hepatectomy and enhanced postoperative care, several authors were able to demonstrate improved perioperative outcomes and long-term survival for well selected BCLC B and in some cases BCLC C patients managed operatively. These successes point to a trend in pushing the limits of the original more conservative guidelines, thereby offering a better survival to those patients deemed to be good candidates for resection. This endeavor however has to be taken cautiously, and patients that offered resection outside class A should be managed at high volume centers and at minimum be discussed at a multispecialty tumor board. With more and more BCLC staging system patients being considered for hepatectomy, the BCLC system should be revised to reflect modern liver surgery safety standards, and BCLC stages B should not be considered as absolute contraindications to surgery [6–10].

#### **3.1 Tumor-node-metastasis staging system**

According to this system, the most important prognostic factors is the extent or vascular invasion (T1 without, T2 with) within the tumor. Another important prognosticator accounted for in the T portion of the TNM staging system is number of tumors (T3) and direct invasion of other organs (T4). Lymph nodes are only seldomly affected with a histologic diagnosis of HCC, therefore only rarely we observe a N1 status on these patients. Naturally, metastatic disease is denoted as M1 [11].

Although the BCLC staging system has been found to be applicable for all stages of HCC limitations of all of the other systems have been identified. For example, the AJCC (TNM) staging system has limited usefulness since a large portion of HCC patients do not undergo surgery. The most comprehensive comparison between HCC prognostic scores has recently been published by Marrero et al., who analyzed a population homogeneously including all HCC disease strata and drew a retrospective comparison between seven HCC staging systems on a prospectively enrolled cohort: the BCLC system proved to offer the best prognostic score [12].

#### **4. Liver function assessment**

An initial assessment of hepatic function involves liver function testing including measurement of serum levels of bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), measurement of prothrombin time (PT) expressed as international normalized ratio (INR), albumin, and platelet count (surrogate for portal hypertension). Other recommended tests include complete blood count (CBC), blood urea nitrogen (BUN), and creatinine to assess kidney function; creatinine is also an established prognostic marker in patients with liver disease. Further assessment of hepatic functional reserve prior to hepatic resection in patients with cirrhosis may be performed with different tools such as US and MRI elastography (which may provide and quantify the degree of cirrhosis-related fibrosis), and seldomly non-focal liver biopsy, and transjugular liver biopsy with pressure measurements.

The Child-Pugh classification has been traditionally used for the assessment of hepatic functional reserve in patients with cirrhosis. The Child-Pugh score incorporates laboratory measurements (i.e., serum albumin, bilirubin, PT) as well as more subjective clinical assessments of encephalopathy and ascites. It provides a general

estimate of the liver function by classifying patients as having compensated (class A) or decompensated (classes B and C) cirrhosis. Advantages of the Child-Pugh score include ease of performance and the inclusion of non-laboratory, clinical parameters.

An important additional assessment of liver function not included in the Child-Pugh score is an evaluation of signs of clinically significant portal hypertension (i.e., esophagogastric varices, splenomegaly, splenorenal shunts and recanalization of the umbilical vein, thrombocytopenia). Evidence of portal hypertension may be evident on axial imaginsg (CT/MRI). Esophageal varices may be evaluated using esophagogastroduodenoscopy (EGD) or contrastenhanced cross – sectional imaging.

The Model for End-Stage Liver Disease (MELD) is another system for the evaluation of hepatic reserve. MELD is a mathematical model based on regression analysis which employees a numerical scale ranging from 6 (best) to 40 (worst) for individuals 12 years or older. It is derived using three laboratory values (serum bilirubin, creatinine, and INR) and was originally devised to provide an assessment of mortality for patients undergoing transjugular intrahepatic portosystemic shunts (TIPS), but has been therefore incorporated as an algorithm of gauging suitability for liver transplants [13].

Which HCC patient is a candidate for resection?

Patients being considered for resection must have a high-performance status and be medically fit for what is a major operation. In general hepatic resection is the procedure of choice as a potentially curative option in patients with good liver function (generally Child-Pugh Class A without – or with mild – portal hypertension), who harbor a solitary mass (regardless of size) albeit, without major vascular invasion. In addition, the future liver remnant should be measured at minimum 20% in patients without cirrhosis and at least 40% with Child-Pugh Class A cirrhosis. Lastly, the future liver remnant should be projected to have adequate vascular and biliary inflow/outflow. Hepatic resection is controversial in patients with limited but multifocal disease and in those where tumors are seen to invade major vessels [13].

#### **5. Partial hepatectomy**

Surgical removal of a portion of a patient's liver (partial hepatectomy) is beneficial in removing the tumor that it harbors and thereby limiting its growth and spread to other organs. Partial hepatectomy for well-selected patients with HCC can nowadays be performed with low operative morbidity (<25%) and mortality (≤2–5%). Results of large retrospective studies have shown 5-year survival rates for patients with preserved liver function and early-stage HCC of approximately 70%.

Since liver resection for patients with HCC includes removal of functional liver parenchyma in the setting of underlying liver disease, careful patient selection, based on patient characteristics as well as characteristics of the liver and the tumor(s), is essential. Beyond functional liver remnant volume and adequacy of vascular inflow & outflow, technical considerations related to tumor and liver anatomy, must be taken into account before a patient is determined to have potentially resectable disease.

Resection is recommended only in the setting of preserved liver function. The Child-Pugh score provides an estimate of liver function, although it has been suggested that it is more useful as a tool to rule out patients for liver resection (i.e., serving as a means to identify patients with substantially decompensated liver disease). An evaluation of the presence of significant portal hypertension is also an important part of the surgical assessment.