*Edited by Kerry Thoirs*

Medical sonography is a medical imaging modality used across many medical disciplines. Its use is growing, probably due to its relative low cost and easy accessibility. There are now many high quality ultrasound imaging systems available that are easily transportable, making it a diagnostic tool amenable for bedside and office scanning. This book includes applications of sonography that can be used across a number of medical disciplines including radiology, thoracic medicine, urology, rheumatology, obstetrics and fetal medicine and neurology. The book revisits established applications in medical sonography such as biliary, testicular and breast sonography and sonography in early pregnancy, and also outlines some interesting new and advanced applications of sonography.

ISBN 978-953-307-947-9

Sonography

Photo by kalus / iStock


**3. Rehabilitation of erectile function after prostatectomy**

Climacturia, an orgasmic dysfunction, is one of the affected aspects of the sexual quality of life of prostatectomized patients. Before it manifests, it has erectile

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**Table 2.**

*Post-prostatectomy sexual disorders.*

*The Pelvic Health Physical Therapy and the Prostatectomy*

lence of orgasm difficulty was 45% (**Table 2**) [19].

within its limitations. This is the beginning of sexual rehab [20].

these frames are taken into account for the treatment [21].

nocturnal erections did that no longer occur [22].

treatment time is over 6 months.

dysfunction (ED), highly prevalent in the second postoperative month, reaching around 90%. Complaints about the quality of erection remain frequent even after 12 months postoperatively in around 75% of patients. At the same time, the preva-

When talking about the post-prostatectomy sexual quality of life, before the complaint of climacturia, which may be presumed that there is penetration, the patient usually presents with a severe erectile dysfunction. The impact on sexual life may be even greater when ED is associated with urinary loss, requiring a much more integral approach to the case, since the patient begins to have an affective distancing from the partner and consequently sexual avoidance. The sexual behavioral approach (**Figure 5**) is necessary in order to keep the patient stimulated sexually,

Once the behavioral framework is handled, the erectile rehabilitation comes to the scene. Because it is a multifactor ED, which begins with neuropraxia of the cavernous nerve, which promotes pro-apoptotic and pro-fibrotic factors in the penile tissue, thus evolving into a veno-occlusive dysfunction, resources that act in

Penile vacuum therapy (**Figure 6**) is a physical rehabilitation resource, therefore a physiotherapy, which aims to promote, through a negative pressure, daily erections with the protective function of the erectile tissue, in the same way that

By getting into the place of nocturnal erections, which were daily, penile vacuotherapy also needs to be performed daily. In the Engel [22] and Raina and collaborators [23] studies, the mean time of application of the therapeutic vacuum is 10 minutes. However it is known that in 5 minutes of use, the arterial blood is equivalent to the venous one, becoming poor in oxygen. It is also known that a rest of 30–60 seconds in the vacuum normalizes penile oxygenation, suggesting that the application should be done intermittently, with rest between one application and another. The therapeutic pressure is between −150 and −200 mmHg [24]. The

Due to the safety of the patient, the prescription and orientation of use should be made by a professional qualified to do so, thus avoiding possible intercurrences during the application, such as penile pain, edema, and hematoma. The patient is taught to perform the penile vacuotherapy, recognizing the perceptions and sensations, regarding the sensitivity, size, and color of the penis, being performed by the

Sound therapy has been used in the treatment of ED. The main treatment currently offered is extracorporeal shock wave therapy. But it is common to the treatment by sound waves; this includes the therapeutic ultrasound, to cause mechanical stress and microtrauma in the place of its application. This vascular stress and microtrauma, which generates a shear stress, induce a cascade of biological

**Sexual function 2 Mo 6 Mo 12 Mo** Poor erections 88% 80% 67% Difficulty with orgasm 62% 51% 45% Erections not firm 90% 84% 75% Erections not reliable 83% 75% 60% Poor sexual function 83% 74% 61% Overall sexuality problem 59% 59% 50%

physiotherapist in the office, in order to make home treatment safe.

*DOI: http://dx.doi.org/10.5772/intechopen.86979*

*Male Reproductive Health*

**Figure 3.**

**Figure 4.**

*Parasacral electrodes.*

**156**

the voiding residue present in the bulbar urethra. The treatment consists of the therapeutic exercises of this musculature, aiming to acquire greater muscular force for the expulsion of the urine of the urethra, after the urination. The urethral milking maneuver and bulbar massage may also be associated, but the effectiveness of

The loss of urine during orgasm, climacturia, is not uncommon in these patients. The inevitable surgical damages generated in the bladder neck, as well as the event in the sympathetic fibers, responsible for contraction of the bladder neck and relaxation of the external sphincter during the ejaculation expulsion phase, are one of the suggested mechanisms for climacturia [18]. And the proposed physiothera-

Climacturia, an orgasmic dysfunction, is one of the affected aspects of the sexual quality of life of prostatectomized patients. Before it manifests, it has erectile

bulbospongiosus muscle contraction is greater [5].

**3. Rehabilitation of erectile function after prostatectomy**

peutic treatment is PFMT.

*Transcutaneous tibial nerve stimulation.*

dysfunction (ED), highly prevalent in the second postoperative month, reaching around 90%. Complaints about the quality of erection remain frequent even after 12 months postoperatively in around 75% of patients. At the same time, the prevalence of orgasm difficulty was 45% (**Table 2**) [19].

When talking about the post-prostatectomy sexual quality of life, before the complaint of climacturia, which may be presumed that there is penetration, the patient usually presents with a severe erectile dysfunction. The impact on sexual life may be even greater when ED is associated with urinary loss, requiring a much more integral approach to the case, since the patient begins to have an affective distancing from the partner and consequently sexual avoidance. The sexual behavioral approach (**Figure 5**) is necessary in order to keep the patient stimulated sexually, within its limitations.

Once the behavioral framework is handled, the erectile rehabilitation comes to the scene. Because it is a multifactor ED, which begins with neuropraxia of the cavernous nerve, which promotes pro-apoptotic and pro-fibrotic factors in the penile tissue, thus evolving into a veno-occlusive dysfunction, resources that act in these frames are taken into account for the treatment [21].

Penile vacuum therapy (**Figure 6**) is a physical rehabilitation resource, therefore a physiotherapy, which aims to promote, through a negative pressure, daily erections with the protective function of the erectile tissue, in the same way that nocturnal erections did that no longer occur [22].

By getting into the place of nocturnal erections, which were daily, penile vacuotherapy also needs to be performed daily. In the Engel [22] and Raina and collaborators [23] studies, the mean time of application of the therapeutic vacuum is 10 minutes. However it is known that in 5 minutes of use, the arterial blood is equivalent to the venous one, becoming poor in oxygen. It is also known that a rest of 30–60 seconds in the vacuum normalizes penile oxygenation, suggesting that the application should be done intermittently, with rest between one application and another. The therapeutic pressure is between −150 and −200 mmHg [24]. The treatment time is over 6 months.

Due to the safety of the patient, the prescription and orientation of use should be made by a professional qualified to do so, thus avoiding possible intercurrences during the application, such as penile pain, edema, and hematoma. The patient is taught to perform the penile vacuotherapy, recognizing the perceptions and sensations, regarding the sensitivity, size, and color of the penis, being performed by the physiotherapist in the office, in order to make home treatment safe.

Sound therapy has been used in the treatment of ED. The main treatment currently offered is extracorporeal shock wave therapy. But it is common to the treatment by sound waves; this includes the therapeutic ultrasound, to cause mechanical stress and microtrauma in the place of its application. This vascular stress and microtrauma, which generates a shear stress, induce a cascade of biological

**Table 2.** *Post-prostatectomy sexual disorders.*

### **Figure 5.** *ICI, intracavernosal injection; VED, vacuum erection device.*

### **Figure 6.** *Vacuum erection device.*

reactions that result in the release of angiogenic factors which in turn triggers tissue neovascularization with subsequent improvement of blood supply [25].

Penile electrical stimulation (**Figure 7**) has also been studied for penile rehabilitation. Its action consists in the endothelial action. The current generates release of endothelium-dependent nitric oxide, leading to increased blood supply and vasodilation. Favoring the action of endothelial progenitor cells, which secrete proangiogenic factors, induces neovascularization, repair, and endothelial function. Endothelial progenitor cells release vascular endothelial growth factor, acting as a mediator of angiogenesis [26].

The erectile latency of the post-prostatectomy patient is variable in duration, and we already know the possible damage to penile erectile tissue. But from the perspective of the striated muscles, it is known that the penis muscles who have a veno-occlusive function, the ischiocavernosus and bulbospongiosus muscles (**Figure 8**), which, besides being in the process of sarcopenia inherent to the senility of the patient, may also pass through atrophy due to disuse.

**159**

function [27].

 Conclusion**

and erectile function.

*The Pelvic Health Physical Therapy and the Prostatectomy*

PFMT, focusing on the penile muscles, acts on the veno-occlusion favoring penile stiffness. However, the ischiocavernosus muscle in particular does not have great relevance in the penile intumescence phase. This suggests that to activate them in a functional PFMT, the penis would need to be at least in its maximum turgidity, considering that this patient has great impairment of the postsurgical erectile

*Perineal muscles ((1) ischiocavernosus muscles, (2) bulbocavernosus muscle, (3) transverse perineal muscle,*

Several studies show the predictive factors for the evolution of both urinary incontinence and erectile dysfunction and that to date no conservative treatment has curative action in both situations. However, it is consensual in the rehabilitation process of the post-prostatectomy patient, offering treatments, based on the levels of evidence and recommendation grade, with the objective of improving the quality of life of the patient and whenever possible accelerate the recovery of continence

*DOI: http://dx.doi.org/10.5772/intechopen.86979*

*The Pelvic Health Physical Therapy and the Prostatectomy DOI: http://dx.doi.org/10.5772/intechopen.86979*

**Figure 7.** *Penile electrodes placement.*

*Male Reproductive Health*

**Figure 5.**

**Figure 6.**

*Vacuum erection device.*

*ICI, intracavernosal injection; VED, vacuum erection device.*

**158**

mediator of angiogenesis [26].

reactions that result in the release of angiogenic factors which in turn triggers tissue

The erectile latency of the post-prostatectomy patient is variable in duration, and we already know the possible damage to penile erectile tissue. But from the perspective of the striated muscles, it is known that the penis muscles who have a veno-occlusive function, the ischiocavernosus and bulbospongiosus muscles (**Figure 8**), which, besides being in the process of sarcopenia inherent to the senility

Penile electrical stimulation (**Figure 7**) has also been studied for penile rehabilitation. Its action consists in the endothelial action. The current generates release of endothelium-dependent nitric oxide, leading to increased blood supply and vasodilation. Favoring the action of endothelial progenitor cells, which secrete proangiogenic factors, induces neovascularization, repair, and endothelial function. Endothelial progenitor cells release vascular endothelial growth factor, acting as a

neovascularization with subsequent improvement of blood supply [25].

of the patient, may also pass through atrophy due to disuse.

PFMT, focusing on the penile muscles, acts on the veno-occlusion favoring penile stiffness. However, the ischiocavernosus muscle in particular does not have great relevance in the penile intumescence phase. This suggests that to activate them in a functional PFMT, the penis would need to be at least in its maximum turgidity, considering that this patient has great impairment of the postsurgical erectile function [27].

### **4. Conclusion**

Several studies show the predictive factors for the evolution of both urinary incontinence and erectile dysfunction and that to date no conservative treatment has curative action in both situations. However, it is consensual in the rehabilitation process of the post-prostatectomy patient, offering treatments, based on the levels of evidence and recommendation grade, with the objective of improving the quality of life of the patient and whenever possible accelerate the recovery of continence and erectile function.

*Male Reproductive Health*


**Development and Validation of an HPLC-DAD Method for the Simultaneous Extraction and Quantification of Bisphenol-A, 4-Hydroxybenzoic Acid, 4-Hydroxyacetophenone and Hydroquinone in Bacterial Cultures of** *Lactococcus lactis*

**Angelos T. Rigopoulos 1, Victoria F. Samanidou 2 and Maria Touraki 1,\***

Received: 8 January 2018; Accepted: 31 January 2018; Published: 6 February 2018

**Abstract:** Bisphenol-A, a synthetic organic compound with estrogen mimicking properties, may enter bloodstream through either dermal contact or ingestion. Probiotic bacterial uptake of bisphenol can play a major protective role against its adverse health effects. In this paper, a method for the quantification of BPA in bacterial cells of *L. lactis* and of BPA and its potential metabolites 4-hydroxybenzoic Acid, 4-hydroxyacetophenone and hydroquinone in the culture medium is described. Extraction of BPA from the cells was performed using methanol–H2O/TFA (0.08%) (5:1 *v*/*v*) followed by SPE. Culture medium was centrifuged and filtered through a 0.45 μm syringe filter. Analysis was conducted in a Nucleosil column, using a gradient of A (95:5 *v*/*v* H2O: ACN) and B (5:95 *v*/*v* H2O: ACN, containing TFA, pH 2), with a flow rate of 0.5 mL/min. Calibration curves (0.5–600 μg/mL) were constructed using 4-*n*-Octylphenol as internal standard (1 > R2 > 0.994). Limit of Detection (LOD) and Limit of Quantification (LOQ) values ranged between 0.23 to 4.99 μg/mL and 0.69 to 15.1 μg/mL respectively. A 24 h administration experiment revealed a decline in BPA concentration in the culture media up to 90.27% while the BPA photodegradation levels were low. Our results demonstrate that uptake and possible metabolism of BPA in *L. lactis* cells facilitates its removal.

**Keywords:** HPLC-DAD; bisphenol A; 4-Hydroxybenzoic Acid; 4-Hydroxyacetophenone hydroquinone; *Lactococcus lactis*

#### **1. Introduction**

Bisphenol-A is the organic synthetic compound 4, 4 -Isopropylidenediphenol, which is widely used in thermal paper industry and as a component of synthetic plastic (vinyl-chloride) due to its mechanical and extreme-temperature resistance [1]. BPA, was found to exhibit estrogenic properties, arising from its structural resemblance to the human 17β-estradiol [2], thus it is also referred to as xenoestrogen or endocrine disruptor. The main routes of human exposure to BPA are the consumption of food in BPA containing packages [3] and through dermal contact with BPA rich materials [4]. Following exposure, BPA is mainly metabolized in humans to BPA-glucuronide through the hepatic glucuronide transferase and excreted from the body [5,6]. However, a percentage of BPA, referred to as "free" or "active" BPA, remains in the blood circulation for up to a week and can interfere with endogenous biological processes [4]. Due to its estrogenic and genotoxic effects and its wide

occurrence, BPA presents a risk to humans and animals and hence it is the object of great environmental concern [7–9].

Although part of bisphenol is degraded via photo-oxidation [10–12], monitoring of its levels is deemed necessary in various environmental sources [13]. To this end, biodegradation has been proposed as an advanced technique for BPA elimination [13–15], since a variety of microorganisms have been identified to decompose BPA [15,16]. Probiotics are bacteria beneficial to the host, which have been recognized as safe for human consumption and are used in the production of food products [17,18]. Recently they have been reported to be ideal against heavy metal toxicity since they bind and sequester metals [19]. Moreover, the probiotic bacteria *Bifidobacterium breve* and *Lactobacillus casei* when administered to rats showed a prophylactic effect against the detrimental effects of BPA, by reducing its intestinal absorption and facilitating its excretion, [18]. Likewise, the ability of *Bacillus* strains [20–22], certain *Lactococcus* strains [23] and *Shingomonas paucimobilis* [24] to degrade bisphenol has been documented. Recently a simplified method for the extraction of bisphenol from bacterial culture suspensions has been reported [25]. Moreover, the compounds 4-hydroxy acetophenone (HAP), 4-hydroxy benzoic acid (HBA) and Hydroquinone (HQ) have been proposed as major bacterial metabolites of BPA bacterial degradation pathways [14,26,27]. However, all these studies employed the determination of bisphenol in the cell free culture supernatant and not in the bacterial cells even though they examine the kinetics of bisphenol.

Analytical methods for the determination of bisphenol in various food samples [3], as well as in biological fluids and environmental samples [28], have been extensively reviewed. The determination of BPA has also been investigated in more complex matrices such as rat tissues [29], human breast milk [30] resin-based dental restorative materials [31] and saliva [32]. However, to the best of our knowledge, no method has been reported for the determination of bisphenol in bacterial cells.

In the present study, the development and validation of an analytical method for the quantification of BPA in bacterial cells is reported for the first time using the widespread probiotic bacterium *Lactococcus lactis* (ATC 11454). The method is also validated in bacterial culture supernatants for the simultaneous determination of the bacterial metabolites reported to be produced during the bacterial major metabolic pathway [14], namely 4-hydroxy-acetophenone (HAP), 4-hydroxy benzoic acid (HBA) and Hydroquinone (HQ). The application of this method demonstrates the uptake of bisphenol by *Lactococcus lactis* cells and allows its quantification.

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

#### *2.1. Chemicals and Reagents*

Acetonitrile was purchased from VWR Chemicals (Paris, France). Methanol and Absolute ethanol were purchased from Fisher Scientific (Loughborough, UK). Trifluoroacetic acid (TFA), KH2PO4 and FeCl3 were purchased from AppliChem GmbH (Darmstadt, Germany), while NaCl, CaCl2, (NH4)2SO4 and MgSO4 were purchased from Merck KGaA (Darmstadt, Germany). All the chemicals used were HPLC Grade, unless specified otherwise.

Bisphenol-A (BPA) and the internal standard 4-*n*-Octylphenol (OP) were purchased from Alfa Aesar (Karlsruhe, Germany). 4-Hydroxyacetophenone (HAP), 4-Hydroxybenzoic Acid (HBA) and Hydroquinone (HQ) were purchased from Acros Organics (Ceel, Belgium).

The bacterial strain from which the liquid culture supernatant and the pellet originated and used for the spike-recovery studies, was *Lactococcus lactis* (ATCC 11454). The liquid culture medium that was used for the growth of *L. lactis* was Brain Heart Infusion broth (BHI broth) and was purchased from AppliChem GmbH (Darmstadt, Germany). An artificial bacterial culture medium was prepared, namely Basal Salts Medium (BSM), composed of KH2PO4 and (NH4)2SO4 1 g/L each, MgSO4 0.1 g/L, NaCl and CaCl2 0.05 g/L each and FeCl3 0.01 g/L. BPA was used as a sole carbon source in BSM in varying concentrations for the feeding experiments. For the retrieval of the bacterial culture supernatant and pellet 100 μL of an *L. lactis* glycerol stock solution were inoculated in a 250 mL

flash with BHI broth. Following a 24-h incubation at 28 ◦C under constant stirring, the culture was centrifuged at 5000 rpm and the precipitate was transferred to a 250 mL flask containing BSM. At the end of the experiment, the culture was centrifuged, the precipitate was placed on a filtering paper and its weight was recorded following removal of excess water. The recorded weight is referred as 'wet cell weight'. The culture supernatant and the wet cell weight were used for the spike-recovery assays.

Solid Phase Extraction columns, frits and C18 sorbent material, 35–75 U were purchased from Grace Davison Discovery Sciences (Bannockburn, IL, USA). Membrane filters with 0.45 μm diameter pores were purchased from Merck KGaA (Darmstadt, Germany).

#### *2.2. Standard Preparation*

#### 2.2.1. Stock Solution Preparation

Stock solutions 500 μg/mL of BPA, HAP, HBA and HQ were prepared by dissolving 25 mg of the analyte in 50 mL methanol. The stock solution of BPA (50 mg/mL) was prepared by dissolving 2.5 g of BPA in 50 mL ethanol. The internal standard stock solution of 500 μg/mL was prepared by dissolving 25 mg of OP in 50 mL methanol.

#### 2.2.2. Working Standard Solution Preparation

Seven-point and six-point working standard solutions were prepared for analytes at concentrations of 0.5, 10, 50, 100, 200, 400, and 600 μg/mL for BPA, at 0.5, 2, 4, 6, 8 and 10 μg/mL for HAP and HQ and at 1, 2, 4, 6, 8 and 10 μg/mL for HBA and the internal standard 4-*n*-octylphenol (75 μg/mL).

#### *2.3. Sample Preparation*

For the bacterial pellet method development, 0.1 g of wet cell weight *L. lactis* was added in a test tube with 8 mL methanol, 2 mL of double distilled water containing 0.08% (*v*/*v*) TFA and 150 μL internal standard from the 500 μg/mL stock. The concentration of the internal standard in the final 1 mL volume was 75 μg/mL. The test tube was transferred to an ice-filled container and was placed under a homogenizer for 5 min. The homogenate was separated in ten Eppendorf centrifuge tubes and was centrifuged for 10 min in 8000 rpm. The supernatant was collected and mixed with an equal volume of ddH2O containing 0.08% TFA. BPA extraction was performed using Solid Phase Extraction (SPE). An SPE cartridge was prepared using 0.5 g of C18 sorbent in an SPE column. The SPE cartridge was conditioned with 10 mL methanol, 5 mL ddH2O and 5 mL ddH2O containing 0.08% TFA. The sample was loaded on the cartridge and a manual flow of 1 mL/min was employed. The cartridge was, then, washed with 5 mL ddH2O and elution was performed using 4 mL methanol. The eluate was collected and placed under a rotary evaporator in a 37 ◦C water bath, to dryness. Finally the sample was reconstituted with 1 mL acetonitrile and stored in an aluminum foil-covered Eppendorf tube at −20 ◦C. All experiments were conducted in dark or covered tubes to avoid photodegradation of BPA.

For the bacterial supernatant, an 8.5 mL sample of the culture was added in a test tube along with 1.5 mL internal standard from the stock solution. The sample was placed in 10 Eppendorf centrifuge tubes (1 mL per tube), and was centrifuged for 10 min in 8000 rpm. The supernatant was collected and filtered through a 0.45 μm diameter pore filter. The filtrate was stored in an aluminum foil-covered Eppendorf tube at −20 ◦C.

#### *2.4. BPA Administration (Feeding) Assay*

For the feeding assays 6 falcon tubes of 50 mL volume were filled with BHI broth and were inoculated with 100 μL of a *L. lactis* glycerol stock solution. The cultures were incubated for 24 h at 28 ◦C under constant stirring. The cultures were, then, centrifuged and the medium was discarded. A total of 50 mL of fresh BSM was added to each culture along with varying quantities from the BPA stock solution (50 mg/mL) so that the final BPA concentration in each culture would be 50, 100, 150, 200, 400 και 500 μg/mL respectively. The cultures were incubated for another 24 h at 28 ◦C

under constant stirring. The remaining BPA in the cultures was quantified using the method and the calibration curves that were described.

#### *2.5. Photodegradation Assay*

To investigate potential photodegradation of BPA, a falcon tube of 50 mL containing BSM and BPA at a concentration of 50 μg/mL was prepared and was incubated under the same conditions as the feeding assays. BPA was quantified at the beginning and the end of the 24 h incubation period. The presence of photodegradation products was examined in the resulting chromatograms.

#### *2.6. HPLC-DAD Conditions and Methods*

The instrument used consisted of an LC20AD pump and an SPD-20A photodiode array detector (DAD) purchased from Shimadzu (Kyoto, Japan). Separation of BPA was achieved on a Nucleosil 100 C18 column (250 × 4.6 mm, 5 μm) purchased by Macherey-Nagel GmbH & Co. (Duren, Germany) with a binary mobile phase system at room temperature. The injection was performed using a 100 μL Hamilton syringe, with an injection volume of 80 μL. Mobile Phase A consisted of a 0.1% TFA in ddH2O solution and Acetonitrile at a ratio of 95:5, *v*/*v*. Mobile Phase B consisted of a 0.08% TFA in ddH2O solution and Acetonitrile at a ratio of 5:95, *v*/*v*. The gradient started at 100% Mobile Phase A which was brought down to 80% over 1 min, 70% over 4 min, 55% at 15 min, 40% at 17 min, 20% at 20 min and finally to 10% over 2 min and then was kept at this level until the end on the analysis. The flow rate was constant at 0.5 mL/min. BPA was detected at 220 nm with a band width of 2.0 nm. HAP, HBA and HQ were detected at 280 nm with a band width of 2.0 nm. Identification was conducted by matching the peak retention times (*Rt*) with those of the pure standards. Internal standard calibration was used by plotting the peak area ratio of the analyte to the peak area of the internal standard against the concentration of the analyte.

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

#### *3.1. Optimization of Sample Preparation*

The homogenization of the sample was performed to lyse the cells and release the adsorbed BPA for the feeding assay. Initially, ethyl acetate was used instead of methanol and a manual homogenization was attempted with a glass homogenizer, but cell lysis was deemed inadequate. Hence methanol was selected as the extraction solvent. The addition of an equal volume ddH2O containing 0.08% (*v*/*v*) TFA after centrifugation aimed to increase the solubility of BPA due to further dilution.

An elaborate and efficient vortex assisted liquid-liquid microextraction with octanol was used for the trace analysis of bisphenol in water and wastewater samples [33]. However, an attempt to use liquid-liquid extraction with ethyl acetate in the samples of the present study resulted to the formation of an emulsion zone between the two phases, a fact that led to greatly varying percentages of BPA recovery due to the loss of the analyte during the procedure. Thus, solid phase extraction was implemented, since SPE is a common step in the extraction procedures of bisphenol from complex matrices, such as rat tissues [29] or human serum [5] and its benefits in the biomonitoring of bisphenol have been demonstrated [28,33]. In the present study a flow rate of 1 mL/min was selected throughout the SPE procedure, as effective and timely favorable, while higher flow rates did not provide full retention of BPA by the C18 material, especially at high BPA concentrations employed and resulted in lower recovery values. A 37 ◦C water bath was selected to facilitate the evaporation of methanol after SPE. The slightly higher evaporation temperatures of 45 ◦C that have been reported for the analysis of bisphenol and 4-nonylphenol [29] resulted in heat-induced decomposition of the internal standard when used in the present study. Our results for BPA extraction are in accordance with a recently published report on a methanol extraction of bisphenols from bacterial cultures and solid matrices [25] in which, however, SPE was not employed and lower concentrations of BPA were used [25], resulting in recoveries up to 95%. The SPE step used in the present study greatly improved recovery which

amounted to 99.38–100.18% in culture supernatants and to 90.8–102.46% in bacterial cells, in the wide range of concentrations employed (Table 1).

**Table 1.** Analyses of the calibration curves for analytes in the cell culture supernatant and the cell culture precipitate.

In an effort to perform the analysis of BPA in the BHI medium, which is recommended for growth of *L. lactis*, the nutrients in the broth resulted in the presence of peaks in the chromatogram that appeared to interfere with the tested bacterial metabolites peaks. The artificial bacterial medium (BSM) was prepared to tackle the problems that arose in the analysis using the reference BHI medium. Moreover, in this manner BPA was the sole carbon source for the bacterium, a fact that contributes in its biotransformation.

#### *3.2. Method Validation*

The results of the linear regression analysis performed using the "Linear Regression" package of Microsoft Excel 2013 software are presented in Table 1, while detection limits and Retention time variance are presented in Table 2. The coefficient of determination values (R2) for the standard curves in BSM supernatant and *L. lactis* cells were 1 and 0.999 respectively, while for HAP, HBA and HQ in BSM supernatant the values exceeded 0.9994, 0.994 and 0.999 respectively, indicating excellent linearity. The Relative Bias was calculated by computing the percentage of the difference of the mean calculated concentration to the nominal concentration divided by the nominal concentration of the analyte. All results are within the accepted range of ±10%. Intra-day precision was calculated by performing a total of three injections of the nominal concentration on the same day, while inter-day precision was calculated by performing six injections of the nominal concentration over three days. Both were computed as % R.S.D. = (S.D./mean) × 100. None of the values exceeded 6.0% for all analytes, with the exception of the values obtained for the lowest nominal value of 0.5 μg/mL of BPA in bacterial cells and in the medium, which might be due to the fact that this nominal value is below LOD (Table 2). Since an elaborate extraction protocol was required for the bacterial cells, the use of an internal standard was deemed necessary, to compensate for sample losses during sample preparation. The internal standard 4-*n*-octylphenol was selected, due to the fact that it is well resolved and it has a structure similar to bisphenol. Moreover, it is not expected to be present in samples, since only 4-*t*-octyphenol has been reported to be found at very low concentrations in environmental samples and human milk [34,35]. Our results, as presented in Table 1, indicate that the method is highly precise and repeatable. Recovery rates were well within range of 10% error, reaching close to 100% recovery, thus further supporting the precision of this method. The recovery results presented in Table 1 are in good correlation with those reported for rat serum (95%) and higher than those reported for rat testis (78.6%) and liver (84.0%) [29].

The nominal concentrations of 0.5 μg/mL BPA in BSM and cell precipitate were below the calculated LOD values (Table 2) and were thus excluded from the estimation of recovery results described above. The values for the Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated from the calibration curves. The LOD was calculated to 3.3 times the ratio of the Standard Error (intercept) to the Coefficients x, while the LOQ was 10 times this ratio. The retention time means, standard deviations and RSDs were calculated using the data from one chromatogram of each of the nominal concentrations used. The value of an LOD of 4.99 μg/mL estimated in the present study for BPA in bacterial cells at the range of 10–600 μg/mL, is higher than the value of 2.8 ng/mL reported for rat tissues, but over a narrower range of concentrations of 0.15–150 μg BPA/mL [29]. A lower value of LOD was found for BPA in the less complex sample of BSM, amounting to 0.64 μg/mL (Table 2), although the same range of concentrations was employed (Table 1), possibly due to lower matrix complexity. Similar low LOD values were estimated for all possible metabolites tested, as shown in Table 2. Although these compounds have been proposed as possible bisphenol bacterial metabolites, they were identified using MS and NMR analysis of the HPLC isolated fractions [27]. In fact, in the studies on the determination of bisphenol degradation using HPLC-DAD [20,23], no validation was included. The method presented in this work allows the simultaneous quantification of bisphenol A and three of its major bacterial metabolites.

#### *3.3. Feeding Results*

A typical chromatogram of HQ, HBA and HAP and BPA is presented in Figure 1. Analyte peaks of HQ, HBA, HAP, BPA and internal standard OP are shown with a retention time (*Rt*) of 10.671, 12.728, 15.579, 26.241 and 40.771 min respectively. The peak that appears at 25.6 (Figure 1) might be attributed to a BPA photodegradation product, since it was also identified during the photodegradation experiment and is presented in Figure 2. Photodegradation of BPA via photooxidation has been confirmed [10,12], and the products of this process presented higher toxicity than the parent compound [11]. Since the feeding experiments were conducted under normal light, it was deemed

appropriate to test if any BPA loss was due to photodegradation. In these experiments the initial BPA concentration was calculated at 48.65 μg/mL and the concentration following the 24 h incubation amounted to 44.18 μg/mL. Hence, the BPA loss due to photodegradation was 9.19%. These values are in accordance with those reported for bisphenol A degradation by UV irradiation in the absence of hydrogen peroxide [36].

**Figure 1.** A chromatogram containing all analytes at a concentration of 10 μg/mL and internal standard at 75 μg/mL in fresh BSM without *L. lactis* inoculation. The first two peaks with *Rt* of 5.7 min and 7.5 min are background noise from acetonitrile solvent and TFA.

**Figure 2.** Chromatogram displaying BPA and its identified photodegradation product with a *Rt* of 26.515 min and 25.610 min respectively. The compounds were detected at a wavelength of 300 nm.

Typical chromatograms of the feeding assays are presented in Figure 3 (bacterial cells) and Figure 4 (bacterial culture medium). The relative blank chromatograms resulting from bacterial cells and from their culture medium any additions are shown in the Supplemental section. The peaks appearing at 10.2 min in both chromatograms does not represent one of the metabolites described in this paper, as confirmed by comparing the absorbance spectra of the peak to those of the pure metabolites analyzed.

**Figure 3.** Chromatogram of cell precipitate originating from the administration of 150 μg/mL BPA in the bacterial cells. The peaks at 26.528 min and 40.822 min correspond to BPA and the internal standard.

**Figure 4.** Chromatogram of culture supernatant originating from the administration of 150 μg/mL BPA in the bacterial cells. The peaks at 26.672 min and 41.604 min correspond to BPA and the internal standard.

Two major pathways have been proposed for the metabolism of bisphenol A by bacteria, the first leading to mineralization, with HBA and HAP as intermediates and the second to hydroxylation [6,16]. However, it has been pointed out that the metabolic pathways might be strain specific [14], since certain metabolites produced by one strain are not detected when another bacterial strain is used. In a similar manner, the analytes HQ, HBA and HAP were not detected in the chromatograms of the feeding assays. Absence of bacterial metabolites has also been reported for *Bacillus pumilus* [20] and certain *Lactococcus* strains [23]. In all these reports, including the present work, the presence of metabolites was examined at 24 h of culture. However, when various *Bacilli* and *Sphingomonas* strains were tested for bisphenol degrading capacity, metabolites were detected for three of them and only from 8–12 h, in a declining manner [37]. These results indicate that the time period of 24 h used in the present paper might be too long, since the metabolites under investigation might have formed and then further transformed. On the other hand, the bacterial metabolites examined in the present paper were

reported to be produced by *Bacillus* sp., yet only after 3 days of incubation in the presence of 20 mM BPA [21]. Moreover, the photodegradation of BPA metabolites has been reported for hydroxybenzoic acid as well as for hydroquinone [38,39]. It is evident that more experiments are needed in the course of bacterial incubation, since metabolism is an ongoing process and each strain presents particular metabolic characteristics.

A depletion of up to 58% in total BPA (μg) was observed in our experiments, in all bacterial cultures as shown in Table 3. Complete degradation of BPA has been reported following a 48 h incubation period [37] or after 9 days of incubation [21]. In our experiments BPA was reduced by 90.27%, 64.16%, 58.60%, 67.74%, 79.83% and 85.05% in the cultures corresponding to initial BPA concentration of 50, 100, 150, 200, 400 and 500 μg/mL. Our results suggest that the initial BPA concentration added has a profound effect on its degradation. Many bacterial strains that can degrade BPA have been reported, and this degrading ability varies among the species [14]. It should also be noted that different initial concentrations of BPA are used in the reports employing bacteria, ranging from 0.1 to 60 mg/L [22,37,40] and for varying incubation periods. Bacteria can clearly employ a variety of metabolic pathways to exploit pollutants as energy sources, under the prerequisite that the pollutants are bioavailable and at levels that could trigger such mechanisms [16].

**Table 3.** Comparative data of BPA administered (μg) and found in feeding assays.

#### **4. Conclusions**

The developed analytical method provides a reliable, robust and sensitive approach for the simultaneous detection and quantification of bisphenol A and its potential metabolites in the complex matrix sample of bacterial cells. Good chromatography separation was achieved on a Nucleosil 100 C18 column with a longer run time, which however allows the detection and separation of metabolites. LC-DAD combined with solid phase extraction and the use of 4-*n*-octylphenol as the internal standard, is capable of operating with a detection limit of 4.99 μg/mL for bisphenol and lower limits for metabolites in bacterial cells. The method was also successfully applied in the analysis of bacterial culture medium with a detection limit of 0.64 μg/mL for bisphenol. The feeding experiments revealed that *L. lactis* is capable to uptake of bisphenol and contribute to its elimination from the culture medium. Further experiments are needed for the detection and quantification of metabolites as a function of culture time and administrated bisphenol concentration.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2297-8739/5/1/12/s1, Figure S1: Blank chromatograms of *L. lactis* cells (A) and cell free bacterial medium (B).

**Acknowledgments:** No additional funding was received in support of the present research work.

**Author Contributions:** M.T. conceived the experiments; M.T. and V.F.S. designed the experiments; A.T.R. performed the experiments; A.T.R. analyzed the data, A.T.R., M.T. and V.F.S. wrote the paper.

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

#### **References**

© 2018 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*


Preface

Matrices have applications in a huge number of scientific fields. In physics, they are used to study physical phenomena, such as the motion of rigid bodies. In computer graphics, they are used to manipulate 3D models and project them onto a 2D screen. In probability theory and statistics, stochastic matrices are used to describe sets of probabilities. Matrices calculus may be used in economics to describe systems of economic relationships. A main part of numerical analysis focuses on the development of efficient algorithms for matrix computations, a subject that is centuries old and is today an expanding area of research. Matrix decomposition meth‐ ods simplify computations, both theoretically and practically. Algorithms that are tailored to particular matrix structures, such as sparse matrices and near-diagonal matrices, expedite computations in the finite element method and other computations. Infinite matrices are present in planetary theory and atomic theory. They are the matrices representing the deriva‐ tive operators, which act on the Taylor series of a function. This new book reviews current research, including applications of matrices and spaces, as well as other characteristics.

The book is divided into two sections. The first section (Chapters 1 and 2) discusses the ap‐ plication of matrices that has become an area of academic research and of great importance in many scientific fields. In Chapter 1, within the framework of the theory of row/column determinants, the determinantal representations (analogs of Cramer's rule) of a partial solu‐ tion to the system of two-sided quaternion matrix equations, *A*1XB1=*C*1, *A*2XB2=*C*2 are ana‐ lyzed. It also gives Cramer's rules for its special cases with one-sided equations and considers the two systems with the first equation *A*1*X*=*C*1 and XB1=*C*1, respectively, and with an unchanging second equation. Cramer's rules for special cases when two equations are one sided, to wit, the system of equations *A*1*X*=*C*1, XB2=*C*2 and the system of the equations *A*1*X*=*C*1, *A*2*X*=*C*2, are studied as well. Chapter 2 introduces and studies a matrix that has the exponential function as one of its eigenvectors and realizes that this matrix represents finite difference derivation of vectors on a partition. This matrix leads to new expressions for finite difference derivatives, which are exact for the exponential function. A number of properties of this matrix, induced derivatives, and its inverse are also found. In addition, the expres‐ sion for the derivative of a product, a ratio, and the inverse of vectors plus the equivalent of the summation by parts theorem of continuous functions are also described. This matrix could be of interest to discrete quantum mechanics theory.

The second section (Chapters 3 to 5) comprises three chapters discussing spaces and linear systems. In Chapter 3, mixing problems are considered since they always lead to linear ODE systems, and the corresponding associated matrices have different structures that deserve to be studied in depth. This structure depends on whether there is recirculation of fluids and if the system is open or closed, among other characteristics such as the number of tanks and their internal connections. Several statements regarding matrix eigenvalues are analyzed for different structures and also some questions and conjectures are posed. Finally, qualitative remarks about differential equation system solutions and their stability or asymptotical sta‐ bility are included. In Chapter 4, special compound ´ magic squares are considered and a dimensional subspace of the nullspace of the ´ squares is determined. All vectors in the subspaces possess the property that the sum of all entries of each vector equals zero. In Chapter 5, a new type of regular matrix generated by Fibonacci numbers is introduced and we shall investigate its various topological properties. The concept of mathematical regulari‐ ty in terms of Fibonacci numbers and phyllotaxy have been discussed.

> **Prof. Dr. Hassan A. Yasser** Thi-Qar University Science College Physics Department Iraq

**Section 1**

**Applications of Matrices**


**5. The context**

**4. The research design**

**Inclusion …**

EI1 is a process

EI2 is aligned to a vision

EI3 requires promotion of locally negotiated responses

EI5 involves increased participation to maximise outcomes

EI7 needs all students to understand and value diversity

EI8 needs adults to value difference as a resource

**Table 1.** Existing indicators (*EIs*) of inclusive school practice.

EI6 needs a learning community that questions and challenges current practice

E19 requires practices that engender feelings of safety and belonging, and E20 requires *supporting and working with students, staff, parents and communities*

EI4 involves changes and modifications

**Figure 2.** Conceptual framework of research design.

tors (EIs) of inclusive school culture (**Table 1**).

40 New Pedagogical Challenges in the 21st Century - Contributions of Research in Education

Maxwell's [29] interactive model of research design was taken into account and consideration given to the reciprocal relationship between the research questions, goals, methods, validity and conceptual framework. **Figure 2**, illustrates the conceptual framework used in this study which draws together Schein's Theory of Organisational Culture [23] with the existing indica-

> The two Primary School case study sites are referred to as PS1 and PS2. Both schools had been identified by regional office staff as having quality outcomes and inclusive environments, and each had a strong sense of identity (**Figure 3**).

> They had similar numbers of students but different demographics. PS1 had above average levels of students with special needs (12% of total enrolment). Primary School 2 (PS2) had an average sized special needs program (9%) but around 50% of students came from backgrounds where English was an additional language or dialect. Both schools had demonstrated strong gains identified by the National Assessment Program.

> Both schools had undertaken a school capacity building process, in partnership with LRI team members, over anumber of years. Each school community had collaboratively developed a strong school vision, well understood and explicitly taught values, and a schoolwide pedagogical

**Figure 3.** PS1's and PS2's vision and SWP.

(SWP) framework [30, 31]. Each of these artefacts emerged as a result of the school engaging with the Innovative Designs for Enhancing Achievements in Schools (IDEAS) Project [22]. Interestingly both school communities adopted the image of a tree as a metaphor fortheir vision, even though each vision was distinctly different. Each vision and SWP is captured in **Figure 3**.

• PHASE 2: Themes linked to stakeholder factors (SF) and existing indicators (EI)

ing staff, parents, students, leaders and system interactions.

*pedagogies for context*

and respond to any concerns raised.

*ily contexts*

• PHASE 3: Theme differences identified, principles articulated and a model created

**PHASE 1**: The Stakeholder focused phase acknowledged that a school is a community of individuals working together. Factors relating to the key stakeholders were summarised according to staff, parent, student, and leadership with management related factors. Although there were context specific nuances, a number of inclusive strategies appeared to be in operation across both schools. These operated as integrated pieces of a whole school approach embrac-

Inclusive Schoolwide Pedagogical Principles: Cultural Indicators in Action

http://dx.doi.org/10.5772/intechopen.70358

43

In both contexts, it had been reported that the principal had placed priority on developing staff capacity to support students with a particular need, and then followed up with additional sessions as required. Collaboration across the school ensured that planning was comprehensive and manageable by all parties inclusive of special support staff, teachers, teacher aides, students, parents and the leadership team. Professional development and time for professional conversations were a priority. Multiple opportunities were provided within the classroom for students to build metacognitive skills through mental processing tasks and 'talk alouds', so students could learn how to express their learning and emotional needs. This was particularly important for PS2 due to their high numbers of students with English as another language.

*Staff focused factors: targeted professional development; professional collaboration; intentional development of metacognitive skills; clear consciousness of varied needs; shared understanding of successful*

Teachers indicated that considerable time was spent by the principal and other members of the leadership team, such as the Head of the Special Education Program, on contacting parents prior to a student's entry into school. Parents confirmed this. PS2 found this challenging as many parents did not speak English but translators were brought into the process. Multiple and varied opportunities were provided for parents to express their concerns and be 'heard'. Sometimes all that was needed was for parents to be reassured that support measures were in place for their child. Where regular consultations with medical practitioners and support personnel were needed, these were arranged and the school assisted parents to understand

*Parent focused factors: prior contact with parents; multiple opportunities for parents to engage; supported interactions with medical or other support services; empathy and understanding of diverse fam-*

Students were encouraged to be leaders of inclusive practice within their classrooms and whilst in the playground. Social skills and values education programs were in place to assist students develop peer relationships and support groups. Student leaders were actively encouraged to be the voice for their peers and were fully engaged in orienting students new to the school and becoming mentors in their initial transition to campus. Teachers were encouraged to provide multiple opportunities within each classroom for students to share their experiences, passions and hobbies in order to find 'triggers' and 'hooks' into learning. At both schools, students were encouraged to build independent learning skills within the classroom. Older students set their own learning goals and planned how to attain these through small achievable steps.


*2.1. Synthesis of PVA Copolymers*

Series of hydrophobically modified PVA copolymers were synthesized by partial acetalization of hydroxyl groups of PVA (average polymerization degree 1600) with acetaldehyde following the procedure described elsewhere [18]. The copolymer composition of obtained PVA–Ac, namely the content of acetal groups, was estimated by using Nuclear Magnetic Resonance (NMR) spectroscopy. **1**H NMR spectra were taken on a Bruker Avance DRX 250 spectrometer (Bruker Corporation, Billerica, MA, USA) in DMSO-d6 as solvent. The extent of hydrophobic modification was evaluated by means of UV-VIS spectroscopy. Transmittance of copolymer aqueous solutions was studied at wavelength of 500 nm at concentration of 5 g/L as a function of temperature. Cloud points (*T*CP) of copolymers solutions were determined from the measured transmittance-vs-temperature curves as the temperature at transmittance level of 50%.

Copolymer solutions of 1 wt.% concentration in mixed water–methanol solvent (20:80 volume ratio) were prepared for thin film deposition process. To obtain hybrid polymer–silica thin films, SiO2 particles were in situ generated in copolymer solutions via the sol-gel method [22,23]. Calculated amount of the precursor tetraethyl orthosilicate (TEOS) was added to the copolymer solution under stirring and mixture was acidified with 1 M HCl to pH 2. The reaction mixture was homogenized by ultra-sonication for 30 min and then TEOS hydrolysis was continued at vigorous stirring on a magnetic stirrer at room temperature for 24 h. The obtained copolymer solution doped with SiO2 particles was used for thin films deposition without further treatment. The condensation of the silica was completed during the annealing of the deposited thin hybrid films.


*4.1. Reagents and Materials*

ZEN, its analogs, and common mycotoxin standards were purchased from Pribolab Pte. Ltd. (Biopolis, Singapore). The BSA and GAM-IgG were supplied by Sigma-Aldrich (St. Louis, MO, USA). The trisodium citrate, HAuCl4, and K2CO3 were bought from Aladdin (Shanghai, China). The NC membranes were from Millipore (Bedford, MA, USA). The absorbent pad, sample pad, polyvinyl chloride (PVC) sheet, and glass-fiber conjugate pad were provided by Jiening Bio. Tech. Co., Ltd. (Shanghai, China). The ZEN antigen (ZEN-BSA) and McAb against ZEN (ZEN-McAb) were supplied by Biosco Biological Tech. Co., Ltd. (Dalian, China). The other reagents were all of analytical grade.

The XYZ3030 dispense platform (Kinbio Tech. Co., Ltd., Shanghai, China) and the HGS201 automatic programmable cutter (Hangzhou Autokun Tech. Co., Ltd., Hangzhou, China) were used in the preparation of the ICA strips. The Tecnai 12 transmission electron microscope (Philips, Eindhoven, Netherlands) was used to scan the diameter and shape of the nanoparticles. The Milli-Q purification system (Millipore, Bedford, MA, USA) was used for preparing the purified water. The TG16-WS high-speed centrifuge was used for centrifuging (Cence Co., Changsha, China). The vortex mixer was provided by North TZ-Biotech Develop. Co., Ltd. (Beijing, China). The results of the ICA for detecting ZEN were confirmed with an Agilent 1200-6460 LC-MS/MS equipped with electrospray ionization (Agilent, Wilmington, DE, USA). The results of the ICA were digitized on an HG-1721 strip reader (Vict Tech. Co., Ltd., Suzhou, China).


**2. Materials and Methods**

The study was carried out in the first half of 2017 in Poland on a population of 500 consumers. It was a pilot study in which 50% of the studied group of respondents were men and 50% were women. The integrity of the study sample was the only parameter of purposeful selection of the study subject matter. Other selection conditions of consumers participating in the study were randomized and comprised of:

Following a demographic analysis of the respondents taking part in the study, regarding their sex, age, education and income, some similarities and relationships were observed between the characteristics. An analysis of concentrations was performed in order to group the respondents

according to uniform or highly similar characteristics [35]. Grouping was intended to focus attention on the groups of consumers participating in the study, which could become representative groups from the point of view of the conclusions drawn from the study. A two-step concentration analysis was applied to divide the study participants into four groups. The Bayesian information criterion (BIC) was used to separate the subgroups, while the distance between the study participants was measured with a credibility logarithm. The two-step concentration analysis facilitated the identification of the following four groups; most homogeneous within a specific group and also most diversified as compared to other groups, based on such variables as sex, age, education, and income:

Table 1 presents the quantitative characteristics of the identified groups.

**Table 1.** Profiles of the four identified groups of respondents.

Source: data developed based on our own study.

A proprietary questionnaire consisting of eight main questions and six identification questions concerning sex, age, education, household size, and income of the respondents (based on the analysis of concentrations, household size and place of residence did not matter from the point of view of respondent grouping) was the study tool. Among the main questions there were six multiple-choice questions and two questions where one needed to assign rank on a 1–6 and 1–5 scale. "1" meant most important, and "5" or "6" least important. The ranked questions concerned those factors most important for consumers when they buy food. They involved such aspects as knowledge of the brands, guarantee of product quality and freshness, low price, best before date, product origin, and ingredients. Moreover, the questions touched upon information that in the consumers' opinions should be placed on food packaging (i.e., information about the producer, the product's energy value, best before date, detailed information about raw materials, and product ingredients).

Collection of empirical materials by means of an interview questionnaire developed by the authors was justified by the specificity of the research and the necessity to reach a wide range of respondents.

A focused group interview (i.e., a focus group), composed of representatives of academic staff and entrepreneurs was involved in the study in order to:

The study used both direct tools (proprietary interviews) and indirect tools (e-mails and Google surveys). The data from the study were collected in an Excel spreadsheet and then summarized and analyzed quantitatively using Statistica (a statistical data-processing software). The following statistical measures were used to analyze the results of the study:

The majority of analyses in the study were based on data in the form of ranks or ordinal variables. Therefore, referring to the characteristics of each statistical measure, when testing hypotheses mentioning different distributions of ranks in different groups, the Kruskal–Wallis test was used. Pearson's Chi-squared test was used for verification of the presence of relationships between the variables according to the hypotheses. The following symbols were used for presenting the study results:


**4. Discussion**

*4.1. Why the Selected Features Are Rational?*

In this study, six sensitive spectral bands (533.5, 659.2, 528.5, 661.7, 968.2, and 523.6 nm) were selected by SPA method to reduce the hyperspectral dimensions, in agreement with previous studies revealing sensitive spectral regions to wheat PM [30]. Among these spectral bands, three wavelengths at around 550 nm are sensitive to photosynthetic pigments and have great potential for early disease monitoring, in accordance with the findings of Maimaitiyiming [47]. When a pathogen infects wheat, the physiological parameters respond accordingly, such as decreased chlorophyll content, which affects the spectral reflectance in the visible light region. As shown in Figure 10, the chlorophyll content of healthy and diseased leaves on different days after infection were analyzed by analysis of variance (*p* < 0.05). At the jointing stage, the chlorophyll content of healthy and diseased leaves differed significantly on day 8 after infection. At the booting stage, the chlorophyll content of healthy and diseased leaves differed significantly on day 6 after infection. Decreased chlorophyll content weakened the ability to absorb light, therefore resulting in higher reflectance in the VIS range.

**Figure 10.** Changes and comparison of chlorophyll content between healthy leaves and diseased leaves infected at jointing stage ( **A**) and booting stage (**B**) on the day after inoculation (DAI). Note: ab represents a difference that is significant (*p* < 0.05).

In the near-infrared range, the reflectance is affected by leaf anatomy, which corresponds to the sensitive wavelength at 968.2nm in this study. As shown in Figure 11, the chloroplast structure (marked by "Ch") in leaves with different DS on 13 DAI was observed using a transmission electron microscope. As the DS increased, the chloroplast in the wheat leaves gradually swelled in the cells. Cavities of varying sizes appeared surrounded by a single layer of membrane, and the chloroplast disintegrated eventually. The cells in the normal leaves form multiple scattering of light resulting in high reflectance, while powdery mildew affects the internal structure of leaves, thus leading to lower reflectance of infected leaves.

**Figure 11.** Chloroplast structure of wheat at different disease severity (DS) after infection.

For texture features, Xie et al. [48] first compared spectral and texture features for detecting different diseases on tomato leaves, and the results obtained by texture features (60.2%) were slightly worse than those obtained by reflectance (97.1%) in classification accuracy. Al-Saddik et al. [23] used hyperspectral images to detect yellowness and esca in grapevines by combining spectral and texture data with an overall accuracy of 99% for both of the diseases. The adoption of NDTI in disease detection of this study is inspired by Zheng [33], who combined NDTIs and VIs to estimate above ground crop biomass with *R*2 of 0.78. However, the features selected in this study are specific to the wheat disease PM. When this methodology is used to monitor diseases on other crops, it would need to re-select the sensitive spectral features and corresponding texture features, as the symptoms (shape and color) of each disease are different.

### *4.2. What Is the Reason of Detection Performance at Varied Growth Stages?*

The detection at the booting stage performed significantly better than that of jointing stage, which might be due to the different rates of disease propagation at two growth stages. Most of the DS values for diseased leaves obtained after infection at the jointing and booting stages were 1–30% and 1–50%, respectively. The factors which affect the development of wheat PM are environment conditions [49], variety resistance [3], and the number of pathogen spores [50]. The varieties, cultivation practices and the number of spores for inoculations used in the two growth stages were the same. Hence, the difference was mainly due to the different climatic conditions during the two inoculation periods. The inoculation interval between the two growth stages differed by 14 days. The inoculation time at the jointing stage was early spring, at this stage the temperature was still low and gradually rising, therefore the development of the disease was slow. At low temperature, the PM fungus tends to reproduce in small quantities and the physiological and biochemical characteristics of diseased plants may not be obvious. However, the disease developed rapidly at the booting stage because of the suitable environment conditions. It was the time of late spring and conditions of high temperature and humidity were optimal for disease development, consistent with the observations of Yao et al. [51]. Such disease favorable environmental conditions enhanced the rate of disease development at the booting stage, thus inducing obvious changes in the biophysical and biochemical parameters of plants which resulted in changed spectral responses [52]. Moreover, high lesion ratios on the leaf surfaces at booting stage changed the texture patterns of wheat leaves more severely thus, ensuring robust and accurate disease detection at booting stage.

### *4.3. How Early to Detect the Disease When the Combined Feature Is Applied?*

This paper focuses on the methodology with regard to the feasibility of detecting the disease at its early stage or even before symptoms appear, at which it is difficult to acquire the weak signal due to the limited bands and course resolution. To the best of our knowledge, the present work is the first attempt to detect wheat PM by combining spectral vegetation indices and texture indices. Wheat PM lesions induce modification of color and surface properties of wheat leaves, and the texture information describes the intensity of the pixel's changes in an image. This phenomenon explains the robustness of the texture approach combined with the spectral approach in the better diagnosis of disease. Previous studies found that the spectral approach performed better when the DS is usually greater than 20%, with classification accuracy of about 87%. Meanwhile, the texture approach performed better in distinguishing DS of less than 20% with classification accuracy of 84%, but the accuracy by using a combination of texture and spectral features can reach up to 90% [53]. Therefore, combining spectral and texture information allows significantly better classification accuracy of diseased and healthy leaves and DS estimation at the early stage of wheat PM.

The early detection of disease on a daily basis after inoculation at different growth stages was carried out by combining the spectral and texture indices, and the classification accuracies of healthy and diseased leaves increased with increasing DS. On 6 DAI at jointing stage, the classification of healthy and diseased leaves was robust and stable, when the average DS was 3.9%. While at the booting stage, disease was recognized well on 3 DAI with an average DS of 1.1%. As the DS increased, the spectral and texture features changed, and the classification accuracies improved as the days passed after inoculation. However, at 9 DAI of booting stage, a slight decrease in classification accuracy was observed. These slight variations in classification accuracies were due to the unbalanced number of healthy and diseased leaves. At the booting stage on 9th DAI, most of the leaves were infected, so the model tended to predict the diseased one, resulting in decreased accuracy of healthy leaves. In another aspect, the variations of chlorophyll contents in leaves are also consistent with the model's performance for disease detection, as the model correctly identified disease on 6 and 3 DAI (Table 4) with an OA of over 85% and chlorophyll contents significantly changed between healthy and diseased leaves on 8 and 6 DAI (Figure 10), at jointing and booting stages, respectively. Therefore, our results well support the idea that HI is an efficient and reliable method for plant disease detection at early stage. It is worth mentioning that ground-based hyperspectral analysis is not suitable for a large area, and it is too expensive to be used by average farmers compared with UAV platforms. Multispectral UAV platforms can monitor disease in large areas with stable and high efficiency but perform poorly at the early disease infection stage. In the future, we believe there would be more hyperspectral equipment mounted on UAV systems, however currently there is only one kind of UHD185 in the community.


*Edited by Masoud Mohammadnezhad and Nafisa Huq*

This edited volume, *"Maternal and Child Health Matters Around the World"*, is a collection of reviewed and relevant research chapters, offering a comprehensive overview of recent developments in the field of medicine. The book comprises single chapters authored by various researchers and edited by an expert active in the medical research area. All chapters are complete in itself but united under a common research study topic. This publication aims at providing a thorough overview of the latest research efforts by international authors on maternal and child health matters around the world, and open new possible research paths for further novel developments.

ISBN 978-1-83968-379-4

Maternal and Child Health Matters Around the World

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