**3.1 Bioactive compounds**

Olive oil glyceridic fraction consists of triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids (FFA). Among them, 80% of them are unsaturated fatty acids. It is particularly rich in essential monounsaturated fatty acids (55–83% of oleic acid) and polyunsaturated fatty acids (2.5–21% of linoleic acid) [12]. The remaining fatty acids, apart from C16: 1, display an average value ranging from 0.3 to 3.5% (**Table 1** and **Figure 3**). Nevertheless, linolenic acid is a minority and its concentration is lower than 1% [12]. A low level of linolenic acid can be used to detect adulteration via some vegetable oils such as rapeseed and soybean oils [13]. Small quantities of saturated fatty acids also compose the triglycerides of olive oil: stearic acid (about 0.5–5%) and palmitic acid (about 7.5–20%). The remaining fatty acids (C17: 0, C17: 1, C20: 0, C20: 1, and C22: 0) are found to be of lower magnitudes. Since their concentrations are below 0.5% (**Table 1**). The unsaponified matter (about 1–2%) contains sterols, triterpene alcohols, tocopherol (mainly α-tocopherol), tocotrienol polyphenols, and squalene. The oil also contains a non-negligible proportion of volatile compounds. The total phytosterols content of VOO ranges between 100 and 200 mg/100 g. Also, 100 mg/100 g represents the inferior

*SFA-Saturated Fatty acids, MUFA-Monounsaturated fatty acids, PUFA-Polyunsaturated fatty acids: Apparent beta-sitosterol: beta-sitosterol +delta-5-avenasterol +delta-5-23-stigmastadienol +clerosterol + sitostanol +delta 5–24-stigmastadienol.*

#### **Table 1.**

*Physicochemical parameters, fatty acids, phytosterols, and tocopherols composition of olive oil.*

*Olive Oil: Extraction Technology, Chemical Composition, and Enrichment Using Natural… DOI: http://dx.doi.org/10.5772/intechopen.102701*

**Figure 3.** *Chromatogram of fatty acids.*

limit set by the international olive council [12]. Apparent beta-sitosterol, (beta-sitosterol + delta-5-avenasterol + delta-5-23-stigmastadienol + clerosterol + sitostanol + delta 5–24-stigmastadienol) are the main compounds in the sterol fraction with a value more than 93% while β-sitosterol has the greatest relative percentage [14, 15] (**Figure 4**). VOO content also includes up to 4.5 g/100 g of total phytosterols [12]. The erythrodiol (5α-olean-12-ene-3β, 28-diol, homo-olestranol) in free and esterified forms and are the major triterpene di-alcohols found in olive oil [14], and their percentage reached up to 4.5% of the total content of sterols [12]. Moreover, four isoforms of tocopherols (α, β, γ, and δ-tocopherol) (**Figure 5**) and four tocotrienols (α, β, γ and δ- Tocotrienol) are present in olive oil. α-tocopherol is the main tocopherol found in olive oil, constituting more than 90% of the total tocopherol fraction [14]. Cunha et al. [16] reported that the proportions of tocopherols and tocotrienols ranged from 100 to 270 mg/kg in Portuguese olive oils [16]. Gharby et al. [47] found that the values of tocopherols varied from 150 to 250 mg/kg in three varieties ('Arbequina', 'Moroccan Picholine', and 'Picual') of olive oil [17]. Moreover, another study, based on the comparison of the tocopherol contents of olive oils from 4 different varieties harvested at different ripening periods found

**Figure 4.** *Chromatogram of sterols.*

#### **Figure 5.**

*Tocopherols chemical structure.*

that the α-tocopherol (major tocopherol) in oils obtained from olives composed of 130.54–180.43 mg/kg [18]. In general, tocopherol and tocotrienol levels in oil fluctuate with several factors such as harvest year, climatic conditions, storage time, extraction method, soil properties and spacing between olive trees [19]. Tocopherols possess a strong antioxidant power [20]. Together with tocopherols and tocotrienols, olive oil contains other antioxidant molecules such as polyphenolic compounds.

Many research works have demonstrated that the content of tocopherols in VOO is lower than that of argan oil [21–23].

The phenolic compounds are endowed to have a large scale of biological functions including stability to auto-oxidation, beneficial effects on human health [24]. About their well-known activities, olive oil polyphenols have been proven to possess an effective role in maintaining the organoleptic properties and the stability of olive oils [25].

Such bioactive compounds are extensively studied for their anti-inflammatory, antioxidant, neuroprotective, cardioprotective, antidiabetic, antimicrobial, and anticancer properties [26–29].

Franco et al. reported that phenolic compounds have a considerable increase during olive fruit growth. However, they are reduced when the fruits reach the maturation stage [30]. Khalatbary documented that the total phenolic content (TPC) in olive oils varies from 190 to 500 mg/kg [31]. In addition, in extra virgin olive oil, TPC commonly varies from 250 to 925 mg/kg [32]. Other factors including climatic conditions, variety, storage time, extraction conditions, soil properties, and analysis of polyphenolic compounds can lead to important variations in TPC [33]. Likewise, several classes of polyphenols are found in olive oils. These are presented as a separate class, to better understand the antioxidant phenolic chemistry of olive oil [33]. Finicelli et al. classify olive oil polyphenols following their chemical structure as follows [34]:

*Olive Oil: Extraction Technology, Chemical Composition, and Enrichment Using Natural… DOI: http://dx.doi.org/10.5772/intechopen.102701*

The volatile fraction of VOOs has been reported to have about 280 different compounds [38]. The majority of volatile compounds are quickly developed during olive milling as a result of the disturbance of olive cells [39]. Although, Nardella et al. reported that most of the volatile compounds typical of olive oils are generated during

**Figure 6.** *Some phenolic alcohols present in olive oil.*

malaxation due to the activation of particular pathways, in which the lipoxygenase (LOX) enzyme plays an essential role in producing a large quantity of C6 aldehydes, esters, and alcohols. These constitute almost all of the positive sensory marks in olive oils [40]. Such changes are initiated when olive tissues are affected, thereby enhancing the liberation of endogenous enzymes like hydroperoxide lyase and lipoxygenase [40].

Besides, several analytical techniques have been used to determine volatiles composition in olive oil. The main important are: GC (gas chromatography), HPLC (high-performance liquid chromatography), HPLC/MS (high-performance liquid chromatography/mass spectroscopy), IRMS (isotope ratio mass spectroscopy), ICP (inductively coupled plasma spectroscopy), NMR (nuclear magnetic resonance), SPME-GC/MS (solid-phase microextraction followed by gas chromatography/mass spectrometry, SNIF/NMR (specific natural isotopic fractionation nuclear magnetic resonance), SCIRA (stable carbon isotope ratio analysis), PTR/MS (proton transfer mass spectrometry) [41]. Fregapane et al. reported that the composition of volatiles may be affected significantly according to many factors such as cropping season, olive variety, harvest time, technological parameters, and agronomic conditions among other factors [42]. Ghanbari et al. reported that several chemical factors such as hydrophobicity, volatility, position, and functional groups type are reported to be directly linked to the odor degree of a given volatile component more than its content [38]. Theodosi et al. investigated correlations between the composition of volatiles of olive oil and altitude variation. The findings demonstrate that the total volatile compounds of 'Koroneiki' olive oil samples and altitude levels are negatively associated. The most important volatile compounds are alcohols, aldehydes, esters, and hydrocarbons [43].


**6.4.3 D-P: particle group**

{*PH*(*v*˜0|*J*)} in Figs 4-5 (g) show six pairs of anti-symmetry distributions: {8 14}, {2 11}, {0 15}, {6 9}, {4 13}, {1 7} four elements are distinguished on the anti-diagonals: {10, 12, 3, 5}. Under this condition, ten classes can be identified.

#### **6.4.4 D-W: wave group**

{*PH*(*v*˜1|*J*)} in Figs 4-5 (h) show six pairs of anti-symmetry distributions: {8 14}, {2 11}, {0 15}, {6 9}, {4 13}, {1 7} four pairs of symmetry elements: {3:5}, {10 : 12}, {2:4}, {11 : 13} are distinguished. Under this condition, twelve classes can be identified.

#### **6.5 Odd and even numbers**

From a group viewpoint, only D-P and D-W need to be reviewed as different groups in symmetry conditions. Anti-symmetry conditions are unremarkable.

It is reasonable to suggest that anti-symmetry operations will be much easier to distinguish under experimental conditions, since sixteen groups in D-P conditions and twelve groups in D-W conditions will have significant differences. However, under the symmetry conditions (only) minor differences can be identified.

#### **6.5.1 Single and double peaks**

Single and Double peaks can be observed in Fig 4(5) (h): {3, 5} for even and odd numbers respectively.

For two other members {10, 12}, (only) single pulse distributions are observed in Figs 4-5 (h) to show the strongest interference results.

#### **6.6 Class numbers in different conditions**

To summarize over the different classes, 16 matrices are shown in different numbers of identified classes as follows:

where Left:Left Path, Right: Right Path, D-P: Double Path for Particles, D-W: Double Path for Waves; SE: Symmetry for Even number, SO: Symmetry for Odd number, AE: Anti-symmetry for Even number, AO: Anti-symmetry for Odd number.

#### **6.7 Polarized effects and double path results**

18 Measurement Systems

{*PH*(*v*˜+|*J*)} elements in Figs 4-5 (e) show that (only) four classes can be distinguished. Elements within these groups members are the same as for symmetry groups in Figs 4-5(a).

{*PH*(*v*˜−|*J*)} elements in Figs 4-5 (f) show that (only) four classes can be distinguished. Elements within these groups are the same as for symmetry groups in Figs 4-5 (b). Their

{*PH*(*v*˜0|*J*)} in Figs 4-5 (g) show six pairs of anti-symmetry distributions: {8 14}, {2 11}, {0 15}, {6 9}, {4 13}, {1 7} four elements are distinguished on the anti-diagonals:

{*PH*(*v*˜1|*J*)} in Figs 4-5 (h) show six pairs of anti-symmetry distributions: {8 14}, {2 11}, {0 15}, {6 9}, {4 13}, {1 7} four pairs of symmetry elements: {3:5}, {10 : 12}, {2:4}, {11 :

From a group viewpoint, only D-P and D-W need to be reviewed as different groups in

It is reasonable to suggest that anti-symmetry operations will be much easier to distinguish under experimental conditions, since sixteen groups in D-P conditions and twelve groups in D-W conditions will have significant differences. However, under the symmetry conditions

Single and Double peaks can be observed in Fig 4(5) (h): {3, 5} for even and odd numbers

For two other members {10, 12}, (only) single pulse distributions are observed in Figs 4-5 (h)

To summarize over the different classes, 16 matrices are shown in different numbers of

13} are distinguished. Under this condition, twelve classes can be identified.

symmetry conditions. Anti-symmetry conditions are unremarkable.

**6.4.1 Left: polarized vertical group**

**6.4.2 Right: polarized horizontal group**

distributions fall within the region [0, 0.5].

{10, 12, 3, 5}. Under this condition, ten classes can be identified.

**6.4.3 D-P: particle group**

**6.4.4 D-W: wave group**

**6.5 Odd and even numbers**

**6.5.1 Single and double peaks**

identified classes as follows:

respectively.

(only) minor differences can be identified.

to show the strongest interference results.

**6.6 Class numbers in different conditions**

Their distributions fall within the region [0.5, 1].

In order to contrast the different polarized conditions, it is convenient to compare distributions {*PH*(*u*˜+|*J*), *PH*(*u*˜−|*J*} and {*PH*(*v*˜+|*J*), *PH*(*v*˜−|*J*} arranged according to the corresponding polarized vertical and horizontal effects. This visual effect is similar to what might be found when using polarized filters in order to separate complex signals into two channels. Different distributions can be observed under synchronous and asynchronous conditions.

#### **6.7.1 Particle distributions and representations**

For all symmetry or non-symmetry cases under ⊕ asynchronous addition operations, relevant values meet 0 ≤ *u*˜0, *v*˜0, *u*˜−, *v*˜−, *u*˜+, *v*˜+ ≤ 1. Checking {*PH*(*u*˜0|*J*), *PH*(*v*˜0|*J*)} series, {*PH*(*u*˜+|*J*), *PH*(*u*˜−|*J*)} and {*PH*(*v*˜+|*J*), *PH*(*v*˜−|*J*)} satisfy following equation.

$$\begin{cases} P\_H(\vec{u}\_0|J) = \frac{P\_H(\vec{u}\_-|J) + P\_H(\vec{u}\_+|J)}{2} \\ P\_H(\vec{v}\_0|J) = \frac{P\_H(\vartheta\_-|J) + P\_H(\vartheta\_+|J)}{2} \end{cases} \tag{16}$$

The equation is true for different values of *N* and *n*.

#### **6.7.2 Wave distributions and representations**

Interference properties are observed in {*PH*(*u*˜+|*J*) = *PH*(*u*˜−|*J*)} conditions. Under + synchronous addition operations, relevant values meet 0 ≤ *u*˜1, *v*˜1, *u*˜−, *v*˜−, *u*˜+, *v*˜+ ≤ 1. Checking {*PH*(*u*˜1|*J*), *PH*(*v*˜1|*J*)} distributions and compared with {*PH*(*u*˜+|*J*), *PH*(*u*˜−|*J*)} and {*PH*(*v*˜+|*J*), *PH*(*v*˜−|*J*)}, non-equations and equations are formulated as follows:

$$\begin{cases} P\_H(\vec{u}\_1|J) \neq P\_H(\vec{u}\_0|J) \\ P\_H(\vec{v}\_1|J) \neq P\_H(\vec{v}\_0|J) \end{cases} \tag{17}$$

Spectra in different cases illustrate wave interference properties. Single and double peaks are shown in interference patterns and these are similar to interference effects in classical double slit experiments.

#### **6.7.3 Non-symmetry and non-anti-symmetry**

However, for the {*PH*(*u*˜+|*J*) �= *PH*(*u*˜−|*J*)} non-symmetry cases, there are significant differences between {*PH*(*u*˜0|*J*), *PH*(*v*˜0|*J*)} and {*PH*(*u*˜1|*J*), *PH*(*v*˜1|*J*)}. Such cases have interference patterns that exhibit greater symmetry than single path and particle distributions.

**7.3 The BCP for a special subset of QM**

**7.4 The EPR contribution on variant construction**

invariant properties.

in the Copenhagen interpretation.

⎧ ⎪⎪⎨

⎪⎪⎩

by Einstein as far back as 1935.

⎧ ⎪⎪⎨

⎪⎪⎩

**7.5 Afshar's experiments on variant construction**

�*S*1, *S*2, *IM*(*S*1, *S*2), *SM*(*S*1, *S*2)� →

are available for future theoretical and experimental exploration.

We may deduce that there is (only) a special subset of QM for which the BCP is satisfied. Under the variant construction there are six distinct logical configurations that can be used to support 0-1 vectors. Of these six, Bohr's approach is suitable for only the two schemes of pure static YES or NO. Meanwhile, the other four variant, invariant and mixed configurations lie outside the BCP framework. From this viewpoint, Bohr offers insight into important special

359 From Conditional Probability Measurements to Global Matrix Representations on Variant

Bohr's QM construction is complete and useful in many theoretical and practical environments for static and static-like systems. However, the variant construction provides a more powerful and general mechanism to handle different dynamic systems with variant and

From EPR proposed experiments and other theoretical considerations, Einstein demonstrated a depth of understanding of weakness inherent in the foundations of the QM approach. He clearly identified two operators with non-communication properties that failed to support simultaneous operations and recognized that this type of mechanism was still not explained

> *IM*(*S*1, *S*2) → {*M*(*u*1), *M*(*v*1), *M*(*u*˜1), *M*(*v*˜1)...}; *SM*(*S*1, *S*2) → {*M*(*u*0), *M*(*v*0), *M*(*u*˜0), *M*(*v*˜0)...}.

From this correspondence, many possible configurations of combinations and their subsets

Using the variant construction, rich configurations can be expressed. From such mapping, it can be seen to be nothing less than astounding that such meta constructions were identified

Afshar's experiments apply anti-symmetry signals making the following correspondence:

*ψ*1 → {*v*+}; *ψ*2 → {*v*1}; *σ*1 → {*PH*(*v*1|*J*)}; *σ*2 → {*PH*(*v*0|*J*)}.

⎧ ⎪⎪⎨

⎪⎪⎩

�{*uβ*, *vβ*, *u*˜*β*, *v*˜*β*...}, {*uβ*, *vβ*, *u*˜*β*, *v*˜*β*...}, {*M*(*u*1), *M*(*v*1), *M*(*u*˜1), *M*(*v*˜1)...}, {*M*(*u*0), *M*(*v*0), *M*(*u*˜0), *M*(*v*˜0)...}�

(18)

(19)

(20)

circumstances of QM rather than provides an all embracing general solution.

Construction – A Particle Model of Intrinsic Quantum Waves for Double Path Experiments

Using the variant construction, EPR devices have the following correspondence:

*S*1 → {*uβ*, *vβ*, *u*˜*β*, *v*˜*β*...}; *S*2 → {*uβ*, *vβ*, *u*˜*β*, *v*˜*β*...};

Four anti-diagonal positions are linked to symmetry and anti-symmetry pairs, twelve other pairs of functions belong to non-symmetry and non-anti-symmetry conditions. Their meta elements can be identified by the relevant variant expressions.


*Article* **Numerical Study on Wave-Ice Interaction in the Marginal Ice Zone**

**Tiecheng Wu 1,2, Wanzhen Luo 1,2,\*, Dapeng Jiang 1,2, Rui Deng 1,2 and Shuo Huang 1,2**

**Abstract:** The effect of waves on ice sheet is critical in the marginal ice zone (MIZ). Waves break large sea ice into small pieces and cause them to collide with each other. Simultaneously, the interaction between sea ice and waves attenuates these waves. In this study, a numerical research is conducted based on a computational fluid dynamics (CFD) method to investigate the response of single ice floe to wave action. The obtained results demonstrate that the sea ice has a violent six degree of freedom (6DoF) motion in waves. Ice floes with different sizes, thicknesses, and shapes exhibit different 6DoF motions under the action of waves. The heave and surge response amplitude operator (RAO) of the sea ice are related to wavelength. Furthermore, the overwash phenomenon can be observed in the simulation. The obtained results are compared with the model test in the towing tank based on artificial ice, and they agree well with test results.

**Keywords:** marginal ice zone; sea ice; wave; six degree of freedom (6DoF) motion; CFD

**Citation:** Wu, T.; Luo, W.; Jiang, D.; Deng, R.; Huang, S. Numerical Study on Wave-Ice Interaction in the Marginal Ice Zone. *J. Mar. Sci. Eng.* **2021**, *9*, 4. https://dx.doi.org/10.3390/ jmse9010004

Received: 27 November 2020 Accepted: 17 December 2020 Published: 22 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 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/).


**2. Radio-binding assay (RBA) and enzyme-linked immunosorbent assay (ELISA)**

In the USA, 1.4 million people have T1D and as many have multiple islet autoantibodies (iAbs) or pre-T1D with normal glucose homeostasis. Of the latter, 84% will progress to clinical diabetes in 15 years with a remarkable consistency across populations [5]. Although T1D is a T-lymphocyte-mediated autoimmune disease with specific destruction of pancreatic islet β-cells, autoantibodies directed against proteins in insulin-producing beta cells [1] are the best biomarkers for risk prediction and clinical diagnosis. These iAbs usually appear years before overt clinical disease, and the presence of ≥2 iAbs [antibodies-directed against insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen 2 (IA-2A) or zinc transporter 8 (ZnT8A)] predicts the development of clinical T1D in nearly all affected children [5]. Children at risk for T1D need to be identified prior to the onset of symptoms to: (1) prevent life-threatening diabetic ketoacidosis, (2) identify individuals for current and upcoming trials to prevent T1D

Appearance of iAbs is currently the most reliable marker of the autoimmune process leading to T1D, and it determines the disease risk and marks the onset of autoimmune beta cell destruction. The presence of iAbs, their number [6–8] and titer [9–11], has being used extensively to stage diabetes risk and as inclusion criteria into T1D prevention trials [12]. Immunoassays to detect iAbs can be classified into four generations. The first generation is the indirect immunofluorescence on cryostat sections of the pancreas for islet cell antibodies (ICA) [13, 14] present in patients' sera to a variety of autoantigens in islet β-cells. This assay requires the pancreas tissue to conduct the measurement and is very limited in the regular screening. The second generation of iAbs immunoassays is a well-established radio-binding assay (RBA) which is based on various biochemically defined autoantigens and plays a major role as the current gold standard assay for all four major islet autoantibodies IAA, GADA, IA-2A and ZnT8A and ELISAs. Traditional ELISA has been proved not to work well for any iAbs with less sensitivity and less specific in multiple workshops of Islet Autoantibody Standardization Program (IASP, previously DASP). However, a modified bridging ELISA with semifluid-phase interaction between the antibody and antigen has achieved sensitivity and specificity equivalent to the RBA for the measurement of GADA, IA-2A and ZnT8A [15–19]. The third generation is a recently developed and extensively validated nonradioactive iAb assay using electrochemiluminescence (ECL) detection with a higher sensitivity and higher disease specificity compared to the RBAs [20–25]. The ECL assay has been demonstrated to be more sensitive and is able to identify the first iAb of "seroconversion" earlier than RBA by years in young children followed from the birth on pre-T1D who were followed to clinical diabetes. More remarkably, ECL assay is able to discriminate high-affinity, high-risk autoantibodies from those "low risk," low-affinity signals generated by RBA in subjects with single iAb who are less likely to progress to T1D. The fourth generation of autoantibody immunoassays is to develop a simple multiplexed assay to fit for the needs of ongoing and future clinical trials to simultaneously screen multiple iAbs and other autoantibodies in one single well, which will facilitate high-throughput autoantibody screening simultaneously for T1D and other multiple relevant autoimmune diseases in large scale of populations. ECL assay has been illustrated as an excellent platform for a simple multiplex assay with a superior advantage of high sensitivity and disease specificity. In this chapter, we mainly focus on the discussion of: (1) two currently most popular assays for iAbs, a gold standard RBA and a modified bridging ELISA; (2) ECL assay and its comparison with RBA and (3) development of a simple multiplex ECL assay with all advantages considered.

and (3) define the onset of islet autoimmunity and its triggers.

190 Autoantibodies and Cytokines

At present, four major biochemically defined β-cell autoantigens were well characterized including IAA [26], GAD65 [27], IA-2 [28] and ZnT8 [29]. Through laboratory proficiency programs [30, 31] and harmonization efforts [32], RBA, a fluid-phase assay using radiolabeled antigens and precipitating antibody-antigen complex by Protein A/G Sepharose, has been well established as a current "gold" standard assay for autoantibodies to all four major antigens [29, 32, 33].

In RBA, recombinant human insulin is labeled with I-125, while GAD, IA-2 and ZnT8 antigens are expressed from their corresponding recombinant human cDNA and labeled with 35S-methionine by coupled in vitro transcription/translation. To assess general implementation of assay methods and to standardize the iAb assays, the Center for Disease Control and Prevention (CDC) and Immunology and Diabetes Society (IDS) started organizing the Diabetes Autoantibody Standardization Program (DASP) workshop in the year of 2000 [34]. Forty-six laboratories in 13 countries participated in the program. The first proficiency evaluation of DASP showed a high concordance in measurement of GADA (r = 0.96; p < 0.0001) and IA-2A (r = 0.89; p < 0.0001) using the new WHO international reference reagent. In contrast, the workshop demonstrated wide variation among IAA assays, with poor overall performance and low sensitivity. IAA assays were improved after the first proficiency evaluation and the median laboratory assigned sensitivity was 26, 36 and 45% in 2002, 2003 and 2005, respectively (p < 0.0001). However, the IAA assay has proven the most difficult to standardize with relatively wide discrepancies between laboratories in the IASP workshop and has not yet achieved a satisfactory level of sensitivity and specificity. There was still remarkable variation between laboratories and the ranking of IAA levels in patient serum samples was concordant to AUC (p < 0.001), and the AUC ranged from 0.36 to 0.91 [30]. To facilitate comparison of quantitative islet autoantibody results between studies, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) set up an Islet Autoantibody Harmonization Committee in 2007 to align measurement and reporting of iAbs in all NIDDK-sponsored studies and several different central laboratories are used [32]. With a few years of combined efforts, both GADA and IA-2A assays were successfully harmonized with great concordance as positive or negative between participating laboratories. The IA-2A assay, using a common threshold of 5 DK units/ml, achieved 64% sensitivity with specificity greater than 99% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. ZnT8 is a most recently identified new islet β-cell autoantigen, and ZnT8A assay has been well established through IASP workshop. In combination of current four iAb assays by the standard RBA, the overall sensitivity is able to cover around 95% in newly diagnosed patients with T1D [29]. To lead to more reproducible identification of individuals at risk of type 1 diabetes and improve monitoring in long-term prospective studies, some recommendations by the Islet Autoantibody Harmonization Committee have been applied to current national and international clinical trials for T1D: (1) confirmation of positive results in a second laboratory is likely to be valuable to identify discrepancies; (2) laboratories should use common calibrators against reference standards; (3) the methods used in multiple centers should be compared and harmonized to eliminate potential factors that will contribute to discrepancies; (4) a common set of samples which can provide knowledge of concordance, sensitivity and specificity including a large number of samples from healthy controls, and patients should be identified to establish working thresholds of positivity that are similar between the participating laboratories.

and extensively validated a new generation of nonradioactive iAb assay using ECL detection, as we described in the previous section, with an excellent sensitivity and specificity [20–25] for both IAA and GADA. The assay is based on the principle that interaction of autoantibodies with antigen molecules is in liquid phase, and the detection signals are directly from the labeled antigen molecules bound to specific autoantibodies without applying the labeled second antibody (labeled anti-human IgG antibody usually used in conventional ELISA) for detection, which usually causes a very high background in autoantibody assays with human samples. As illustrated for ECL assay protocol in **Figure 1**, autoantibodies in serum help bridge the Sulfo-tagged antigen molecule to the biotinylated antigen molecule, which will be captured on the solid phase of the streptavidin-coated plate. Detection of plate-captured Sulfo-tagged antigen is accomplished with electrochemiluminescence. The fact that the ECL assay is able to capture all immunoglobulin subgroups no matter IgG, IgM, IgA or IgE allows this assay a higher sensitivity than current standard RBA based on IgG detection. With the new ECL assays, we have analyzed thousands of participants in Diabetes Autoimmunity Study in the Young (DAISY) [21], TrialNet Pathway to Prevention subjects [23] and very recently TEDDY subjects (unpublished data). Compared with gold standard RBA, the ECL assays, especially ECL-IAA assays, were demonstrated more sensitive. In DAISY longitudinally follow-up study, we analyzed 427 sequential samples from 63 pre-T1D who were closely followed to clinical T1D. Nearly all of these children (62/63) were detected ECL-IAA years before disease onset, including 10 children who were completely IAA negative by RBA-IAA during the follow-up. Remarkably, 25% of these early longitudinally followed samples during

Development of a Simple Multiplex Electrochemiluminescence (ECL) Assay for Screening Pre…

http://dx.doi.org/10.5772/intechopen.75515

193

**Figure 1.** Illustration of the bivalent plate capture ECL assay. The antibody in serum will link the Sulfo-tagged antigen to the biotinylated antigen, which will be captured on the solid phase of the streptavidin-coated plate. Detection of plate-

captured Sulfo-tagged antigen is accomplished with electrochemiluminescence.

T1D is now able to be predicted by measuring iAbs present in the peripheral blood, and these iAbs have been used to determine disease risk and onset of autoimmune beta cell destruction. Almost all children with two or more of these iAbs will progress to clinical diabetes with a remarkable consistency across populations, while progression to diabetes in children persistently positive for a single iAb is only 15% in 15 years [30], as most of these children have a low-affinity iAb not associated with disease [35–37].

In summary, the current method most commonly used for screening iAbs is the "gold" standard RBA, a fluid-phase assay using radiolabeled antigens and immunoprecipitation, to quantify four major iAbs named IAA, GADA, IA-2A and ZNT8A. These four RBAs are currently able to cover 95% of sensitivity in newly diagnosed patients with T1D. Through the efforts of iAb proficiency programs and harmonization consortia, current standard RBA for iAbs have been greatly improved, but there are still work to be done, especially (1) IAA is not achieved its sensitivity and specificity and (2) there is a low risk prediction in subjects with single iAb positivity.

A direct enzyme-linked immunosorbent assay (ELISA) format (binding of antigen to plate and detection of bound autoantibody with labeled anti-antibodies) has proven difficult to develop. To date, only one ELISA-based ElisaRSR™ for GADA, IA-2A and ZnT8A, distributed by Kronus that utilizes capture of solution-phase antigen by one chain of immunoglobulin (Ig) while being bound by its other chain to plate-bound antigen has demonstrated sensitivity and specificity similar to the fluid-phase RBA (www.rsrltd.com). But there is no any ELISAbased assay that works well for IAA measurement according to IASP workshop [30]. ELISA assays were found only to detect insulin antibodies induced by exogenous insulin injection [38], but not natural insulin autoantibodies. Our group reported a high sensitive ELISA-based competition Europium IAA assay in 2009 [39], and it worked very well for mouse IAA assay, but unfortunately it did not work at all for human samples, although RBA-IAA assay works equally well for both human and mouse samples [33].


**An Innovative Systematic Approach to Financial Portfolio Management via PID Control**

Gino Gandolfi1, Antonella Sabatini1,2 and Monica Rossolini3 *1University of Parma 2M.I.T. 3"Banking and Finance" Tor Vergata University, SDA Bocconi 1,3Italy 2USA*


**3. Haematology of Tiger**

#### **3.1. Erythrocytes**

230 Blood Cell – An Overview of Studies in Hematology

chemistry and urine analysis.

(Gopal, 1993).

squeeze cage.

**2. Blood collection and investigation**

drugs. It has been experienced that the values of serum enzymes increased after 72 hrs interval of 2nd immobilization by Ketamine and Xylazine mixture (personal communication, Shrivastav 2012). Sign of anemia such as pale mucous membranes weakness, fatigue and tachycardia may be observed depending on the severity of anemia. A variety of abnormalities may be noticed by analysis of blood, bone marrow cytology, serum

Wild felids are commonly injured in territorial fight or sometimes serious injuries and internal hemorrhages occur during hunting. If blood loss is above the 50% of total volume in short period may be fetal and tiger may die due to hypovolumeic shock. Information on haematology and blood biochemistry is meagre in wild animals. However, several studies on selected haematological parameters of exotic species of captive Felids have been reported. Currier and Russell (1982) studied the higher pack cell volume in wild and captive mountain lions (*Felis concolor*) and Fowler (1986) has reviewed the haematological and biochemical profile of Felids including captive tigers whereas Jain (1986) reviewed the information of the genera *Panthera, Felis, Uncia* and *Acinonyx* concluded that blood parameters were almost similar to that of domestic cat with exception of higher concentration of plasma protein and Pack cell volume (PCV). Seal et al. (1987) have studied the haematological and biochemical profile of captive Bengal tigers with emphasis of anaesthetic effect on blood parameters. Chandranaik et.al. (2006) also studied the haematology of physically restrained tigers that were kept in squeeze cages without using anaesthetics. However, the haematological and biochemical studies were made in twelve

apparently healthy tigers in free ranges of Central India (Shrivastav et.al. 2011).

Health monitoring, assessment of health during treatment and disease diagnosis in free range tigers needs baseline data on haemato-biochemical parameters. This baseline data is important especially for comparative health assessment of felids during out breaks of diseases between sylvatic and domestic cycle vice versa. It is also required, as the tiger is on top of the sylvatic food chain and to be protected for maintaining balances in ecosystem

The collection of blood for laboratory investigations is comparatively difficult in both free range and captive tigers and only possible when animal is sedated or restrained properly in

Withstanding facts, chemical capture is comparatively safe, if accomplished by trained and experienced wildlife veterinarians. There are several drugs available for sedation. Each drug works in a different manner and is more suited to some species only. The time required for a drug to have an effect depends upon the factors such as route of administration, absorption rate, concentration and physiological status of the animal while it is difficult to generalize the choice of drug and doses (WII, 1985). It depends upon circumstances like species of the animal, age, sex, weight, location, temperature regimes in season, time of the day and The morphology of erythrocytes varies with 2 to 7.6, 7.3 ± 0.45 µm in size; appears circular, discoid, central pallor with slight anisocytosis whereas the rouleaux formation(Plate- 2) is common in tiger's blood. Chandranaik, et.al. (2006) also reported the mild anisocytosis in physically restrained tigers. However, the range and mean (with one standard deviation) of total erythrocyte count (TEC) was 4.66 to 9.15, 7.9± 1.42 million /µl. Likewise haemoglobin concentration (Hb) was obtained 9.8 to13.5, 12.8 ±1.65 mg/dl in male and 7.8 to11.5, 10.8±1.05 mg/dl in female tigers (Shrivastav et.al. 2011).

Jain (1986) defined that the rouleaux formation is associated with erythrocyte sedimentation rate (ESR) and useful for evaluation of the disease status. Shrivastav et. al. (2011) encountered ESR (14 to 26, 21 ± 4.21 /hour) and PCV (36 to 45, 38 ± 4.45 %) in free range tigers (Table 1). The consequences of ESR and PCV up and downs mostly confined to

erythrocyte osmotic fragility that increased in case of immune-mediated hemolytic anemia. Taketa et. al. (1967) have assessed the oxygen affinity of the haemoglobin is much lower in felines than that of other mammals including humans.

Tigers Blood: Haematological and Biochemical Studies 233

pathogenetic mechanism and bone marrow erythroid response ( Jain, 1986).In wild animals the clinical signs and their magnitude depends on habitat and availability of nutritive materials. Prolonged nutritional deficiencies of protein vitamins and minerals essential for erythrocytes production lead to anaemia. The type of anaemia varies with the nutritional deficiency, blood loss and the animal species involved. Despite the nutritional consequences the blood loss may be encountered through traumatic injuries, complication in blood vascular system, thrombocytopenia, and coagulation disorders. A normocytic – nonchromatic, non responsive anaemia is commonly found in association with chronic infections, chronic infectious inflammatory conditions and some type of malignancies though microcytic-

Several blood sucking parasites produce blood loss anaemia in tigers like Ancylostomes, Toxoscaris that may cause haemolytic anaemia while Trypanosomes, Babesia and Haemobartonella (*Mycoplasm haemofelis*) may alter the total blood as well as plasma volumes with acute blood loss. Chronic blood loss may lead to gastrointestinal lesions, ulcers, heavy parasitism like coccidiosis, neoplasm with bleeding into body cavity, deficiency of Vitamin

hypochromic is the sign of iron deficiencies (Jain and Kono 1975).

**Plate 1.** Tiger Blood smear stained with Modified Wright Stain x1000.

K and prothrembin etc.

**Table 1.** Haematological and Biochemical Values of Bengal tigers (*Panthera tigris tigris*)

Jain (1986) reviewed the haematological parameters of big cats including *Panthera, Felis, Uncia*  and *Acinonyx* and found that the blood composition were almost similar. Among all cats few erythrocytes had single refractile structure (Heinz body) when stained with new methylene blue stain. The Heinz body appearance in erythrocytes is the unique feature of the family Felidae (Plate -1) while they are not visible usually in blood films with Romanowsky stain (Jain,1986). The reduction in erythrocyte count (TEC) and haemoglobin concentration (Hb) are generally associated with anaemia and classified on the basis of erythrocyte morphology, pathogenetic mechanism and bone marrow erythroid response ( Jain, 1986).In wild animals the clinical signs and their magnitude depends on habitat and availability of nutritive materials. Prolonged nutritional deficiencies of protein vitamins and minerals essential for erythrocytes production lead to anaemia. The type of anaemia varies with the nutritional deficiency, blood loss and the animal species involved. Despite the nutritional consequences the blood loss may be encountered through traumatic injuries, complication in blood vascular system, thrombocytopenia, and coagulation disorders. A normocytic – nonchromatic, non responsive anaemia is commonly found in association with chronic infections, chronic infectious inflammatory conditions and some type of malignancies though microcytichypochromic is the sign of iron deficiencies (Jain and Kono 1975).

Several blood sucking parasites produce blood loss anaemia in tigers like Ancylostomes, Toxoscaris that may cause haemolytic anaemia while Trypanosomes, Babesia and Haemobartonella (*Mycoplasm haemofelis*) may alter the total blood as well as plasma volumes with acute blood loss. Chronic blood loss may lead to gastrointestinal lesions, ulcers, heavy parasitism like coccidiosis, neoplasm with bleeding into body cavity, deficiency of Vitamin K and prothrembin etc.

232 Blood Cell – An Overview of Studies in Hematology

5Erythrocyte sedimentation

7Differential leukocyte

**Blood plasma biochemistry**

5Blood urea nitrogen

7Aspertate

6Alanine Aminotransferase

count

felines than that of other mammals including humans.

erythrocyte osmotic fragility that increased in case of immune-mediated hemolytic anemia. Taketa et. al. (1967) have assessed the oxygen affinity of the haemoglobin is much lower in

 **Haematology Unit Range Mean SE (±)**  1 Red blood corpuscles (TEC) ×106/µl 4.66–9.15 7.90 1.42 2 Total Leukocytes Count (TLC) ×103/µl 6.2–11.05 8.50 1.42 3 Haemoglobin (Hb) g/dl 7.8–13.8 12.8 1.65 4 Haematocrit (PCV) Ratio 36–45 38 2.54

rate (ESR) Hours 14–26 21 4.21 6 Icterus index (II) u/l 2– 5 2 1.51

i Neutrophils – 57–75 60 5.08 ii Lymphocytes – 18–35 30 4.56

iii Monocytes – 2– 6 0.5 1.21 iv Eosinophils – 2–6 0.4 1.30 v Basophils – 0–4 0.1 1.21

1 Albumin (ALB) g/dl 2.1–4.6 3.50 0.99 2 Total protein ( TPROT) g/dl 3.7–8.7 6.40 1.88 3 Total bilirubin TBIL) mg/dl 0.4–3.2 1.90 1.21 4 Creatinine (CRE) mg/dl 1.6–4.6 2.90 1.03

(BUN) mg/dl 6.5–48.2 27.90 13.77

(ALT) IU/L 21.2–109.0 67.88 27.84

Aminotransferase (AST) IU/L 14.4–84.0 57.96 17.27

Jain (1986) reviewed the haematological parameters of big cats including *Panthera, Felis, Uncia*  and *Acinonyx* and found that the blood composition were almost similar. Among all cats few erythrocytes had single refractile structure (Heinz body) when stained with new methylene blue stain. The Heinz body appearance in erythrocytes is the unique feature of the family Felidae (Plate -1) while they are not visible usually in blood films with Romanowsky stain (Jain,1986). The reduction in erythrocyte count (TEC) and haemoglobin concentration (Hb) are generally associated with anaemia and classified on the basis of erythrocyte morphology,

**Table 1.** Haematological and Biochemical Values of Bengal tigers (*Panthera tigris tigris*)

%

**Plate 1.** Tiger Blood smear stained with Modified Wright Stain x1000.

Tigers Blood: Haematological and Biochemical Studies 235

neutrophil numbers while eosinophil commonly not affected. Meyers-Wallen et. al. (1984) observed the young cats normally have high lymphocyte counts and hence a greater tendency to develop lymphocytosis than the adults. This observation may also be attributed in the case of tigers as they belong to the member of same family with wild habitat as an escape behavior. Increases in neutrophil numbers due to physiologic influences are more pronounced in felines than in canines because of the difference in the intravascular distribution of neutrophils. Prasse et. al.(1973) have observed 3 times mean marginal pool of neutrophils of clinically healthy cats than the circulating pool whereas in dog it was about

Neutrophils considered as first line of defense against microbial infections and are important participants in inflammatory reactions. Shrivastav, et.al. (2011) encountered 57 to 75, 60 ±5.08 % with multi-lobed nuclei of 3-5 lobes while sometimes mono-lobed nuclei with pale to slightly pink granules in the cytoplasm in free range tigers( Plate3). Chandranaik, et.al. (2006) has also reported the segmented or multi lobed nuclei while Jain (1986) studied

The changes in blood neutrophil differential count (Haden, 1935) is associated with many consequences related to infectious diseases. Several functions have been suggested for the contents of granules, as neutrophils are phagocytic cells and regulating adhesiveness and aggression hydroxyl radical formation and generation of compliment derived chemotactic factors while azurophilic granules are involved in modulation of inflammatory process (Gallin, et. al.1982). Condensation of nuclear chromatin leads to formation of darker-staining plaques separated by delicate, light-staining areas with slight brown colour cytoplasm.

Shrivastav et.al. (2011) observed eosinophils contained small, uniformly round bright eosinophilic granules almost occupying the entire and clear cytoplasm (Plate 3). These cells were encountered 2 to 6, 4 ±1.21 % in free range tigers (Table 1). The nuclei of the cells were generally less lobulated than those of the neutrophils. The eosinophils are slightly larger than neutrophils. Chandranaik, et.al. (2006) also observed the larger eosinophils larger than neutrophils and lobulated nuclei with orange cytoplasm in tigers. Jain (1986) reported the granules of the eosinophil are rod-like in domestic cats and Cheetah (*Acinonyx jubatus*) while round granules in the eosinophils of Lion and Leopard. The eosinophils are commonly seen

The basophile is a numerically insignificant but functionally important leukocyte that resemble with mast cells and it believed to share similar function as it is associated with allergic reaction, inflammatory process and immunocompitancy to the body fluids. Galli et.

the sex chromatin in few neutrophils as the drumstick lobe in the female cats.

equal or slightly greater.

**4.1. The neutrophils**

**4.2. The eosinophils**

**4.3. The basophiles**

in prolonged parasitic infections or allergic disorders.

**Plate 2.** Tiger Blood smear stained with Modified Wright Stain x1000.


**4. Current approaches to rebuild primary care**

It has long been apparent that system reform will be required in the U. S. before primary care can grow and thrive at its foundation. Our non-system has been taken over by corporate stakeholders dancing to the tune of Wall Street investors, not the needs of patients, families and taxpayers. Before we can move back to a traditional service ethic in health care based on the public interest, major reforms will be needed. As the distinguished medical historian, Rosemary Stevens, observed 20 years ago:

*The most important impediment to a clear-cut role for family practice has been the lack of a formal administrative structure for primary care practice on a nationwide basis in the United States [28].*


**Abstract**

Since green revolution, chemical fertilizers are deemed an indispensable input of modern crop production systems, but these have associated environmental and ecological consequences. Loss of nutrients from agricultural fields in the form of leaching and gaseous emissions has been the leading cause of environmental pollution and climate change. Ensuring the sustainability of crop production necessitates exploring other sources of nutrients and modifying prevalent nutrient sources. Nanotechnology, which utilizes nanomaterials of less than 100 nm size, may offer an unprecedented opportunity to develop concentrated sources of plant nutrients having higher-absorption rate, utilization efficacy, and minimum losses. Nanofertilizers are being prepared by encapsulating plant nutrients into nanomaterials, employing thin coating of nanomaterials on plant nutrients, and delivering in the form of nano-sized emulsions. Nanopores and stomatal openings in plant leaves facilitate nanomaterial uptake and their penetration deep inside leaves leading to higher nutrient use efficiency (NUE). Nanofertilizers have higher transport and delivery of nutrients through plasmodesmata, which are nanosized (50–60 nm) channels between cells. The higher NUE and significantly lesser nutrient losses of nanofertilizers lead to higher productivity (6–17%) and nutritional quality of field crops. However, production and availability, their sufficient effective legislation, and associated risk management are the prime limiting factors in their general adoption as plant nutrient sources.

**Keywords:** controlled release fertilizers, eutrophication, nanogels, encapsulated nutrients, slow released fertilizers

### **1. Introduction**

Intensive farming practices introduced and evolved since the inception of green revolution have been deemed unsustainable as the utilization efficacy of applied chemicals including mineral fertilizers has remained below 30% [1]. Fertilizers have taken axial role with respect to boosting crops yield and nutritional quality especially after the development of fertilizer responsive crop varieties. Among mineral nutrients, nitrogen is the first and foremost nutrient

required for crop plants as it is the constituent of chlorophyll and many proteins and enzymes and thus plays a significant role during the vegetative growth of crops. Nitrogen is absorbed by the plants in the form of nitrate (NO− 3) and ammonium (NH+ 4) [2]. Nitrogen is lost through the processes of nitrate leaching, de-nitrification and ammonia volatilization. Loss of mineral nutrients through leaching and runoff to surface and ground water along with abundant volatilization constitute growing concerns owing to economic losses and environmental pollution. Conventional application techniques are resulting in seriously overdosing of chemical fertilizers which has become evident through the phenomenon of eutrophication (algal growth on the surface of water bodies due to nutrients enriched water, which hampers oxygen supply to fish) in many European and North American countries. Moreover, nitrogen volatilization results in the release of nitrous oxides and thus being the greenhouse gases, contribute to the global warming. It is really unfortunate that modern profit-oriented farming systems encompass nitrogenous fertilizers use efficiency of only 45–50%, while the corresponding figure for phosphorous fertilizers has been reported to be only 10–25% [3].

It is also pertinent to mention that ammonium ions react with alkaline rain water which leads to the formation of ammonia gas that escapes into the atmosphere and thus becoming a source of environmental pollution. Whenever, there is excess of nitrogen, more and more nitrates and ammonium ions get accumulated in the leaves of crops especially leafy vegetables and become detrimental to human health. In addition, nitrate rich diets have been reported to be associated with numerous human diseases such as bladder and gastric cancer as well as methemoglobinemia [4]. It is being stressed to deliver the required quantities of active agents only where they are direly needed. Environmentalists and consumers call for reducing the use of synthetic fertilizers to decrease pollution and residue effect on form produces along with conserving agro-ecosystems.

Nanotechnology is a promising field of research which has the potential to offer sustainable remedies to pressing challenges confronted to modern intensive agriculture. Nanotechnology employs nanomaterials which typically have the size of 1–100 nm and this small size imparts unique characteristics and benefits to nanomaterials. In addition to numerous other benefits, large surface area offers opportunity for better and effective interaction of nanoparticles to target sites. Nanofertilizers hold potential to fulfill plant nutrition requirements along with imparting sustainability to crop production systems and that too without compromising the crops yield [5].

This chapter entails and attempts to fulfill the need to periodically compile and review the present state and advances on nanofertilizers and to spur interest for conducting further in-depth research. The ultimate goal is to synthesize and assess the role of nanofertilizers in boosting nutrients uptake and nutrients use efficiency, reducing losses through leaching and gaseous emissions along with reducing the risk of nutrient toxicity for ensuring food security achieved through higher productivity and economic turn outs by practicing the sustainable farming practices. This chapter briefly sheds light on the critical role of nanotechnology pertaining to modern farming practices, its potential in developing smart fertilizers, nanofertilizers and their different types of formulations, biological mechanism of nanofertilizers in plants, numerous advantages offered by nanofertilizers and field evidences of superior performances of nanoparticles in imparting critical characteristics to crop plants leading to higher productivity. Lastly, few limitations pertaining to the development and use of nanoparticles as plant nutrient source have also been described.

**295**

*Nano-Fertilizers for Sustainable Crop Production under Changing Climate: A Global Perspective*

Nanotechnology encompasses controlling matter at 1–100 nm dimensions for utilization in taking images, measurements and preparing models for making virtual predictions along with manipulation of matter at nanoscale. Like all other fields, the solid impact of nanomaterials is also being felt in agriculture sector. Previously, nanoencapsulation entailing encapsulation of active agents by microspheres of starch on a matrix having nanopores proved its resilience in accurately delivering the active agents to target sites [6]. These nanocapsules or micro-beads become attached to heir of bees in the similar fashion to pollens and keep parasites at bay owing to slow release of active agents gradually and slowly. Thus, nanoencapsulation resulted in minimum use of active agents and offered the maximum protection to bees against parasites. On the similar fashion, nanogels were developed which assist in controlled release of pheromones from insects to offer them protection against diversified pests. Nanoencapsulation has also yielded encouraging results for improving the fertilizer

**2. Critical applications of nanomaterials in agriculture**

use efficacy with significant reduction of active ingredients use [7].

In order to detect pathogen and to prolong the shelf life of packaged foods, nanosensors and nanobiosensors have given encouraging results. However, development of nanomaterials using nanotechnology is an evolving field of research and future is destined to witness extensive and multidimensional benefits in food production and preservation. In future, it will be impossible to ensure food and nutritional security without developing nanomaterials based technologies for food

**3. Nanotechnology's strategic potential in developing fertilizers of**

**4. Nanoscale fertilizers and their formulations**

Modern intensive farming systems utilize organic and mineral manures in order to supply essential plant nutrients, but this approach has resulted in serious deterioration of ecosystems and environment [8]. Loss of nitrogen as nitrous oxide and nitrates leaching has resulted in eutrophication and manifesting the impacts of global warming and climate change. Phosphate fertilizers have even lesser nutrient use efficacy (NUE) that has been reported to be below 20% [9]. Nanofertilizers have the potential to enhance NUE owing to higher nutrients uptake caused by smaller surface area of nanomaterials which increases nutrient-surface interaction. Along with boosting crops yield on sustainable basis, nanofertilizers hold potential to put a halt to environmental pollution caused by fertilizers. Slow release fertilizers (chemical compounds having slight solubility in water or other solvents and get broken down gradually and slowly by soil microbial population) coated with nanoparticles significantly reduced nitrate leaching and de-nitrification [10]. Moreover, controlled releasing fertilizers (have higher solubility in contrast to slow release fertilizers but are coated with materials which significantly reduce the exposure of active ingredient with the solvent resulting in controlled liberation of nutrients through diffusion) coated with nanomaterials for reducing surface area my provide excellent of source of supplying plant nutrients in times to come.

Different fertilizers inputs have been reported to be resized into smaller fractions through mechanical means or by employing specific chemical methods,

*DOI: http://dx.doi.org/10.5772/intechopen.89089*

production and agriculture.

**future**

*Nano-Fertilizers for Sustainable Crop Production under Changing Climate: A Global Perspective DOI: http://dx.doi.org/10.5772/intechopen.89089*


**4. Material culture of Paleolithic Ryukyu Islands**

In contrast to the relatively good preservation of human bone, few artifacts have been found from the Paleolithic sites of the Ryukyu Islands. Small amorphous flakes made of chert, quartz, and shales and sandstone cobble tools were found at several sites. Notably, several types of flake tools, which are often discovered at Paleolithic sites in mainland Japan, are restricted to islands north of Tokunoshima (**Table 1**). No chipped stone tools of Paleolithic age have been found from Okinawa or other southern islands.

All the stone materials were available at surrounding area of each site, and no obvious oversea transportation of stone material is known in the Paleolithic Ryukyu Islands. It seems a strange phenomenon since Paleolithic people came across the sea to the Ryukyu Islands and probably were familiar with maritime technologies, including oversea voyage [1, 11]. In mainland Japan, Paleolithic people translocated obsidians beyond the sea [53]. Although the possible oversea transportation of wild pigs more than 20,000 years ago is suggested in the southern and the central Ryukyus [31, 54], it is controversial as discussed later.

The small amorphous flakes and cobble tools are not suitable for typological study or analysis of usage. The Paleolithic and Neolithic cultures of the northern Ryukyus were closely related to those of Kyusyu Island, Japan, as mentioned above. However, the cultures of the central and southern Ryukyus have little in common with them. In the Holocene, the Jomon and subsequent mainland Japanese cultures influenced the prehistoric cultures of the central Ryukyus. In the southern Ryukyus, endemic prehistoric culture continued until 1000 years ago. After the tenth century, the inhabitants eventually constructed a cultural network

**139**

**Figure 4.**

*The Migration, Culture, and Lifestyle of the Paleolithic Ryukyu Islanders*

of the difficulty in oversea voyage between the islands.

throughout the Ryukyus. Cultural exchange across islands was difficult in prehistoric times. In summary, the Paleolithic people and culture of the central and the southern Ryukyus seem different from those of mainland Japan, probably because

The material culture of the Paleolithic Ryukyu Islands was a mystery until the authors and colleagues found a variety of shell artifacts and stone tools in the Paleolithic sediments of Sakitari Cave, Okinawa Island (**Figure 4**) [16, 55, 56]. The shell artifacts uncovered from the 20,000 to 23,000-year-old sediments (layer II) of Sakitari Cave consist of fishhooks, two types of scrapers, and two types of beads. The fishhooks were made of *Trochus* shells, similarly to the hooks found in the Paleolithic site of Timor [19]. Therefore, the fishhook technology distributed around the

western Pacific might be related to each other, though the locations of the sites differ; Sakitari Cave is a riverside cave and a seasonal campsite for seeking freshwater animal resources such as crabs and snails [16], while the Asitau kuru (Jerimalai Shelter), Timor, is a coastal site for marine resources such as fish and shellfish [5, 19, 57]. The Sakitari assemblage is unique in its lack of stone tools. There were three 13,000-year-old amorphous quartz flakes [55] and a 23,000-year-old tiny fragment of sandstone, which probably was used to grind fishhooks [16]. The Paleolithic stone artifacts of Sakitari Cave consist of only these four items, while many more shell artifacts were unearthed from the same sediments (**Figure 4**). Paleolithic shell flakes were reported in *Homo sapiens* sites of Philippines [58] and Indonesia [59] dating back to 30,000 years ago. It is known that pre-sapiens hominin also uses shellfish tools [60], so it may be difficult to talk about the relevance of culture with shellfish tools alone. But the Paleolithic culture of ISEA and Okinawa seems similar

However, Sakitari assemblage is quite unique in the dominance of shell artifacts. It probably is linked to the distribution of stone and shell materials on the island. There is no good stone material for making tools except the low-quality chert and small-sized quartz, which are available only in the northern half of Okinawa Island. In contrast to this stone distribution, shells are abundantly available on the island, especially in the coastal area. Sakitari Cave is located at the southern end of

*Isolated human teeth and artifacts obtained from layer II (20,000–23,000 years ago; a–p) and layer I (13,000–16,000 years ago; q–v) of Sakitari cave. (a) Human tooth (right lower third molar), (b) shell fishhook, (c–i) bivalve shell scraper (*Callista chinensis*), (j, k) bivalve shell scraper (*Septifer bilocularis*), (l, m) bivalve shell bead (*Sunetta kirai*), (n–p) Scaphopoda bead, (q) human tooth (right upper deciduous canine),*

*(r–t) quartz flake, (u, v) snail shell bead (*Pyrene testudinaria*).*

*DOI: http://dx.doi.org/10.5772/intechopen.92391*

in terms of shell flakes and fishhooks.

#### *The Migration, Culture, and Lifestyle of the Paleolithic Ryukyu Islanders DOI: http://dx.doi.org/10.5772/intechopen.92391*

*Pleistocene Archaeology - Migration, Technology, and Adaptation*

Cave indicates that Paleolithic people occupied Okinawa Island almost continuously from 30,000 to 13,000 years ago (**Figure 3**). Studies of the other Paleolithic individuals excavated from Shiraho-Saonetabaru Cave and the human bones recently discovered in Sakitari Cave near the Minatogawa site will provide insight into this

*Section of trench I and pit 1 of Sakitari cave. Partial human remains and animal remains consumed by humans were obtained from layers I to III. Artifacts were unearthed from layers I and II (see also Figure 4).*

In contrast to the relatively good preservation of human bone, few artifacts have been found from the Paleolithic sites of the Ryukyu Islands. Small amorphous flakes made of chert, quartz, and shales and sandstone cobble tools were found at several sites. Notably, several types of flake tools, which are often discovered at Paleolithic sites in mainland Japan, are restricted to islands north of Tokunoshima (**Table 1**). No chipped stone tools of Paleolithic age have been found from Okinawa or other

All the stone materials were available at surrounding area of each site, and no obvious oversea transportation of stone material is known in the Paleolithic Ryukyu Islands. It seems a strange phenomenon since Paleolithic people came across the sea to the Ryukyu Islands and probably were familiar with maritime technologies, including oversea voyage [1, 11]. In mainland Japan, Paleolithic people translocated obsidians beyond the sea [53]. Although the possible oversea transportation of wild pigs more than 20,000 years ago is suggested in the southern and the central

The small amorphous flakes and cobble tools are not suitable for typological study or analysis of usage. The Paleolithic and Neolithic cultures of the northern Ryukyus were closely related to those of Kyusyu Island, Japan, as mentioned above. However, the cultures of the central and southern Ryukyus have little in common with them. In the Holocene, the Jomon and subsequent mainland Japanese cultures influenced the prehistoric cultures of the central Ryukyus. In the southern Ryukyus, endemic prehistoric culture continued until 1000 years ago. After the tenth century, the inhabitants eventually constructed a cultural network

**138**

problem in the near future.

**Figure 3.**

southern islands.

**4. Material culture of Paleolithic Ryukyu Islands**

Ryukyus [31, 54], it is controversial as discussed later.

throughout the Ryukyus. Cultural exchange across islands was difficult in prehistoric times. In summary, the Paleolithic people and culture of the central and the southern Ryukyus seem different from those of mainland Japan, probably because of the difficulty in oversea voyage between the islands.

The material culture of the Paleolithic Ryukyu Islands was a mystery until the authors and colleagues found a variety of shell artifacts and stone tools in the Paleolithic sediments of Sakitari Cave, Okinawa Island (**Figure 4**) [16, 55, 56]. The shell artifacts uncovered from the 20,000 to 23,000-year-old sediments (layer II) of Sakitari Cave consist of fishhooks, two types of scrapers, and two types of beads. The fishhooks were made of *Trochus* shells, similarly to the hooks found in the Paleolithic site of Timor [19]. Therefore, the fishhook technology distributed around the western Pacific might be related to each other, though the locations of the sites differ; Sakitari Cave is a riverside cave and a seasonal campsite for seeking freshwater animal resources such as crabs and snails [16], while the Asitau kuru (Jerimalai Shelter), Timor, is a coastal site for marine resources such as fish and shellfish [5, 19, 57].

The Sakitari assemblage is unique in its lack of stone tools. There were three 13,000-year-old amorphous quartz flakes [55] and a 23,000-year-old tiny fragment of sandstone, which probably was used to grind fishhooks [16]. The Paleolithic stone artifacts of Sakitari Cave consist of only these four items, while many more shell artifacts were unearthed from the same sediments (**Figure 4**). Paleolithic shell flakes were reported in *Homo sapiens* sites of Philippines [58] and Indonesia [59] dating back to 30,000 years ago. It is known that pre-sapiens hominin also uses shellfish tools [60], so it may be difficult to talk about the relevance of culture with shellfish tools alone. But the Paleolithic culture of ISEA and Okinawa seems similar in terms of shell flakes and fishhooks.

However, Sakitari assemblage is quite unique in the dominance of shell artifacts. It probably is linked to the distribution of stone and shell materials on the island. There is no good stone material for making tools except the low-quality chert and small-sized quartz, which are available only in the northern half of Okinawa Island. In contrast to this stone distribution, shells are abundantly available on the island, especially in the coastal area. Sakitari Cave is located at the southern end of

#### **Figure 4.**

*Isolated human teeth and artifacts obtained from layer II (20,000–23,000 years ago; a–p) and layer I (13,000–16,000 years ago; q–v) of Sakitari cave. (a) Human tooth (right lower third molar), (b) shell fishhook, (c–i) bivalve shell scraper (*Callista chinensis*), (j, k) bivalve shell scraper (*Septifer bilocularis*), (l, m) bivalve shell bead (*Sunetta kirai*), (n–p) Scaphopoda bead, (q) human tooth (right upper deciduous canine), (r–t) quartz flake, (u, v) snail shell bead (*Pyrene testudinaria*).*

Okinawa Island, a stone-poor region. At present, it is about 2 km inland from the nearest coast, and it was 5–6 km away from the coast around 20,000–23,000 years ago. Therefore, the Paleolithic people of Sakitari Cave were able to access the coast easily and collect shells and other marine products such as fish. The unique material culture of Sakitari Cave was suitable for the island environment, and it represents the behavioral plasticity of the Paleolithic people and their capacity to adapt to the stone-poor, shell-rich island environment of Okinawa.


**1. Introduction**

The currently observed climate warming is unquestionable and evident among others through an increase in mean global air temperature [1]. The result is a decrease in the number of cold days and cold waves, although the changes are not as intensive as in the case of an increase in the frequency of hot days in the summer period. Current research conducted in Poland also points to a decrease in the number of cold days [2–4], although more evident changes are observed in the case of frequency of occurrence of days with strong frosts [5,6]. Despite the observed changes, during numerous winter seasons, several-day periods with very low air temperature have been recorded in Poland in recent years, for example in January 2016 and 2017 and February 2018 [7–9].

The meteorological conditions of the city and suburban areas are considerably different because of transformation of the environment. Due to a small contribution of natural plant surfaces, numerous vertical surfaces, as well as human activity, substantial amounts of heat are accumulated within the city during the day. They are then released to the atmosphere causing their slower cooling than in the surrounding areas [10]. Therefore, air temperature in the city is usually higher than in suburban areas.

In recent years, teledetection data have been applied in research on thermal conditions in urban areas increasingly frequently. An example of such research can be the analysis of relations between surface temperature and air temperature in the central part of Japan [11]. Voogt and Oke [12] emphasized that the urban heat island observed from thermal remote sensing data is the surface urban heat island (SUHI). Analyses of the urban heat island based on teledetection data have been conducted among others in Brno [13], Kraków [14], Madrid [15], and Pozna ´n [16]. Moreover, satellite teledetection data are increasingly frequently used for the analysis of lake surface temperature [17,18] and dates of the beginning of the vegetative season [19].

The study of the temperature field in urban areas at a resolution of 30 × 30 m allows distinguishing part of the city exposed to very high [20] and low values of air temperature. These thermal conditions are the cause of thermal stress both in the summer and winter seasons. In this study, the obtained maps allow to compare the different types of land cover according to Urban Atlas in terms of their thermal conditions. This paper constitutes a continuation of research on thermal conditions in the city during extreme air temperatures [20]. In the context of forecasts of the UN [21] showing an increase in the number of population living in cities, it is desirable to thoroughly investigate thermal conditions in the city both in the case of high and low temperatures. Taking this into consideration, the objective of the paper is: