**3. Research method**

Basically, tax is a representative resource mobilization tool of the advanced welfare state. Therefore, the level of tax burden in terms of public revenue should be discussed in relation to the fiscal sustainability of the welfare state. In order to cover welfare expenditures, a certain level of tax burden must be guaranteed, but if the tax burden is too high, it is not easy to increase the burden level [19, 65]. There are many reasons for the increase in incentives for tax avoidance and tax evasion. On the other hand, too low level of tax burden can also negatively impact fiscal sustainability. This is because there is a high possibility that sufficient financial

In addition, the structural characteristics of tax, especially tax equity, should be considered as the main factors. Because taxation inevitably violates the private ownership of a member, a lack of reasonable grounds for who owes taxes can lead to tax resistance and promote social conflict and division. Therefore, taxes must be imposed on the basis of justifiable grounds to secure political support for welfare states [12]. Indeed, the views on the taxation of the public are determined not by the level of burden but by the fairness of burden [27, 66]. The fairness of taxation can be defined as the principle of the ability to pay and the benefit principle. The former is the view that members of society are obliged to pay taxes regardless of the benefits they receive from the state as a member of the state. Accordingly, it is fair and desirable to pay taxes according to the ability to pay or to charge. On the other hand, the principle of benefit attaches importance to the exchange of benefits from tax and public goods, with the view that the taxpayer will pay the benefits of the provision of national services. In other words, it is fair to pay fair compensation for benefits.

Tax on the basis of each principle can have a different impact on the fiscal sustainability of a welfare state. First, in relation to the principle of ability to pay, direct taxation with a high tax rate can have a negative impact on economic growth by lowering incentives for labor and high tax evasion in the high-income class. On the basis of this, the enhancement of tax progressivity may hinder the fiscal sustainability of the welfare state. However, it is also true that people are not always opposed to high-level taxation [67]. In addition, the Progressive Tax System can be designed to lower income inequality by designing the higher income group to pay a higher tax burden than the low-income group, thus contributing to social sustainability by preventing

conflicts between taxpayers and beneficiaries due to worsening income distribution.

the level of equity is raised to the level of horizontal equity.

Meanwhile, the horizontal equity, one part of ability to pay, is also considered. Related to this, the possibility of taxation of capital and property is lowered due to the intensification of tax competition caused by globalization [68], and advanced welfare countries have shown a tendency to rely on a consumption tax rather than an income tax. Unlike in the past, the gap between the labor and the capital is significantly increasing, while the gap between the labor and the consumption is significantly decreasing. Recalling that vertical equity and horizontal equity are inseparable, and that inequity on one side is not offset by the achievement of equity through other principles [69], the inhibition of confidence that the tax burden is fairly distributed can make it difficult and may not only lead to a lack of financial resources to support the welfare state, but also to difficulties in obtaining political support. Thus, the widening gap between tax sources have a negative effect on the fiscal sustainability of the welfare state as

On the other hand, social security contributions and the contributions of the private sector are closely related to the principle of benefit. This is mainly used for specific social security

resources are not available for public expenditure.

104 Taxes and Taxation Trends

#### **3.1. Analysis target and timing**

The analysis of national finances should incorporate careful selection of the analysis target because analysis results may be different depending on which country is analyzed. Because, there is a huge gap between the high- and low-income countries' socioeconomic development levels, especially in terms of the level of public expenditures, the taxation capacity, and the tax structure, so it is necessary to analyze the two groups separately. This chapter analyses the 17 OECD countries, and considering the possibility of data access and the analysis of OECD major countries is reasonable in order to draw implications in the establishment of a welfare state with a financial balance. Specifically, the analysis includes Austria, Belgium, Canada, Denmark, Finland, France, Germany, Greece, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, the United Kingdom, and the United States.

Next, this chapter analyzes the fiscal sustainability of welfare states over the course of 28 years, from 1986 to 2013, while the independent variables, including tax structure and welfare expenditures, are based on the period from 1985 to 2012, lagged term (*t* − 1), considering temporal precedence as a requirement for causality.3 Those OECD countries have undergone a series of welfare and tax reforms to alleviate the burden of national financing, having experienced severe economic downturns during the mid to late 1970s. Since the effects of reform are not immediately

3 This is based on the fact that the expenditures for that year are carried out in accordance with the previous year's budget plan. The analysis is also conducted by adding value from 5 years prior to reflect the medium-term fiscal plan in highincome countries as a 5-year plan. In the determinants of fiscal sustainability of the welfare state, the correlation between welfare expenditures and fiscal capacity may not be reflected within a short time frame. In particular, the impact of fiscal capacity on welfare expenditures is likely to be seen in the medium term, because in high-income countries, the level of public expenditures is usually determined through the medium- and long-term financial management of the country.

apparent, but, rather, come after a certain period of time, this study has limited its analysis to late 1980s, specifically since 1986 (independent variables since 1985). In addition, until early 1990s, most advanced welfare states demonstrated a relatively moderate increase in national debt. However, since mid-1990s, sovereign debt has soared, and concerns about the national debt have become more widespread since the 2008 global financial crisis. Therefore, it is possible to derive timely policy implications for ensuring the fiscal sustainability of the welfare state by analyzing the period when the national debt had soared and a widespread financial crisis occurred.

an important role in resource mobilization to cover future spending, as well as cushioning against unexpected risks [77, 78]. In other words, the issue of fiscal space is a question of whether countries can finance their obligations, including social security, without sacrificing economic growth and stability based on fiscal sustainability [5, 50]. Therefore, fiscal space can

How Does a Welfare State achieves Fiscal Sustainability? A Study of the Impact of Tax Equity

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In order to derive the abovementioned fiscal space as shown in **Figure 1**, it is necessary to estimate the fiscal reaction function and select the appropriate gap between the interest rate and the growth rate [57]. This is because it is necessary to determine the debt limit of each country on the basis of the intersection of the estimated base on the fiscal reaction function and the interest repayment schedule. This study estimates the fiscal reaction function through pooled time series analysis and uses a vector autoregressive model for estimation to establish the gap between the appropriate interest rate and the growth rate. The description of variables used

Next, one of the most important points to be considered in determining the national debt limit, along with the estimation of the fiscal reaction function, is how to define the long-term

similar to the works of Polito and Wickens [62, 63]. This is because it not only avoids arbitrary problems, but also reflects the endogenous relationship between the interest rate and the national debt level (**Table 2**). In this study, the autoregressive model is used to model the endogenous relationship between the interest rate and the national debt, adding government revenue, government spending, debt, the economic growth rate, the inflation rate, and short- and long-term interest rates [62, 63]. The gap between these estimates and the average real growth rates of the countries from 1985 to 2013 are used to calculate the debt limit and determine fiscal space based on this. The contents and data sources of the variables used for

The second dependent variable is public welfare expenditures. This is the level of public (general government) social welfare spending that reflects public welfare efforts or the level of benefits enjoyed by the public. Total public welfare expenditures divided by the gross domestic product is used to control differences in the welfare expenditure level according to the

The dependent variable is the primary balance, and the independent variables are the financial factors (national debt, public welfare expenditures, output gap, inflation rate), the economic structural factors (unemployment rate, service industry ratio, portion of involuntary part-time work, economic openness, aging rate, future old age portion), and political and financial institutional factors (election, change of ideology, mandatory political system, concentration index, fiscal rule index). In this study, it is based on the works of Ostry et al. [56] and Ghosh et al. [57], but some variables are

Ostry et al. [56] determined long-term interest rates in two ways. The first assumes that the observed interest rate itself reflects the perceived probability of bankruptcy of a country, so the current market rate is used as the long-term interest rate. In this case, it is possible to overestimate the maximum value of sustainable debt by overlooking the fact that the interest rate rises as the debt level approaches its limit, and the risk of bankruptcy increases. An alternative method of overcoming this limitation is to use the interest rate, which is calculated by taking into account the endogenous relationship between debt levels and interest rates. Specifically, they used the calculated interest rate, assuming a recovery rate of 90% when bankruptcy occurred. Alternative methods which they used help to accurately estimate fiscal space by reflecting endogenous relationships between interest rates and macroeconomic variables. However, the abovementioned study does not provide a clear basis for assumptions used in interest rate estimation. Therefore, it is not free of the problems caused by an arbitrary definition of the recovery rate [80]. In order to overcome these limitations, this study

4

This study estimates the interest rate through vector autoregulation (VAR),

be a useful tool in examining the fiscal sustainability of the welfare state.

for estimating the fiscal reaction function is shown in **Table 1**.

estimating the fiscal reaction function are shown in **Table 3**.

level of economic scale by country.

excluded in consideration of multicollinearity.

uses the estimates through VAR.

4

5

interest rate [56].5


**5.2.2 Particle tracking and retention**

166 Hydrodynamics – Natural Water Bodies

Fig. 7. Depth-averaged current field maps for (a) Wreck Bay and (b) Sand Hills Bay during a dominant falling tide combined with sea-breeze regime. Current vectors depict well-formed,

closed looping circulation on the up-shore reef arm (circled), causing both bays to be

expanded beyond the reef.

Under only the rising tide regime, 19 % particles remained in Sand Hills Bay after 9 hrs. The rising tide combined with land-breeze regime increased the remaining particles to 22 % after 9 hrs. When the sea-breeze dominated, however, combined with the rising tide the retention dropped to 2 % in 9 hrs. Therefore particles were likely to remain trapped in Sand Hills Bay the longest when introduced at the beginning of the rising tide cycle during a land-breeze regime and were likely to be flushed out the quickest if introduced during the sea-breeze with mid-falling tide.

Fig. 8. Reef gyre extension measurements for Wreck Bay and Sand Hills Bay during 18 hrs (1.5 tidal cycles) of highest Y-component current speeds recorded in Wreck Bay. Tracks are displayed as time progresses in 3-hr increments for new particles introduced into the bay every three hours. Gyres undergo expansion and contraction but are always present.

Under only the falling tide regime, 36 % particles remained in Wreck Bay after 6 hrs. The falling tide combined with land-breeze or sea-breeze regime decreased the remaining particles to 6 % and 10 % respectively after 6 hrs. Therefore particles were likely to remain trapped in Wreck Bay the longest if introduced at the beginning of the falling tide cycle and were likely to be flushed out the quickest if introduced at the beginning of the rising tide.


2.2.2. Satellite Data

To monitor the growth stages of major crop types cultivated from spring to autumn in the study area, we examined Sentinel-1 and Sentinel-2 data acquired in the time period from late March to early October. After discarding Sentinel-2 acquisitions with too much cloud cover (more than 20%), there were in total eight Sentinel-2 acquisitions left. The available number of the Sentinel-1 and Sentinel-2 acquisitions for each month is summarized in Table 4.

The employed Sentinel-1 data is the level-1 interferometric wide swath (IW) single look complex (SLC) data. In total, 18 acquisitions were used (Table 5).

**Table 5.** Metadata of the data stack of Sentinel-1 (S1) interferometric wide swath (IW) single look complex (SLC) data using the parameters from the first image. These values remain very close for all subsequent acquisitions.

The Sentinel-2 data used in this study is the MSI Level-1C data (Table 6). In total, eight acquisitions were used, spanning the time from 9 May 2018 to 11 October 2018.

**Table 6.** Central wavelength and bandwidth of different spectral bands of Sentinel-2 data used in this research.


**Author details**

Monia Renzi\*

Address all correspondence to<EMAIL_ADDRESS>
Department of Environmental Science, Via Mattioli, University of Siena, Italy

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**Section 3**

**Treatment**

**Section 3**


**Yeonchul Cho 1, Kihun Kim 1, Jaewoo Ahn 1,\* and Jaeheon Lee 2**

Received: 26 May 2020; Accepted: 25 June 2020; Published: 28 June 2020

**Abstract:** In order to manufacture lithium carbonate to be used as a raw material for a secondary lithium battery, lithium sulfate solution is used as a precursor, and the concentration of lithium is required to be 10 g/L or more. Electrodialysis (ED) was used as a method of concentrating lithium in a low-concentration lithium sulfate solution, and multistage concentration (MSC) electrodialysis was used to increase the concentration ratio (%). When MSC was performed using a raw material solution containing a large amount of sodium sulfate, the process lead time was increased by 60 min. And the concentration ratio (%) of lithium decreased as the number of concentration stages increased. In order to remove sodium sulfate, methanol was added to the raw material solution to precipitate sodium sulfate, and when it was added in a volume ratio of 0.4, lithium was not lost. Using a solution in which sodium sulfate was partially removed, fourth-stage concentration ED was performed to obtain a lithium sulfate solution with a lithium concentration of 10 g/L.

**Keywords:** electrodialysis (ED); lithium; lithium-ion battery; lithium sulfate; multistage concentration (MSC)


**4.2 Educational and research usage of videolaryngoscopy**

Videolaryngoscopy is an excellent tool for teaching because it allows unhindered supervision of the intubation procedure [60]. In practice, it proves to be a particularly useful tool for introducing beginners to intubation procedures. Moreover, depending on the choice of blade and type of device, it allows the education of different advanced intubation techniques comparable to direct laryngoscopy, including a combination of different techniques [61]. The supervisor follows the procedure performed by the student in a real time with possibility to guide the student verbally or manually with prompt feedback of what has been done. To gain an expertise in situations of unexpected difficult airway, it is necessary to learn how to manage expected difficult airway during routine intubations. Evidence suggests that using videolaryngoscopy during intubation significantly helps in mastering the intubation technique by the trainees even in the most sensitive populations for intubation such as newborns [62, 63].

As a relatively new technique with plenty of new innovative devices, videolaryngoscopy is currently a fruitful subject for the researchers interested in airway management. However, as the indications for its use spread, videolaryngoscopy may be used as an auxiliary tool for the other static and dynamic researches that include visualization of oropharynx and the upper airway.

#### **4.3 Medicolegal usage of videolaryngoscopy**

The possibility of recording the videolaryngoscopy procedure and subsequent reviewing of the recorded material offers the possibility of a thorough analysis of the procedure, which itself lasts a limited short time. The first such application was described in 1987 with the purpose of recording the vocal cords during vocal therapies [64]. Recorded material during videolaryngoscopy can be reproduced multiple times, which can be useful in subsequent analyzes that can be done for various medicolegal purposes (Video 1, Video 2, Video 3, and Video 4). It is especially useful to use archived images and videos as the part of preoperative preparation in patients for whom difficult intubation is expected. Another practical indication could be the examination of vocal cords after thyroid surgery and recording for medicolegal issues.


*Article* **Intelligent Safety Assessment of Prestressed Steel Structures Based on Digital Twins**

**Zhansheng Liu 1,2,\*, Guoliang Shi 1,2, Zedong Jiao 1,2 and Linlin Zhao 1,2**

**Abstract:** In the development process of intelligent construction, the safety assessment of prestressed steel structures as an important research direction has become more and more attractive in academia. Digital twins (DTs) is the key technology to realize intelligent construction. The virtual and real interaction of the DTs can provide an efficient management and control mechanism for the construction process. This research proposes an intelligent safety assessment method of prestressed steel structures based on DTs. In this research method, the structural safety assessment is divided into two aspects: performance analysis and maintenance. By analyzing the characteristics of the construction safety assessment, a DTs framework for construction safety assessment is built. Driven by the DTs framework, a physical space model and a virtual space model are constructed. On the basis of virtual and actual interaction, multidimensional information fusion of time and space is carried out to realize the analysis of structural safety performance. On this basis, the paper establishes a Bow-tie model for the maintenance modeling of unsafe construction events. Moreover, the theoretical method formed is applied to the construction of a symmetrical structure (wheel–spoke cable truss). The validity of the method is verified by comparing the cable force calculated by the theoretical method and measured on site. The assessment method driven by the DTs ensures the structural safety and improves the intelligence level of safety management and control of the structure construction.

**Keywords:** digital twin; prestressed steel structure; construction process; safety assessment; intelligent construction


**3.4. Stage 4 (remodelling/maturation phase)**

This is the fourth and final stage of wound healing and it might extend up to 2 years from the time of tissue trauma. During this stage, the raised and reddish scar becomes more flat, smooth and lighter in colour. This relates to a reduction in the blood supply. Mature scars are hairless, avascular and do not contain sweat or sebaceous glands.

Collagen fibres are reorganised to maximise tensile strength, a process called remodelling and it is stimulated by macrophages [8].

Hypertrophic scar and keloid formation are two known abnormalities associated with this stage. While the former takes place after initial repair, the latter occurs sometime after healing is completed and continues to grow afterwards [9]. Keloid formation occurs 10 times more commonly in the Black Afro-Caribbean population in comparison to Caucasian population [9] **Table 1**.


**4.3 Suspected cases and confounding factors during liver transplantation**

MH syndrome exhibits a wide range of symptoms including tachycardia, progressive elevation of the exhaled CO2, arrhythmias, hyperthermia, profuse sweating, fever up to 40ºC, cyanosis, poor skin perfusion and blood pressure instability31. The only physical sign typical of MH is muscular rigidity, although it may be hard to detect due to the limited access for physical evaluation. MMR may be observed upon anesthetic induction and is predictive of the syndrome32-33.

However, tachycardia, arrhythmias, poor skin perfusion, blood pressure instability and other subtle manifestations of the initial phase of HM are commonly observed during liver transplantation, as it involves large volume paracentesis, manipulation of major vessels, massive bleeding and reperfusion syndrome116. Inadequate anesthetic depth and pyrogenic reaction can mimic some of those symptoms.

Most of liver transplant patients are maintained normothermic with the aid of forced warm air mattresses. After the reperfusion, the graft begins to produce heat by its exothermic metabolic reactions and the addition of this new source of heat may lead to hyperthermia. However this temperature rise only begins lately in the course of the surgery, usually is minimized by turning off the mattresses and rarely exceeds 39ºC. Another potential source of confusion in the diagnosis of MH is the use of defective equipment for patient heating (leading to overheating and sweating) or poorly calibrated temperature monitors.

Some situations can induce severe intraoperative Systemic Inflammatory Response Syndrome (SIRS) during liver transplantation, like bacteremia/sepsis, acute rejection and graft non-function. Although SIRS may show up with hyperthermia117, other MH symptoms like severe hypercapnia are not usually present in these cases.

Hypercapnia may have several causes during liver transplantation, like intrinsic pulmonary diseases, accumulation of lung secretions in the airway, lung compression by retractors, inappropriate mechanical ventilation, exhaustion of soda lime and faulty carbon dioxide monitoring. However, after checking and solving all these issues, the maintenance of a progressive rise on end-tidal carbon dioxide becomes a strong indicator of malignant hyperthermia.

To make diagnosis even problematic, most early laboratory manifestations of malignant hyperthermia, such as respiratory and lactic acidosis and hyperkalemia, are also commonly observed in anesthesia for liver transplantation. The reasons for respiratory acidosis were described above. Lactic acidosis frequently results from the combination of tissue hypoperfusion and decreased hepatic clearance of lactate during the anhepatic phase. Hyperkalemia during liver transplantation may have several reasons, like poor baseline renal function, large and rapid transfusion of red cells and high-potassium content of preservation solutions. Nonetheless, mixed venous oxygen saturation (SvO2) may have a lower value when MH is suspected. Due to severe increase in cellular oxygen consumption,

MH syndrome exhibits a wide range of symptoms including tachycardia, progressive elevation of the exhaled CO2, arrhythmias, hyperthermia, profuse sweating, fever up to 40ºC, cyanosis, poor skin perfusion and blood pressure instability31. The only physical sign typical of MH is muscular rigidity, although it may be hard to detect due to the limited access for physical evaluation. MMR may be observed upon anesthetic induction and is

However, tachycardia, arrhythmias, poor skin perfusion, blood pressure instability and other subtle manifestations of the initial phase of HM are commonly observed during liver transplantation, as it involves large volume paracentesis, manipulation of major vessels, massive bleeding and reperfusion syndrome116. Inadequate anesthetic depth and pyrogenic

Most of liver transplant patients are maintained normothermic with the aid of forced warm air mattresses. After the reperfusion, the graft begins to produce heat by its exothermic metabolic reactions and the addition of this new source of heat may lead to hyperthermia. However this temperature rise only begins lately in the course of the surgery, usually is minimized by turning off the mattresses and rarely exceeds 39ºC. Another potential source of confusion in the diagnosis of MH is the use of defective equipment for patient heating

Some situations can induce severe intraoperative Systemic Inflammatory Response Syndrome (SIRS) during liver transplantation, like bacteremia/sepsis, acute rejection and graft non-function. Although SIRS may show up with hyperthermia117, other MH symptoms

Hypercapnia may have several causes during liver transplantation, like intrinsic pulmonary diseases, accumulation of lung secretions in the airway, lung compression by retractors, inappropriate mechanical ventilation, exhaustion of soda lime and faulty carbon dioxide monitoring. However, after checking and solving all these issues, the maintenance of a progressive rise on end-tidal carbon dioxide becomes a strong indicator of malignant

To make diagnosis even problematic, most early laboratory manifestations of malignant hyperthermia, such as respiratory and lactic acidosis and hyperkalemia, are also commonly observed in anesthesia for liver transplantation. The reasons for respiratory acidosis were described above. Lactic acidosis frequently results from the combination of tissue hypoperfusion and decreased hepatic clearance of lactate during the anhepatic phase. Hyperkalemia during liver transplantation may have several reasons, like poor baseline renal function, large and rapid transfusion of red cells and high-potassium content of preservation solutions. Nonetheless, mixed venous oxygen saturation (SvO2) may have a lower value when MH is suspected. Due to severe increase in cellular oxygen consumption,

(leading to overheating and sweating) or poorly calibrated temperature monitors.

like severe hypercapnia are not usually present in these cases.

**4.3 Suspected cases and confounding factors during liver transplantation**

drugs to administer to these patients115.

predictive of the syndrome32-33.

hyperthermia.

reaction can mimic some of those symptoms.

Local anesthetics, nondepolarizing muscle relaxants, barbiturates, benzodiazepines, droperidol, ketamine, nitrous oxide, opioids, propofol and vasoactive drugs are all safe the SvO2 of patients with MH is usually low. Such low values are not usually seen in liver transplantation, since cirrhotic patients generally have systemic shunts and a hyperdynamic circulation, yielding high values of SvO2118.

### **4.4 Intraoperative differential diagnosis**

Several disorders share similarities with MH and may be confused with the syndrome. Neuroleptic malignant syndrome is characterized by hyperthermia, acidosis, hyperkalemia and myoglobinuria following use of a wide variety of neuroleptics, especially haloperidol. Patients taking mono-amino-oxidase inhibitors who receive meperidine may present with hyperthermia, acidosis and an increase in creatine kinase concentration, what may become fatal. Other conditions that may resemble the MH situation include – but are not limited to – : iatrogenic overheating, thyroid storm in thyrotoxicosis, hypothalamic lesions, heat illness, pheochromocytoma, and intrathecal injection of high osmolar contrast agents, cocaine or ecstasy overdose, hypoxic encephalopathy and sudden cardiac arrest in a patient with occult myopathy119. None of these disorders, however, is frequent in liver transplant patients.

#### **4.5 Investigation of suspected cases**

Investigation of susceptibility should begin upon clinical suspicion during preoperative evaluation.

Creatine kinase (CPK) dosage during rest has been suggested as a component of a clinical grading scale to predict malignant hyperthermia susceptibility for patients with positive family history; increased resting values could suggest myopathy and MH susceptibility120. The use of this test is not recommended for the general population as it would yield an unacceptably high rate of false positive results. Cirrhotic patients habitually have reduced muscle mass and decreased exercise-tolerance, thus high CPK results in patients with positive family history are suggestive.

Caffeine-Halothane Contracture Test (CHCT) is considered the gold standard for the diagnosis of MH. This test, which uses a small piece of live muscle from biopsy, assesses the muscular contractility in response to increasing concentrations of halothane and caffeine exposure. It has a 97% sensitivity and 78% specificity121. Even in typical cases, CHCT is beneficial to guide the necessity of investigation of relatives. MH is inherited in an autosomal dominant pattern, meaning that if one of the parents has the disease, the risk of his or her passing it down to sons or daughters is 50 per cent122. Muscle biopsy for CHCT should be avoided in patients whose weight is less than 20 kg, patients under chronic dantrolene or calcium channel blocker therapy and in the first three months after a MH crisis, because muscle lesion may still be present123. A muscle sample is removed from the *vastus lateralis* or *medialis* or *rectus abdominis*. As the tests have to be finished up to five hours after its collection, patients have to be transported to specialized MH centers. The procedure is performed under general or regional anesthesia, obviously avoiding potentially trigerring agents and keeping dantrolene immediately available123. CHCT is indicated in patients preoperatively deemed at risk for MH, postoperatively in patients with a typical MH crisis during liver transplantation and the relatives of the patients with a positive CHCT.


*2.2. Synthesis of Activated Carbon*

Eucalyptus leaves were collected from the surrounding of NIT Warangal, India. Eucalyptus leaves were pre-treated with DI water and dried, oil was extracted from the eucalyptus leaves using steam distillation. After extracting oil, the left over bio-waste (eucalyptus residue) was dried at 80 ◦C for 8 h. The dried biomass was crushed in fine powder by using a mortar and pestle and sieved with a mesh size of 60 to 80. The resultant eucalyptus leaves powder (ELP) was carbonized in tubular furnace under an inert atmosphere (nitrogen) with an inlet flow rate of 75 mL min−1 . The furnace consist of PID temperature controller with long tube of length 160 cm and the diameter of 40 mm. Inside the tubular furnace a quartz boat is used to hold the sample. The ELP was carbonized at 300 ◦C for 1 h. @ 5 ◦C min−1 . Next, the samples were mixed with ZnCl2 in the proportion of 1:4 (ZnCl2: sample). The obtained mixture was agitated to create a homogenous slurry and dried for 12 h and at 100 ◦C. The resultant products were then carbonized to 400–800 ◦C for 1 h. @ 5 ◦C min−1 under an inert atmosphere. The product was dried at 80 ◦C for 10 h. After the process was completed, the material was cooled and taken from the tubular furnace. The carbonized material was then rinsed with 1 M HCl till the pH reached 7 and then filtered and dried for 12 h at 100 ◦C. The resultant product of this post-treatment was activated carbon, abbreviated as AC. The AC was labelled as AC-x, where x (*x* = 400 to 800 ◦C) represents the carbonization temperature and stored AC-x materials in sample bottles.


*2.8. Riboprobe Synthesis*

mRNA probes to identify NfuNT-6 mRNA were synthetized by in vitro transcription (IVT) using MAXIscript™ SP6/T7 in vitro transcription kit (Invitrogen by Thermo Fisher Scientific–Catalogue number AM1312, Carlsbad, CA, USA) and following the manufacturer's instructions. 1 μg of DNA template was transcribed to RNA in 20 μL volume reaction, using NfuNT6 primer associated with the T7 promoter sequence (left 5- -TGGTCCTGCTCTTCCTGATC-3- ; T7 right 5- -GGTAATACGACTCACTATAGG\_GTGTGTTTGAAGCTGCTCGA-3- ) and a DIG RNA Labeling Mix, 10× conc (Roche, Basel, Switzerland) containing digoxigenin labeled uracil. After IVT reaction, product was briefly centrifuged and incubated at 37 ◦C for 1 h. Then, 1 μL of turbo DNase 1 was added, sample was mixed well and incubated for 15 min at 37 ◦C. 1 μL of EDTA 0.5 M was added to stop the reaction. Reaction product was analyzed by gel electrophoresis and quantized.