3. Staphylococcus aureus

S. aureus is a Gram-positive coccus with a diameter of 0.5–1.5 μm, grouped as single cells, in pairs, tetrads, short chains, or forming a conglomerate in a cluster of grapes. This microorganism was first described in the year 1880, in Aberdeen, Scotland, by the surgeon Alexander Ágoston. The name comes from the Greek σταφυλόκοκκος, which is composed of the terms "staphylé," meaning cluster, and coccus, meaning grain or grape, and from the Latin "aureus" which means golden, that is to say "cluster of golden grapes."

They are non-motile bacteria, not sporulated, with no capsule (although there are some strains that develop a slime capsule); they are facultative anaerobes. Most staphylococci produce catalase (enzyme capable of dismutating hydrogen peroxide in H2O+O2), characteristic that is used to differentiate its sort from others like Streptococcus and Enterococcus. In 1961, the first report was made on the existence of a methicillin-resistant Staphylococcus aureus [18].

### 3.1 Staphylococcus aureus and endothelial cell

S. aureus is a pathogen that causes significant morbidity and mortality worldwide [2]. It is the leading pathogen associated with life-threatening bloodstream infections [19].

Although S. aureus is mainly known as an extracellular pathogen, it has been shown to invade and survive within endothelial cells, both within vacuoles and free in the cytoplasm, which implies that the bacteria can escape from the phagolysosome. S. aureus tends to infect endovascular tissue. It is believed that this ability contributes to causing a persistent endovascular infection with endothelial destruction.

#### 3.2 Endothelial cell and Staphylococcus aureus ingestion

On the other hand, the death of endothelial cells after the invasion of S. aureus occurs at least in part by apoptosis, as demonstrated by DNA fragmentation and changes in nuclear morphology. Apoptotic changes are observed as early as 1 h after infection of endothelial cells [18]; they are considered to function as nonprofessional phagocytes, being able to ingest S. aureus [20, 21] following the adhesion of this to endothelial cell monolayers; invasion can occur through ingestion by endothelial cells.

For the internalization of S. aureus, adherence seems to be necessary, since the use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. Studies show that living intracellular S. aureus induces apoptosis of endothelial cells and that this depends on a factor associated with viable organisms, since dead S. aureus (by ultraviolet light) also internalized does not induce it [18]. The process has been observed through electron transmission micrographs of bovine aortic endothelial cell monolayers infected with S. aureus, showing phagocytosis following a sequence of events: (I) adhesion of S. aureus to the endothelial cell, (II) formation of cupshaped processes on the surface of the endothelial cell underlying the adherent bacteria, and (III) elongation of the cup and engulfment of bacteria within a phagosome [19].

### 3.3 Heme prosthetic group and Staphylococcus aureus

To colonize a vertebrate host, S. aureus requires numerous nutrients, such as the prosthetic group heme. The requirement can be met through two distinct mechanisms: importing exogenous heme through dedicated machinery or synthesizing endogenous heme from own metabolic precursors. These two mechanisms are necessary for a complete virulence of S. aureus [22, 23]. Once acquired, heme is used for several cell processes. The intact heme is used as a cofactor for enzymes [24], including cytochromes in the electron transport chain, catalase for the detoxification of reactive oxygen species, and bacterial nitric oxide synthase (bNOS).

Although the S. aureus requires heme, its excess is toxic to the germ, so it has a mechanism for hem detoxification through a hem sensor system (HssRS) that induces the expression of a hem regulator transporter (hrtAB) [25]. The suppression of the components of this route affects the virulence of S. aureus. This ability to detoxify heme is critical to survive in the host. Also, the synthesis of nitric oxide is important for the bacteria to survive. Bacteria encode genes similar to nitric oxide synthetase in mammals, which leads to the characterization of the nitric oxide synthase hemoprotein (bNOS) [26].

#### 3.4 Staphylococcus aureus as a pro-inflammatory agent

The S. aureus contains molecules such as peptidoglycan and lipoteichoic acid, potent stimulants for the production of cytosines such as TNF-α, IL-4, IL-6, IL-8, IL-12, IL-1be, growth-regulated oncogene (GRO) alpha, and regulated upon activation, normal T-cell expressed, and secreted (RANTES). RANTES has a chemotactic function to perform leukocyte recruitment to areas of infection in addition to inducing tumor necrosis factor alpha and interleukin 1. Elevated levels can persist for 7–14 days [27]. As we can observe, S. aureus activates in a very important way the process of inflammation.

#### 3.5 Staphylococcus aureus and blood stream infections in infective endocarditis

Circulatory blood stream infections (positive blood cultures) occur in patients with intravascular prosthetic devices as the most common source of infections related to health care [28]. MRSA was the most frequent pathogen in these types of infections with a consistent increase in the isolates of MRSA [29–31]. In the EU, epidemiological surveillance data on bloodstream infections show a marked variability among the member countries that make up a proportion of S. aureus that is resistant to methicillin, ranging from less than 1% to more than 50%. In addition to infections associated with health care, new MRSA strains have emerged in their communities as human pathogens associated with livestock [32].

#### 3.6 Endocardial endothelium and myocardial capillary endothelium

The anatomical and physiological barriers of cardiac protection such as the endothelium can be compromised in its structure when areas of turbulence and injury are generated, producing an area exposed to infection. The intracardiac cavities have a cell layer called endocardial endothelium (EE) that covers the endocardium of the atria, ventricles, and all their anatomical components (papillary muscles, chordae tendineae, and heart valves). The EE acts as an active mechanism of biological heart-blood barrier, since it interacts dynamically with cardiomyocytes allowing direct communication and signaling between both types of cells. This

Infective Endocarditis: Inflammatory Response, Genetic Susceptibility, Oxidative Stress… DOI: http://dx.doi.org/10.5772/intechopen.84908

electrochemical communication between the cells of the EE and the cardiomyocytes allows a rapid intracellular electrochemical propagation and amplification of the functional properties of the EE.

Signaling between cardiac endothelial cells (EE and myocardial capillary endothelium) and cardiomyocytes influences cardiac growth, contractile performance, and rhythmicity. The network of Purkinje fibers and the subendocardial neural plexus (parasympathetic nervous system) is immediately below the endocardial endothelium (EE) and participates in the endothelial control of cardiac rhythm. Endothelin-1 (Et-1), nitric oxide (ON), prostaglandins (PGI2), prostacyclin (AI and AII), angiotensin I and II, and vascular endothelial growth factor (VEGF) are involved in these processes.

The endothelium that covers cardiac structures is at the vascular level, the myocardial capillary, and the endocardium; its activation includes changes in the endothelial phenotype as part of the physiological adaptive response to several possible injuries and stressors. The dysfunction of the endothelium implies a deregulated response that is not useful and that can be permanent.

One of the clinical disorders that selectively damage the endocardium and subendocardial interstitial tissue is endocarditis. This entity causes activation of the vascular and endocardial endothelial system, as well as poor adaptation or failure characterized by hemodynamic abnormalities, neurohormonal imbalance, cytokine expression, and endothelial dysfunction [33].

Infective endocarditis is an anatomoclinical entity characterized by microbial infection of the valvular or parietal endothelium or both; it is located predominantly on the left side of the heart, although it can also occur in the right (e.g., endovenous drug), which produces inflammation, exudation, and proliferation of the endocardium. The most characteristic lesion is the vegetation, constituted by an amorphous mass of platelets and fibrin, of variable size, which contains multiple microorganisms and scarce inflammatory cells (fibrinoplaquetary thrombus) [34]. This type of lesions generates metastatic infection in other anatomical territories, for example, the central nervous system, apostematous meningitis, myocarditis, pyelonephritis, and splenic abscesses which are at risk of rupture [35, 36].

#### 4. Clinical manifestations

The clinical manifestations of infective endocarditis are acute rapidly progressive or subacute; the pathophysiological processes of both are explained by immunological and vascular phenomena, such as inflammatory response, mediators of inflammation triggered by a maladaptive response to an infectious process, aggregation of immune complexes, infectious vasculitis, and peripheral microembolism [34, 37]. Depending on the affected cardiac cavity (right/left) or valvular system, the clinical manifestations will be due to the aforementioned processes [38] (Table 1).

#### 4.1 Anatomopathological changes

The anatomopathological changes due to the formation of vegetations in the valvular ring and/or in the leaflets cause an anatomical alteration. If this anatomical alteration generated by a vegetation prevents valvular closure, it will be expressed as a murmur of valvular insufficiency and in severe cases such as microembolisms septic and non-septic and cardiac failure [34, 37].

#### Table 1.

Clinical signs and complications of infective endocarditis.

#### 4.2 Considerations on the cardiac cavity affected by infective endocarditis

The standard reference to corroborate the clinical diagnosis of IE is transesophageal echocardiography since the transthoracic echocardiogram, even when limited to native valves, decreases the diagnostic probability of IE [39].

Right and left endocarditis are two distinct entities that require different clinical and surgical approaches. The diagnosis of endocarditis on the right side requires a high index of clinical suspicion. It can occur with a history of intravenous drug use, fever, and pulmonary infiltrates, although intravenous drug abuse is also a cause of IE on the left side of the heart [36]. The information provided by echocardiography is of prognostic and therapeutic value.

If the vegetation is <1.0 cm in diameter, it can be expected that antibiotic therapy will resolve the infection; if the size of the vegetation determined by echocardiography is ≥1.0 cm without response to treatment, surgical intervention should be considered [40].

Surgical treatment in IE on the left side of the heart, for example, the mitral valve, is indicated in patients with severe mitral regurgitation, even in the absence of congestive heart failure, with mitral annular abscess, large vegetation >10 mm, uncontrolled sepsis, and multiple embolisms [41]. Mitral valve (MV) replacement has traditionally been considered as the standard treatment for MV endocarditis that does not respond to antibiotic treatment.

However, the pioneering work of Dreyfus et al. surgery for repair of the mitral valve with IE can be performed safely and is often associated with a better outcome compared to mitral valve replacement [42, 43].


**1. Introduction**

Animals, including wild animals, are guided by the senses of smell and taste when selecting food [1]. The taste and aroma of potential food sources play a secondary role only in extreme situations, such as drought, deep snow cover, or the risk of hunger. Such extreme conditions persisted in Poland in the winters of 1962/63, 1978/79, 1986/87, and 2005/06, when roe deer, red deer, elk, as well as hares foraged on the young and green plant parts protruding above the deep snow cover. In those years, animals caused considerable damage to young forests and nurseries of forests, orchards, and ornamental trees and shrubs. Cervids also cause considerable browsing damage in years with less

**Citation:** Budny, M.; Zalewski, K.; Lahuta, L.B.; Stolarski, M.J.; Stryi ´nski, R.; Okorski, A. Could the Content of Soluble Carbohydrates in the Young Shoots of Selected Willow Cultivars Be a Determinant of the Plants' Attractiveness to Cervids (Cervidae, Mammalia)? *Agriculture* **2021**, *11*, 67. https://doi.org/10.3390/ agriculture11010067

Received: 20 November 2020 Accepted: 12 January 2021 Published: 15 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional clai-ms in published maps and institutio-nal affiliations.

**Copyright:** © 2021 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/).

severe weather conditions, which forces forest managers to implement costly protective measures. In 2018, the total area affected by the foraging and browsing behavior of game animals, including red deer, fallow deer, roe deer, wild boars, and hares, in Poland was estimated at 56,300 ha, including 27,500 ha of agricultural land, 21,500 ha of young forests, and 7300 ha of mature forests. According to State Forests data [2], around €44.8 million was spent on protecting forests against foraging animals in 2019. Chemical and mechanical protection of seedlings and fencing generate the highest costs. In 2018, seedlings were protected in a total area of 91,500 ha, and 22,600 ha of forest nurseries were fenced. In Germany, €11 million was spent on forest nurseries and forest protection, of which €3.2 was dedicated to combating woodworm infestations. Considerable funds are also allocated to the protection of young forests against wild animals [3].

European foresters and hunters have long expressed an interest in experiments where alternative food sources for cervids, including mixed willow, sycamore, beech, oak, and linden stands, as well as pure willow stands, are grown in food plots, unused forest enclaves, browse plots, and on fallow land by forest and orchard nurseries [4]. The cultivation of willow browse is relatively problem-free. Willows (genus *Salix*) can be planted in a wide variety of soils, but only selected cultivars, such as *S. alba* cv. Tristis, *S. integra* cv. Hakuro Nishiki, *S. purpurea* cv. Gracilis, *S. purpurea* cv. Nana, or other cultivars of *S. purpurea* perform well on sandy and dry soils [5,6]. *S. acutifolia* is the most droughtresistant cultivar that is used to stabilize sand dunes on the Baltic coast and in the Bł ˛edów desert in Poland [7,8]. However, its attractiveness to foraging animals has never been studied. Some of the above willow cultivars (*S. acutifolia*) proved to be highly invasive, and their cultivation was discontinued.

The attractiveness of willow browse is a complex problem that is influenced by various factors. In a study performed by Drogoszewski and Wlazełko [9], only 6 out of 133 tested willow cultivars were not browsed by roe deer. Four of those were *S. purpurea* cultivars (out of the analyzed 18). Willow cultivars that are not attractive to foraging animals usually contain bitter inorganic salts, tannins, salicin, alkaloids, and other phenols in the leaves and bark. It is also possible that wild animals are able to detect the presence of substances with medicinal properties in willow shoots [10,11]. On the other hand, plants contain attractants such as complex carbohydrates—starches, soluble sugars, vitamins, mineral salts with essential macronutrients and micronutrients, proteins, fat, and fiber. Not all animal species have a preference for the same plant substances. For example, aspartame, an artificial sweetener composed of two amino acids, tastes sweet to humans, but not to mice [1]. Bears, monkeys, dogs, wild boars, squirrels, ants, and other animals have a preference for sweet foods. Cervids' preferences for sweet-tasting foods have never been investigated, and the extent to which the chemical composition of trees and shrubs contributes to their attractiveness to wild animals remains unknown. On the other hand, getting to know the preferences of cervids with regard to the feeding attractiveness to various species, varieties, or clones of willow might be for them an indicator of the quantity and quality of substances contained in these plants. In view of the above, the aim of this study was to determine the content and composition of soluble carbohydrates in ten willow cultivars, and to evaluate their attractiveness to cervids.

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

#### *2.1. Experimental Plots*

In the spring of 2015, a willow thicket was established at the research station of the Polish Hunting Association in Czempi ´n near Pozna ´n, Poland. Willow cuttings were obtained from experimental nurseries in the Resko Forest Inspectorate (Region of Western Pomerania), and field-plots established at experimental stations (owned by the University of Warmia and Mazury in Olsztyn) in Obory, Poland (Region of Pomerania), and Bałdy, Poland (Region of Warmia and Mazury) (Table 1). Weeds were removed only mechanically and manually in July of 2015 and 2016. The survival of willow cuttings and shoot growth were evaluated in September 2015. The willow thicket in Czempi ´n, Poland, was cut

in the spring of 2016 and in early March 2017, and one-year-old shoots were used to establish three experimental browse plots near Grzybno, Poland (X: 482950.30; Y: 351700.42), Bieczyny, Poland (X: 484298.21; Y: 345303.04), and Słonin, Poland (X: 475485.18; Y: 344974.35) (Figure 1).

Willow varieties, breeding lines, and cultivars (collectively referred to as cultivars in the study) for the establishment of the willow thicket at the research station in Czempi ´n, Poland, were selected based on their availability and the results of previous research on willow browse [3,8,9]. Based on the numerous studies investigating willow browse and the medicinal properties of willows, *S. purpurea* 1126, which was completely or partly ignored by wild animals during previous observations, was introduced as the control cultivar on account of its high salicin content [8,9,12–15].

**Table 1.** Cultivar designations used later in the work in the tables and figures.

**Figure 1.** Location of the experimental willow plots.

Willows were planted in 2017, with inter-row spacing of 75 cm and intra-row spacing of 30 cm. The area of the plot established in 2015 in Czempi ´n was 2500 m2, where 5045 cuttings were planted. Respectively, the plots established in spring 2018: Bieczyny—800 m2 and 2800 cuttings; Grzybno—870 m2 and 3080 cuttings; and Słonin—920 m2 and 3430 cuttings. The number of individual cultivars planted in a plot depended on their availability on the market in 2015 and was as follows (according to the numbering from Table 1): Cultivar No. 1-50; 2-1000; 3-1000; 4-1000; 5-1000; 6-110; 7-400; 8-45; 9-40; and 10-400 cuttings. Each of the cultivars numbered 2, 3, 4, 5, 7, and 10 were randomly planted in several rows. Thus, e.g., *S. amygdalina* cv. Krakowianka was planted in 5 rows separated by other cultivars. The planting of cuttings on the mother plot was completely random, as well as on the occlusal plots established two years later.

Browse plots were fenced with wire mesh to a height of 2.5 m, for two years, and weeds were removed mechanically each year in July. The performance of the willow thicket was evaluated in September of 2017 and 2019, and the survival of the willow cuttings in browse plots was evaluated in September 2019.

#### *2.2. Browsing Damage Assessment*

In the fall of 2017 and 2019, the damage caused by wild animals in browse plots after fencing had been removed was assessed on a 5-point scale proposed by Bukiewicz [12], with some modifications, where 1 point—absence of damage, no traces of shoot, bud or leaf browsing; coefficient for calculating browsing damage—0.00 (0%); 2 points—minor damage to selected shrubs, with full recovery; coefficient for calculating browsing damage—0.05 (5%); 3 points—moderate damage to the top part of the main shoot or side shoots; signs of foliage browsing; bark stripping on short stem segments; absence of terminal buds, which can inhibit shoot growth and weaken willow shrubs; coefficient for calculating browsing damage—0.15 (15%); 4 points—considerable damage to the main shoot, side shoots and buds; complete or partial loss of foliage; damage to stump sprouts; bark stripping on more than half of stem length, which can significantly inhibit shrub growth and lead to tissue necrosis in stumps under unfavorable conditions; coefficient for calculating browsing damage—0.50 (50%); and 5 points—absence of shoots, damaged and necrotized shoots; coefficient for calculating browsing damage—1.00 (100%).

When assessing the browsing damage, five repetitions were made, each with 20 consecutive seedlings in a row. For cultivars with less than 200 plants per plot (cultivars No. 1, 6, 7, and 8), all seedlings were assessed.

#### *2.3. Analysis of the Content of Soluble Carbohydrates*

At the end of July 2017, ten one-year old shoots with leaves grown from two-year-old stool were obtained from each willow cultivar (three replicates) in the thicket in Czempi ´n, Poland, and the harvested material was analyzed for the content of soluble carbohydrates. A total of 30 samples of 10 cm tops of one-year-old shoots were analyzed. Each sample of 10 stems was analyzed separately. Since *S. pentederana* and *S. amygdalina* cv. Krakowianka did not survive in the three-year-old thicket in Czempi ´n, Poland, shoot samples from these cultivars were obtained from the experimental plots in Bałdy, Poland.

Shoot samples (10 cm tips with the buds of one-year old shoots) were weighed, frozen in liquid nitrogen, and lyophilized. Dry tissues were crushed in a mixer mill MM200 (Retsch, Katowice, Poland). Soluble carbohydrates were extracted from 40 mg of dry material using 800 μL of 50% ethanol (containing 100 μg of xylitol, as an internal standard) and analyzed by gas chromatography method on the ZEBRON ZB-1 capillary column (Phenomenex, CA, USA), as described previously [16]. The carbohydrate content was calculated using the internal standard method. Standards of carbohydrates (xylitol, fructose, galactose, glucose, *myo*-inositol, sucrose, galactinol, raffinose, 1-kestose, stachyose, and verbascose) were purchased from Sigma (USA). The amount of unknown carbohydrates with retention times (Rt) of 6.52 and 7.39 was calculated based on the nearest known standards (1-kestose and stachyose, respectively).

#### *2.4. Experiment with Farmed Red Deer and Fallow Deer*

In January 2020, twenty one-year-old shoots grown from leafless three-year-old stool were harvested from each willow cultivar (in three replicates) and fed to European red deer at the research station in Czempi ´n, Poland, and red deer and fallow deer at the research station of the Polish Academy of Sciences in Kosewo Górne, Poland. Both deer species were kept separately. The shoots came from the plot located in Bieczyny, Poland. One hundred and twenty shoots were cut from each cultivar and divided into 6 bunches, three for each town (Czempi ´n and Kosewo Górne). All bunches from all cultivars were collected at the same time and their fresh weight was determined. All bunches were tied to the fence where the animals were kept. The decrease in shoot mass caused by browsing was determined after 3 days. The degree of browsing damage was determined after the loss of fresh weight. All tests were subjected to the same atmospheric conditions.

The analysis of correlations between the relative sweetness of the total carbohydrates and severity of browsing damage on willow shoots was performed. The sweetness of the individual sugars and total carbohydrates was determined by multiplying the sugar content of the willow tissues (mg g−1 DM) by the relative sweetness (RS) indicators (Table 2) given in the literature. Galactinol and sucrose are non-reducing disaccharides because both galactose and *myo*-inositol have α-(1-1)-glycosidic bonds; therefore, an RS index of 0.6 was adopted for galactinol [17–19].

**Table 2.** Sweetness of the selected carbohydrates relative to sucrose (see the source in the reference list).

#### *2.5. Statistical Analysis*

The results were processed statistically in the Statistica program (v. 13.1, Dell Inc., Tulsa, OK, USA). The least significant difference was calculated at *p* ≤ 0.01, with the use of Tukey's test. The relationships between the carbohydrate content of the shoots and the severity of browsing damage caused by cervids were determined based on the calculated values of Pearson's linear correlation coefficient R (*p* ≤ 0.05; *p* ≤ 0.01).

### **3. Results**

#### *3.1. Soil Analysis*

Soil samples for laboratory analyses were collected from the experimental plots where the evaluated willow cultivars were grown. The soil acidity (KCl), content of organic carbon, total nitrogen, and major macronutrients (P, K, and Mg) were determined in 100 g soil samples (Table 3). Soil samples from the willow thicket in Czempi ´n, Poland, were acidic, moderately abundant in phosphorus, highly abundant in potassium, and moderately abundant in magnesium. The content of total organic carbon was determined at 1.15%, which, when converted to humus content (1.15 × 1.7 = 1.96), is a satisfactory (average) result for slightly loamy sand. The experimental plot in Słonin, Poland, was established on slightly alkaline soil with a moderate phosphorus and potassium content, which was highly permeable due to an 80 cm-deep layer of loose sand. The experimental plots were established in the following types of habitats: Czempi ´n, Poland—fresh forest established on former agricultural land (Category 2); Bieczyny, Poland—dehydrated humid forest established on former agricultural land (Category 2); Grzybno, Poland—fresh mixed coniferous forest with low groundwater levels (Category 1); Słonin, Poland—fresh forest with very low groundwater levels (Category 1). Categories 1 and 2 characterize the degree of moisture in the soil in which the forest grows, where Category 1 indicates poor soil irrigation and Category 2 good soil irrigation.

**Table 3.** Soil parameters in the experimental willow plots.

#### *3.2. Willow Performance (Survival)*

Willow survival in the first and third year of growth were high (above 60%) in the thicket and in the experimental plots in Bieczyny and Grzybno, Poland (Figure 2). In Słonin, Poland (Figure 2D), the survival exceeded 80% in three cultivars only. Two years after establishment, numerous stumps were lost in all plots due to drought, and the highest losses were noted in Słonin, Poland (Figure 2D). Losses ranged from 80% to 95%, depending on the cultivar (applies to Cultivars No. 1, 2, 4, and 8). In 2017, 2018, and 2019, the Wielkopolska Region in Poland was affected by soil drought, which led to a massive loss of willow stumps in browse plots and partial losses in the thicket. Willow survival in the plots was evaluated for the second time in September in the third year of growth, whereas the thicket was assessed three times during the experiment. Browsing damage was most extensive in the experimental plot in Słonin, Poland, where willows were grown on sandy and highly permeable soil. *S. laurina* 220/225 (cv. No. 8) was nearly completely eliminated, and less than 5% of the *S. amygdalina* 1102 (cv. No. 4), *S. amygdalina* cv. Dunajec (cv. No. 1), and *S. amygdalina* cv. Krakowianka (cv. No. 2) shrubs survived (Figure 2). In the browse plot in Grzybno, Poland, *S. amygdalina* cv. Dunajec (cv. No. 1) was infested with dock bugs (*Coreus marginatus* L.), which damaged nearly 80% of the leaves and developing buds. The above cultivar survived the infestation.

#### *3.3. Analysis of Shoot Browsing*

The quality of willow cultivars and the survival differed across the plots after fencing had been removed (Figure 3). Willow performance was largely influenced by the type of habitat, in particular in dry years. The plot in Słonin, Poland, was established on highly permeable soil of the lowest quality (classes IV–VI). In the north, the plot was bound by a fresh mixed forest and a fresh forest composed of pines, birches, and, sporadically, oaks, with a dense undergrowth of black cherry and hazel. Signs of foraging by fallow deer and roe deer (which permanently inhabited the region), as well as mouflons (which were periodically observed in the region) were noted (Figure 3).

In comparison with the remaining two plots, the plot in Bieczyny, Poland offered the optimal habitat for red deer and roe deer. The plot in Grzybno, Poland, was characterized by medium-quality soils, and it was visited mainly by roe deer. Signs of foraging and tracks of other animal species were observed sporadically after fencing had been removed. In the willow thicket, two cultivars (*S. amygdalina* cv. Krakowianka and *S. pentederana*) were also lost due to drought, but only in the fourth and fifth year of growth (Figure 2).

**Figure 2.** Willow survival (%) in the experimental plots in the first (2015), second (2017), and third (2019) year of vegetation in (**A**) Czempi ´n-backwoods; (**B**) Bieczyny; (**C**) Grzybno; and (**D**) Słonin. All stumps were counted. For the cultivar explanation, see Table 1.

**Figure 3.** Severity of browsing damage to the analyzed willows cultivated in (**A**) Bieczyny; (**B**) Grzybno; and (**C**) Słonin. The mean values for three localization (**A**–**C**) are shown in box (**D**). Data were processed by analysis of variance. Bars with the same letters did not differ significantly (*p* ≤ 0.01) in Tukey's test. For the cultivar explanation, see Table 1.

#### *3.4. Content of Soluble Carbohydrates*

The shoots of all analyzed willow cultivars contained the same soluble carbohydrates: glucose, fructose, sucrose, *myo*-inositol, galactinol, and raffinose. Total carbohydrate content ranged from 21.31 (*S. amygdalina* 1045) to 69.37 mg/g−1 DM (*S. purpurea*).

Glucose was the predominant sugar in most cultivars, accounting for more than 50% of the identified soluble carbohydrates (Figure 4). Glucose levels were highest in *S. purpurea*

(45.93 mg/g−1 DM), *S. pentaderana* (35.08 mg/g−1 DM), and *S. fragilis* (29.19 mg/g−1 DM on average), and lowest in *S. amygdalina* 1045 (11.61 mg/g−1 DM on average). The fructose content of the shoots was approximately two-fold lower than their glucose content in all willow cultivars, whereas the galactose content was approximately 10-fold lower than the fructose content. Most cultivars contained even less sucrose than galactose, excluding *S. purpurea* 1126 (0.55 mg/g−1 DM). Smaller differences were noted in the content of *myo*-inositol, which ranged from 4.61 (*S. amigdalina* 1045) to 8.26 mg/g−1 DM (*S. fragilis* cv. Kamon/Resko). The phloem of all willow species also contained small quantities of galactinol (0.07–0.55 mg/g−1 DM) and trace amounts of raffinose (0–0.13 mg/g−1 DM). Raffinose was not detected in Cultivars No. 2, 3, 4, and 6 (Figure 4).

**Figure 4.** The content of soluble carbohydrates in the shoots of the analyzed willow cultivars: (**A**) total soluble carbohydrates; (**B**) glucose; (**C**) fructose; (**D**) *myo*-inositol; (**E**) galactose; (**F**) sucrose; (**G**) galactinol; and (**H**) raffinose. Values marked with different letters (a to e) are significantly different at *p* ≤ 0.01. For the cultivar explanation, see Table 1.

#### *3.5. Shoot Sweetness and Browsing Damage*

The extent to which browsing damage was correlated with the sweetness of the evaluated willow cultivars was evaluated in a statistical analysis using Pearson's linear correlation coefficient R (*p* ≤ 0.05; *p* ≤ 0.01).

The analysis revealed weak negative correlations between the RS of total carbohydrates (R = −0.28) (Figure 5A), the RS of glucose (R = −0.26) (Figure 5B), and the RS of fructose (R = −0.27) (Figure 5C) vs. the severity of browsing damage on willow shoots. No such correlations were observed for the remaining carbohydrates that were less abundant in willow tissues (Figure 5D–H).

**Figure 5.** The analysis of correlations between the relative sweetness of the total carbohydrates and severity of browsing damage on willow shoots: (**A**) total soluble carbohydrates; (**B**) glucose; (**C**) fructose; (**D**) *myo*-inositol; (**E**) galactose; (**F**) sucrose; (**G**) galactinol; and (**H**) raffinose. R—Pearson's linear correlation coefficient.

Farmed red deer and fallow deer did not express particular interest in one-year-old shoots harvested from the studied willow cultivars (Table 4). The animals had the greatest preference for *S. cordata* and *S. laurina* (red deer), as well as *S. cordata*, *S. amygdalina* cv. Dunajec, *S. amygdalina* cv. Krakowianka, and *S. laurina* (fallow deer), but intensive foraging behavior was not observed. It should also be noted that the content of soluble sugars in overwintered shoots increased by 52.3% relative to the shoots harvested in the middle of the growing season (July). An analysis of *S. cordata* tissue samples also demonstrated changes in the proportions of soluble carbohydrates. The content of fructose decreased, whereas sucrose levels increased between July and January (Table 5).

Red deer expressed varied interest in willow shoots, which generally differed between the studied locations. The animals in both stations had a greater preference for *S. cordata*, but fallow deer were less discriminating in their choice of willow cultivar than red deer (Table 4).

**Table 4.** The attractiveness of the analyzed willow cultivars to red deer and fallow deer at the research station of the Polish Hunting Association in Czempi ´n and the Cervid Farm of the Polish Academy of Sciences in Kosewo Górne, based on the average decrease in shoot mass (%) of duplicate measurements.

**Table 5.** Average sugar content (mg/g−1 DM) of *Salix cordata* shoots harvested in July and January (*n* = 3 for each species and sugar) with the standard deviation.

#### **4. Discussion**

Human interventions in reducing damage to forests, such as the use of fencing and chemicals, should be minimized or completely eliminated in order to preserve the biodiversity of natural habitats. Artificial structures and chemicals should be replaced with trees and shrubs that, at least partially, protect valuable crops from damage and reduce crop losses to an economically acceptable level.

The attractiveness of willow shoots to herbivores is determined by various factors, in particular the chemical composition of willow cultivars and their growth stage. The survival and growth rates of willows and, to a certain extent, the chemical composition of the shoots is correlated with the soil quality and soil moisture content (Figures 2 and 4; Table 3). In the present study, the willow thicket and the experimental plots differed significantly in soil parameters. The soil in Słonin, Poland, was susceptible to dehydration due to low water-holding capacity and the absence of capillary action. In this experimental plot, the performance of willows was completely dependent on precipitation, in particular in the dry years 2017–2019. The survival was very low in the first year after planting, and only 21.7% of the planted cuttings survived the dry years 2018 and 2019. *S. laurina* was the least resistant to drought (only 3.2% of stumps survived), whereas *S. amygdalina* 1045, *S. amygdalina* cv. Kamon/Resko, and *S. pantederana* were least susceptible to water deficit (50.1% of stumps survived). The results of the study clearly demonstrate that soil parameters and habitat type significantly influence root establishment and sprouting.

The content and composition of soluble carbohydrates in willows remain insufficiently investigated. Aliferis et al. [22] identified glucose, fructose, galactose, *myo*-inositol, trehalose, and sugar alcohols—arabitol, mannitol, and glycosides—in fully developed leaves of 6- to 8-week-old *S. purpurea* L. (cv Fish Creek) plants. Sucrose, galactinol, and raffinose were not detected. Similar to this study, the predominant sugars in the leaves of hydroponically grown *S. viminalis* were sucrose, glucose, and fructose (Figure 4) [23]. However, the sucrose, glucose, and fructose concentrations were more than twice higher in the cited experiment than in this study. In the leaves harvested from the top shoots of two *S. viminalis* × *S. schwerinii* hybrid cultivars grown in a field, the glucose and sucrose concentrations were similar and significantly higher than the fructose levels [24]. The sugar concentrations were lower in older than in younger plants, and similar observations were made in the hybrid poplars *Populus deltoides* × *P. nigra* [25]. Corol et al. [24] also identified small quantities of raffinose and stachyose in the leaves of *S. viminalis* × *S. schwerinie*. In the present study, raffinose (but not stachyose) was detected in most willow cultivars (excluding *S. amigdalina*), and it was most abundant in *S. cordata* (0.05 mg g−1 DM). The presence of galactinol (a precursor of raffinose and its higher homologs) in all willow species indicates this oligosaccharide is synthesized in tissues in response to water and temperature stress (drought) [26]. Unlike glucose and fructose, galactinol does not appear to increase the sweetness of willow shoots, due to its low content. However, galactinol acts as a donor of the galactosyl groups for other sugars, and therefore its presence can enhance sweetness [27]. The statistical analysis revealed significant differences in the concentrations of individual soluble carbohydrates and in the total sugar content between the evaluated willow cultivars. The differences in the content of soluble carbohydrates between the cultivars studied here could be associated with the rate of the plant's growth, and/or rate of shoot tip elongation. In our study, the much higher concentration of monosaccharides (glucose and fructose) (Figure 5B,C) than that of sucrose, found for all cultivars, could be an indirect confirmation of the role of the shoot tips as the major sink tissues of willow plants, as in other species [28]. Sucrose is the major photoassimilate distributed from the leaves (sources) to the sinks [29], where it is hydrolyzed (presumably by invertases) into monosaccharides and serving as the carbon and energy sources for fast-growing tissues.

According to research, sweet taste perception differs considerably among animal species [30]. Foods rich in soluble carbohydrates are attractive to many animals and promote foraging behavior. The presence of sugars is responsible for sweet taste perception, but perceived sweetness can also be enhanced or reduced by phenols and other compounds. These compounds can exert a greater effect on sweetness in combination than alone.

In the current study, an experiment involving farmed red deer and fallow deer was carried out to determine whether the concentration of soluble sugars influences the attractiveness of willow shoots to cervids. One-year-old shoots of the analyzed willow cultivars harvested in mid-January were less attractive to the animals than the shoots harvested in spring and summer. Red deer and fallow deer had the greatest preference for *S. cordata*

and *S. laurina* (signs of browsing damage were observed on up to 30% of the shoots). Sugar content could play a role in the foraging preferences of cervids because shoots harvested in January contained significantly more soluble carbohydrates (approx. 52% in *S. cordata*) than those harvested in late July. However, the statistical analysis revealed no significant correlations between the severity of browsing damage in spring and summer and the content of soluble sugars in the evaluated willow cultivars. These findings suggest that soluble carbohydrates do not act as typical attractants for red deer and fallow deer. Further research is needed to determine whether a sweet taste is not perceived by fallow deer and red deer due to the absence of the respective receptors [31], too low sugar concentrations in willow shoots, or the presence of other compounds, such as phenols, that mask the sweet taste of willow shoots.

#### **5. Conclusions**

The data presented in this work show that success in the cultivation of willow depends on the chosen cultivar and soil quality, especially its hydration. Young shoots of the analyzed cultivars contained from 2.1% to 6.9% soluble sugars, among which quantitatively dominated glucose. These sugars did not have a significant impact on the attractiveness of willows for cervids, which means that other chemical compounds are such a factor. It is possible that there are quantitative relationships between soluble sugars, phenolics, and other compounds that determine food attractiveness for cervids. Roe deer, fallow deer, and European deer do not have identical preferences for all willow cultivars, but they are those that are eaten very willingly by all of these animal species. This fact should be taken into account when selecting species for established plantations.

**Author Contributions:** Conceptualization, K.Z. and M.B.; methodology, K.Z., L.B.L. and M.B.; software, M.B. and A.O.; validation, M.B. and K.Z.; formal analysis, M.B. and K.Z.; investigation, M.B.; resources, M.J.S.; data curation, M.B., A.O., M.J.S. and K.Z.; writing—original draft preparation, M.B.; writing—review and editing, K.Z., R.S.; visualization, A.O.; supervision, K.Z.; project administration, K.Z. and M.B.; funding acquisition, K.Z. and M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Warmia and Mazury in Olsztyn, grant number 12.610.012-110; and the Main Board of the Polish Hunting Association, grant number 1/PZŁ/2016.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study because in the test system used, animals were fed with no additives or chemical modifications. The animals received food that occurs naturally in the environment, and the care of the animals was provided under the status of the Research Station of Polish Hunting Association in Czempin, Poland, where these animals were kept.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**1. Introduction**

Requirements prioritization (RP) is an important activity of requirements management, however, this activity can become a complex process in a family of products projects, due to common requirements, increasing dependencies, and diversity of stakeholders involved. In most prioritization method, such as Hundred Dollar, MoSCoW and Numerical Assignment Technique (NAT), the participation of stakeholders are essential to provide the prioritization criteria based on their expertise [1]. In this respect, Hujainah et al. [2] suggest the exclusion of users from tasks that can be automated, and include them only in important tasks that generate value.

In the latest years, the application of AI techniques in several stages in Software Engineering has been increasing and will continue growing [3]. We argue that it is possible to take advantage of these techniques to exploit information and discover new criteria, to decrease the stakeholder's participation.

In this paper, we focus mainly on those activities that can be automated for identifying a set of prioritization criteria and generating a list of ranked requirements. We also analyzed the available datasets and discuss their main limitations. In the next section, the proposed process is shown in detail.

### **2. A Semi-Automated Data-Driven Requirements Prioritization Process**

The proposed process consists of two phases, Criteria Identification Phase and Requirements Prioritization Phase. A summary of the proposal is shown in Figure 1.

**Citation:** Limaylla, M.I.; Condori-Fernandez, N.; Luaces, M.R. Towards a Semi-Automated Data-Driven Requirements Prioritization Approach for Reducing Stakeholder Participation in SPL Development. *Eng. Proc.* **2021**, *7*, 27. https://doi.org/10.3390/engproc<PHONE_NUMBER>

Academic Editors: Joaquim de Moura, Marco A. González, Javier Pereira and Manuel G. Penedo

Published: 13 October 2021

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

**Figure 1.** Data sources and AI techniques used for prioritizing requirements of SPL projects.

#### *2.1. Criteria Identification Phase*

In this first phase, multiple prioritization criteria are identified with the minimum stakeholder participation. This phase starts with the data sources selection carried out by the analyst, and, optionally, loading new requirements and criteria. Then, the data is automatically collected by extracting and analyzing data from several sources, like reviews from App Marketplaces and requirements' formal documents. After collection, Natural Language Processing (NLP) techniques can be used to identify features (features are distinctive characteristics or properties of a family of systems) and associating them with existing features in feature models. Feature models are diagrams in SPL projects that show features in a hierarchical structure and conceptual relationships among features [4]. These features can be previously prioritized and new prioritization criteria can be obtained when associating the new features with the existing ones. Moreover, thanks to the use of sentiment analysis, we aim to identify sentiment and deontic in user reviews, which can provide another type of prioritization criteria. A (supervised or non-supervised) classification algorithm is used to perform the classification in FR/NFR. This classification can be used as other criteria, due to the importance of some NFRs like security or performance, considered crucial to the quality of systems. All these criteria can be obtained automatically, without the participation of stakeholders.

#### *2.2. Requirements Prioritization Phase*

In the Second Phase, a requirements prioritization is performed based on criteria previously identified. All these criteria require to be unified and summarized in order to provide more understandable information. At this point, stakeholders can review the prioritization criteria, by confirming those that are relevant for the project. Once the criteria are selected, the prioritization is performed automatically by means of a machine learning algorithm. Algorithms such as Machine-Learned ranking, classification algorithms like Decision Tree or Random Forest, and even Deep Learning algorithms in combination with others algorithms can be used in this process. Finally, the output of this process is a list of ranked requirements. This will be saved as historical data for future use.

#### *2.3. Datasets*

Datasets are an essential component of any machine learning model. PROMISE [5] is a dataset used in most of the research for FR/NFR classification. This dataset has 625 requirement sentences, with 255 identified as functional and 370 as non-functional requirements. The NFR is labeled with the following types: Availability, Legal, Look and feel, Maintainability, Operational, Performance, Scalability, Security, Usability, Fault tolerance, and Portability. However, it presents unbalanced data in the categories of NFR. The unbalanced data can affect the precision and recall metrics of several classification algorithms, and generate a biased model. There are several ways to address this problem. Down-sampling in the majority classes is one technique, but it could lose valuable data. Synthetic data generation (Up-sampling) is another technique, that using some algorithms to create data that follow the tend of the minority classes. Balanced ensemble learning refers to the use of multiple learning machines and combines their outputs to obtain a better prediction.

For requirements prioritization methods based on supervised algorithms, RALIC [6] is a dataset used for some research. RALIC dataset contains several data about ratings and recommendations of requirements by stakeholders. This dataset is used in traditional methods and in machine learning methods for predicting the value of a rating from stakeholders.

Both datasets are in the English language. These datasets are good references but more datasets, especially in Spanish, are needed. This implies collecting historical requirements and carrying out their labeling, get balanced and standardized data and ensure enough quantity for training, testing and validation.

#### **3. Conclusions**

In this article, we presented a data-driven requirements prioritization process that can be used in SPL projects. The proposed prioritization process aims to reduce mainly the stakeholder participation through the identification of additional criteria to avoid some risks like disagreement between stakeholders and lack of time. We rely on AI techniques, like NLP and Machine Learning algorithms, to optimize mainly the criteria identification by exploiting information from different data sources. We also review two datasets that are used for FR/NFR classification and for requirements prioritization. As a result of this review, some of their limitations (e.g., imbalanced datasets), and the necessity of new datasets were identified.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Publicly available PROMISE dataset [7] and RALIC dataset [6] were analyzed in this study.


**Advanced Support Technologies in Roadway**

Editors

**Guoming Liu Xiangming Hu**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Guoming Liu Shandong University of Science and Technology China

Xiangming Hu Shandong University of Science and Technology China

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Applied Sciences* (ISSN 2076-3417) (available at: https://www.mdpi.com/journal/applsci/special issues/Advanced Support Technologies Roadway).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-5775-5 (Hbk) ISBN 978-3-0365-5776-2 (PDF)**

© 2022 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.


**3. Ascorbic acid as an electron donor**

Ascorbic acid is a reducing agent and thus an electron donor. The electron donation ability of ascorbic acid is responsible for its biochemical and physiological roles [14]. The double-bond carbon between the second and third of the 6-carbon molecules of ascorbic acid give two electrons. Ascorbic acid is often referred to as an antioxidant owing to the fact that it prevents other molecules from being consumed by donating electrons [15]. Ascorbic acid as an antioxidant and its interaction with reactive nitrogen species and singlet oxygen has been extensively documented [16]. Ascorbic acid has been utilized as an antioxidant for the stabilization of oxidationprone medications. These medications include vitamin A, morphine, cholecalciferol, rifampin, promethazine, and sulphacetamide [17].

Electrons from ascorbic acid can react with metals such as copper and iron, leading to the generation of superoxide and hydrogen peroxide, which may later result in the formation of ROS. Consequently, in certain situations, ascorbic acid will give rise to an oxidizing agent by way of its operation as a reducing agent. This reaction occurs in the biological system when different pharmacological doses of ascorbic acid are absorbed in the plasma and extracellular fluid compartment as well as low doses of ascorbic acid in cell culture media containing metals [18].

When vitamin C donates electrons, the electrons are lost sequentially. The product formed after the loss of one electron is a free radical, semidehydroascorbic acid, or ascorbyl radical. When compared with other free radicals, the ascorbyl radical is different because its half-life can be measured in many seconds or even minutes depending on the absence or the presence of oxygen or electron acceptors, especially iron [19]. Ascorbyl radical is relatively stable and fairly reactive. This attribute explains why ascorbic acid may be a preferred antioxidant. In other words, a reactive and a harmful free radical can react with ascorbic acid [20]. The reduction of a reactive free radical with the generation of a less-reactive product is sometimes called free radical scavenging or quenching. Therefore, ascorbic acid is a good free radical scavenger due to its chemical composition [21]. For instance, under some conditions, ascorbyl radical could be estimated in blood and extracellular fluid samples [22].

Upon the loss of second electron, a more stable species, dehydroascorbic acid is formed when compared with ascorbyl free radical. The stability of dehydroascorbic acid depends on temperature and pH of the medium, but is often only in minutes. Dehydroascorbic acid has affinity for facilitated glucose transporters and is transported by a number of them [23]. Variety of oxidants in biological system mediate the formation of both ascorbyl radical and dehydroascorbic acid which includes molecular oxygen, superoxide, hydroxyl radical, hypochlorous acid, reactive nitrogen species, and trace metals like iron and copper. Both dehydroascorbic acid and ascorbyl radical could be reversibly broken down to ascorbic acid *via* different metabolic pathways and also through oxidizing compounds inside the body system including glutathione [24]. All the ascorbic acids consumed by human beings cannot be regained because there exists just fragmental depletion back to ascorbic acid.

**Figure 2.** *Redox chemistry of vitamin C.*

In case of nonreduction of dehydroascorbic acid to ascorbic acid, it will be broken down to 2, 3-diketogulonic acid irreversibly due to the rupture of the lactone ring structure that is a component of ascorbic acid, ascorbyl radical, and dehydroascorbic acid. The 2, 3-diketogulonic acid is metabolized further into other compounds such as xylose, xylonite, lyxonate, and oxalate [25]. The development of oxalate is clinically significant because some persons with hyperoxaluria (excessive oxalate excretion) develop oxalate kidney stones (**Figure 2**).


**1. Introduction**

Living creatures on this earth need water as an essential element. Almost 70% of the human body is made up of water. Access to clean drinking water is not merely required for human existence but is also considered one of the basic human rights [1]. However, the manifestation of several organic and inorganic toxins, such as pesticides, phenol, chlordane, arsenic fluoride, nitrate, copper, etc., cause the population to consume poor quality water, which is perilous to their health [2]. According to UNICEF and the WHO, all over the world, approximately 2.2 billion people lack access to clean and innocuous drinking water sources. However, about 2 billion people live in high-water-stress countries [3]. According to the WHO, by 2025, almost half of the entire world's population will be forced to live in areas with scarce water sources. Moreover, 6.8 billion individuals worldwide have access

**Citation:** Aziz, A.; Akram, K.; Abrar ul Haq, M.; Hawaldar, I.T.; Rabbani, M.R. Examining the Role of Clean Drinking Water Plants in Mitigating Drinking Water-Induced Morbidity. *Sustainability* **2022**, *14*, 9644. https:// doi.org/10.3390/su14159644

Academic Editor: Marc A. Rosen

Received: 9 May 2022 Accepted: 1 August 2022 Published: 5 August 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to basic drinking water services, while 785 million people around the globe lack access to these services [4].

Contaminated drinking water poses a serious threat to human life across the world. Diseases transmitted due to drinking contaminated water include cholera, diarrhea, typhoid, and dysentery [5]. Waterborne diseases, especially diarrhea, kill approximately one million people globally, and, mostly, children under five years of age are at a higher risk of diarrhea [6]. According to estimates by the World Bank [6], all over the world, because of diarrhea, a well-known disease allied with poor quality of drinking water, about 2.5–3.5 million people are infected, while 485,000 die yearly. Moreover, almost 80 percent of children annually become water-related syndrome victims. Several non-diarrheal illnesses are also associated with the unavailability of adequate and safe drinking water sources. Numerous chemicals (i.e., organic and inorganic) also have an adversative effect on human health in several diseases such as cancer, vomiting, skin rashes, and nausea [6]. Contaminated water causes about 4% of all deaths and 7.5% of all illnesses worldwide [7].

Similarly, in Pakistan, sources of clean drinking water are diminishing swiftly, and drinking water quality is viewed as a grave issue [8]. Poor and indecorous arrangements of the water supply and sewage ejection systems result in the sewage and mess being directly released into drains which ultimately flows into the sea, rivers, or canals and adversely damages water quality [9]. According to a World Bank report [6], 64 percent of Pakistan's population lacks access to clean and innocuous drinking water sources. Only 36 percent of Pakistan's population has access to safely managed and clean drinking water. Therefore, the environmental performance index ranked Pakistan at 140 out of 180 countries regarding safe water and sanitation. Moreover, a report by the World Bank highlighted that 25 percent of the total population in Pakistan is at high risk of consuming arseniccontaminated water [6]. Pakistan is among the top 10 countries that lack access to clean drinking water, where rich people have more access to safe drinking water sources than poor households [9].

Likewise, the public faces problems regarding access to safe and suitable drinking water in Pakistan. Pollution of drinking water causes a greater threat to Pakistan's public health. Out of 122 nations, Pakistan is in 80th place in terms of following the drinking water quality standards [10]. According to the World Bank report [6], about 53,000 children die annually in Pakistan due to severe diarrhea or waterborne illness. Moreover, drinking contaminated water infected 4 out of 10 children in the country [6]. Moreover, from 2010 to 2019, approximately 250,000 deaths occurred in Pakistan due to drinking contaminated water [11]. Additionally, arsenic, a hazardous pollutant and chemical, has been cited as a key cause of waterborne diseases in several areas of the country. The high level of arsenic in drinking water endangered the health of more than 60 million inhabitants, especially in Punjab, Pakistan [12].

To cope with this problem, the local government has instigated a platform to install safe drinking water filtration plants in each city in Pakistan. The reason for the installation of these plants is to meet the Millennium Development Goal (M.D.G.) by delivering nontoxic drinking water to the public by the end of 2030 [13]. To attain this tenacity, the government of Pakistan, in the middle of 2005, instigated an initiative termed the Clean Drinking Water Initiative (CDWI) to fix about 452 WPEPs (Water Purification Filtration Plants) practically in every tehsil of the Punjab province. Conversely, instead of enormous exertions to provide access to safe drinking water from WPFPs, the community is still facing several problems, for instance, a long-time shutdown of plants due to huge load shedding or closure of plants during holidays, and the non-functionality of a few plants because of a lack of supervision [14].