**4. Hybridization of carbon atoms**

The recent materials discoveries at nano scale open new horizons in all science and engineering fields. One of the most important point of view reported in several literatures is depending on classify carbon materials according to their hybridization characteristics [24, 25].

The electron hybridization determines the ability of carbon atoms to arrange themselves in a wide variety of structures with linear, planar and tetrahedral symmetry forming different allotropes, such as carbyne/carbyte, graphene/ graphite and diamond. So, depending on hybridization type carbon structures can be classified from sub-molecular to macroscopic scales into three general families: carbyne- general family (sp1 family), this family includes carbon atoms with sp1 hybridization state from its nanostructured like carbyne or cyclo-carbon to its macrostructural crystalline form [26]. The second class called graphene general family (sp2 family), this family includes carbon atoms with sp2 hybridization state in their nano as well as macro-crystal structures with hexagonal and rhombohedral structures like graphene and graphite. The third class called diamond general family (sp3 family), the carbon within this family existing sp3 hybridization state like diamond structure in its nano and macro scales [27].

For more complexation, carbon atoms can be exist with other hybridization states called intermediate hybridizations with different degrees. The degree of hybridization in this case caused by the curvature of the sp2 hybridized structure, leading to produce strained C–C bonds. Usually carbon atoms with pure sp2 hybridization state shows an ideal flat structure. While in the case of atoms with curved structure, their hybridization degree should be >2 as in the case of fullerene carbon [14, 27].

On the other side, usually carbon atoms in sp2 hybridization state arrange themselves in hexagonal rings as well as in a various other polygonal rings. The non-hexagonal rings can leads to curving the flat sheet or keeping its flatness if the polygon arrangement fulfils certain symmetry rules [14].

**Figure 3.**

*The ternary carbon allotropy phase diagram based on hybridization type (reuse with permission Elsevier [14]).*

The pentagon rings induce a positive curvature while heptagon or octagon rings induce a negative curvature. Therefore, carbon materials with sp2 hybridization state can be exist into three types: positive curved fullerene-type, which includes carbon atoms with hexagonal and pentagonal rings and negative curved schwarzite-type, this type includes carbon atoms with hexagonal and either heptagonal or octagonal rings and the last one called zero-curvature graphene-type which includes carbon atoms with hexagonal rings only [14]. This procedure of classification carbon based- nanomaterials had been presented by a triangular carbon allotropes phase diagram, see **Figure 3**. In which, hypothetical carbon allotropes located at the corner of the diagram, and allotropes with intermediate hybridization states located at the edge of the diagram while carbon existing mixed hybridization states located inside the triangular diagram [14].

## **5. Fullerene and nanodiamond**

The recent discoveries of the nano-sized carbon materials leads to expand the list of carbon allotropes. In association with the ability of carbon atoms to form wide range of structures, carbon based- nanomaterials become widespread in the fields of nanoscience and nanotechnology [28].

Generally, Carbon-based nanomaterials possess effective physicochemical properties make them as a powerful tool in medicine. For example, graphene possess many promising properties due to its high surface area and high functionalization ability that make it suitable for drug delivery treatment, along with its high mechanical properties, graphene had been recommended for tissue engineering

**91**

*Fullerenes and Nanodiamonds for Medical Drug Delivery*

field. Carbon nano tubes CNTs had been suggested for different in vivo applications due to its strong optical absorption in the specific wave length and as an active tool for bi0 imaging and drug delivery applications. Recently, fullerene and nanodiamond had been investigated and received much attention to use as a drug delivery

In spite of the promising benefits of using carbon based- nanomaterial as medical tools for the treating of difficult to treat diseases, several challenges are involved within this technology such as, their toxicity, diffusion and distribution abilities throughout the body which may leading to unpredictable effects. So, in-depth and carefully studies around their nature and behavior in human body regarded the most important factors in this field. One of the great promises of nanotechnology in medicine is the local or targeted delivery of drugs. Efficient targeting would allow for a reduced systemic dosage meaning also a reduced toxicity while resulting in relatively higher or more efficient dosage at the desired

Several ideas, suggestions and observations in addition to physical and chemical experiments of clustered molecules were led to the way for discovery of C60 in 1985. In 1966, David Jones discussed the possibility of creating balloons made up from carbon atoms. Then in 1970, this idea was progressed by Eiji Osawa when he revealed the possibility of preparing molecule made up of 60 carbon atoms knowm as C60 molecule in a condensed icosahedron structure [30]. After that in 1971, Eiji Osawa and Zensho Yoshida enumerated the possible aromatic properties of the structured C60 molecule. Later, Bochva and co-worker studied the electronic structure of this molecule. Then in 1980, Davidson characterize the closed- hollow structure of this molecule using different theoretical techniques. Subsequently, in 1985, Kroto and Smalley and their team obtained carbon cluster through scientific experiment to study the suitable conditions at which carbon atoms nucleates in the atmosphere of the red gait star. The mass spectrometer analysis of the obtained clusters indicates that most of them had 60 carbon atoms

This carbon allotrope become the heart of nanotechnology and attracted significant attention of scientists. For that, in 1996, Kroto, Curl and Smalley were

Fullerene derived its name in the honor of the geodesic domes designer the artchitect Buckminster Fuller. Fullerene family usually represented by a formula of Cn, where n refers to the existing carbon atoms in the cage structure which can be up to several hundred atoms, the number of the carbon atoms existing within fullerene structure has a great influence on its structural geometry and its properties. C60 is the most dominant molecules within fullerene family [31].

The structure of C60 has truncated icosahedrons made up of 20 hexagonal rings located at the center of the icosahedral faces and a 12 pentagons located around the apexes. It is the most symmetric molecule. It has 2.fold, 3.fold and 5.fold rotational symmetry. The first one is through the edge center of 2- hexagons, the second one is from the center of 2-hexagons which facing each other, while the last one is through

Furthermore, fullerene molecules can be exist as a spherical, ellipsoid, tubular shapes consisting hexagonal, pentagonal and sometimes heptagonal rings. C60

rewarded by Nobel Prize in chemistry for their discovery of fullerene [31].

two pentagons centers which facing each other [30, 31].

*DOI: http://dx.doi.org/10.5772/intechopen.97867*

carriers [29].

target site [29].

**6. Fullerene**

**6.1 History and structure**

and some of them had 70 ones [31].

*Fullerenes and Nanodiamonds for Medical Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.97867*

field. Carbon nano tubes CNTs had been suggested for different in vivo applications due to its strong optical absorption in the specific wave length and as an active tool for bi0 imaging and drug delivery applications. Recently, fullerene and nanodiamond had been investigated and received much attention to use as a drug delivery carriers [29].

In spite of the promising benefits of using carbon based- nanomaterial as medical tools for the treating of difficult to treat diseases, several challenges are involved within this technology such as, their toxicity, diffusion and distribution abilities throughout the body which may leading to unpredictable effects. So, in-depth and carefully studies around their nature and behavior in human body regarded the most important factors in this field. One of the great promises of nanotechnology in medicine is the local or targeted delivery of drugs. Efficient targeting would allow for a reduced systemic dosage meaning also a reduced toxicity while resulting in relatively higher or more efficient dosage at the desired target site [29].

### **6. Fullerene**

*Materials at the Nanoscale*

The pentagon rings induce a positive curvature while heptagon or octagon rings

state can be exist into three types: positive curved fullerene-type, which includes carbon atoms with hexagonal and pentagonal rings and negative curved schwarzite-type, this type includes carbon atoms with hexagonal and either heptagonal or octagonal rings and the last one called zero-curvature graphene-type which includes carbon atoms with hexagonal rings only [14]. This procedure of classification carbon based- nanomaterials had been presented by a triangular carbon allotropes phase diagram, see **Figure 3**. In which, hypothetical carbon allotropes located at the corner of the diagram, and allotropes with intermediate hybridization states located at the edge of the diagram while carbon existing mixed hybridization

The recent discoveries of the nano-sized carbon materials leads to expand the list of carbon allotropes. In association with the ability of carbon atoms to form wide range of structures, carbon based- nanomaterials become widespread in the

Generally, Carbon-based nanomaterials possess effective physicochemical properties make them as a powerful tool in medicine. For example, graphene possess many promising properties due to its high surface area and high functionalization ability that make it suitable for drug delivery treatment, along with its high mechanical properties, graphene had been recommended for tissue engineering

hybridization

induce a negative curvature. Therefore, carbon materials with sp2

*The ternary carbon allotropy phase diagram based on hybridization type (reuse with permission*

states located inside the triangular diagram [14].

fields of nanoscience and nanotechnology [28].

**5. Fullerene and nanodiamond**

**90**

**Figure 3.**

*Elsevier [14]).*

#### **6.1 History and structure**

Several ideas, suggestions and observations in addition to physical and chemical experiments of clustered molecules were led to the way for discovery of C60 in 1985.

In 1966, David Jones discussed the possibility of creating balloons made up from carbon atoms. Then in 1970, this idea was progressed by Eiji Osawa when he revealed the possibility of preparing molecule made up of 60 carbon atoms knowm as C60 molecule in a condensed icosahedron structure [30]. After that in 1971, Eiji Osawa and Zensho Yoshida enumerated the possible aromatic properties of the structured C60 molecule. Later, Bochva and co-worker studied the electronic structure of this molecule. Then in 1980, Davidson characterize the closed- hollow structure of this molecule using different theoretical techniques. Subsequently, in 1985, Kroto and Smalley and their team obtained carbon cluster through scientific experiment to study the suitable conditions at which carbon atoms nucleates in the atmosphere of the red gait star. The mass spectrometer analysis of the obtained clusters indicates that most of them had 60 carbon atoms and some of them had 70 ones [31].

This carbon allotrope become the heart of nanotechnology and attracted significant attention of scientists. For that, in 1996, Kroto, Curl and Smalley were rewarded by Nobel Prize in chemistry for their discovery of fullerene [31].

Fullerene derived its name in the honor of the geodesic domes designer the artchitect Buckminster Fuller. Fullerene family usually represented by a formula of Cn, where n refers to the existing carbon atoms in the cage structure which can be up to several hundred atoms, the number of the carbon atoms existing within fullerene structure has a great influence on its structural geometry and its properties. C60 is the most dominant molecules within fullerene family [31].

The structure of C60 has truncated icosahedrons made up of 20 hexagonal rings located at the center of the icosahedral faces and a 12 pentagons located around the apexes. It is the most symmetric molecule. It has 2.fold, 3.fold and 5.fold rotational symmetry. The first one is through the edge center of 2- hexagons, the second one is from the center of 2-hexagons which facing each other, while the last one is through two pentagons centers which facing each other [30, 31].

Furthermore, fullerene molecules can be exist as a spherical, ellipsoid, tubular shapes consisting hexagonal, pentagonal and sometimes heptagonal rings. C60

belongs to the spherical fullerene class which looks like a soccer ball, while C70 belongs to the ellipsoidal class which looks like rugby ball. In addition, several efforts have been reported to produce fullerene with high yields, in 1990, a method was discovered for producing macroscopic amounts of this distinctive material and this breakthrough allowed scientists to understand its chemistry and explore its properties [31].

Generally, fullerene can be classified into classical fullerene and non-classical fullerene. The first one containing 12 pentagons and any number of hexagons, while the second fullerene class can have heptagons, octagons, and an additional number of pentagons or squares [30, 31]. Due to the unique structure and properties of C60, scientists showed high interesting in synthesis both larger and smaller fullerenes. Therefore, the family of fullerenes has been expanded involving fullerene molecules with a wide range of carbon atoms number. Larger fullerenes that have an icosahedral- symmetry also can be constructed. While, the carbon cages structure smaller than C60 consist of adjacent pentagons. These smallest fullerenes are predicted to have unusual physical as well as mechanical properties due to the high curvature of their molecular surface. The smallest fullerene molecules is a dodecahedron consisting of 20 carbon atoms with only pentagon rings. The fundamental understanding of the size dependence of the closed carbon cage structures is important for tailoring these systems for possible nanotechnology applications [31].

Due to the electronegative nature of fullerene, fullerene can form different compounds with different structures. One of the most important fullerene species is derived from the cage- like structure in which there is an ability of trapping metal atom inside the cage and forming specific endohedral fullerene known as metallofullerene. Exohedral fullerene is another type of fullerene with enhanced solubility property, obtained due to the chemical reaction with chemical groups. On the other side, when one or more carbon atoms within cage structure are substituted by specific hetero atom a hetrofullerene is produced [31].

#### **6.2 Synthesis routes**

Synthesis of graphenic materials like fullerene have been studied and reported in many literatures. Different techniques have been adopted for fullerene synthesis, such as arc discharge technique [32], vapor deposition of carbon atoms technique [33] and laser technique using graphite [34]. The cage- like structure of fullerene was identified for the first time by Kroto and Smalley in 1985, their experiment depending on applying an intense pulsed laser on a rotating graphitic disk to vaporize carbon atoms in the presence of helium atmosphere, then the condensed material had been collected, some of these routes are shown in **Figure 4** [35].

In 1990, another method was used to prepare fullerene reported by Ajie and co-workers, this method depending on the principle of the resistive heating of carbonic rods in a partial helium atmosphere which leads to evaporating carbonic atoms and then condensing it into fullerene structure [36].

Another method was discussed in U.S Patent in 1991, using electric arc technique, through this process an electric arc is generated between graphitic rods in inert atmosphere leading to produce soot- like product in which fullerene molecules extracted from the soot using suitable solvents [37].

The previous methods were associated with producing fullerene with low yields and there is no temperature controlling zone which is required for graphitization step. To overcome these two limitations, in 1994, Smalley discussed a laser vaporization technique to prepare fullerene from graphite materials using a focused laser beam. This technique involving evaporating carbon atoms and retained it in a

**93**

**Figure 4.**

*Some synthesis routes of Fullerene.*

*Fullerenes and Nanodiamonds for Medical Drug Delivery*

temperature- controlled- zone for sufficient time in order to complete the growth

Furthermore, another method was reported by Boorm and co-workers in 2001 called the direct method. This method depending on using polycyclic aromatic hydrocarbons with fullerene- like- framework. Through this method fullerene was synthesized directly due to rolling- up of the hydrocarbons structure into fullerene structure via laser irradiation process under flash vacuum pyrolysis conditions [30]. Whereas, in 1993, fullerene has been synthesized in high yields via ablation technique of graphite rod with solar irradiation using solar- furnace. Through this technique graphite was vaporized under the action of direct exposure to high- fluxsolar- irradiation. Then, the carbonic vapor was entrained by helium flow and

Since the discovery of fullerene as an important carbonic allotrope, tremendous development have been made in nanoscience and nanotechnology in order to fill the urgent need for this materials in a wide variety of applications. But, the formation mechanism of the cage- like structure still as a mystery and not well known [36]. One attempt to explain fullerene formation mechanism was reported by Paul and co-workers in 2012. Through their experiment, they depending on the principle of the bottom- up technique to explain the growth mechanism of fullerene

by integration with carbon atoms and C2 using laser irradiation technique. A carbonic target made up from graphite or 13C amorphous carbon with fullerene content exposed to a single laser strike that leads to vaporizing carbonic target into atomic carbon and C2while fullerene molecules desorbed into carbonic vapor zone under the action of helium flow. Then an interaction takes place between C60 fullerene (98.9% 12C and 1.1% 13C) and enriched 13C carbonic vapor which involves the exchanging of the original 12C atom of fullerene structure with 13C atom of carbonic vapor [40]. Each ingesting of 13C atom into fullerene structure occurs parallel with ejecting 12C atom from its structure and this interaction will spurring bond rearrangement with fullerene structure. Then, the produced species leaves clustering zone and undergo to a supersonic expansion step. Hence, different fullerene isomer such as C70 can be formed and the ejected 12C atoms may be

Furthermore, the growth mechanism of fullerene via different techniques that rely on the interaction between carbon atom and C2 affected by several parameters such as the density of carbonic vaper, exposure time, the flow rate of

and annealing process of the produced structure [38].

cooled into dark- water- zone to form fullerene structure [39].

ingested or exchanged with another fullerene structure [36].

*DOI: http://dx.doi.org/10.5772/intechopen.97867*

*Fullerenes and Nanodiamonds for Medical Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.97867*

#### **Figure 4.** *Some synthesis routes of Fullerene.*

*Materials at the Nanoscale*

properties [31].

applications [31].

**6.2 Synthesis routes**

belongs to the spherical fullerene class which looks like a soccer ball, while C70 belongs to the ellipsoidal class which looks like rugby ball. In addition, several efforts have been reported to produce fullerene with high yields, in 1990, a method was discovered for producing macroscopic amounts of this distinctive material and this breakthrough allowed scientists to understand its chemistry and explore its

Generally, fullerene can be classified into classical fullerene and non-classical fullerene. The first one containing 12 pentagons and any number of hexagons, while the second fullerene class can have heptagons, octagons, and an additional number of pentagons or squares [30, 31]. Due to the unique structure and properties of C60, scientists showed high interesting in synthesis both larger and smaller fullerenes. Therefore, the family of fullerenes has been expanded involving fullerene molecules with a wide range of carbon atoms number. Larger fullerenes that have an icosahedral- symmetry also can be constructed. While, the carbon cages structure smaller than C60 consist of adjacent pentagons. These smallest fullerenes are predicted to have unusual physical as well as mechanical properties due to the high curvature of their molecular surface. The smallest fullerene molecules is a dodecahedron consisting of 20 carbon atoms with only pentagon rings. The fundamental understanding of the size dependence of the closed carbon cage structures is important for tailoring these systems for possible nanotechnology

Due to the electronegative nature of fullerene, fullerene can form different compounds with different structures. One of the most important fullerene species is derived from the cage- like structure in which there is an ability of trapping metal atom inside the cage and forming specific endohedral fullerene known as metallofullerene. Exohedral fullerene is another type of fullerene with enhanced solubility property, obtained due to the chemical reaction with chemical groups. On the other side, when one or more carbon atoms within cage structure are

Synthesis of graphenic materials like fullerene have been studied and reported in many literatures. Different techniques have been adopted for fullerene synthesis, such as arc discharge technique [32], vapor deposition of carbon atoms technique [33] and laser technique using graphite [34]. The cage- like structure of fullerene was identified for the first time by Kroto and Smalley in 1985, their experiment depending on applying an intense pulsed laser on a rotating graphitic disk to vaporize carbon atoms in the presence of helium atmosphere, then the condensed material had been collected, some of these routes are shown in **Figure 4** [35]. In 1990, another method was used to prepare fullerene reported by Ajie and co-workers, this method depending on the principle of the resistive heating of carbonic rods in a partial helium atmosphere which leads to evaporating carbonic

substituted by specific hetero atom a hetrofullerene is produced [31].

atoms and then condensing it into fullerene structure [36].

molecules extracted from the soot using suitable solvents [37].

Another method was discussed in U.S Patent in 1991, using electric arc technique, through this process an electric arc is generated between graphitic rods in inert atmosphere leading to produce soot- like product in which fullerene

The previous methods were associated with producing fullerene with low yields and there is no temperature controlling zone which is required for graphitization step. To overcome these two limitations, in 1994, Smalley discussed a laser vaporization technique to prepare fullerene from graphite materials using a focused laser beam. This technique involving evaporating carbon atoms and retained it in a

**92**

temperature- controlled- zone for sufficient time in order to complete the growth and annealing process of the produced structure [38].

Furthermore, another method was reported by Boorm and co-workers in 2001 called the direct method. This method depending on using polycyclic aromatic hydrocarbons with fullerene- like- framework. Through this method fullerene was synthesized directly due to rolling- up of the hydrocarbons structure into fullerene structure via laser irradiation process under flash vacuum pyrolysis conditions [30]. Whereas, in 1993, fullerene has been synthesized in high yields via ablation technique of graphite rod with solar irradiation using solar- furnace. Through this technique graphite was vaporized under the action of direct exposure to high- fluxsolar- irradiation. Then, the carbonic vapor was entrained by helium flow and cooled into dark- water- zone to form fullerene structure [39].

Since the discovery of fullerene as an important carbonic allotrope, tremendous development have been made in nanoscience and nanotechnology in order to fill the urgent need for this materials in a wide variety of applications. But, the formation mechanism of the cage- like structure still as a mystery and not well known [36].

One attempt to explain fullerene formation mechanism was reported by Paul and co-workers in 2012. Through their experiment, they depending on the principle of the bottom- up technique to explain the growth mechanism of fullerene by integration with carbon atoms and C2 using laser irradiation technique. A carbonic target made up from graphite or 13C amorphous carbon with fullerene content exposed to a single laser strike that leads to vaporizing carbonic target into atomic carbon and C2while fullerene molecules desorbed into carbonic vapor zone under the action of helium flow. Then an interaction takes place between C60 fullerene (98.9% 12C and 1.1% 13C) and enriched 13C carbonic vapor which involves the exchanging of the original 12C atom of fullerene structure with 13C atom of carbonic vapor [40]. Each ingesting of 13C atom into fullerene structure occurs parallel with ejecting 12C atom from its structure and this interaction will spurring bond rearrangement with fullerene structure. Then, the produced species leaves clustering zone and undergo to a supersonic expansion step. Hence, different fullerene isomer such as C70 can be formed and the ejected 12C atoms may be ingested or exchanged with another fullerene structure [36].

Furthermore, the growth mechanism of fullerene via different techniques that rely on the interaction between carbon atom and C2 affected by several parameters such as the density of carbonic vaper, exposure time, the flow rate of inert gas, therefore, there is a possibility of controlling the growth mechanism by these parameters [36].


**2. Results**

#### *2.1. Predicted Concentrations of Antimicrobials in WWTPs*

According to sales data for 2018 (published by the Polish National Health Fund (NFZ)), among detected antimicrobials, the highest sale in Poland was noted for clarithromycin (up to 1509 kg/month), sulfamethoxazole (up to 1358 kg/month), ofloxacin (up to 829 kg/month), ciprofloxacin (up to 821 kg/month), and clindamycin (737 kg/month). We compared the predicted load of antimicrobials in the WWTP1 (PLoad) (Poland) with the load calculated based on the measured concentrations of the drugs in the influent (water phase) and in the primary sludge (Load W+S) (Table A1, Online Resource). For most of the tested antimicrobials, the Load W+S to PLoad ratio was low (up to 20%) because the high metabolism in the human body results in a lower level of parent compound, e.g., the biotransformation ratio of clindamycin is 85% [30]. Moreover, the measured load of fluoroquinolones (ciprofloxacin and norfloxacin) was very close to that of the predicted load, and unlike other antimicrobials that are primarily present in the water phase, ciprofloxacin and norfloxacin are distributed evenly between water and the primary sludge. For six antimicrobials (erythromycin, metronidazole, oxytetracycline, tetracycline, sulfathiazole, and vancomycin), the Load W+S to PLoad ratio was very high and exceeded 1000% because of the low and very low predicted load value. Note that almost all antimicrobials in Poland are available by prescription and most of them are reimbursed; however, some, such as vancomycin, are primarily used in hospitals, while tetracyclines (oxytetracycline and tetracycline) and sulfathiazole are primarily used in veterinary medicine and are not reported by NFZ.

#### *2.2. Antimicrobial Concentrations in Influents of WWTPs*

The concentrations of 26 antimicrobials in influents from WWTP1 and WWTP2 and receiving waterbodies are shown in Table 1. The significant differences between the concentrations of antimicrobials in the samples collected in di fferent sampling periods were observed. The di fferences were due to seasonal variations in antimicrobial use. According to National Health Fund (NFZ) database, in summer the consumption of antimicrobials was low (Table A1). In autumn and winter, the consumption increased significantly, probably due to numerous infections occurring each year in that period [31].

CFR (270 ng/L), FLRX (3.1 ng/L), LOM (0.9 ng/L), NAL (6.1 ng/L), PEF (12 ng/L), SDD (3.7 ng/L) were not detected. Their MDLs (method detection limits) are provided in parentheses. 1 Presented MDLs and method quantitation limits (MQLs) were calculated for influents. The values for effluents are about two times lower and for surface water about four times lower.

Nineteen and 18 out of 26 analyzed antimicrobials were detected in the wastewater from WWTP1 and WWTP2, respectively. In most cases, the antimicrobial levels were higher in wastewater of WWTP2 than in wastewater of WWTP1. The mean values of antimicrobials' concentration in analyzed samples ranged from <MDL (method detection limit) to 7400 ng/L. In influent from WWTP1, the average concentrations of two antimicrobials, i.e., ciprofloxacin and sulfamethoxazole, were higher than 1000 ng/L and concentrations of 10 antimicrobials was higher than 100 ng/L. While in influent from WWTP2, the concentration of four antimicrobials, i.e., ciprofloxacin, metronidazole, sulfamethoxazole, and vancomycin, exceeded the level of 1000 ng/L and concentrations of 12 antimicrobials exceeded the level of 100 ng/L. The highest average concentrations of antimicrobials in influent from WWTP2 were recorded for metronidazole (7400 ng/L), followed by ciprofloxacin (4300 ng/L), vancomycin (u 3200 ng/L), and sulfamethoxazole (3000 ng/L). In other countries, the concentration of metronidazole was lower than 1000 ng/L, that of ciprofloxacin was up to 3800 ng/L [32] but frequently below 400 ng/L [33], and that of sulfamethoxazole was up to 7900 ng/L [34]. There are not much data on vancomycin occurrence in the environment, and it has not been detected in very high concentrations to date as it is primarily used intravenously to treat severe infections in hospitals [35]. The differences in the concentrations of antimicrobials in the influent between two WWTPs can be due to various consumption of antimicrobials in the sampling period and various sources of antimicrobials in these regions. As an example, near WWTP1 is a hospital (449 beds), whereas near WWTP2 is a hospital (458 beds), poultry plant, and galenic laboratory.

#### *2.3. Antimicrobial Concentrations in E*ffl*uent of WWTPs*

The concentrations of 26 antimicrobials in effluents from WWTP1 and WWTP2 are shown in Table 1. In effluents from both WWTPs, the highest average concentrations were observed for azithromycin (up to 650 ng/L), sulfamethoxazole (up to 770 ng/L), ciprofloxacin (up to 312 ng/L), and clindamycin (up to 290 ng/L). In effluent from WWTP2, a higher mean concentration of azithromycin but lower of metronidazole was observed. The percentage contribution of the analyzed antimicrobials in influent and effluent of both WWTPs are presented in Figure 1. To summarize, among antimicrobials tested, ciprofloxacin and sulfamethoxazole were the most dominant antimicrobials in influent and effluent from both WWTPs.

Comparing our data from the literature, the results obtained were similar. The concentration of azithromycin was up to 380 ng/L [36] in Switzerland, that of sulfamethoxazole was up to 1300 ng/L in Spain [21], and that of clindamycin was up to 5 ng/L in Australia [32].

#### *2.4. Antimicrobial Concentrations in Receiving Waters*

In order to assess the impact of antimicrobials on the receiving water bodies, samples were taken upstream and downstream from the WWTPs' discharge (see Section 3.1). The concentrations of 26 antimicrobials in receiving water are shown in Table 1. The river upstream of WWTP1 was more polluted with analyzed antimicrobials than WWTP2 upstream river (Table 1). The highest concentration of sulfamethoxazole (average value: 644 ng/L) was detected upstream of WWTP1. While in the upstream of WWTP2, the highest concentration of clarithromycin (average value: 12.2 ng/L) was detected. The wastewater discharge from WWTP2 resulted in a significant increase of antimicrobials' concentration in the river. This fact can be explained by the high concentrations of antimicrobials in the case of effluent from WWTP2 (Table 1). In the case of WWTP1, taking into account all antimicrobials, no statistical differences between upstream and downstream were observed. Despite this, the average concentration of azithromycin in WWTP1 downstream was more than 15 times higher compared to the concentration in the upstream. The concentrations of antimicrobials in the receiving waters depend on the concentration of antimicrobials in effluent, distance of sampling, or the different flow rate. In our study, WWTP2 effluent effect on antimicrobials' concentrations in the receiving surface water was observed.

**Figure 1.** Percentage contribution (%) of tested antimicrobials in influent and effluent of both WWTPs, *n* = 3.

## *2.5. Antimicrobials' Removal E*ffi*ciency*

The removal efficiency (calculated based on equations described in Section 3.3) of tested antimicrobials was similar for both WWTPs (*p* > 0.05). The average removal efficiency was above 50% for 12 and 10 out of 20 detected antimicrobials for WWTP1 and WWTP2, respectively (Figure 2). Among the antimicrobials from the sulfonamide group, four were detected in effluents and their removal rate ranged from 17% to 80% with an average of 52%. Certain inconsistencies exist in literature about sulfonamide removal, as per the review by Le-Minh et al. [37]. Some researchers have reported an effective removal of sulfonamide [38], although others have mentioned the opposite results [39]. Based on the literature data, this fact might be explained by the differences in operational conditions of each WWTP.

The removal efficiency of trimethoprim was 37% in WWTP1 and 76% in WWTP2 (*p* = 0.2) (the treatment process used in the WWTPs are presented in Section 3.1). Regarding fluoroquinolones, three of them, i.e., norfloxacin, ciprofloxacin, and ofloxacin, were detected in influent of both WWTPs with the average removal being above 80%. The plausible mechanism for the removal of fluoroquinolones from water is sorption to sediment because their concentration was detected as very high in the sludge [40–42] and a slow biodegradation rate was reported [43]. The average removal efficiency for fluoroquinolone antimicrobials estimated by Wang et al. [44] was about 50%. Rodayan et al. [45] reported values of around 60%, while for WWTPs in Switzerland, higher removal efficiencies were observed, reaching up to 87% (for norfloxacin) [46]. In the study of Gao et al. [47], the removal efficiencies of WWTPs for fluoroquinolone antimicrobials ranged from 48% to 72%.

**Figure 2.** Removal efficiency (%) of selected antimicrobials in WWTPs calculated based on equations described in Section 3.3.

In this study, a negative removal rate for azithromycin, clindamycin, lincomycin, and thiabendazole was observed (Figure 2). Moreover, for these antimicrobials, a high variance in removal rate was noted, which agrees with previous studies in which certain antimicrobials were reported to be more abundant in effluents than influents [6,8,10,25,33,48].

The negative removal rate has been detected in the literature. For example, Chunhui et al. [49] reported that the removal efficiencies for macrolide antimicrobials in WWTPs was –4%. Similarly, Gao et al. [47] reported that the removal rate of macrolide antimicrobials was higher, and reached from –34% to 69%. The negative removal rate is explained in many ways. First, certain metabolites can return to the parent pharmaceutical during primary and secondary treatment because of glucuronide conjugate hydrolytic cleavage. Second, pharmaceuticals that sorb to organic matter and particles, as well as accumulate in sediments and biofilms, might be resuspended in wastewater during storm water events. Typically, deconjugation or resorption from particles is observed for fluoroquinolones (ciprofloxacin, ofloxacin, and norfloxacin), lincomycin, tetracycline, trimethoprim, and sulfamethoxazole. This issue is particularly relevant to pharmaceuticals that are mainly excreted with bile and feces-like macrolides. During wastewater treatment, they are redissolved, and, therefore, their concentrations in water increase [10,33].

HRT (hydraulic retention time) is one of the major factors influencing the antimicrobial removal efficiency, particularly for compounds that are readily biodegradable and have a low Kd (low tendency to absorb to sludge) [50]. In this study, HRT was 12 h in WWTP1 and 24 h in WWTP2. Other factors influencing the removal efficiency are adsorption coefficients and persistence of the antimicrobials. Lower levels in effluents could be also interpreted as the removal of antimicrobials because of biodegradation and/or chemical and physical transformations, e.g., hydrolysis or sorption to solid matter. Biodegradation/biotransformation and sorption are the two primary mechanisms occurring in the WWTPs. The sorption to sewage sludge is the primary removal mechanism for certain antimicrobials in the samples, e.g., the percentage of the daily load of antimicrobials was >40% for azithromycin, ciprofloxacin, ofloxacin, and oxytetracycline in sludge (Table 2), while the lowest value was observed for sulfamethoxazole and lincomycin. The differences between the sorption of antimicrobials depend on their physicochemical properties, such as charge and lipophilicity, the properties of the sludge-like chemical nature, and other factors such as pH and redox potential [51]. Sorption may occur by hydrophobic interactions with lipophilic cell membranes of the microorganisms or the lipid fractions

of the suspended solids and electrostatic interactions of positively charged groups of chemicals with negatively charged surfaces of microorganisms or other components of the sludge [33]. Figure 3 shows differences between the concentrations of antimicrobials in wastewater and sludge (separation observed by principal component 1 (PC1) accounted for 45% of total variance). Two groups were designated by the various amounts of oxytetracycline and norfloxacin in the samples. The relative percentage estimated for the antimicrobial was significantly higher in the sludge than in the wastewater. In wastewater, the relative percentage of metronidazole, lincomycin, and sulfamethoxazole was higher compared to that in the sludge. Similarly, a higher contribution of metronidazole and erythromycin in wastewater and ciprofloxacin in sludge was observed by Ostman et al. [24].

**Figure 3.** Differences in antimicrobials' concentrations between the wastewater, sludge samples, and leachate collected from WWTP1 and WWTP2. Principal component analysis (PCA) of the all samples (**A**). Variables of a data matrix are represented as arrows. Visualization of the observed relations on the heat map (**B**). ES, excessive sludge; FS, fermented sludge; In, influent; LS, leachate; Out, effluent; PS, primary sludge; RS, residual sludge.

Our results showed that leachate from WWTP1 contained a significant amount of antimicrobials. The mean concentrations of nine antimicrobials were above 100 ng/L (Table 1). The concentrations of azithromycin, ciprofloxacin, norfloxacin, ofloxacin, sulfamethoxazole, and vancomycin were comparable to those in the WWTP1 influent. The results obtained suggest that the antimicrobials are strongly sorbed to solids (Table A1). The antimicrobials bound to solids can be leached to the environment, which should be taken into consideration when conducting the risk assessment. In the Table A1, the Kd values for tested antimicrobials are presented. Generally, Kd is used to estimate the mobility and distribution of the pharmaceuticals in the environment [41,52]. Compounds with low Kd are potentially mobile from soil to the water (through leaching or runoff). According to our study, the most mobile antimicrobials are trimethoprim (log Kd = 5.8), sulfamethoxazole (log Kd = 5.1), lincomycin (log Kd = 5.2), clarithromycin (log Kd = 5.7), clindamycin (log Kd = 5.7), and thiabendazole (log Kd = 5.9).

#### *2.6. Antimicrobials' Concentrations in Sewage Sludge from WWTPs*

Fifteen antimicrobials in the primary (WWTP1-PS) and excessive sludge (WWTP1-ES) from WWTP1 as well as in the fermented (WWTP2-FS) and residue sludge (WWTP2-RS) from WWTP2 were detected (Table 2). Results obtained indicated that a part of the antimicrobials were adsorbed to the sludge along the whole technological process.

**Table 2.** Mean and standard deviation (*n* = 3) of target antimicrobial concentration (ng/g dry weight) in sewage sludge samples in two wastewater treatment plants (WWTPs) located in Poland at Silesian (WWTP1) and Warmian-Masurian Voivodship (WWTP2).

CFR (350 ng/g), FLRX (12 ng/g), LOM (7.9 ng/g), NAL (3.3 ng/g), MTZ (25 ng/g), RIF (150 ng/g), ST (30 ng/g), SDD (39 ng/g) were not detected. Their MDLs (method detection limits) are provided in parentheses; MQL, method quantitation limit; WWTP-PS, primary sludge; WWTP-ES, excessive sludge; WWTP-FS, fermented sludge, WWTP-RS residual sludge; 1calculated based to the lowest Kd observed and concentration of antimicrobials in leachate.

In our study the highest concentrations for fluoroquinolones, such as ciprofloxacin (up to 57 μg/g), norfloxacin (up to 9.5 μg/g), ofloxacin (up to 2.0 μg/g), and azithromycin (up to 2.3 μg/g), were determined in the sludge samples (data not presented). In China, the concentration of norfloxacin, ofloxacin, and ciprofloxacin in sludge was determined from all the major provincial cities, ranging ranged from 0.1 to 15.7 μg/g, from 0.3 to 7.9 μg/g, and from 0.1 to 4.7 μg/g, respectively [53]. In Switzerland (up to 3.3 μg/g) and Sweden (up to 4.2 μg/g), the maximum concentration of norfloxacin was lower than that in China and presented in Poland. Furthermore, the maximum level of other fluoroquinolones was higher in Poland than in Switzerland (up to 0.9 μg/g for ciprofloxacin) and Sweden (up to 4.8 μg/g for ciprofloxacin and up to 2.0 μg/g for ofloxacin) [54,55]. The concentration of azithromycin in the activated sludge was up to 0.16 μg/g and 0.056 μg/g in Germany and Switzerland, respectively, and the values were lower compared to those for Poland (up to 0.71 μg/g) [56]. Recently, a study from Germany reported the concentration of azithromycin and ciprofloxacin in sediments to be up to 0.33 μg/g and 0.71 μg/g [41], respectively.

#### *2.7. Antimicrobial Resistance Risk Assessment*

The risk factor of antimicrobial resistance selection (named as antimicrobial resistance risk, defined as preferential outgrowth of antimicrobial-resistant bacteria) in wastewater was high for the following antimicrobials: azithromycin, ciprofloxacin, clarithromycin, metronidazole, and trimethoprim (Figure 4A). As expected, this factor was the highest in influent from both WWTPs. Similar results were obtained by Ostman et al. [24], who observed a high risk of resistance selection in influents in Swedish WWTPs because of the prevalence of ciprofloxacin and metronidazole. No risk for azithromycin was detected in their study.

**Figure 4.** Estimation of risk quotients of resistance selection for the antimicrobials occurring in wastewater (**A**), sludge (**B**), and sludge-amended soil (**C**). Risk quotient below 0.1 indicates minimal risk (green area), between 0.1 and 1 is medium risk (orange area), and over 1 is high risk (red area). ES, excessive sludge; FS, fermented sludge; In, influent; LS, leachate; Out, effluent; PS, primary sludge; RS, residual sludge.

It is interesting, the notable antimicrobial resistance risk was also predicted upstream of both WWTPs. High antimicrobial resistance risk for ciprofloxacin and medium risk for azithromycin, clarithromycin, and clindamycin was observed in the case of WWTP1, while medium antimicrobial resistance risk for ciprofloxacin was noted in the case of WWTP2. Downstream WWTP1, on top of the previous risk, the risk for metronidazole turned medium, whereas the risk for azithromycin rose to high. In the case of WWTP2, the risk for ciprofloxacin rose from medium to high and the medium risk for ofloxacin, clarithromycin, and clindamycin appeared (data not presented).

The antimicrobial resistance risk was high for azithromycin, ciprofloxacin, clarithromycin, norfloxacin, trimethoprim, ofloxacin, and tetracycline in sludge (Figure 4B). Interestingly, residual sludge could pose a risk to the environment, particularly because of the presence of fluoroquinolones and macrolides. Mean PECsoil (predicted environmental concentration in soil) was mainly lower than 1 ng/g and higher concentrations were observed for ciprofloxacin (up to 41 ng/g), norfloxacin (up to 7.8 ng/g), ofloxacin (up to 1.2 ng/g), and azithromycin (up to 2.3 ng/g) (Table 3). When RQ was analyzed, a high risk for soil was noted for ciprofloxacin (Figure 4C). The data on risk assessment on the antimicrobial resistance selection in sludge and sludge-amended soil presented in this paper are the first.

#### *2.8. Environmental Risk Assessment*

The high risk was observed for both cyanobacteria (Figure 5A) and eukaryotic species (Figure 5B) due to azithromycin, clarithromycin and sulfamethoxazole in effluents. Furthermore, medium risk was predicted for chronic exposure of cyanobacteria to ciprofloxacin, erythromycin, norfloxacin, and ofloxacin (in effluents). The same level of risk was evaluated for chronic exposure of eukaryotic organisms to ciprofloxacin, erythromycin, and roxithromycin (in effluents). Verlicchi et al. [33] published a review, which reported that six antimicrobials posed a high environmental risk for eukaryotes, i.e., erythromycin, ofloxacin, sulfamethoxazole, clarithromycin, tetracycline, and azithromycin. Harrabi et al. [6] observed a high risk for ofloxacin, ciprofloxacin, azithromycin, sulfamethoxazole, and trimethoprim for eukaryotic species; however, clarithromycin was not evaluated. In their studies, the predicted no effect concentration (PNEC) was, however, calculated differently compared to our study. The authors obtained PNEC values 1000 times lower than the toxicity values found for the most sensitive eukaryotic species that were assayed [6,33]. In our study, other factors, i.e.1000, 100, 50 or10, were used to calculate PNEC. Thus, RQ values obtained for the same concentrations of antimicrobials were lower.

In the case of upstream of WWTP1, there was high risk for cyanobacteria posed by azithromycin, norfloxacin, and sulfamethoxazole, whereas medium risk was observed for ciprofloxacin and clarithromycin. The risk for eukaryote was also observed but the level of the risk was different for the antimicrobials, e.g., for clarithromycin high risk was estimated, and for azithromycin, ciprofloxacin, and sulfamethoxazole medium risk was noted.

None of the antimicrobials posed high risk from upstream of WWTP2. However, medium risk was posed by azithromycin, clarithromycin for cyanobacteria, and medium risk for eukaryote posed by clarithromycin were observed. The medium risk for cyanobacteria posed by ciprofloxacin, ofloxacin, clarithromycin, and sulfamethoxazole in WWTP2 downstream was noted. The risk of toxicity due to azithromycin content increased from medium to high comparing the upstream and downstream. Two more antimicrobials, i.e., azithromycin and ciprofloxacin, were predicted to cause the medium risk for eukaryote.

The RQ calculated for cyanobacteria exposed to sludge was medium for azithromycin (primary sludge up to 0.36, excessive sludge up to 0.12, fermented sludge up to 0.39) and clarithromycin (primary sludge up to 0.13, fermented sludge up to 0.16). No data on risk assessment on antimicrobial presence in sludge was found in literature to compare with our data.

Antimicrobials are designed to act on prokaryotic organisms; thus, environmental bacteria are more likely to be adversely affected compared to other environmental species such as aquatic invertebrates and vertebrates. However, compared to cyanobacteria, certain microalgae and macrophytes are more sensitive to certain antifolate and quinolone antimicrobials [27].

**Figure 5.** Estimation of risk quotients of chronic exposure of cyanobacteria (**A**) and eukaryotic organisms (**B**) to the antimicrobials occurring in effluents and receiving water. Calculations were based on the measured concentration of antimicrobials in wastewater and PNEC calculated based on [27]. Risk quotient below 0.1 indicates minimal risk (green area), between 0.1 and 1 is medium risk (orange area), and over 1 is high risk (red area).

#### **3. Materials and Methods**

#### *3.1. Description of WWTPs and Sample Collection*

The raw influent, final effluent, leachate, and sludge samples (i.e., primary sludge and excessive sludge (WWTP1) or fermented sludge and residual sludge (WWTP2)) were collected from two Polish WWTPs during the three sampling campaigns: (1) In June/July 2018, (2) in October 2018, and (3) in December 2018. Simultaneously, the receiving river water samples from upstream and downstream (WWTP1, 0.25 km; WWTP2, 1.8 km) of the outlet were collected. Each sample was collected in triplicate. The rivers' average annual flow is 3.5 m3/s (WWTP1) and 3.7 m3/s (WWTP2).

WWTP1 is located in one of the cities from Metropolitan Association of Upper Silesia, one of the largest urban areas in the EU and the center of Poland's industries, particularly coal and metal,

with a density of 1600 people per km2 [57], geographical coordinates: N 50◦ 5 35.881, E 19◦ 3 32.202. WWTP2 is located in the forested and agriculture area of Warmian-Masurian Voivodship in the city with a density of 1960 people per km2 [57], geographical coordinates: N 53◦ 48 46.700, E 20◦ 26 55.800.

In 2018, WWTP1 had an equivalent population of 189,332 inhabitants, the average flow rate was 26,830 m3/d, and the plant was operated with a hydraulic retention time (HRT) of ~12 h and a solid retention time (SRT) of 25 days, while WWTP2 served a population equivalent of 250,000 inhabitants, and had a flow rate of 32,130 m3/d, HRT of 24 h, and SRT of 87 days. Both WWTPs receive domestic, hospital, and industrial wastewaters and treat wastewater with a mechanical-biological system with elevated removal of nutrients. The biological section of WWTP1 included sequential reactors activated sludge chambers (the system of chambers of di fferent oxygen conditions: anaerobic, anoxic, and aerobic), secondary settling tank, and anoxic chamber. In the case of WWTP2, the biological part included a pre-denitrification chamber, phosphorus removal tank, nitrification/denitrification chambers, and secondary settling tanks. Sewage sludge produced in the WWTP1 was subjected to a thickening process, methane fermentation, and dehydration. The leachate produced during sludge fermentation (WWTP1-FS) was returned to the pumping station and again treated. The sewage sludge from WWTP2 was used as a soil improver. The detailed description and technical parameters of both WWTPs are presented by Buta et al. [58].

All samples were collected in triplicate and placed in the sterile glass bottles in volumes of 1–2 liters. Sludge samples were collected after mechanical concentration (primary sludge, WWTP1-PS), after gravity concentration (excessive sludge, WWTP1-ES), after an open fermentation pool (fermented sludge, WWTP2-FS), and after all processes (residue sludge, WWTP2-RS), i.e., sludge for management. Then the samples were transported to the laboratory on the same day and stored at a temperature of 4 ◦C until analysis.

#### *3.2. Analysis of Antimicrobials' Concentrations*


**3. Hybrid Three-Tier Model**

In this section, we propose a hybrid three-tier model to tackle the issue of steering renewable energy in favor of energy system reliability. First, we present the conceptual design of such a model. Then, we demonstrate the model's power with an abstract and highly stylized example. Finally, we discuss what needs to be accounted for when the model is transformed from a concept to application in a real case.