**Industrial Applications of Advanced Oxidation Technologies: Past and Future**

Editors

**Gassan Hodaifa Rafael Borja Mha Albqmi**

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

*Editors* Gassan Hodaifa Department of Molecular Biology and Biochemical Engineering Pablo de Olavide University Seville Spain

Rafael Borja Instituto de la Grasa Spanish National Research Council Seville Spain

Mha Albqmi Chemistry Department Jouf University Sakaka Saudi Arabia

*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 *Catalysts* (ISSN 2073-4344) (available at: www.mdpi.com/journal/catalysts/special\_issues/industrial\_AOPs\_ catalysis).

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 978-3-0365-8193-4 (Hbk) ISBN 978-3-0365-8192-7 (PDF)**

© 2023 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.

 He received his doctoral degree from the University of Jaén. He joined the research group Bioprocesses TEP-138 (Junta de Andalucía), and works in the areas of microalgae, the treatment of wastewater, chemical oxidation, membrane technology, adsorption, pesticide removal, olive oil processing, enzyme biotechnology, and nematode biotechnology. He has participated in 27 research projects (8 as IP) and 4 European programs (1 PRIMA Project, and 3 in collaboration with M.A.P.A. and the INFAOLIVA and UNAPROLIVA companies), with which he has signed 16 research contracts (4 as IP). He has been contracted twice as Adviser for the mega Japanese company "Asahi Kasei Corporation", and has had six projects financed by J. Andalucía (two of them as IP). He has completed five projects with the National Research Plan, one project for the Madrid Community, and the rest he carried out with companies. As a result of his research work, he has had 64 articles published, with 56 articles published in journals included in the JCR (31 in Q1, 18 in Q2, and 7 in Q3). Of these articles, more than 10 articles have been published as a collaboration with authors from different countries on all continents. He has published six total articles in open access journals, four articles in journals with an impact factor different from that listed in the JCR, and five other articles in outreach magazines. He has two patents for the water purification of olive oil mill wastewater, and has written two books and 46 book chapters that have been published by prestigious publishers such as the Academic Press (Elsevier), CRC Press, Taylor and Francis Group, Springer International Publishing AG, etc. He was the Editor in Chief of three Special Issues in *J. Chemistry* and *Catalysts*. He has participated in 125 congresses. He is currently a member of 15 Editorial Boards of international journals such as *Catalysts* and *Heliyon*.

### **Rafael Borja**

Rafael Borja is a Doctor (PhD) in Chemical Sciences. Since 1986, he has been working as a researcher at the Instituto de la Grasa (CSIC, Spanish National Research Council) in Seville, after obtaining an FPI grant funded by the Andalusian Government for his doctoral thesis. He received the San Alberto Magno Prize for Doctoral Theses, which was awarded by the National Association of Spanish Chemists (ANQUE), Delegation of Andalusia, in 1991. During the period of September 1992–September 1994, he carried out a postdoctoral stay at the "Environmental Technology Centre" belonging to the "Department of Chemical Engineering" of the University of Manchester's Institute of Science and Technology (UMIST) in the United Kingdom. In 1997 he obtained the position of Tenured Scientist of the CSIC, and since then he has developed several lines of research related to the use and treatment of wastewater and solid wastes from agro-food industries. In 2008 he obtained the position of Research Scientist at the CSIC. He has directed six doctoral theses and is a co-author of eight patents. He has published 302 articles in different scientific journals that are included in the "Science Citation Index (SCI)".

He has been the principal investigator of 12 projects funded by the R&D National Plan and by the European Union, as well as several R&D contracts developed at the Instituto de la Grasa (CSIC) in Seville. He currently is a member of the Editorial Board of the scientific journals *Process Biochemistry* (Elsevier), *Catalysts* (MDPI), and *Emerging Science Journal* (Ital Publication). During the period 2013–2015, he was the vice-president of the association, "International Association of the Mediterranean Agro-industrial Wastes (IAMAW)", which is located in Perugia, Italy, and from 2016 to 2019 he was a member of the "Audit Committee" of this association.

### **Mha Albqmi**

Dr. Mha Albqmi currently holds the position of Assistant Professor at Jouf University and serves as the Director of the Olive Research Center at Jouf University, Saudi Arabia. Her educational background includes a Ph.D. in Chemistry from Oklahoma State University, Stillwater, Oklahoma, USA, a Master of Science Degree in Chemistry from Lamar University, Texas, USA, and a Bachelor of Science Degree in Chemistry from Taif University, Taif, Saudi Arabia.

Dr. Mha Albqmi's great passion lies in the field of catalysis, and studies its applications in various industrial chemical processes. Particularly, throughout her Ph.D. program, she made contributions to the development of new procedures that enabled the direct conversion of organic compounds into their resulting products, eliminating the requirement for solvents and effectively reducing waste generation.

Her current research interests lie in environmental chemistry, the recycling of waste materials, and the prevention of pollution. Additionally, she actively valorizes the olive waste residue on certain plants experiencing heavy-metal toxicity. She is involved in the study of nanotechnology applications, such as water purification and photocatalytic degradation processes. Furthermore, she is researching the direct conversion method of organic compounds using catalysts as well as the potential of natural products for the treatment of metal-overload diseases.

## **Preface to "Industrial Applications of Advanced Oxidation Technologies: Past and Future"**

In recent years, climate change has become more evident due to the high values of the carbon footprints and the hydraulic footprints registered by industries. Additionally, the availability of drinking water is scarce in many countries around the world. Technological advances have permitted the establishment of large industries in the last century. Most of these industries are characterized by a high consumption of drinking water that is transformed into wastewater as it enters and leaves these industrial processes. Unfortunately, a large part of this wastewater is not recovered because it is difficult to treat due to the presence of a high organic load with persistent, toxic, and inhibitory compounds. The capabilities of conventional urban wastewater treatment plants (CUWTPs) make them unable to meet the standards set by local, national, or international legislation. On the other hand, society is unwilling to give up many of the comforts offered by technological developments, highlighting the need for effective solutions to minimize or reduce climate change, which has clearly made its presence felt. In response to this challenge, a green and sustainable pre-treatment, or the treatment of industrial wastewater prior to its discharge from CWWTPs, is mandatory for industries.

The use of Advanced Oxidation Technologies (AOTs) for wastewater treatment is an important area of research which has not yet been fully exploited at an industrial level and has significant potential in the disposal of many industrial effluents. In particular, this includes effluents that are difficult to treat by conventional biological treatment processes. Given the positive response from researchers to our first Special Issue entitled "Photocatalysis in the Wastewater Treatment", this new Special Issue represents a continuation and an extension of the work carried out in the previous Special Issue and aims to explain the importance of advanced oxidation technologies and how their incorporation into the industrial sector as green and clean technologies can improve the current situation of the Earth's ecosystems and environment. In this sense, it is worth mentioning treatments based on photolysis, TiO2/solar light, ozone/ultraviolet irradiation, oxidant/ultraviolet irradiation, oxidant/catalyst/ultraviolet irradiation, high-energy electron beam irradiation (E-beam), sonication/photocatalysis, etc. This Special Issue includes works with new perspectives on catalytic ozonation for organic removal, photo-based advanced oxidation processes for pharmaceutical degradation, etc.

This reprint is addressed to all readers interested in the manufacture of catalysts and chemical oxidation-based treatment processes, whether they are academics or professionals. Finally, the Editors would like to thank the authors who have participated in the writing of this interesting book, which will be of great interest at the industrial, academic, and research level.

> **Gassan Hodaifa, Rafael Borja, and Mha Albqmi** *Editors*

## *Article* **Comprehensive Study on Environmental Behaviour and Degradation by Photolytic/Photocatalytic Oxidation Processes of Pharmaceutical Memantine**

**Sandra Babi´c 1,\* , Davor Ljubas 2 , Dragana Mutavdži´c Pavlovi´c 1 , Martina Bioši´c 1 , Lidija Curkovi´c ´ 2,\* and Dario Dabi´c 1,3**

**Abstract:** Memantine is a pharmaceutical used to treat memory loss, one of the main symptoms of dementia and Alzheimer's disease. The use of memantine is expected to continue to grow due to the increasing proportion of the elderly population worldwide. The aim of this work was to conduct a comprehensive study on the behaviour of memantine in the environment and the possibilities of its removal from wastewater. Abiotic elimination processes (hydrolysis, photolysis and sorption) of memantine in the environment were investigated. Results showed that memantine is stable in the environment and easily leached from river sediment. Therefore, further investigation was focused on memantine removal by advanced oxidation processes that would prevent its release into the environment. For photolytic and photocatalytic degradation of memantine, ultraviolet (UV) lamps with the predominant radiation wavelengths of 365 nm (UV-A) and 254/185 nm (UV-C) were used as a source of light. TiO2 in the form of a nanostructured film deposited on the borosilicate glass wall of the reactor was used for photocatalytic experiments. Photodegradation of memantine followed pseudo-first-order kinetics. The half-life of photocatalytic degradation by UV-A light was much higher (46.3 min) than the half-life obtained by UV-C light (3.9 min). Processes degradation efficiencies and evaluation of kinetic constants were based on the results of HPLC-MS/MS analyses, which also enable the identification of memantine oxidation products. The acute toxicity of the reaction mixture during the oxidation was evaluated by monitoring the inhibition of the luminescence of *Vibrio fischeri* bacteria. The results showed that memantine and its oxidation products were not harmful to *Vibrio fischeri*.

**Keywords:** memantine; hydrolysis; photolysis; sorption; photocatalysis; sol-gel TiO2 film; degradation products; toxicity

### **1. Introduction**

With the increase in the aging population worldwide, Alzheimer's disease and other dementias have become a rapidly increasing public health concern, with an estimated 50 million people currently living with dementia [1]. The prevalence of Alzheimer's disease is approximately 0.6% at the age of 60 but it doubles every five years, so the prevalence is about 40% at the age of 90 [2]. Currently, no cure exists for Alzheimer's disease but there are treatments that temporarily slow down the development of symptoms and improve cognitive functions. In Europe and the USA two symptomatic treatments are approved, the use of acetylcholinesterase (AChE) inhibitors and *N*-methyl-*D*-aspartate receptor antagonist memantine (3,5-dimethyladamantan-1-amine) [1]. Once in the organism, memantine is

**Citation:** Babi´c, S.; Ljubas, D.; Mutavdži´c Pavlovi´c, D.; Bioši´c, M.; Curkovi´c, L.; Dabi´c, D. ´ Comprehensive Study on Environmental Behaviour and Degradation by Photolytic/ Photocatalytic Oxidation Processes of Pharmaceutical Memantine. *Catalysts* **2023**, *13*, 612. https://doi.org/ 10.3390/catal13030612

Academic Editors: Gassan Hodaifa, Rafael Borja and Mha Albqmi

Received: 20 February 2023 Revised: 15 March 2023 Accepted: 16 March 2023 Published: 17 March 2023

**Copyright:** © 2023 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/).

poorly metabolized and most (57–82%) of the administered dose is excreted unchanged in urine [3]. Memantine is a compound highly soluble in water (29.4 mg/L, [4]).

In view of this, it is reasonable to expect that memantine will end up in wastewater and, without proper treatment, in environmental waters. Memantine was detected in rivers and sewage treatment plant (STP) influents and effluents in Japan at high frequency (>70%), with a maximal measured environmental concentration in seven rivers of 47.4 ng/L. The measured concentration of memantine in STP was lower than 1 µg/L with an average removal rate in three STPs lower than 20% [5]. Memantine was detected in effluent wastewater from a wastewater treatment plant (WWTP) located near Barcelona (Spain) at a concentration ranging from 0.028 to 0.134 µg/L for samples taken on 10 different days [6]. Memantine was also detected in sewage effluents in three Sweden STPs in concentrations ranging from 10 to 14 ng/L. It is also detected in the plasma of fishes exposed to sewage effluents (from <LOQ (0.5 ng/mL) to 2.3 ng/mL) [7]. Kårelid et al. [8] studied the adsorption of pharmaceuticals on granular activated carbon (GAC) and powdered activated carbon (PAC) at three Swedish wastewater treatment plants and observed that, among 22 investigated pharmaceuticals, only memantine showed removal lower than 95%. Despite the evidence that memantine is present in the environment, data on the fate and behaviour of memantine in the environmental are scarce.

The incomplete removal of pharmaceuticals in conventional wastewater treatment plants clearly indicates the need for the development of innovative technologies such as advanced oxidation processes (AOPs). AOPs have been proposed as a tertiary treatment for wastewater [9,10]. Among different AOPs, heterogeneous photocatalysis is a promising method for removing organic micropollutants (OMPs), including pharmaceuticals [11–13]. The most commonly used semiconductor photocatalyst is TiO2 with the potential for the total mineralization of OMPs, resulting in the formation of non-toxic compounds (CO2, H2O and the corresponding mineral acids). TiO2 can be used in the form of TiO2 powder suspension (slurry) or it can be immobilized by different techniques on different substrates such as borosilicate glass [14,15], alumina foam [16], alumina ring and borosilicate ring. Immobilization of TiO2 on the adequate reactor walls eliminates the need to separate the photocatalyst from the treated water. Among different deposition techniques (sol-gel, thermal treatment, pulsed laser deposition, reactive evaporation, physical vapour deposition (PVD), chemical vapour deposition (CVD), electrodeposition, sol-spray, hydrothermal deposition, etc.), the sol-gel technique offers many advantages: relatively low cost, low processing temperature, simple deposition, relatively simple control of composition, possibility of various forming processes, and ability to prepare nano-sized thin films and to produce fine structures [14,17]. Sol-gel films can be generally deposited by two methods—the dip coating and the spin coating technique [18].

The aim of this study was to investigate the environmental behaviour of memantine and possibility of its degradation by advanced oxidation processes. Environmental behaviour was investigated by studying abiotic elimination processes: hydrolysis, photolysis and sorption. For investigation of photolytic/photocatalytic oxidation of memantine, a photoreactor with UV-A and UV-C lamps, and TiO2 in a form of a nanostructured film deposited on borosilicate glass wall of the reactor was used. Process degradation efficiencies and evaluation of kinetic constants were based on the results of HPLC-MS/MS analyses, which also enable identification and monitoring of memantine degradation products. In addition, the acute toxicity of the reaction mixture during the degradation experiment was evaluated by monitoring the inhibition of the luminescence of *Vibrio fischeri* bacteria.

### **2. Results and Discussion**

### *2.1. Environmental Behaviour*

2.1.1. Hydrolytic and Photolytic Degradation

Hydrolytic degradation of memantine was investigated according to the procedure described in OECD 111 [19]. The results of hydrolytic degradation experiments showed that memantine is persistent to hydrolytic degradation with the degree of hydrolytic

degradation between 1.5% and 2.8% under the applied conditions (Figure 1A). Given that hydrolytic degradation of 10% at 50 ◦C corresponds to a half-life of approximately 30 days, which is equivalent to the half-life of 1 year at 25 ◦C [1], memantine was considered stable and no further investigation is required. hydrolytic degradation of 10% at 50 °C corresponds to a half-life of approximately 30 days, which is equivalent to the half-life of 1 year at 25 °C [1], memantine was considered stable and no further investigation is required.

Hydrolytic degradation of memantine was investigated according to the procedure described in OECD 111 [19]. The results of hydrolytic degradation experiments showed that memantine is persistent to hydrolytic degradation with the degree of hydrolytic degradation between 1.5% and 2.8% under the applied conditions (Figure 1A). Given that

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2.1.1. Hydrolytic and Photolytic Degradation

**2. Results and Discussion**  *2.1. Environmental Behaviour*

Photolytic degradation was investigated with memantine solution in MilliQ water (direct photolysis) and in the presence of substances commonly present in environmental waters: humic acids (HA), chloride and nitrate (indirect photolysis). The concentrations of inorganic ions and humic acids were typical for the environment. The presence of HA and nitrates resulted in a lower concentration of memantine, while chlorides did not affect photolytic degradation of memantine. The photolysis due to the presence of HA and nitrate can be attributed to the formation of highly reactive hydroxyl radicals [20,21]. However, the observed decrease in the concentration of memantine was not significant (less than 15% after 24-h exposure to simulated solar radiation, Figure 1B), which points to the conclusion of its persistence during exposure to artificial solar radiation. Blum et al. [22] Photolytic degradation was investigated with memantine solution in MilliQ water (direct photolysis) and in the presence of substances commonly present in environmental waters: humic acids (HA), chloride and nitrate (indirect photolysis). The concentrations of inorganic ions and humic acids were typical for the environment. The presence of HA and nitrates resulted in a lower concentration of memantine, while chlorides did not affect photolytic degradation of memantine. The photolysis due to the presence of HA and nitrate can be attributed to the formation of highly reactive hydroxyl radicals [20,21]. However, the observed decrease in the concentration of memantine was not significant (less than 15% after 24-h exposure to simulated solar radiation, Figure 1B), which points to the conclusion of its persistence during exposure to artificial solar radiation. Blum et al. [22] reported similar results of memantine photolytic persistence with a degree of degradation of less than 10%.

reported similar results of memantine photolytic persistence with a degree of degradation of less than 10%. According to the available literature, similar environmental behaviour was not observed for other pharmaceuticals detected in environmental waters. They are usually sus-According to the available literature, similar environmental behaviour was not observed for other pharmaceuticals detected in environmental waters. They are usually susceptible to photolytic [20,21,23–25] or hydrolytic [26,27] degradation or to both elimination processes [28,29].

#### ceptible to photolytic [20,21,23–25] or hydrolytic [26,27] degradation or to both elimina-2.1.2. Sorption

#### tion processes [28,29]. Kinetics of Sorption and Desorption

2.1.2. Sorption Kinetics of Sorption and Desorption Based on previously published papers [30,31], there is already some information Based on previously published papers [30,31], there is already some information about the tendency of memantine to sorption on soil and sediment particles, namely that sorption is definitely not a dominant process in its case. In this context, our goal was to compare from a kinetic point of view how much memantine is sorbed to the sediment particles and how much memantine is desorbed from the same sediment studied.

about the tendency of memantine to sorption on soil and sediment particles, namely that sorption is definitely not a dominant process in its case. In this context, our goal was to compare from a kinetic point of view how much memantine is sorbed to the sediment particles and how much memantine is desorbed from the same sediment studied. From Figure 2A, it can be seen that the "faster" sorption of memantine to the sediment sample occurs in the first 6 h, after which further sorption of memantine occurs slowly over the observed 24-h period. At the same time, desorption of the previously sorbed memantine takes place during the same time intervals (Figure 2B). It should be noted that, as the concentration of memantine increases, the amount of memantine sorbed and desorbed decreases, so that the largest difference between the amount sorbed and desorbed was obtained at the lowest initial concentration of memantine tested (0.1 mg/L). To investigate the control mechanisms of the sorption [32] and desorption processes, experiments were

performed at different time periods, i.e., a kinetic study was performed at three concentration levels (0.1, 0.5 and 2.0 mg/L), in contrast to the other six concentrations at which the sorption experiments will be carried out. periments were performed at different time periods, i.e., a kinetic study was performed at three concentration levels (0.1, 0.5 and 2.0 mg/L), in contrast to the other six concentrations at which the sorption experiments will be carried out.

From Figure 2A, it can be seen that the "faster" sorption of memantine to the sediment sample occurs in the first 6 h, after which further sorption of memantine occurs slowly over the observed 24-h period. At the same time, desorption of the previously sorbed memantine takes place during the same time intervals (Figure 2B). It should be noted that, as the concentration of memantine increases, the amount of memantine sorbed and desorbed decreases, so that the largest difference between the amount sorbed and desorbed was obtained at the lowest initial concentration of memantine tested (0.1 mg/L). To investigate the control mechanisms of the sorption [32] and desorption processes, ex-

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**Figure 2.** Kinetics of (**A**) sorption and (**B**) desorption for memantine on the sediment Studena, *T* = **Figure 2.** Kinetics of (**A**) sorption and (**B**) desorption for memantine on the sediment Studena, *T* = 25 ◦C.

The sorption and desorption data were analysed using three different kinetic models; Lagergren pseudo-first-order, pseudo-second-order and the intraparticle diffusion (IPD) model. All kinetic models are presented in Table 1 where *q*e and *q*t are the amounts of memantine (μg/g) adsorbed/desorbed on investigated sediment samples at equilibrium and at time *t*; *k*1 (1/min) is the rate constant of the pseudo-first-order adsorption; *k*2 (g/μg min) is the rate constant of the pseudo-second-order sorption and *k*pi (μg/g min1/2) is the intraparticle diffusion rate parameter of stage *i*. *C*i, the intercept of stage *i*, gives an indi-The sorption and desorption data were analysed using three different kinetic models; Lagergren pseudo-first-order, pseudo-second-order and the intraparticle diffusion (IPD) model. All kinetic models are presented in Table 1 where *q*e and *q*t are the amounts of memantine (µg/g) adsorbed/desorbed on investigated sediment samples at equilibrium and at time *t*; *k*1 (1/min) is the rate constant of the pseudo-first-order adsorption; *k*2 (g/µg min) is the rate constant of the pseudo-second-order sorption and *k*pi (µg/g min1/2) is the intraparticle diffusion rate parameter of stage *i*. *C*i , the intercept of stage *i*, gives an indication of the thickness of the boundary layer, i.e., the larger the intercept, the larger the boundary layer effect.

cation of the thickness of the boundary layer, i.e., the larger the intercept, the larger the **Table 1.** Kinetic models.

25 °C.

Ho's pseudo-second-order ௧ ൌ 1 ଶ ଶ The sorption and desorption rate constants *k*1, *k*2 and *q*e,cal as well as the correlation coefficients (*R* 2 ) for the pseudo-first and pseudo-second models are shown in Table 2.

From the results, it can be concluded that the pseudo-second-order kinetic model perfectly describes both the kinetics of memantine sorption and the kinetics of desorption of previously sorbed memantine, since very high correlation coefficients (*R* 2 > 0.999) were obtained in both cases. These results suggest that the sorption/desorption capacity is regulated by the number of available active sites on the sediment. According to this model, the maximum concentration absorbed at equilibrium (*q*e) on the Studena sediment was approximately 10.11 µg/g, which corresponds to the maximum sorption capacity of this sediment for memantine in the experiments performed. However, if we consider the results of desorption according to the same model, it follows that the maximum concentration desorbed at equilibrium on the Studena sediment was about 4.40 µg/g, which practically corresponds to almost half of the amount of memantine previously sorbed. This ratio of sorbed/desorbed memantine from the sediment studied depends, of course, on the memantine concentration in contact with the sediment, so that Table 2 shows that, at concentrations of 0.1 mg/L and 0.5 mg/L, almost one-third is desorbed, which is less than the previously mentioned concentration of 2.0 mg/L, at which almost half was desorbed. The higher the concentration of memantine in contact with the sediment, the more memantine is washed out of the sediment, which is consistent with what was said before, i.e., that the sorption/desorption capacity is regulated by the number of available active sites on the sediment. The desorption rate constants according to the pseudo-second-order kinetic model are much higher than the sorption constants under the same conditions. Such a result is even more discouraging because, no matter how little memantine is sorbed on a sediment, it is still quite a lot and is rapidly desorbed, posing a risk of water contamination by memantine.

The kinetics of sorption and desorption can also be described from a mechanical point of view. The whole process of sorption and desorption can be controlled by one or more steps, such as surface diffusion, pore diffusion, external diffusion and sorption/desorption at the pore surface. When the sorbent is porous, as in the case of sediments, intraparticle diffusion often plays a major role. During rapid stirring, the diffusion mass transport can be related to the diffusion coefficient, which describes well the experimental sorption/desorption data. Results of the IPD model are shown in Table 3.

**Table 3.** Intraparticle diffusion model constants and correlation coefficients for memantine on the sediment Studena at different initial concentrations.

From these results, it is evident that the process of sorption and desorption of memantine on the studied sediment is multilinear, indicating that the sorption and desorption process occurs in three phases [33]: (i) initial boundary layer diffusion or adsorption/desorption at the outer surface, (ii) gradual intraparticle diffusion or diffusion in the pores, where the degree of intraparticle diffusion is rate-controlled, and (iii) equilibrium stage showing saturation of the sorbent surface.

This multilinearity of the sorption and desorption processes suggests that IPD was not the only rate-controlling step [34] but that multiple steps occur at this microlevel. All the kinetic results obtained are of great importance, especially the desorption information, which plays an important role in evaluating the behaviour of memantine in the environment.

### Sorption Isotherms

The sorption of memantine was tested on three sediment samples (Pakra, Petrinjˇcica and Studena) and described by two sorption isotherms: the Linear and Freundlich sorption isotherms, and results are presented in Table 4. All presented values are expressed by the average value of three determinations. Achieved relative standard deviations are lower than 10%.

**Table 4.** Sorption coefficients (*K*d), Freundlich and Dubinin-Radushkevich sorption isotherm parameters in 0.01 M CaCl2 at initial pH values.

From the obtained regression coefficients *R* 2 , it can be seen that only the linear isotherm describes the sorption process with *R* 2 > 0.99 in all cases, while the range of regression coefficients for the Freundlich isotherm is 0.89–0.92. The values of the Freundlich exponent, *n*, range from 1 to 10, indicating favourable sorption [35]. The Dubinin–Radushkevich model shows the worst agreement with the experimental data since *R* 2 ranges from 0.6451 to 0.7082. Since the values of sorption energy, *E* (obtained from the D-R isotherm model), are from 3.24 to 4.18 kJ/mol for the investigated sediments, it could be said that the sorption of memantine on the investigated sediments is of a physical nature.

In addition to these three sorption models, many other sorption isotherm models were tried in the preliminary experiment to obtain more information about sorption of memantine, but without success. For example, when trying to applied the Langmuir isotherm we either obtained negative values for the Langmuir isotherm constants or the *R* 2 were extremely low (*R* 2 < 0.25). This indicates that the Langmuir sorption isotherm is not suitable to explain the sorption process of memantine on the sediment samples studied, since these Langmuir constants indicate the binding surface energy and monolayer coverage.

The values of the distribution coefficient *K*d from the linear isotherm and the adsorption capacity *K*F from the Freundlich isotherm indicate a slightly weaker binding of memantine to the sediments of Studena and Pakra compared to the sediment of Petrinjˇcica. However, it should be noted that high values of sorption coefficients were not expected at all, since, according to previous studies, a low tendency of memantine to sorption on soil and sediment particles was observed [30,31]. In any case, the results obtained in this study support the fact that memantine poses a major threat to natural waters because it is easily leached from sediment samples and thus has high mobility in these sediments.

Numerous previous studies clearly show that pH is one of the most important factors affecting the sorption mechanism and rate [36,37]. This is supported by the fact that whether the observed molecule behaves as a cation, anion or neutral molecule depends on the pH of the medium, but also that the activity/presence of metal ions present in the sediment changes depending on it. Since memantine as a molecule is characterized by a p*K*a constant (10.27), it can be concluded that it also occurs in ionic form. However, based on the natural pH value of the sediments studied, it could be concluded that memantine occurs exclusively as a neutral molecule in all sediments studied [30].

In addition to the effect of pH on the distribution of the ionic/molecular species of the memantine under environmental conditions, the effect of pH on their sorption at three different pH values (pH 5, 7 and 9) was also investigated (Figure 3A).

43].

occurs exclusively as a neutral molecule in all sediments studied [30].

different pH values (pH 5, 7 and 9) was also investigated (Figure 3A).

**Figure 3.** The influence of (**A**) pH and (**B**) ionic strength on the sorption capacity of memantine in studied sediments (T = 25 °C). **Figure 3.** The influence of (**A**) pH and (**B**) ionic strength on the sorption capacity of memantine in studied sediments (T = 25 ◦C).

Numerous previous studies clearly show that pH is one of the most important factors affecting the sorption mechanism and rate [36,37]. This is supported by the fact that whether the observed molecule behaves as a cation, anion or neutral molecule depends on the pH of the medium, but also that the activity/presence of metal ions present in the sediment changes depending on it. Since memantine as a molecule is characterized by a p*K*a constant (10.27), it can be concluded that it also occurs in ionic form. However, based on the natural pH value of the sediments studied, it could be concluded that memantine

In addition to the effect of pH on the distribution of the ionic/molecular species of the memantine under environmental conditions, the effect of pH on their sorption at three

In these experiments, an inverse relationship between sorption and pH was observed for memantine [38,39], i.e., with higher acidity, the sorption coefficient decreases. In all three sediments studied, differences in *K*d values are observed with the change in pH. While the changes in distribution coefficient from neutral to the alkaline pH range are easily visible, the change in distribution coefficient from the neutral to the acidic pH range is much less pronounced. From these results, it can be clearly concluded that the influence of pH on the sorption of memantine dominates. In these experiments, an inverse relationship between sorption and pH was observed for memantine [38,39], i.e., with higher acidity, the sorption coefficient decreases. In all three sediments studied, differences in *K*d values are observed with the change in pH. While the changes in distribution coefficient from neutral to the alkaline pH range are easily visible, the change in distribution coefficient from the neutral to the acidic pH range is much less pronounced. From these results, it can be clearly concluded that the influence of pH on the sorption of memantine dominates.

In addition to the influence of pH, the influence of ionic strength on the sorption of memantine was also investigated. It was found that the sorption coefficients decreased with increasing ionic strength (Figure 3B). The obtained results indicate a possible surface complexity between the memantine and the studied sediments. For all sediments studied, the highest *K*d coefficient values were obtained at the lowest concentration of CaCl2 solution tested. The influence of ionic strength on sorption could be related to the fact that the thickness of the charged surface of the "electric double layer" is reduced, resulting in decrease in surface charge and fewer interactions between the ionic form of the drug and the sediment surface [40]. Of course, this theory is also supported by the fact that memantine is in the form of a neutral molecule [30] in all experiments performed, which makes a possible interaction even less likely. A similar trend was observed in a previous study of the sorption of memantine and in other studies of the sorption of pharmaceuticals [30,41– In addition to the influence of pH, the influence of ionic strength on the sorption of memantine was also investigated. It was found that the sorption coefficients decreased with increasing ionic strength (Figure 3B). The obtained results indicate a possible surface complexity between the memantine and the studied sediments. For all sediments studied, the highest *K*d coefficient values were obtained at the lowest concentration of CaCl2 solution tested. The influence of ionic strength on sorption could be related to the fact that the thickness of the charged surface of the "electric double layer" is reduced, resulting in decrease in surface charge and fewer interactions between the ionic form of the drug and the sediment surface [40]. Of course, this theory is also supported by the fact that memantine is in the form of a neutral molecule [30] in all experiments performed, which makes a possible interaction even less likely. A similar trend was observed in a previous study of the sorption of memantine and in other studies of the sorption of pharmaceuticals [30,41–43].

### *2.2. Photolytic and Photocatalytic Oxidation of Memantine in Aqueous Solution*

*2.2. Photolytic and Photocatalytic Oxidation of Memantine in Aqueous Solution*  The photocatalytic activity of sol-gel TiO2 film was evaluated through the degrada-The photocatalytic activity of sol-gel TiO2 film was evaluated through the degradation of memantine aqueous solution (10 mg/L) under ultraviolet (UV) lamps with the predominant radiation wavelengths of 365 nm (UV-A) and 254/185 nm (UV-C).

tion of memantine aqueous solution (10 mg/L) under ultraviolet (UV) lamps with the predominant radiation wavelengths of 365 nm (UV-A) and 254/185 nm (UV-C). In order to investigate the kinetics of the photocatalytic degradation of memantine by photolytic and photocatalytic processes, the pseudo-first-order kinetic model was used. The linear form of the pseudo-first-order kinetic model is [44]:

$$
\ln \frac{\mathbf{C}\_0}{\mathbf{C}\_t} = -k\_1 \cdot t \tag{1}
$$

The half-life time *t*1/2 was calculated using the following expression [16]:

$$t\_{1/2} = \frac{\ln \text{ (2)}}{k} \tag{2}$$

where *Ct* (mg/L) is the concentration of memantine at time *t* (min), *C*0 (mg/L) is the initial memantine concentration and *k*1 (1/min) is the degradation rate constant.

The first-order degradation rate constant (*k*1, 1/min) from equation 1 can be calculated by the slope of the straight line obtained from plotting linear regression of −ln (*Ct*/*C*0) versus irradiation time (*t*) (Figure 4A). Table 5 shows the pseudo-first-order kinetic constant (*k*1, 1/min), coefficient of determination (*R* 2 ), half-life time (*t*1/2, min) and efficiency (*η*, %)

for memantine removal by photolysis and photocatalysis. It is noticed that the pseudofirst-order model has an *R* 2 > 0.96, which confirms that the memantine removal follows a pseudo-first-order model. memantine removal by photolysis and photocatalysis. It is noticed that the pseudo-firstorder model has an *R*2>0.96, which confirms that the memantine removal follows a pseudo-first-order model.

In order to investigate the kinetics of the photocatalytic degradation of memantine by photolytic and photocatalytic processes, the pseudo-first-order kinetic model was

> ଵ/ଶ ൌ ሺ2ሻ

The first-order degradation rate constant (*k1*, 1/min) from equation 1 can be calculated by the slope of the straight line obtained from plotting linear regression of -ln(*Ct*/*C*0) versus irradiation time (*t*) (Figure 4A). Table 5 shows the pseudo-first-order kinetic constant (*k1*, 1/min), coefficient of determination (*R*2), half-life time (*t*1/2, min) and efficiency (*η, %)* for

where *C*t (mg/L) is the concentration of memantine at time *t* (min), *C*0 (mg/L) is the initial

ൌ െଵ ∙ (1)

(2)

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 8 of 17

used. The linear form of the pseudo-first-order kinetic model is [44]:

 ௧

The half-life time *t*1/2 was calculated using the following expression [16]:

memantine concentration and *k*1 (1/min) is the degradation rate constant.

**Figure 4.** (**A**) Photolytic and photocatalytic degradation of memantine under UV-A (365 nm) and UV-C (254/185 nm) radiation by sol-gel nanostructured TiO2 film as a function of irradiation time, C0 (memantine) = 10 mg/L. Inset: linear transform of −ln (Ct/C0) versus *t*. (**B**) Photolytic and photocatalytic degradation efficiency. All experiments were triplicated with the standard deviation from the average value ± 4%. **Figure 4.** (**A**) Photolytic and photocatalytic degradation of memantine under UV-A (365 nm) and UV-C (254/185 nm) radiation by sol-gel nanostructured TiO2 film as a function of irradiation time, *C*0 (memantine) = 10 mg/L. Inset: linear transform of −ln (*Ct*/*C*0 ) versus *t*. (**B**) Photolytic and photocatalytic degradation efficiency. All experiments were triplicated with the standard deviation from the average value ± 4%.

4A,B) after 45 min of irradiation, complete degradation of memantine is achieved (100%

efficiency). Conversely, the photocatalytic experiments under UV-C light (Figure 4A,B) From the photocatalytic experiments, it is observed that, under UV-A light (Figure 4A,B) after 45 min of irradiation, complete degradation of memantine is achieved (100% efficiency). Conversely, the photocatalytic experiments under UV-C light (Figure 4A,B) show that, after 120 min of irradiation, 85% of memantine removal is obtained. The photocatalytic degradation rate of memantine in the "UV-C + TiO2 film" experiment is much faster (0.1779 min−1 ) than that in the "UV-A + TiO2 film" experiment (0.0159 min−1 ). Similar behaviour was also observed in a recently published study of memantine oxidation [45]. In addition, photolytic oxidation of memantine by UV-A and UV-C light radiation was investigated. Memantine showed no degradation after 120 min of exposure to UV-A light alone, which is attributed to the low energy level of this type of UV light. Memantine degradation efficiency under exposure to UV-C light is the same as photocatalytic degradation by TiO2 film irradiated with UV-C light (100% efficiency after 45 min, Table 5) but a rate constant of memantine degradation is two times lower for UV-C photolysis than for photocatalysis in the "TiO2 film + UV-C" experiment (Table 5). The half-life of photolytic memantine degradation by UV-C light was almost two times higher (7.6 min) than the half-life obtained in the "TiO2 film + UV-C" photocatalytic degradation experiment (3.9 min). It was found that the half-life of photocatalytic degradation by UV-A light was much higher (46.3 min) than the half-life obtained by UV-C light (3.9 min).

Švagelj et al. [16] published their findings on the photocatalytic degradation of memantine using a sol-gel TiO2 film deposited on alumina foam substrate irradiated by UV-A light. The application of sol-gel TiO2 film deposited on alumina foam substrate resulted in a larger specific surface area and therewith fast degradation of memantine can be obtained. in a larger specific surface area and therewith fast degradation of memantine can be obtained. The diffuse reflectance spectroscopy (DRS) result and the Tauc plot are shown in Fig-

Švagelj et al. [1*6*] published their findings on the photocatalytic degradation of me-

mantine using a sol-gel TiO2 film deposited on alumina foam substrate irradiated by UV-A light. The application of sol-gel TiO2 film deposited on alumina foam substrate resulted

higher (46.3 min) than the half-life obtained by UV-C light (3.9 min).

show that, after 120 min of irradiation, 85% of memantine removal is obtained. The photocatalytic degradation rate of memantine in the "UV-C+TiO2 film" experiment is much faster (0.1779 min–1) than that in the "UV-A+ TiO2 film" experiment (0.0159 min–1). Similar behaviour was also observed in a recently published study of memantine oxidation [45]. In addition, photolytic oxidation of memantine by UV-A and UV-C light radiation was investigated. Memantine showed no degradation after 120 min of exposure to UV-A light alone, which is attributed to the low energy level of this type of UV light. Memantine degradation efficiency under exposure to UV-C light is the same as photocatalytic degradation by TiO2 film irradiated with UV-C light (100% efficiency after 45 min, Table 5) but a rate constant of memantine degradation is two times lower for UV-C photolysis than for photocatalysis in the "TiO2 film +UV-C" experiment (Table 5). The half-life of photolytic memantine degradation by UV-C light was almost two times higher (7.6 min) than the half-life obtained in the "TiO2 film + UV-C" photocatalytic degradation experiment (3.9 min). It was found that the half-life of photocatalytic degradation by UV-A light was much

The diffuse reflectance spectroscopy (DRS) result and the Tauc plot are shown in Figure 5A,B. It was found that prepared TiO2 only absorbs photons at wavelengths shorter than 400 nm. Based on the DRS, the Tauc plots can be obtained to determine the energy bandgap of TiO2 (Figure 5B). It is observed that prepared TiO2 presents a lower energy bandgap (2.99 eV) in comparison to commercial TiO2 P25 (3.20 eV) [46]. ure 5A,B. It was found that prepared TiO2 only absorbs photons at wavelengths shorter than 400 nm. Based on the DRS, the Tauc plots can be obtained to determine the energy bandgap of TiO2 (Figure 5B). It is observed that prepared TiO2 presents a lower energy bandgap (2.99 eV) in comparison to commercial TiO2 P25 (3.20 eV) [46].

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 9 of 17

**Figure 5.** (**A**) DRS spectra and (**B**) Tauc plot for energy bandgap determination of TiO2. **Figure 5.** (**A**) DRS spectra and (**B**) Tauc plot for energy bandgap determination of TiO2 .

### *2.3. Oxidation Products of Memantine*

*2.3. Oxidation Products of Memantine*  Compared to the chromatogram of the memantine before oxidation, five new peaks were observed corresponding to the possible oxidation products of memantine. All five degradation products have lower retention times than memantine (Table 6), indicating that they are more polar. Tentative structures of memantine oxidation products (Table 6) Compared to the chromatogram of the memantine before oxidation, five new peaks were observed corresponding to the possible oxidation products of memantine. All five degradation products have lower retention times than memantine (Table 6), indicating that they are more polar. Tentative structures of memantine oxidation products (Table 6) were proposed based on the retention times, *m/z*-values and fragmentation patterns obtained through HPLC-MS/MS analysis. Mass spectra of memantine and its oxidation products are shown in Supplementary Materials, Figure S1.

were proposed based on the retention times, *m/z*-values and fragmentation patterns obtained through HPLC-MS/MS analysis. Mass spectra of memantine and its oxidation products are shown in Supplementary Materials, Figure S1. Despite the different kinetics, all five degradation products were detected in all oxi-Despite the different kinetics, all five degradation products were detected in all oxidation experiments, except photolysis UV-A light when degradation was not achieved. The same oxidation products after UV-C/H2O2 and UV-A/TiO2 treatment were recently reported in [40].

#### dation experiments, except photolysis UV-A light when degradation was not achieved. *2.4. Toxicity of the Mixture of Memantine and Its Degradation Products*

The same oxidation products after UV-C/H2O2 and UV-A/TiO2 treatment were recently reported in [40]. Generally, some compounds do not show toxicity to a specific species, but this does not necessarily mean that they are not toxic or harmful to the environment or humans or another organism tested for toxicity [47,48]. Papac et. al. [45] determine that memantine was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment of the acute toxicity of memantine and its oxidation products during TiO2 photocatalysis by UV-C light was carried out using *Vibrio fischeri* bacteria. Luminescence inhibition (%) was measured in triplicate for each tested sample and mean values and standard deviations (*s*) were calculated. The results presented in Table 7 indicate that memantine (10 mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied experimental conditions.

NH2

NH2

NH2

**Table 6.** Proposed chemical structures of memantine oxidation products. *Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17 *Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17 *Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 10 of 17

not necessarily mean that they are not toxic or harmful to the environment or humans or another organism tested for toxicity [47,48]. Papac et. al. [45] determine that memantine was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment *2.4. Toxicity of the Mixture of Memantine and Its Degradation Products 2.4. Toxicity of the Mixture of Memantine and Its Degradation Products 2.4. Toxicity of the Mixture of Memantine and Its Degradation Products*  **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light.

#### was measured in triplicate for each tested sample and mean values and standard deviations (*s*) were calculated. The results presented in Table 7 indicate that memantine (10 experimental conditions. experimental conditions. toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment toxicity is not always more sensitive than acute toxicity. Keeping that in mind, assessment **3. Environmental Relevance**

mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied experimental conditions. **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. **Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. **Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. **Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 of the acute toxicity of memantine and its oxidation products during TiO2 photocatalysis by UV-C light was carried out using *Vibrio fischeri* bacteria. Luminescence inhibition (%) was measured in triplicate for each tested sample and mean values and standard deviations (*s*) were calculated. The results presented in Table 7 indicate that memantine (10 mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied experimental conditions. **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. of the acute toxicity of memantine and its oxidation products during TiO2 photocatalysis by UV-C light was carried out using *Vibrio fischeri* bacteria. Luminescence inhibition (%) was measured in triplicate for each tested sample and mean values and standard deviations (*s*) were calculated. The results presented in Table 7 indicate that memantine (10 mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied experimental conditions. **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. of the acute toxicity of memantine and its oxidation products during TiO2 photocatalysis by UV-C light was carried out using *Vibrio fischeri* bacteria. Luminescence inhibition (%) was measured in triplicate for each tested sample and mean values and standard deviations (*s*) were calculated. The results presented in Table 7 indicate that memantine (10 mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied experimental conditions. **Table 7.** Luminescence inhibition during the TiO2 photocatalysis by UV-C light. The fate and behaviour of pharmaceuticals in the environment is controlled by their physicochemical properties and the characteristics of the environment. Once in the environment, the pharmaceutical can be distributed between different compartments of the environment (such as water, soil, air and biota) and be exposed to different biotic and abiotic elimination processes that can potentially lead to lowering of their environmental concentration. On the other hand, the results of elimination processes can lead to the formation of new compounds—degradation products—with different physicochemical and toxic properties.

mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied

mg/L) and its oxidation products were not harmful to *Vibrio fischeri* under the applied

was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic

was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic

was toxic to *Daphnia magna* (EC50 = 7.19 mg/L). Blaschke et al. [49] demonstrate that chronic

**Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 **Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 **Exposure Time, min** 0 10 20 30 45 60 180 **Luminescence inhibition ±** *s***, %** 0.86 ± 0.02 1.04 ± 0.03 1.40 ± 0.05 0.76 ± 0.03 0.67 ± 0.04 0.73 ± 0.04 1.98 ± 0.07 Memantine is highly soluble in water (Table S1). From the results of in silico prediction of memantine biodegradability using different QSAR models provided by EPISuit [50], memantine can be considered a persistent compound because it does not biodegrade fast (biowin 2 < 0.5 or biowin 6 < 0.5) and its ultimate biodegradation timeframe prediction is longer than months (biowin 3 < 2.25) [51] (Table S1).

The results of our research showed that memantine is persistent to hydrolytic degradation. Although some photolytic degradation was observed in the presence of humic acids and nitrates, the degree of photodegradation was insignificant. Due to the absence of chromophores in the molecule of memantine, such results are expected. For persistent compounds, such as memantine, it is important to investigate their potential mobility in the environment determined by the compound's water solubility and sorption properties. Results of sorption/desorption experiments showed that memantine has a low tendency to sorption and is easily leached from river sediments. Considering this and its high solubility in water, it is possible to conclude that memantine will not be eliminated by natural processes and has the potential to be transported from the release site.

Such compounds, persistent and mobile in the environment, are of great concern for water quality since they are highly polar and are not removed from water by sorption. They can therefore end up in drinking water, posing a potential risk to human health [52]. Conventional wastewater treatment, based on microbial degradation and sorption, is expected to be ineffective for the removal of persistent and mobile compounds, since they are neither biodegradable nor sorbed substantially [52]. Therefore, it is of great importance to prevent their release into the environment by developing advanced and effective wastewater treatment processes.

Today, water treatment technology is trying to introduce processes that will be efficient and cheap, but also in accordance with ecological principles. One of the methods for degradation of such persistent and mobile compounds in water that is close to meeting these requirements is photocatalytic oxidation, with the use of titanium(IV) oxide (TiO2) as a photocatalyst [53]. In addition to TiO2, the presence of a suitable source of UV radiation that starts the process and oxygen dissolved in water are also necessary. It is a process that is included in the so-called advanced oxidation processes (AOPs). For the photocatalytic oxidation process, it is not necessary to add any additional chemicals except a solid photocatalyst (in the form of particles or nanostructured films on the reactor walls) and oxygen, while ensuring irradiance in UV spectra [54]. The use of solar radiation as a process activator (i.e., a source of UV radiation) and oxygen from the air around the reactor contribute to approaching the ecological principles of this technology. Photocatalytic oxidation of memantine resulted with the occurrence of five degradation products. According to the shorter chromatographic retention times compared to memantine, it is assumed that the oxidation products are more polar than memantine. This may indicate better solubility in water and a weaker tendency for sorption. Although oxidation products of memantine do not show inhibition of bioluminescence of *Vibrio Fischeri*, future studies on the current topic are suggested to assess the cytotoxicity and genotoxicity of memantine and its oxidation products in surface waters and wastewaters, as well as toxicological risks to ecosystems and human health. Furthermore, experimental data on the biodegradation of memantine and its oxidation products should be gathered.

### **4. Experimental Section**

### *4.1. Materials and Chemicals*

Analytical standard of memantine hydrochloride (CAS number: 41100-52-1) (Sigma Aldrich, St. Louis, MO, USA) of high purity (>98%) was used in this study. Memantine stock solution concentration of 1000 mg/L was prepared by weighing the accurate mass of memantine standard and dissolving it in methanol.

Acetonitrile, formic acid, citric acid, ascorbic acid and inorganic salts were of analytical grade and supplied by Kemika (Zagreb, Croatia). For buffer preparation, analytical grade reagents were used. Ultra-pure water was prepared using a Millipore Simplicity UV system (Millipore Corporation, Billerica, MA, USA).

For toxicity evaluation, freeze-dried and liquid luminescent bacteria *Vibrio fischeri* NRRL-B-11177 (LCK484, LUMINStox LUMISmini, Hach Lange, Varaždin, Croatia) were used. The bacterial reagents as well as reconstitution reagents were purchased from Kemika (Zagreb, Croatia).

For the preparation of TiO2 sol (colloidal solution), the following components were used: titanium (IV) isopropoxide (Ti(C3H5O12)4, TTIP, 97%, Sigma-Aldrich, St. Louis, MO, USA), i-propanol (C3H7OH, Grammol, Croatia), acetylacetone (CH3(CO)CH2(CO)CH3 ≥ 99%, Honeywell, Charlotte, NC, USA), nitric acid (HNO3, Carlo Erba Reagents, Barcelona, Spain) and polyethylene glycol (H(OCH2CH2)nOH, Mr = 5000–7000, Sigma-Aldrich, St. Louis, MO, USA). All these chemicals were analytical grade reagents.

### *4.2. Sediment Samples*

Samples of river or fluvial sediments were collected on the territory of the Republic of Croatia and in the following areas: in Sisak-Moslavina County on the Petrinjˇcica River in the town of Petrinja, in Požega-Slavonia County on the Pakra River in the town of Pakrac and in Primorsko-Goranska County on the Studena River in the surroundings of the city Rijeka. In all locations, the samples were collected outside of human activities, which provides some assurance that the samples are not contaminated, especially in the case of pharmaceuticals, and in the summertime when it is easier to reach the area and collect samples due to dryness. All samples were air-dried, crushed, sieved through a 2-mm sieve and characterized according to the proposed procedure [55]. The physicochemical properties of used samples can be seen in the previously published work [31].

### *4.3. Hydrolytic Degradation Experiments*

Hydrolytic degradation was conducted at (50 ± 0,1) ◦C (Incubator shakers KS 3000 i control, IKA, Staufen, Germany) for 5 days and at three pH values (4, 7 and 9) in capped glass vials under dark conditions. A buffer solution with a pH value of 4 was prepared by mixing 38.55 mL of 0.2 M K2HPO4 and 61.45 mL of 0.1 M citric acid. A pH value buffer solution of 7 was prepared by mixing 29.63 mL of 0.1 M NaOH, 50 mL of 0.1 M KH2PO4 and 20.37 mL of MilliQ water, and the pH 9 buffer solution was prepared by mixing 21.30 mL of 0.1 M NaOH, 50 mL of 0.1 M H3BO3 in 0.1 M KCl and 28.70 mL of MilliQ water. The pH of each buffer solution was checked with pH meter S20 SevenEasy (Mettler Toledo, Greifensee, Switzerland). Memantine solutions were prepared in appropriate buffer solutions at a concentration of 10 mg/L. Concentration of memantine solutions after hydrolytic degradation experiment were determined by HPLC-MS/MS. All experiments were performed in three replicates.

### *4.4. Photolytic Degradation Experiments at Environmentally Relevant Conditions*

Direct photolysis experiment was performed with memantine solution in MilliQ water (10 mg/L). To test the possibility of indirect photolysis, solutions of memantine (10 mg/L) were prepared in solutions of Cl− ions (10 mg/L), NO3 − ions (3 mg/L) and humic acids (3 mg/L). Forty millilitres of test solution were irradiated in quartz vessels (diameter 4.6 cm) placed in Suntest CPS+ simulator (Atlas, Linsengericht, Germany). Suntest CPS+ is equipped with a temperature sensor and a xenon lamp as a source of artificial sunlight in the wavelength range of 300–800 nm. The distance between the liquid surface and the lamp was 14 cm. During the experiments, the radiation intensity was maintained at 500 W m−2 and the reaction temperature was kept at (25 ± 2) ◦C. In all photolysis experiments, dark control experiments were performed under the same conditions but protected from the radiation. Control samples with the same composition as test solutions were used to establish that memantine degradation was a consequence of the irradiation. Aliquots of irradiated memantine solution were analysed by HPLC-MS/MS. All experiments were performed in three replicates.

### *4.5. Sorption Experiments*

Batch sorption experiments were performed according to the OECD 106 procedure [56]. The procedure is performed in triplicate by shaking on a laboratory shaker (Innova 4080 Incubator Shaker, NewBrunswick Scientific, Edison, NJ, USA), which allows continuous contact of the sediment samples with the memantine solution. To avoid photolytic degradation, all experiments were performed in the dark and, to avoid microbiological activities, all sediment samples were sterilized beforehand.

It is very important to choose a good ratio between sediment (sorbent) and memantine solution. Since previous studies [30,31] show that memantine does not have excessive sorption potential to sediment or soil samples, all experiments were performed with a 1:10 (*w*/*v*) sediment/memantine solution ratio. In a previously published paper [30], it was determined that 24 h was sufficient for memantine to reach sorption equilibrium, so all experiments were conducted with 24 h of shaking. The procedure consisted of adding 10 mL known concentration (0.1–2.0 mg/L) of memantine solution in 50 mL of laboratory glass to 1 g of air-dried sediment. The prepared suspension was shaken in a shaker (at 200 rpm) for 24 h at room temperature (25 ◦C), filtered through a 0.45 µm syringe filter and transferred to HPLC vials. Blank samples containing the same amount of sediment and soil in contact with 10.0 mL of 0.01 M CaCl2 solution were also included in the analysis. They served as controls to detect interfering compounds or contaminated sediment.

In order to investigate the influence of pH and ionic strength on the sorption of memantine on the sediments studied, a series of experiments were performed in which one of the factors was changed while the others remained constant. The effect of pH was monitored using three series of experiments with different pH values of the studied memantine solutions (pH values 5, 7 and 9). All these experiments were performed in a 0.01 M CaCl2 solution. To determine the effect of ionic strength, the pH of the memantine solution must be adjusted to the initial value (pH 7.0). These experiments were performed with three different concentrations of CaCl2 solution (0.001, 0.01 and 0.1 mol/L).

Since the experiments to determine the required contact time (24 h) were the basis for the determination of sorption kinetics, we were able to start immediately with the determination of the kinetics of memantine desorption from the sediments studied. Desorption kinetics were studied for three memantine solutions in 0.01 mol/L CaCl2 (0.1, 0.5 and 2.0 mg/L) using a decanting and refilling technique. After 24 h of shaking, the memantine solutions in contact with the sediment samples were replaced with fresh 0.01 mol/L CaCl2. Memantine solution was removed using a disposable glass pipette. The residual solution that could not be removed before the desorption experiment was determined gravimetrically, and the same amount of 0.01 mol/L CaCl2 as the removed memantine solution was weighed and added to the residual solution. Samples were then shaken at 25 ◦C for various periods (10, 20, 30, 40 and 50 min, and 1, 2, 4, 6, 12, 18 and 24 h).

### *4.6. Photolytic and Photocatalytic Oxidation Experiments*

All experiments were carried out with 10 mg/L memantine solution in two borosilicate glass tubes (200 mm in height, 30 mm in diameter, 0.11 L) with continuous purging with air (O2), at (25 ± 0.2) ◦C: (i) with the TiO2 film, for photocatalytic experiments and (ii) without the TiO2 film, for photolytic experiments. UV lamps (UV-A and UV-C) were placed in the middle of each reactor. Detailed experimental set-up is published elsewhere [17]. UV lamps used in experiments were 15 W mercury UV lamps: (i) model Pen-Ray CPQ-7427, UV-A with *λ*max = 365 nm and (ii) model Pen-Ray 90-0004-07, UV-C with *λ*max = 254/185 nm, manufactured by UVP (Upland, CA, USA). Both lamps were used with the same electrical source PS-4, *I* = 0.54 A, from UVP, too. The experimental set-up was described in detail in [17]. The reaction temperature was controlled by the circulation of cooling water. Total irradiation time for each oxidation test was 120 min. Aliquots of 1 mL were collected in defined time intervals and stored in the dark at 4 ◦C until HPLC-MS/MS analysis.

Nanostructured TiO2 film was deposited on a borosilicate glass substrate by the solgel process using the dip-coating method. TiO2 colloidal solution (sol) was prepared by mixing titanium(IV) isopropoxide (Ti-iPrOH) as a precursor, i-propyl alcohol (iPrOH) as a solvent, acetylacetone (AcAc) as a chelating agent, nitric acid (NA, 0.5 M) as a catalyst and polyethylene glycol as an organic/polymer additive in the amount of 2 g. The molar ratio of these reactants was Ti-iPrOH:iPrOH:AcAc:NA=1:35:0.63:0.015. The film was dried at 100 ◦C for 1 h prior to the deposition of the next layer. After the deposition of the three

layers, the deposited film was heat-treated at 550 ◦C for 4 h [14]. The procedure for the film preparation as well as its characterization was described in detail elsewhere [14,17].

Energy bandgap (Eg) of prepared TiO2 was calculated from diffuse reflectance spectroscopy (DRS) measurements, which were performed on a QE Pro High-Performance Spectrometer (Ocean Insight, Orlando, FL, USA) equipped with an integrating sphere and a DH 2000 deuterium–halogen source in the analysis range 200–1000 nm with a resolu-tion of 1 nm and integration time of 10 s.

### *4.7. HPLC-MS/MS Analysis*

Samples from photolytic and hydrolytic degradation experiments as well as samples from AOP experiments were analysed using an Agilent Series 1200 HPLC system (Santa Clara, CA, USA) coupled with an Agilent 6410 triple-quadrupole mass spectrometer equipped with an ESI interface (Santa Clara, CA, USA). Chromatographic separation was performed on an Kinetex C18 column (100 mm × 2.1 mm, 2.6 µm) (Phenomenex, Torrance, CA, USA) using mobile phase comprising MilliQ water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B. The composition of 50% organic phase (B) was maintained at flow rate of 0.2 mL/min throughout the analysis. An injection volume of 5 µL was used in all analyses. The analyses were done in positive ion mode under the following conditions: drying gas temperature 350 ◦C; capillary voltage 4.0 kV; drying gas flow 11 L/min and nebulizer pressure 35 psi. Instrument control, data acquisition and evaluation were done with Agilent MassHunter 2003–2007 Data Acquisition for Triple Quad B.01.04 (B84) software (Santa Clara, CA, USA).

The residual concentration of memantine in the remaining liquid phase after sorption was analysed using UHPLC-MS (Agilent 6490 coupled with Agilent Infinity UHPLC System Triple Quadrupole Mass Spectrometer, Santa Clara, CA, USA) with electrospray ionization. The chromatographic column Shim pack XR ODS II (50 mm × 2 mm i.d., 1.6µm) (Shimadzu, Duisburg, Germany) was used at 30 ◦C with an injection volume of 1 µL. The mobile phase consisted of two eluents: eluent A (0.1% formic acid in MilliQ water) and B (0.1% formic acid in acetonitrile) and was performed in gradient elution mode. The gradient started with a 0.1-min linear gradient from 100% A to 10% B, followed by a 1.0-min linear gradient to 98% B, followed by a 0.5-min linear gradient back to 100% A held for 0.4 min. The flow rate was 0.2 mL/min. All analyses were performed in positive ion mode under the following parameters: drying gas temperature 200 ◦C, capillary voltage 3.0 kV, drying gas flow rate 15 L/min and nebulizer pressure 20 psi. Memantine was analysed by MRM, using the two highest characteristic precursor ion/product ion transitions (*m*/*z* 291.25→230.2; *m*/*z* 291.25→123.0).

### *4.8. Assessment of Acute Toxicity by Vibrio Fischeri*

Acute toxicity assessment toward *Vibrio fischeri* culture was performed on standard solutions of memantine (10 mg/L), a mixture of memantine with its degradation products and finally the degradation products themselves without the detectable presence of memantine. Acute toxicity assessment was performed according to the method described in detail in [29]. In brief, sample solutions for toxicity measurements were prepared by serial dilutions in linear progression with addition of 2% NaCl. The experiments were conducted in a test tube by combining each volume of initial or diluted sample (1.5 mL) and 0.5 mL of bacterium *Vibrio Fischeri* suspension. The inhibition of luminescence was measured before and after 30 min of exposure of the sample to *Vibrio fischeri* on a luminometer (LUMIStox 300 Hach Lange, Düsseldorf, Germany) at 15 ◦C. In order to control bacteria performance, the reference substances ZnSO4 x (OH2)7 (109.9 µg/mL) and K2Cr2O7 (22.6 µg/mL) were used.

### **5. Conclusions**

This research provides a comprehensive picture of memantine behaviour in the aquatic environment. The results showed that memantine is resistant to hydrolytic and photolytic (i.e., irradiated by solar light) degradation, has a low tendency to sorption and is easily desorbed from river sediments. For such compounds, which are persistent and mobile in the environment, it is of great importance to prevent their release into the environment by effective wastewater treatment.

Investigation of memantine oxidation by photolytic/photocatalytic oxidation by UV-A and UV-C light showed that memantine could be completely oxidized within 30 min during photocatalytic and within 50 min during photolytic oxidation processes by UV-C light. Photolytic degradation by UV-A light did not occur, while photocatalytic degradation by UV-A light did occur although the degradation rate was lower and memantine degradation was not completed even after 120 min. A kinetic study showed that the oxidation in all experiments, in which oxidation occurred, followed pseudo-first-order reaction kinetics.

As a result of the oxidation, five oxidation products were formed, which were identified using high performance liquid chromatography coupled with a triple quadrupole mass spectrometer. The same oxidation products were identified in all investigated processes in which oxidation occurred. The results of the acute toxicity assessment of memantine and its mixture with oxidation products indicate that the resulting products are not harmful.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/catal13030612/s1, Figure S1: Mass spectra of memantine and its oxidation products; Table S1: Results of EPISuite biodegradability prediction for memantine.

**Author Contributions:** Conceptualization, S.B. and D.L.; methodology, S.B., D.L., D.M.P., M.B., L.C. and D.D.; formal analysis, M.B., D.M.P. and D.D.; investigation, S.B., D.L., D.M.P., M.B., L. ´ C. ´ and D.D.; resources, S.B., D.L., D.M.P. and L.C.; data curation, S.B., D.L., D.M.P., M.B., L. ´ C. and ´ D.D.; writing—original draft preparation, S.B., L.C. and D.M.P.; writing—review and editing, S.B., ´ D.L., D.M.P., M.B., L.C. and D.D.; visualization, L. ´ C., D.M.P. and M.B.; supervision, S.B.; funding ´ acquisition, S.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Croatian Science Foundation under the project Fate of pharmaceuticals in the environment and during advanced wastewater treatment (PharmaFate) (IP-09-2014-2353).

**Data Availability Statement:** The data presented in this study are available upon reasonable request from the corresponding author.

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

### **References**

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## *Article* **Development of Nanomedicine from Copper Mine Tailing Waste: A Pavement towards Circular Economy with Advanced Redox Nanotechnology**

**Amrita Banerjee 1,2, Ria Ghosh 3 , Tapan Adhikari 4 , Subhadipta Mukhopadhyay 1 , Arpita Chattopadhyay 5,\* and Samir Kumar Pal 3,\***

**Abstract:** Copper, the essential element required for the human body is well-known for its profound antibacterial properties, yet salts and oxides of copper metals in the copper mine tailings are reported to be a big burden in the modern era. Among other copper oxides, CuO, in particular, is known to have beneficial effects on humans, while its slight nanoengineering viz., surface functionalization of the nanometer-sized oxide is shown to make some paradigm shift using its inherent redox property. Here, we have synthesized nanometer-sized CuO nanoparticles and functionalized it with a citrate ligand for an enhanced redox property and better solubility in water. For structural analysis of the nanohybrid, standard analytical tools, such as electron microscopy, dynamic light scattering, and X-ray diffraction studies were conducted. Moreover, FTIR and UV-VIS spectroscopy studies were performed to confirm its functionalization. The antibacterial study results, against a model bacteria (*S. hominis*), show that CuO nanohybrids provide favorable outcomes on antibiotic-resistant organisms. The suitability of the nanohybrid for use in photodynamic therapy was also confirmed, as under light its activity increased substantially. The use of CuO nanoparticles as antibiotics was further supported by the use of computational biology, which reconfirmed the outcome of our experimental studies. We have also extracted CuO nanogranules (top-down technique) from copper mine tailings of two places, each with different geographical locations, and functionalized them with citrate ligands in order to characterize similar structural and functional properties obtained from synthesized CuO nanoparticles, using the bottom-up technique. We have observed that the extracted functionalized CuO from copper tailings offers similar properties compared to those of the synthesized CuO, which provides an avenue for the circular economy for the utilization of copper waste into nanomedicine, which is known to be best for mankind.

**Keywords:** copper nanohybrid; citrate functionalized CuO; nano-medicine; *S. hominis* infection control; photodynamic therapy

### **1. Introduction**

Copper was the first metal discovered in the history of human civilization during the chalcolithic or copper age, around 6000 years ago. This marvel metal was well known in ancient times, not only for its ability to enhance the strength of the tools, but also for its impressive healing capacity. The ancient Indians, Greeks, and Egyptians all used copper containers for water purification, treatment of wounds, and lung ailments. Copper cooking

**Citation:** Banerjee, A.; Ghosh, R.; Adhikari, T.; Mukhopadhyay, S.; Chattopadhyay, A.; Pal, S.K. Development of Nanomedicine from Copper Mine Tailing Waste: A Pavement towards Circular Economy with Advanced Redox Nanotechnology. *Catalysts* **2023**, *13*, 369. https://doi.org/10.3390/ catal13020369

Academic Editors: Gassan Hodaifa, Rafael Borja and Mha Albqmi

Received: 10 January 2023 Revised: 1 February 2023 Accepted: 3 February 2023 Published: 7 February 2023

**Copyright:** © 2023 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/).

utensils were employed throughout the Roman empire to stop the spread of disease [1]. In recent times, the US Environmental Protection Agency (EPA) has classified copper and its derivatives as antibacterial materials [2]. On the other hand, the presence of copper in the human body is essential for healthy development, cardiovascular and lung functionality, neovascularization, neuroendocrine function, and iron metabolism [3].

Metallic copper, cupric oxide (CuO), and cuprous oxide (Cu2O) nanoparticles are attracting considerable research interest nowadays due to their widespread applications in catalysts and therapeutic domains [4–10] compared to their bulk counterparts, by virtue of their nano dimensional higher surface area to volume ratio. Specifically, copper oxide nanoparticles (CuO NPs) display broad-spectrum antibacterial and photocatalytic properties [11] and have the potential to be used as an alternative to antibiotics. The design and development of new compounds as antibiotics possess an emergent need, as the overuse and misuse of existing antibiotics are responsible for the growing episodes of antibiotic-resistant infections and deaths globally [12].

The antibacterial activity of CuO NPs against Gram-positive bacteria, such as *S. aureus* and *B. subtilis* as well as Gram-negative bacteria, such as *E. coli* and *P. aeruginosa* are reported in various studies [13–17]. Metallic Cu and CuO NPs are also found to exhibit multi-toxicity on multi-drug resistant bacterial species, such as the methicillin-resistant S. aureus (MRSA) [18]. In the case of CuO NPs, it is suggested that its antibacterial effect might be associated with cell membrane dissociation and reactive oxygen species (ROS) production [19]. Various simultaneous mechanisms of action of CuO nanoparticles against bacteria make it almost impossible for the microbes to develop resistance, as the bacterial cell would be required to generate multiple simultaneous gene mutations to develop this resistance [20]. Copper oxide nanoparticles are obtaining growing attention for their cheaper price and abundance in comparison with other noble and expensive metals, such as silver and gold, and their competent potential application as microbial agents [15,21,22].

The size, morphology, and solubility play a significant role in the antibacterial activities of Cu, CuO, and Cu2O [23–25]. However, the major limitation of metallic CuO NPs in the nano-size range is the lack of significant stability in dispersions, due to their strong tendencies to aggregate and the formation of larger clusters that reduce the energy associated with their high surface area [26–28]. The formation of clusters results in sedimentation leading to loss of reactivity and antimicrobial performance, in which a nanometric size is essential [28]. Further surface modifications of CuO NPs using a post functionalization approach, not only enhances their colloidal stability, yet can also introduce unique physical and chemical properties, including the possible enhancement of their antimicrobial activities. Functionalization, or capping of an inorganic nanoparticle with an organic ligand-like citrate or folate, is evidenced to produce nanohybrids that have unique therapeutic potentials [29].

In the current study, we have explored the effect of citrate-capped CuO NPs on a Grampositive *Staphylococcus hominis* (SH) bacterial strain. Indeed, *S. hominis* is a commensal bacteria that resides on human skin [30]. Although it is apparently harmless, it has been reported that one of its subspecies, novobiosepticus, is multidrug-resistant and causes nosocomial infections, such as sepsis, alongside bloodstream infections in neonates and immunocompromised patients [31], and various opportunistic infections of humans [32]. *S. hominis* is also well known for its ability to generate pungent body odor in humans [33]. In the present study, we have reported the synthesis or extraction, characterization, and antimicrobial activity of citrate functionalized CuO NPs on the Staphylococcus hominis bacterial strain. CuO NPs were synthesized and capped using a precipitation technique [34] and grafting method [35], respectively, in a bottom-up approach. Similarly in a topdown method, CuO NPs were extracted, and citrate functionalized from two types of copper-containing stones from copper mines. The structural properties of both synthesized and extracted CuO NPs were examined by X-ray diffraction (XRD), and field emission scanning electron microscopy (FESEM), which were equipped with an energy dispersive X-ray spectroscopy (EDS) and found to be similar. Dynamic light scattering (DLS) and zeta potential studies were also employed for estimating the hydrodynamic diameter

and solubility assessment of the synthesized nanohybrid. The surface functionalization of CuO NPs by citrate ligands was confirmed by FTIR and UV-vis spectroscopy. The antimicrobial activity of citrate-CuO NPs was examined in the *S. hominis* bacteria strain. The citrate functionalized NPs were found to generate reactive oxygen species (ROS) upon photoexcitation, which is responsible for their antimicrobial action because ROS has the ability to destroy the active substances in the bacterial inner and outer membranes [36–38]. This phenomenon establishes the credentials of citrate CuO NPs for applications including antibacterial photodynamic therapy (PDT) with enhanced efficacy. We have also used computational biology strategy in order to rationalize the antibiotic-resistant bacterial remediation found in our experimental studies.

Immediately after the establishment of the CuO nanohybrid as a potential antibacterial agent, we also explored the use of copper mine tailings as a source of raw materials used for the nanohybrid synthesis, in order to simultaneously cater to low-cost antibiotics to a wider population across the globe and to remediate the burden from the copper mines. We have also developed a prototype FMCG product (talcum powder) for the remediation of bacteria *S. hominis* which are responsible for the generation of several human disorders.

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

### *2.1. Materials*

Copper acetate, citric acid, sodium hydroxide, and sodium citrate were purchased from Sigma Aldrich (St. Louis, MO, USA), California. All solvents and all other used chemicals were procured from Merck (New Jersey, USA), unless otherwise stated. 2,7 dichlorodihydrofluorescein diacetate (DCFH-DA) was bought from Calbiochem to estimate the reactive oxygen species (ROS) production. Similarly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma (St. Louis, MO, USA) to monitor the antioxidant activity in the samples. All reagents were analytical grade and used without any further purification. Nanopore water, with a resistivity value ≥ 18 MΩ cm, from the Milli-Q system (Millipore GmbH, Germany) was used in all experiments. For bacterial studies, LB top agar and Luria broth (LB) medium were purchased from HIMEDIA. The Gram-positive bacteria, Staphylococcus hominis (*S. hominis*) strain, was procured from ATCC.

### *2.2. Synthesis of Functionalized CuO Nanoparticles*

In this study, the CuO NPs were synthesized following the reported precipitation method by Zhu et al. [34]. The citrate ligands generated in the process provided satisfactory passivation against aggregation and sufficient stability to the NPs in colloidal suspension. Briefly, 150 mL of deionized water was used to dissolve 0.54 g of copper acetate. Next, 0.52 g of citric acid was added, and the mixture was vigorously stirred while being heated to boiling at 100 ◦C. Once the mixture's pH attained a value of 6–7, 0.7 gm or 0.015 mol of sodium hydroxide (NaOH) was added quickly, causing a significant amount of dark brown precipitate to form at the same time. The blue color solution was immediately converted to brown, indicating the production of CuO NPs. The liquid was cooled to room temperature while being stirred after 5 additional minutes of reflux. The CuO-NPs were subsequently separated by centrifugation (4000 rpm, 10 min), and washed twice with water and another two times with ethanol. The supernatant, which contains citrate-capped CuO NPs, was then separated.

Using the citric acid grafting procedure, the CuO NPs were further functionalized. In a water-to-ethanol ratio of 8:2, suspensions of 200 mM acetic acid and 65 mM CuO were performed. The produced citric acid solution was combined with the CuO suspension, and the pH of the resulting combination was raised to 12 by adding 6 M NaOH. Next, the mixture was refluxed for three hours. The product was then centrifuged and properly cleaned three times to remove the extra citric acid. To obtain citrate-capped CuO (C-CuO) from the two copper tailings in the mines of Peru and Bhopal (India), a similar procedure was followed. Only 160 mg of each stone dust was mixed initially with 150 mL of deionized water before the addition of citric acid and boiling occurred.

### *2.3. Characterization Tools and Techniques*

Optical absorbance spectra of samples were measured in a double-beam UV-vis spectrophotometer (model UV-2600, Shimadzu, Japan) in the 200−800 nm wavelength range. The room temperature steady-state emission spectra were recorded using a Fluorolog Model LFI-3751 (Horiba-Jobin Yvon, Edison, NJ, USA) spectrofluorometer equipped with a microchannel plate−photomultiplier tube (MCP−PMT, Hamamatsu, Japan). All fluorescence spectra were corrected for variations with a wavelength in source intensity, photomultiplier response, and monochromator throughput. The X-ray diffraction (XRD) pattern of synthesized CuO nanoparticles was measured in a PANalytical X'PertPRO (Malvern Panalytical Ltd., Malvern, UK) diffractometer, with Cu Kα radiation (at 40 mA and 40 kV) generating at a rate of 0.02 ◦ s −1 in the 2θ range from 20 ◦ to 80 ◦ . The liquid CuO nanoparticles were subjected to lyophilization for the XRD analysis. We have performed Fourier transform infrared spectroscopy (FTIR) on the liquid samples and the spectra were obtained using a JASCO FTIR-6300 spectrometer instrument (Oklahoma city, OK, USA). A NanoS Malvern (Zeta-seizer) instrument equipped with a 4 mW He:Ne laser (λ = 632.8 nm) and a thermostat coupled sample chamber was employed for dynamic light scattering (DLS) and ζ potential measurements. Quartz cuvettes of 10 mm path length were used to execute all spectroscopic experiments. The structural morphologies and chemical compositions of the synthesized citrate CuO nanohybrids from different sources were analyzed using scanning electron microscopy (SEM) and EDAX methods. Before scanning in a field emission scanning electron microscope (Quanta FEG 250: source of electrons, FEG source; operational accelerating voltage, 200 V to 30 kV; resolution, 30 kV under low vacuum conditions: 3.0 nm; detectors, large field secondary electron detector for the low vacuum operation), the coverslips containing samples were coated with gold.

### *2.4. Antioxidant Activity*

The free radical scavenging capability of the samples of interest was determined using the DPPH assay method. A 0.15 mM DPPH solution was prepared in methanol and 0.5 mL citrate CuO nanoparticles of various concentrations were added to 2.5 mL of the freshly prepared DPPH solution. The characteristic absorption maxima of DPPH at 535 nm were selected to monitor the degradation process with DPPH in the presence and absence of light at room temperature. The absorption spectra of DPPH were recorded in the interval of 2 s for an hour using SPECTRA SUITE software provided by Ocean Optics.

### *2.5. Quantification and Characterization of ROS*

For the purpose of quantifying the generated ROS, we used 20 ,70 -dichlorofluorescein (DCFH), which is a well-known reagent. The DCFH was prepared via a de-esterification reaction from DCFH-DA at room temperature, following a standardized protocol described in previous studies [39,40]. The oxidation of DCFH, in the presence of light, leads to the production of DCF emitting fluorescence [41,42]. In this study, the ROS generated in the aqueous citrate functionalized CuO NPs convert DCFH into DCF, which has a characteristic emission maximum of 522 nm upon excitation at 488 nm. The DCF emissions were recorded in the Fluorolog Model LFI-3751 (Horiba-Jobin Yvon, Edison, NJ) spectrofluorometer. The ROS experiments were performed in the dark and in the light for 30 min.

### *2.6. Bacterial Strain and Culture Conditions*

The antibacterial activity of the synthesized and extracted samples have been investigated against a strain of Staphylococcus hominis bacteria. The Gram-positive *S. hominis* strain was procured from ATCC. For the antibacterial assay, fresh *S. hominis* bacteria have been cultured using sterilized Luria–Bertani (LB) medium in a shaker incubator at a temperature of 37 ◦C for 24 h. All used glassware, suction nozzle, and culture medium were sterilized in an autoclave at a high pressure of 0.1 MPa and a temperature of 120 ◦C for 30 min prior to the experiments beginning. The treatment of bacteria was performed on LB agar plates using the colony forming unit (CFU) assay method under dark and light

illumination conditions. The freshly grown original *S. hominis* bacterial suspension was firstly washed twice with phosphate-buffered saline (PBS; pH 7.4) solution and further diluted 106 times before the test samples were added. Then, they were incubated with the respective nanoparticles for 1 h. The resulting bacterial PBS suspensions (200 µL) were uniformly spread over gelatinous LB agar plates and cultured at 37 ◦C for 24 h to obtain the CFUs. To quantify the antibacterial activity, the number of survival colonies was manually counted and presented as a bar diagram.

Detection of microbial growth was also performed in the cuvette system for different citrate CuO NPs under various conditions. For this bacterial mortality study, a bacterial solution of 108 CFU/mL was considered and incubated with test solutions of 1 mM concentrations, initially for 3 h with photoactivation.

For the cuvette-based detection of microbial growth, the cells were cultured in an LB medium under an incubator shaker at 37 ◦C for 24 h. The optical density of the freshly grown overnight culture was fixed to 0.1 in LB medium initially. The culture was then put into a cuvette, mixed with the test samples, and incubated at 37 ◦C for 9 h with shaking under dark and illumination conditions. The absorbance was taken at 1 h intervals and plotted against time, with baseline correction for studying the growth curves.

For the microscopic studies, the bacteria cells after proper incubations with the nanoparticles were stained with DAPI and PI. DAPI stains all cells, while PI only stains the membrane-disrupted cells. The (1-red/blue) ratio was obtained to assess the viability of the *S. hominis*. The tests were repeated three times. The samples (15 µL) were observed under a fluorescence microscope (Leica digital inverted microscopes DMI8).

### *2.7. Statistical Analysis*

All the data in this current work are represented as mean ± standard deviation (SD), unless otherwise stated. An unpaired 2-tailed *t*-test was used for comparison between the groups. A value of *p* < 0.05 was considered significant. GraphPad Prism (v8.0) software was used for all statistical tests.

### *2.8. Method of Computational Biology*

The web resource STITCH (http://stitch.embl.de/; accessed on 24 December, 2022) was utilized to predict the chemical–protein (CP) interaction networks of CuO NP in *S. hominis*. The STITCH database can forecast around 960,000 proteins and 430,000 compounds from the 2031 eukaryotic and prokaryotic genomes [43,44]. The confidence score of a chemical–protein interaction can be used to predict the relationship, with a higher value indicating a stronger interaction. For the purposes of this investigation, a medium confidence score of 0.4 was taken into account.

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

X-ray diffraction (XRD) characterization of the synthesized and extracted CuO NPs was carried out to estimate the precise elemental composition, particle size, and superficial morphology, as depicted in Figure 1a. It was determined that all CuO NPs were in a monoclinic geometry with a space group of C2/C. No characteristic peaks of any other impurities were detected, suggesting the preparation of high-quality CuO NPs. Moreover, the obtained χ 2 value of 1.82 for the Le Bail fitting indicates excellent agreement with the previously reported literature [45,46]. The crystallite size is estimated from the XRD pattern using Debye Scherrer's Equation (1) [47]:

$$\mathbf{D} = \mathbf{K}\lambda / \beta \cos \theta \tag{1}$$

where *K* = 0.94 is the shape factor, λ is the X-ray wavelength of Cu Kα radiation (1.541 Å), θ is the Bragg diffraction angle, and β is the full width at half maxima (FWHM) of the respective diffraction peak. The crystallite size corresponding to the highest peak observed in XRD was found to be 34.4 nm. The presence of sharp structural peaks in XRD patterns and a crystallite size less than 100 nm corresponds to the nanocrystalline nature of

synthesized CuO NPs. The peaks at 32.5, 35.4, 35.5, 38.7, 38.9, 46.2, 48.8, 51.3, 53.4, and 56.7 in 2θ correspond to the different CuO planes [48]. Similarly, as shown in Figure 1a, the CuO obtained from Peru and Bhopal mine samples exhibited peaks corresponding to the synthesized CuO NPs. The peaks at the same 2θ positions for the extracted CuO nanoparticles from both Peru and Bhopal mine samples are referred to here and confirm the extraction of CuO from the mine samples. The additional peaks appear due to the attribution of elements such as Au, Cr, etc., which are present in the mine samples, as revealed from the SEM EDAX analysis (Figure 2). 56.7 in 2θ correspond to the different CuO planes [48]. Similarly, as shown in Figure 1a, the CuO obtained from Peru and Bhopal mine samples exhibited peaks corresponding to the synthesized CuO NPs. The peaks at the same 2θ positions for the extracted CuO nanoparticles from both Peru and Bhopal mine samples are referred to here and confirm the extraction of CuO from the mine samples. The additional peaks appear due to the attribution of elements such as Au, Cr, etc., which are present in the mine samples, as revealed from the SEM EDAX analysis (Figure 2).

X-ray diffraction (XRD) characterization of the synthesized and extracted CuO NPs was carried out to estimate the precise elemental composition, particle size, and superficial morphology, as depicted in Figure 1a. It was determined that all CuO NPs were in a monoclinic geometry with a space group of C2/C. No characteristic peaks of any other impurities were detected, suggesting the preparation of high-quality CuO NPs. Moreover, the obtained χ2 value of 1.82 for the Le Bail fitting indicates excellent agreement with the previously reported literature [45,46]. The crystallite size is estimated from the XRD pat-

where = 0.94 is the shape factor, is the X-ray wavelength of Cu K radiation (1.541 Å), is the Bragg diffraction angle, and is the full width at half maxima (FWHM) of the respective diffraction peak. The crystallite size corresponding to the highest peak observed in XRD was found to be 34.4 nm. The presence of sharp structural peaks in XRD patterns and a crystallite size less than 100 nm corresponds to the nanocrystalline nature of synthesized CuO NPs. The peaks at 32.5, 35.4, 35.5, 38.7, 38.9, 46.2, 48.8, 51.3, 53.4, and

D = / cos (1)

*Catalysts* **2023**, *13*, 369 6 of 17

tern using Debye Scherrer's Equation (1) [47]:

**3. Results and Discussion**

**Figure 1. (a)** XRD of the synthesized and extracted (from Peru and Bhopal samples) CuO nanoparticles. (**b**) FTIR spectra of different citrate-capped CuO samples and citric acid in the range between 0 and 4000 cm−1, (**c**) DLS of the citrate-capped CuO from supernatants of C-CuO samples, (**d**) zeta **Figure 1.** (**a**) XRD of the synthesized and extracted (from Peru and Bhopal samples) CuO nanoparticles. (**b**) FTIR spectra of different citrate-capped CuO samples and citric acid in the range between 0 and 4000 cm−1 , (**c**) DLS of the citrate-capped CuO from supernatants of C-CuO samples, (**d**) zeta potential distribution of the synthesized citrate functionalized CuO NP. The insets (i), (ii), and (iii) of Figure 1d are the synthesized C-CuO, the Peru mine tailing, and the Bhopal mine sludge, respectively.

To ensure the surface functionalization of the synthesized and extracted CuO NPs by citrate, FTIR analysis was performed on citric acid and the nanoparticles (Figure 1b). The absorption band, around 620.9 cm−1 in synthesized citrate CuO (C-CuO), C-CuO from mine sample 1 (Peru), and C-CuO from mine sample 2 (Bhopal) can be attributed to the vibrations of the Cu−O group [49,50]. Furthermore, the absence of other molecular vibrations, due to the calcination of the synthesized C-CuO, confirms the formation of a pristine surface. The citric acid was found to exhibit an absorption band occurring at around 1718 cm−1 , which is attributed to the C=O stretching, while the band occurring at 1394 cm−1 is attributed to the C−O stretching [51]. Moreover, the signature of the O−H stretching from the tertiary alcohol of citric acid can be witnessed at around 1099 cm−1 , and the bending of the O−H group from the carboxylic acid portion can be observed at 1383 cm−1 . Finally, the absorption band at 3299 cm−1 is due to the presence of the O−H bond from the citric acid. In the CuO FTIR spectrum, the C−O and C=O stretching can be seen at 1370 cm−1 and around 1600 cm−1 , while the tertiary alcohol band is at 1080 cm−1 . The shift in C=O and

C−O indicates that the CA is bonded to the CuO via the chemisorption of the carboxylate group. Therefore, the weakening of C=O causes a shift in frequency from 1700 to 1600 cm−1 . This characterization confirmed the success of the surface modification of CuO with citric acid. All the FTIR spectra shown in Figure 1b are baseline corrected; however, the apparent baseline shift may be due to the excess concentration of the synthesized citrate CuO nanoparticles in comparison with the extracted CuO NPs from the copper mine tailings. *Catalysts* **2023**, *13*, 369 7 of 17 potential distribution of the synthesized citrate functionalized CuO NP. The insets (i), (ii), and (iii) of Figure 1d are the synthesized C-CuO, the Peru mine tailing, and the Bhopal mine sludge, respectively.

**Figure 2.** FESEM images of (**a**) synthesized citrate-capped CuO NPs. The inset shows the EDAX parameters of the nanoparticles. (**b**) Extracted and citrate-capped CuO from Peru mine stone sample. The inset shows the EDAX parameters of the nanoparticles. (**c**) Extracted and citrate-capped CuO from the Bhopal mine stone sample. The inset shows the EDAX parameters of the nanoparticles. **Figure 2.** FESEM images of (**a**) synthesized citrate-capped CuO NPs. The inset shows the EDAX parameters of the nanoparticles. (**b**) Extracted and citrate-capped CuO from Peru mine stone sample. The inset shows the EDAX parameters of the nanoparticles. (**c**) Extracted and citrate-capped CuO from the Bhopal mine stone sample. The inset shows the EDAX parameters of the nanoparticles.

The hydrodynamic diameter of the synthesized CuO nanohybrid, C-CuO mine sample 1 (Peru) and 2 (Bhopal) were estimated to be 78.8 nm,106 nm and190 nm, respectively, from the dynamic light scattering (DLS) studies (Figure 1c), which are close enough to each other. The results of the DLS corroborate with the size obtained from the XRD (34.4 nm) and FESEM analyses (38.1 nm). Dynamic light scattering (DLS) accounts for the hydrodynamic diameter of the functionalized nanomaterial, which comprises core CuO and citrate ligands at the surface, along with associated water molecules and some possible aggregation of the functionalized nanoparticles in the aqueous solution. The electron micrograph of the nanoparticles shows the inorganic nanomaterial, as a whole, contains crystalline and amorphous inorganic core substrate. On the other hand, the size from the XRD accounts for only the crystalline materials in the core inorganic particles. Thus, it is obvious that DLS overestimates the size, while XRD reveals the crystalline nanoparticles in the core [52]. Additionally, the citrate CuO NPs exhibited a ζ-potential of magnitude −10.38 mV, assuring moderate solubility of the NPs (Figure 1d). The ζ-potential for the Peru and Bhopal samples were obtained to be −11.2 mV and −8 mV, respectively. These results lower the possibility of instability and particle agglomeration or precipitate tendencies of the CuO NPs out of the solutions. The full curve for a sample is only provided by the instrument software, while the titration experiment was performed because we obtained a synthesized pure citrate-capped CuO nanohybrid. However, for the extracted citrate CuO nanoparticles from Peru and Bhopal mine samples, only zeta potential measurements were carried out, for which only values in the mV unit are provided because, as mentioned previously, no full curves were available from the instrument. The insets in i, ii, and iii of Figure 1d are the synthesized C-CuO, the Peru mine tailing, and the Bhopal mine sludge, respectively.

Figure 2a–c shows the FESEM images of the C-CuO NPs. The inset of Figure 2a depicts the EDAX spectrum of the synthesized CuO NPs. The EDAX analysis of the extracted citrate CuO from the Peru and Bhopal mine tailings are shown in the insets of Figure 2b,c. The EDAX result shows that there are no other elemental impurities present in the prepared CuO NPs. However, trace amounts of Au and Cr, etc., were found in the extracted CuO NPs from the EDAX method. The peak of silicon appearing in the EDAX graphs of Figure 2 is attributed to the substrate. For EDAX analysis, the test sample was a drop cast on a Si wafer substrate and the thickness of the drop casted layer may not be uniform throughout the substrate. In the FESEM images of C-CuO, the synthesized nanohybrids are seen to consist of clustered spherical particulates with approximate diameters between 38 and 46 nm. The average diameter of the CuO NPs was calculated by measuring over 100 particles in a random field of FESEM view. The SEM-EDAX analysis demonstrated that the atomic compositions of the Cu and O elements were 38.41% and 61.59%, respectively. The mean ratio of Cu and O was, therefore, 38.41:61.5, and an accurate compound formula based on this atomic ratio of Cu and O can, thus, be given as Cu1.2O or CuO0.8. Therefore, it can be ensured that most of the synthesized nanoparticulate sample was indeed CuO.

The UV-vis spectra of the citrate-capped CuO samples (Figure 3a) exhibited a broad absorbance peak at 285 nm, which is characteristic of a surface plasmon resonance of the CuO nanoparticles [53]. A weaker peak at around 350 nm, signifies the d–d transition of CuO nanoparticles due to citrate functionalization and the quantum confinement effect of the CuO NPs on the citrate functionalization [54]. Similarly, the peaks observed at 285 nm and 350 nm, after the capping of Peru and the Bhopal samples, signified the presence of citrate-capped CuO NPs within the system.

The dark and photoinduced ROS generation capability of the citrate CuO NPs is illustrated using a well-known nonfluorescent probe: DCFH (Figure 3b). DCFH is oxidized to fluorescent dichlorofluorescein (DCF) by ROS, exhibiting an emission near 522 nm upon excitation at 488 nm. Thus, the enhancement of the ROS generation level is indicated by the increase in the emission intensity at 522 nm [37]. The oxidation of only DCFH control and CuO NPs are monitored for 35 min in the dark and then under irradiation of UV light (365 nm). In the dark, there is a considerable enhancement of emission intensity at 522 nm, indicating the presence of dark ROS generation. However, with light exposure, a greater

increase in emission intensity is observed for the citrate CuO nanohybrid compared to the control (Figure 3b). This confirms the ROS generation capability of the synthesized nanohybrid under light exposure making it suitable to apply for photodynamic therapy. and 350 nm, after the capping of Peru and the Bhopal samples, signified the presence of citrate-capped CuO NPs within the system.

uniform throughout the substrate. In the FESEM images of C-CuO, the synthesized nanohybrids are seen to consist of clustered spherical particulates with approximate diameters between 38 and 46 nm. The average diameter of the CuO NPs was calculated by measuring over 100 particles in a random field of FESEM view. The SEM-EDAX analysis demonstrated that the atomic compositions of the Cu and O elements were 38.41% and 61.59%, respectively. The mean ratio of Cu and O was, therefore, 38.41:61.5, and an accurate compound formula based on this atomic ratio of Cu and O can, thus, be given as Cu1.2O or CuO0.8. Therefore, it can be ensured that most of the synthesized nanoparticulate sample

The UV-vis spectra of the citrate-capped CuO samples (Figure 3a) exhibited a broad absorbance peak at 285 nm, which is characteristic of a surface plasmon resonance of the CuO nanoparticles [53]. A weaker peak at around 350 nm, signifies the d–d transition of CuO nanoparticles due to citrate functionalization and the quantum confinement effect of the CuO NPs on the citrate functionalization [54]. Similarly, the peaks observed at 285 nm

*Catalysts* **2023**, *13*, 369 9 of 17

was indeed CuO.

**Figure 3.** (**a**) Absorbance spectra of synthesized and extracted citrate-CuO nanohybrids. (**b**) DCFH oxidation (monitored at 522 nm) with time in the presence and absence of citrate CuO NPs under dark and illumination conditions. (**c**) The DPPH catalytic activity of the synthesized CuO NPs in dark conditions upon the addition of DPPH in periodic intervals of time (i) (ii) (iii) and (iv). (**d**) The DPPH assay of the synthesized CuO NPs under UV light illumination conditions. Inset shows the **Figure 3.** (**a**) Absorbance spectra of synthesized and extracted citrate-CuO nanohybrids. (**b**) DCFH oxidation (monitored at 522 nm) with time in the presence and absence of citrate CuO NPs under dark and illumination conditions. (**c**) The DPPH catalytic activity of the synthesized CuO NPs in dark conditions upon the addition of DPPH in periodic intervals of time (i) (ii) (iii) and (iv). (**d**) The DPPH assay of the synthesized CuO NPs under UV light illumination conditions. Inset shows the decay of absorbance of DPPH radical at 535 nm with multiple light exposures of 10 min intervals.

decay of absorbance of DPPH radical at 535 nm with multiple light exposures of 10 min intervals. The antioxidant activity of the citrate functionalized CuO NPs was evaluated using the DPPH assay method (Figure 3c,d). The absorbance of DPPH at 535 nm was observed to decrease with time, establishing the presence of antioxidant properties of the CuO NPs under dark conditions and with a visual transition of the solution from purple to yellow. Now, upon further addition of 50 uM DPPH, after a periodic interval of time, it is observed that the free radical scavenging activity remains almost intact in the same 100 uM C-CuO sample. The study of antioxidant activity in the presence of UV light (365 nm) is a prerequisite to a full understanding of its potential antioxidant properties. As shown in Figure 3d, the rate of absorbance at 535 nm decreases (from 1541.51sec to 1117.01sec to 1016.05sec) with UV exposure of 10 mins for each case, keeping the concentrations of DPPH and C-CuO NPs to be constant. The results corroborate the enhancement of antioxidant activities with light exposure. The same sample of C-CuO NPs is capable of providing catalytic activities after getting recharged by light exposure.

The antibacterial activities of the C-CuO NPs, synthesized and extracted from nine samples were investigated against Gram-positive *S. hominis* bacteria by analyzing their

growth curves. Figure 4a reveals the growth pattern of the bacteria in terms of absorbance at 600 nm with one-hour intervals. The synthesized C-CuO, under illumination as well as dark conditions, is observed to almost nullify the growth, with respect to the controls where only the bacteria are present in LB media. The C-CuO extracted from the Peru and Bhopal mine samples are observed to exhibit better antimicrobial activity in presence of light. The copper chloride salt used for the synthesis of CuO NPs, and sodium citrate used for the capping purpose were found to have almost no effect on bacterial growth, establishing the fact that the entire antimicrobial activities are solely generated by the C-CuO NPs. *Catalysts* **2023**, *13*, 369 11 of 17

**Figure 4.** (**a**) Effect of C-CuO NPs on the growth curves of *S. hominis* bacteria under dark and illumination conditions. (**b**) Bacterial viability after treatment with different citrate CuO NPs in the presence and absence of light irradiation. (**c**) Agar plate colony count assay. '\*\*\*\*' indicates *p*-value < 0.0001. (**d**) Bright-field microscopic images and fluorescence micrographs of bacteria stained with DAPI, and fluorescence micrographs of bacteria stained with PI. (**e**) The ratio of live and dead cells **Figure 4.** (**a**) Effect of C-CuO NPs on the growth curves of *S. hominis* bacteria under dark and illumination conditions. (**b**) Bacterial viability after treatment with different citrate CuO NPs in the presence and absence of light irradiation. (**c**) Agar plate colony count assay. '\*\*\*\*' indicates *p*value < 0.0001. (**d**) Bright-field microscopic images and fluorescence micrographs of bacteria stained with DAPI, and fluorescence micrographs of bacteria stained with PI. (**e**) The ratio of live and dead cells obtained from microscopy.

obtained from microscopy. The antimicrobial activity of the synthesized and extracted CuO NPs after citrate functionalization was investigated against *S. hominis* growth to explore the antibiotic potential against bacterial infections in the agar plate assay method. To probe the antibacte-The antimicrobial activity of the synthesized and extracted CuO NPs after citrate functionalization was investigated against *S. hominis* growth to explore the antibiotic potential against bacterial infections in the agar plate assay method. To probe the antibacterial action of the nanoparticles, they were used for incubating the culture for 3 h. As shown

rial action of the nanoparticles, they were used for incubating the culture for 3 h. As shown in Figure 4b and c, minimal colonies were observed (the bacterial growth was found to

in the CFU under dark conditions. The effect of two different mine-extracted copper nanohybrids under dark and light irradiation conditions was then studied further upon the growth of *S. hominis* bacteria (Figure 4b). In the case of the Peru and Bhopal samples, the bacterial growth is found to be decreased by 79% CFU and 80% CFU, respectively, in dark in Figure 4b,c, minimal colonies were observed (the bacterial growth was found to have decreased by 96.9% in the CFU from the control plate) for the nanohybrid under UV light illumination conditions. The bacterial growth is found to be decreased by 85% only in the CFU under dark conditions. The effect of two different mine-extracted copper nanohybrids under dark and light irradiation conditions was then studied further upon the growth of *S. hominis* bacteria (Figure 4b). In the case of the Peru and Bhopal samples, the bacterial growth is found to be decreased by 79% CFU and 80% CFU, respectively, in dark conditions. On the other hand, a huge decrement in bacterial growth is observed for both the Peru and Bhopal samples after citrate-capping on UV illumination. The bacterial growth was reduced by 90% and 91% for the citrate-capped Peru and Bhopal samples, respectively, on the UV illumination. To nullify the effect of the salt and the capping agent (sodium citrate), their antibacterial effects were studied. No significant change in the number of colonies in the negative control group was found, indicating a low antibacterial effect of these salts on *S. hominis*. Thus, the antibacterial effect found is solely due to the citrate-capped CuO NP. From these results, it is evident that the nanohybrid is itself an antibacterial agent and its efficiency enhances many folds upon white light exposure, which triggers an overall huge antibacterial effect.

To study the effect of the citrate CuO nanohybrid, bacterial cultures were performed five times for each group (control in dark and light, citrate CuO in dark and light, different concentrations of the nanohybrid, etc.) and their differences were calculated to identify their significance levels. The sample size for each of the groups was five. The *p*-value was calculated using an unpaired 2-tailed *t*-test and a value of *p* < 0.05 was considered to be significant. The statistical difference between the control and treatment is designated by '\*'. The '\*' represents a *p*-value < 0.1 and '\*\*\*\*' represents a *p*-value < 0.0001 [55].

The antimicrobial activity of the NPs was further investigated using optical microscopy. We used *S. hominis*, the Gram-positive bacteria, as the model biological system. *S. hominis* were incubated with both synthesized and extracted CuO samples for two hours in LB broth and then stained with DAPI and PI. While DAPI stains all the cells in the medium, PI is specific to dead cells as it cannot cross intact cell membranes. Therefore, the ratio of DAPI-stained cells to PI-stained cells reveals the viability of the cells. Figure 4d,e shows the results of the microscopic microbial studies. The first row of Figure 4d contains the microscopic images of the control cells after staining with DAPI and PI. As expected, the number of viable cells was more than the dead cells (ratio ~6.6). The microscopic images of the synthesized C-CuO NPs (Figure 4d, second row) incubated with bacteria showed a reduced ratio–i.e., the dead cells outnumbered the live cells (ratio ~2.1) compared to the control. Interestingly, the group of bacteria treated with C-CuO NPs extracted from the Bhopal and Peru mine samples (Figure 4d, rows 3 and 4) also showed higher viability compared to the control. The ratio for these C-CuO NP-treated groups was similar to that of the synthesized C-CuO-treated group, as shown in Figure 4e. Therefore, the microscopic studies revealed that the C-CuO NPs exerted toxic effects and effectively killed the *S. hominis*. The therapeutic action of the CuO NPs against *S. hominis* bacteria occurs primarily via the generation of reactive oxygen species, which mainly includes the formation of hydroxyl radicals (HO.\* ) and superoxide radicals (O2 −\* ). Initially, electron-hole (e-/h+ ) pairs are formed when the electromagnetic radiation of energy (hυ) is either greater than or equal to the bandgap energy (Eg) of the CuO NPs. This phenomenon excites the electron from the valence band (VB) to the conduction band (CB), leaving holes behind in the VB. These photoexcited electrons reduce the surface adsorbed O2 to O2 −., while the holes oxidize H2O or HO- to OH\* [56–58], as described in the graphical abstract. This in situ production of the reactive radicals starts off by attacking the bacterial population and eliminates them through the production of toxic by-products, resulting from ROS-mediated damage to the cellular system.

$$\text{CuO} + \text{h}\nu = \text{CuO} \left(\text{e}^- + \text{h}^+\right).$$

$$\text{O}\_2 + \text{e}^- = \text{O}\_2{}^{-\*}.$$

$$\mathrm{H\_2O + h^+ = H^+ + HO^\*}.$$

In the absence of electromagnetic radiation, the antimicrobial activity may be attributed to the ligand-to-metal charge transfer (LMCT) of the C-CuO NPs. The LMCT bands originate due to the interaction of the Cu1+/ 2+ centers in the NP with the surface-bound citrate ligands. In the dark condition, the origin of the antibacterial activity might be due to the conversion of the Cu1+ to Cu2+ states at the center, accompanied by the direct injection of the electrons into the CB of the C-CuO NPs and followed by the reduction of the surface adsorbed O2 to O2 −. *Catalysts* **2023**, *13*, 369 13 of 17 injection of the electrons into the CB of the C-CuO NPs and followed by the reduction of the surface adsorbed O2 to O2−. Cu1+ - O = Cu+2-O\*+ e−

$$\text{Cu}^{1+} - \text{O} = \text{Cu}^{+2} - \text{O}^{\*} + \text{e}^{-}$$

$$\text{O}\_{2} + \text{e}^{-} = \text{O}\_{2}^{-}$$

The enhanced efficacy of the citrate functionalized CuO NPs, in the presence of light for more bacterial remediation, establishes its credentials for the application of APDT (antibacterial photodynamic therapy). for more bacterial remediation, establishes its credentials for the application of APDT (antibacterial photodynamic therapy). The extraordinary effect of the synthesized copper nanohybrid may be hypothesized

The extraordinary effect of the synthesized copper nanohybrid may be hypothesized using predictive biological interactions. To compare our findings with the existing research and related predictive models, the previously mentioned STITCH database was used. It generated an interaction network (Figure 5) between the ligand CuO and its effect on various proteins of *S. hominis*. To understand how CuO affects the survival of the bacteria, a comprehensive table (Table 1) was populated with the protein names and their respective activities. The table suggests the negative impact of CuO on key proteins of *S. hominis*, which helps the organism deal with environmental stress, energy metabolism, GTP binding, and translation regulation, etc. using predictive biological interactions. To compare our findings with the existing research and related predictive models, the previously mentioned STITCH database was used. It generated an interaction network (Figure 5) between the ligand CuO and its effect on various proteins of *S. hominis*. To understand how CuO affects the survival of the bacteria, a comprehensive table (Table 1) was populated with the protein names and their respective activities. The table suggests the negative impact of CuO on key proteins of *S. hominis*, which helps the organism deal with environmental stress, energy metabolism, GTP binding, and translation regulation, etc.

**Figure 5.** Compound protein interactions network of citrate CuO nanohybrid on *S. hominis* bacterial strain. **Figure 5.** Compound protein interactions network of citrate CuO nanohybrid on *S. hominis* bacterial strain.

Pyridine nucleotide-disulfide oxidoreductase family pro-

oxidoreductase

**Name Activity**

tein Controls mitochondrial function. [60]

Catalase, an antioxidant enzyme found in all aerobic organisms, facilitates the transformation of H2O2 into water and oxygen under environmental stress. [59]

**Table 1.** Effect of citrate functionalized CuO NPs on *S. hominis* bacterial strain.

HMPREF0798\_00999 Pyridine nucleotide-disulfide

HMPREF0798\_01742

**Identifier Corresponding Protein**

**Table 1.** Effect of citrate functionalized CuO NPs on *S. hominis* bacterial strain.

### **4. Conclusions**

In this study, pure-grade citrate-capped CuO NP was synthesized using simple precipitation and grafting methods. From two types of copper mine tailings, CuO NPs were also extracted and functionalized with citrate. The XRD spectrum confirmed the formation of monoclinic crystals of CuO NPs with space group C2/C. FESEM and EDAX revealed the morphology of CuO NPs. The average SEM diameter of CuO NPs was around 38.1 nm, which agreed fairly well with the XRD and DLS data. FTIR and UV-vis spectroscopy confirmed the surface functionalization of the CuO NPs with the citrate ligand. The synthesized nanohybrid was found to generate ROS under UV light exposure in the DCFH assay and showed excellent antimicrobial activity against *S. hominis* bacterial strains in a triparted study consisting of growth curve analysis, agar plate assay method, and microscopic studies after staining using DAPI and PI. The C-CuO NPs were also found to possess antioxidant properties in the DPPH assay. Consequently, citrate-functionalized CuO NPs can, therefore, be used as an antibacterial agent in surface coatings, on a variety of substrates to stop microbes from adhering to them, colonizing and growing on them, and producing biofilms, such as in habitational medical equipment. This study suggests that the mechanisms of the antimicrobial response of a citrate-CuO nanohybrid in different species of bacteria should be further explored. The use of copper mine tailings as a source of raw materials for nanohybrid synthesis as a means of, simultaneously, catering low-cost antibiotics to a wider population across the globe and remediating the burden from the copper mines was also explored.

**Author Contributions:** A.B.: (Conceptualization: Lead; Data curation: Lead; Formal analysis: Lead; Investigation: Lead; Methodology: Lead; Project administration: Lead; Resources: Lead; Software: Lead; Validation: Lead; Visualization: Supporting; Writing—original draft: Lead; Writing—review & editing: Supporting); R.G.: (Data curation: Equal; Formal analysis: Supporting; Investigation: Supporting; Methodology: Equal; Validation: Supporting; Writing—review & editing: Supporting); T.A. (Data curation: Supporting; Formal analysis: Supporting; Validation: Supporting; Writing review & editing: Supporting); S.M.: (Resources: Equal; Writing—review & editing: Supporting); A.C.: (Investigation: Supporting; Methodology: Supporting; Writing—review & editing: Supporting, Resources: Supporting); S.K.P.: (Conceptualization: Lead; Investigation: Supporting; Methodology: Equal; Supervision: Lead; Writing—review & editing: Supporting Formal analysis: Equal; Project

administration: Lead; Resources: Equal). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** All data are available on request to the corresponding author.

**Acknowledgments:** SKP wants to thank the Indian National Academy of Engineering (INAE) for the Abdul Kalam Technology Innovation National Fellowship, INAE/121/AKF.

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

### **References**

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