Fungal-Based Remediation in the Treatment of Anthropogenic Activities and Pharmaceutical-Pollutant-Contaminated Wastewater

: Pharmaceutical personal care products (PPCPs) have increased in consumption due to the worldwide post-pandemic situation, marking them as chemical and pathogenic pollutants in signiﬁcantly higher concentrations than ever in the ecosystem. Considering the inexplicable levels of these chemical residues discharged into the environment, concerns have been raised regarding their probable ecotoxicity to marine and terrestrial life. A further concern is the potential for developing and spreading antibiotic-resistant microorganisms and genes in aquatic ecosystems due to antibiotic exposure. Hence, knowing how these compounds impact aquatic ecosystem functioning is imperative, and thus is a critical area of research. The ecological risk analysis of PPCPs in aquatic ecosystems has been carried out using various strategies


Introduction
Water resources are becoming scarcer, and the quality of water bodies is seriously challenged by several contaminants that influence both human health and aquatic environments due to their long-lasting nature, resistance to degradation, and toxicity [1,2]. Emerging pollutants are frequently detected in water environments, including fire retardants, pharmaceuticals, and skin care products [3]. Pharmaceuticals and personal care products (PPCPs) are one major category of the recently emerging organic micropollutants causing much concern [4].
The US Environmental Protection Agency defines pharmaceuticals and personal care products (PPCPs) as "any product used by persons for personal health or cosmetic purposes or used by agribusiness to increase growth or health of livestock" (USEPA). This description covers the wide range of chemicals used in cosmetics, fragrances, over-the-counter drugs, and veterinary treatments ( Figure 1).

Introduction
Water resources are becoming scarcer, and the quality of water bodies is seriously challenged by several contaminants that influence both human health and aquatic environments due to their long-lasting nature, resistance to degradation, and toxicity [1,2]. Emerging pollutants are frequently detected in water environments, including fire retardants, pharmaceuticals, and skin care products [3]. Pharmaceuticals and personal care products (PPCPs) are one major category of the recently emerging organic micropollutants causing much concern [4].
The US Environmental Protection Agency defines pharmaceuticals and personal care products (PPCPs) as "any product used by persons for personal health or cosmetic purposes or used by agribusiness to increase growth or health of livestock" (USEPA). This description covers the wide range of chemicals used in cosmetics, fragrances, over-thecounter drugs, and veterinary treatments ( Figure 1). Since they are designed to have the most substantial effect at low concentrations, PPCPs significantly impact the environment and people, even in trace quantities [5]. As a result, concerns have been raised concerning the potential damage PPCPs could bring to people and ecosystems, notably by promoting the spread of antibiotic-resistant genes. In addition to improving daily life, PPCPs are used to prevent and treat illnesses in animals and humans. Under typical circumstances, PPCPs might not evaporate quickly, even though they might easily dissolve in water. Because of these characteristics, PPCPs can access water sources in various ways [6]. PPCPs are frequently discovered in Since they are designed to have the most substantial effect at low concentrations, PPCPs significantly impact the environment and people, even in trace quantities [5]. As a result, concerns have been raised concerning the potential damage PPCPs could bring to people and ecosystems, notably by promoting the spread of antibiotic-resistant genes. In addition to improving daily life, PPCPs are used to prevent and treat illnesses in animals and humans. Under typical circumstances, PPCPs might not evaporate quickly, even though they might easily dissolve in water. Because of these characteristics, PPCPs can access water sources in various ways [6]. PPCPs are frequently discovered in concentrations between ng/L and g/L in water and wastewater samples. In aquatic environments, PPCPs have detrimental ecological effects that affect the ecosystem and human health [7].
Different methods, including photo-Fenton, electrochemical treatment and activated carbon methods and emerging technologies such as graphene-based nano-systems, ZnO nanostructures, and zirconia nanostructures, have been utilized as effective adsorbents for the elimination of pharmaceutical compounds [8][9][10]. However, despite using these physicochemical technologies for pharmaceutical waste treatment, they have certain drawbacks, such as producing by-products, high operational costs, and labor-intensive processes [11]. Further assistance is required to address these limitations. Moreover, most wastewater treatment facilities need help to eradicate the new micropollutants. Various physicochemical and biological approaches to micropollutant (PPCP) treatment have been investigated, including the advanced oxidation process and fungi-induced activated sludge methods. PPCPs are pollutants that are generated in the form of industrial waste.
Microbes such as fungi, which promote the breakdown of organic substances at this stage, are crucial [12]. Indeed, high biodegradation rates of PPCPs have been observed in activated sludge chambers.
Fungi are microorganisms that break down the bulk of organic chemicals in the environment. Fungi have been utilized in water and soil bioremediation techniques since the 1980s [13]. In recent years, using white-rot fungi for the biological treatment of wastewater containing pharmaceuticals and personal care products (PPCPs) has emerged as a promising approach [14]. Various studies conducted have demonstrated through batchscale experiments that white-rot fungi are capable of secreting abundant enzymes and degrading a wide range of environmental pollutants, including pharmaceuticals, dyes, heavy metals, and organic contaminants which disrupt the endocrine system [15][16][17]. Moreover, research has indicated that fungi can also employ biosorption to remove pharmaceutical and personal care substances [18]. Several reports have shown the efficient removal of pharmaceutical compounds via filamentous fungi, achieving removal rates ranging from 78% to 100% in synthetic wastewater media [19][20][21][22]. However, previous studies have often exhibited slow degradation rates, with treatment durations exceeding 26 days [14,23,24]. Nonetheless, recent investigations have involved the white-rot fungus Trametes versicolor [25] and species of Aspergillus, Mucor, Penicillium, Rhizopus, Trametes, and Trichoderma [26,27]. They have demonstrated the significant degradation of pharmaceutical compounds with high removal rates achieved within a shorter timeframe, ranging from 4 h to 10 days [28][29][30][31]. Fungi provide several advantages over traditional physicochemical treatment procedures, including being highly effective, having a cheap cost, and being eco-friendly [32]. Furthermore, they can act in various pH circumstances and have a variety of enzymatic abilities for degradation ( Figure 2). Additionally, the hyphal forms of fungus can aid in the biosorption process of water purification by allowing PPCPs and other chemicals to cling to their surface or be ingested inside the cell, where they are stored rather than transferred in the water [33]. The previous cases have reported the presence of PPCPs from the industrial effluents and the coastal water followed by their treatment through fungal strains [34].
Fungi have demonstrated remarkable capability in degrading diverse pharmaceutical and personal care compounds, making them highly valuable for the bioremediation of emerging contaminants in wastewater. This review will provide a comprehensive overview of the recent research, focusing specifically on using fungi in the bioremediation of PPCPs. The review examines cellular mechanisms, treatment processes, an overview of the fungi employed, their specific applications for different pollutants, and the beneficial byproducts generated through fungal activity. Furthermore, the review will address the challenges encountered in this field, highlight prospects, and explore the potential of innovative technologies to harness and enhance fungi's remediation abilities in treating PPCPs.
The details which contributed to the studies of PPCPs as significant contributors to contamination and their removal through physical, chemical, biological, and integrated green approaches, explicitly focusing on fungal strains, hybrid fungal and other algal or bacterial strains, are mentioned in Table 1. Further, the new techniques through AOR approaches, including monitoring and devices, are also categorized in Table 1. Fungi have demonstrated remarkable capability in degrading diverse pharmaceutical and personal care compounds, making them highly valuable for the bioremediation of emerging contaminants in wastewater. This review will provide a comprehensive overview of the recent research, focusing specifically on using fungi in the bioremediation of PPCPs. The review examines cellular mechanisms, treatment processes, an overview of the fungi employed, their specific applications for different pollutants, and the beneficial byproducts generated through fungal activity. Furthermore, the review will address the challenges encountered in this field, highlight prospects, and explore the potential of innovative technologies to harness and enhance fungi's remediation abilities in treating PPCPs.
The details which contributed to the studies of PPCPs as significant contributors to contamination and their removal through physical, chemical, biological, and integrated green approaches, explicitly focusing on fungal strains, hybrid fungal and other algal or bacterial strains, are mentioned in Table 1. Further, the new techniques through AOR approaches, including monitoring and devices, are also categorized in Table 1. Presence of PPCPs and merging resources as sources of contamination in effluent and water bodies at the global level in waterbodies.

PPCP as Pollutants
Pollutants from pharmaceuticals and personal care products (PPCPs) have emerged due to their nature of causing diverse physiological impacts in humans, even at nominal doses. Numerous studies conducted in recent years have confirmed the presence of different PPCPs in various environmental niches, primarily due to the indiscriminate use of antimicrobials and other organic compounds to treat bacterial and fungal infections, cardiac and other major surgeries in the treatment of cancer, and patients undergoing organ transplant. In addition, the use of similar compounds in personal care products raises concerns about their potential adverse effects on humans and wildlife [98][99][100]. These products constitute a large and diverse group of organic compounds, including prescription and non-prescription medications, nutritional supplements, diagnostic agents, and daily personal care products (PCPs) such as soaps, lotions, toothpaste, fragrances, and cosmetics, together with their metabolites and transformation products [101]. In the USA, approximately two out of three prescription medications were reported to be either redundant or unused. It was also found that the total estimated cost of these wasted prescriptions ranged anywhere from USD 59,264.20 to USD 152,014.89. All healthcare centers are responsible for 3% of the world's pharmaceutical waste contribution, followed by tablets and injectables [100]. Microbeads in a diversity of PPCP cosmetics, including shower gels, toothpaste, nail polishes, and other daily care products, account for the environmental plastic waste grouped as microplastics or nano plastics. Most of these microplastics cannot be treated in wastewater treatment plants (WWTPs). Therefore, they are discarded directly into the environment, causing health hazards; it is estimated that over 3 trillion microbeads are released via WWTPs. These plastic microbeads attract organic pollutants and chemicals linked to chemical hazards, including cancer [89]. At this hour, reliable, relevant, and valid scientific data are required to guide regulatory and policy responses to address possible human, environmental, and ecology hazards from PPCP pollutants. The government, academics, cosmetics manufacturers, pharmaceutical corporations, and consulting firms engaged in scientific research or policy and management activities are the main stakeholders in the management of these pollutants [1,102].

Sources and Routes
Sewage treatment facilities have mostly been found as reservoirs of trace antibiotics and PPCPs (upto µg/L). Most often, concentrations in treatment plants in China have ranged between not-detected (ND) and 7910 ng/L (influent) and between not-detected and 9460 ng/L (effluent) (54). Doxycycline, fluoroquinolones, and penicillin comprise most antimicrobial agents used among chemotherapeutics needed to prevent deadly illnesses in both people and animals [103]. Pharmaceutical industries are compelled to scale up the production of these antimicrobials to keep up with the rising demand for these medications. Although some drugs are used internally, they remain unchanged in urine and feces and are then deposited in sewage treatment facilities. Several antibiotics and their metabolic products [101], hormones [38], and other pharmaceuticals were frequently detected in sewage samples [2]. Such pharmaceutically active molecules include fluoroquinolones, macrolides, azoles, cephalexin, erythromycin, and its metabolic products (e.g., Roxithromycin). These sewage treatment plants also contain acetaminophen; caffeine; hormones such as estrogen and progesterone; glucocorticoids; and others. The two primary mechanisms influencing the PPCPs' fates in the environment are adsorption and biodegradation. Penicillin, amoxicillin, tetracycline, quinolones, novobiocin, and gentamicin are the antibiotics most utilized when treating animals. The most common illnesses treated in animals used for meat production include diarrhea, shipping fever, and bovine pneumonia [39]. These compounds have been discovered in manure and soil that is heavily contaminated with manure. Antimicrobial agents and PCPs have been utilized in animal husbandry, aquaculture, and agriculture as growth promoters and to boost feed conversion efficiency. These practices include animal farming [36,49]. PPCPs are ubiquitously found in several environmental niches, such as in excretion products (urine and feces), and hence are most typically detected in WWTPs. They subsequently enter aquatic system components in medicines and household items which include estrogenic endocrine-disrupting chemicals (e-EDCs), toxic metals, and nanoparticles, and their subsequent discharge in rivers represents a significant risk to human and livestock health [91]. The distribution and hazardous outcomes of PPCPs are discussed in Table 2.

Mechanism of Action and Hazards Caused to Human Health
Pharmaceutical substances employ toxic or static action toward the microbial cell. This cytotoxic or static mechanism enables the immune system to eradicate pathogens. The inhibition of cell wall production, the synthesis of DNA/RNA, the synthesis of protein, ribonucleic acid (RNA) synthesis, protein synthesis, and membrane disorganization and the disruption of membranes are all mechanisms used by antibiotics to kill or inhibit the growth of bacteria or fungi [98]. Since antimicrobials are efficient against a wide variety of microbial strains, they are used worldwide to treat various infections. This widespread use has led to ubiquitous distribution in the different environmental milieus.
It is now established that PPCPs pose a significant threat to human health and wildlife and should be immediately addressed. Chronic exposure to these chemicals increases the risk of developing testicular germ line and breast cancer, even at low concentrations. Prolonged exposure to these compounds also causes hypospadias, cryptorchidism, and infertility due to decreased sperm count [98,115]. Experts believe that investments should be made in technologies aiding advanced wastewater treatment and educating medical professionals to reduce the overprescription of antibiotics. These steps can reduce the environmental burden of PPCPs. This requires support from pharmaceutical companies and governments to incorporate pharmaceutical-return programs in corroboration with education and asking all municipalities to adhere to minimum secondary wastewater treatment [115]. The administration should implement these strategies to reduce the burden of human-derived PPCPs on the environment.

Fungal Genera and Enzymes for the Remediation of PPCP
Bioremediation minimizes the pollutant level by converting recalcitrant and xenobiotic components to a less toxic or acceptable form [58]. Due to their great metabolic activity, microorganisms are omnipresent and can survive various environmental conditions. Their universality, diversity, colossal population and biomass, anaerobic activity, survival capacity in extreme conditions, and stupefied catalytic mechanisms make them suitable for bioremediation [55]. They have compelled scientists and researchers all over the globe to dive deep into understanding their biochemistry and genetics for their utilization in various applications. Bioremediation, accomplished by biodegradation, involves biotic and abiotic transformations which microorganisms achieve. It is a complex activity involving the disruption of the different organic compounds into their inorganic components. The physical and chemical interaction of microorganisms with various polluting factors leads to the complete or partial degradation of the molecule of interest. Pharmaceuticals and personal care products (PPCPs) mostly contain endocrine-disrupting chemicals and trace organic compounds that cause bioaccumulation, are highly toxic to aquatic life, and adversely affect human health. Although traditional wastewater treatment processes such as activated sludge and membrane bioreactors are adequate for removing most organic compounds, nutrients, and pathogens, certain hydrophilic compounds and compounds containing functional groups with strong electron-withdrawal capacities are barely eliminated. Thus, PPCPs demand specialized treatment processes other than conventional techniques. The details of the fungal enzymatic bioremediation of PPCPs and their resources through different fungal stains in different bioreactors are mentioned in Tables 3 and 4.  Fungi have been utilized in bioremediation for more than three decades. The rich morphology; diverse metabolic capacity; and ability to thrive, survive, colonize, and propagate in diverse habitats make fungi one of the most suitable candidates for detoxifying PPCPs. Additionally, fungi are comparatively more effective for bioremediation than bacteria because of their various processes and enzymatic activities, as well as their efficiency in functioning under a wide range of pH [32,51,72,76]. Several factors, such as the composition of media, pH, spore suspension concentration, and incubation factors, including duration, mixing speed, etc., are the key factors influencing mycelium growth and enzyme production. Other crucial factors include various operational parameters such as cultivation time, the concentration of organic and inorganic constituents, stationary or submerged cultures, aeration, the concentration of inducers, and protease activation [76]. White-rot fungi (WRF), belonging to the phylum Basidiomycota, refer to a collection of an eco-physiological group of fungal species such as Pleurotus ostreatus, Phanerochaete chrysosporium, Trametes versicolor, Ganoderma lucidum, and Irpex lacteous that possess a lignin modification enzyme (LME) and are capable of lignin degradation. The fungi are named so because of their lignin degradation capacity. Woods attacked by them acquire a bleached appearance with a fibrous texture since dark-colored lignin gets eliminated in its presence. The non-specific WRF enzymes, which are non-stereoselective and extensively diverse, facilitate their utilization for the bioremediation of PPCPs [53]. WRF degrades lignin along with its aromatic compounds. The lignin-modifying system (LMS) possessed by WRF refers to the assembly of oxidoreductive metalloenzymes, heme peroxidases, laccases, secondary metabolites, and surfactants. Lignin peroxidases (LiPs), manganese-dependent peroxidases (MnPs), and laccases are the three major groups of enzymes secreted by WRF [81]. However, all three classes of enzymes may not be secreted by the same fungal strain. After the degradation or fragmentation of lignin, the surfeit of the fragments thus formed is taken up by the hyphae and passed on to the intercellular network for further metabolism [59].
Fungi, more specifically WRF, may utilize four distinct pathways or mechanisms for the bioremediation of PPCPs that can be efficient both independently and synergistically. The four pathways are (1) utilizing their hyphae for the bioabsorption and immobilization of various PPCPs, (2) involving the extracellular enzymes, peroxidases, and phenolases, (3) involving the intracellular enzyme complexes such as cytochrome P450, and (4) producing reactive oxygen species (ROS). The enzymes secreted by WRF, being extensively oxidative, do not require the compound's internalization and subsequent toxicity, but instead can potentially act intracellularly or extracellularly [57].
The vegetative fungal body comprises masses of long or thread-like structures known as hyphae, a defining property of filamentous fungi such as WRF that aids in rapid colonization. This structure is involved in various functions, a crucial one among them being absorption. Various chemicals, including PPCPs, adhere to the surfaces of hyphae of WRF or might be internalized within the cell, thereby getting absorbed by the fungi. This absorption mechanism stops the PPCPs from flowing along with the effluent, proving the efficiency of WRF in bioabsorption and immobilization [51].
Lignin peroxidase is an enzyme belonging to the oxidoreductase family. It can be broadly categorized as a ligase enzyme capable of degrading lignin, a highly resistant biopolymer. Manganese-dependent peroxidase is another ligninolytic enzyme that aids in lysing lignin. Laccases constitute a family of copper-containing oxidase enzymes; the copper ions present in the enzyme's active site undergo a cycle of oxidation and reduction reactions during the catalytic process in which two water molecules are formed on the concomitant electron loss of a single oxygen molecule. This abstracted electron further leads to the oxidation of various benzene-ring-containing compounds capable of degrading various aromatic compounds, including phenols or amines [60,65]. Trametes versicolor, Phanerochaete chrysosporium, and Phlebia tremellosa are some of the most prominent WRFs in bioremediation. T. versicolor is a versatile species that secretes all three extracellular ligninolytic enzymes; however, laccase is the predominant enzyme secreted by the strain ATCC 7731. It also secretes the cytochrome P450 complex. This species has relatively more significant redox potential and is stable in crude and purified forms. However, its bioremediation efficiencies in terms of PPCPs are majorly dependent on factors such as cultivation conditions, substrates, and the chemical components present in the PPCP effluent [52]. PPCPs, including steroids, nonylphenol, and octocrylene, which can lead to the dysfunctioning of the endocrine system, have been reported to be removed at a rate of 70-99% by T. versicolor. However, pharmaceutical compounds containing strong electron-withdrawing functional groups such as ciprofloxacin, salicylic acid, azithromycin, tetracycline, and carbamazepine have been reported to be removed at the rate of 35%, 0-5%, 26%, 0-5%, and 0-90%, respectively. Additionally, this fungal strain has been reported to be effective for removing compounds such as triclosan and oxybenzone, apart from octocrylene and nonylphenol found in personal care products. Some of the common drugs removed by T. versicolour are naxopren, carbamazepine, and ibuprofen. Among other WRF species, Phanerochaete chrysosporium, Bjerkandera adusta, Dichomitus squalene, and Pleurotus ostreatus have been investigated to be effective in the bioremediation of PPCPs such as ibuprofen, diclofenac, and triclosan owing to their LMEs [92]. Genetic engineering techniques targeting microbes can also be used in the treatment of wastewater [48]. P. chrysosporium is capable of degrading chlorinated xenobiotics, whereas B. adusta spp. TBB-03 effectively removes acetaminophen and bisphenol under varied conditions of temperature and pH [54]. Moreover, both fungal strains successfully degrade antidepressants such as citalopram and fluoxetine. Besides LMEs, WRF secrete natural mediators that enable crude enzymes to achieve better results in removing PPCPs than purified enzymes. Crude extracellular enzymes potentially degrade PPCPs, including oxytetracycline and tetracycline, at 84% and 72%, respectively, in batch and continuous cultures. Additionally, investigations on cell cultures of WRF have shown that submerged whole-cell cultures are more promising than solid media cultures.
In addition to the extracellular enzymatic machinery of WRF, several intracellular enzymes have also been investigated to be efficient in detoxifying xenobiotic compounds. The cytochrome P450 monooxygenase superfamily is one such complex with heme as a cofactor, capable of degrading PPCPs, including chlorinated hydrocarbons and polycyclic aromatic hydrocarbons. This enzyme complex aids in catalyzing deamination, heteroatom oxygenation, dehalogenation, dealkalinalization, and hydroxylation reactions, ultimately leading to the mineralization and degradation of organic pollutants [15]. Organic pollutants can be degraded through metal-carbon hybrid material-mediated PS-AOPs for wastewater decontamination [8]. In order to overcome these challenges, alternative approaches with excellent performance have been looked for [6,8].
The LME machinery of WRF makes these fungal species potential producers of superoxide anion radicals (O 2•− ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO • ), which are reactive oxygen species (ROSs). ROSs are chemically reactive molecules with oxygen. Superoxide radicals are formed when an oxygen molecule gets reduced by gaining an electron. The volatile oxygen molecule in this form tries to get rid of the electron, which makes that molecule reactive when taken up by another molecule [51]. The dismutation of superoxide, thus formed, produces hydrogen peroxide, which in turn may get partially reduced to a hydroxyl radical or wholly reduced to water following the Fenton reaction. These ROSs oxidize various compounds to a high degree. The laccase oxidation and dismutation activity by Pleurotus eryngii on hydroquinone and other aromatic aldehydes enhances the ROS production process.
Hence, WRF can be considered promising and efficient weapons in combating PPCPs, which are extensively harmful contaminants. The efficiency of various fungal strains in detoxifying chemical and biochemical compounds makes this 'fungal rescue' path effective.

Mechanistic Approaches Using Fungal Strains to Remove PPCPs
Mycodegradation refers to the decomposition or degradation of various chemical compounds and other materials via fungi. It is a fact that fungi are capable of efficient degradation, since it has been witnessed in many circumstances that fungi destroy several kinds of materials such as leather, paper, wood, textiles, and plastic. Pleurotus ostreatus and Irpex lacteus successfully break down PAH present in polluted industrial soil. The cultures of white-rot fungus are very capable of not only decomposing PAH but also eliminating different PAHs. Several different kinds of enzymes such as laccases, manganese peroxidase (MnP), and lignin peroxidase (LiP) are produced by fungi, and these can very effectively be employed in the decomposition of hydrocarbons, pesticides, polychlorinated biphenyls, dyes, chlorinated and phenolic compounds, etc. [95].
An array of enzymes can be synthesized by fungi, such as laccase, esterase, manganese peroxidase, lipase, and unspecific peroxygenases (UPOs). These enzymes can remediate organic pollutants such as pharmaceuticals, pesticides, microplastics, recalcitrant PAHs, and personal care products. In addition to enzyme-mediated methods, a range of other approaches can be used with fungi, such as biotransformation, bioabsorption, and biominer-alization (bio-precipitation), which helps them counteract recalcitrant toxic compounds [80]. As an analytical study exhibited that there was a decline in the activity of catalase as an increment in the concentration of oil occurred when the bioremediation of contaminated soil was underway, this indicated that catalase activity had the potential to be employed as a tool to monitor the efficacy of bioremediation. Hence, if the essentiality of catalase in making fungi tolerant to heavy metals is considered, fungi are a viable option for the remediation of areas contaminated with metals [70].
For the purification of contaminated water effluents polluted with phenolic elements and their derivatives, the decolorization of dyes used in the textile industry, the breakdown of polyaromatic hydrocarbons (PAHs) and pesticides, and for the effective removal of H 2 O 2 residues from bleaching effluents, the enzyme catalase is an excellent option. Catalase also keeps track of the physiological response of organisms, especially when environmental pollutants are present [73].
Fungal laccases provide many benefits of immense value for biotechnological uses of commercial effluent treatment. Since these enzymes show broad specificity concerning the substrate, they are well suited for bleaching Kraft pulp or rendering agricultural byproducts, including wastes from olive mills or coffee pulp. Effective decolorization was observed when the effluent from a Kraft paper mill bleach plant was treated with laccase from a fungal isolate of Flavodon flavus. Bisphenol A, an endocrine-disrupting chemical, was successfully degraded by laccase purified from a white-rot basidiomycete named Trametes villosa. It has also been documented that laccases isolated from aquatic hyphomycetes called Clavariopsis aquatica break down xenoestrogen nonylphenol. Apart from its possible use in organic degeneration in freshwater ecosystems, the enzyme laccase also presents novel ideas for biotechnological utilities, such as treating contaminated water [62]. Initially, the laccases derived from fungal species were classified as enzymes that degrade lignin (lignin-degrading). Later, it was discovered that they aid the coupling of the reduction of O 2 with the oxidation of an array of substrates of organic nature, which include phenols, aromatic amines, aliphatic amines, polyphenols, and phenols that have -OCH 3 substitution. Earlier works have indicated the role of fungal laccases in the decomposition of various upcoming pollutants, such as bisphenol A (BPA), sulfamethoxazole, atrazine, estrogens (e.g., 17α-ethinylestradiol (EE2), estrogen (E1), and 17β-estradiol (E2)), and other recalcitrant organic pollutants [74].
Depending on the origin and functional nature, peroxidases are classified as manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP). Of these, lignin and manganese peroxidases are heme peroxidases and need hydrogen peroxidases and manganese's presence for functionality. These peroxidases are mainly known for the breakdown of toxins via white-rot fungi and fungi belonging to Basidiomycetes. When it comes to versatile peroxidases, they have the potential to oxidize both phenolic as well as non-phenolic compounds and are essential for bioremediation purposes. Notably, versatile peroxidase (VP) has broad substrate-specificity [75].
Specific peroxidases cannot be adjusted into the abovementioned classification; they are dye-decolorizing peroxidases (DyPs) and unspecific peroxygenases (UPOs). They are heme peroxidases and utilize hydrogen peroxide to catalyze the oxidation of several non-phenolic lignin model compounds and other organic substances [63].
Lately, to improve the stability under acidic conditions (even at a pH as low as 2), a manganese peroxidase enzyme derived from C.subvermispora was engineered. The improved Mn 2+ -oxidizing nature and enhanced acid stability were incorporated by analyzing its crystalline structure as a scaffold. A stable enzyme was synthesized, which could oxidize veratryl alcohol and Reactive Black 5 [63].
In order to render the toxic elements in the environment non-toxic, fungi have sophisticated oxidative and hydrolytic enzymatic machinery [66,71]. Apart from this machinery, some fungi also have intracellular networks which comprise the genome, constituted by cytochrome (CYP) P450 monooxygenases and glutathione transferases for combating an array of contaminants.
The genome comprises the members of detoxification pathways, usually from multigenic families such as cytochrome P450 monooxygenases and glutathione transferases [56].
The fungal cytochrome P450 system can act as a versatile catalyst for the region-and stereospecific oxidation of non-activated hydrocarbons. It also can serve as an ideal substitute for chemical catalysts [78]. Mutagenic/carcinogenic fused-ring high-molecular-weight PAHs (HMW-PAHs), crude oil aliphatic hydrocarbon n-alkanes, and endocrine-disrupting long-chain alkylphenols (APs) were oxidized via CYP63A2 P450monooxygenase isolated from the white-rot fungus, i.e., P. chrysosporium [79]; the mechanism is summarized in Figure 3. The different mechanisms for treating pharmaceutics through fungal degradation are shown in Figure 4.  Fungal biodegradation can be carried out against diverse types of micropollutants due to the ability of fungi to produce a wide range of enzymes that can break down different types of pollutants. Different types of fungi are used depending on the specific micropollutant being targeted, and factors such as pH, temperature, and nutrient availability can also affect the efficiency of fungal biodegradation [64]. The mechanisms of fungal biodegradation are diverse and can be applied to various micropollutants, including pharmaceuticals, pesticides, and industrial chemicals. For example, studies have shown that certain species of fungi, such as Phanerochaete chrysosporium and Trametes versicolor, can degrade a range of micropollutants, including pharmaceuticals, pesticides, and personal care products. The key to successful biodegradation lies in optimizing fungal growth and activity conditions, such as pH, temperature, and nutrient availability. One example of the successful fungal biodegradation of micropollutants is using white-rot fungi to degrade pharmaceuticals in wastewater. In this case, researchers isolated various white-rot fungi and conducted culture experiments to determine which strains were most effective at breaking down specific pharmaceuticals. This method allowed for the targeted and efficient removal of micropollutants in the wastewater [64,67].    [29]. (C-H) shows CAR's removal efficiency (%) at pH 5.5 and 6.3 from synthetic wastewater media with the degradation percentages following 24 h (G) and 7 days (H) for each strain tested. The figures are adapted from [22]. (I-K) The pharmaceutical removal efficiency of laccase enzymes (I) and removal of acetaminophen related to laccase activities (J) and sugar concentrations (K); decreases in acetaminophen, sulfamethoxazole, and carbamazepine concentrations due to the activities of crude laccase. The colors indicate nonwoody and woody lignocellulosic biomasses in (J,K). The figures are adapted from [19]. * This indicates significantly different concentrations compared to the control are marked by * based on a t-test (* p-value < 0.05).

Current Methods of Fungal Biodegradation: Of PPCPs
Nowadays, several methods of fungal modification have been opted for and invented by researchers [69,97]. The genetic modification of fungi for enhanced biodegradation involves identifying and manipulating genes responsible for the degradation pathways of specific micropollutants. This can be achieved through gene knockout, gene overexpression, and gene editing using CRISPR/Cas9. However, there are still challenges in achieving the efficient and stable expression of modified genes in fungi and ensuring the safety and environmental impact of genetically modified organisms. Therefore, research in this area is ongoing, with the intention to develop more effective and sustainable methods of fungal biodegradation. Using fungal consortia for biodegradation is another promising approach to micropollutant removal. This involves combining different types of fungi with complementary degradation pathways, allowing for a broader range of pollutants to be targeted. Studies have shown that using fungal consortia can enhance the efficiency and stability of biodegradation compared to using single strains alone. Fungus-based biofilters are becoming increasingly popular in wastewater treatment plants as a low-cost and sustainable micropollutant method [69,97].
Genetic modification, nanobiotechnological approaches, and fungal consortia are promising approaches to improving the efficiency and sustainability of fungal biodegradation [41,45]. However, challenges remain in achieving the stable expression of modified genes and ensuring the safety and environmental impact of genetically modified organisms.
Additionally, fungus-based biofilters are becoming increasingly popular in wastewater treatment plants as a low-cost and sustainable method for micropollutant removal. Nonetheless, the issue of how genetically modified fungi can become dominant strains in environments remains a challenge. For example, a study showed that a fungal consortium of five strains could efficiently degrade a mixture of pesticides and herbicides in contaminated soil [47]. Each strain had a specific degradation pathway, allowing for the breakdown of different pollutants. Using the fungal consortium resulted in higher degradation rates and increased stability than using each strain alone.
Overall, various novel methods are available for treating pharmaceutical waste, including chemical, physical, and biological approaches [96]. Recent studies have shown the effectiveness of using fungal consortia for degrading pesticide and herbicide contaminants in soil, which could have potential applications in pharmaceutical waste treatment [47]. Chemical methods involve oxidizing or reducing agents to break down the waste. Physical methods (Sinha, 2018) include filtration, evaporation, and distillation. Biological methods use microorganisms to degrade the waste [42][43][44]47]. However, these methods have limitations such as high cost and low efficiency. Recently, new devices have been developed for pharmaceutical waste treatment using advanced oxidation processes (AOPs) and nanotechnology. AOPs use reactive species, such as hydroxyl radicals, to break down the waste [12]. Nanotechnology involves the use of nanoparticles to enhance the efficiency of AOPs. Commercial devices such as cavitation and ultrasound devices were utilized and developed in various cases of pharmaceutical wastewater treatment [90]. These new devices have shown promising results with high degradation rates and low costs. However, further research is needed to optimize these devices for large-scale applications and ensure their safety for human health and the environment.

Environmental Regulations and Initiatives in the Removal of PPCP
The unintended accumulation of PPCPs in different aquatic and environmental niches (such as freshwater, sediments, and lifeforms) at amounts capable of deteriorating humanity and the ecosystem has come to our attention in recent years. PPCPs are often found in freshwater environments at relatively low concentrations. However, many of them and their intermediates are physiologically active and can potentially affect aquatic animals that are not their intended biological targets [116]. Due to the actual usage of PPCPs, rising applicability for human and veterinary medicine, and the ongoing leakage of these substances into the environment, this has become a severe issue. The European Union (EU) and the United States Environmental Protection Agency (USEPA) have created priority pollutant categories that highlight a wide range of compounds found in wastewater and the environment that may be detrimental. The Environment Agency (EA) of England and Wales developed a classification scheme for these compounds' estimated hazard ratios to identify compounds with a significantly high potential to orchestrate concern for the aquatic environment and other biological lifeforms. This classification scheme combines conventional risk assessment techniques with propensity, biomagnification, and toxicological effects (PBT) standards. According to this process, the top 10 compounds were thioridazine, clotrimazole, lofepramine, procyclidine, dextropropoxyphene, paracetamol, tramadol, mebeverine, tamoxifen, and aminophylline [117]. Pharmaceuticals and personal care products were also detected wastewater from municipal and the marine water bodies [35].
In a related expedition, an alternative selection of priority intoxicants was recognized utilizing the Oil Spill Prevention, Administration, and Response (OSPAR) sampling and categorization method for hazardous material, which included lofepramine. Perfluoroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs), and PCBs are examples of environmental contaminants with toxicities. These contaminants were primarily designed to optimize their physiological activity at low concentrations and to specifically target biochemical, enzymatic, or cell-signaling mechanisms [94].
Nevertheless, the cytotoxicity of PPCPs circulating in the environment has extended beyond the immediate effects fostered by therapeutic agents. Recent scientific expedi-tions and studies around the globe have shown that the orchestration of PPCP toxicity may vary depending upon the organism being exposed, the time duration of exposure, the concentration metric of the contaminant, and the route through which exposure occurs [77]. Moreover, the detrimental effects of acute exposure traces, especially at specific concentrations, are explanative enough for the physiological abnormalities observed in non-specific organisms than those recorded for an acutely higher concentration of dose exposure. The efficacious management and overall permissible circulation of PPCPs seem implausible without a global consensus furbishing innovative ideas to create and install a well-structured, sustainable Environmental Risk Assessment (ERA) protocol for these lethal contaminants. Pre-existing procedures and predicaments must be upgraded and evolved to acclimatize to the potential consequences of these toxic compounds in the environment [93].

Challenges and Future Directions
Fungi could now be considered a valuable tool for the remediation of contaminants from pharmaceutical and personal care origin. They make ideal biological agents to incorporate in wastewater treatment procedures due to their versatility in degrading a wide range of resistant compounds and ease of handling. However, fungus performance has usually been studied maximally in sterile batches. Since bacterial contamination can significantly impair fungal function, few experiments have been carried out in continuousflow reactors under non-sterile circumstances. More work is therefore needed to address this limitation. The added limitation of the fungal strain treatment process is washing out the mediators and the enzymes with treated effluent. This presents a particular challenge for enzymatic reactors that use enzymes rather than fungi.
Moreover, the remediation pathways of a few PPCPs, such as ibuprofen, carbamazepine, naproxen, and diclofenac, have been described in the literature. The metabolites/intermediate products are more hazardous than the parent chemical. Therefore, efforts should be made to characterize and illuminate the intermediate product formation during the treatment of PPCPs via fungus strains, which would further help to ascertain the degradation pathways/mechanisms of PPCPs. Many enzymatic conversion/full-conversion gaps in pathways are also unresolved, obstructing fungal use in the treatment process. Genetic manipulation/engineering could help offset the mismatch, representing a vital step towards improving bioremediation via fungal systems. In this regard, it is essential to develop efficient strategies for genetic manipulation that would be supported for all fungi.
Moreover, to develop a potent tool usable in wastewater treatment plants to prevent PPCPs from entering ecosystems, many investigations on the physiology and functional genomics in representatives of various groups still require a complete understanding of the mechanisms of the metabolism of PPCPs to ensure the highest efficiency for pharmaceutical and personal care products' removal. New genetic engineering expertise should be introduced to choose and amplify the best fungal strains to eliminate PPCPs. Essential metabolic genes are present in several bacteria and may be transferred to other organisms. Since these microorganisms can remove pharmaceuticals from wastewater and allow for their continuous use, considering legal restrictions and a practical market and cost rationale, genetically modified fungi will probably have a significant future in this field.