Bioremediation of Endocrine Disruptors (EDs): A Systematic Review of Fungal Application in ED Removal from Wastewater

Abstract

Endocrine disruptors (EDs), including natural estrogens, such as 17β-estradiol (E2) and synthetic chemicals (e.g., bisphenol A (BPA) and per- and polyfluoroalkyl substances (PFAS)), pose environmental and human health risks due to their ability to interfere with hormonal systems, even at trace concentrations and can lead to developmental, reproductive, and carcinogenic effects. These persistent compounds often escape removal in conventional wastewater treatment processes, leading to environmental contamination and human exposure. Given their widespread presence in wastewater and resistance to conventional treatments, the use of fungi offers a promising bioremediation strategy. This review explores the potential of fungal biodegradation, particularly using the white-rot fungus Trametes versicolor, in mitigating the estrogenic activity of EDs in wastewater. Laccase, an oxidative enzyme produced by white-rot fungus, shows high efficiency in degrading EDs, positioning fungal treatment as an eco-friendly alternative to conventional technologies. This systematic literature review was conducted using the Methodi Ordinatio, a multi-criteria decision-making methodology that allows for a structured selection of relevant studies and underscores the significant potential of fungal-based systems in addressing the global challenge of ED contamination in water environments.

1. Introduction

Endocrine disruptors (EDs) are emerging pollutants of increasing concern due to their potential interference with the endocrine systems of both humans and wildlife, even at low concentrations [1,2]. Natural and synthetic hormones, as well as pharmaceuticals (e.g., anti-inflammatory drugs), parabens, Ultraviolet (UV) filters, phthalates, bisphenol A (BPA), per- and polyfluoroalkyl substances (PFASs), alkylphenols, and organochlorinated pesticides are examples of the EDs commonly found in soil and water [2,3,4,5,6,7]. These compounds draw significant attention due to their persistence in the environment and potential to cause adverse health effects, including reproductive disorders and cancer [1]. Yet, there is still a lack of specific environmental legislation regulating the maximum allowable concentrations of many of the EDs in water and soil, despite their widespread presence and clear harm to human and animal life [3].

Wastewater treatment plants (WWTPs) often fail to completely remove these pollutants, resulting in their discharge into aquatic ecosystems [5]. This highlights the urgent need for more effective and sustainable treatment methods to mitigate the environmental impact of EDs. Additionally, it is essential to address not only the EDs themselves but also their degradation byproducts, which may still exhibit estrogenic activity.

Physical–chemical treatments, including reverse osmosis and advanced oxidation processes (such as ozonation, heterogeneous photocatalysis, and electrochemical techniques) have demonstrated varying degrees of effectiveness at removing EDs. However, these methods often present significant drawbacks, such as high energy requirements, expensive installation costs, and continuous operational and maintenance expenses, along with the use of potentially harmful chemicals [2,5].

In recent years, bioremediation has emerged as a promising alternative to conventional treatment technologies [2]. This approach leverages the metabolic abilities of microorganisms to break down or transform pollutants into less harmful forms, often with fewer adverse effects compared to chemical treatments [2]. Fungi, particularly white-rot fungi, have demonstrated significant potential in the removal of EDs due to their ability to produce extracellular oxidative enzymes, such as laccases and peroxidases, which can effectively degrade a wide range of contaminants, including complex organic pollutants with estrogenic activity [8,9,10,11,12,13,14,15,16]. In addition to fungi, certain bacterial strains, such as Pseudomonas, Sphingomonas, Arthrobacter, Bacillus, Burkholderia, Pseudomonas, Kocuria, Archromobacter, Sphingomonas, and Chromohalobacter, have also shown the ability to degrade EDs through different metabolic pathways, including dehydrogenation, dehydrochlorination, and hydroxylation, leading to complete degradation or mineralization, even under axenic and anoxic conditions [17,18,19]. While fungi, especially Trametes versicolor, excel in breaking down complex organic EDs due to their versatile oxidative enzymes, some bacterial species are particularly efficient at degrading simpler compounds and metabolizing specific EDs, such as BPA [15,16]. Comparatively, fungi often display broader substrate specificity and are particularly effective at degrading complex organic molecules with estrogenic activity, due to their enzymatic repertoire [10,19,20,21,22]. Unlike fungi, bacterial systems may struggle with the degradation of more complex EDs, and the efficiency of biodegradation is often highly strain dependent [14,15,16,17,18,19]. Additionally, bacterial strains often have a narrower spectrum of activity and may not be as effective at removing highly persistent ED. Fungi produce extracellular enzymes, which are secreted into the surrounding environment, enabling them to degrade a broad spectrum of pollutants, including complex and recalcitrant EDs, without needing to take up the pollutants into their cells [11,12,19,20,21,22]. This extracellular enzymatic activity allows fungi to break down large, insoluble molecules and persistent organic contaminants, making them highly efficient in bioremediation processes where other microorganisms might struggle [10,18,22]. In contrast, bacteria typically rely on intracellular enzymes for degradation, meaning they need to take the pollutants up into their cells for transformation. While some bacteria are also capable of producing extracellular enzymes, their enzymatic range is generally more restricted when compared to fungi. Additionally, bacterial systems often struggle to degrade large or complex organic molecules, such as those found in many EDs, which require more specialized enzymatic capabilities [23]. Archaea are recognized for their contribution to bioremediation in various applications where bacteria are also involved and play an important role in the degradation of specific pollutants that may challenge bacterial metabolism [10,23].

Another key advantage of fungi is their greater resistance to environmental toxicity, which is particularly important when dealing with EDs and other harmful pollutants [10,23]. Fungi are generally more resilient to toxic compounds and harsh environmental conditions, such as extremely acidic pH levels, high temperatures, or the presence of toxic byproducts. This resilience is due to their robust cell walls, which provide a protective barrier against environmental stressors, and their ability to adapt to adverse conditions. In fact, many fungi can thrive in contaminated environments that may be inhibitory to bacterial growth, allowing them to survive and degrade pollutants in environments with high levels of toxicity [19,22,23]. On the other hand, while certain bacterial strains, such as Pseudomonas and Sphingomonas, exhibit a remarkable tolerance for specific toxic substances, bacteria are often more sensitive to environmental stressors. High concentrations of pollutants or extreme environmental conditions may inhibit bacterial growth and activity, reducing their effectiveness in bioremediation processes. This is particularly true when dealing with EDs that are persistent and toxic to microbial life. Both microorganisms have significant potential in the bioremediation of EDs, but their complementary roles can be maximized by integrating fungal and bacterial systems.

Given EDs widespread presence in wastewater and their resistance to standard treatment methods, the use of fungi presents a promising bioremediation strategy, particularly due to their extracellular enzymatic activity, and they have demonstrated potential in the removal of EDs. However, there is a lack of comprehensive reviews specifically addressing the effectiveness of fungi in ED removal and the factors influencing their bioremediation capabilities. This gap highlights the need for further research and systematic analyses to fully understand and optimize fungal-based treatments for wastewater remediation and guiding future research in this critical area of wastewater treatment. This manuscript presents a systematic review of the role of fungi in removing EDs from contaminated wastewater, using the Methodi Ordinatio approach to analyze key studies on fungal bioremediation. It aims to provide a comprehensive overview of current research, highlighting the mechanisms by which fungi, particularly through the action of oxidative enzymes such as laccases, contribute to estrogenic activity removal and address the challenges of applying them in wastewater treatment systems. By synthesizing the existing research, this article advances the understanding of sustainable and effective ED treatment through fungal bioremediation.

2. Methodology

For this systematic literature review, a bibliographic search was conducted in three databases: Science Direct, Scopus, and Web of Science (Figure 1). Combinations of descriptors and Boolean operators were used to identify publications relevant to this research. The search employed combinations of the terms “microorganisms” AND “biodegradation” AND “endocrine disruptors” AND “wastewater”, with filters applied to include only scientific articles and exclude review articles. A total of 145 results were obtained: 127 from Science Direct, 10 from Scopus, and 8 from Web of Science. Following the database search, the articles were downloaded in BibTex format and imported into the Mendeley Reference Manager 2100.0 for reading and screening. The screening process was conducted within the Mendeley software, version 1.19.8, by removing duplicate articles (n = 17) and reviewing the abstracts of the remaining 128 articles. Based on the review, articles were selected or excluded based on their relevance to the research objectives (n = 21), with manuscript unavailability (n = 3) resulting in 104 retained for further analysis. These remaining articles were then classified using the Methodi Ordinatio approach to systematic literature reviews, which helps assess their scientific relevance for the systematic literature review. The method for excluding articles based on relevance employs the InOrdinatio equation, which considers three key factors in a scientific article: the impact factor (IF), the year of search (YR = 2024), the year of publication (YP), and the number of citations (nCis) [23].

InOrdinatio = (IF/1000) + (α × (10 − (YR − YP))) + (nCi)

IF was obtained by consulting the Journal Citation Reports or Researchify database. For this study, α was set to 10 to prioritize newer publications. Ci was obtained from the Google Scholar platform.

The application of the InOrdinatio equation resulted in 29 articles with scores ranging from 376,008 to 52,004, published between 2009 and 2023 (Table S1, Supplementary Information). The scores were used to prioritize recent, high-impact studies, ensuring that the selected articles align with the objectives of this review. It is important to note that the nCis played a more significant role in determining the final score, reflecting each article’s scientific impact within the field. These articles were thoroughly analyzed to extract key information related to the topic under study. Additionally, other articles providing fundamental and more generalized information were cited to support the broader context and enhance the understanding of key concepts.

To ensure that all selected papers met the established criteria and aligned with the review’s objectives, VOSviewer software (version 1.6.20) was used to conduct a term frequency analysis based on the titles and abstracts of the selected studies.

3. Results and Discussion

3.1. Overview of Key Findings

The bibliometric analysis conducted using VOSviewer software provided a comprehensive overview of the key themes within the literature on fungal-mediated degradation of various EDs. Figure 2 shows a network which consists of nodes, each representing a specific concept, and edges that link related concepts. The keyword network is typically divided into clusters, each containing a set of terms related to various aspects of residential satisfaction, and these clusters are usually represented by different colors to aid in visual differentiation. However, in this study, the clusters are not clearly or distinctly separated by color, indicating potential overlap or ambiguity between them.

The size of the nodes corresponds to their frequency in the literature, reflecting their relative importance within the field. As illustrated in Figure 2, the term “laccase” emerged as a central focus, i.e., it presents with a larger node in the lexical network which indicates a higher frequency of occurrence of the keyword, reflecting its significant role in the bioremediation of EDs, particularly in wastewater treatments. The strong association between the term “lac” (laccase) and “estrogenic activity”, as well as the terms “laccase”, “EE2” (17α-ethinylestradiol), and “BPA”, underscores the enzyme’s central role in addressing this class of pollutants. Additionally, the frequent occurrence of terms such as “BPA” and “EE2” highlights their prevalence and the concern of the research community about these compounds, and further emphasizes their status as high-priority pollutants due to their potent endocrine-disrupting effects. It is also linked to several key concepts, including “removal”, and “biotransformation”, which highlights the enzyme’s critical role in the elimination of pollutants from the environment. The concept of “biotransformation” reflects the enzyme’s ability to convert hazardous pollutants into less toxic substances, enhancing its relevance in environmental remediation strategies. Additionally, laccase is connected to “wastewater”, emphasizing its application in the treatment of water contaminated by various pollutants. This analysis emphasizes the key role of laccase and the focus on specific EDs in the current research on fungal bioremediation, which led to the inclusion of a dedicated section in this article to further exploring the role of these enzymes in the degradation of EDs.

On the other hand, the “toxicity” node, connected to “humans” highlights the importance of assessing the potential harmful effects of these compounds, emphasizing the need to identify and develop efficient treatment processes. Moreover, the closed terms “detoxification” and “wastewater” suggest a focus on the removal of EDs from wastewater through bioremediation processes. This connection highlights the potential of various bioremediation strategies, particularly those involving fungi, and laccase, to detoxify the harmful compounds present in contaminated water. Lastly, the concept of “environmental risk”, located near the “aquatic ecosystem” node, emphasizes the need for a thorough evaluation of the ecological impact of EDs on these systems.

3.2. Fungal Applications for the Removal of Endocrine Disruptors

Fungi, particularly white-rot fungi, have garnered considerable interest in recent years for their potential in wastewater treatment [8,13,16,19,20,21,22,23]. The key advantages of fungi in this context include their broad substrate range, which enables them to metabolize a wide variety of organic pollutants such as phenols, phthalates, hormones, and pharmaceutical residues [10,19,20,21,22,23]. These organisms produce extracellular oxidative enzymes, including laccases, manganese peroxidases, and lignin peroxidases, which are secreted into their surrounding environment. This allows them to effectively degrade complex compounds outside their cells, including those that are resistant to conventional biological treatments [11,13,19,20,21,22,23,24]. Fungi are also highly adaptable to harsh environments, thriving in diverse and extreme conditions such as low pH and high salinity environments, making them particularly suitable for treating various types of wastewaters, including industrial effluents [10,19].

Fungi can be applied directly in bioreactors or constructed wetlands for in situ treatment, or their enzymes can be extracted and used in enzymatic treatment systems [19,20,21,22,23,24,25,26]. However, one limitation of using fungi for wastewater treatment is that fungal growth tends to be slower compared to bacteria, particularly under suboptimal conditions. Fungi generally require longer incubation times for effective pollutant degradation, which may not always be suitable for high-throughput wastewater treatment processes [27]. Furthermore, fungi may require specific environmental conditions, such as an appropriate temperature, pH level, and moisture content, to grow efficiently. In some cases, fungal systems may also encounter challenges related to nutrient availability, and their activity can be inhibited by certain pollutants or toxic substances present in the wastewater.

Numerous fungi have the capability to degrade EDs, with Trametes versicolor standing out as the most thoroughly researched, particularly due to its promising applications in large-scale wastewater treatment systems (Table 1). Nearly 40% of the reviewed studies employed Trametes versicolor or its commercial enzymes, emphasizing its effectiveness at reducing estrogenic activity in contaminated waters. For instance, studies by Shreve et al. [28] and Lloret et al. [29] reported near-total reductions in estrogenic activity through fungal biodegradation. These studies, which involved a variety of estrogen-mimicking pollutants such as atrazine, BPA, carbamazepine, and triclosan, all of which are compounds known for their persistence in conventional wastewater treatment plants and harmful effects on aquatic life, demonstrate the broad-spectrum efficacy of fungal enzymes in mitigating multiple contaminants simultaneously. Karp et al. [30] found that a laccase from Trametes versicolor could degrade over 80% of atrazine in contaminated water within 72 h, indicating that fungal bioremediation could be a promising solution for agricultural runoff treatment. The enzymatic process primarily involves breaking down the complex aromatic structures of pesticides into simpler, less toxic byproducts, which can then be further degraded by microbial consortia. In addition to addressing estrogenic compounds, Trametes versicolor, along with its enzymes, has demonstrated a broad-spectrum efficacy against a wide variety of the EDs commonly found in wastewater. In the study by Pezzella et al. [16], the potential of Trametes versicolor was tested alongside Pleurotus ostreatus and Phanerochaete chrysosporium for the ability to degrade five EDs including phenols, parabens, and phthalates. Trametes versicolor was selected for its superior efficiency in removing these contaminants. Using a fed-batch bioreactor approach, Trametes versicolor successfully removed all EDs across five consecutive degradation cycles without the need for external nutrient supplementation. Trametes versicolor exhibited adaptability to stressful conditions, such as the absence of nutrients, while maintaining high degradation efficiency. This suggests its capability to metabolize EDs or stored internal compounds for metabolism. Notably, the efficiency observed when the same fungal biomass was reused for multiple cycles demonstrated that the process is both sustainable and cost-effective. Authors concluded that Trametes versicolor is a promising candidate for large-scale wastewater treatment, as its biomass can be recycled for continuous use, it operates efficiently under non-sterile conditions, and it does not require additional nutrients. These advantages make Trametes versicolor a viable option for integration into conventional wastewater treatment processes. The optimal pH range for Trametes versicolor is reported to be between 4.5 and 6.5, with slightly acidic to neutral conditions being ideal for growth and enzyme production. Its growth thrives at temperatures between 25 °C and 30 °C, although it can tolerate a broader range, albeit with slower growth at extreme temperatures [22,30,31,32]. Trametes versicolor grows best under static or low-agitation conditions, but when excessive agitation conditions persist it can disrupt the formation of its mycelial network. However, mild agitation can aid oxygen transfer in liquid cultures [22,30,31]. Trametes versicolor produces hyphae, which are crucial for breaking down organic matter and interacting with pollutants. The extensive hyphal network enables the secretion of extracellular enzymes to degrade complex molecules, including EDs. Additionally, the hyphae enhance the fungus’s ability to adhere to and endure harsh conditions, and adapt to changes in temperature, pH, and nutrient availability [22,33]. Their hyphal network, along with their biomass and prolonged life cycle, also contributes to fungi being more effective than bacteria in the bioremediation of polluted environments [22,33]. Additionally, the laccase enzymes produced by Trametes versicolor have been noted for having the highest redox potential among laccases, recorded at 785 mV compared to the standard hydrogen electrode [34]. This elevated redox potential is significant because it is associated with increased laccase activity, making this enzyme particularly appealing for diverse biotechnological uses, including the degradation of EDs.

Other fungi and their enzymes have also shown potential for the degradation of ED compounds. For instance, the study by Benitez et al. [35] compared the efficiency of single cultures and a fungal consortium for the treatment of polychlorinated biphenyls (PCBs). Among them, Pleurotus pulmonarius LBM 105, in single culture, accomplished the highest PCBs removal (95.4%) and toxicity reduction, outperforming both Trametes sanguinea LBM 023 and a binary consortium, which only degraded about 55% of PCBs. These results indicate that P. pulmonarius LBM 105 is more effective in bioremediating PCBs-contaminated transformer oil, with notable changes in ligninolytic enzyme secretion observed in the co-culture. Another example is the study by Acevedo et al. [36], which investigated the degradation of polycyclic aromatic hydrocarbons (PAHs) by the white-rot fungus Anthracophyllum discolor in both a liquid medium and contaminated soil. The fungus effectively degraded the PAHs in autoclaved soil, with the highest removal percentage (75%) observed for benzo(a)pyrene, which was associated with the production of manganese peroxidase. However, bioaugmentation in non-autoclaved soil did not enhance PAH removal, highlighting the need for further research to optimize fungal bioaugmentation conditions in non-sterile environments. Marinho et al. [37] evaluated the ability of Aspergillus niger AN 400 to tolerate atrazine concentrations up to 30 mg/L and its capacity to biodegrade atrazine in 3 L batch reactors with a dispersed fungal biomass. The effect of supplementing glucose as a co-substrate in atrazine biodegradation was also examined. Aspergillus niger achieved an atrazine degradation of (40 ± 3)% without a co-substrate, but the addition of glucose at 3 g/L doubled the degradation rate and improved removal by 30%. However, at higher glucose levels (4–5 g/L), degradation rates declined, likely due to substrate competition and glucose-induced gene repression. The laccase-producing white-rot fungus Trametes hirsuta La-7 demonstrated high efficiency in biologically transforming BPA under both in vivo and in vitro conditions, achieving over 80% BPA removal within 6 h using an extracellular crude laccase solution and homogenized mycelium [38]. The biotransformation involved six metabolic mechanisms, including polymerization, hydroxylation, and dehydrogenation, and was not affected by the presence of BPA in a radish seed germination test. This strain also efficiently metabolized other EDs, such as estrone and 17β-estradiol (E2), suggesting its potential for environmental bioremediation of this class of compounds. The biological treatment achieved 46% detoxification as measured by the Allium test. The white-rot fungus Phanerochaete chrysosporium has also shown the ability to degrade various pollutants, including chlorinated organics, pharmaceuticals, and endocrine disruptors [39]. For instance, it successfully transformed 6:2 fluorotelomer alcohol (6:2 FTOH), a short-chain PFAS commonly used in manufacturing, into 5:3 fluorotelomer carboxylic acid (5:3 FTCA), which is more easily transformed in the environment compared to the environmentally persistent perfluorocarboxylic acids (PFCA). Cajthaml et al. [40] investigated the ability of Trametes versicolor and seven other ligninolytic fungal strains, namely Irpex lacteus, Bjerkandera adusta, Phanerochaete chrysosporium, Phanerochaete magnoliae, Pleurotus ostreatus, Pycnoporus cinnabarinus, and Dichomitus squalens, to biodegrade common EDs and suppress estrogenic activity. The targeted EDs included 4-n-nonylphenol, technical 4-nonylphenol, bisphenol A, 17α-ethinylestradiol, and triclosan. Over 14 days of cultivation, most of the tested fungi exhibited the capacity to degrade these compounds, with I. lacteus and P. ostreatus being the most efficient, achieving over 90% and 80% degradation in just 7 days. Both fungi successfully degraded 4-nonylphenol, bisphenol A, and 17α-ethinylestradiol below detection limits within the first three days. However, despite the degradation, the study also revealed residual estrogenic activity in some cultures, particularly for I. lacteus, P. ostreatus, and P. chrysosporium, in which 28–85% of the initial estrogenic activity of 17α-ethinylestradiol remained. Interestingly, Bjerkandera adusta showed a temporary increase in estrogenic activity during the degradation of 4-nonylphenol, suggesting the formation of estrogen-active intermediates which were subsequently degraded. In addition to evaluating ED degradation, the researchers assessed the effects of EDs on the ligninolytic enzyme activities of the fungi. The results showed variable enzyme responses, with no clear correlation between enzyme activity and the degradation extents. While some strains, i.e., T. versicolor, P. ostreatus, and P. cinnabarinus, exhibited increased laccase activity in the presence of nonylphenols, in most cases, enzyme activities were suppressed by 4-n-nonylphenol and triclosan. In the study by Rajendran et al. [41], the yeast Candida rugopelliculosa RRKY5 was able to degrade the common endocrine-disrupting pollutant 4-(1,1,3,3-tetramethylbutane)-phenol under aerobic conditions, with a maximum degradation rate constant of 0.107 d⁻¹, utilizing both alkyl side chain and aromatic ring cleavage pathways.

A recent study by Merino et al. [42] investigated how different nutrients affect the enzymes involved in the biotransformation of 6:2 FTOH and the production of x:3 FTCA versus PFCA. The study examined the impact of factors such as lignocellulose presence, glucose levels, varying sulphate concentrations, and different nitrogen sources. The results indicated that conditions favoring cellulose breakdown (i.e., using yeast extract), as well as limited glucose, led to the most efficient transformation of 6:2 FTOH into 5:3 FTCA, rather than PFCA. The presence of metal chelator nitrilotriacetic acid or low sulfate conditions were detrimental to fungal growth and led to increased PFCA production. The research also suggested that cytochrome P450 enzymes play a role in the early stages of the degradation process, while peroxidases are involved in the subsequent reactions.

All these studies feature the fact that fungal bioremediation provides a versatile solution capable of tackling multiple contaminants simultaneously, offering a significant advantage over conventional treatment methods, which are often pollutant-specific. Furthermore, fungi, particularly those with extracellular ligninolytic enzymes, can degrade the pollutants that are resistant to bacterial treatments [22]. Notably, fungal biodegradation is effective even for remediation at low pollutant concentrations, which is crucial for treating EDs that can cause harmful effects even at trace levels. In wastewater treatment, fungi can be applied in different configurations, such as biofilters, bioreactors, or in combination with other treatment technologies [10]. Additionally, fungal systems are adaptable to diverse environmental conditions, such as varying pH levels and temperatures, making them suitable for treating a wide range of wastewaters [10,43]. This adaptability positions fungi as a promising tool for industrial wastewater treatment, where pollutants are often more diverse and present at higher concentrations compared to municipal wastewater [44].

3.3. Laccase-Mediated Degradation of Endocrine Disruptors

Laccases, as multicopper oxidases, are among the most studied fungal enzymes for the degradation of EDs [45,46,47,48,49,50]. They are capable of catalyzing oxidation reactions involving a broad range of organic compounds, including complex and persistent pollutants such as hormones, pharmaceuticals, and pesticides [48]. By transforming these compounds into less toxic or more biodegradable forms, laccases play a crucial role in ED degradation. These enzymes function under mild conditions, such as an ambient temperature and neutral pH, and require oxygen as a co-factor [10,45,48,49,50]. This makes them an environmentally friendly alternative to other enzymes like peroxidases, which require toxic chemicals like hydrogen peroxide (H₂O₂) for activation. Furthermore, laccase-producing fungi are easy to cultivate, enhancing their potential for large-scale application in wastewater treatment technologies [10,45,48,49,50]. Laccases generally oxidize a broad spectrum of substrates converting them into unstable products and free radicals. These unstable products and highly reactive radical species attack the complex structures of ED molecules, leading to a cleavage of aromatic rings, demethylation, and the oxidation of side chains [45]. These transformations often result in the formation of less harmful compounds that are easier to mineralize or are less bioactive, thereby reducing their endocrine-disrupting potential [45].

Studies have demonstrated the high efficacy of laccases in degrading a broad spectrum of EDs. For instance, Sun et al. [46] reported a 79% reduction in estrogenic activity following the treatment of 17β-estradiol (E2) and EE2 with laccase in a fluidized bed reactor. Similarly, Lzaod and Dutta [8] achieved 68% biodegradation of 4,4′-dihydroxybiphenyl in a separate reactor system. These findings underscore the strong potential of laccases as efficient “green biocatalysts”, characterized by their favorable redox potential, thermodynamic efficiency, and operational versatility. The studies conducted by Lloret et al. [11], Lloret et al. [29], Lloret et al. [51], and Chappell et al. [52] emphasized the utility of laccase enzymes, albeit derived from different fungal sources, in reducing estrogenic activity and removing pharmaceuticals from wastewater. The broad applicability of laccases, whether derived from Trametes versicolor or Lentinula edodes, underlines their versatility as biocatalysts. Despite these advantages, Lzaod and Dutta [8] found that laccase immobilization reduced the catalytic efficiency of the enzyme, affecting its kinetic performance. These contrasting findings suggest a trade-off between the stability benefits of immobilization and the potential reduction in enzymatic activity, which merits further optimization in future applications. Laccase immobilization has been explored as a strategy to enhance operational stability and reusability. Immobilized enzymes can be incorporated into continuous flow systems, which offer enhanced efficiency for large-scale applications. This further supports their potential for use in wastewater treatment and other industrial processes, where sustained enzyme activity and scalability are critical for an optimal performance [46,47,52,53]. Sun et al. [46] found that immobilization improved enzyme stability under varying pH and temperature conditions. Zofair et al. [53] advanced the field by applying laccases immobilized on silver nanoparticles (AgNP-PLL) to degrade β-estradiol, achieving a remarkable 95.3% reduction in the hormone’s estrogenic activity.

While most of the studies on ED enzymatic degradation have been conducted with model solutions, it is crucial to evaluate their effectiveness with real wastewater. Becker et al. [54] tackled this issue by investigating the enzymatic degradation of hormones and EDs in both artificial mixtures and real wastewater using laccases from Trametes versicolor and Myceliophthora thermophila. The results showed that even at very low enzyme concentrations (2.8 U/L for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic 166 acid)), laccases were effective at breaking down hormones and EDs. In artificial mixtures, the primary removal mechanism was adsorption onto the immobilization supports. For binary mixtures, immobilized laccase from T. versicolor was the most effective, achieving 83% removal within 6 h, while the enzyme from M. thermophila achieved only 7%. Yet, after 24 h, adsorption became the dominant mechanism, leading to 99% removal of the contaminants. A similar pattern was observed in the assays with wastewater. Although there was reduced adsorption, due to less carrier material, the immobilized laccase maintained its high efficiency, removing 82% of estrogenic activity within 24 h, and 99% of androgenic activity within just 6 h. These findings underscore the promising potential of fungal enzyme-based treatments for real wastewater applications, which will be discussed further below.

The kinetics and degradation pathways of EDs used by laccase have been extensively studied, revealing that the enzyme’s efficiency can vary depending on the chemical structure of the target compound. Lloret et al. [29] demonstrated the rapid biodegradation of estrogens, achieving a complete removal within 30 min. However, pharmaceuticals such as diclofenac and naproxen were degraded more slowly, likely due to their more complex molecular structures, which make them more resistant to breakdown. These differences highlight the importance of substrate specificity in influencing the reaction kinetics of laccase-mediated processes. Natural mediators like syringaldehyde have been found to significantly enhance laccase’s catalytic activity, improving the degradation efficiency of these more resistant compounds. As an example, in the work of Lloret et al. [11] the addition of syringaldehyde significantly improved laccase’s catalytic activity, boosting the removal efficiency of diclofenac and naproxen to 70–100% within 6 h. This mediator-assisted approach promotes the formation of the reactive radicals that aid in breaking down complex pollutants, pointing out the potential of laccase-mediated treatments in environmental applications.

The degradation mechanisms generally involve the oxidation of the pollutant molecule by laccase, followed by subsequent reactions that form various byproducts [42,43,44,45,46,47,48,49,50,51]. These byproducts may undergo further degradation either by the laccase itself, and/or through microbial processes, and/or via side chemical reactions [54]. For example, in the case of estrogens, laccase oxidizes the phenolic hydroxyl group, causing a cleavage of the aromatic ring and the formation of metabolites that are less estrogenic or non-estrogenic. The specific degradation pathways can differ based on the pollutant [54]. For diclofenac, oxidative decarboxylation and hydroxylation are the main pathways driven by laccase. Naproxen, on the other hand, undergoes demethylation and oxidation. These pathways lead to smaller, more biodegradable fragments that are less toxic and more easily mineralized by the microbial communities in wastewater systems [54].

A key concern highlighted in the literature is the economic feasibility of using high concentrations of commercial laccase enzymes for large-scale applications. While Lloret et al. [51] achieved a near-complete estrogenic activity removal in membrane reactors using enzyme concentrations of up to 2000 U/L, concerns arose regarding the high costs associated with commercial enzyme use. Chappell et al. [52] addressed this issue by cultivating Lentinula edodes for in situ laccase production, an enzyme capable of achieving 51% removal of EE2 within just one day. This approach highlights the potential for more cost-effective solutions through fungal cultivation, thereby reducing the reliance on expensive commercial enzymes. Other strategies, such as enzyme immobilization, have been explored to enhance the efficiency and reusability of laccases, further improving their economic viability. Immobilizing laccase on various supports (e.g., activated carbon or silica gel) facilitates its repeated use, thereby reducing the need for continuous enzyme replenishment. For example, Cabana et al. [47] investigated the immobilization of laccase from Coriolopsis polyzona on a diatomaceous earth support (Celite R-633) and used it in a packed bed reactor to treat BPA, nonylphenol, and triclosan at a concentration of 5 mg/L. Over 99% removal was achieved in less than 200 min, with the biocatalyst maintaining consistent performance across five treatment cycles. This demonstrates the strong potential for ED degradation in continuous systems that can be integrated into existing wastewater infrastructure, thereby improving the overall efficiency of contaminant removal. Employing renewable materials for enzyme immobilization not only reduces waste disposal but also lowers the cost of enzyme immobilization, contributing to the development of a circular economy [55,56]. Furthermore, the development of genetically modified fungi with enhanced laccase production capabilities could help reduce the costs associated with enzyme extraction [56]. As such, fungal-based enzymatic treatments have the potential to become a more economically viable option for large-scale wastewater treatment, provided that these challenges are addressed through advances in enzyme production, immobilization techniques, and bioprocess optimization.

3.4. Applications of Fungi and Their Enzymes in Real Wastewater Treatment

Despite the promising potential of fungi and their enzymes, and the significant progress being made, several challenges remain in scaling up these processes for real wastewater treatment. One key challenge is maintaining operational stability, as consistent enzyme activity and fungal growth in large-scale bioreactors can be hindered by the presence of the inhibitory substances present in real wastewater, such as heavy metals and high organic loads [54]. An additional critical concern is the potential toxicity of degradation byproducts. While fungi are effective at degrading EDs, the intermediates formed during the ED biodegradation process may still pose toxicological risks. Therefore, it is crucial to monitor and evaluate the toxicity of these byproducts to ensure the safety of treated wastewater. For instance, Cajthaml et al. [40] demonstrated that although ligninolytic fungi can effectively degrade EDs, and reduce estrogenic activity, the formation of potentially harmful degradation intermediates warrants further investigation. These findings stress the need for additional research to identify degradation products, gain a deeper understanding of the mechanisms involved in ED biodegradation, and assess the associated toxicological risks.

Another concern for the application of fungal bioremediation in real wastewaters is that some EDs are resistant to biodegradation, requiring specific fungal strains or optimized conditions to achieve an effective transformation, which can limit the general application of fungi across different contaminants. In the study by Beck et al. [57], while laccase was effective at oxidizing several estrogenic compounds, bisphenol S (BPS) was resistant to degradation, indicating that not all EDs are equally susceptible to fungal enzyme treatment. Similarly, Liu et al. [58] observed a reduction in the efficiency of β-estradiol removal in the presence of humic acid precursors, highlighting the need for a further exploration of the environmental factors that may influence enzymatic efficiency. In contrast, studies with a straw substrate containing the fungal biomass of the white-rot fungus Pleurotus ostreatus HK 35 demonstrated its high efficiency in degrading BPA and estradiol, with degradation extents exceeding 90% within 12 days [59].

Pleurotus ostreatus HK 35 was effective in both laboratory and real-world wastewater treatment systems, including a pilot-scale trickle-bed reactor, where it removed over 76% of EDs and suppressed estrogenic activity. The study highlights the potential for scaling up the proposed bioremediation process using a spent mushroom substrate, emphasizing the need for further optimization and testing under real environmental conditions.

A batch fluidized bed bioreactor employing Trametes versicolor pellets was operated to treat hospital wastewater and remove pharmaceutical active compounds (PhACs) and EDs [60], both under sterile and non-sterile conditions. In this system, Trametes versicolor was able to partially or completely remove 46 out of the 51 detected PhACs and EDs, achieving total elimination extents of 83.2% under sterile conditions, and 53.3% under non-sterile conditions. Diclofenac, a notoriously persistent compound in conventional wastewater treatment plants, was completely removed during this process. Additionally, toxicity reduction was observed over time, further confirming the effectiveness of the fungal treatment. These promising results suggest that fungal bioreactors could serve as a pre-treatment step for hospital wastewater, preventing the dilution of highly contaminated hospital effluents with urban wastewater, and improving overall contaminant removal before conventional treatment.

Scaling up fungal bioremediation also requires addressing the issues related to enzyme production, cost-effectiveness, and the long-term stability of fungal systems [61,62]. While the cost of producing fungal enzymes on a large-scale, particularly in purified forms, remains high, the ongoing research is focusing on optimizing the production methods and exploring cheaper substrates, like agricultural waste, for fungal cultivation [63]. Also, advances in enzyme immobilization, the genetic engineering of fungi for enhanced enzyme production, and hybrid systems combining fungal treatment with other technologies could overcome these challenges [64,65]. Moreover, bioreactor design and operating conditions, including mixing, hydraulic retention times, and biomass concentrations, are crucial factors influencing the overall treatment performance [19,66].

One of the emerging trends in the application of fungi in wastewater treatment is the integration of fungal-based systems with conventional or advanced treatment technologies such as membrane filtration, activated sludge, adsorption, or advanced oxidation, to overcome individual limitations and improve overall wastewater treatment efficiency [19]. These hybrid systems leverage the enzymatic degradation capabilities of fungi while combining them with other processes. For example, hybrid membrane bioreactors combined with fungal enzymes have been proposed to enhance the removal of micropollutants in wastewater [67].

Table 1. Fungal applications for the removal of estrogenic activity and endocrine disruptors in wastewater treatment.

Fungus	EDs	Conditions	Main Results	Ref.
Trametes versicolor, Pleurotus ostreatus, Phanerochaete chrysosporium	PhOH, Parabens, Phthalates	Fed-batch and starvation strategies reduced fresh biomass input and external nutrients. The fungus operated in two bioreactors over one week with five consecutive degradation cycles of EDs.	Best results with T. versicolor It efficiently removed all EDs without additional nutrients, showing potential for repeated cycles in bioreactors. Biotransformation was the primary removal mechanism, with minimal biosorption.	[16]
Trametes versicolor NRRL 66313	E2, single A mixture of EDs: E1, E2, EE2, BPA, ATZ, CBZ, DEET, OBZ, TCS	Fungus was grown in glucose-amended, sterile wastewater (5 g/L). Removals performed in aerated Erlenmeyer incubated for 8 days at room temperature (25 ± 2 °C) and spiked with 5 mg/L of E2 or the mixture of EDCs (350 μg/L each). Abiotic and heat-killed fungus controls were also tested.	T. versicolor reduced E2 from 5 mg/L to below detection levels within 5 h, with E1 as a metabolite, which was subsequently removed. For the mixture of EDs, 62–100% removal was achieved within 3.5 h, and estrogenic activity reduced by 77% (compared to 4–8% in controls). After 12 h, estrogenic activity reduction exceeded 98% (vs. 24–42% for controls).	[28]
Trametes versicolor	ATZ, BPA, CBZ, TCS	Commercial enzymes, biodegradation of estrogenic pollutants in wastewater.	Near-total reduction in estrogenic activity. >80% of atrazine in contaminated water within 72 h by laccase. High efficiency across 5 degradation cycles without external nutrients. Laccase. The process breaks down complex aromatic pesticide structures into simpler, less toxic byproducts, which were further degraded by microbial consortia.	[30]
Pleurotus pulmonarius LBM 105 Trametes sanguinea LBM 023	PCBs	Single culture vs. consortium in bioremediation of PCB-contaminated transformer oil.	Pleurotus pulmonarius LBM 105 showed the highest PCB degradation 95.4% PCB removal, outperforming Trametes sanguinea LBM 023 and fungal consortium.	[35]
Anthracophyllum discolor	PAHs B[a]P	Biodegradation in liquid medium and autoclaved contaminated soil.	75% PAH removal in soil. Manganese peroxidase production linked to degradation. Lower efficiency in non-autoclaved soils.	[36]
Aspergillus niger AN 400	ATZ	Batch reactors with dispersed fungal biomass, glucose as co-substrate.	40% ATZ removal without co-substrate, doubled efficiency with glucose addition at 3 g/L. Higher glucose levels reduced degradation due to competition.	[37]
Trametes hirsuta La-7	BPA, E1, E2	In vivo and in vitro degradation using extracellular laccase and mycelium.	>80% BPA removal within 6 h. Metabolized EDs through six mechanisms, unaffected by BPA presence in plant test.	[38]
Phanerochaete chrysosporium	6:2 FTOH	Transformation of PFAS in bioreactors with Kirk medium with and without glucose, supplemented with organic nutrients like lignocellulosic powder.	Phanerochaete chrysosporium biotransformed 6:2 FTOH into perfluorocarboxylic acids (PFCAs), polyfluorocarboxylic acids, and intermediates within 28 days. Main product was 5:3 FTCA, making up 32–43% of the initial 6:2 FTOH, with minor amounts of PFCAs (5.9%). Efficient EDs degradation, but with some residual estrogenic activity.	[39]
Trametes versicolor, Irpex lacteus, Bjerkandera adusta, Phanerochaete chrysosporium, Phanerochaete magnoliae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Dichomitus squalens	NP, n-NP, BPA, EE2, TCS	Biodegradation in static conditions at 28 °C, malt extract–glucose medium.	I. lacteus and P. ostreatus were the most efficient degraders, >90% and >80% in 7 days, respectively. Both fungi degraded pollutants below detection limit within the first 3 days. Estrogenic activities decreased with advanced degradation, but residual activity was observed in cultures of I. lacteus, P. ostreatus, and P. chrysosporium (28–85% of initial). B. adusta showed an increase in estrogenic activity during NP degradation, suggesting endocrine-active intermediates. Ligninolytic enzyme activity was affected by the ED, indicating potential stimulation or suppression during biodegradation.	[40]
Pleurotus ostreatus HK 35	BPA, E2	Trickle-bed reactor, lab and real wastewater treatment.	Degraded >90% of EDs in 12 days, >76% ED removal in pilot reactor.	[60]
Trametes versicolor (pellets)	PhACs, EDs	Fluidized bed bioreactor treating hospital wastewater under sterile and non-sterile conditions.	Removed 46 out of 51 detected EDs and PhACs. 83.2% removal in sterile conditions; 53.3% in non-sterile environments. Complete removal of DIF.	[61]

Notes: ATZ—atrazine; B[a]P—Benzo[a]pyrene; BPA—bisphenol A; CBZ—carbamazepine; DEET—N,N-diethyl-3-methylbenzamide; DIF—Diclofenac; EDs—Endocrine Disruptors; E1—estrone; E2—17β-estradiol; EE2—17α-ethynylestradiol; 6:2 FTOH—6:2 fluorotelomer alcohol; 5:3 FTCA—5:3 fluorotelomer carboxylic acid; NP—4-nonylphenol; n-NP—4-n-nonylphenol; OBZ—oxybenzone; PAHs—Polyhydroxyalkanoates; PCBs—polychlorinated biphenyls; PhAC—Pharmaceutical active compounds; PhOH—phenols; PFCAs—perfluorocarboxylic acids; TCS—Triclosan.

Table 1. Fungal applications for the removal of estrogenic activity and endocrine disruptors in wastewater treatment.

Fungus	EDs	Conditions	Main Results	Ref.
Trametes versicolor, Pleurotus ostreatus, Phanerochaete chrysosporium	PhOH, Parabens, Phthalates	Fed-batch and starvation strategies reduced fresh biomass input and external nutrients. The fungus operated in two bioreactors over one week with five consecutive degradation cycles of EDs.	Best results with T. versicolor It efficiently removed all EDs without additional nutrients, showing potential for repeated cycles in bioreactors. Biotransformation was the primary removal mechanism, with minimal biosorption.	[16]
Trametes versicolor NRRL 66313	E2, single A mixture of EDs: E1, E2, EE2, BPA, ATZ, CBZ, DEET, OBZ, TCS	Fungus was grown in glucose-amended, sterile wastewater (5 g/L). Removals performed in aerated Erlenmeyer incubated for 8 days at room temperature (25 ± 2 °C) and spiked with 5 mg/L of E2 or the mixture of EDCs (350 μg/L each). Abiotic and heat-killed fungus controls were also tested.	T. versicolor reduced E2 from 5 mg/L to below detection levels within 5 h, with E1 as a metabolite, which was subsequently removed. For the mixture of EDs, 62–100% removal was achieved within 3.5 h, and estrogenic activity reduced by 77% (compared to 4–8% in controls). After 12 h, estrogenic activity reduction exceeded 98% (vs. 24–42% for controls).	[28]
Trametes versicolor	ATZ, BPA, CBZ, TCS	Commercial enzymes, biodegradation of estrogenic pollutants in wastewater.	Near-total reduction in estrogenic activity. >80% of atrazine in contaminated water within 72 h by laccase. High efficiency across 5 degradation cycles without external nutrients. Laccase. The process breaks down complex aromatic pesticide structures into simpler, less toxic byproducts, which were further degraded by microbial consortia.	[30]
Pleurotus pulmonarius LBM 105 Trametes sanguinea LBM 023	PCBs	Single culture vs. consortium in bioremediation of PCB-contaminated transformer oil.	Pleurotus pulmonarius LBM 105 showed the highest PCB degradation 95.4% PCB removal, outperforming Trametes sanguinea LBM 023 and fungal consortium.	[35]
Anthracophyllum discolor	PAHs B[a]P	Biodegradation in liquid medium and autoclaved contaminated soil.	75% PAH removal in soil. Manganese peroxidase production linked to degradation. Lower efficiency in non-autoclaved soils.	[36]
Aspergillus niger AN 400	ATZ	Batch reactors with dispersed fungal biomass, glucose as co-substrate.	40% ATZ removal without co-substrate, doubled efficiency with glucose addition at 3 g/L. Higher glucose levels reduced degradation due to competition.	[37]
Trametes hirsuta La-7	BPA, E1, E2	In vivo and in vitro degradation using extracellular laccase and mycelium.	>80% BPA removal within 6 h. Metabolized EDs through six mechanisms, unaffected by BPA presence in plant test.	[38]
Phanerochaete chrysosporium	6:2 FTOH	Transformation of PFAS in bioreactors with Kirk medium with and without glucose, supplemented with organic nutrients like lignocellulosic powder.	Phanerochaete chrysosporium biotransformed 6:2 FTOH into perfluorocarboxylic acids (PFCAs), polyfluorocarboxylic acids, and intermediates within 28 days. Main product was 5:3 FTCA, making up 32–43% of the initial 6:2 FTOH, with minor amounts of PFCAs (5.9%). Efficient EDs degradation, but with some residual estrogenic activity.	[39]
Trametes versicolor, Irpex lacteus, Bjerkandera adusta, Phanerochaete chrysosporium, Phanerochaete magnoliae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Dichomitus squalens	NP, n-NP, BPA, EE2, TCS	Biodegradation in static conditions at 28 °C, malt extract–glucose medium.	I. lacteus and P. ostreatus were the most efficient degraders, >90% and >80% in 7 days, respectively. Both fungi degraded pollutants below detection limit within the first 3 days. Estrogenic activities decreased with advanced degradation, but residual activity was observed in cultures of I. lacteus, P. ostreatus, and P. chrysosporium (28–85% of initial). B. adusta showed an increase in estrogenic activity during NP degradation, suggesting endocrine-active intermediates. Ligninolytic enzyme activity was affected by the ED, indicating potential stimulation or suppression during biodegradation.	[40]
Pleurotus ostreatus HK 35	BPA, E2	Trickle-bed reactor, lab and real wastewater treatment.	Degraded >90% of EDs in 12 days, >76% ED removal in pilot reactor.	[60]
Trametes versicolor (pellets)	PhACs, EDs	Fluidized bed bioreactor treating hospital wastewater under sterile and non-sterile conditions.	Removed 46 out of 51 detected EDs and PhACs. 83.2% removal in sterile conditions; 53.3% in non-sterile environments. Complete removal of DIF.	[61]

Notes: ATZ—atrazine; B[a]P—Benzo[a]pyrene; BPA—bisphenol A; CBZ—carbamazepine; DEET—N,N-diethyl-3-methylbenzamide; DIF—Diclofenac; EDs—Endocrine Disruptors; E1—estrone; E2—17β-estradiol; EE2—17α-ethynylestradiol; 6:2 FTOH—6:2 fluorotelomer alcohol; 5:3 FTCA—5:3 fluorotelomer carboxylic acid; NP—4-nonylphenol; n-NP—4-n-nonylphenol; OBZ—oxybenzone; PAHs—Polyhydroxyalkanoates; PCBs—polychlorinated biphenyls; PhAC—Pharmaceutical active compounds; PhOH—phenols; PFCAs—perfluorocarboxylic acids; TCS—Triclosan.

4. Methodologies for Assessing Estrogenic Activity

Various methodologies have been employed to assess estrogenic activity in environmental samples, particularly in the studies focused on EDs [68]. Among the most used methods are bioassays, which provide insights into the biological activity of estrogen-like compounds. Key assays include the “Yeast Estrogen Screen (YES)”, the “Recombinant Yeast Assay (RYA)”, and the “T47D-KBluc Reporter Gene Assay” [69]. These tests are widely accepted in research due to their sensitivity, reproducibility, and ability to quantify estrogenic activity in a variety of sample matrices.

The YES assay, in particular, is widely used for in vitro bioassays that employ genetically modified yeast cells expressing the human estrogen receptor (hER). The yeast cells are also engineered to contain a reporter gene (usually lacZ) linked to an estrogen-responsive promoter. In the presence of estrogenic compounds, the activated estrogen receptor binds to the estrogen-responsive elements, triggering the expression of the reporter gene. This gene expression can be quantified by colorimetric or fluorescent methods, providing a measurable indication of estrogenic activity. Studies by Lloret et al. [50], Shreve et al. [28], and Rajendran et al. [68] have used the YES assay to quantify estrogenic activity in samples contaminated with EDs. Their results emphasize the assay’s sensitivity to a broad spectrum of estrogenic compounds, including pharmaceuticals, industrial chemicals, and the natural estrogens present in wastewater. The YES assay stands out for its cost-effectiveness, ease-of-use, and versatility, making it suitable for assessing a variety of environments, including water, sediments, and sludge.

The RYA is another yeast-based bioassay that shares similarities with the YES assay but incorporates different genetic constructs to detect estrogenic activity. Typically, it employs yeast strains engineered with a reporter gene (such as GFP or luciferase) that responds to the activation of estrogen receptors. This assay has been successfully applied to detect estrogenic activity in environmental matrices, particularly those containing a complex mixture of chemicals. For example, Badia-Fabregat et al. [69] and Llorca et al. [70] applied RYA to assess the estrogenic potential of wastewater samples both before and after biological treatments aimed at degrading EDs. Their studies demonstrated that RYA is a reliable tool for monitoring the efficacy of bioremediation processes, including those involving fungi and bacteria. The assay is praised for its robustness in detecting low levels of estrogenic compounds even in complex matrices, making it highly suitable for environmental monitoring.

Another powerful tool for assessing estrogenic activity is the T47D-KBluc assay, which employs the human breast cancer cell line T47D-KBluc. This cell line is stably transfected with a luciferase reporter gene, under the control of estrogen-responsive elements. Upon exposure to estrogenic compounds, the estrogen receptor in T47D cells activates the transcription of the luciferase gene, producing a luminescent signal that can be quantitatively measured. Kasonga et al. [9] employed the T47D-KBluc reporter gene assay to evaluate estrogenic activity in samples treated with fungal enzymes. Their study highlighted the high sensitivity and specificity of this assay for detecting estrogens, making it particularly useful for testing the efficacy of biotechnological treatments designed to reduce ED concentrations. Unlike yeast-based assays, the T47D-KBluc assay uses mammalian cells, offering a more physiologically relevant system for assessing estrogen receptor activity in a human cellular context.

These assays, YES, RYA, and T47D-KBluc, are invaluable tools for detecting estrogenic activity in environmental samples, playing a key role in assessing the effectiveness of various bioremediation treatments. While the YES and RYA assays are simple, cost-effective, and suitable for high-throughput screening, the T47D-KBluc assay provides greater physiological relevance due to its use of human cells. Each assay offers distinct advantages, depending on the complexity of the sample and the specific research objectives. Collectively, the studies by Kasonga et al. [9], Shreve et al. [28], Lloret et al. [51], Rajendran et al. [68], Badia-Fabregat et al. [69], and Llorca et al. [70] underscore the versatility of these assays in detecting changes in estrogenic activity, particularly in samples undergoing fungal enzymatic treatments to degrade EDs.

Recently, Slaby et al. [67] introduced the Dicentrarchus labrax estrogen screen (DLES) test, a reporter gene assay that uses nuclear estrogen receptors from Dicentrarchus labrax (sbEsr1; sbEsr2a; sbEsr2b), aiming to diversify the bioassays for detecting estrogen-like EDs. The proposed test complements the existing assays by incorporating non-human estrogen receptors. Positive responses were observed for all three receptors, with sbEsr2b demonstrating the highest sensitivity. When applied to wastewater treatment plant extracts, the DLES test demonstrated greater sensitivity than the YES test, particularly with sbEsr2b, underscoring the need for broader EDs screening to better assess their environmental impact on marine species

The selection of the most appropriate assay depends on various factors, including the sample type, the expected concentrations of EDs, and the need for physiological relevance. Together, these methodologies provide a comprehensive framework for assessing the estrogenic activity of pollutants and evaluating the success of the treatment processes designed to mitigate their environmental and health impacts.

5. Critical Assessment of Research Gaps and Limitations

Despite promising laboratory results, the transition to full-scale wastewater treatment using fungal bioremediation remains underexplored. Further research is required to test the effectiveness of fungi, such as Trametes versicolor, in real-world conditions where varying pollutant loads, environmental stressors, and microbial competition can significantly impact performance. Additionally, while current studies demonstrate that fungal enzymes, particularly laccase, are effective at degrading EDs, scaling up enzyme production to meet the demands of industrial-scale treatment is a significant challenge. Maintaining enzyme stability during prolonged treatment processes is critical for ensuring continuous ED degradation. There is a need for further research on enzyme immobilization techniques and the application of natural mediators to enhance stability and catalytic efficiency. Another major challenge is the economic feasibility of fungal bioremediation on a larger scale, especially considering the costs associated with enzyme production and process maintenance. Future studies should explore cost-reduction strategies, such as optimizing fungal growth conditions or utilizing waste products as substrates to boost enzyme yields.

Current studies on the fungal bioremediation of EDs have several limitations. For instance, while many focus on the removal efficiency of EDs, few evaluate the toxicity of the degradation byproducts. Future research should prioritize investigating the environmental impact and potential risks associated with these byproducts. Additionally, the lack of standardized experimental conditions across studies—such as differences in pollutant concentrations, fungal strains, and treatment times—makes it difficult to compare results directly. Standardizing the methods and parameters would improve comparability and provide clearer insights into the overall efficacy of fungal bioremediation. Another limitation is the geographic bias in the research, with most studies conducted in specific regions. Expanding the research to diverse geographic locations with varying wastewater compositions would provide a more comprehensive understanding of fungal capabilities.

The methodology used in this manuscript offers a strong framework for selecting and analyzing the research on the biodegradation of EDs in wastewater using microorganisms. However, some limitations should be considered for future studies. Firstly, while the use of three major databases (Science Direct, Scopus, and Web of Science) is thorough, excluding others like PubMed or regional databases may limit the scope of relevant studies. Including additional databases could broaden the literature base. Secondly, excluding review articles may have omitted valuable syntheses that highlight the gaps and trends in the field. Therefore, including key reviews would provide a more comprehensive view. The emphasis on newer studies through the InOrdinatio equation might overlook older, foundational research that remains relevant. A balance between recent and landmark studies should be considered. Also, relying on the impact factor and citation count could bias the selection toward well-known journals, missing high-quality studies in niche ones. Screening based solely on abstracts may also result in overlooking significant studies that do not fully present their relevance in the abstract. Extending the screening to include the full text for borderline cases could improve accuracy. Lastly, VOSviewer’s term frequency analysis, while useful, may miss nuanced themes in the full text. Using qualitative analysis tools or thematic coding could provide deeper insights into the reviewed articles.

In conclusion, while Methodi Ordinatio provides an efficient and structured way to prioritize research, future studies should consider complementary frameworks like PRISMA, which emphasizes systematic review processes and ensures a comprehensive coverage of the literature. By addressing these limitations, we can enhance the reproducibility, transparency, and inclusiveness of the review process, ensuring a more balanced and comprehensive analysis.

6. Conclusions and Future Perspectives

Endocrine Disrupting Compounds (EDs) are persistent micropollutants that threaten both environmental and human health by disrupting hormonal systems. Fungal bioremediation, particularly using Trametes versicolor and its laccase enzymes, shows great potential for addressing these pollutants in wastewater treatment, offering a sustainable and cost-effective alternative to conventional treatment processes. However, key challenges remain in scaling up fungal bioremediation for full-scale applications. These include optimizing enzyme production, maintaining stability during treatment, and improving economic feasibility. Enzyme immobilization and the application of natural mediators, such as syringaldehyde, may significantly improve the efficiency of the process, but further research is needed. Looking ahead, future research directions should focus on advancing enzyme immobilization techniques and integrating natural mediators like syringaldehyde to boost laccase efficiency. Additionally, exploring the role of mixed microbial cultures in stabilizing fungal bioremediation within complex wastewater environments is necessary to enhance the overall performance. Investigating fungal bioremediation as a complementary method alongside conventional treatment processes could provide a more comprehensive solution for the removal of EDs and other micropollutants, paving the way for more effective wastewater management strategies.