Exploring Sustainable Remediation Options: The Mycodegradation of Halogenated Nitroaromatic Compounds by Caldariomyces fumago

Abstract

Chlorinated and fluorinated nitrophenols (HNCs) are widely used in agriculture and industry, with a global market valued at USD $25 billion, one which is expected to grow by 5% by 2030. However, these compounds pose significant environmental risks; they are classified as toxic by the International Agency for Research on Cancer (IARC). Existing treatment methods include advanced oxidation, adsorption, and bioremediation, though to date, there has been only limited research on fungal remediation of these halogenated pollutants. This study aims to explore a sustainable approach by using fungi’s potential to degrade HNCs in minimal media. Ten fungi were selected through literature screening; Caldariomyces fumago and Curvularia sp. were highly effective, degrading over 50% of 2-chloro-4-nitrophenol (2C4NP) and 80% of 5-fluoro-2-nitrophenol (5F2NP) within 24 and 48 h, respectively. Additionally, five strains showed degradation potential for fluorinated compounds. Further studies revealed C. fumago could degrade up to 1 mM of chlorinated compounds and 12 mM of fluorinated compounds, far exceeding any known environmental concentrations of HNCs; importantly, ecotoxicology tests demonstrated reductions in toxicity of 77% and 85%, respectively. This work highlights fungi’s underexplored ability to degrade toxic HNCs, offering a sustainable mycoremediation strategy and positioning mycology as a critical tool for future environmental remediation efforts.

1. Introduction

Halogenated nitroaromatic compounds (HNCs), such as chlorinated and fluorinated nitrophenols, have been extensively used as intermediates in the synthesis of various industrial chemicals, including dyes, polymers, pesticides, insecticides, fungicides, and explosives. Many of these compounds have been shown to have significant impacts upon their release into the environment, adversely affecting ecosystem and human health. The original organophosphate insecticides have been largely replaced by carbamate alternatives, including neonicotinoids like acetamiprid and imidacloprid, following the former’s classification as “probably carcinogenic to humans” by the International Agency for Research on Cancer (IARC) [1,2]. Acetamiprid and imidacloprid use 4-nitrophenol to create the halogenated structure of C₁₀H₁₁ClN₄ and C₉H₁₀ClN₅O₂ [3]. These neonicotinoids became highly valued in the agriculture industry due to their high selectivity for insects; however, there is now growing evidence of these compounds’ persistence in the environment and their potential impacts on human health. Additionally, acetamiprid is classified as moderately toxic to bees, while imidacloprid is classified as highly toxic [4].

Global production volumes for chlorinated nitrophenols are not readily published; however, neonicotinoids are the leading insecticides used worldwide, accounting for 27% of the global insecticide market [5]. According to global market insights, the market for neonicotinoids is valued at USD 5.1 billion and expected to have a compounding annual growth rate (CAGR) of 5.4% between 2024 and 2032 [6].

The major use of neonicotinoids has been centered around Asia and North and South America. There are 120 countries registered to use these products on over 140 different crops [7]. Up to 75% of neonicotinoids applied to soil remain available for transport through surface water and leaching into groundwater [7]. In addition, appropriate mitigation strategies must be used on the application equipment as this process can lead to offsite run-off and environmental damage. Neonicotinoids have been detected in groundwater in the U.S. and Europe [7].

Production data for fluorinated nitrophenols are also not readily accessible. However, according to a report published by Research Report World, the global fluorinated-compound market was valued at USD 19.35 billion, with a projected demand in 2030 of USD $27 billion, and with a CAGR of 4.9% [8]. To date, these compounds have not been classified as toxic by governmental agencies. However, Gershon’s studies on various isomers of halophenols and halogenated nitrophenols indicate that 5-halo-2-nitrophenols are the most toxic to fungi, likely due to nucleophilic reactions targeting specific sites, with the halogen reactivity sequence being observed as F > I > Br > Cl [9].

While HNCs are beneficial in terms of agricultural purposes, their presence poses significant risks to human and environmental health, making their careful use and prompt removal from the environment a matter of urgent importance. Several options are available for the removal of HNCs from environmental matrices. Examples of treatment technologies include advanced oxidation processes, reduction and adsorption technologies, and membrane-related technologies [10,11]. However, these technologies are energy-intensive processes that are not always effective and are expensive to operate [10,11].

In contrast, bioremediation technologies potentially represent an environmentally friendly and low-capital solution. The microbial degradation of chlorinated nitrophenols with bacterial species such as Cupriavidus sp. CNP-8, Ralstonia eutropha JMP134, Bacillus sp. strain MW-1, Enterobacter cloacae, Exiguobacterium sp. PMA, Arthrobacter sp. SJcon, Burkholderia sp. RKJ 800, and Rhodococcus imtechensis RKJ300 has been reported, with strains reported to be able to degrade the chlorinated isomers of the contaminant. Experiments have shown the ability of bacteria to cleave the chloride bond, forming amino-hydroquinone, hydroquinone, hydroxyquinol, and mineralization of the compound [12,13,14].

Fungal degradation of 2-chloro-4-nitrophenol (2C4NP) (Figure 1A) was reported by Čapek in 1969, and those studies used strains resistant to this compound. The strains used for this study were Trichophyton mentagraphytes, Microsporum gypseum, and Aspergillus niger [15]. To the author’s knowledge, there are currently no published studies on the ability of additional fungi to degrade chlorinated or fluorinated HNCs such as 5-fluoro-2-nitrophenol (5F2NP) (Figure 1B); searches using keywords such as ‘halogenated compounds’, ‘chlorinated compounds’, ‘chlorinated nitrophenols’, ‘fluorinated compounds’, ‘fluorinated nitrophenols’, ‘bioremediation’, ‘biotransformation’, ‘mycotransformation’, and ‘mycoremediation’ produced no results within the Web of Science, PubMed, PubChem, and American Chemical Society databases. This is perhaps surprising, as fungi are notable for their ability to produce various enzymes such as lignin and manganese peroxidases and laccases which have been proven to break down recalcitrant organic pollutants. For example, Phanerochaete chrysosporium is a highly researched fungus, well-known for its ability to degrade lignin, in addition to common environmental contaminants such dioxins, polychlorinated biphenols, chlorobenzenes, dichloro-diphenyl-trichloroethane (DDT), phenooxyherbicides, chlorophenols, and trichloroethane (TCE) [16].

Fungi represents a diverse and unique kingdom, encompassing seven major phyla: Ascomycota, Basidiomycota, Glomeromycota, Zygomycota, Chytridiomycota, Blastocladiomycota, and Neocallimastigomycota; the kingdom includes an estimated 11.7 to 13.2 million species [17]. Among them, filamentous fungi offer a distinct advantage due to their extensive mycelial networks, which enable them to penetrate soils and other substrates. This capability facilitates the immobilization of toxic substances through mechanisms such as bioadsorption [18,19,20,21]. This makes fungi highly valuable in bioremediation/mycoremediation efforts.

The aim of the research was to identify fungi capable of degrading halogenated nitrophenols (HNCs) under aerobic conditions, which was achieved by assessing their ability to significantly reduce the toxicity of these harmful compounds. By harnessing fungi, the study advanced sustainable bioremediation and thereby highlights the crucial role of mycoremediation in innovative environmental management solutions.

2. Materials and Methods

2.1. Fungal Strain Selection

The selected fungi represent 3 different classes, 5 orders, and 6 families across the 10 fungal candidates. Caldariomyces fumago/Leptoxyphium fumago (ICMP5613) and Phanerochaete chrysosporium (ICMP 15693) were purchased from Land Care New Zealand. Trametes versicolor, Pleurotus ostreatus, and Cordyceps militaris were donated by a private company. Curvularia sp. was kindly donated by the University of Queensland. Lentinus brumalis, Aspergillus fumigatus, Aspergillus niger, Aspergillus galacus, and Penicillium roquetforti were obtained from RMIT University, Bundoora, VIC, Australia.

Each fungal candidate was screened for its ability to mycoremediate or mycotransform halogenated xenobiotic compounds.

Table 1. Fungal candidates and basis for selection.

Class	Order	Family	Mycobank Name	Basis for Selection
Agaricomycetes	Agaricales	Pleurotaceae	Pleurotusostreatus	Bioremediation of diclofenac [22]
Agaricomycetes	Polyporales	Phanerochaeteaceae	Phanerochaete	Bioremediation of aliphatic halocarbons [23]
Agaricomycetes	Polyporales	Polyporaceae	Lentinus brumalis (Polyporus brumalis)	Poly R-478 decolorization [24]
Agaricomycetes	Polyporales	Polyporaceae	Trametes versicolor	Biotransformation of chloramphenicol [25]
Dothideomycetes	Capnodiales	Capnodiaceae	Leptoxyphium fumago (Caldariomyces Fumago)	Fluorophenol oxidation by chloroperoxidase [26]
Dothideomycetes	Pleosporales	Pleosporaceae	Curvularia	Vanadate-dependent chloroperoxidase [27]
Eurotiomycetes	Eurotiales	Aspergillaceae	Aspergillus fumigats mut. Fumigatus	Mycoremediation of Rhodamine B [28]
Eurotiomycetes	Eurotiales	Aspergillaceae	Aspergillus glaucus	Biomineralization of fipronil [29]
Eurotiomycetes	Eurotiales	Aspergillaceae	Aspergillus niger	Bioremediation of diclofenac [22]
Eurotiomycetes	Eurotiales	Aspergillaceae	Penicillium roquefortii	Bioremediation of diclofenac [22]

Data verified by www.mycobank.org, accessed on 8 October 2024.

provides a list of the strains selected for the study and the rationales behind the selections.

2.2. Growth of Fungal Cultures

Potato dextrose agar (PDA) (Merck, Truganina, VIC, Australia) was prepared according to the manufacturer’s recommendations and autoclaved at 121 °C for 15 min at 15 PSI. For plate inoculation, a 1 cm² subculture was aseptically taken and placed at the center of a 90 mm Petri plate. Inoculated plates were incubated at 25 °C for 10 days (L. fumago, 21 days).

2.3. Model Compound Preparation

Quantities of 0.3 M stock solutions of 2-chloro-4-nitrophenol (2C4NP) (Sigma, Truganina, VIC, Australia, C₆H₄ClNO₃) and 5-fluoro-2-nitrophenol (5F2NP) (Sigma, C₆H₄FNO₃) (Figure 1) were prepared in methanol, and the required concentration was added prior to filter sterilization (0.22 µm, Millipore, Truganina, VIC, Australia) [30].

2.4. Media Preparation

A fungal minimal-salts medium was prepared in MillQ H₂O; the medium contained nitrate salts, trace elements, and thiamine (Sigma). The nitrate salts consisted of sodium nitrate (Sigma), potassium chloride (Chem-Supply, Gillman, SA, Australia), magnesium sulfate heptahydrate (Sigma), and monopotassium phosphate (Merk). Trace elements contained included zinc sulfate heptahydrate (Sigma), boric acid (Sigma), manganese (II) chloride tetrahydrate (Sigma), iron (III) sulfate heptahydrate (Sigma), cobalt chloride hexahydrate (Sigma), cupric sulfate pentahydrate (Sigma), sodium molybdate dihydrate (Chem Supply), and tetrasodium EDTA (Sigma) [31].

A 50% glucose solution was filter sterilized by being passed through a 0.22 µM filter and then appropriately [12] diluted to a 1% final solution in the autoclaved minimal-salts media.

2.5. Degradation Studies

To monitor degradation, absorbance measurements were conducted at 420 nm, using standard calibration curves prepared with HNC concentrations of 0.4, 0.2, 0.1, 0.04, 0.02, and 0.01 mM. Calibration curves for 2-chloro-4-nitrophenol (2C4NP) and 5-fluoro-2-nitrophenol (5F2NP) yielded R² values of 0.9923 and 0.9999, respectively (Figure 2). Based on the R² values, 420 nm was selected as the appropriate wavelength for subsequent degradation studies [12,30].

To conduct the degradation studies using the two HNCs, minimal media (150 mL) was added to an Erlenmeyer flask (250 mL); a small square (1 cm²) of fungal culture was removed and homogenized using a glass homogenizer, and then the aliquot (1 mL) was introduced in the media. The flasks were covered with aluminum foil, incubated at 27 °C ± 2 °C, and shaken at 110 RPM. Aseptic sampling (5 mL) was conducted regularly throughout the 72 h incubation. Samples were centrifuged at 5000 rpm, at 20° C for 5 min. Following removal of the supernatant, the measurement of absorbance at 420 nm was recorded using a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). Decolorization studies on HNCs have demonstrated that this process effectively reflects the utilization and depletion of the compound [12,32].

2.6. Detection of Chloride and Fluoride by Ion Chromatography

To detect chloride and fluoride release, ion chromatography was carried out using a IC930 Compact Flex with 919 IC Autosampler Plus, Methrom (Herisau, Switzerland). The column used was a Metrosep A Supp 17–150/4.0 and a 4 mm Guard Column: Metrosep A Supp 17 Guard 4.0. Stock solutions of sodium chloride (NaCl) (Merck) and sodium fluoride (NaF) (Merck) were used to create a standard calibration curve. Supernatant samples were pH-adjusted to 6.2 using 1 M NaOH (ChemSupply), filtered (0.22 µm, Merck), and diluted to a 1:1 ratio (MillQ:supernatant); 3 mM samples were adjusted to 0.3 mM and diluted appropriately. The following concentrations (mM) of NaCl were used for calibration: 4, 3, 2, and 1 mM; for NaF, the values were a0.1, 0.3, 0.5, and 0.8 mM. R² values for NaCl and NaF were 0.9998 and 0.9961, respectively.

2.7. Bioluminescence Inhibition Testing (MicroTox Test)

Toxicity analysis was conducted using a Modern Water Microtox LX analyzer (York, UK); using 0.3 mM of both 2C4NP and 5F2NP, their respective supernatants were analyzed for toxicity. The manufacturer’s recommendation for analysis was used [33]. Culture supernatants from fungal incubations in minimal-salts media containing 2C4NP and 5F2NP were diluted to a 1:1 ratio (supernatant: MillQ Water) for further analysis. The medium for reconstitution was supplied by JW Industrial Instruments Pty. Ltd. (Belrose, NSW, Australia), and the acute Microtox reagent by Modern Water Microtox ^® (York, UK). The reconstituted freeze-dried marine bacteria Aliivibrio fischeri was allowed to stabilize in a 4 °C Microtox^® Analyser before the test was begun. A 2% sodium chloride (NaCl) solution served as the diluent, while a 22% NaCl solution was used to adjust the osmotic pressure during the test. Inhibition of bioluminescence was measured at the 5 min time point and expressed as EC₅₀ using the provided software. Conversion of EC₅₀ values to toxicity units was calculated using the following formula: TU = (1/EC₅₀) × 100 [34]. The scale of toxicity is as follows: 0 = not toxic, <1 ‘slightly toxic’, 1–10 ‘toxic’, 11–100 ‘very toxic’, and >100 ‘extremely toxic’ [35,36].

3. Results and Discussion

3.1. Degradation of 2-Chloro-4-nitrophenol and 5-Fluoro-2-nitrophenol (0.3 mM)

The objective of these degradation studies was to identify fungi capable of degrading HNCs under aerobic conditions. Fungal candidates were selected based on their ability to remediate xenobiotic organohalogens, based on reports in the published literature (Table 1). Previous studies on the degradation of chlorinated nitrophenols (HNCs) have been focused on bacterial strains. Several bacterial species have demonstrated the ability to degrade various isomers of chlorinated nitrophenols [12,13,14]. The degradation was quantitatively measured at an absorbance of 420 nm using spectrophotometry, based on Arora’s bacterial degradation studies [12].

C. fumago and Curvularia sp. were able to degrade more than 80% of 2C4NP over a 72 h period. A lag period (8 h) was observed prior to the observed degradation (Figure 3A). C. fumago could degrade the compound by more than 50% after 24 h. A similar level of degradation by Curvularia sp. was observed after 32 h (Figure 3A). The results from these degradation studies are comparable to those previously reported for bacterial degradation; for example, Enterobacter cloacae, was reported to degrade 0.25 mM 4C2NP after 50 h [37]. Similarly, Arthobacter sp SJcon, under aerobic conditions, was able to transform 0.3 mM of 2C4NP to chlorohydroquinone, and then to maleylacetate within 48 h [38]. Finally, Rhodococcus imtechensis RKJ300, under aerobic conditions, completely degraded 0.3 mM 2C4NP within 10–12 h. The proposed degradation pathway involved the conversion of chloro-hydroquinone to hydroquinone, followed by transformation to γ-hydroxymuconic semialdehyde [39,40]. In the current study, degradation studies were similarly conducted with P. roquefortii, T. versicolor, P. brumalis, P. ostreatus, A. fumigatus, A. glaucus, A. niger, and P. chrysosporium; however, no significant degradation was observed.

As for 5F2NP, several fungi could degrade this compound (Figure 3B). C. fumago and Curvularia sp. were both capable of degrading the fluorinated nitrophenol compound (Figure 3B). C. fumago achieved 84% degradation of 0.3 mM 5F2NP within 24 h; Curvularia sp. required 48 h to reach the same % degradation. T. versicolor and P. brumalis also degraded the fluorinated compound; however, they required 56 h to reach a degradation above 80% (Figure 3B). P. roquefortii required 72 h to achieve greater than 80% degradation. Five additional strains were also assessed in terms of their ability to degrade the fluorinated compound: P. ostreatus, A. fumigatus, A. glaucus, A. niger, and P. chrysosporium were unable to degrade 5F2NP. To the best of the author’s knowledge, this is the first known biodegradation study to be conducted on 5F2NP.

Based on the ability of C. fumago (Figure 4) to degrade both HNCs, this organism was selected for further studies.

3.2. Effect of the Concentration of HNCs on Degradation by C. fumago

With environmental concentrations of HNCs in the environment ranging from 2.75 × 10⁻⁵ to 1 × 10⁻³, and due to their increased use and resistance to degradation, they are likely to accumulate in environmental matrices [41]. This accumulation could further complicate bacterial degradation of xenobiotic compounds. However, filamentous fungi, such as C. fumago, have demonstrated their potential for degrading these compounds more efficiently by penetrating and decomposing HNCs within complex environmental matrices. The ability of C. fumago to degrade higher concentrations of the HNCs was therefore investigated.

Figure 5A demonstrates the ability of C. fumago to degrade 1 mM of 2-chloro-4-nitrophenol. C. fumago was also exposed to 2, 3, 6, 9, and 12 mM of the chlorinated compound, but was unable to significantly degrade 2C4NP at these concentrations. C. fumago was able to degrade 80% of 1 mM of 2C4NP within 96 h, representing the highest reported degradation of this compound by any organism. Work by Čapek (0.3 mM 2C4NP) has demonstrated fungal resistance to 2C4NP, identifying four metabolites. This work did not identify the compounds; however, only trace amounts of the parent compound were identified [15]. A. niger was used in the current degradation study in a MSM environment, but was unable to degrade 2C4NP, suggesting that the degradation of A. niger reported by Čapek may have been a result of growth in a nutrient-rich medium (Sabouraud) rather than the use of 2C4NP as an energy source.

The bacteria Cupriavidus sp. has previously been shown to degrade 0.6 mM 2-chloro-5-nitrophenol; Ralstonia eutropha JMP134 was shown to degrade the same compound at an initial exposure of 0.46 mM and an additional 0.35 mM. Arthobacter sp. SJcon, Burkholderia sp. RKJ 800sp. PMA, and Rhodococcus imtechensis RKJ 300 were shown to use 2C4NP as their energy source [14,38,39,40,42,43]. In their study the highest reported degradation was 0.4 mM by Burkholderia sp., RKJ 800, with degradation complete after 96 h [12,44,45]. The three bacteria were able to transform the compound, cleaving both the nitrogen and halogen groups. In addition, the bacteria Exiguobacterium sp. strain MW-1 was also reported to degrade 0.6 mM 2-chloro-4-nitrophenol [32]. The current work is the first scientific report of an organism capable of degrading a 1 mM concentration of 2-chloro-4-nitrophenol, confirming the potential of this fungus for use in bioremediation.

The ability of the fungus C. fumago to degrade a range of concentrations (0.3–12 mM) of 5F2NP was assessed (Figure 5B). C. fumago achieved 50% degradation for 0.3, 3, and 6 mM within the first 8 h of incubation, reaching approximately 85% degradation after 12 h. A period of adaptation, as seen in a lag period, was required when C. fumago was grown at elevated concentrations of 5F2NP (9 and 12 mM) (Figure 3B). Degradation at 9 and 12 mM concentrations was observed after 16 h; however, it took an additional 24 h for the degradation to reach over 90% (Figure 5B).

Metabolite analysis using gas chromatography mass spectrometry (GC-MS) and thin layer chromatography (TLC) did not yield conclusive results for identifying a degradation pathway for 2C4NP and 5F2NP by C. fumago. This outcome likely reflects the complexity of these compounds and the intricate metabolic process of fungi, which may require additional or alternative analytical approaches to fully elucidate. Advanced analytical techniques, coupled with molecular studies, could provide the clarity required to understand the breakdown mechanisms of these compounds. Therefore, while a definitive pathway could not be proposed in this study, this research lays a foundation for future work to investigate potential degradation pathways in greater detail.

3.3. Detection of Chloride and Fluoride Ions

To confirm halogen cleavage, ion chromatography was used to detect chloride and fluoride ions. Chloride and fluoride were detected in the culture supernatant at the end of the incubation, confirming that dehalogenation had occurred. A mass balance estimated 0.25 mg/L of chloride from minimal media. Full cleavage of 2C4NP and 5F2NP would result in 10.6 mg/L of chloride and 5.7 mg/L fluoride, respectively. The concentrations detected in supernatant were 1.063 mg/L and 0.584 mg/L for 2C4NP and 5F2NP, respectively, representing approximately 10% of the total amount of halogen ions produced if complete dehalogenation had occurred (Figure 6A,B). Additional halogen degradation products may be located intracellularly, or it is possible that incomplete dehalogenation could be attributed to using a minimal media, namely, using 1% (w/v) glucose compared to 4% fructose (w/v), as in the studies conducted by Pickard [46]. Further investigation as to the optimization of the degradation process may enhance the dehalogenation efficiency. To the best of the authors’ knowledge, this is the first reported degradation of a halogenated nitrophenol by C. fumago. Previous studies used purified enzyme extracts for halogenated phenols.

Although the current literature does not provide extensive information on additional enzymes produced by C. fumago, it is widely recognized that fungi produce a diverse array of enzymes capable of degrading complex compounds [47,48].

3.4. Assessment of the Impact of the Degradation of HNC on Ecotoxicity

In terms of bioremediation, it is critical that, in addition to monitoring degradation of the pollutant, the impact of the treatment on the ecotoxicity of the degradation be assessed. Ecotoxicity assessment is regarded as a highly valuable tool for determining environmental effects [49]. This assay employs bioluminescent bacteria (A. fischeri) to gauge the toxicity of various substances, including environmental samples, chemicals, and wastewater. A decrease in light emission from the bacteria signifies that their metabolic activity has been compromised by toxic agents in the sample. The extent of light reduction correlates directly with the level of toxicity present in the sample, with greater reductions indicating higher toxicity.

In this study Microtox analysis was conducted to determine the ecotoxicity of 2C4NP and 5F2NP and their degraded products. Figure 7A,B confirms that the degradation of both HNCs by C. fumago resulted in a significant decrease in the toxicity of the degraded product. Figure 7A,B highlights the ability of C. fumago to reduce toxicity within a 72 h period.

C. fumago was tested with 0.3 mM (52 mg/L) of 2C4NP and successfully transformed approximately 82% of the parent compound within 72 h. The toxicity units (TU) of the initial 2C4NP amounted to 35; following degradation, the supernatant had a TU of 8, reflecting a 77% reduction in toxicity (p = 0.0099). C. fumago was also tested against 5F2NP (0.3 mM); this resulted in an 85% transformation of the parent compound within 24 h. The initial TU of 29 for 5F2NP was reduced to 5.23 in the supernatant within a 72 h period, representing an 83% reduction in toxicity (p ≤ 0.0001).

Previous studies on the degradation of chlorinated nitrophenol compounds lacked information on the ecotoxicity of the daughter products. To the best of the authors’ knowledge, this is the first reported study on the ecotoxicity of the parent and daughter products of 2C4NP and 5F2NP compounds. The significant decreases in ecotoxicity, 77% and 85%, respectively, highlight C. fumago’s ability to be used in mycoremediation efforts. Pilot-scale testing in both aqueous and soil matrices should be considered for environments contaminated with HNCs to further test this fungus’s ability. Understanding the progress of degradation is a principal factor to consider in bioremediation strategies. Previous studies have reported that daughter products from the degradation of an environmental contaminant have led to increased toxicity; for example, the degradation products of neonicotinoids like acetamiprid and imidacloprid produced by T. versicolor have been reported to be 6-chloronicotinic acid and hydroxyl-imidacloprid. Microtox analysis showed increased toxicity compared to the parent compound. The toxicity of the daughter products of imidacloprid and acetamiprid increased from 0.54 to 3 and from 0 to 0.86, respectively [1].

4. Conclusions

The market demand for HNCs is anticipated to grow significantly in the coming years, with a substantial portion of neonicotinoids persisting in soils and leaching into ground water. Consequently, environmental concentrations of these compounds are expected to increase, potentially surpassing the degradation capacities of bacterial systems. Bacterial studies demonstrate potential in degrading chlorinated nitrophenols, though the toxicity levels of breakdown products remain unknown. Furthermore, no bacterial species have been documented to degrade fluorinated nitrophenols, indicating a need for new strategies and further research in this area.

The presented body of work highlights C. fumago, a filamentous fungus, and showcases its potential to be used in mycoremediation applications. This fungus was able to degrade elevated concentrations of each compound and, notably, significantly reduce the toxicity of the parent compound. The observed reduction in toxicity may potentially allow further degradation by microbial communities.

Additionally, C. fumago was able to dehalogenate both compounds in an aerobic environment, highlighting the potential of fungal systems to degrade highly recalcitrant compounds. With their expansive mycelial networks, filamentous fungi, including C. fumago, offer a promising approach to sustainable bioremediation. These networks not only facilitate substrate penetration but also increase enzyme–pollutant interaction times, enhancing degradation efficiency in complex environments. This unique advantage over bacterial degradation affirms the role of fungi as valuable organisms in bioremediation and mycoremediation strategies for halogenated nitrophenol compounds.