Bioremediation of Persistent Organic Pollutant—Oxybenzone with Pleurotus djamor

Abstract

Oxybenzone, a common sunscreen ingredient, has been widely detected in various environmental matrices, posing significant ecological and health risks. The present study demonstrates, for the first time, the capacity of Pleurotus djamor to degrade oxybenzone in in vitro cultures. After 14 days of mycelial incubation, oxybenzone concentrations in the medium decreased from 25 mg to 1.5394 ± 0.095 mg. The final amount of oxybenzone in the mycelium after lyophilization was 6.2067 ± 0.2459 mg. Furthermore, oxybenzone addition significantly reduced biomass growth from 2.510 ± 0.6230 g to 1.4697 ± 0.0465 g. The transformation products in the dry mycelium and medium were assessed and identified using UPLC-Q-tof based on monoisotopic molecular mass and fragmentation spectra. In processes initiated by P. djamor, mainly acylated derivatives of oxybenzone were formed. Additionally, compounds with thiol and amino groups were identified. Alterations in antioxidant profiles (L-tryptophan, 6-methyl-D,L-tryptophan, p-hydroxybenzoic acid, ergosterol, lovastatin, phenylalanine, and ergothioneine) in response to oxybenzone exposure were observed. Our findings reveal significant changes in the antioxidant levels and biomass growth inhibition, underscoring the potential toxicological risks associated with oxybenzone. The observed reduction in oxybenzone concentration highlights the potential of P. djamor as an effective and environmentally friendly strategy for mitigating this pollutant.

1. Introduction

Oxybenzone (Benzophenone-3, BP-3) is commonly found in sunscreens, coatings, and paints as a photoprotectant. BP-3 absorbs UV radiation in the 280–315 nm range and short-wavelength UVA radiation in the 315–355 nm range. It then dissipates this energy without emitting radiation, through a process called photoinduced keto-enol tautomerization. This process converts the absorbed UV energy into heat, which is then harmlessly released. This mechanism not only makes it an effective, photostable sunscreen but also contributes to its relatively long persistence in surface waters, lasting from weeks to months [1,2,3]. In the United States and the European Union, the maximum concentration of BP-3 in sunscreens and cosmetics is 6%, in Australia 10%, and in Japan 5% [4]. The global consumption of oxybenzone is estimated to be around 3000 tons per year. Oxybenzone is registered under the REACH regulation and is either manufactured or imported into the European Economic Area in amounts ranging from 100 to 1000 tons annually [5]. According to Euromonitor, the combined usage of benzophenone-class UV filters in the U.S. is approximately 11.4 mg per capita per day, totaling 1386 tons annually [6].

The widespread use of benzophenone UV filters and their incomplete removal by sewage treatment processes have resulted in their presence in various environmental matrices, including water, soil, sediments, sludge, and biota. They are prevalent in coastal environments, either through direct application or via wastewater discharge. In recent years, increasing attention has been paid to the harmful effects of BP-3 on marine ecosystems, particularly coral reefs [7,8]. Oxybenzone is genotoxic to corals, especially after exposure to sunlight [9]. The compound causes endocrine and developmental disruption in vertebrates and invertebrates. The presence of BP-3 is routinely confirmed in environmental samples. BP-3 concentrations in water samples collected in Hanauma Bay (Oahu, HI, USA) ranged from 136 to 27,880 ng/L [10]. In drains flowing into the Yellow River in Ningxia, China, the measured BP-3 concentration was 477.28 ng/L [11]. The main source of water contamination with UV filters is their washing off from the skin of water recreationists, but also sewage treatment plant effluents and surface runoff from nearby beach showers [10,12]. The average BP-3 concentration in sand samples collected from showers on three Hawaiian Islands (Maui, Oahu, HI, USA) was 5619 ng/g [4]. Due to the confirmed harmful effects of oxybenzone in causing coral reef bleaching, BP-3 has been banned in some places around the world, such as Hawaii, Key West, the U.S. Virgin Islands, Palau, and Bonaire [13].

The pervasive occurrence of BP-3 in the environment results in continuous human exposure to this compound from multiple sources. Benzophenones have also been detected in fresh water, tap drinking water, indoor dust and air, textiles, and seafood [14]. BP-3 has been also approved by the US Food and Drug Administration as an indirect food additive and may be found, for example, in food packaging [15]. According to the opinion of the Scientific Committee on Consumer Safety of the European Commission, the use of BP-3 as a UV filter in cosmetic products is safe for consumers provided that its concentration does not exceed 2.2%. This decision is based on concerns regarding reproductive toxicity, potential carcinogenic properties, and effects on fetal and neonatal development [16]. BP-3 has estrogenic and anti-androgenic activity. Numerous studies have confirmed that oxybenzone is absorbed through human skin, with absorption in some cases reaching up to 10% of the applied dose [9,17]. The presence of BP-3 has been confirmed in various biological matrices such as urine, serum, semen, and even amniotic fluid and breast milk [18]. Oxybenzone undergoes phase I and phase II biotransformation, and some of its metabolites may be more toxic than BP-3 itself. Examples include 4-hydroxybenzophenone and benzophenone-1 (2,4-dihydroxybenzophenone), which have higher estrogenic activity than BP-3 itself. Urine is the primary route of excretion of BP-3 and its metabolites [15,17,19]. Exposure to BP-3 is positively correlated with diseases such as endometriosis, reproductive disorders, cancer, and osteoarthritis [14].

Organic UV filters do not degrade quickly in the natural environment and thus constitute persistent organic pollutants. Moreover, conventional purification processes used in wastewater treatment plants are ineffective at removing these substances [20]. Mycoremediation is a form of bioremediation that involves the use of extracellular ligninolytic enzymes produced by fungi, such as laccase, lignin peroxidase, and manganese peroxidase, to remove or neutralize environmental pollutants and toxins [20,21,22]. Laccases belong to a large family of enzymes referred to as “multicopper oxidases”. These enzymes are characterized by low substrate specificity and high oxidation capacity for various phenolic and non-phenolic compounds [23]. The evaluation of the removal efficiency of three organic UV filters (2-ethylhexyl salicylate (EHS), homosalate (HMS), and ethylhexyl methoxycinnamate (EHMC)) using Spent Mushroom Composts (SMCs) from four different mushrooms (P. eryngii, P. djamor, P. ostreatus, and Auricularia polytricha) revealed that P. djamor showed the highest efficiency in removing EHS and HMS [20]. Therefore, P. djamor was selected for further study. The species P. djamor, commonly known as pink oyster mushrooms, is renowned for its diverse applications in medicine and cosmetics due to its immunostimulatory, anti-cancer, and anti-aging properties. P. djamor is an excellent candidate for the mycoremediation of oxybenzone due to its adaptability to various substrates and environmental friendliness. This species has demonstrated significant ligninolytic enzyme production crucial for breaking down organic pollutants.

Given the ability of P. djamor to degrade compounds with similar chemical structures, there is potential for its use in the decomposition of BP-3. Currently, to the best of our knowledge, the presence of biologically active compounds in the mycelia derived from in vitro cultures of P. djamor following the addition of oxybenzone has not been previously explored. Consequently, we seek to evaluate the efficiency of oxybenzone elimination from the culture medium and to analyze the structure and toxicity of the resulting bioremediation products. Additionally, our study aims to investigate the profile of biologically active substances in the mushroom material produced.

2. Materials and Methods

2.1. Reagents

Oxybenzone, utilized as a pharmaceutical secondary standard, was acquired from Sigma-Aldrich Corp. (St. Louis, MO, USA). HPLC-grade methanol, acetonitrile, 98% formic acid, chloramphenicol, dichloromethane, glucose, maltose extract, casein hydrolysate, L-asparagine, adenine, and yeast extract were supplied by Merck (Darmstadt, Germany). Ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride anhydrous, iron(III) chloride, manganese sulfate monohydrate, and zinc sulfate hexahydrate were sourced from PPH Golpharm (Kraków, Poland). Quadruple-distilled water, with a conductivity of less than 1 μS cm⁻¹, was obtained using an S2-97A2 distillation apparatus (ChemLand, Stargard Szczeciński, Poland). All substances were of analytical purity.

2.2. Mushroom Material

The Pleurotus djamor strain, (strain number PL24), commercially obtained from Grzybnia Płynna (Wola Rafałowska, Poland), was used to generate in vitro cultures. Representative samples of the material are preserved at the Department of Medicinal Plant and Mushroom Biotechnology, Jagiellonian University Medical College in Kraków, Poland.

2.3. Preparation of Pleurotus djamor Mycelial Cultures

The mycelia from in vitro cultures grown on solid media were transferred to 250 mL of a modified liquid Oddoux medium to optimize biomass production for subsequent analyses. The medium contained glucose and maltose extract as carbon sources; casein hydrolysate, L-asparagine, and adenine as nitrogen sources; and macroelements supplied by aqueous solutions of NH₄Cl, KH₂PO₄, MgSO₄·7H₂O, and CaCl₂·6H₂O. Microelements were provided by aqueous solutions of FeCl₃, MnSO₄·H₂O, and ZnSO₄, along with yeast extract. The liquid cultures were incubated at 23 ± 2 °C under a 12 h light (900 lx)/12 h dark cycle and agitated at 140 rpm on a rotary shaker (ALTEL, Kraków, Poland). P. djamor cultures were maintained under these conditions for three weeks to generate sufficient biomass for the study.

2.4. Experimental Mycelial Cultures of Pleurotus djamor

During the mycoremediation trials, 25 mg of oxybenzone powder was introduced into 250 mL Erlenmeyer flasks containing a sterile liquid medium, which had been autoclaved at 121 °C for 20 min and subsequently inoculated with P. djamor mycelium. Control samples without oxybenzone were also prepared. Following inoculation, the cultures were incubated on a shaker at 23 ± 2 °C for 21 days. Once the incubation period ended, the P. djamor biomass was separated from the medium, rinsed with redistilled water, and freeze-dried using a lyophilizer (FreeZone 4.5, Labconco). The culture medium was also evaporated to dryness using the same lyophilization process. The freeze-dried biomass and medium residues were then weighed and ground, and prepared for further analysis.

2.5. Sample Preparation for Analysis

The fungal biomass in powdered form underwent extraction with 125 mL of methanol in a 49 kHz ultrasonic bath (Sonic-2, Polsonic, Warszawa, Poland) for 20 min. After each extraction cycle, the mixture was filtered via paper filter and funnel, with the methanol extract collected in a crystallizer. This procedure was replicated nine times per sample. Pooled extracts from all cycles were air-dried at ambient temperature (23 ± 2 °C), yielding an average of 1.2 g extract per 4 g starting biomass. Both dried extracts and dehydrated culture media were redissolved in HPLC-grade methanol, then passed through 0.22 µm PTFE membrane filters (ChemLand, Poland) into sterilized glass vials (Witko, Poland). While the media samples were analyzed undiluted, the biomass-derived extracts required 10-fold methanol dilution prior to RP-HPLC/UPLC analysis owing to elevated oxybenzone levels.

2.6. HPLC Analysis

The content of organic compounds was analyzed using the RP-HPLC-DAD method, which relied on standard calibration curves and assumed a linear relationship between peak area and reference standard concentration.

Indole compounds, phenolic compounds, lovastatin, ergothioneine, and L-phenylalanine concentrations were determined via RP-HPLC. All information regarding the identification of the studied compounds has been described or cited in previous work [24]. UV detection was set at 280 nm for indole compounds, 238 nm for lovastatin, and 257 nm for ergothioneine. The calibration equations and R² values for the analyzed compounds are as follows: L-tryptophan: y = 3 × 10⁷x − 236,885, R² = 0.9983; 6-metylo-D,L-tryptofan: y = 3 × 10⁷x − 236,885, R² = 0.9983; p-hydroxybenzoic acid: y = 2 × 10⁸x − 71,530, R² = 0.9998; ergosterol: y = 5 × 10⁷x − 3 × 10⁶, R² = 0.9861; lovastatin: y = 5 × 10⁷x + 760,417, R² = 0.9964; ergothioneine: y = 7 × 10⁷x + 493,847, R² = 0.9985; L-phenylalanine: y = 2 × 10⁶x − 4982.9, R² = 0.9999. Oxybenzone concentration was measured using RP-HPLC following the method by Chawla et al. [25]. The separation was performed on a Hitachi HPLC system (Merck, Tokyo, Japan) with an L-7100 pump and a Purospher^® RP-C18 column (200 mm × 4 mm, 5 μm), using a methanol–water (95:5, v/v) mobile phase at a flow rate of 1 mL/min, with UV detection at 280 nm. Qualitative analysis of oxybenzone involved comparing sample peak retention times with those of the standard. To confirm oxybenzone presence, a standard solution was added to the samples, and an increase in peak height at the corresponding retention time indicated its presence. Additionally, an MS/MS analysis was conducted. The quantification of oxybenzone was performed using a calibration curve constructed with standard solutions in the concentration range of 0.025 to 0.25 mg/mL, described by the equation y = 3 × 10⁸x + 374,701 and an R² value of 0.9989. Phenolic compounds, L-phenylalanine, and sterols were analyzed using a Hitachi-Merck HPLC VWR liquid chromatograph (Darmstadt, Germany) as previously described. All compound quantities were reported in milligrams per 100 g of dry weight (d.w.).

2.7. UPLC Analysis

After lyophilizing 100 mL of the solution, 20 mL of methanol (for LC-MS LiChrosolv^®; Supelco, St. Louis, MO, USA) was added to the samples and shaken for 30 min. The methanol solutions were then centrifuged at 4000 RPM for 20 min. The supernatants were analyzed immediately using the UPLC method with an ACQUITY UPLC I Class System (Waters), utilizing an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 50 mm) and a QTof detector (Xevo G2-XS, Waters, Milford, MA, USA). Detailed chromatographic separation parameters are provided in

Table 1. Detailed data for the analytical procedure and the mobile phase compositions.

Component A	H2O (for LC-MS Chromasolv®; Fluka-Analytical, Buchs, Switzerland), with 0.01% HCOOH (98–100%, for LC-MS, LiChropur®; Sigma-Aldrich, St. Louis, MO, USA)
Component B	CH3CN (for LC-MS LiChrosolv®; Sigma-Aldrich, St. Louis, MO, USA) with 0.01% HCOOH
Gradient (min; % A)	0.0–90, 6.0–40, 8.0–10, 8.5–10, 9.0–90, 10.0–90
Flow rate	0.300 mL min−1
Sample volume	0.5 and 1.0 µL
Column temperature	35 °C
Source	ES+
Scan time	0.1 s
Start mass	50.0 Da
End mass	600.0 Da
Maximum mass error	0.5 mDa
Operating mode	ms and ms/ms
Collision energy	0–25 eV
Reference compound	Leucine Enkephalin single point (ms)
Software	MassLynx v4.1

.

2.8. Statistical Analysis

Statistical analyses were conducted using Statistica v.8.0 (StatSoft Inc., Tulsa, OK, USA). Each sample underwent triplicate testing. The results are expressed as means with standard deviations (SD). A one-way ANOVA with Tukey’s post hoc test for multiple comparisons determined significance, with p-values of less than 0.05 considered significant. The toxicity risk profile of biotransformation products was predicted in silico using OSIRIS Property Explorer (available at: https://www.organic-chemistry.org/prog/peo/, accessed on 20 December 2024). The predicted chronic toxicity of biotransformation products was assessed using the Ecological Structure Activity Relationships Predictive Model provided in the ECOSAR Operation Manual v2.2 (available at: https://www.epa.gov/tsca-screening-tools/ecological-structure-activity-relationships-program-ecosar-operation-manual, accessed on 20 December 2024).

3. Results

3.1. Profile of Organic Compounds with Health Benefits

The chemical profile of biomass obtained from in vitro cultures of P. djamor supplemented with oxybenzone was analyzed, with particular emphasis on selected organic compounds with potential health benefits. For comparative purposes, the chemical composition of biomass obtained under standard cultivation conditions was also examined. The results are presented in

Table 2. Antioxidant compounds determined in mycelium from in vitro cultures of Pleurotus djamor [mg/100 g dry weight].

Analyzed Compounds	Mycelium from In Vitro Cultures	Mycelium from In Vitro Cultures with BP-3
Indole compounds
L-Tryptophan	–	8.47 ± 0.344 *
6-metylo-D,L-tryptophan	1.52 ± 0.06	4.56 ± 0.55 *
Phenolic compounds
p-Hydroxybenzoic acid	–	5.90 ± 0.08 *
Sterols
Ergosterol	0.15 ± 0.03	8.88 ± 0.11 *
Other organic compounds
Lovastatin	–	–
Ergothioneine	3.96 ± 0.17	1.58 ± 0.11 *
Phenylalanine	54.03 ± 4.3	176.33 ± 5.85 *

Values in a row followed by * are significantly different at p ≤ 0.05 (statistical analysis: one-way analysis of variance (ANOVA), followed by Tukey’s test). Each value represents the mean of three replicates ± standard deviation.

.

A comparison of the quantified ergothioneine content in P. djamor biomass cultivated under standard conditions (3.96 mg/100 g dry weight) and in the presence of oxybenzone (1.58 mg/100 g dry weight) revealed a significant reduction in the levels of this compound. This decline suggests that oxybenzone may influence fungal metabolic activity, particularly the biosynthetic pathways of ergothioneine. Oxybenzone is known to generate reactive oxygen species (ROS) and disrupt oxidative homeostasis. Studies have demonstrated that increased ROS levels may lead to reduced ergothioneine production as this compound serves as an antioxidant protecting cells from oxidative stress. However, as ROS levels were not measured in this study, this remains a hypothesis requiring further verification. Additionally, the observed reduction in ergothioneine may be linked to metabolic shifts favoring the synthesis of alternative protective compounds [26].

The reduction in ergothioneine content may also result from a metabolic shift towards the biosynthesis of alternative protective compounds. Studies on another Pleurotus species, Pleurotus citrinopileatus, have demonstrated that stress factors can lead to increased production of polyphenols, terpenoids, or other antioxidants. It is conceivable that in response to oxybenzone exposure, P. djamor mycelium undergoes adaptive metabolic modifications, compensating for the decrease in ergothioneine with other bioactive compounds. However, the specific compounds involved in this compensation require further investigation [27].

The comparison of ergosterol content in P. djamor biomass revealed a substantial increase from 0.15 mg/100 g to 8.88 mg/100 g following oxybenzone supplementation. Ergosterol is a crucial sterol in fungal cell membranes, affecting their fluidity and integrity. Its biosynthesis is a complex process requiring multiple enzymatic steps. Various factors, such as oxygen availability, inhibitors, and precursor accessibility, have been reported to influence this process. However, there are no direct studies on the effect of oxybenzone on ergosterol biosynthesis in P. djamor. It is plausible that oxybenzone modulates the expression of sterol biosynthesis-related genes, alters precursor availability, or induces stress responses that lead to increased ergosterol production. Nevertheless, these mechanisms require further experimental confirmation [28,29].

A significant increase in phenylalanine content in P. djamor biomass following oxybenzone treatment (from 54.03 mg/100 g dry weight to 176.34 mg/100 g dry weight) indicates a strong impact of this compound on aromatic amino acid metabolism. Phenylalanine is a key product of the shikimate pathway, leading to the biosynthesis of aromatic amino acids (phenylalanine, tyrosine, tryptophan) and secondary phenolic compounds. Given the phenolic structure of oxybenzone, it may have influenced enzymes involved in this pathway, such as EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) and chorismate mutase-prephenate dehydratase, which play crucial roles in converting prephenate to phenylalanine and tyrosine [30].

The analysis of hydroxybenzoic acid content in P. djamor biomass indicates that its presence exclusively in cultures supplemented with oxybenzone may result from shikimate pathway activation or metabolic transformations leading to its synthesis. Oxybenzone may have influenced the activity of enzymes such as prephenate dehydratase or chorismate decarboxylase, facilitating the production of hydroxybenzoates. The increased biosynthesis of hydroxybenzoic acid could represent a protective mechanism in P. djamor against oxybenzone-induced oxidative stress [31]. The concentration of oxybenzone used in this study was higher than typical environmental levels, allowing for testing of remediation capacity and assessing the upper limits of the metabolic and remediation capabilities of P. djmor.

The comparison of the final average dry matter content of P. djamor mycelium obtained from in vitro cultures, which were supplemented with oxybenzone powder at a final concentration of 0.1 mg/mL and without oxybenzone (control), is shown in Figure 1. The addition of oxybenzone significantly inhibited the growth of P. djamor mycelium (one-way ANOVA followed by Tukey’s test, p < 0.05, n = 3).

Figure 2 compares the average milligrams of BP-3 absorbed by the mycelium of P. djamor after two weeks of growth with the final amount of 25 mg of oxybenzone added into Oddoux liquid medium. P. djamor significantly metabolized oxybenzone, reducing its initial concentration in the medium (p < 0.05, n = 3). After incubation, oxybenzone levels dropped from 25 mg to 1.5394 ± 0.0952 mg. Post-lyophilization, the mycelium contained 6.2067 ± 0.2406 mg of oxybenzone. Despite effective BP-3 degradation, P. djamor showed significantly lower growth compared to the control.

3.2. Identification of Biodegradation Products of Oxybenzone and Toxicity Evaluation

In this study, extracts from in vitro cultures of P. djamor were analyzed to determine the biotransformation products. The cultures were maintained with and without oxybenzone supplementation. Biotransformation products of oxybenzone were identified by comparing QToF detector chromatograms from samples incubated with (Figure 3A) and without (Figure 3B) oxybenzone, similar to previous studies on oxybenzone [25]. The protonated monoisotopic molecular masses (M+H)⁺ and their fragmentation spectra (with fragmentation energies ranging from 10 to 25 eV) were determined for peaks corresponding to oxybenzone transformation products. The structural formulas of the identified compounds were determined using the ChemDraw Std v.20.0 with Analysis package (CambridgeSoft). Low-molecular aliphatic reaction products were not identified. The identified probable products formed after oxybenzone incubation with P. djamor in vitro cultures are presented in Figure 4 and

Table 3. Detailed data of LC-MS/MS analysis of solutions after Pleurotus djamor incubation.

Retention Time (tr) (min)	Symbol in Figure	Summary Formula [M+H+]	Daughter Ions [m/z]; Formula
2.52	A	C16H17O4S	287.0738; C16H15O3S 269.0633; C16H13O2S 241.0684; C15H13OS
2.65	B	C16H17O4S	255.0478; C15H11O2S 227.052; C14H11OS 211.0576; C14H11S 209; C14H9S
2.89	C	C15H11O4	237.0552; C15H9O3 227.0707; C14H11O3 199.0758; C13H11O2 181.0650; C13H9O 137.0239; C7H5O3
3.01	D	C16H13O5	270.0524; C15H10O5 229.0863; C14H13O3
3.40	E	C13H11O3	137.0235; C7H5O3 105.0342; C7H5O
3.50	F	C15H15O2	91.0548; C7H7
3.56	G	C16H14NO4S	274.0538; C14H12NO3S 259.0305; C13H9NO3S 237.0094; C10H7NO4S 105.0336; C7H5O
4.02	H	C14H13O3	151.0400; C8H7O3 105.0345; C7H5O
4.48	I	C15H12NO3S	208.0072; C9H6NO3S 180.0120; C8H6NO2S 152.0164; C7H6NOS
4.78	J	C15H15O2	91.0548; C7H7
5.36	K	C15H15O3	165.0551; C9H9O3 105.0345; C7H5O
6.11	Oxybenzone	C14H13O3	151.0400; C8H7O3 105.0345; C7H5O
6.46	L	C15H15O2	151.0399; C8H7O3 91.0548; C7H7
7.08	M	C15H15O2	151.0399; C8H7O3 91.0548; C7H7

. In processes initiated by P. djamor, mainly acylated derivatives of oxybenzone are formed, which are products of esterification with low-molecular-weight compounds. The identified carbocations indicate that these are decomposition products of larger molecules with unstable molecular ions. Additionally, compounds with introduced thiol (sulfur) and amino (nitrogen) groups were identified. These may be condensation products of oxybenzone derivatives with medium components. Except for one product, 2,4-dihydroxybenzophenone (product E), the identified biodegradation products were completely different from those in the analogous process conducted for L. edodes [25]. Demethylation of the methoxy substituent (O-demethylation) leads to the formation 2,4-dihydroxybenzophenone—benzophenone 1. It is ‘light stabilizer’ and ‘UV absorber, and it protects products from UV-induced degradation. 2,4-Dihydroxybenzophenone has been detected in various environmental matrices, indicating its widespread presence and potential ecological impact. In groundwater samples, BP-1 concentrations have been reported to reach up to 0.22 µg/L. Surface water analyses have shown BP-1 levels as high as 3.72 µg/L, particularly in areas downstream of wastewater discharge points. Additionally, BP-1 has been identified in human biological fluids, including urine, placenta, and breast milk, suggesting its bioaccumulation and potential for human exposure [32,33].

Finally, the evaluation of toxicity risks associated with oxybenzone and its mycoremediation products was conducted for the biotransformation products of oxybenzone. Predictions were generated using the OSIRIS Property Explorer tool (accessed on 20 February 2025 from https://www.organic-chemistry.org/prog/peo/tox.html). The prediction of the mutagenic, tumorigenic, irritant, and reproductive effects of Products A-M was conducted (

Table 4. Toxicity risk assessment of oxybenzone and its biodegradation products using Pleurotus djamor mycelium generated by OSIRIS Property Explorer.

Compound	Mutagenic	Tumorigenic	Irritant	Reproductive Effects
Oxybenzone	known to be mutagenic	known to be tumorigenic	–	high-risk fragment
Product A	medium-risk fragment	–	–	–
Product B	–	–	medium-risk fragment	–
Product C	–	–	medium-risk fragment	–
Product D	–	–	–	–
Product E	high-risk fragment	medium-risk fragment	high-risk fragment	medium-risk fragment
Product F	–	–	medium-risk fragment	–
Product G	–	–	medium-risk fragment	–
Product H	–	–	–	–
Product I	medium-risk fragment	–	–	–
Product K	–	–	–	–
Product L	high-risk fragment	–	medium-risk fragment	high-risk fragment
Product M	–	–	–	–

). According to OSIRIS, the highest toxicity risk was identified for degradants E and L. No toxicity risk was indicated for products D, H, and M.

The predicted ecotoxicity of biodegradation products generated during incubation of P. djamor mycelium with oxybenzone was unexpectedly high (Figure 5). For instance, the compound labeled “L” exhibited an ecotoxicity more than five times greater than that of oxybenzone. The predicted ecotoxicity of products I and J was higher than that of oxybenzone, while products C, D G, H and K had ecotoxicity levels lower than those of the initial compound.

4. Conclusions

The analysis of P. djamor biomass grown under standard and oxybenzone-supplemented conditions revealed significant alterations in the profile of organic compounds with potential health benefits. Notably, exposure to oxybenzone resulted in a marked reduction in ergothioneine content, indicating a substantial impact on fungal metabolic activity. The observed shift towards increased synthesis of polyphenols and terpenoids suggests an adaptive metabolic response to the presence of this contaminant.

In total, thirteen oxybenzone degradation products were identified, several of which exhibited high predicted ecotoxicity. These findings highlight the complex interplay between environmental pollutants and fungal metabolism, demonstrating the sensitivity and adaptability of P. djamor. Importantly, the identification of biodegradation products with notable ecotoxicity emphasizes the need to thoroughly evaluate the ecological and health risks not only of the parent compound but also of its transformation products.