Toxicity of organic (benzophenone-3) and inorganic (titanium dioxide and zinc oxide nanoparticles) ultraviolet filters on growth and metabolic activity of fungi cultured from the marine shallow-water hydrothermal vents of Kueishan Island, Taiwan

Toxicity of organic (benzophenone-3) and inorganic (titanium dioxide and zinc oxide nanoparticles) ultraviolet filters on growth and metabolic activity of fungi cultured from the marine shallow-water hydrothermal vents of Kueishan Island, Taiwan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Toxicity of organic (benzophenone-3) and inorganic (titanium dioxide and zinc oxide nanoparticles) ultraviolet filters on growth and metabolic activity of fungi cultured from the marine shallow-water hydrothermal vents of Kueishan Island, Taiwan Lam Kong, Wing-Fai Lu, Ka-Lai Pang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6210800/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Ecotoxicology → Version 1 posted 9 You are reading this latest preprint version Abstract The increased awareness of the damaging effects of ultraviolet radiation from the sun has promoted the use of sunscreen products. The active ingredients of sunscreen lotion, i.e. benzophenone-3 (BP-3), titanium dioxide (TiO 2 ) nanoparticles (NPs), and zinc oxide (ZnO) NPs, can pollute the marine environment through runoff or human activities such as swimming. Early studies have revealed the toxic effects of these sunscreen active ingredients on aquatic animals, however, their effects on the marine decomposer community are less known, especially on fungi. This study investigated the effect of BP-3, TiO 2 NPs, and ZnO NPs on growth and metabolic activity of selected fungi isolated from the marine shallow-water hydrothermal vent ecosystem at Kueishan Island, Taiwan. Growth inhibition was observed for the majority of the tested fungi (especially on Aspergillus spp.) by increased concentrations of ZnO NPs (0-100 mg/L). In contrast, TiO 2 NPs and BP-3 exerted little effect on fungal growth. The differences in toxicity between ZnO NPs and TiO 2 NPs might be attributed to variations in their solubility, size, and shape. Surprisingly, BP-3 exhibited the least toxicity on fungal growth, despite its known effects on other marine organisms at very low concentrations. The toxicity of ZnO NPs (12.5 mg/L) on metabolic activity of the growth-inhibited fungi, using Biolog FF MicroPlate, was also examined, i.e. Aspergillus tubingensis NTOU5277, A. terreus NTOU5276 and A. terreus NTOU4989. A significant reduction in average well colour development (AWCD) was observed in the presence of ZnO NPs, suggesting an overall reduction in metabolic activity. Interestingly, the average well turbidity development (AWTD) of A. tubingensis NTOU5277 in the presence of ZnO NPs was higher than that of the control group without ZnO NPs. In terms of carbon utilization, D-galactose, γ-hydroxy-butyric acid, and L-proline were not utilized by A. tubingensis NTOU5277 in the presence of ZnO NPs, with the latter two compounds being related to the tricarboxylic acid (TCA) cycle. Aspergillus terreus isolates NTOU5276 and NTOU4989 showed a reduction in the utilization of L-phenylalanine and β-hydroxy-butyric acid in the presence of ZnO NPs, respectively. These results suggest the potential toxic effects of ZnO NPs on energy production and metabolism in fungi and highlight the prospect of using Biolog FF MicroPlate for assessing metabolic effects of other anthropogenic pollutants on fungi. UV-filters Nanoparticle Fungi Microbial ecotoxicology Growth inhibition Biolog FF MicroPlate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Sunscreen lotion contains ultraviolet (UV) filters to shield harmful UV rays in sunlight when applied to the skin, preventing sunburn, aging, and skin cancer (Kim and Choi, 2014 ; Schneider and Lim, 2019 ; Zhang et al., 2021 ). Generally, the UV filters can be divided into inorganic metal oxide nanoparticles (NPs) which include zinc oxide (ZnO) and titanium oxide (TiO 2 ), while the organic UV filters include benzophenone and its derivatives. Inorganic UV filters (e.g. ZnO and TiO 2 NPs) are coated with a surface layer, which absorb and reflect solar UV radiation, thus preventing UV from reaching the skin (Tovar-Sánchez et al., 2013 ; Yuan et al., 2022 ), whereas organic UV filters absorb and stabilize solar UV radiation (Sambandan and Ratner, 2011 ; Kim and Choi, 2014 ; Stiefel and Schwack, 2015 ). Over 10,000 tons of UV filters are estimated to be produced every year and incorporated into sunscreen products (Zhang et al., 2021 ). These UV filters can be released into the aquatic environment directly through washing off from skin during recreational activities or indirectly through the discharge of wastewater (Du et al., 2017 ; Zhang et al., 2017 ; Chatzigianni et al. 2022 ). UV filters have been detected in the water of rivers, lakes, seashores, and/or sewage treatment plants (Labille et al. 2020 ). Sunscreen concentrations (including both organic and inorganic UV filters) at an Atlantic beach ranged from 10 to 96.7 mg/L in the unfiltered fraction and up to 75.7 mg/L in the dissolved fraction (Tovar-Sánchez et al., 2020 ). On beaches of the Mediterranean Coast, concentrations of TiO 2 NPs in the surface water layer (top 1 cm) of the bathing zone and the water column below were 100–900 and 20–50 µg/L, and those of ZnO NPs were 10–15 and 1–3 µg/L, respectively (Labille et al., 2020 ). Benzophenone-3 (BP-3) concentrations in seawater collected at sites of the US Virgin Islands and Hawaii were 75 µg/L-1.4 mg/L and 0.8–19.2 µg/L, respectively (Downs et al., 2016 ). In Taiwan, BP-3 was detected in seawater of seven sampling sites at Kenting National Park in southern Taiwan, and its concentration ranged from 9.3 ± 6.8 ng/L at Houbihu to 514.6 ± 282.0 ng/L at Baisha Beach (Ku et al., 2020 ). However, the concentration of BP-3 in other coastal areas of Taiwan is not known. Also, no information is available on the concentration of TiO 2 and ZnO NPs in the coastal waters of Taiwan. UV filters pose a potential threat to marine life and were reported to exert acute and chronic effects on marine organisms. For example, the LC50 of coral planulae exposed to benzophenone-3 (BP-3) in the light for an 8h and 24h exposure was 3.1 mg/L and 139 µg/L, respectively while that in darkness were 16.8 mg/L and 779 µg/L (Downs et al., 2016 ). The EC50 of ZnO NPs in the alga Dunaliella tertiolecta , the bioluminescent bacterium Vibrio fischeri , and the crustacean Artemia salina was 2.2 mg/L, 17 mg/L, and 58 mg/L, respectively (Schiavo et al., 2018 ). TiO 2 NPs induced oxidative stress and was bioaccumulated in Artemia salina after exposure for 48 hours (Bhuvaneshwari et al., 2018 ) Kueishan Island, situated at the southernmost part of the Okinawa Trough in the western Pacific Ocean, is a region of both economic and ecological significance in Taiwan. The area features approximately 50 shallow-water hydrothermal vents at depths ranging from 10 to 80 meters (Yang et al., 2005 ; Pang et al., 2020 ; Wang et al., 2022 ), which continuously emit hydrothermal fluids and volcanic gases (Yang et al., 2005 ; Wang et al., 2022 ), making the seawater so called ‘the milky sea’. During the summer months in the last few years, human water activities have increased near/at the hydrothermal vent area and sunscreen on skin may wash off from the visitors. Kueishan Island is only 10 km away from the Yilan coast, chemical pollution from the main Taiwan island may also enter the hydrothermal vent ecosystem. Although the toxicological effects of UV filters on marine vertebrates and invertebrates and some algae have been widely studied, their impact on microbial decomposers such as fungi remain largely unknown. Fungi are an integral part of the eukaryotic degrader community of the sea, playing a pivotal role in degrading recalcitrant substrate and providing various nutrients to the marine food web (O’Rorke et al., 2014 ; Jones et al., 2019 ; Cunliffe, 2023 ), yet little attention has been given to how UV filters influence their growth and metabolic activity. Given that UV filters can induce oxidative stress and disrupt cellular processes, they may pose a significant treat to marine fungal communities. Additionally, these UV filters, introduced by the increasing human activity, may disrupt the proper ecosystem functioning at the hydrothermal vents of Kueishan Island. To address this gap, this study investigated the effects of inorganic (TiO 2 and ZnO NPs) and organic (benzophenone-3) UV filters on growth and metabolic activity of fungi cultured from substrates collected at/near the hydrothermal vents of Kueishan Island (Pang et al., 2019 ). By employing the Biolog FF Microplate assay, we also assessed how these compounds impact fungal metabolism and carbon utilization, shedding light on the potential ecological consequences of UV filter pollution in the marine environment. 2. Materials and methods Fungal cultures The fungi isolated (Table 1 ) from the vent crab Xenograpsus testudinatus and sediment samples collected at/near the hydrothermal vent system of Kueishan Island (121˚57’6.01" E, 24˚50’30.98" N) between 2015 and 2017 were kept on CMAS (17.0 g/L cornmeal agar, 34 g/L sea salt) agar plates (Pang et al., 2019 ). Table 1 Culture number and species name of selected fungi isolated from Kueishan Island. Substrate Culture number Species name Yellow sediment NTOU4989 Aspergillus terreus NTOU4992 Penicillium sp. NTOU5414 Chondrostereum sp. NTOU5435 Acremonium brunnescens NTOU5440 Trichoderma harzianum Black sediment NTOU5275 Parengyodontium album NTOU5276 Aspergillus terreus NTOU5277 Aspergillus tubingensis NTOU5278 Aspergillus versicolor NTOU5282 Microascales sp. NTOU5284 Aspergillus versicolor The vent crab Xenograpsus testudinatus NTOU5287 Hypocreales sp. NTOU5289 Hortaea werneckii NTOU5290 Tritirachium sp. NTOU5292 Microascus brevicaulis Chemical preparation and characterization ZnO nanoparticles (particle size < 5 µm, 99.9% purity), TiO 2 nanoparticles (particle size < 25 nm, 99.7% purity), and BP-3 (98% purity) were purchased from Sigma-Aldrich. The stock solution (100 mg/L) of ZnO and TiO 2 was prepared by adding 1% malt extract (Bacto™, Sparks, USA) seawater broth (34 PSU) and was dispersed by sonication (Delta DC150H; 150 W; 40 kHz) at 25°C for 30 min. The stock solution (100 mg/L) of BP-3 was dissolved in 0.01% dimethyl sulfoxide (DMSO, Sigma-Aldrich, purity > 99%) and in 1% malt extract seawater broth, stored in the dark at 4°C. Transmission electron microscopy (Hitachi HT7700) was performed by placing 8 µL drop of stock solution onto a formvar-coated grid. After air-drying, the sample was examined at an accelerating voltage of 75 kV to assess the particle size and morphology of the obtained ZnO and TiO 2 NPs. Using Image J software, the mean size distribution of both ZnO and TiO 2 NPs was determined by measuring 100 randomly selected particles. Growth study A series of concentrations (0, 3.13, 6.25, 12, 5,25, 50, 100 mg/L) of the three compounds was prepared from the stock solutions and dispensed (180 µL) into wells of a microtiter plate (Corning, Maine, USA). A solvent control (0.01% DMSO) was included for BP-3 growth test. Suspensions of hyphae and spores were made by adding 3 mL of 0.1% Tween 80 (Honeywell Fluka, Seelze, Germany) on the top of the fungal colonies and the plates were gently shaken or scrubbed to dislodge the spores or hyphae. The suspensions were counted using a hemocytometer (Bright-Line hemocytometer, Hausser Scientific, Horsham, PA, USA) and adjusted to 2×10 4 spores(hyphae)/mL. Spore/hyphal suspension (20 µL) was added to the wells of a microtiter plate, preloaded with the different concentrations of ZnO NPs, TiO 2 NPs and BP-3. Six replicates were inoculated for each treatment. The plates were placed in the incubator (Yih Der LM 570RD, Taiwan) at 25°C for two weeks without shaking. The absorbance was measured periodically at 630 nm by the multi-detection microplate readers (Synergy HT, BioTek) within a 14-day period. Metabolic profiling of fungi exposed to ZnO nanoparticles Aspergillus terreus NTOU4989, A. terreus NTOU5276, and A. tubingensis NTOU5277 (based on the results of the growth experiment) were subcultured on CMAS agar plates for 10 days at 25°C. Spore suspensions were prepared by adding 3 mL of a solution containing 0.1% Tween 80, 3.4% sea salt, and 12.5 mg/L ZnO NPs (the lowest observed effect concentration (LOEC) for the three fungi) on the top of the fungal colonies. The plates were gently shaken or scrubbed to dislodge the spores and the spore suspensions were counted using a hemocytometer and adjusted to 2×10 5 spores/mL. The spore suspensions in 100 µL were added to the wells of the Biolog FF MicroPlate™. Three replicates were done for each isolate and the plates were incubated at 25°C. The absorbance was measured every 24 h at 490 nm and 750 nm by the multi-detection microplate readers for 7 days. The reading at 490 nm was subtracted from that at 750 nm to determine the corrected redox value (CRV), which represents metabolic capability, for the reduction of iodonitrotetrazolium (INT) in each well. Functional diversity was determined by the number of different substrates utilized, which was calculated by counting all positive optical densities (OD) readings with a threshold of ≥ 0.25. Average well color development (AWCD; Garland and Mills, 1991 ) and average well turbidity development (AWTD; Klimek and Niklińska, 2007 ) represent average metabolic capability and average mycelial production, respectively. The calculation of AWCD and AWTD is described below: $$\:AWCD=\frac{\sum\:_{i=1}^{n}\left({C}_{i}-R\right)}{n}$$ 1 C i : CRV of each well R: CRV of control well n: number of carbon-containing substrates (total 95) in the Biolog FF MicroPlate™ $$\:AWTD=\:\frac{\sum\:_{i=1}^{n}{(C}_{i}-R)}{n}$$ 2 C i : OD 750 nm reading of each well R: OD 750 nm reading of control well n: number of carbon-containing substrates (total 95) in the Biolog FF MicroPlate™ 2.5. Statistical analyses Origin 9.1 (OriginLab Corporation, USA) was used to fit the growth model. The sigmoidal function, logistic I, had the best fit for the data. The formula of this generalized logistic model is $$\:\text{y}=\frac{\text{a}}{1+{e}^{-k\left(x-{x}_{c}\right)}}$$ 3 where, y represents the observed optical density at time x; a, the upper asymptote; xc, the point of inflection on the x-axis; k, growth rate constant; e, the base of the natural logarithm. Statistical analyses were performed using SPSS 25 (IBM Corp., Armonk, New York, USA). Shapiro-Willk and Levene’s tests were utilized to assess normality and homogeneity of variances across all groups. One-way ANOVA and Tukey test were used to assess the significance of the absorbance on the last incubation day for the growth inhibition test and t-tests were conducted to assess the significance between with and without ZnO NPs exposure in AWCD and AWTD for the metabolic profiling test. 3. Results Characterization of ZnO and TiO 2 nanoparticles TEM images revealed that ZnO NPs predominantly exhibited a rod-like morphology, with a mean length of 189.6 ± 63.6 nm and width of 75.7 ± 22.8 nm (Fig. 1 a). Similarly, the rod-shape TiO 2 NPs appeared relatively uniform, with a mean length of 37.6 ± 7.1 nm and width of 18.5 ± 3.6 nm (Fig. 1 b ) . Both ZnO and TiO 2 NPs showed some degree of agglomeration, as individual particles tended to clump together in seawater. Growth study The growth curves of the 15 fungi incubated for 14 days under 0 mg/L to 100 mg/L of ZnO NPs are shown in Fig. 2 . Generally, all fungi exhibited an increased growth trend over time at all concentrations, except for Aspergillus tubingensis NTOU5277, where no/little growth was observed at 50 mg/L and 100 mg/L. Eight fungal isolates ( Aspergillus terreus NTOU4989, Penicillium sp. NTOU4992, Aspergillus terreus NTOU5276, Aspergillus tubingensis NTOU5277, Microascales sp. NTOU5282, Aspergillus versicolor NTOU5284, Chondrostereum sp. NTOU5414, and Trichoderma harzianum NTOU5440) exhibited lower growth rates at higher ZnO NPs concentrations (50 mg/L and 100 mg/L). Aspergillus terreus NTOU4989 and Trichoderma harzianum NTOU5440 reached growth saturation on Day 6 at lower concentrations (3.13 mg/L and 6.25 mg/L) of ZnO NPs, whereas growth saturation was not reached at higher concentrations throughout the experimental period. In contrast, A. versicolor NTOU5284 only reached growth saturation at higher concentrations of ZnO NPs. A slower growth rate was observed at higher concentrations in Penicillium sp. NTOU4992, A. terreus NTOU5276, A. tubingensis NTOU5277, Microascales sp. NTOU5282, and Chondrostereum sp. NTOU5414, and growth did not reach saturation. Aspergillus versicolor NTOU5278 displayed a slower growth rate in the first 7 days at higher concentrations, but its growth rate was similar to other concentrations after 7 days. Figure 3 illustrates the final growth (optical density) of the fungi on Day 14 under the different ZnO NPs concentrations. The growth at 50 mg/L and 100 mg/L was significantly lower ( p < 0.05) than the control in A. terreus NTOU4989, A. terreus NTOU5276, A. tubingensis NTOU5277, A. versicolor NTOU5284, Chondrostereum sp. NTOU5414, and Trichoderma harzianum NTOU5440. Although lower growth at higher concentrations was observed in Penicillium sp. NTOU4992 and Microascales sp. NTOU5282, the differences were not statistically significant. Conversely, no significant dose-dependent response was observed in the remaining fungal isolates. Growth rates for the majority of the tested fungi under the different concentrations of TiO 2 NPs were similar, except for Penicillium sp. NTOU4992 and Aspergillus versicolor NTOU5278, which showed a decreased growth rate at 6.25 mg/L to 100 mg/L and 25 mg/L to 100 mg/L, respectively (Fig. 4 ). Similarly, no significant dose-dependent response was observed in the remaining fungal isolates. The growth of Penicillium sp. NTOU4992, A. versicolor NTOU5278 and Microascales sp. NTOU5282 was significantly lower ( p < 0.05) than the control at higher concentrations (Fig. 5 ). No statistically significant difference was observed between the absorbance of solvent control (0.01% DMSO) and 0 mg/L BP-3 (results not shown). Similar growth rate was observed for most of the tested fungi under all concentrations of BP-3 over 14 days except for Penicillium sp. NTOU4992 and A. versicolor NTOU5278 (Fig. 6 ). No significant dose-dependent response was observed in the remaining fungal isolates. The growth of Penicillium sp. NTOU4992, Aspergillus versicolor NTOU5278 and Microascus brevicaulis NTOU5292 was significantly lower ( p < 0.05) at higher concentrations of BP-3 (Fig. 7 ). Metabolic activity of fungi after exposure to ZnO nanoparticles Metabolic activity of Aspergullus tubingensis NTOU5277, A. terreus NTOU5276, and A. terreus NTOU4989 were studied using the Biolog FF MicroPlate with or without ZnO NPs (12.5 mg/L). In the absence of ZnO NPs, the percentage utilization of carbon sources was 88.4% (84/95 substrates), 88.4% (84/95), and 87.3% (83/95) for A. tubingensis NTOU5277, A. terreus NTOU5276, and A. terreus NTOU4989, respectively; in the presence of ZnO NPs, they were 85.2% (81/95), 87.3% (83/95), and 88.4% (84/95). The AWCD was significantly higher without ZnO NPs exposure in A. tubingensis NTOU5277, A. terreus NTOU5276, and A. terreus NTOU4989 (Fig. 8 a). On the other hand, the AWTD of A. terreus NTOU4989 was significantly higher in the control group, while the AWTD of A. tubingensis NTOU5277 was significantly lower ( p < 0.05) in the control group; the AWTD of A. terreus NTOU5276 remained similar after 7 days of exposure to ZnO NPs (Fig. 8 b). The carbon source utilization pattern is shown in Fig. 9 . The three isolates of Aspergillus exhibited low utilization (CRV < 0.25) of L-fucose, D-galacturonic acid, glucuronamide, γ-amino-butyric acid, bromosuccinic acid, fumaric acid, sebacic acid, succinamic acid, succinic acid mono-methyl ester, N-acetly-L-glutamic acid, and L-pyroglutamic acid, with or without ZnO NPs exposure. D-galactose, γ-hydroxy-butyric acid, and L-proline were poorly utilized (CRV < 0.25) by A. tubingensis NTOU5277 in the presence of ZnO NPs. L-phenylalanine and β-hydroxy-butyric acid were the only carbon sources poorly utilized by A. terreus NTOU5276 and A. terreus NTOU4989, respectively. 4. Discussion Toxicological effect of ZnO NPs, TiO₂ NPs, and BP-3 on marine fungi In recent years, tourism in the water around Kueishan Island has been steadily increasing, leading to greater anthropogenic impacts on this economically and ecologically important territory. The rise in tourist numbers, driven primarily by favorable weather, the unique colour of the ‘milk sea’, and increased awareness of UV protection, likely results in increased application of sunscreen products on the skin which may be washed off and suspend/settle in the sea and impose adverse effects on marine organisms. Among the three UV filters, ZnO NPs had the strongest inhibition effect on the growth of Aspergillus terreus NTOU4989, A. terreus NTOU5276, A. tubingensis NTOU5277, A. versicolor NTOU5284, Chondrostereum sp. NTOU5414 and Trichoderma harzianum NTOU5440. Aspergillus terreus NTOU4989 and other Aspergillus species was found to be adaptive to changes in environmental conditions in the marine environment, with their genetic capability (Pang et al. 2020 ). Aspergillus species may represent one of the key decomposers in the marine environment (Chou et al. 2022 ), while ZnO NPs may interfere them from fulfilling that role. The fungicidal activity of ZnO NPs was previously known as it was also used as an effective fungicide in food safety, agriculture, and clean water (Sun et al., 2018 ; Paraguay-Delgado et al., 2022 ). The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) of Penicillium expansum were found to be 798 mg/L and 147 mg/L, respectively; the MIC of Aspergillus niger was 2.5 mg/L ZnO NPs as a fungicide (Gondal et al., 2012 ; Sardella et al., 2018 ). ZnO NPs are able to interact with fungal cell membranes, altering their permeability and causing leakage of intracellular components, which leads to cell death (Adams et al., 2006 ). However, the impact of ZnO NPs is not limited to fungi as they also exhibit varying levels of toxicity toward other marine microorganisms, including cyanobacteria and microalgae. A recent study on Synechococcus sp. demonstrated that exposure to ZnO NPs at concentrations as low as 1.4 mg/L led to increased reactive oxygen species (ROS) production, disruption of the photosynthetic apparatus, and a decline in chlorophyll and phycoerythrin content (Shoman et al., 2025 ). ZnO NPs were found to be the most toxic among the nanoparticles, exhibiting significantly higher toxicity compared to TiO 2 and other metal oxide nanoparticles (Aruoja et al., 2009 ; Wang et al., 2009 ; Miller et al., 2010 ; Dasari et al., 2013 ; Bhuvaneshwari et al., 2017 ). For example, the EC50 of ZnO NPs and TiO 2 NPs on the microalga Pseudokirchneriella subcapitata was 0.042 mg/L and 5.83 mg/L, respectively (Aruoja et al., 2009 ). ZnO caused a significant reduction in growth rate in marine phytoplankton whereas TiO 2 did not (Miller et al., 2010 ). ZnO NPs release free zinc ions (Zn 2+ ) in water and can rapidly dissolve in seawater (Aruoja et al., 2009 ; Miller et al., 2010 ; Bhuvaneshwari et al., 2017 ; Yan et al., 2023 ). These free Zn 2+ ions induce the generation of ROS which can cause cell damage such as DNA damage, lipid peroxidation, and protein denaturation (Nel et al., 2006 ; Xia et al., 2008 ; Hou et al., 2018 ). The release of Zn 2+ from uncoated zinc NPs (8.9 mg/L) was more efficient than zinc oxide NPs (5.5 mg/L), and the former exhibited higher toxicity towards Artemia salina (Ates et al., 2013 ). Thus, the toxicity of ZnO NPs on the fungi in this study may result from two mechanisms: direct contact with the nanoparticles and the dissolution of Zn 2+ ions. In contrast, the toxicity of TiO 2 NPs is likely caused only by direct nanoparticle contact, as TiO 2 NPs are insoluble in seawater and requires strong acids for dissolution. Although both ZnO and TiO 2 NPs can generate reactive oxygen species (ROS), their toxicity level differs due to differences in solubility (Blake et al., 1999 ; Hanley et al., 2008 ; Xia et al., 2008 ; Miller et al., 2010 ; Singh et al., 2020 ; Alabdallah et al., 2024 ). Moreover, TiO 2 NPs aggregate more rapidly in seawater than ZnO NPs, thus reducing their toxicity toward marine organisms (Bhuvaneshwari et al., 2017 ). The toxicity of nanomaterials is closely linked to their physicochemical properties, such as size and shape (Hou et al., 2018 ; Jin et al., 2021 ). For instance, the rod-like ZnO NPs are more toxic than the spherical ZnO NPs by having a large contact area (Hou et al., 2018 ). Our TEM analysis confirmed that ZnO NPs in this study were predominantly rod-shaped, which may have contributed to their toxicity. The toxicity between of inorganic and organic UV filters has rarely been discussed. In our study, growth inhibition was not observed for most of the tested fungi after BP-3 exposure. It is interesting to note that a very low concentration (µg/L to ng/L) of BP-3 could cause various toxicological effects to marine animals and microorganisms such as genotoxicity (Almeida et al., 2019 ), oxidative stress (Liu et al., 2015a ; Zhang et al., 2021 ), and growth inhibition (Paredes et al., 2014 ; Liu et al., 2015b ; Mao et al., 2017 ). However, only 2 out of 27 bacterial isolates belonging to the phyla Bacteroidetes and Proteobacteria were growth-exhibited under 1 mg/L BP-3 (Lozano et al., 2020 ). Microorganisms including fungi and bacteria may possess unique physiological or metabolic mechanisms that can counteract the presence of BP-3, such as specialized transport systems, detoxification, or enzymatic degradation pathways that mitigate the effects of such pollutants (Lozano et al., 2020 , 2021 ). The underlying biological processes that enable these microorganisms to withstand environmental stressors posed by organic UV filters will require further study. Metabolic profiling of fungi exposed to ZnO NPs After 7 days of exposure to ZnO NPs (12.5 mg/L), all Aspergillus spp. were able to metabolize 80–90% of the tested carbon sources in the Biolog FF MicroPlate, but the average utilization of carbon sources (AWCD) was significantly lower in the control when no ZnO NPs was added. Interestingly, despite lower metabolic capability, the mycelial production (AWTD) varied among species. For example, A. tubingensis NTOU5277 had higher mycelial production even though its AWCD was lower in the presence of ZnO NPs. This result is similar to the findings by Chou et al. ( 2022 ), in which A. terreus NTOU4989, incubated at 25°C and pH 3, had lower metabolic activity (AWCD) but higher biomass production (AWTD) compared to incubation at pH 7. The higher mycelial production in A. tubingensis NTOU5277 may be attributed to the utilization of D-gluconic acid and glycogen. The utilization of these carbon sources was not significantly affected by ZnO NPs exposure, and may have provided an alternative energy source, allowing the fungus to sustain growth even when other metabolic pathways were disrupted. Aspergillus tubingensis NTOU5277 may possess a selective carbon utilization strategy that enables it to compensate for unfavorable growth conditions caused by ZnO NPs. In addition, Wang et al. ( 2016 ) found that Fusarium kyushuense exposed to fungicides Azoxystrobin and Kresoxim-methyl showed a reduced metabolic capacity for these substances. γ-hydroxybutyric acid and L-proline are key compounds in the tricarboxylic acid (TCA) cycle by contributing to ATP production and cellular respiration. The inability to metabolize these compounds under ZnO NPs exposure suggests a disruption in the energy-generating pathways, which may reduce the fungus’s capacity to cope with oxidative stress, and may render the organisms more vulnerable to environmental changes (Fuentes and Quiñones, 2016 ; Wang et al., 2016 ). However, the lack of utilization of D-galactose, γ-hydroxybutyric acid, and L-proline did not seem to impede the growth in A. tubingensis NTOU5277 as AWTD was higher than the control. Aspergillus tubingensis NTOU5277 may be able to survive in environments with limited carbon sources by significantly increasing the utilization of specific carbon sources when faced with unfavorable conditions. In contrast, A. terreus NTOU5276 and A. terreus NTOU4989 exhibited only minor reductions in the metabolism of L-phenylalanine and β-hydroxybutyric acid, respectively, in the presence of ZnO NPs. These species maintain relatively stable metabolic activity even under ZnO NPs exposure, suggesting a higher resilience to Zn²⁺ toxicity. This result aligns with that of Klimek and Niklińska ( 2007 ) who also observed a low impact of Zn²⁺ (300 mg/L) on the metabolic activity of fungi. The ability to sustain metabolic function despite nanoparticle stress may contribute to their higher recovery potential in contaminated environments. Potential environmental implications of UV filter exposure While the concentration of ZnO NPs used in this study (12.5 mg/L) is higher than the typical levels detected in open seawater, the potential for localized accumulation should not be overlooked. ZnO NPs tend to aggregate and settle in marine sediments, where their concentrations can be significantly higher than in the surrounding water column (Labille et al. 2020 ). In heavily impacted coastal areas, such as those with high tourism activity, sunscreen concentrations were reported to reach up to 96.7 mg/L (Tovar-Sánchez et al., 2020 ). Given that the fungi examined in this study were originally isolated from sediments near hydrothermal vents at Kueishan Island, where ZnO NPs can settle and accumulate. Sediment-associated fungal communities may be at an elevated risk of exposure to ZnO NPs, potentially facing greater toxic effects than pelagic microbial populations. Such prolonged exposure could disrupt fungal-mediated organic matter decomposition and nutrient cycling, processes that are critical in benthic ecosystems (Cunliffe, 2023 ). Comparing the effects of the three tested UV filters, our results align with previous studies indicating that ZnO NPs generally exhibit higher toxicity than TiO 2 NPs across a wide range of marine organisms (Aruoja et al., 2009 ; Schiavo et al., 2016 ; Schiavo et al., 2018 ; Corinaldesi et al., 2018 ; Yuan et al., 2023 ). While there is a growing movement toward eco-friendly sunscreens, the use of organic-based UV filters such as oxybenzone and octinoxate has been heavily restricted or outright banned in multiples regions, including Hawaii, Palau, and parts of the European Union, due to their well-documented role in coral reef bleaching and disruption of marine ecosystems (Miller et al., 2021 ; Chatzigianni et al. 2022 )). This shift in regulation has led to an increased reliance on inorganic-based (ZnO and TiO 2 ) alternatives (Hojerov´a et al., 2011 ; Blasco et al., 2020 ). However, despite their low impact on corals, the continued use of ZnO and TiO 2 still raises concerns regarding their potential impact on marine microbial ecosystems. Marine fungi and other sediment-associated microorganisms play essential roles in organic matter decomposition and nutrient cycling, prolonged exposure to these compounds could have cascading ecological effects. As research continues to explore the environmental consequences of inorganic UV filters, developing alternative sunscreen formulations that replace ZnO with less toxic yet effective UV-blocking agents may be a viable future direction for reducing the unintended impacts of sunscreen pollution on marine ecosystem. The observed metabolic responses further highlight the potential of fungal communities as bioindicators for ZnO NP contamination. ZnO NPs exposure resulted in reduced metabolic, particularly in carbon utilization pathways, similar metabolic profiling approaches could be applied in environmental monitoring programs to assess the extent of contamination in marine ecosystems. This study provides the first insight into the effects of inorganic and organic UV filters on the growth and metabolic activity of marine fungi. Future research should focus on long-term exposure studies at environmentally relevant concentrations, particularly in sediment and biofilm environments where ZnO NPs are likely to persist. Additionally, investigating the combined effects of multiple UV filters, as well as their interaction with natural environmental factors such as organic matter and salinity, would provide a more comprehensive understanding of their ecological risk. 5. Conclusion This is the first study to report the potential effects of inorganic and organic UV filter exposure on the growth and metabolic activity of marine fungi. Growth for most of the fungi was not adversely affected by TiO 2 NPs and BP-3 exposure, whereas ZnO NPs had a significant growth inhibition for some fungi, especially on Aspergillus spp. Further metabolic profiling of A. tubingensis NTOU5277, A. terreus NTOU5276, and A. terreus NTOU4989 using Biolog FF MicroPlate in the presence of ZnO NPs indicated a reduction in metabolic capacity, with variations in mycelial growth and carbon source utilization. The variations in physiological response to the three UV filters highlights the different sensitivities of the fungi towards these pollutants. It will be interesting in the future to study the effect of mixed UV filters on the growth of fungi, their degradation of organic substrates, ROS generation, and transcriptome changes so as to provide a more comprehensive understanding of how UV filters affect the ecological functioning of fungal community in the marine environment. Declarations Author Contribution Lam Kong: Methodology, Investigation, Data curation, Writing – original draft. Ka-Lai Pang: Methodology, Investigation, Supervision, Writing – review & editing. Wing fai Lu: Conceptualization, Investigation, Methodology. Acknowledgement This study was funded by the National Science and Technology Council of Taiwan (110-2621-M-019-002-, 111-2621-M-019-002-, and 112-2621-M-019-003-). The authors thank the Electron Microscopy Center of Institute of Marine Biology at National Taiwan Ocean University for technical assistance. References Adams, L. K., Lyon, D. Y., & Alvarez, P. J. (2006). Comparative eco-toxicity of nanoscale TiO 2 , SiO 2 , and ZnO water suspensions. Water research, 40 (19), 3527–3532. Alabdallah, N.M., Alluqmani, S.M., Almarri, H.M., AL-Zahrani, A.A., 2024. Physical, chemical, and biological routes of synthetic titanium dioxide nanoparticles and their crucial role in temperature stress tolerance in plants. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6210800","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":428068793,"identity":"fb5ee3db-5dea-4e25-9a0c-f28672676988","order_by":0,"name":"Lam Kong","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Lam","middleName":"","lastName":"Kong","suffix":""},{"id":428068794,"identity":"9a0d3a24-775c-4749-81ad-065ee7a01d6b","order_by":1,"name":"Wing-Fai Lu","email":"","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Wing-Fai","middleName":"","lastName":"Lu","suffix":""},{"id":428068795,"identity":"83788169-d435-41a6-8d88-ca856523bd25","order_by":2,"name":"Ka-Lai Pang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACCXYwZcPAwMzcABM0wK+FGUylAbUwArUkwLQkENRyGIiJ1SLZzHvwc8Gv89H87YwNzIU/bBIb2Ju3STD+OIxTizQzX7L0zL7buTMOA7XMSEhLbOA5VibBkIBbixwzj4E0b8/t3AaQFp6Ew4kNEjlmQC238Wkx/s3bcy53PlyL/Bv8WqSZecykeX4cyN2AsIUHvxbJZr40a96G5NyNQC2HedLSjNt40ootEtL+49Qicbz38G2eP3a5884fPviYx8ZGtp/98MYbH2zScGphYOABxkgbhHkARLCBiAQ8GsBaGP7gVTEKRsEoGAUjHQAAdhxPRdtQWDsAAAAASUVORK5CYII=","orcid":"","institution":"National Taiwan Ocean University","correspondingAuthor":true,"prefix":"","firstName":"Ka-Lai","middleName":"","lastName":"Pang","suffix":""}],"badges":[],"createdAt":"2025-03-12 09:38:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6210800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6210800/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10646-025-02971-z","type":"published","date":"2025-09-25T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78652011,"identity":"ae69ebc6-d98c-41c6-8f3e-92e983370e25","added_by":"auto","created_at":"2025-03-17 08:39:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":786135,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of ZnO\u003csub\u003e \u003c/sub\u003e(a) and TiO\u003csub\u003e2\u003c/sub\u003e (b) NPs from stock solution (100 mg/L).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/ace233df910b3cb6385bc1ad.png"},{"id":78651125,"identity":"8fadf2f9-d5b7-44e4-a52f-8e3a79dc184d","added_by":"auto","created_at":"2025-03-17 08:31:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1190109,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth curves of tested fungi under different ZnO NPs concentrations are based on fitting of the generalized logistic model, logistic I.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/5935dd660495b6ca88ca82e2.png"},{"id":78651127,"identity":"cfe4469f-a467-4756-8a5e-9828787d18a1","added_by":"auto","created_at":"2025-03-17 08:31:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1320271,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of tested fungi under different ZnO NPs concentrations based on the absorbance of Day 14 at the stationary phase of growth or the growth did not reach the stationary phase. Data are presented as mean ± standard deviation. Different letters (a, b, c) indicate significant differences among concentrations identified by Tukey test at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/9c87c6513a2b2a91575ae751.png"},{"id":78652492,"identity":"5abd5f4d-7f35-4982-9fe7-7bf819cd6077","added_by":"auto","created_at":"2025-03-17 08:47:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1108364,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth curves of tested fungi under different TiO\u003csub\u003e2\u003c/sub\u003e NPs concentrations are based on fitting of the generalized logistic model, logistic Ⅰ.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/afff29aba086058f71026bb2.png"},{"id":78652491,"identity":"35dc303f-fe1c-49a4-a9c0-85c0203c8a98","added_by":"auto","created_at":"2025-03-17 08:47:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1265630,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of tested fungi under different TiO\u003csub\u003e2\u003c/sub\u003e NPs concentrations based on the absorbance of Day 14 at the stationary phase of growth or the growth did not reach the stationary phase. Data are presented as mean ± standard deviation. Different letters (a, b, c) indicate significant differences among concentrations identified by Tukey test at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/a0b4e8b2b328eb096653c311.png"},{"id":78651130,"identity":"811d4131-df33-468f-a2a1-52ae657c4730","added_by":"auto","created_at":"2025-03-17 08:31:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1098959,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth curves of tested fungi under different BP-3 concentrations are based on fitting of the generalized logistic model, logistic Ⅰ.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/fcf0c7a8e78975aae3d10fcd.png"},{"id":78652022,"identity":"6555d4b3-d48c-4acd-81cf-4b5f484a679e","added_by":"auto","created_at":"2025-03-17 08:39:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1254503,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of tested fungi under different BP-3 concentrations based on the absorbance of Day 14 at the stationary phase of growth or the growth did not reach the stationary phase. Data are presented as mean ± standard deviation. Different letters (a, b, c) indicate significant differences among concentrations identified by Tukey test at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/7e8bac64668fb30deb2eb316.png"},{"id":78651142,"identity":"1e002567-b787-4cf1-bc75-99f65434c2a6","added_by":"auto","created_at":"2025-03-17 08:31:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":729534,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The AWCD (Metabolic capability) and (b) the AWTD (Mycelial production) of \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989 after 7 days of incubation in ZnO NPs (12.5 mg/L) in both the control and treatment groups. Data are presented as mean ± standard deviation. Asterisks (*) indicate significant differences between the control and treatment groups identified by Tukey test at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/0a1d1d893a5deefcf1e6ebed.png"},{"id":78651154,"identity":"b80b2caf-2646-469e-8f0c-4670d7bcaa6b","added_by":"auto","created_at":"2025-03-17 08:31:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1295441,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap representing the utilization profile of\u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989 for each carbon source, as determined by FF MicroPlates after 7 days of incubation in ZnO NPs (12.5 mg/L) in both the control and treatment groups. The color scale in the heatmaps is measured in terms of corrected redox value (CRV).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/59ec7bceeabbc5c18bce6700.png"},{"id":92431155,"identity":"1e9da767-a53b-4d99-8f6c-3de709b9079f","added_by":"auto","created_at":"2025-09-29 16:08:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10979339,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6210800/v1/bd607a44-7545-4292-9358-60df1e6a4e52.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Toxicity of organic (benzophenone-3) and inorganic (titanium dioxide and zinc oxide nanoparticles) ultraviolet filters on growth and metabolic activity of fungi cultured from the marine shallow-water hydrothermal vents of Kueishan Island, Taiwan","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSunscreen lotion contains ultraviolet (UV) filters to shield harmful UV rays in sunlight when applied to the skin, preventing sunburn, aging, and skin cancer (Kim and Choi, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Schneider and Lim, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Generally, the UV filters can be divided into inorganic metal oxide nanoparticles (NPs) which include zinc oxide (ZnO) and titanium oxide (TiO\u003csub\u003e2\u003c/sub\u003e), while the organic UV filters include benzophenone and its derivatives. Inorganic UV filters (e.g. ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs) are coated with a surface layer, which absorb and reflect solar UV radiation, thus preventing UV from reaching the skin (Tovar-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), whereas organic UV filters absorb and stabilize solar UV radiation (Sambandan and Ratner, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kim and Choi, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Stiefel and Schwack, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Over 10,000 tons of UV filters are estimated to be produced every year and incorporated into sunscreen products (Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These UV filters can be released into the aquatic environment directly through washing off from skin during recreational activities or indirectly through the discharge of wastewater (Du et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chatzigianni et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUV filters have been detected in the water of rivers, lakes, seashores, and/or sewage treatment plants (Labille et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sunscreen concentrations (including both organic and inorganic UV filters) at an Atlantic beach ranged from 10 to 96.7 mg/L in the unfiltered fraction and up to 75.7 mg/L in the dissolved fraction (Tovar-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On beaches of the Mediterranean Coast, concentrations of TiO\u003csub\u003e2\u003c/sub\u003e NPs in the surface water layer (top 1 cm) of the bathing zone and the water column below were 100\u0026ndash;900 and 20\u0026ndash;50 \u0026micro;g/L, and those of ZnO NPs were 10\u0026ndash;15 and 1\u0026ndash;3 \u0026micro;g/L, respectively (Labille et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Benzophenone-3 (BP-3) concentrations in seawater collected at sites of the US Virgin Islands and Hawaii were 75 \u0026micro;g/L-1.4 mg/L and 0.8\u0026ndash;19.2 \u0026micro;g/L, respectively (Downs et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In Taiwan, BP-3 was detected in seawater of seven sampling sites at Kenting National Park in southern Taiwan, and its concentration ranged from 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 ng/L at Houbihu to 514.6\u0026thinsp;\u0026plusmn;\u0026thinsp;282.0 ng/L at Baisha Beach (Ku et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the concentration of BP-3 in other coastal areas of Taiwan is not known. Also, no information is available on the concentration of TiO\u003csub\u003e2\u003c/sub\u003e and ZnO NPs in the coastal waters of Taiwan.\u003c/p\u003e \u003cp\u003eUV filters pose a potential threat to marine life and were reported to exert acute and chronic effects on marine organisms. For example, the LC50 of coral planulae exposed to benzophenone-3 (BP-3) in the light for an 8h and 24h exposure was 3.1 mg/L and 139 \u0026micro;g/L, respectively while that in darkness were 16.8 mg/L and 779 \u0026micro;g/L (Downs et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The EC50 of ZnO NPs in the alga \u003cem\u003eDunaliella tertiolecta\u003c/em\u003e, the bioluminescent bacterium \u003cem\u003eVibrio fischeri\u003c/em\u003e, and the crustacean \u003cem\u003eArtemia salina\u003c/em\u003e was 2.2 mg/L, 17 mg/L, and 58 mg/L, respectively (Schiavo et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). TiO\u003csub\u003e2\u003c/sub\u003e NPs induced oxidative stress and was bioaccumulated in \u003cem\u003eArtemia salina\u003c/em\u003e after exposure for 48 hours (Bhuvaneshwari et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eKueishan Island, situated at the southernmost part of the Okinawa Trough in the western Pacific Ocean, is a region of both economic and ecological significance in Taiwan. The area features approximately 50 shallow-water hydrothermal vents at depths ranging from 10 to 80 meters (Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which continuously emit hydrothermal fluids and volcanic gases (Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), making the seawater so called \u0026lsquo;the milky sea\u0026rsquo;. During the summer months in the last few years, human water activities have increased near/at the hydrothermal vent area and sunscreen on skin may wash off from the visitors. Kueishan Island is only 10 km away from the Yilan coast, chemical pollution from the main Taiwan island may also enter the hydrothermal vent ecosystem.\u003c/p\u003e \u003cp\u003eAlthough the toxicological effects of UV filters on marine vertebrates and invertebrates and some algae have been widely studied, their impact on microbial decomposers such as fungi remain largely unknown. Fungi are an integral part of the eukaryotic degrader community of the sea, playing a pivotal role in degrading recalcitrant substrate and providing various nutrients to the marine food web (O\u0026rsquo;Rorke et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jones et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cunliffe, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), yet little attention has been given to how UV filters influence their growth and metabolic activity. Given that UV filters can induce oxidative stress and disrupt cellular processes, they may pose a significant treat to marine fungal communities. Additionally, these UV filters, introduced by the increasing human activity, may disrupt the proper ecosystem functioning at the hydrothermal vents of Kueishan Island. To address this gap, this study investigated the effects of inorganic (TiO\u003csub\u003e2\u003c/sub\u003e and ZnO NPs) and organic (benzophenone-3) UV filters on growth and metabolic activity of fungi cultured from substrates collected at/near the hydrothermal vents of Kueishan Island (Pang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By employing the Biolog FF Microplate assay, we also assessed how these compounds impact fungal metabolism and carbon utilization, shedding light on the potential ecological consequences of UV filter pollution in the marine environment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cb\u003eFungal cultures\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe fungi isolated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) from the vent crab \u003cem\u003eXenograpsus testudinatus\u003c/em\u003e and sediment samples collected at/near the hydrothermal vent system of Kueishan Island (121˚57\u0026rsquo;6.01\" E, 24˚50\u0026rsquo;30.98\" N) between 2015 and 2017 were kept on CMAS (17.0 g/L cornmeal agar, 34 g/L sea salt) agar plates (Pang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCulture number and species name of selected fungi isolated from Kueishan Island.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCulture number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eYellow sediment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU4989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAspergillus terreus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU4992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePenicillium\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eChondrostereum\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAcremonium brunnescens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTrichoderma harzianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eBlack sediment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eParengyodontium album\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAspergillus terreus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAspergillus tubingensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAspergillus versicolor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicroascales sp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAspergillus versicolor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eThe vent crab\u003c/p\u003e \u003cp\u003e\u003cem\u003eXenograpsus testudinatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHypocreales sp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHortaea werneckii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTritirachium\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTOU5292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMicroascus brevicaulis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical preparation and characterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eZnO nanoparticles (particle size\u0026thinsp;\u0026lt;\u0026thinsp;5 \u0026micro;m, 99.9% purity), TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles (particle size\u0026thinsp;\u0026lt;\u0026thinsp;25 nm, 99.7% purity), and BP-3 (98% purity) were purchased from Sigma-Aldrich. The stock solution (100 mg/L) of ZnO and TiO\u003csub\u003e2\u003c/sub\u003e was prepared by adding 1% malt extract (Bacto\u0026trade;, Sparks, USA) seawater broth (34 PSU) and was dispersed by sonication (Delta DC150H; 150 W; 40 kHz) at 25\u0026deg;C for 30 min. The stock solution (100 mg/L) of BP-3 was dissolved in 0.01% dimethyl sulfoxide (DMSO, Sigma-Aldrich, purity\u0026thinsp;\u0026gt;\u0026thinsp;99%) and in 1% malt extract seawater broth, stored in the dark at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy (Hitachi HT7700) was performed by placing 8 \u0026micro;L drop of stock solution onto a formvar-coated grid. After air-drying, the sample was examined at an accelerating voltage of 75 kV to assess the particle size and morphology of the obtained ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs. Using Image J software, the mean size distribution of both ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs was determined by measuring 100 randomly selected particles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA series of concentrations (0, 3.13, 6.25, 12, 5,25, 50, 100 mg/L) of the three compounds was prepared from the stock solutions and dispensed (180 \u0026micro;L) into wells of a microtiter plate (Corning, Maine, USA). A solvent control (0.01% DMSO) was included for BP-3 growth test. Suspensions of hyphae and spores were made by adding 3 mL of 0.1% Tween 80 (Honeywell Fluka, Seelze, Germany) on the top of the fungal colonies and the plates were gently shaken or scrubbed to dislodge the spores or hyphae. The suspensions were counted using a hemocytometer (Bright-Line hemocytometer, Hausser Scientific, Horsham, PA, USA) and adjusted to 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e spores(hyphae)/mL. Spore/hyphal suspension (20 \u0026micro;L) was added to the wells of a microtiter plate, preloaded with the different concentrations of ZnO NPs, TiO\u003csub\u003e2\u003c/sub\u003e NPs and BP-3. Six replicates were inoculated for each treatment. The plates were placed in the incubator (Yih Der LM 570RD, Taiwan) at 25\u0026deg;C for two weeks without shaking. The absorbance was measured periodically at 630 nm by the multi-detection microplate readers (Synergy HT, BioTek) within a 14-day period.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolic profiling of fungi exposed to ZnO nanoparticles\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 (based on the results of the growth experiment) were subcultured on CMAS agar plates for 10 days at 25\u0026deg;C. Spore suspensions were prepared by adding 3 mL of a solution containing 0.1% Tween 80, 3.4% sea salt, and 12.5 mg/L ZnO NPs (the lowest observed effect concentration (LOEC) for the three fungi) on the top of the fungal colonies. The plates were gently shaken or scrubbed to dislodge the spores and the spore suspensions were counted using a hemocytometer and adjusted to 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e spores/mL. The spore suspensions in 100 \u0026micro;L were added to the wells of the Biolog FF MicroPlate\u0026trade;. Three replicates were done for each isolate and the plates were incubated at 25\u0026deg;C. The absorbance was measured every 24 h at 490 nm and 750 nm by the multi-detection microplate readers for 7 days. The reading at 490 nm was subtracted from that at 750 nm to determine the corrected redox value (CRV), which represents metabolic capability, for the reduction of iodonitrotetrazolium (INT) in each well. Functional diversity was determined by the number of different substrates utilized, which was calculated by counting all positive optical densities (OD) readings with a threshold of \u0026ge;\u0026thinsp;0.25. Average well color development (AWCD; Garland and Mills, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and average well turbidity development (AWTD; Klimek and Niklińska, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) represent average metabolic capability and average mycelial production, respectively. The calculation of AWCD and AWTD is described below:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:AWCD=\\frac{\\sum\\:_{i=1}^{n}\\left({C}_{i}-R\\right)}{n}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eC\u003csub\u003ei\u003c/sub\u003e: CRV of each well\u003c/p\u003e \u003cp\u003eR: CRV of control well\u003c/p\u003e \u003cp\u003en: number of carbon-containing substrates (total 95) in the Biolog FF MicroPlate\u0026trade;\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:AWTD=\\:\\frac{\\sum\\:_{i=1}^{n}{(C}_{i}-R)}{n}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eC\u003csub\u003ei\u003c/sub\u003e: OD\u003csub\u003e750 nm\u003c/sub\u003e reading of each well\u003c/p\u003e \u003cp\u003eR: OD\u003csub\u003e750 nm\u003c/sub\u003e reading of control well\u003c/p\u003e \u003cp\u003en: number of carbon-containing substrates (total 95) in the Biolog FF MicroPlate\u0026trade;\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analyses\u003c/h2\u003e \u003cp\u003eOrigin 9.1 (OriginLab Corporation, USA) was used to fit the growth model. The sigmoidal function, logistic I, had the best fit for the data. The formula of this generalized logistic model is\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{y}=\\frac{\\text{a}}{1+{e}^{-k\\left(x-{x}_{c}\\right)}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere, y represents the observed optical density at time x; a, the upper asymptote; xc, the point of inflection on the x-axis; k, growth rate constant; e, the base of the natural logarithm.\u003c/p\u003e \u003cp\u003eStatistical analyses were performed using SPSS 25 (IBM Corp., Armonk, New York, USA). Shapiro-Willk and Levene\u0026rsquo;s tests were utilized to assess normality and homogeneity of variances across all groups. One-way ANOVA and Tukey test were used to assess the significance of the absorbance on the last incubation day for the growth inhibition test and t-tests were conducted to assess the significance between with and without ZnO NPs exposure in AWCD and AWTD for the metabolic profiling test.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of ZnO and TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003enanoparticles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTEM images revealed that ZnO NPs predominantly exhibited a rod-like morphology, with a mean length of 189.6\u0026thinsp;\u0026plusmn;\u0026thinsp;63.6 nm and width of 75.7\u0026thinsp;\u0026plusmn;\u0026thinsp;22.8 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similarly, the rod-shape TiO\u003csub\u003e2\u003c/sub\u003e NPs appeared relatively uniform, with a mean length of 37.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 nm and width of 18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Both ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs showed some degree of agglomeration, as individual particles tended to clump together in seawater.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe growth curves of the 15 fungi incubated for 14 days under 0 mg/L to 100 mg/L of ZnO NPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Generally, all fungi exhibited an increased growth trend over time at all concentrations, except for \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277, where no/little growth was observed at 50 mg/L and 100 mg/L. Eight fungal isolates (\u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989, \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992, \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU5276, \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277, Microascales sp. NTOU5282, \u003cem\u003eAspergillus versicolor\u003c/em\u003e NTOU5284, \u003cem\u003eChondrostereum\u003c/em\u003e sp. NTOU5414, and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e NTOU5440) exhibited lower growth rates at higher ZnO NPs concentrations (50 mg/L and 100 mg/L). \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989 and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e NTOU5440 reached growth saturation on Day 6 at lower concentrations (3.13 mg/L and 6.25 mg/L) of ZnO NPs, whereas growth saturation was not reached at higher concentrations throughout the experimental period. In contrast, \u003cem\u003eA. versicolor\u003c/em\u003e NTOU5284 only reached growth saturation at higher concentrations of ZnO NPs. A slower growth rate was observed at higher concentrations in \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, Microascales sp. NTOU5282, and \u003cem\u003eChondrostereum\u003c/em\u003e sp. NTOU5414, and growth did not reach saturation. \u003cem\u003eAspergillus versicolor\u003c/em\u003e NTOU5278 displayed a slower growth rate in the first 7 days at higher concentrations, but its growth rate was similar to other concentrations after 7 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the final growth (optical density) of the fungi on Day 14 under the different ZnO NPs concentrations. The growth at 50 mg/L and 100 mg/L was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the control in \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. versicolor\u003c/em\u003e NTOU5284, \u003cem\u003eChondrostereum\u003c/em\u003e sp. NTOU5414, and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e NTOU5440. Although lower growth at higher concentrations was observed in \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992 and Microascales sp. NTOU5282, the differences were not statistically significant. Conversely, no significant dose-dependent response was observed in the remaining fungal isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGrowth rates for the majority of the tested fungi under the different concentrations of TiO\u003csub\u003e2\u003c/sub\u003e NPs were similar, except for \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992 and \u003cem\u003eAspergillus versicolor\u003c/em\u003e NTOU5278, which showed a decreased growth rate at 6.25 mg/L to 100 mg/L and 25 mg/L to 100 mg/L, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, no significant dose-dependent response was observed in the remaining fungal isolates. The growth of \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992, \u003cem\u003eA. versicolor\u003c/em\u003e NTOU5278 and Microascales sp. NTOU5282 was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the control at higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo statistically significant difference was observed between the absorbance of solvent control (0.01% DMSO) and 0 mg/L BP-3 (results not shown). Similar growth rate was observed for most of the tested fungi under all concentrations of BP-3 over 14 days except for \u003cem\u003ePenicillium\u003c/em\u003e sp. NTOU4992 and \u003cem\u003eA. versicolor\u003c/em\u003e NTOU5278 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). No significant dose-dependent response was observed in the remaining fungal isolates. The growth of \u003cem\u003ePenicillium sp.\u003c/em\u003e NTOU4992, \u003cem\u003eAspergillus versicolor\u003c/em\u003e NTOU5278 and \u003cem\u003eMicroascus brevicaulis\u003c/em\u003e NTOU5292 was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at higher concentrations of BP-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolic activity of fungi after exposure to ZnO nanoparticles\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMetabolic activity of \u003cem\u003eAspergullus tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989 were studied using the Biolog FF MicroPlate with or without ZnO NPs (12.5 mg/L). In the absence of ZnO NPs, the percentage utilization of carbon sources was 88.4% (84/95 substrates), 88.4% (84/95), and 87.3% (83/95) for \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989, respectively; in the presence of ZnO NPs, they were 85.2% (81/95), 87.3% (83/95), and 88.4% (84/95).\u003c/p\u003e \u003cp\u003eThe AWCD was significantly higher without ZnO NPs exposure in \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). On the other hand, the AWTD of \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989 was significantly higher in the control group, while the AWTD of \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the control group; the AWTD of \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276 remained similar after 7 days of exposure to ZnO NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe carbon source utilization pattern is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The three isolates of \u003cem\u003eAspergillus\u003c/em\u003e exhibited low utilization (CRV\u0026thinsp;\u0026lt;\u0026thinsp;0.25) of L-fucose, D-galacturonic acid, glucuronamide, γ-amino-butyric acid, bromosuccinic acid, fumaric acid, sebacic acid, succinamic acid, succinic acid mono-methyl ester, N-acetly-L-glutamic acid, and L-pyroglutamic acid, with or without ZnO NPs exposure. D-galactose, γ-hydroxy-butyric acid, and L-proline were poorly utilized (CRV\u0026thinsp;\u0026lt;\u0026thinsp;0.25) by \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 in the presence of ZnO NPs. L-phenylalanine and β-hydroxy-butyric acid were the only carbon sources poorly utilized by \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276 and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cb\u003eToxicological effect of ZnO NPs, TiO₂ NPs, and BP-3 on marine fungi\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn recent years, tourism in the water around Kueishan Island has been steadily increasing, leading to greater anthropogenic impacts on this economically and ecologically important territory. The rise in tourist numbers, driven primarily by favorable weather, the unique colour of the \u0026lsquo;milk sea\u0026rsquo;, and increased awareness of UV protection, likely results in increased application of sunscreen products on the skin which may be washed off and suspend/settle in the sea and impose adverse effects on marine organisms. Among the three UV filters, ZnO NPs had the strongest inhibition effect on the growth of \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. versicolor\u003c/em\u003e NTOU5284, \u003cem\u003eChondrostereum\u003c/em\u003e sp. NTOU5414 and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e NTOU5440. \u003cem\u003eAspergillus terreus\u003c/em\u003e NTOU4989 and other \u003cem\u003eAspergillus\u003c/em\u003e species was found to be adaptive to changes in environmental conditions in the marine environment, with their genetic capability (Pang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eAspergillus\u003c/em\u003e species may represent one of the key decomposers in the marine environment (Chou et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while ZnO NPs may interfere them from fulfilling that role.\u003c/p\u003e \u003cp\u003eThe fungicidal activity of ZnO NPs was previously known as it was also used as an effective fungicide in food safety, agriculture, and clean water (Sun et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Paraguay-Delgado et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) of \u003cem\u003ePenicillium expansum\u003c/em\u003e were found to be 798 mg/L and 147 mg/L, respectively; the MIC of \u003cem\u003eAspergillus niger\u003c/em\u003e was 2.5 mg/L ZnO NPs as a fungicide (Gondal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sardella et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ZnO NPs are able to interact with fungal cell membranes, altering their permeability and causing leakage of intracellular components, which leads to cell death (Adams et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, the impact of ZnO NPs is not limited to fungi as they also exhibit varying levels of toxicity toward other marine microorganisms, including cyanobacteria and microalgae. A recent study on \u003cem\u003eSynechococcus\u003c/em\u003e sp. demonstrated that exposure to ZnO NPs at concentrations as low as 1.4 mg/L led to increased reactive oxygen species (ROS) production, disruption of the photosynthetic apparatus, and a decline in chlorophyll and phycoerythrin content (Shoman et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZnO NPs were found to be the most toxic among the nanoparticles, exhibiting significantly higher toxicity compared to TiO\u003csub\u003e2\u003c/sub\u003e and other metal oxide nanoparticles (Aruoja et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Miller et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dasari et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bhuvaneshwari et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, the EC50 of ZnO NPs and TiO\u003csub\u003e2\u003c/sub\u003e NPs on the microalga \u003cem\u003ePseudokirchneriella subcapitata\u003c/em\u003e was 0.042 mg/L and 5.83 mg/L, respectively (Aruoja et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). ZnO caused a significant reduction in growth rate in marine phytoplankton whereas TiO\u003csub\u003e2\u003c/sub\u003e did not (Miller et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). ZnO NPs release free zinc ions (Zn\u003csup\u003e2+\u003c/sup\u003e) in water and can rapidly dissolve in seawater (Aruoja et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Miller et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bhuvaneshwari et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These free Zn\u003csup\u003e2+\u003c/sup\u003e ions induce the generation of ROS which can cause cell damage such as DNA damage, lipid peroxidation, and protein denaturation (Nel et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hou et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The release of Zn\u003csup\u003e2+\u003c/sup\u003e from uncoated zinc NPs (8.9 mg/L) was more efficient than zinc oxide NPs (5.5 mg/L), and the former exhibited higher toxicity towards \u003cem\u003eArtemia salina\u003c/em\u003e (Ates et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, the toxicity of ZnO NPs on the fungi in this study may result from two mechanisms: direct contact with the nanoparticles and the dissolution of Zn\u003csup\u003e2+\u003c/sup\u003e ions. In contrast, the toxicity of TiO\u003csub\u003e2\u003c/sub\u003e NPs is likely caused only by direct nanoparticle contact, as TiO\u003csub\u003e2\u003c/sub\u003e NPs are insoluble in seawater and requires strong acids for dissolution. Although both ZnO and TiO\u003csub\u003e2\u003c/sub\u003e NPs can generate reactive oxygen species (ROS), their toxicity level differs due to differences in solubility (Blake et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Hanley et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Miller et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Alabdallah et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, TiO\u003csub\u003e2\u003c/sub\u003e NPs aggregate more rapidly in seawater than ZnO NPs, thus reducing their toxicity toward marine organisms (Bhuvaneshwari et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The toxicity of nanomaterials is closely linked to their physicochemical properties, such as size and shape (Hou et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For instance, the rod-like ZnO NPs are more toxic than the spherical ZnO NPs by having a large contact area (Hou et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our TEM analysis confirmed that ZnO NPs in this study were predominantly rod-shaped, which may have contributed to their toxicity.\u003c/p\u003e \u003cp\u003eThe toxicity between of inorganic and organic UV filters has rarely been discussed. In our study, growth inhibition was not observed for most of the tested fungi after BP-3 exposure. It is interesting to note that a very low concentration (\u0026micro;g/L to ng/L) of BP-3 could cause various toxicological effects to marine animals and microorganisms such as genotoxicity (Almeida et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), oxidative stress (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and growth inhibition (Paredes et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e; Mao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, only 2 out of 27 bacterial isolates belonging to the phyla Bacteroidetes and Proteobacteria were growth-exhibited under 1 mg/L BP-3 (Lozano et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Microorganisms including fungi and bacteria may possess unique physiological or metabolic mechanisms that can counteract the presence of BP-3, such as specialized transport systems, detoxification, or enzymatic degradation pathways that mitigate the effects of such pollutants (Lozano et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The underlying biological processes that enable these microorganisms to withstand environmental stressors posed by organic UV filters will require further study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolic profiling of fungi exposed to ZnO NPs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 7 days of exposure to ZnO NPs (12.5 mg/L), all \u003cem\u003eAspergillus\u003c/em\u003e spp. were able to metabolize 80\u0026ndash;90% of the tested carbon sources in the Biolog FF MicroPlate, but the average utilization of carbon sources (AWCD) was significantly lower in the control when no ZnO NPs was added. Interestingly, despite lower metabolic capability, the mycelial production (AWTD) varied among species. For example, \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 had higher mycelial production even though its AWCD was lower in the presence of ZnO NPs. This result is similar to the findings by Chou et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in which \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989, incubated at 25\u0026deg;C and pH 3, had lower metabolic activity (AWCD) but higher biomass production (AWTD) compared to incubation at pH 7. The higher mycelial production in \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 may be attributed to the utilization of D-gluconic acid and glycogen. The utilization of these carbon sources was not significantly affected by ZnO NPs exposure, and may have provided an alternative energy source, allowing the fungus to sustain growth even when other metabolic pathways were disrupted. \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277 may possess a selective carbon utilization strategy that enables it to compensate for unfavorable growth conditions caused by ZnO NPs. In addition, Wang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that \u003cem\u003eFusarium kyushuense\u003c/em\u003e exposed to fungicides Azoxystrobin and Kresoxim-methyl showed a reduced metabolic capacity for these substances. γ-hydroxybutyric acid and L-proline are key compounds in the tricarboxylic acid (TCA) cycle by contributing to ATP production and cellular respiration. The inability to metabolize these compounds under ZnO NPs exposure suggests a disruption in the energy-generating pathways, which may reduce the fungus\u0026rsquo;s capacity to cope with oxidative stress, and may render the organisms more vulnerable to environmental changes (Fuentes and Qui\u0026ntilde;ones, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the lack of utilization of D-galactose, γ-hydroxybutyric acid, and L-proline did not seem to impede the growth in \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277 as AWTD was higher than the control. \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277 may be able to survive in environments with limited carbon sources by significantly increasing the utilization of specific carbon sources when faced with unfavorable conditions.\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276 and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989 exhibited only minor reductions in the metabolism of L-phenylalanine and β-hydroxybutyric acid, respectively, in the presence of ZnO NPs. These species maintain relatively stable metabolic activity even under ZnO NPs exposure, suggesting a higher resilience to Zn\u0026sup2;⁺ toxicity. This result aligns with that of Klimek and Niklińska (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) who also observed a low impact of Zn\u0026sup2;⁺ (300 mg/L) on the metabolic activity of fungi. The ability to sustain metabolic function despite nanoparticle stress may contribute to their higher recovery potential in contaminated environments.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePotential environmental implications of UV filter exposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile the concentration of ZnO NPs used in this study (12.5 mg/L) is higher than the typical levels detected in open seawater, the potential for localized accumulation should not be overlooked. ZnO NPs tend to aggregate and settle in marine sediments, where their concentrations can be significantly higher than in the surrounding water column (Labille et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In heavily impacted coastal areas, such as those with high tourism activity, sunscreen concentrations were reported to reach up to 96.7 mg/L (Tovar-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given that the fungi examined in this study were originally isolated from sediments near hydrothermal vents at Kueishan Island, where ZnO NPs can settle and accumulate. Sediment-associated fungal communities may be at an elevated risk of exposure to ZnO NPs, potentially facing greater toxic effects than pelagic microbial populations. Such prolonged exposure could disrupt fungal-mediated organic matter decomposition and nutrient cycling, processes that are critical in benthic ecosystems (Cunliffe, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComparing the effects of the three tested UV filters, our results align with previous studies indicating that ZnO NPs generally exhibit higher toxicity than TiO\u003csub\u003e2\u003c/sub\u003e NPs across a wide range of marine organisms (Aruoja et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Schiavo et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Schiavo et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Corinaldesi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While there is a growing movement toward eco-friendly sunscreens, the use of organic-based UV filters such as oxybenzone and octinoxate has been heavily restricted or outright banned in multiples regions, including Hawaii, Palau, and parts of the European Union, due to their well-documented role in coral reef bleaching and disruption of marine ecosystems (Miller et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chatzigianni et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)). This shift in regulation has led to an increased reliance on inorganic-based (ZnO and TiO\u003csub\u003e2\u003c/sub\u003e) alternatives (Hojerov\u0026acute;a et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Blasco et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, despite their low impact on corals, the continued use of ZnO and TiO\u003csub\u003e2\u003c/sub\u003e still raises concerns regarding their potential impact on marine microbial ecosystems. Marine fungi and other sediment-associated microorganisms play essential roles in organic matter decomposition and nutrient cycling, prolonged exposure to these compounds could have cascading ecological effects. As research continues to explore the environmental consequences of inorganic UV filters, developing alternative sunscreen formulations that replace ZnO with less toxic yet effective UV-blocking agents may be a viable future direction for reducing the unintended impacts of sunscreen pollution on marine ecosystem.\u003c/p\u003e \u003cp\u003eThe observed metabolic responses further highlight the potential of fungal communities as bioindicators for ZnO NP contamination. ZnO NPs exposure resulted in reduced metabolic, particularly in carbon utilization pathways, similar metabolic profiling approaches could be applied in environmental monitoring programs to assess the extent of contamination in marine ecosystems. This study provides the first insight into the effects of inorganic and organic UV filters on the growth and metabolic activity of marine fungi. Future research should focus on long-term exposure studies at environmentally relevant concentrations, particularly in sediment and biofilm environments where ZnO NPs are likely to persist. Additionally, investigating the combined effects of multiple UV filters, as well as their interaction with natural environmental factors such as organic matter and salinity, would provide a more comprehensive understanding of their ecological risk.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis is the first study to report the potential effects of inorganic and organic UV filter exposure on the growth and metabolic activity of marine fungi. Growth for most of the fungi was not adversely affected by TiO\u003csub\u003e2\u003c/sub\u003e NPs and BP-3 exposure, whereas ZnO NPs had a significant growth inhibition for some fungi, especially on \u003cem\u003eAspergillus\u003c/em\u003e spp. Further metabolic profiling of \u003cem\u003eA. tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276, and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989 using Biolog FF MicroPlate in the presence of ZnO NPs indicated a reduction in metabolic capacity, with variations in mycelial growth and carbon source utilization. The variations in physiological response to the three UV filters highlights the different sensitivities of the fungi towards these pollutants. It will be interesting in the future to study the effect of mixed UV filters on the growth of fungi, their degradation of organic substrates, ROS generation, and transcriptome changes so as to provide a more comprehensive understanding of how UV filters affect the ecological functioning of fungal community in the marine environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLam Kong: Methodology, Investigation, Data curation, Writing \u0026ndash; original draft. Ka-Lai Pang: Methodology, Investigation, Supervision, Writing \u0026ndash; review \u0026amp; editing. Wing fai Lu: Conceptualization, Investigation, Methodology.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was funded by the National Science and Technology Council of Taiwan (110-2621-M-019-002-, 111-2621-M-019-002-, and 112-2621-M-019-003-). The authors thank the Electron Microscopy Center of Institute of Marine Biology at National Taiwan Ocean University for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams, L. K., Lyon, D. Y., \u0026amp; Alvarez, P. J. (2006). Comparative eco-toxicity of nanoscale TiO\u003csub\u003e2\u003c/sub\u003e, SiO\u003csub\u003e2\u003c/sub\u003e, and ZnO water suspensions. Water research, \u003cem\u003e40\u003c/em\u003e(19), 3527\u0026ndash;3532.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlabdallah, N.M., Alluqmani, S.M., Almarri, H.M., AL-Zahrani, A.A., 2024. Physical, chemical, and biological routes of synthetic titanium dioxide nanoparticles and their crucial role in temperature stress tolerance in plants. 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Toxicol. 235, 105835. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquatox.2021.105835\u003c/span\u003e\u003cspan address=\"10.1016/j.aquatox.2021.105835\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"UV-filters, Nanoparticle, Fungi, Microbial ecotoxicology, Growth inhibition, Biolog FF MicroPlate","lastPublishedDoi":"10.21203/rs.3.rs-6210800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6210800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increased awareness of the damaging effects of ultraviolet radiation from the sun has promoted the use of sunscreen products. The active ingredients of sunscreen lotion, i.e. benzophenone-3 (BP-3), titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e) nanoparticles (NPs), and zinc oxide (ZnO) NPs, can pollute the marine environment through runoff or human activities such as swimming. Early studies have revealed the toxic effects of these sunscreen active ingredients on aquatic animals, however, their effects on the marine decomposer community are less known, especially on fungi. This study investigated the effect of BP-3, TiO\u003csub\u003e2\u003c/sub\u003e NPs, and ZnO NPs on growth and metabolic activity of selected fungi isolated from the marine shallow-water hydrothermal vent ecosystem at Kueishan Island, Taiwan. Growth inhibition was observed for the majority of the tested fungi (especially on \u003cem\u003eAspergillus\u003c/em\u003e spp.) by increased concentrations of ZnO NPs (0-100 mg/L). In contrast, TiO\u003csub\u003e2\u003c/sub\u003e NPs and BP-3 exerted little effect on fungal growth. The differences in toxicity between ZnO NPs and TiO\u003csub\u003e2\u003c/sub\u003e NPs might be attributed to variations in their solubility, size, and shape. Surprisingly, BP-3 exhibited the least toxicity on fungal growth, despite its known effects on other marine organisms at very low concentrations. The toxicity of ZnO NPs (12.5 mg/L) on metabolic activity of the growth-inhibited fungi, using Biolog FF MicroPlate, was also examined, i.e. \u003cem\u003eAspergillus tubingensis\u003c/em\u003e NTOU5277, \u003cem\u003eA. terreus\u003c/em\u003e NTOU5276 and \u003cem\u003eA. terreus\u003c/em\u003e NTOU4989. A significant reduction in average well colour development (AWCD) was observed in the presence of ZnO NPs, suggesting an overall reduction in metabolic activity. Interestingly, the average well turbidity development (AWTD) of \u003cem\u003eA. tubingensis \u003c/em\u003eNTOU5277 in the presence of ZnO NPs was higher than that of the control group without ZnO NPs. In terms of carbon utilization, D-galactose, γ-hydroxy-butyric acid, and L-proline\u003cem\u003e \u003c/em\u003ewere not utilized by \u003cem\u003eA. tubingensis \u003c/em\u003eNTOU5277 in the presence of ZnO NPs,\u003cem\u003e \u003c/em\u003ewith the latter two compounds being related to the tricarboxylic acid (TCA) cycle. \u003cem\u003eAspergillus terreus\u003c/em\u003e isolates NTOU5276 and NTOU4989 showed a reduction in the utilization of L-phenylalanine and β-hydroxy-butyric acid in the presence of ZnO NPs, respectively. These results suggest the potential toxic effects of ZnO NPs on energy production and metabolism in fungi and highlight the prospect of using Biolog FF MicroPlate for assessing metabolic effects of other anthropogenic pollutants on fungi.\u003c/p\u003e","manuscriptTitle":"Toxicity of organic (benzophenone-3) and inorganic (titanium dioxide and zinc oxide nanoparticles) ultraviolet filters on growth and metabolic activity of fungi cultured from the marine shallow-water hydrothermal vents of Kueishan Island, Taiwan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 08:31:04","doi":"10.21203/rs.3.rs-6210800/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-12T02:49:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-14T11:26:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-25T06:14:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175265221221072681681540209972319015222","date":"2025-04-11T09:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55129006349417594188253697958419075966","date":"2025-04-11T08:06:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T03:32:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-13T01:15:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-13T01:15:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ecotoxicology","date":"2025-03-12T09:32:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"989f7b81-6d0a-427d-9156-0aef4ebc1438","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-6210800","link":"https://doi.org/10.1007/s10646-025-02971-z","journal":{"identity":"ecotoxicology","isVorOnly":false,"title":"Ecotoxicology"},"publishedOn":"2025-09-25 15:57:03","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2025-03-17 08:31:04","video":"","vorDoi":"10.1007/s10646-025-02971-z","vorDoiUrl":"https://doi.org/10.1007/s10646-025-02971-z","workflowStages":[]},"version":"v1","identity":"rs-6210800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6210800","identity":"rs-6210800","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}