Metabolic disruptions in marine environments: synthetic eternal chemicals PFBS impact widespread microbial eukaryotes

Metabolic disruptions in marine environments: synthetic eternal chemicals PFBS impact widespread microbial eukaryotes | 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 Article Metabolic disruptions in marine environments: synthetic eternal chemicals PFBS impact widespread microbial eukaryotes Michael Lintner, Irina Polovodova Asteman, Ketil Hylland, Joachim Sturve, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7460059/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Pollution caused by per- and polyfluoroalkyl substances (PFAS) is an urgent environmental issue, as these substances are widespread, highly persistent and toxic to many organisms. Nevertheless, the effects of PFAS on microbial eukaryotes have hardly been analysed yet. In this study, we investigated the influence of perfluorobutane sulfonic acid (PFBS) in different concentrations (0, 0.1, 1, 10, and 100 µg/L) on the metabolism of five benthic foraminifera taxa: Bulimina marginata , Cassidulina laevigata , Eubuliminella sp. , Globobulimina turgida and Nonionella sp. T1. In laboratory experiments, foraminifera were incubated with the pollutant for three days while maintaining natural physical parameters from the sampling site in the Gullmar Fjord, Sweden. Our results show that even at low PFBS concentrations, a disruption in the metabolic activity, detected through feeding with isotopically ( 13 C and 15 N) labelled algae, was observed. Even the lowest concentrations of PFBS (0.1 µg/L) completely inhibited metabolic cycles in these protists. With the exception of one species ( C. laevigata ), which exhibited generally very low metabolic activity during incubation, all species demonstrated significant decreases in food uptake, suggesting species-specific impact of PFBS. Since a reduction in metabolic activity was evident in all studied taxa, we conclude that PFBS is highly toxic to microbial eukaryotes. Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Earth and environmental sciences/Ocean sciences Figures Figure 1 Figure 2 Figure 3 1. Introduction 1.1. General introduction The introduction of pollutants into the environment by humans has increased rapidly over the last few decades. The group of per- and polyfluoroalkyl substances (PFAS) has in particular attracted general concern in recent years. The PFAS group includes today about 15 000 different compounds of similar properties (SSNC, 2024). These exclusively synthetic chemicals are characterized by an aliphatic carbon in which hydrogen has been replaced by fluorine (Abunada et al., 2020 ). If hydrogen has been completely replaced, the compound is called per- and by partial substitution poly-fluoroalkyl (Abunada et al., 2020 ). Such chemicals have been used since the 1940s in various areas of industry like textile waterproofing, fire-fighting foam, non-sticking frying pans and cosmetics but can also be found in ammunition, nylon guitar strings or artificial turf (SSNC, 2024; Glüge et al., 2020 ; Kissa, 2001 ). The problem with these compounds is that they are highly persistent, so that they are sometimes referred to as eternal chemicals (SSNC, 2024). It means they can accumulate in the environment for a long time and have a negative impact on the health of organisms (De Silva et al., 2021 ). Today, PFAS have been found in waters, soils, living organisms and even air (e.g. Giesy and Kannan, 2001 ). In addition, PFAS are found in all aqueous matrixes, such as rain, snow, groundwater, seawater, lakes and rivers (Fabrega et al., 2014; Liu et al., 2017 ; Moghadasi et al., 2023 ; Mumberg et al., 2024 ). Based on lifestyle, people consume about 0.17–0.21 ng/kg body weight of PFAS daily (Gellrich et al., 2013 ). In the environment, concentrations of 7,000–290,000 ng/L PFAS have been found in soils (Eriksson et al., 2013 ). Analysis of tap water showed values ​​of 0.62 ng/L in Japan (Mak et al., 2009 ) but extreme levels of up to 2,000 ng/L in Oakdale (USA) have been reported (Abunada et al., 2020 ). In some river inlets like the Shuangtaizi Estuary (China) a concentration of 66.2 to 185 ng/L PFAS were found in the surface water (Shao et al., 2016 ). In marine coastal, surface waters, the highest PFAS concentrations are found in the Indian Ocean (41.1 ng/L) followed by the North Pacific Ocean (12.8 ng/L), the North Atlantic Ocean (4.0 ng/L), the South Atlantic Ocean (2.6 ng/L), the Arctic Ocean (1.1 ng/L), the South Pacific Ocean (0.5 ng/L) and, finally, the Southern Ocean (< 0.1 ng/L) (Khan et al., 2023 ). Similarly, PFAS has also been detected in the marine sediments of North Pacific Ocean (4.61 ng/g), Southern Ocean (4.56 ng/g), South Pacific (3.16 ng/g), North Atlantic (2.29 ng/g) and the Arctic (1.54 ng/g) oceans (Kahn et al., 2023). In this study we investigated effects of the pollutant PFBS (perfluorobutane sulfonic acid), which is a short-chain PFAS compound. Compared to long-chain PFAS, PFBS has been suggested as an alternative with lower propensity to bioconcentrate in tissues relative to other PFAS (Ivantsova et al., 2024 ). PFBS concentrations of 1.21–46.06 ng/L were measured in Jiulong River estuary, China, up to 4.9 ng/L in groundwater from Ganges River, India, or 7.89–29.1 ng/g in wastewater sludge in Germany (Zhang et al., 2018 ; Sharma et al., 2016 ; Gomez-Canela et al., 2012). However, in the last few years some studies found a relationship between a toxic effect or pathological development of humans and the presence of PFBS (Wang et al., 2017 ), and thus there is an urgent need to improve our understanding how PFBS interact with organisms. 1.2. PFAS and their effect on organisms Once pollutants have entered the aquatic environment, it does not take long for them to interact with organisms there. The easiest way to transmit them is through direct ingestion by aquatic organisms. Many studies have previously focused on long-chain PFOS (perfluorooctanesulfonic acid), which can accumulate to between 9 and 55 ng/g dry weight in fish, and it has been shown that these compounds are more likely to accumulate in liver tissues than in muscle tissue (Kahn et al., 2023). A particularly high value was found in the liver of dusky flathead (135 ng/g ww) (Kahn et al., 2023). But PFAS can also bioaccumulate in other aquatic organisms. Beale et al., ( 2022 ) investigated freshwater turtles ( Emydura macquarii macquarii ) and found 235 times higher PFAS concentration in the turtle’s serum (PFOS; 889 ± 56 ng/mL) than in the water where the animals lived (ΣPFAS 32.0 µg/L). However, the bioaccumulation of PFAS does not only affect aquatic organisms. In 2023, Lettoof et al., investigated 35 tiger snakes ( Notechis scutatus ) and they found PFOS concentrations up to 322 ± 193 µg/kg animal. Even in birds and their eggs elevated PFOS concentrations (8 to 608 ng/g ww) have been observed (Kahn et al., 2023). All these examples illustrate that PFAS is widely distributed in nature and both terrestrial and aquatic food chains, and therefore potentially poses a risk to humans. The daily intake of PFAS in humans is assumed to be in the range of 72–1810 pg·kg bw − 1 ·day − 1 (Poothong et al., 2020 ). Most studies have focused on bioaccumulation of PFAS is in vertebrates, whilst studies on invertebrates or microbial eukaryotes remain limited. For example, mean values ​​for PFOS in Mediterranean mussels were 60.03 ng/g ww, which was found comparable to other animals (Khan et al., 2023 ). Accumulations of PFAS have also been reported for lower trophic levels, such as plankton (Pan et al., 2021 ). Although some of the microbes like bacteria are involved in the degradation of PFAS, based on few studies, these pollutants can be just as toxic to unicellular organisms. In a laboratory study, the protists Tetrahymena pyriformis that belong to Ciliophoran were exposed to PFOS concentrations of 0–5000 µM and cellular motility, division and function were measured (Lim, 2022 ). Even at 5 µM PFOS, a decrease in the vitality of the organisms was observed, as compared to the control group incubated without PFOS (Lim, 2022 ). To expand our knowledge about the interaction of PFAS and microbial eukaryotes this study focus on the change of the metabolic behaviour on benthic foraminifera if PFBS is present in the seawater. Foraminifera are mostly marine protists, which can be found in all marine habitats on Earth (Sen Gupta, 2003 ). Due to their high abundance, foraminifera play a major role in global marine biogeochemical cycles and are also important ecosystem engineers (Hallock et al., 2003 ; Piña-Ochoa et al., 2010 ; Salonen et al., 2019 ; Langlet et al., 2023; Glock et al., 2024). Foraminifera can be used as bioindicators as they react fast and sensitive to different pollutants (e.g. Prazeres et al., 2020 ; Frontalini et al., 2011). Heavy metals were shown to cause a disruption in foraminifera metabolism (Lintner et al., 2025a , 2021 ), likely explaining lower diversity or even foraminifera-barren sediments in regions heavily polluted with metals (e.g. Ferraro et al., 2006 ; Polovodova Asteman et al., 2015 ). Yet, much less is known about the effects of organic pollutants on foraminifera. Some initial studies have shown that the presence of sunscreens, pesticides or antibiotics in seawater greatly reduces or even completely inhibits the metabolism of foraminifers (Lintner et al., 2022 , 2024 , 2025b). This study aims to investigate the influence of PFBS on foraminifera. To do this, we use different concentrations of PFBS in laboratory experiments to test a concentration-dependent reduction in metabolism through feeding with labelled algae. The first hypothesis (H1) is that at higher concentrations of PFBS, the metabolism of foraminifera is reduced. Further we hypothesize (H2) that the pollutant has different effects on the different species and that metabolism is inhibited in a species-dependent manner. To test a species-specific impact of PFBS, we are testing the response of five different species to the presence of the pollutant. 2. Material and Methods 2.1. Sediment sampling and sampling area Sediment sampling was performed on 3nd of July 2024 aboard of R/V Alice (University of Gothenburg) by using a box corer at Station 3 (Fig. 1 ) in the outer part of the Gullmar Fjord, Sweden (58°15.538’N, 11°27.483’E and 40 m water depth). The Gullmar Fjord, located on the west coast of Sweden, is a true fjord with a maximum water depth of 118.6 m. It is one of the world’s most well-studied marine settings, with the first hydrographic observations being taken as early as 1869 (Ekman, 1870). As a sill fjord, the Gullmar Fjord has remarkably high sediment accumulation rates of 0,7–1.4 cm per year (Filipsson and Nordberg, 2004) and low tidal activity providing a high-resolution paleo-environmental archive (e.g. Harland et al., 2013; Polovodova Asteman et al, 2018). The sill of the fjord has a depth of 42 m and results in a fjord basin being stratified with respect to temperature and salinity. The fjord deepest basin > 50 m water depth has a water mass derived from the Skagerrak (S = 34–35 PSU) and is prone to stagnation and seasonal oxygen depletion, which became more frequent since the early 1980s driven by the variability of the North Atlantic Oscillation (Nordberg et al., 2000; Bjork and Nordberg, 2003). The fjord areas shallower than sill depth are influenced by the brackish water from the Baltic Current mixed with the Skagerrak and Kattegat water, as well as local runoff and supply from the Örekilsälven river in the inner part of the fjord (Arneborg et al., 2004). The Gullmar Fjord became the first marine conservation area in Sweden in 1983 due to its high biodiversity, and it currently has no anthropogenic impact. The closest impact sources are the Lysekil harbor, the Preem Oil Refinery, located in the adjacent Brofjord, and the European route E6 passing by next to the fjord head (Fig. 1 ). In parallel to the sediment sampling, environmental data (temperature, salinity, and oxygen) were taken on board by using a CTD probe. The T, S and O 2 observations proximal to the sea floor showed 14.9°C, salinity of 33 and 4.1 ml O 2 L − 1 , respectively. The retrieved sediment surface in the box corer was intact and covered by water, which was gently siphoned out by using a plastic tube. The top 5 cm of the sediment were transferred to a bucket and taken to the lab at Kristineberg Centre for Marine Research and Innovation for further experiments. There, the sediment was gently sieved by using a 63 and 1000 µm sieves using ambient fjord water to concentrate living foraminifera and remove larger macrofauna. The resulting sediment fraction (63–1000 µm) was collected in a plexiglass tank to establish a permanent culture, which was cultivated under 10°C in the darkness to mimic natural conditions and to acclimatize foraminifera to laboratory conditions. 2.2. Experimental Setup After three days of acclimatization, the sediment was washed again over a 150 µm sieve and the foraminifera, which had a characteristic brown-yellow color of the cytoplasm, were picked out in a Petri dish by using a very fine brush. Following that, foraminifera were cleaned with a brush, all adhering particles were carefully removed and clean foraminifera were transferred to a crystallization dish. Fifty individuals were placed in the center of a dish, and left for one day to test their vitality by a so called “crawling test.” After one day, only the actively moving individuals were selected for further experiments. To accurately identify foraminifera on species level, secondary electron microscopy (SEM) images were taken of individuals that were first coated with gold. For feeding and isotopic uptake experiments, 20 individuals (with three replicates) of each of the five foraminifera species ( Bulimina marginata, Cassidulina laevigata, Eubuliminella sp., Globobulimina turgida and Nonionella sp. T1) were placed per separate crystallization dish and covered with 100 ml artificial seawater water with the same salinity as at the sampling sites. These cultures served as control samples, where no additional interference factor (PFBS) was added. As interference factor, PFBS was used in four different concentrations (100 µg/L; 10 µg/L; 1 µg/L and 0.1 µg/L). As PFBS is largely insoluble in water, a 10 mg/mL stock solution in methanol was prepared and added to artificial seawater. The lowest concentration (0.1 µg/L) is in the range of natural contamination (see examples in Introduction). To test the effect of future PFBS contamination, three increased levels of the pollutant were also applied, each elevated by a factor 10. To observe the change in the metabolic activity of the foraminifera, isotopically labelled diatoms ( Chaetoceros simplex var. calcitrans ) were added to the cultures. Theses diatoms naturally occur in the Gullmar Fjord (Hallfors, 2004), where the foraminifera come from. Diatoms were grown in a nutrient medium enriched with 13 C and 15 N and therefore themselves contained an increased concentration of isotopes (for method details see Lintner et al., 2020). After consuming these algae, the foraminifera themselves become enriched with isotopes. Generally, the higher the proportion of isotopes in the foraminifera after cultivation, the lower the influence of the disturbing factor (here, PFBS), as indicated by higher feeding intensity of the foraminifera, used here as a measure to assess foraminiferal metabolism. 2.3. Sample and data processing After incubation with the isotopically enriched algae foraminifera were removed from the culture and cleaned from any adhering particles with a brush. To measure the isotopic uptake, 20 specimens of Bulimina marginata were transferred to separate pre-weighted Sn-capsules. In total 300 (5 concentrations x 20 individuals x 1-time points x 3 replicates) specimens of B. marginata were cultured for this study. In addition, only the highest concentration (100 µg/L) of PFBS was also applied to four other foraminifera taxa ( Nonionella sp. T1, Cassidulina laevigata, Globobulimina turgida and Eubuliminella sp. ) with the same number of foraminifera and replicates. The capsules including foraminifera were dried for 3 days at room temperature and the calcitic test was removed by adding 12 µL 4% hydrochloric acid (HCl) to each capsule. Finally, the capsules were dried at 50°C for three days and sent to the University of Vienna for further measurements. The isotope mass ratio was measured at the Stable Isotope Laboratory for Environmental Research (SILVER – University of Vienna), and the calculation of the amount of phytodetrital 13 C (pC) and 15 N (pN) was done according to Lintner et al. (2020). One-way ANOVAs (level of significance p = 0.05) using PAST 4.0 software (Hammer and Harper, 2001 ) were performed to test if the concentration of the pollutant significantly affected the food uptake of the foraminifera. Two-way ANOVA (level of significance p = 0.05) was applied to test differences between tested species and concentrations. 3. Results The carbon uptake (pC) differs between the tested species (p < 0.001). Detailed analysis (post-hoc) between the species is given in Table 1 and Fig. 2 . Two tested species ( Eubuliminella sp. ), p = 0.394 and C. laevigata , p = 0.059 showed no significant change in the carbon uptake during incubation with PFBS, although for C. laevigata a (p = 0.0585) a trend towards lower amount of pC could was evident. All other species ( B. marginata , p = 0.044; G. turgida , p = 0.028 and Nonionella sp. T1, p = 0.002) reacted highly sensitively to the presence of PFBS in the culture medium, significantly reducing their carbon uptake. The nitrogen uptake (pN) was also significantly different (p < 0.001) between the species. Except for C. laevigata (p = 0.116) all tested species showed a clear reduction of nitrogen uptake in the cultures spiked with PFBS ( F. complanata , p < 0.001; B. marginata , p = 0.038; G. turgida , p = 0.037 and Nonionella sp. T1, p = 0.004). The concentration effect was more closely investigated by B. marginata (Fig. 3 ). Carbon uptake differs significantly (p = 0.001, df = 4) between the groups and except for the lowest concentration (0.1 µg/L PFBS) all applied levels of PFBS reduce pC significantly during incubation (Table 2 ). Additionally, the nitrogen uptake (pN) was significantly reduced in B. marginata (p = 0.001, df = 4), due to the increased level of PFBS. Similar to pC, the lowest concentration of PFBS does not affect pN (p = 0.096), but all other concentrations significantly reduce the nitrogen assimilation (Table 2 ). Table 1 Post-hoc test of pC (yellow) and pN (green) for different foraminifera species. Significant values are in bold. Species G. turgida Eubuliminella sp. C. laevigata Nonionella sp. T1 B. marginata G. turgida 0.964 0.769 0.020 < 0.001 Eubuliminella sp. 0.307 0.985 0.005 < 0.001 C. laevigata 0.117 0.978 0.001 < 0.001 Nonionella sp. T1 0.026 < 0.001 < 0.001 < 0.001 B. marginata < 0.001 < 0.001 < 0.001 0.010 Table 2 Post-hoc test for carbon (pC) and nitrogen (pN) uptake at different PFBS concentrations (0, 0.1, 1, 10 and 100 µg/L). Significant values are in bold. Concentrations 0 0.1 1 10 100 0 0.245 0.002 0.002 0.012 0.1 0.096 0.065 0.055 0.324 1 0.002 0.162 1.000 0.800 10 0.002 0.119 1.000 0.746 100 0.006 0.428 0.946 0.878 Plate 1: SEM images of investigated foraminifera. A : Eubuliminella sp. ; B : Bulimina marginata ; C : Cassidulina laevigata , D : Globobulimina turgida , E : Nonionella sp. T1; All scale bars are 100 µm. 4. Discussion 4.1. The influence of PFAS on the metabolism of organisms PFAS exposure has earlier been shown to impact the health of humans and wildlife. Studies found a link between exposure of this pollutants and various health problems like cancers, elevated cholesterol levels, decreased immune functionalities or increasing birth defects (Bonefeld-Jorgensen et al., 2014). The microbial eukaryotes (foraminifera) examined here are protists, which means that they only have one cell and cannot have disease symptoms similar to humans or higher organisms. Yet, foraminifera can clearly be affected by the presence of PFAS. We showed that low concentrations of PFBS (1 µg/L) lead to a reduction in the metabolism of the foraminifera. In 2022, Lim examined the influence of PFOS on the ciliate Tetrahymena pyriformis and, while they found only minor toxic effects at 5 µM (2,500 µg/L), significant toxicity was observed at PFOS concentrations > 50 µM. Our disruptive factor was PFBS, which is about 3/5 of the molecular weight of PFOS. Yet, the first negative trend (p = 0.096) in the carbon uptake of the foraminifera was seen already at 0.1 µg PFBS/L, which corresponds to a molecular concentration of 0.3 nM. For most of the foraminifera tested herein, the toxic concentration of PFAS was between 0.1 and 1 µg/L. The same PFAS range (0.8 µg/L) was found to be toxic for water flea, Daphnia magna , which is a commonly used water quality indicator (Boudreou et al., 2003). Based on recent literature, PFAS causes mitochondrial dysfunction through several mechanisms, including attacks on oxidative phosphorylation, calcium signalling and structural integrity. Consequently, organisms with impaired metabolism and lacking energy fail (e.g., Hofmann et al., 2023 ; Liu et al., 2023 ). The negative effect of PFAS depends primarily on how long the organisms stay in contact with the pollutant. Increased mortality and reproductive defects were found in other water flea species, D. carinata , after 21 days of incubation at 1 µg/L PFOS (Logeshwaran et al., 2021 ). The foraminifera in our experiments were exposed to the pollutants only for three days, but no increased mortality was observed. In the future, it must be assumed that further enrichments of PFAS in drinking water, groundwater and surface water in eutrophic areas will lead to increased concentrations of PFAS in seawater (Ma et al., 2022 ). These increased concentrations of PFAS will strongly influence marine microbial eukaryotes such as foraminifera, highlighting that further studies are necessary to clarify which PFAS compounds have a particularly negative impact on marine microbial eukaryotes, so that the most toxic pollutants can be avoided in the future. 4.2. How harmful is PFBS compared to other pollutants? In the past, foraminifera were often incubated with organic or inorganic pollutants to study their effect on foraminiferal activity. Due to their diversity and sensitivity, foraminifera are considered useful bioindicators for determining the presence of pollutants in marine habitats (e.g. O’Brien et al., 2021; Bouchet et al., 2018 ). Our results show that in four out of five species tested, either carbon or nitrogen cycling (or both) was significantly reduced when the foraminifera were incubated with PFBS, C. laevigata was the only species unaffected by PFBS. It should be mentioned at this point that the amount of food consumed by C. laevigata (control group) is extremely low at an average of 0.004 µg C/mg or 0.001 µg N/mg. If C. laevigata was incubated with PFBS, even lower average values ​​of 0.001 µg C/mg or 0.0005 µg N/mg were obtained. Based on the small amount of food consumed by C. laevigata , we can assume that either C. calcitrans is not a preferred food source for this species, or that C. laevigata did not thrive well in laboratory conditions, which resulted in the widely scattered measured values. Other laboratory experiments with C. laevigata also confirmed that this species is difficult to cultivate under stressful conditions (Mojtahid et al., 2023 ). However, this in turn leads to the assumption that the control group and the foraminifera incubated with PFBS do not differ significantly, although food intake (based on the mean values ​​of pC and pN) is much lower as soon as PFBS is in the culture medium. Depending on the species, the presence of PFBS leads to a decrease in carbon assimilation of 30–70%. Other studies showed that the presence of trace metals such as Cd, Zn, Pb or Cu cause a reduction of the carbon assimilation of foraminifera ( Ammonia confertitesta ) by approximately 50–60% (Lintner et al., 2025a ). Larger benthic foraminifera ( Amphistegina lobifera ) incubated with nanoparticles (TiO 2 and ZnO) also showed a reduced carbon uptake of around 75% when TiO 2 is present or 90% when ZnO is present in the culture water (Lintner et al., 2025b). Based on this comparison, the toxicity of PFBS on foraminifera can be roughly compared to that of trace metals. However, the far more interesting fact is that the nitrogen assimilation of foraminifera incubated with PFBS was also reduced by 60–70%. In another experiment, when foraminifera were incubated with pollutants such as trace metals, the nitrogen content often increased, which suggests an increased production of stress proteins (Lintner et al., 2025a ). Similar result was found in ciliate T. pyriformis , which showed an increased proportion of proteins during their incubation with PFOS (Lim, 2022 ). However, we observed a decrease in pC and pN in the foraminifera species we studied, which suggests no increased production of proteins. Only when exposed to highly toxic pollutants such as ZnO was the nitrogen cycle inhibited in correlation with the carbon cycle (Lintner et al., 2025a ). This observation suggests two conclusions: (1) PFBS could be a compound that is equally toxic as ZnO, however, its effect does not occur within two days as they do with ZnO, but may be slower; (2) It is possible that PFBS causes a disruption in protein biosynthesis in foraminifera, simultaneously causing an inhibition of the stress protein production. If this is the case, this underlines the serious nature of PFAS pollution because stress proteins are evolutionary intended to protect eukaryotic cells from damage under stressful conditions (e.g. Wan et al., 2020). Dysregulation of stress proteins in humans is associated with many fatal diseases including cancer, stroke as well as neurodegenerative and infectious diseases (Wan et al., 2020 and references therein). Dysregulation of stress proteins in protists is also known but more research is necessary to understand this complex process (Kahn et al., 2015). Further studies are necessary to investigate these aspects in detail, as it can be expected that in the future the influence of PFAS on foraminifera (and other microbial eukaryotes) will increase due to an increase in PFAS pollution in aquatic environments. 5. Conclusions Our study clearly showed that PFAS, or more precisely PFBS, negatively effects the metabolism of foraminifera, with implications for other microbial eukaryotes. Our first hypothesis (H1) that the concentration of the pollutant is an important factor, could not be completely verified based on our experiments, as we could not determine a significant concentration effect within the range of 0.1 to 100 µg/L in Bulimina marginata . Nevertheless, certain trends emerged indicating a decrease in foraminiferal metabolic activity as the PFBS concentration increased (see Table 2 ). We could demonstrate that foraminiferal metabolism was significantly reduced (p = 0.002) even at low (1 µg/L) PFBS concentrations, which confirms the highly toxic potential of this pollutant. The second hypothesis (H2) was fully confirmed showing that response to PFBS was species-specific. Certainly, the metabolism of the five studied foraminiferal species reacted differently to PFBS and some species ( B. marginata , G. turgida ) have more limitations in their metabolism as compared to others ( Eubuliminella sp. , Nonionella sp. T1). It can generally be postulated that PFBS is a pollutant with the potential to disrupt foraminifera in their natural habitat. Future studies should focus on investigating the effects of PFBS on foraminifera and other eukaryotes, as these pollutants appear to block metabolic pathways that are unaffected by other pollutants, such as trace metals. Declarations Acknowledgements We thank crew of the R/V Alice and personal of the Kristineberg Research and Innovation Center (Fiskebäckskil, Sweden) for assistance with sampling and experimental setup. For the purpose of Open Access, ML has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. Funding declaration This research is part of the project No. UMO-2022/47/P/ST10/01013 co-funded by the Polish National Science Centre (NCN) and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 945339 to ML. Research stay at the Kristineberg Research and Innovation Centre in summer 2024 was funded by the Birgit and Birger Wåhlströms Memorial Fund (ML and IPA). Author contributions ML planned and conducted the experiments, evaluated the measured values using appropriate statistics, and wrote the first manuscript draft. IPA obtained funding (together with ML), organised and performed sampling at sea and assisted with drafting the first version of the manuscript. KH and JS provided the interference factor and assisted with experimental design. WW organized the isotope measurements of the samples. JG and JT assisted with sample processing in the laboratory. All authors carefully read the manuscript and contributed to writing. Competing interests The authors have no competing interests to declare. Data availability Data will be made available upon request to the corresponding author (Irina Polovodova Asteman). References Abunada, Z., Alazaiza, M. Y. & Bashir, M. J. An overview of per-and polyfluoroalkyl substances (PFAS) in the environment: Source, fate, risk and regulations. Water 12(12), 3590, (2020). Beale, D. J. et al. Bioaccumulation and metabolic response of PFAS mixtures in wild-caught freshwater turtles (Emydura macquarii macquarii) using omics-based ecosurveillance techniques. Sci. 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Exposure to perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at ppb concentration induces chronic toxicity in Daphnia carinata . Sci. Total Environ. 769 , 144577 (2021). Ma, T., Ye, C., Wang, T., Li, X. & Luo, Y. Toxicity of per-and polyfluoroalkyl substances to aquatic invertebrates, planktons, and microorganisms. Int. J. Environ. Res. Public Health . 19 (24), 16729 (2022). Mak, Y. L. et al. Perfluorinated compounds in tap water from China and several other countries. Environ. Sci. Technol. 43 (13), 4824–4829 (2009). Mojtahid, M. et al. Assessing the impact of different carbonate system parameters on benthic foraminifera from controlled growth experiments. Chem. Geol. 623 , 121396 (2023). Moghadasi, R., Mumberg, T. & Wanner, P. Spatial Prediction of Concentrations of Per-and Polyfluoroalkyl Substances (PFAS) in European Soils. Environ. Sci. Technol. Lett. 10 (11), 1125–1129 (2023). Mumberg, T., Ahrens, L. & Wanner, P. Managed aquifer recharge as a potential pathway of contaminants of emerging concern into groundwater systems – A systematic review. Chemosphere 364 , 143030. https://doi.org/10.1016/j.chemosphere.2024.143030 (2024). Ivantsova, E., Lu, A. & Martyniuk, C. J. Occurrence and toxicity mechanisms of perfluorobutanoic acid (PFBA) and perfluorobutane sulfonic acid (PFBS) in fish. Chemosphere 349 , 140815 (2024). Pan, C. G., Xiao, S. K., Yu, K. F., Wu, Q. & Wang, Y. H. Legacy and alternative per-and polyfluoroalkyl substances in a subtropical marine food web from the Beibu Gulf, South China: Fate, trophic transfer and health risk assessment. J. Hazard. Mater. 403 , 123618 (2021). Piña-Ochoa, E. et al. Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida. Proceedings of the National Academy of Sciences , 107(3), 1148–1153, (2010). Polovodova Asteman, I., Hanslik, D. & Nordberg, K. An almost completed pollution-recovery cycle reflected by sediment geochemistry and benthic foraminiferal assemblages in a Swedish–Norwegian Skagerrak fjord. Mar. Pollut. Bull. 95 (1), 126–140 (2015). Poothong, S., Papadopoulou, E., Padilla-Sánchez, J. A., Thomsen, C. & Haug, L. S. Multiple pathways of human exposure to poly-and perfluoroalkyl substances (PFASs): From external exposure to human blood. Environ. Int. 134 , 105244 (2020). Prazeres, M., Martínez-Colón, M. & Hallock, P. Foraminifera as bioindicators of water quality: The FoRAM Index revisited. Environ. Pollut. 257 , 113612 (2020). Salonen, I. S., Chronopoulou, P. M., Bird, C., Reichart, G. J. & Koho, K. A. Enrichment of intracellular sulphur cycle – associated bacteria in intertidal benthic foraminifera revealed by 16S and aprA gene analysis. Sci. Rep. 9 (1), 11692 (2019). Schlitzer, R. Ocean data view. (2019). http://odv.awi.de Sen Gupta, B. K. Introduction to modern foraminifera in Modern foraminifera (ed. Sen Gupta, B.K.) 3–6Springer Dordrecht, (2003). Shao, M. et al. Occurrence and distribution of perfluoroalkyl substances (PFASs) in surface water and bottom water of the Shuangtaizi Estuary, China. Environ. Pollut. 216 , 675–681 (2016). Sharma, B. M. et al. Perfluoroalkyl substances (PFAS) in river and ground/drinking water of the Ganges River basin: emissions and implications for human exposure. Environ. Pollut. 208 , 704–713 (2016). Wang, B. et al. Perfluoroalkyl substances and endometriosis-related infertility in Chinese women. Environ. Int. 102 , 207–212 (2017). Zhang, X., Hu, T., Yang, L. & Guo, Z. The investigation of perfluoroalkyl substances in seasonal freeze–thaw rivers during spring flood period: a case study in Songhua River and Yalu River, China. Bull. Environ Contam. Toxicol. 101 , 166–172 (2018). Plates Plates 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Plate1.png Plate 1: Scanning Electron Microscopy images of investigated foraminifera. A: Eubuliminella sp. ; B: Bulimina marginata ; C: Cassidulina laevigata , D: Globobulimina turgida , E: Nonionella sp. T1; All scale bars are 100 µm. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 May, 2026 Reviewers agreed at journal 16 May, 2026 Reviews received at journal 23 Feb, 2026 Reviewers agreed at journal 23 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers invited by journal 19 Jan, 2026 Editor assigned by journal 27 Aug, 2025 Submission checks completed at journal 26 Aug, 2025 First submitted to journal 26 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:27:54","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120416,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/2a9e6a2b74745a258cb329e0.html"},{"id":94253412,"identity":"45669895-3d03-4286-a977-9fae36c511a4","added_by":"auto","created_at":"2025-10-24 07:19:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":364573,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the location of the study area (Gullmar Fjord) and the sampling site (star). The map was created using Ocean Data View (ODV) software – version 5.2.0, Schlitzer, 2019; http://odv.awi.de.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/4875073d1c9c1f1600a4f547.png"},{"id":94254184,"identity":"4183c468-eb40-4882-a518-3d12a1d478ca","added_by":"auto","created_at":"2025-10-24 07:27:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173408,"visible":true,"origin":"","legend":"\u003cp\u003eCarbon (pC) and nitrogen (pN) uptake of different foraminiferal species during incubation with PFBS. The concentration c0 indicates the control (where no PFBS was added), and c100 means 100 µg/L PFBS.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/5fd11eec00b070b9679f3ef0.png"},{"id":94253409,"identity":"6e47fd19-4131-4380-8ec3-3c3a55f58e8f","added_by":"auto","created_at":"2025-10-24 07:19:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101797,"visible":true,"origin":"","legend":"\u003cp\u003eCarbon (pC) and nitrogen (pN) uptake of \u003cem\u003eB. marginata\u003c/em\u003e during incubation with different concentrations (0, 0.1, 1, 10 and 100 µg/L) of PFBS.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/5cb5881c9275691f35929c7d.png"},{"id":94255723,"identity":"441c5169-048d-472b-9c4c-436e2f66a7af","added_by":"auto","created_at":"2025-10-24 07:43:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1467031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/6f98efde-aa90-4903-b7c2-257b65862ca1.pdf"},{"id":94254181,"identity":"fbdbbb3a-faf6-4bcf-bd37-2cb10f2873d9","added_by":"auto","created_at":"2025-10-24 07:27:53","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":375913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlate 1: \u003c/strong\u003eScanning Electron Microscopy images of investigated foraminifera. A: \u003cem\u003eEubuliminella sp.\u003c/em\u003e; B: \u003cem\u003eBulimina marginata\u003c/em\u003e; C: \u003cem\u003eCassidulina laevigata\u003c/em\u003e, D: \u003cem\u003eGlobobulimina turgida\u003c/em\u003e, E: \u003cem\u003eNonionella \u003c/em\u003esp. T1; All scale bars are 100 µm.\u003c/p\u003e","description":"","filename":"Plate1.png","url":"https://assets-eu.researchsquare.com/files/rs-7460059/v1/d1275fff2ee424433f5e5dce.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic disruptions in marine environments: synthetic eternal chemicals PFBS impact widespread microbial eukaryotes","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1. General introduction\u003c/h2\u003e\u003cp\u003eThe introduction of pollutants into the environment by humans has increased rapidly over the last few decades. The group of per- and polyfluoroalkyl substances (PFAS) has in particular attracted general concern in recent years. The PFAS group includes today about 15 000 different compounds of similar properties (SSNC, 2024). These exclusively synthetic chemicals are characterized by an aliphatic carbon in which hydrogen has been replaced by fluorine (Abunada et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). If hydrogen has been completely replaced, the compound is called per- and by partial substitution poly-fluoroalkyl (Abunada et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Such chemicals have been used since the 1940s in various areas of industry like textile waterproofing, fire-fighting foam, non-sticking frying pans and cosmetics but can also be found in ammunition, nylon guitar strings or artificial turf (SSNC, 2024; Gl\u0026uuml;ge et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kissa, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The problem with these compounds is that they are highly persistent, so that they are sometimes referred to as eternal chemicals (SSNC, 2024). It means they can accumulate in the environment for a long time and have a negative impact on the health of organisms (De Silva et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eToday, PFAS have been found in waters, soils, living organisms and even air (e.g. Giesy and Kannan, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In addition, PFAS are found in all aqueous matrixes, such as rain, snow, groundwater, seawater, lakes and rivers (Fabrega et al., 2014; Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Moghadasi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mumberg et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on lifestyle, people consume about 0.17\u0026ndash;0.21 ng/kg body weight of PFAS daily (Gellrich et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the environment, concentrations of 7,000\u0026ndash;290,000 ng/L PFAS have been found in soils (Eriksson et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Analysis of tap water showed values ​​of 0.62 ng/L in Japan (Mak et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) but extreme levels of up to 2,000 ng/L in Oakdale (USA) have been reported (Abunada et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In some river inlets like the Shuangtaizi Estuary (China) a concentration of 66.2 to 185 ng/L PFAS were found in the surface water (Shao et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In marine coastal, surface waters, the highest PFAS concentrations are found in the Indian Ocean (41.1 ng/L) followed by the North Pacific Ocean (12.8 ng/L), the North Atlantic Ocean (4.0 ng/L), the South Atlantic Ocean (2.6 ng/L), the Arctic Ocean (1.1 ng/L), the South Pacific Ocean (0.5 ng/L) and, finally, the Southern Ocean (\u0026lt;\u0026thinsp;0.1 ng/L) (Khan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, PFAS has also been detected in the marine sediments of North Pacific Ocean (4.61 ng/g), Southern Ocean (4.56 ng/g), South Pacific (3.16 ng/g), North Atlantic (2.29 ng/g) and the Arctic (1.54 ng/g) oceans (Kahn et al., 2023).\u003c/p\u003e\u003cp\u003eIn this study we investigated effects of the pollutant PFBS (perfluorobutane sulfonic acid), which is a short-chain PFAS compound. Compared to long-chain PFAS, PFBS has been suggested as an alternative with lower propensity to bioconcentrate in tissues relative to other PFAS (Ivantsova et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). PFBS concentrations of 1.21\u0026ndash;46.06 ng/L were measured in Jiulong River estuary, China, up to 4.9 ng/L in groundwater from Ganges River, India, or 7.89\u0026ndash;29.1 ng/g in wastewater sludge in Germany (Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sharma et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gomez-Canela et al., 2012). However, in the last few years some studies found a relationship between a toxic effect or pathological development of humans and the presence of PFBS (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and thus there is an urgent need to improve our understanding how PFBS interact with organisms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.2. PFAS and their effect on organisms\u003c/h2\u003e\u003cp\u003eOnce pollutants have entered the aquatic environment, it does not take long for them to interact with organisms there. The easiest way to transmit them is through direct ingestion by aquatic organisms. Many studies have previously focused on long-chain PFOS (perfluorooctanesulfonic acid), which can accumulate to between 9 and 55 ng/g dry weight in fish, and it has been shown that these compounds are more likely to accumulate in liver tissues than in muscle tissue (Kahn et al., 2023). A particularly high value was found in the liver of dusky flathead (135 ng/g ww) (Kahn et al., 2023). But PFAS can also bioaccumulate in other aquatic organisms. Beale et al., (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) investigated freshwater turtles (\u003cem\u003eEmydura macquarii macquarii\u003c/em\u003e) and found 235 times higher PFAS concentration in the turtle\u0026rsquo;s serum (PFOS; 889\u0026thinsp;\u0026plusmn;\u0026thinsp;56 ng/mL) than in the water where the animals lived (ΣPFAS 32.0 \u0026micro;g/L). However, the bioaccumulation of PFAS does not only affect aquatic organisms. In 2023, Lettoof et al., investigated 35 tiger snakes (\u003cem\u003eNotechis scutatus\u003c/em\u003e) and they found PFOS concentrations up to 322\u0026thinsp;\u0026plusmn;\u0026thinsp;193 \u0026micro;g/kg animal. Even in birds and their eggs elevated PFOS concentrations (8 to 608 ng/g ww) have been observed (Kahn et al., 2023). All these examples illustrate that PFAS is widely distributed in nature and both terrestrial and aquatic food chains, and therefore potentially poses a risk to humans. The daily intake of PFAS in humans is assumed to be in the range of 72\u0026ndash;1810 pg\u0026middot;kg bw\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Poothong et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMost studies have focused on bioaccumulation of PFAS is in vertebrates, whilst studies on invertebrates or microbial eukaryotes remain limited. For example, mean values ​​for PFOS in Mediterranean mussels were 60.03 ng/g ww, which was found comparable to other animals (Khan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Accumulations of PFAS have also been reported for lower trophic levels, such as plankton (Pan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although some of the microbes like bacteria are involved in the degradation of PFAS, based on few studies, these pollutants can be just as toxic to unicellular organisms. In a laboratory study, the protists \u003cem\u003eTetrahymena pyriformis\u003c/em\u003e that belong to Ciliophoran were exposed to PFOS concentrations of 0\u0026ndash;5000 \u0026micro;M and cellular motility, division and function were measured (Lim, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Even at 5 \u0026micro;M PFOS, a decrease in the vitality of the organisms was observed, as compared to the control group incubated without PFOS (Lim, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo expand our knowledge about the interaction of PFAS and microbial eukaryotes this study focus on the change of the metabolic behaviour on benthic foraminifera if PFBS is present in the seawater. Foraminifera are mostly marine protists, which can be found in all marine habitats on Earth (Sen Gupta, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Due to their high abundance, foraminifera play a major role in global marine biogeochemical cycles and are also important ecosystem engineers (Hallock et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Pi\u0026ntilde;a-Ochoa et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Salonen et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Langlet et al., 2023; Glock et al., 2024). Foraminifera can be used as bioindicators as they react fast and sensitive to different pollutants (e.g. Prazeres et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Frontalini et al., 2011). Heavy metals were shown to cause a disruption in foraminifera metabolism (Lintner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), likely explaining lower diversity or even foraminifera-barren sediments in regions heavily polluted with metals (e.g. Ferraro et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Polovodova Asteman et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Yet, much less is known about the effects of organic pollutants on foraminifera. Some initial studies have shown that the presence of sunscreens, pesticides or antibiotics in seawater greatly reduces or even completely inhibits the metabolism of foraminifers (Lintner et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, 2025b). This study aims to investigate the influence of PFBS on foraminifera. To do this, we use different concentrations of PFBS in laboratory experiments to test a concentration-dependent reduction in metabolism through feeding with labelled algae. The first hypothesis (H1) is that at higher concentrations of PFBS, the metabolism of foraminifera is reduced. Further we hypothesize (H2) that the pollutant has different effects on the different species and that metabolism is inhibited in a species-dependent manner. To test a species-specific impact of PFBS, we are testing the response of five different species to the presence of the pollutant.\u003c/p\u003e\u003c/div\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sediment sampling and sampling area\u003c/h2\u003e\u003cp\u003eSediment sampling was performed on 3nd of July 2024 aboard of R/V Alice (University of Gothenburg) by using a box corer at Station 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in the outer part of the Gullmar Fjord, Sweden (58\u0026deg;15.538\u0026rsquo;N, 11\u0026deg;27.483\u0026rsquo;E and 40 m water depth).\u003c/p\u003e\u003cp\u003eThe Gullmar Fjord, located on the west coast of Sweden, is a true fjord with a maximum water depth of 118.6 m. It is one of the world\u0026rsquo;s most well-studied marine settings, with the first hydrographic observations being taken as early as 1869 (Ekman, 1870). As a sill fjord, the Gullmar Fjord has remarkably high sediment accumulation rates of 0,7\u0026ndash;1.4 cm per year (Filipsson and Nordberg, 2004) and low tidal activity providing a high-resolution paleo-environmental archive (e.g. Harland et al., 2013; Polovodova Asteman et al, 2018). The sill of the fjord has a depth of 42 m and results in a fjord basin being stratified with respect to temperature and salinity. The fjord deepest basin\u0026thinsp;\u0026gt;\u0026thinsp;50 m water depth has a water mass derived from the Skagerrak (S\u0026thinsp;=\u0026thinsp;34\u0026ndash;35 PSU) and is prone to stagnation and seasonal oxygen depletion, which became more frequent since the early 1980s driven by the variability of the North Atlantic Oscillation (Nordberg et al., 2000; Bjork and Nordberg, 2003). The fjord areas shallower than sill depth are influenced by the brackish water from the Baltic Current mixed with the Skagerrak and Kattegat water, as well as local runoff and supply from the \u0026Ouml;rekils\u0026auml;lven river in the inner part of the fjord (Arneborg et al., 2004). The Gullmar Fjord became the first marine conservation area in Sweden in 1983 due to its high biodiversity, and it currently has no anthropogenic impact. The closest impact sources are the Lysekil harbor, the Preem Oil Refinery, located in the adjacent Brofjord, and the European route E6 passing by next to the fjord head (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn parallel to the sediment sampling, environmental data (temperature, salinity, and oxygen) were taken on board by using a CTD probe. The T, S and O\u003csub\u003e2\u003c/sub\u003e observations proximal to the sea floor showed 14.9\u0026deg;C, salinity of 33 and 4.1 ml O\u003csub\u003e2\u003c/sub\u003e L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The retrieved sediment surface in the box corer was intact and covered by water, which was gently siphoned out by using a plastic tube. The top 5 cm of the sediment were transferred to a bucket and taken to the lab at Kristineberg Centre for Marine Research and Innovation for further experiments. There, the sediment was gently sieved by using a 63 and 1000 \u0026micro;m sieves using ambient fjord water to concentrate living foraminifera and remove larger macrofauna. The resulting sediment fraction (63\u0026ndash;1000 \u0026micro;m) was collected in a plexiglass tank to establish a permanent culture, which was cultivated under 10\u0026deg;C in the darkness to mimic natural conditions and to acclimatize foraminifera to laboratory conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental Setup\u003c/h2\u003e\u003cp\u003eAfter three days of acclimatization, the sediment was washed again over a 150 \u0026micro;m sieve and the foraminifera, which had a characteristic brown-yellow color of the cytoplasm, were picked out in a Petri dish by using a very fine brush. Following that, foraminifera were cleaned with a brush, all adhering particles were carefully removed and clean foraminifera were transferred to a crystallization dish. Fifty individuals were placed in the center of a dish, and left for one day to test their vitality by a so called \u0026ldquo;crawling test.\u0026rdquo; After one day, only the actively moving individuals were selected for further experiments. To accurately identify foraminifera on species level, secondary electron microscopy (SEM) images were taken of individuals that were first coated with gold.\u003c/p\u003e\u003cp\u003eFor feeding and isotopic uptake experiments, 20 individuals (with three replicates) of each of the five foraminifera species (\u003cem\u003eBulimina marginata, Cassidulina laevigata, Eubuliminella sp., Globobulimina turgida\u003c/em\u003e and \u003cem\u003eNonionella\u003c/em\u003e sp. T1) were placed per separate crystallization dish and covered with 100 ml artificial seawater water with the same salinity as at the sampling sites. These cultures served as control samples, where no additional interference factor (PFBS) was added. As interference factor, PFBS was used in four different concentrations (100 \u0026micro;g/L; 10 \u0026micro;g/L; 1 \u0026micro;g/L and 0.1 \u0026micro;g/L). As PFBS is largely insoluble in water, a 10 mg/mL stock solution in methanol was prepared and added to artificial seawater. The lowest concentration (0.1 \u0026micro;g/L) is in the range of natural contamination (see examples in Introduction). To test the effect of future PFBS contamination, three increased levels of the pollutant were also applied, each elevated by a factor 10.\u003c/p\u003e\u003cp\u003eTo observe the change in the metabolic activity of the foraminifera, isotopically labelled diatoms (\u003cem\u003eChaetoceros simplex\u003c/em\u003e var. \u003cem\u003ecalcitrans\u003c/em\u003e) were added to the cultures. Theses diatoms naturally occur in the Gullmar Fjord (Hallfors, 2004), where the foraminifera come from. Diatoms were grown in a nutrient medium enriched with \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN and therefore themselves contained an increased concentration of isotopes (for method details see Lintner et al., 2020). After consuming these algae, the foraminifera themselves become enriched with isotopes. Generally, the higher the proportion of isotopes in the foraminifera after cultivation, the lower the influence of the disturbing factor (here, PFBS), as indicated by higher feeding intensity of the foraminifera, used here as a measure to assess foraminiferal metabolism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Sample and data processing\u003c/h2\u003e\u003cp\u003eAfter incubation with the isotopically enriched algae foraminifera were removed from the culture and cleaned from any adhering particles with a brush. To measure the isotopic uptake, 20 specimens of \u003cem\u003eBulimina marginata\u003c/em\u003e were transferred to separate pre-weighted Sn-capsules. In total 300 (5 concentrations x 20 individuals x 1-time points x 3 replicates) specimens of \u003cem\u003eB. marginata\u003c/em\u003e were cultured for this study. In addition, only the highest concentration (100 \u0026micro;g/L) of PFBS was also applied to four other foraminifera taxa (\u003cem\u003eNonionella\u003c/em\u003e sp. T1, \u003cem\u003eCassidulina\u003c/em\u003e laevigata, \u003cem\u003eGlobobulimina turgida\u003c/em\u003e and \u003cem\u003eEubuliminella sp.\u003c/em\u003e) with the same number of foraminifera and replicates. The capsules including foraminifera were dried for 3 days at room temperature and the calcitic test was removed by adding 12 \u0026micro;L 4% hydrochloric acid (HCl) to each capsule. Finally, the capsules were dried at 50\u0026deg;C for three days and sent to the University of Vienna for further measurements. The isotope mass ratio was measured at the Stable Isotope Laboratory for Environmental Research (SILVER \u0026ndash; University of Vienna), and the calculation of the amount of phytodetrital \u003csup\u003e13\u003c/sup\u003eC (pC) and \u003csup\u003e15\u003c/sup\u003eN (pN) was done according to Lintner et al. (2020). One-way ANOVAs (level of significance p\u0026thinsp;=\u0026thinsp;0.05) using PAST 4.0 software (Hammer and Harper, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) were performed to test if the concentration of the pollutant significantly affected the food uptake of the foraminifera. Two-way ANOVA (level of significance p\u0026thinsp;=\u0026thinsp;0.05) was applied to test differences between tested species and concentrations.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe carbon uptake (pC) differs between the tested species (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Detailed analysis (post-hoc) between the species is given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Two tested species (\u003cem\u003eEubuliminella sp.\u003c/em\u003e), p\u0026thinsp;=\u0026thinsp;0.394 and \u003cem\u003eC. laevigata\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.059 showed no significant change in the carbon uptake during incubation with PFBS, although for \u003cem\u003eC. laevigata\u003c/em\u003e a (p\u0026thinsp;=\u0026thinsp;0.0585) a trend towards lower amount of pC could was evident. All other species (\u003cem\u003eB. marginata\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.044; \u003cem\u003eG. turgida\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.028 and \u003cem\u003eNonionella\u003c/em\u003e sp. T1, p\u0026thinsp;=\u0026thinsp;0.002) reacted highly sensitively to the presence of PFBS in the culture medium, significantly reducing their carbon uptake. The nitrogen uptake (pN) was also significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the species. Except for \u003cem\u003eC. laevigata\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.116) all tested species showed a clear reduction of nitrogen uptake in the cultures spiked with PFBS (\u003cem\u003eF. complanata\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eB. marginata\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.038; \u003cem\u003eG. turgida\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.037 and \u003cem\u003eNonionella\u003c/em\u003e sp. T1, p\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e\u003cp\u003eThe concentration effect was more closely investigated by \u003cem\u003eB. marginata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Carbon uptake differs significantly (p\u0026thinsp;=\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;4) between the groups and except for the lowest concentration (0.1 \u0026micro;g/L PFBS) all applied levels of PFBS reduce pC significantly during incubation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, the nitrogen uptake (pN) was significantly reduced in \u003cem\u003eB. marginata\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;4), due to the increased level of PFBS. Similar to pC, the lowest concentration of PFBS does not affect pN (p\u0026thinsp;=\u0026thinsp;0.096), but all other concentrations significantly reduce the nitrogen assimilation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\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\u003ePost-hoc test of pC (yellow) and pN (green) for different foraminifera species. Significant values are in bold.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eG. turgida\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEubuliminella sp.\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eC. laevigata\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eNonionella sp. T1\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eB. marginata\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eG. turgida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.964\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.020\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEubuliminella sp.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.307\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. laevigata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNonionella sp. T1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.026\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. marginata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.010\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePost-hoc test for carbon (pC) and nitrogen (pN) uptake at different PFBS concentrations (0, 0.1, 1, 10 and 100 \u0026micro;g/L). Significant values are in bold.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentrations\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.012\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.096\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.324\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.800\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.119\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.746\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.428\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.946\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlate 1: SEM images of investigated foraminifera. \u003cb\u003eA\u003c/b\u003e: \u003cem\u003eEubuliminella sp.\u003c/em\u003e; \u003cb\u003eB\u003c/b\u003e: \u003cem\u003eBulimina marginata\u003c/em\u003e; \u003cb\u003eC\u003c/b\u003e: \u003cem\u003eCassidulina laevigata\u003c/em\u003e, \u003cb\u003eD\u003c/b\u003e: \u003cem\u003eGlobobulimina turgida\u003c/em\u003e, \u003cb\u003eE\u003c/b\u003e: \u003cem\u003eNonionella\u003c/em\u003e sp. T1; All scale bars are 100 \u0026micro;m.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.1. The influence of PFAS on the metabolism of organisms\u003c/h2\u003e\u003cp\u003ePFAS exposure has earlier been shown to impact the health of humans and wildlife. Studies found a link between exposure of this pollutants and various health problems like cancers, elevated cholesterol levels, decreased immune functionalities or increasing birth defects (Bonefeld-Jorgensen et al., 2014). The microbial eukaryotes (foraminifera) examined here are protists, which means that they only have one cell and cannot have disease symptoms similar to humans or higher organisms. Yet, foraminifera can clearly be affected by the presence of PFAS. We showed that low concentrations of PFBS (1 \u0026micro;g/L) lead to a reduction in the metabolism of the foraminifera. In 2022, Lim examined the influence of PFOS on the ciliate \u003cem\u003eTetrahymena pyriformis\u003c/em\u003e and, while they found only minor toxic effects at 5 \u0026micro;M (2,500 \u0026micro;g/L), significant toxicity was observed at PFOS concentrations\u0026thinsp;\u0026gt;\u0026thinsp;50 \u0026micro;M. Our disruptive factor was PFBS, which is about 3/5 of the molecular weight of PFOS. Yet, the first negative trend (p\u0026thinsp;=\u0026thinsp;0.096) in the carbon uptake of the foraminifera was seen already at 0.1 \u0026micro;g PFBS/L, which corresponds to a molecular concentration of 0.3 nM.\u003c/p\u003e\u003cp\u003eFor most of the foraminifera tested herein, the toxic concentration of PFAS was between 0.1 and 1 \u0026micro;g/L. The same PFAS range (0.8 \u0026micro;g/L) was found to be toxic for water flea, \u003cem\u003eDaphnia magna\u003c/em\u003e, which is a commonly used water quality indicator (Boudreou et al., 2003). Based on recent literature, PFAS causes mitochondrial dysfunction through several mechanisms, including attacks on oxidative phosphorylation, calcium signalling and structural integrity. Consequently, organisms with impaired metabolism and lacking energy fail (e.g., Hofmann et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The negative effect of PFAS depends primarily on how long the organisms stay in contact with the pollutant. Increased mortality and reproductive defects were found in other water flea species, \u003cem\u003eD. carinata\u003c/em\u003e, after 21 days of incubation at 1 \u0026micro;g/L PFOS (Logeshwaran et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The foraminifera in our experiments were exposed to the pollutants only for three days, but no increased mortality was observed.\u003c/p\u003e\u003cp\u003eIn the future, it must be assumed that further enrichments of PFAS in drinking water, groundwater and surface water in eutrophic areas will lead to increased concentrations of PFAS in seawater (Ma et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These increased concentrations of PFAS will strongly influence marine microbial eukaryotes such as foraminifera, highlighting that further studies are necessary to clarify which PFAS compounds have a particularly negative impact on marine microbial eukaryotes, so that the most toxic pollutants can be avoided in the future.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.2. How harmful is PFBS compared to other pollutants?\u003c/h2\u003e\u003cp\u003eIn the past, foraminifera were often incubated with organic or inorganic pollutants to study their effect on foraminiferal activity. Due to their diversity and sensitivity, foraminifera are considered useful bioindicators for determining the presence of pollutants in marine habitats (e.g. O\u0026rsquo;Brien et al., 2021; Bouchet et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our results show that in four out of five species tested, either carbon or nitrogen cycling (or both) was significantly reduced when the foraminifera were incubated with PFBS, \u003cem\u003eC. laevigata\u003c/em\u003e was the only species unaffected by PFBS. It should be mentioned at this point that the amount of food consumed by \u003cem\u003eC. laevigata\u003c/em\u003e (control group) is extremely low at an average of 0.004 \u0026micro;g C/mg or 0.001 \u0026micro;g N/mg. If \u003cem\u003eC. laevigata\u003c/em\u003e was incubated with PFBS, even lower average values ​​of 0.001 \u0026micro;g C/mg or 0.0005 \u0026micro;g N/mg were obtained. Based on the small amount of food consumed by \u003cem\u003eC. laevigata\u003c/em\u003e, we can assume that either \u003cem\u003eC. calcitrans\u003c/em\u003e is not a preferred food source for this species, or that \u003cem\u003eC. laevigata\u003c/em\u003e did not thrive well in laboratory conditions, which resulted in the widely scattered measured values. Other laboratory experiments with \u003cem\u003eC. laevigata\u003c/em\u003e also confirmed that this species is difficult to cultivate under stressful conditions (Mojtahid et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, this in turn leads to the assumption that the control group and the foraminifera incubated with PFBS do not differ significantly, although food intake (based on the mean values ​​of pC and pN) is much lower as soon as PFBS is in the culture medium.\u003c/p\u003e\u003cp\u003eDepending on the species, the presence of PFBS leads to a decrease in carbon assimilation of 30\u0026ndash;70%. Other studies showed that the presence of trace metals such as Cd, Zn, Pb or Cu cause a reduction of the carbon assimilation of foraminifera (\u003cem\u003eAmmonia confertitesta\u003c/em\u003e) by approximately 50\u0026ndash;60% (Lintner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Larger benthic foraminifera (\u003cem\u003eAmphistegina lobifera\u003c/em\u003e) incubated with nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003e and ZnO) also showed a reduced carbon uptake of around 75% when TiO\u003csub\u003e2\u003c/sub\u003e is present or 90% when ZnO is present in the culture water (Lintner et al., 2025b). Based on this comparison, the toxicity of PFBS on foraminifera can be roughly compared to that of trace metals. However, the far more interesting fact is that the nitrogen assimilation of foraminifera incubated with PFBS was also reduced by 60\u0026ndash;70%. In another experiment, when foraminifera were incubated with pollutants such as trace metals, the nitrogen content often increased, which suggests an increased production of stress proteins (Lintner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Similar result was found in ciliate \u003cem\u003eT. pyriformis\u003c/em\u003e, which showed an increased proportion of proteins during their incubation with PFOS (Lim, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, we observed a decrease in pC and pN in the foraminifera species we studied, which suggests no increased production of proteins. Only when exposed to highly toxic pollutants such as ZnO was the nitrogen cycle inhibited in correlation with the carbon cycle (Lintner et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). This observation suggests two conclusions: (1) PFBS could be a compound that is equally toxic as ZnO, however, its effect does not occur within two days as they do with ZnO, but may be slower; (2) It is possible that PFBS causes a disruption in protein biosynthesis in foraminifera, simultaneously causing an inhibition of the stress protein production. If this is the case, this underlines the serious nature of PFAS pollution because stress proteins are evolutionary intended to protect eukaryotic cells from damage under stressful conditions (e.g. Wan et al., 2020). Dysregulation of stress proteins in humans is associated with many fatal diseases including cancer, stroke as well as neurodegenerative and infectious diseases (Wan et al., 2020 and references therein). Dysregulation of stress proteins in protists is also known but more research is necessary to understand this complex process (Kahn et al., 2015). Further studies are necessary to investigate these aspects in detail, as it can be expected that in the future the influence of PFAS on foraminifera (and other microbial eukaryotes) will increase due to an increase in PFAS pollution in aquatic environments.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur study clearly showed that PFAS, or more precisely PFBS, negatively effects the metabolism of foraminifera, with implications for other microbial eukaryotes. Our first hypothesis (H1) that the concentration of the pollutant is an important factor, could not be completely verified based on our experiments, as we could not determine a significant concentration effect within the range of 0.1 to 100 \u0026micro;g/L in \u003cem\u003eBulimina marginata\u003c/em\u003e. Nevertheless, certain trends emerged indicating a decrease in foraminiferal metabolic activity as the PFBS concentration increased (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We could demonstrate that foraminiferal metabolism was significantly reduced (p\u0026thinsp;=\u0026thinsp;0.002) even at low (1 \u0026micro;g/L) PFBS concentrations, which confirms the highly toxic potential of this pollutant. The second hypothesis (H2) was fully confirmed showing that response to PFBS was species-specific. Certainly, the metabolism of the five studied foraminiferal species reacted differently to PFBS and some species (\u003cem\u003eB. marginata\u003c/em\u003e, \u003cem\u003eG. turgida\u003c/em\u003e) have more limitations in their metabolism as compared to others (\u003cem\u003eEubuliminella sp.\u003c/em\u003e, \u003cem\u003eNonionella\u003c/em\u003e sp. T1). It can generally be postulated that PFBS is a pollutant with the potential to disrupt foraminifera in their natural habitat. Future studies should focus on investigating the effects of PFBS on foraminifera and other eukaryotes, as these pollutants appear to block metabolic pathways that are unaffected by other pollutants, such as trace metals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank crew of the R/V Alice and personal of the Kristineberg Research and Innovation Center (Fiskebäckskil, Sweden) for assistance with sampling and experimental setup. For the purpose of Open Access, ML has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is part of the project No. UMO-2022/47/P/ST10/01013 co-funded by the Polish National Science Centre (NCN) and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 945339 to ML. Research stay at the Kristineberg Research and Innovation Centre in summer 2024 was funded by the Birgit and Birger Wåhlströms Memorial Fund (ML and IPA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eML planned and conducted the experiments, evaluated the measured values using appropriate statistics, and wrote the first manuscript draft. IPA obtained funding (together with ML), organised and performed sampling at sea and assisted with drafting the first version of the manuscript. \u0026nbsp;KH and JS provided the interference factor and assisted with experimental design. WW organized the isotope measurements of the samples. JG and JT assisted with sample processing in the laboratory. All authors carefully read the manuscript and contributed to writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request to the corresponding author (Irina Polovodova Asteman).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbunada, Z., Alazaiza, M. Y. \u0026amp; Bashir, M. J. 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The investigation of perfluoroalkyl substances in seasonal freeze\u0026ndash;thaw rivers during spring flood period: a case study in Songhua River and Yalu River, China. \u003cem\u003eBull. Environ Contam. Toxicol.\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e, 166\u0026ndash;172 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Plates","content":"\u003cp\u003ePlates 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7460059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7460059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePollution caused by per- and polyfluoroalkyl substances (PFAS) is an urgent environmental issue, as these substances are widespread, highly persistent and toxic to many organisms. Nevertheless, the effects of PFAS on microbial eukaryotes have hardly been analysed yet. In this study, we investigated the influence of perfluorobutane sulfonic acid (PFBS) in different concentrations (0, 0.1, 1, 10, and 100 \u0026micro;g/L) on the metabolism of five benthic foraminifera taxa: \u003cem\u003eBulimina marginata\u003c/em\u003e, \u003cem\u003eCassidulina laevigata\u003c/em\u003e, \u003cem\u003eEubuliminella sp.\u003c/em\u003e, \u003cem\u003eGlobobulimina turgida\u003c/em\u003e and \u003cem\u003eNonionella\u003c/em\u003e sp. T1. In laboratory experiments, foraminifera were incubated with the pollutant for three days while maintaining natural physical parameters from the sampling site in the Gullmar Fjord, Sweden. Our results show that even at low PFBS concentrations, a disruption in the metabolic activity, detected through feeding with isotopically (\u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN) labelled algae, was observed. Even the lowest concentrations of PFBS (0.1 \u0026micro;g/L) completely inhibited metabolic cycles in these protists. With the exception of one species (\u003cem\u003eC. laevigata\u003c/em\u003e), which exhibited generally very low metabolic activity during incubation, all species demonstrated significant decreases in food uptake, suggesting species-specific impact of PFBS. Since a reduction in metabolic activity was evident in all studied taxa, we conclude that PFBS is highly toxic to microbial eukaryotes.\u003c/p\u003e","manuscriptTitle":"Metabolic disruptions in marine environments: synthetic eternal chemicals PFBS impact widespread microbial eukaryotes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 07:19:49","doi":"10.21203/rs.3.rs-7460059/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"311781665789968129663322005534672796333","date":"2026-05-17T08:04:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168631311308001739975481241664055343781","date":"2026-05-16T07:32:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T11:21:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5524417717538937135444306249273455009","date":"2026-01-23T07:12:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197953746427553668257696190630294249800","date":"2026-01-22T08:39:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T20:39:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-27T07:46:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-26T11:43:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-26T07:36:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41fcfd1e-d77c-4716-a241-14abb0be13f5","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"311781665789968129663322005534672796333","date":"2026-05-17T08:04:34+00:00","index":59,"fulltext":""},{"type":"reviewerAgreed","content":"168631311308001739975481241664055343781","date":"2026-05-16T07:32:55+00:00","index":55,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54927018,"name":"Biological sciences/Ecology"},{"id":54927019,"name":"Earth and environmental sciences/Ecology"},{"id":54927020,"name":"Earth and environmental sciences/Environmental sciences"},{"id":54927021,"name":"Biological sciences/Microbiology"},{"id":54927022,"name":"Earth and environmental sciences/Ocean sciences"}],"tags":[],"updatedAt":"2026-01-19T20:53:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-24 07:19:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7460059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7460059","identity":"rs-7460059","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}