Enantioselective toxicity of propranolol on marine diatoms: Assessing growth, energy metabolism and oxidative damage in Phaeodactylum tricornutum

Enantioselective toxicity of propranolol on marine diatoms: Assessing growth, energy metabolism and oxidative damage in Phaeodactylum tricornutum Marina Arenas a,* , Eduardo Feij˜ ao b,c , Irina A. Duarte b , Vanessa F. Fonseca b,d , Irene Aparicio a , Esteban Alonso a , Bernardo Duarte b,e a Departamento de Química Analítica, Escuela Polit´ ecnica Superior, Universidad de Sevilla, C/Virgen de ´ Africa, 7, E–41011 Seville, Spain b MARE—Marine and Environmental Sciences Centre & ARNET—Aquatic Research Network Associated Laboratory, Faculdade de Ciˆ encias da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal c BioISI—Instituto de Biossistemas e Ciˆ encias Integrativas, Departamento de Biologia Vegetal, Faculdade de Ciˆ encias da Universidade de Lisboa, Campo Grande, 1749016 Lisbon, Portugal d Departamento de Biologia Animal, Faculdade de Ciˆ encias da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal e Departamento de Biologia Vegetal, Faculdade de Ciˆ encias da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal ARTICLE INFO Keywords: Diatom Ecotoxicology Pharmaceuticals Enantiomers Photochemistry Marine pollution ABSTRACT Concern about the presence of pharmaceuticals in aquatic systems has increased in recent years owing to their continued release and the impact they may have on non-target organisms. Over half of these pharmaceuticals are chiral, with enantiomers that may have different pharmacokinetics and effects. However, most studies on their toxicity in marine biota have used racemic mixtures, ignoring the effects of isolated enantiomers. This work examines the potential enantioselective toxic effects of the chiral β-blocker propranolol, widely prescribed for cardiovascular diseases and migraines, and increasingly concerning due to its long-term use and raising consumption. This study used the diatom Phaeodactylum tricornutum as a model organism to assess the effect of each enantiomer on growth, photosynthesis, energy metabolism, and oxidative damage. The results showed that exposure of diatoms to R-propranolol induced growth inhibition due to deficiencies in photochemical metabolism, which was reflected in changes in the photosynthetic pigment profile. Oxidative stress also occurred in cells, resulting in lipid oxidation and DNA damage. In contrast, such effects were not observed for the S-enantiomer at the tested concentrations. This work shows the importance of considering enantiomer-specific effects in ecotoxicological assessments, as the two PRO enantiomers exhibit different toxicities in marine diatoms. 1. Introduction Beta-adrenoceptor antagonists, also known as β-blockers, are commonly prescribed for treating various cardiovascular conditions such as ischemic heart disease, hypertension, and migraines (Pandya et al., 2021). Propranolol (PRO) is one of the β-blockers most widely consumed and with the largest accumulated clinical experience and indications (Finn et al., 2012; Martínez-Milla et al., 2019). PRO is a nonselective β-blocker frequently prescribed for hypertension as well as for other pathologies such as migraine and anxiety (Messerli et al., 2023). It is relatively persistent (Bendz et al., 2005), bioaccumulative (Maurer et al., 2007), and highly water-soluble (Xu et al., 2019) and posses low volatility (Breton and Boxall, 2003). Because of these hydrophilic properties, PRO can persist in the aquatic environment, which it reaches mainly through wastewater discharges. PRO is a chiral pharmaceutical, marketed as a racemic mixture, despite the significant differences in metabolism, pharmacokinetics, and pharmacological effects of its enantiomers. In particular, the efficacy on β-receptors is approximately 100 times higher for S-(−)-PRO (Pham-Huy et al., 1994). Nevertheless, most of the studies reported to date on the environmental presence and risks of chiral pharmaceuticals do not consider their enantiomeric composition (Arenas et al., 2023). Considering total PRO concentration, without distinction within enantiomers, concentrations have been reported to be up to 2.9 ng L −1 in open sea (Adenaya et al., 2024), 60 ng L −1 in estuaries (McKenzie et al., 2020), 590 ng L −1 in rivers (Sumpter et al., 2021), 1900 ng L −1 in sewage effluents (Huggett * Corresponding author. E-mail address: [email protected] (M. Arenas). Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul https://doi.org/10.1016/j.marpolbul.2025.117751 Received 27 November 2024; Received in revised form 12 February 2025; Accepted 24 February 2025 Marine Pollution Bulletin 214 (2025) 117751 Available online 4 March 2025 0025-326X/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). et al., 2002) and 6500 ng L −1 in hospital effluents (G´ omez et al., 2006). The β-adrenergic receptors affected by PRO are conserved in organisms throughout the animal kingdom (Sumpter et al., 2021). Consequently, the presence of PRO in aquatic environments has led to concerns regarding its adverse effects on exposed organisms. Several authors have evaluated the ecotoxicity of PRO in different aquatic organisms, such as fish (Finn et al., 2012), mollusks (Franzellitti et al., 2011), crustaceans (Jeong et al., 2018), and bacteria (Ferrari et al., 2004). Only a limited number of studies have investigated these organisms considering PRO chirality. Stanley et al. (2006) (Stanley et al., 2006) conducted acute and chronic studies with Daphnia magna and Pimephales promelas to determine enantiospecific toxicity of PRO for a model aquatic invertebrate and vertebrate. Sun et al. (2014) also studied enantioselective toxicity of PRO in zebrafish embryos and larvae. However, organisms belonging to other taxonomic groups, such as algae and plants, have not been extensively investigated. Understanding the impact of environmental pollutants on autotrophic organisms is crucial as they form the basis of marine food webs with a direct impact on vertebrate and invertebrate animals. The β-adrenergic receptors of plants and algae are different from those of animals but are functionally similar and may also be affected by non-specific β-blockers such as PRO (Galindo-Trigo et al., 2016). In phototrophic organisms, these receptors are implicated in processes such as the regulation of reactive oxygen species (ROS), sensory functions, developmental processes, and innate immunological responses (Schulze et al., 2015). Some authors have conducted toxicity studies on PRO using various algal species as model organisms, reporting that PRO is the most toxic β-blocker to aquatic life (Bonnineau et al., 2010; Maszkowska et al., 2014), particularly affecting marine primary producers such as diatoms (Duarte et al., 2020) which are often the most sensitive non-target organisms (Claessens et al., 2013). However, these studies did not account for the chirality of the compound because they did not evaluate R-PRO and S-PRO separately. Therefore, information on the possible enantioselective effects of PRO on marine phototrophs is limited. Diatoms are among the marine phototrophic organisms most widely employed as models in biological stress and ecotoxicological studies. Their prevalence places them as one of the main groups of microalgae, building the basis of marine and estuarine food webs worldwide (Malviya et al., 2016). They contribute approximately 20 % of the world's primary productivity (Domingues et al., 2012), making them a significant marine carbon sink and crucial oxygen producer, vital for sustaining marine heterotrophic life (Benoiston et al., 2017). In ecotoxicological studies, physiological and biochemical features commonly assessed include photochemical feedback (Cabrita et al., 2018; Duarte et al., 2019) and responses to oxidative stress (Pires et al., 2021). These characteristics could serve as in vitro biomarkers for ecotoxicology, aiding the understanding of the mechanisms of action of emerging pollutants. Employing toxicophenomic tools, such as phenotyping techniques such as chlorophyll fluorescence analysis, allows for the collection of extensive physiological data over time without sacrificing organisms or causing alterations or interferences in the assays (Cabrita et al., 2018; Duarte et al., 2019). The combination of classical biochemical tools with non-invasive phenotyping methods has been effective in ecotoxicological research on marine diatoms, contributing to a better understanding of how pollutants affect these organisms and their mechanisms of action (Duarte et al., 2020). To the best of our knowledge, the majority of existing studies on the toxicity of pharmaceutical residues in marine biota have been focused on racemic mixtures containing both enantiomers. Given the enantioselective behaviour of these molecules in human metabolism, their excretion could also be influenced. This fact can result in a higher release of one of the enantiomers to the environment through wastewater. With this in mind, the present work aims to fill the knowledge gap regarding the potential enantioselective toxic effects of the β-blocker PRO on marine primary producers. P. tricornutum was used as a model organism, as a representative of one of the most abundant phytoplankton groups (diatoms) and due to its wide distribution and ecological relevance as a cosmopolitan microalga (Cabrera et al., 2025), which is also fastgrowing species and can be cultured under controlled laboratory conditions (Zhao et al., 2024). This diatom species has been previously used by many authors to assess the toxicity of pharmaceuticals, such as salicylic acid (Zhao et al., 2024), oxytetracycline (Sharma et al., 2024; Siedlewicz et al., 2020) and cytostatic drugs (Jureczko and Przysta´ s, 2024). In this work, the effects of each PRO enantiomer were evaluated on growth, photosynthesis, energy metabolism, and oxidative damage. 2. Materials and methods 2.1. Experimental design For the present study, the monoclonal P. tricornutum Bohlin (Bacillariophyceae) axenic cell culture was supplied by Instituto Portuguˆ es do Mar e da Atmosfera (IPMA) (strain IO 108–01). It was maintained under asexual reproduction conditions in f/2 medium (Guillard and Ryther, 1962). All the procedures were performed in a laminar flow hood to ensure aseptic conditions. The axenic conditions of the cultures were verified using routine microscopic examination. The exposure experiments were conducted in a phytoclimatic chamber at 18 ◦C with continuous aeration. The cultures were subjected to a day/night cycle of 14/10 h (RGB 1:1:1, maximum PAR 80 μ mol photons m −2 s −1 ), and natural sunrise and sunset conditions were simulated using a sinusoidal function (Feij˜ ao et al., 2018). Exposure assays followed the Organisation for Economic Cooperation and Development (OECD) guidelines for algae assays (OECD, 2011), with slight adjustments, starting with an initial cell density of 1.5 ×10 5 cells mL −1 . Aeration with room air was the source of carbon for the cultures. Given the rapid growth of this strain, which enters the stationary phase and shows ageing effects after 72 h, (Cabrita et al., 2018; Pires et al., 2021; Feij˜ ao et al., 2018), the exposure duration was limited to 48 h. Single enantiomer standards of R- (+)-PRO and S-(−)-PRO were purchased from Sigma-Aldrich (Steinheim, Germany). After a 48-h acclimation period, the isolated PRO enantiomers were added to the diatom cultures to achieve the desired concentration levels (0, 0.5, 50, 200 and 400 μ g L −1 ). For each level, three separate 250 mL batch cultures (replicates) were prepared. The selected concentrations represented a gradient that reflected current environmental levels, along with a higher concentration to consider the rising use of PRO (Vashistha and Kumar, 2020). Concentration levels tested were selected considering maximum concentrations reported in surface water and urban and hospital wastewater and were extended to high levels to ensure that the study covers possible fluctuations in environmental exposure and assesses worst-case scenarios. In addition, the inclusion of higher concentrations allows to better establish doseresponse relationships. The cells were collected after a 48-h exposure by centrifugation at 4000 ×g for 15 min at 4 ◦C. The diatoms, aggregated into pellets after centrifugation, were quickly frozen using liquid nitrogen and stored at −80 ◦C until biochemical analysis. 2.2. Cell growth rates P. tricornutum cells (1 mL samples) were counted under an improved Neubauer counting chamber and an Olympus BX50 inverted microscope (Tokyo, Japan) at 400×magnification. The growth of the cultures was determined using the average specific growth rate (SGR), calculated as the difference between the logarithmic cell densities at the beginning and end of the exposure period (Santos-Ballardo et al., 2015). The doubling time and number of divisions per day were determined based on the specific growth rate. The growth inhibition percentage for each treatment was calculated using the cell density of the control cultures as a reference. Finally, the growth inhibition percentages at each concentration level were used to estimate the concentration that led to a decrease in the growth rate of 50 %, denoted IC50 (OECD, 2011), using the drc package in Ritz et al. (2015) (Ritz et al., 2015). The lowest M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 2 observed effect concentration (LOEC) and no observed effect concentration (NOEC) were described from the experimental data, with LOEC being the lowest tested concentration at which the substance was observed to have a statistically significant reducing effect on growth and NOEC being the test concentration immediately below the LOEC. 2.3. Chlorophyll-a pulse amplitude modulated fluorometry Pulse amplitude modulated (PAM) chlorophyll-a fluorometry was used for the bio-optical analysis. Fluorometric measurements were performed using FluorPen FP100 (Photo System Instruments, Brno, Czech Republic). The cell density was monitored daily under non-actinic light to minimize fluorescence over time (Ft). On the last day of the exposure and prior to pellet collection, 1 mL samples of each replicate were adapted to darkness for 15 min to ensure reoxidation of all available and functional photosystem II (PSII) reaction centers. The darkadapted samples were then analyzed for chlorophyll transient light curves using a previously based on fast chlorophyll a fluorescence induction (OJIP acronym refers to the 4 distinct phases observed in a typical Kautsky curves and that refer to specific photobiological events) (Duarte et al., 2019). Table 1 lists the parameters resulting from these measurements, determined using the protocol (Strasser et al., 2004). 2.4. Pigments analysis For pigment extraction, the cell sample pellets were treated with 100 % acetone. To ensure full disaggregation of the cell material, the cells were placed in a cold ultrasonic bath for 2 min. The extraction process was performed at −20 ◦C for 24 h in complete darkness to avoid degradation (Cabrita et al., 2018; Feij˜ ao et al., 2018; Cabrita et al., 2016). The samples were then centrifuged at 4000 ×g at 4 ◦C for 15 min. The supernatants were scanned using a dual-beam spectrophotometer from 350 nm to 750 nm in sections of 0.5 nm. Gauss-Peak Spectra (GPS) fitting library was used in the SigmaPlot Software to introduce the absorbance spectrum. Chlorophyll a and c, Pheophytin a, β-carotene, Fucoxanthin, Diadinoxanthin and Diatoxanthin were detected using an algorithm designed by Küpper et al. (2007). 2.5. Cellular damage biomarkers To assess the potential cellular damage caused by oxidative stress from exposure to PRO enantiomers, two oxidative stress biomarkers were analyzed in the cultures. Quantification of lipid peroxidation products was carried out by a prior homogenization of the cells with 10 % (v/v) trichloroacetic acid and brief sonication (Duarte et al., 2020; Feij˜ ao et al., 2018). The reaction of the homogenate with 0.5 % (v/v) thiobarbituric acid was performed for 30 min at 95 ◦C and it was immediately stopped on ice and centrifuged at 3000 ×g for 5 min at 4 ◦C (Duarte et al., 2015). Absorbance values at 532 nm and 600 nm were recorded. The concentration of thiobarbituric acid reactive substances (TBARS), expressed as malondialdehyde (MDA) equivalents, was calculated using the molar extinction coefficient of 155 mM −1 cm −1 (Heath and Packer, 1968). DNA damage was assessed using an adaptation of DNA alkaline precipitation assay (De Lafontaine et al., 2000), originally developed by Olive P. in 1988 (Olive, 1988). Intact singleand double-stranded DNA were precipitated using sodium dodecyl sulfate (SDS), while nucleic acids that were damaged (double-strand breaks) remained in the supernatant. Damaged DNA was stained with Hoechst dye (1 μ g/mL bis-benzimide, Sigma-Aldrich) and quantified by measuring fluorescence at a wavelength of 360/460 nm (Lemos et al., 2024). The results were expressed as nanograms of damaged DNA per million cells, with calf thymus DNA (Sigma-Aldrich, USA) used as a standard. 2.6. Statistical analysis All statistical analyses were conducted using R Studio 1.4.1717. Since neither normality (Shapiro-Wilk test), nor homoscedasticity (regression residues), requirements were met, non-parametric Kruskal–Wallis tests with Bonferroni post hoc comparisons were performed using the ‘agricolae’ package (De Mendiburu and Simon, 2015) to compare the variable values between exposure treatments. Spearman correlation coefficients between exposure concentration and biological features were determined using the ‘corrplot’ package (Wei and Simko, 2021). Volcano plots were constructed to assess potential biomarkers based on the correlation of biological features with the nominal exposure dose. Two thresholds were established to evaluate the potential biomarker candidate variables by selecting only variables with Spearman correlation coefficients above 0.6 or below −0.6 with a significant p-value (p <0.05). 3. Results 3.1. Growth-related parameters Diatom growth was evaluated after a 48-h exposure period to single PRO enantiomers, revealing significant effects along with notable differences between isomers. Cultures exposed to R-PRO showed considerably lower cell densities in diatoms exposed to 200 μ g L −1 and 400 μ g L −1 , while for the S-enantiomer, growth inhibition effects were only observed at the highest concentration tested, 400 μ g L −1 (Fig. 1A). Growth rates were also significantly lower than those of the control for concentrations above 50 μ g L −1 for R-PRO. For S-PRO, only cultures exposed to 400 μ g L −1 were found to be significantly different (Fig. 1B). The reductions in cell density were probably due to the decreasing number of divisions per day (Fig. 1C) and the increasing doubling time (Fig. 1D), both significant for 200 and 400 μ g L −1 exposures to the Renantiomer and only at 400 μ g L −1 for S-PRO. Considering growth inhibition percentage after exposure for 48 h (Fig. 1E), IC50 concentrations for both PRO enantiomers were calculated for the tested conditions, being 223.4 μ g L −1 for R-PRO and 828.7 μ g L −1 for S-PRO. Since the attained value for S-PRO is above the tested range of concentrations, this value was extrapolated assuming that the toxicological pattern follows the one observed within the tested range. This also allows to use the same methodology used for R-PRO IC50 calculation, allowing a comparison between the two tested enantiomers. The experimental NOEC and LOEC values were both lower for the R-enantiomer (50 and 200 μ g L −1 , respectively) than for S-PRO (200 and 400 μ g L −1 , respectively) (Fig. 1F). 3.2. Diatom photochemistry The maximum photochemical efficiency of photosystem II (Fv/Fm) was compromised by exposure of diatoms to PRO (Fig. 2A). Under the control conditions, Fv/Fm ranged from 0.675 to 0.689. In contrast, diatoms exposed to R-PRO exhibited significantly reduced Fv/Fm values, with a more pronounced decrease in cultures subjected to 200 μ g L −1 concentrations (average Fv/Fm =0.633), and most drastically for those exposed to a concentration of 400 μ g L −1 (average Fv/Fm =0.589). However, for the S-enantiomer, the Fv/Fm values were not significantly Table 1 Description of fluorometric analysis parameters. OJIP-Test Parameter description F v /F m Maximum quantum yield of the PSII ABS/CS Absorbed energy flux per cross-section TR/CS Trapped energy flux per cross-section ET/CS Electron transport energy flux per cross-section DI/CS Dissipated energy flux per cross-section RC/CS Number of available reaction centres per cross-section P G Grouping probability between the two PSII units PI Performance index M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 3 Fig. 1. Growth curves (A), specific growth rate (SGR, B), doubling time (C), number of divisions per day (D), growth inhibition percentages (E) and IC50, NOEC and LOEC (F) of diatom cultures exposed to different R-PRO and S-PRO concentrations (average ±standard error, N =3, letters denote differences at p <0.05). Fig. 2. Maximum quantum yield (Fv/Fm, A), grouping probability (P G , B), absorbed (ABS/CS, C), trapped (TR/CS, D), transported (ET/CS, E) and dissipated (DI/CS, F) energy fluxes, number of available reaction centres per cross-section (RC/CS, G) and performance index (PI, H) values assessed for the diatom cultures exposed to different R-PRO and S-PRO concentrations (average ±standard deviation, N =3, different letters indicate significant differences at p <0.05). M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 4 reduced, compared to those of the control cultures, in any of the treatments. The grouping probability of PSII units greatly increased in cultures exposed to the highest concentration of R-PRO (Fig. 2B). The four main energetic fluxes associated with the overall photochemical process from electron transport during light harvesting were analyzed. Cultures exposed to increasing concentrations of PRO showed notable differences. For R-PRO, the amount of energy absorbed by the PS II antennae (ABS/CS) (Fig. 2C) and the effectively trapped inside PS II (TR/CS) (Fig. 2D), transported within the electronic transport chain (ET/CS) (Fig. 2E), and dissipated (Fig. 2F) energy fluxes were considerably reduced for exposure concentrations higher than 50 μ g L −1 . In contrast, no significant reduction in these parameters was observed by SPRO exposure. A similar trend was observed for the number of oxidized PS II reaction centers (RC/CS) (Fig. 2G). The overall functionality of the electron flow through PSII was quantified using the performance index (PI) (Fig. 2H), which was significantly reduced in cultures treated with R-PRO concentrations above 50 μ g L −1 . The S-enantiomer did not reduce the performance at any of the concentrations tested. 3.3. Pigment profiles The composition of the light-harvesting pigments and carotenoids was also evaluated (Fig. 3). The presence of R-PRO at 200 and 400 μ g L −1 induced changes in Chlorophyll a, Chlorophyll c, and Pheophytin a levels (Fig. 3A). There was an increase in total Chlorophyll content with higher concentrations of Chlorophyll c, leading to a reduction in the Chlorophyll a/Chlorophyll c ratio, which was most significant at the 400 μ g L −1 concentration (Fig. 3B). Simultaneously, the Pheophytin a level showed an increase. However, exposure to this enantiomer did not significantly affect the levels of Xanthophyll cycle pigments (Diadinoxanthin and Diatoxanthin), Fucoxanthin, or β-carotene. Analysis of the pigment profile of diatoms subjected to different S-PRO concentrations (Fig. 3) indicated that the content of none of the pigments was severely altered. 3.4. Stress biomarkers The TBARS assay results (Fig. 4A) revealed that MDA production derived from lipid peroxidation was significantly enhanced in cultures exposed to 50 μ g L −1 R-PRO compared to the controls, and it was strongly increased at higher concentrations. None of the cultures exposed to S-PRO showed an increase in the production of lipid peroxidation products. The damaged DNA (Fig. 4B) concentration was found to be significantly higher in cultures treated with R-PRO concentrations above 200 μ g L −1 . In contrast, S-PRO did not cause any significant increase in DNA damage in the cultures, even at the highest concentrations. 3.5. Enantioselective candidate biomarkers Considering all of the biomarkers assessed in this study, their correlation with the concentration of PRO enantiomers was evaluated. Fig. 5 shows the coefficient of determination (R 2 ) between the enantiomer concentration and biomarker response versus the statistical significance of the observed correlation. Biomarkers above the horizontal line show highly significant changes, but if R 2 is below 0.6 (direct correlations) or above −0.6 (indirect correlations), the variation in biomarker response cannot be explained by contaminant concentration. Concerning R-PRO, the most useful biomarkers to monitor its effects in the toxicity study, with a direct correlation, were doubling time, growth inhibition percentage, and TBARS. The specific growth rate, number of divisions per day, and performance index were the most suitable biomarkers with indirect correlations. For S-PRO, however, none of the biomarkers showed a sufficient correlation, essentially because this enantiomer did not produce significant effects in the cultures. 4. Discussion A previous study carried out in the laboratory with racemic PRO (Duarte et al., 2020) showed that the effective concentration producing a growth inhibition effect on 50 % (IC50) of the population was 380.9 μ g L −1 , which is above the IC50 value of 288 μ g L −1 reported by some authors (Claessens et al., 2013). The concentrations tested in this work for each individual PRO enantiomer included a range of concentrations between those detected in the environment and those known to have a significant effect on P. tricornutum. The results of this study show marked differences in the influence of each enantiomer on diatom growth. For RPRO, the IC50 was found to be 223.4 μ g L −1 , what is lower than in studies carried out with the racemic mixture, while the S-enantiomer did not inhibit growth in the concentration range tested (IC50 was extrapolated to 828.7 μ g L −1 , as indicated in growth-related parameters results section). The significant reductions found in the primary photochemical metabolism of diatoms can explain the inhibition of growth induced by R-PRO. A decrease in the PSII quantum yield (Fv/Fm) of cells exposed to concentrations above 50 μ g L −1 showed that certain impairments in Fig. 3. Pigment composition (A) and Chlorophyll a/Chlorophyll c ratio (B) of diatom cultures exposed to different R-PRO and S-PRO concentrations (average ± standard deviation, N =3, different letters indicate significant differences at p <0.05). M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 5 energy metabolism had occurred (Fig. 2A). A lower Fv/Fm is associated with an increase in the grouping probability (P G ) (Fig. 2B), indicating a connectivity loss between the PS II antennae and a possible disruption of energetic transport (Duarte et al., 2016) in diatoms exposed to R-PRO. Further analysis of the photochemical data revealed an overall decrease in the efficiency of photosynthesis, which started with a reduction in the absorbed energy flux (ABS/CS) (Fig. 2C) Consequently, the trapped (TR/ CS) and dissipated (DI/CS) energies are reduced (Fig. 2D and E). The amount of trapped energy entering the electron transport chain (ET/CS) was also lower when exposed to R-PRO (Fig. 2F). Given these results, it can be concluded that the reduction in photosynthetic efficiency originated in the first stage of the energy flow transduction pathway, with the initial reduction in the absorbed energy flux (ABS/CS) being the main cause of these changes. Energy absorption impairment is mostly due to a Fig. 4. Lipid peroxidation products (TBARS, A) and DNA damage (DNAd, B) in diatom cultures exposed to different R-PRO and S-PRO concentrations (average ± standard deviation, N =3, different letters indicate significant differences at p <0.05). Fig. 5. Spearman correlation volcano plot between the photochemical and biochemical traits analyzed and the exogenous applied concentration in diatom cultures exposed to R-PRO and S-PRO (N =3). Significant correlations (p <0.05) higher than 0.60 or bellow −0.60 are highlighted in green or red respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 6 reduction in the number of oxidized reaction centers (RC/CS) available to trap incident photons (Fig. 2G). All processes involved in energy transduction pathways can be numerically evaluated using an integrative performance index (PI) (Duarte et al., 2016) (Fig. 2H). This value was severely reduced in cultures exposed to 200 and 400 μ g L −1 R-PRO, reflecting the effects of exposure to this enantiomer on all the abovementioned processes of photochemical metabolism, thus reinforcing the impairment of photochemical metabolism in these organisms. Photochemical efficiency is intrinsically related to photosynthetic pigments. The results of pigment analysis in this study indicate a negative correlation between the light-harvesting pigments Chlorophyll a and Chlorophyll c and reduced photosynthetic efficiency (Fig. 3A). This may be interpreted as a compensatory mechanism to cope with the decreased efficiency of PSII by increasing the levels of light-harvesting pigments (Feij˜ ao et al., 2018). In addition, the Chlorophyll a/Chlorophyll c ratio was reduced in the presence of R-PRO (Fig. 3B). These changes in the proportion of photosynthetic pigments occur in response to different stress conditions (Kuczynska et al., 2015), such as reduced light availability, in an adaptive attempt to optimize light harvesting and photosynthetic efficiency. Alterations in Chlorophyll content serve as one of the earliest markers of physiological conditions in microalgae (Baker, 2008). Consequently, Chlorophyll fluorescence can be a sensitive bioindicator of plant stress caused by various contaminants (Cabrita et al., 2016). The reduction in photosynthetic capacity is also evidenced by an increase in the levels of Pheophytin a, a degradation product of Chlorophyll a (Franzitta et al., 2020). Rising levels of Pheophytin a suggest that Chlorophyll a undergoes degradation, which may indicate that diatoms are experiencing physiological stress. Regarding stress biomarkers, the results showed a positive correlation between cell lipid peroxidation and exogenous R-PRO concentration (Fig. 4A). This suggests that the activity of ROS-scavenging enzymes was insufficient to prevent free radicals from producing oxidative stress in diatoms, and although these activities were not determined in this study, they have been previously reported in a study carried out with racemic PRO (Duarte et al., 2020). However, in this case, the R-enantiomer induced greater oxidative stress, whereas S-PRO did not have a significant effect (Fig. 4A). Lipid peroxidation is regarded as the major mechanism through which ROS induces cell damage, affecting the polyunsaturated fatty acids of the diatom cellular and plastidial membranes (Heath and Packer, 1968; De Lafontaine et al., 2000), ultimately disrupting essential energy, biosynthesis, and compartmentalization functions (Chia et al., 2021). DNA damage showed a trend similar to that of lipid peroxidation, with significant DNA strand breaks induced by ROS in cultures exposed to R-PRO concentrations above 50 μ g L −1 (Fig. 4B). This DNA damage may have compromised the viability and function of P. tricornutum cells, particularly affecting cell duplication, because DNA integrity is essential for asexual reproduction in diatoms. This fact is in line with the results of this study, where the cultures that displayed higher DNA damage values also presented a lower number of divisions per day. Considering all the results from the different metabolic and biochemical traits evaluated, the greater toxicity of the R-enantiomer was evidenced by the presence of highly significant effects strongly correlated with the dose of R-PRO applied exogenously. More specifically, three candidate enantioselective biomarkers were highlighted, corresponding to different metabolic traits evaluated, including growth (SGR) and photochemistry (diadinoxanthin cellular concentration and PI), reinforcing the negative effects of this enantiomer on diatom fitness (Fig. 5). On the other hand, and again reinforcing the abovementioned results, non-candidate enantioselective biomarkers fulfilled the selection criteria, in line with the lack of toxicity described above for cultures exposed to this compound. Although the specific mechanisms underlying the higher toxicity of the R-PRO enantiomer remain unknown, enantiomers differ structurally in the spatial orientation of key functional groups, which affects their binding interactions with β-adrenergic receptors. This stereoselectivity is crucial, as the S-enantiomer exhibits higher affinity and therapeutic activity, whereas the R-enantiomer may engage in non-specific interactions that contribute to its potential toxicity. Given that the S-enantiomer has the highest affinity for β-receptors (Pandya et al., 2021; Pham-Huy et al., 1994), and it is therefore more absorbed and metabolized in humans, R-PRO enantiomer is expected to be released into the environment at higher concentrations because of its lower metabolization rate. Combining the results of this work, with the enantioselective preference of human metabolism, the higher concentrations of R-PRO expected in marine systems, particularly those close to urban wastewater discharge sites, pose a serious threat to diatom metabolism, the most relevant group of marine primary producers, potentially leading to cascading inevitable effects on the ecosystem. 5. Conclusion From an ecotoxicological perspective, the β-adrenergic receptor blocker PRO has an impact on marine diatoms. Although this type of pharmaceutical is designed for target specific human receptors, it can also affect receptors that are conserved in other taxonomic groups, including algae. In addition, PRO is a chiral compound, and the enantioselective toxicity tests carried out in this study showed marked differences in the effects of the enantiomers on the model diatom P. tricornutum. Exposure of diatoms to R-PRO, especially at high concentrations, resulted in growth inhibition. This fact is due to deficiencies in photochemical metabolism, which was reflected in changes in the photosynthetic pigment profile. Oxidative stress in the cells also occurred, resulting in lipid oxidation and DNA damage. In contrast, such effects were not reported for the Senantiomer at the tested concentrations. This study highlights the importance of considering enantiomer-specific effects in ecotoxicological assessments, as the two enantiomers of PRO exhibit notably different toxicities in marine diatoms. These variations underline the need for more detailed risk assessments of chiral compounds including the impacts of each enantiomer on non-target organisms in the environment. CRediT authorship contribution statement Marina Arenas: Writing – original draft, Investigation, Formal analysis. Eduardo Feij˜ ao: Writing – review & editing, Methodology, Investigation. Irina A. Duarte: Writing – review & editing, Investigation, Formal analysis. Vanessa F. Fonseca: Writing – review & editing, Validation, Conceptualization. Irene Aparicio: Writing – review & editing, Supervision, Resources. Esteban Alonso: Supervision, Project administration, Funding acquisition. Bernardo Duarte: Writing – review & editing, Software, Methodology, Data curation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements M. Arenas acknowledges the Spanish Ministerio de Universidades for a FPU predoctoral contract (grant number FPU20/00540) and the funding received for an international research internship (grant number EST24/00093). The authors would like to thank FCT for funding the Marine and Environmental Sciences Centre (MARE, http://doi.org/10 .54499/UIDB/04292/2020 and http://doi.org/10.54499/UIDP/04 292/2020) and the Aquatic Research Network Associated Laboratory (ARNET, http://doi.org/10.54499/LA/P/0069/2020). M. Arenas et al. Marine Pollution Bulletin 214 (2025) 117751 7 Data availability Data will be made available on request. References Adenaya, A., Quintero, R.R., Brinkhoff, T., Lara-Martín, P.A., Wurl, O., Ribas-Ribas, M., 2024. Vertical distribution and risk assessment of pharmaceuticals and other micropollutants in southern North Sea coastal waters. Mar. Pollut. Bull. 200, 116099. https://doi.org/10.1016/j.marpolbul.2024.116099. 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