# Gender and organ responsiveness in Dreissena polymorpha exposed to the antidepressant sertraline: from uptake to effects

Linda Lumor, Anne Vignet, Céline Passignat, Vincent Felten, Céline Simon, Marie Zaffino, Guillaume Grosjean, Laure Giambérini, Laetitia Minguez

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Linda Lumor, Anne Vignet, Céline Passignat, Vincent Felten, Céline Simon, et al.. Gender and organ responsiveness in Dreissena polymorpha exposed to the antidepressant sertraline: from uptake to effects. Environmental Research, 2025, 289, pp.123422. 10.1016/j.envres.2025.123422. hal-05385829

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# Gender and organ responsiveness in *Dreissena polymorpha* exposed to the antidepressant sertraline: from uptake to effects

Linda Lumor<sup>a,∗</sup> <sup>●</sup>, Anne Vignet<sup>b</sup>, Céline Passignat<sup>b</sup>, Vincent Felten<sup>a</sup>, Céline Simon<sup>a</sup>, Marie Zaffino<sup>a,●</sup>, Guillaume Grosjean<sup>a,●</sup>, Laure Giambérini<sup>a</sup>, Laetitia Minguez<sup>a,∗∗</sup> <sup>●</sup>

## ARTICLE INFO

Keywords:

Pharmaceutical

Ecotoxicology

Bioaccumulation

Biomarkers

Aquatic ectotherms

Gender

## ABSTRACT

Sessile bivalves like Zebra mussels are effective bioindicators of aquatic contaminants due to their ability to bioaccumulate various pollutants. In the aquatic environment, pharmaceuticals like selective serotonin reuptake inhibitor (SSRI) have been reported to exert effect on bioaccumulation, metabolism and physiological functions. In this study, the gender-specific absorption, distribution, and metabolism of sertraline, a toxic SSRI and its effect on physiological functions in the digestive gland and gonad tissues of mussels was evaluated. The findings revealed a clear concentration-dependent accumulation of sertraline, with females exhibiting higher metabolic conversion to its active metabolite, norsertraline, particularly at intermediate doses than males. This suggests a potential sex-specific differences in cytochrome P450 enzyme activity and hormonal influence on drug metabolism. Sertraline exposure disrupted vital physiological processes like filter feeding, energy metabolism and mitochondria function. Interestingly, males showed more pronounced mitochondrial and metabolic disruptions, including a shift from aerobic to anaerobic respiration, and altered energy reserves. Females, while less sensitive, exhibited compensatory metabolic adaptations at higher doses. Hemolymph osmolality decreased significantly in both genders, implying impaired osmoregulation potentially linked to serotonergic pathway disruptions. Moreover, sertraline exposure modulated monoamine oxidase (MAO) activity, reduced total antioxidant capacity (TAC), and altered markers of oxidative stress, inflammation and apoptosis, especially at intermediate to high concentrations. These findings highlight sertraline's potential to induce neurotoxic, immunotoxic, and metabolic stress responses in non-target freshwater organisms, emphasizing the need for further ecotoxicological assessments of SSRIs in aquatic environments.

## 1. Introduction

Antidepressants are among the plethora of pharmaceutical residues (constitute about 4 %) found in the aquatic environment, with compounds, like sertraline, fluoxetine, paroxetine been found in surface waters at trace amounts (ng/L-µg/L range) (Santos et al., 2010). Often these residues find their way into the aquatic ecosystem due to inappropriate disposal method and incomplete removal during treatment. As they are highly soluble, they become readily bioavailable (60–100 %) to aquatic organisms and are often bioaccumulated in their tissues (Richmond et al., 2018). Antidepressants are biological neuroactive compounds manufactured for altering mood, treating anxiety and depression related conditions by targeting brain dopaminergic,

adrenergic and serotonergic systems (Khushboo, 2017). These neurohormones and targets have been conserved through evolution and most aquatic ectotherms rely on the neuroendocrine systems to maintain their homeostatic processes (Canesi et al., 2022). Hence, a better understanding of the bioaccumulation and ecotoxic properties of these compounds on invertebrates is crucial.

Bivalves, a key invertebrate group of aquatic ecosystems (high biomass and important ecological function), display features that suggest they may be particularly exposed and sensitive to these antidepressants. As filter-feeders and sedentary organisms, they can accumulate high amounts of contaminants through direct internalization via the gills and food ingestion via the digestive tract. Bivalves provide accurate and reliable biological endpoints (e.g., biochemical,

<footnote><sup>a</sup> Université de Lorraine, CNRS, LIEC, F-57000, Metz, France</footnote>

<footnote><sup>b</sup> LABÉO Manche, 1352 avenue de Paris - CS 33608, F-50008, Saint-Lô Cédex, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: linda.adzigbli@univ-lorraine.fr (L. Lumor), laetitia.minguez@univ-lorraine.fr (L. Minguez).</footnote>
cellular, physiological parameters) that can be measured in their tissues. These biomarkers have been used successfully for evaluating contaminant-induced physiological stress (André and Gagné, 2017; Maranho et al., 2015). In bivalves, serotonin is involved in gametogenesis and spawning, oocyte maturation and sperm motility, regulates heart function and other important physiological functions (Canesi et al., 2022; Fong and Ford, 2014). Since the development of the bivalve gonad is linked to the functioning of its digestive gland, any stimulation of reproduction could also disrupt digestive gland physiology (Gao et al., 2022). Serotonin and its receptors have also been detected in the digestive gland of several bivalve species, suggesting it could be impaired by neuro-disrupting drugs (Canesi et al., 2022). This observation has also been reported in patients treated with SSRIs with symptoms involving the digestive system being the most common adverse sertraline reactions, leading to low compliance (Oliva et al., 2021). To our knowledge, no studies have assessed tissue-dependent effects of these antidepressants on bivalves. Additionally, the serotonin system has been reported to exhibit gender-specific differences in humans, with men and women exhibiting distinct responses to antidepressant drugs (Canesi et al., 2022; Ma et al., 2019). Similar gender differences in serotonin reuptake inhibitor response are also seen in tests using mammals, with little knowledge of the underlying mechanisms (Fernández-Guasti et al., 2017). As these serotonin system and targets are evolutionarily conserved in aquatic ectotherms, gender differences in pharmacokinetic and pharmacodynamic mechanisms are also expected in invertebrates. In 2007, McClellan-Green et al. (McClellan-Green et al., 2007) advised to consider gender in ecotoxicological studies focusing on neurondocrine active chemicals, but since pooling organisms (regardless of their gender) is still the norm in studies using bivalves, this advice have not yielded much data. Given the consequences in terms of significant ecological effects if males and females appear to have different sensitivities, a better understanding of the mechanisms used by the different genders to cope with SSRIs is necessary. Hence, an evaluation of the gender-specific stress response to antidepressants, which has, to the best of our knowledge, never been performed on bivalves was conducted.

Antidepressants, depending on the considered molecules, can alter
several physiological and behavioural processes like reproduction, im-
mune response, locomotion, burrowing, at concentrations currently
found in the environment (Fong and Ford, 2014). Most previous studies
assessed the effects of only a few antidepressants (mainly fluoxetine and
venlafaxine) on marine and freshwater mussels (Ford et al., 2018;
Mennigen et al., 2011; Fong et al., 2015). Sertraline (SER), one of the
most toxic (LC = 0.02–0.04 mg/L) SSRI antidepressants (Minguez et al.,
2014; Gilroy et al., 2017) has only gained attention recently (Gilroy
et al., 2017; Xie et al., 2015). The surface water concentrations of ser-
traline are quite low (from 1 to 120 ng/L), but still can be a potential
threat to aquatic organisms due to their high biological activity. In
extreme cases, sertraline can be found around wastewater treatment
plant and untreated sewage at 600 ng/L and 17 µg/L, respectively (de
Solla et al., 2016). That notwithstanding, given its hydrophobic (log
K<sub>OW</sub> = 5.3, log K<sub>d</sub> ≥ 4) and ionizable properties (pKa = 9.16), SER can
easily partition into sediments and organism tissues, like most of the
antidepressants (de Solla et al., 2016; Santobuono et al., 2025; Passignat
et al., 2025), suggesting that sertraline quantifications in only water
samples are unsuitable for assessing environmental impregnation and
risk. In addition to determining sertraline body burden in organisms, it is
also important to study its metabolism to better understand the effects
observed (Santobuono et al., 2025). The main SER's metabolite is
N-Desmethyl-sertraline, also called norsertraline, known to be less
active on serotonin system than sertraline. Considering the limited
knowledge of sertraline effects on freshwater bivalves, more studies
with environmentally relevant concentration are necessary in assessing
and improving the understanding of the effects of SER on bivalves.

In this study, the focus was on investigating the gender-specific
changes in bioaccumulation, metabolism and various physiological

functions (i.e., related to neuroendocrine system, antioxidant defences, osmoregulation and energy metabolism) to SER exposure in a freshwater bivalve, the Zebra mussel *Dreissena polymorpha*. This species is a valuable sentinel model for ecotoxicological studies with wide geographical distribution and abundance, and high filtration activity. This bivalve also possess the ability to transfer pollutants through food web hence its utilization as a model species for freshwater biomonitoring over the years (André and Gagné, 2017; Gagné et al., 2007; Schäfer et al., 2012). In this study, various biomarkers were measured in male and female Zebra mussels and in two tissues; digestive gland and gonad after exposure to 4 concentrations of SER (50, 500, 1000 and 5000 ng/L, for 0.16, 1.6, 3.2 and 16 nM) for 28d. These tissues were selected because the digestive gland, a metabolically active organ with a significant role in the physiology and survival of mussels has been proposed as a significant tissue for screening physiological effects of neuro-endocrine disruptors both in field and laboratory studies (Faggio et al., 2018) and the gonad because of the effect of antidepressants on reproduction. For the measured concentration, they were selected based on previous studies on SER concentrations in the environment and chronic effects on invertebrates (Shi et al., 2020; Neuparth et al., 2019). The concentration of 500 ng/L is slightly higher than the predicted no effect concentration from the NORMAN ecotoxicology database (sertraline hydrochloride: 460 ng/L) (NORMAN, 2024). Hence, findings from this study will reveal the potential effects of sertraline exposure beyond the no effect concentrations on aquatic species like the Zebra mussels. Additionally, the results from this study will help in streamlining the potential tolerance concentration beyond which the presence of sertraline in freshwater can be lethal to freshwater mussels.

**2. Materials and methods**

2.1. *Animal collection and preparation*

Zebra mussels *D. polymorpha* were sampled at end of May 2022 (during gamete emission period) from the canal of the Moselle River at Jouy-aux-arches. All bivalves were adults (shell length: 24 ± 2 mm). On arrival to the laboratory, the shells of the mussel were cleaned and the mussels transferred into an aquarium (100 individual per condition, divided into 2 aquaria) and acclimated at 16 °C for 11d under semi-static condition. During the acclimation period, the field water was gradually replaced with spring water Cristaline ® Aurèle and the mussels were fed every other day with 5 mL algae *Chlorella vulgaris* (2 × 10⁴ cell/mussel/ d) with water renewal and continuous aeration. Mussels were kept under similar conditions of temperature (16 °C), and pH (7.2), and natural photoperiod as its habitat.

## 2.2. *Chemical preparation and exposures*

Sertraline hydrochloride (CAS No. 79559-97-0) was purchase from Enzo Life Sciences (Villeurbanne, France) at analytical grade (purity ≥98 %). A stock solution was prepared in ultrapure water at 320 µM (100 mg/L). The stock solution was further diluted in spring water to 3.2 µM on the day of a renewal (1 mg/L). Exposure media at concentrations 0.16 nM (50 ng/L; SER0.16), 1.6 nM (500 ng/L; SER1.6), 3.2 nM (1000 ng/L; SER3.2), and 16 nM (5000 ng/L; SER16) were then prepared by diluting the 3.2 µM solution. Water in the exposure tanks were totally renewed every 2d and the Zebra mussels were exposed to the various concentrations (four sertraline concentrations and one control) for a total of 28d.

## 2.3. *Sertraline quantification in the water and, its accumulation and metabolism by the organisms*

Nominal sertraline concentrations in exposure media were validated using ultra high-performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS) (Passignat et al., 2025). The samples
(n = 5 pools of 2 individuals per gender and condition) were freeze-dried for 48h and finely ground before sertraline quantification (Passignat et al., 2025). The method to quantify norsertraline follows the one of sertraline, with optimum multiple reaction mode MS transitions equal to 292.1 (m/z) for the precursor ion, 159 and 275 (m/z) for the production ion as quantitative and qualitative ion, respectively.

## 2.4. Biomarker analyses

The survival of bivalves was checked every day. Mussels were considered dead or nearly dead when they were completely open or if they had difficulty closing. No mortality was recorded throughout the exposure period. The effects of sertraline were assessed at the individual and subcellular levels.

### 2.4.1. Biomarker measurement at individual level

After the 28d exposure period, the maximum length (L, maximum antero-posterior axis), width (w, maximum lateral axis) and height (h, maximum dorsoventral axis) of all shells were measured with a calliper rule to the nearest 0.01 mm. The soft tissues were weighted after removing excess body fluids (m). A condition index (Cond.Ind) expressed in g mm⁻³ was calculated as follows: $Cond.Ind = m/(L \times w \times h)$. Clearance rate (CR in L/h⁻/g dw) and Respiration rate (RR in mg O₂/h/g dw) were measured simultaneously on 10 bivalves per condition, and the tissues and hemolymph of these bivalves were later extracted for subcellular biomarker analyses (see section 2.4.2 "Biomarker measurement at cellular and subcellular level"). Briefly, mussels were placed individually in 180 mL-glass beakers containing exposure media and 1 mL of a suspension of *Chlorella vulgaris* ($6 \times 10^7$ cells/mL) of known caloric content. Initial and final (i.e., after 100 min) algae concentration were measured in each beaker by flow cytometry. During these 100 min in the dark, oxygen consumption was also assessed with fiber-optic oxygen microsensors (PreSens) connected to an oxygen meter (OXY-4 SMA trace, PreSens) with integrated processing software (PreSens Measurement Studio 2, PreSens). During each run of oxygen analysis, one beaker with 180 mL Crystalline® water and 1 mL of algae but without bivalve was added to assess the effect of algae on the measured oxygen concentrations (Lachaux et al., 2025). CR and RR were then transformed into absorbed (AE) or respired (RE) energy before calculating scope for growth (SfG, in J/h/g dw), an index of the whole-animal energy status, as follows: $SfG = AE - RE$. Reserves, as total carbohydrates (TS), lipids (TL) and proteins (TP) were quantified in 10 other bivalves per condition, following similar methods used by (Lachaux et al., 2025). Glucose, olive oil and bovine serum albumin were used as standards for carbohydrate, lipid and protein assays, respectively (Lachaux et al., 2025).

### 2.4.2. Biomarker measurement at cellular and subcellular level

Hemolymph was collected from the adductor muscle of mussel individuals used to measure clearance rate and respiration rate using 2 mL syringes fitted with 27G x 5/8-inch needles. Hemolymph osmolality (Osmo), indicator of organism' osmoregulation capacities, was immediately measured in 30 nL hemolymph with an Otago nano-osmometer (Otago, New Zealand) using a 100 mOsm/kg standard. Bivalves were then opened, and a small piece of gonad was removed to determine the mussel's gender by a gonadal smear under optical microscope. The remaining gonad and digestive gland tissues were excised, and immediately snap frozen in liquid nitrogen and stored at -80 °C until further analysis. Both gonad and digestive gland tissue were investigated for sertraline-induced changes in enzymatic activities. Collectively, 9 subcellular biomarkers related to the neuroendocrine system [monoamine oxidase (MAO) and cyclooxygenase (Cycloox) activities], oxidative stress response [total antioxidant capacity (TAC), lipid hydroperoxide concentrations (LOOH) and Caspase-3 (Csp3) activities], and energy metabolism [cytochrome c oxidase (COX), citrate synthase (CS), electron transport system (ETS) and lactate dehydrogenase (LDH) activities], were investigated in both gonad and digestive gland using an

automated spectrophotometer (Konelab 20-XTi, ThermoScientific) and a spectrofluorometer (SAFAS Xenius, Monaco). The two devices allow measurements on very small volumes, enabling the measurement of the whole set of parameters on each digestive gland and gonad. The detailed methods used for sample preparation and measurements are available in (Passignat et al., 2025; Lachaux et al., 2025). MAO and Cycloox activities were quantified following experimental protocols outlined by (André and Gagné, 2017; Gagné et al., 2007) respectively. Remaining tissues were fixed in Bouin's fixative (48 h) and processed to obtain 5 µm-thick sections through classic histology slide preparations (rinsing in water, dehydration in graded series of ethanol-ROTIBiHistol, and staining in Gill II hematoxylin/eosin). These sections, showing the residual undissected digestive gland and gonad, allowed for the presence of parasites to be checked and the gonadal stage of the individuals identified (Minguez et al., 2009).

## 2.5. Statistical analysis

In this study, both univariate and multivariate analysis were conducted. All data acquired from the study were subjected to Shapiro normality test. Due to the limitation with sex determination before sampling, most treatment groups had unequal number of replicates for each gender despite having equal replicates for all treatment groups. Hence, to evaluate the dose-dependent effects on male and female tissues, both parametric (Dunnett's multiple comparison) and non-parametric (Kruskal-Wallis post-hoc) test were used for pairwise comparison of treatment groups in the SER and NorSER bioaccumulation data and for the individual biomarkers investigated. All univariate analyses were conducted using IBM SPSS Statistics 22.0.0.0 (IBM Corp, Armonk, NY, USA) and GraphPad Prism ver 7.02 (GraphPad Software Inc. La Jolla, CA, USA). For a comprehensive understanding of the gender-specific biomarker response to sertraline exposure, all biomarkers were integrated to perform a Partial Least Square Discriminant Analysis (PLS-DA) using R programming software version 3.6.3. PLS-DA aims to highlight biomarkers that differentiate groups, and discern biomarker patterns associated with different experiment conditions. For a straightforward interpretation of the PLS-DA model, Variable Importance in Projection (VIP) scores were used. VIP scores are summarized in the supplementary file. Biomarkers with a VIP score greater than 0.8 are considered significant to the model. Validity of each model was checked using a Hotelling T² test, a method based on permutation testing ($p < 0.05$). To determine the magnitude of experimental effects (difference between exposed groups and the untreated one, control), mean effect sizes (Hedges' g) and their 95 % confidence intervals were calculated for each biomarker.

An effect size is considered significant when its confidence interval does not include 0. A given effect size is considered as small if $0.2 \le g < 0.5$; moderate if $0.5 \le g < 0.8$; large if $0.8 \le g < 1.3$; and very large if $1.3 \le g$ (Sullivan and Feinn, 2012). To provide both a graphical synthesis of VIP biomarkers along the concentration gradient, and a numeric value integrating all these responses at once, the integrated biomarker response (IBR), using the CalIBRi web interface (Devin et al., 2023) was calculated, with coefficient values equal to 1 for each biomarker. Throughout the manuscript, the evidence-based language (Muff et al., 2022) was used to describe the results. The statistical differences were considered significant if the probability of type II error $P$ was <0.05, with the individual thresholds described as follows: $P \le 0.05$ (little or no evidence of effect), $P = 0.049-0.011$ (moderate evidence), $P = 0.01-0.001$ (strong evidence), $P < 0.001$ (very strong evidence of effect).

# 3. Results and discussion

## 3.1. Mortality and bioaccumulation of sertraline in Zebra mussels

No mortality was recorded throughout the exposure period and no
parasites were seen on histological slides. Exposure of Zebra mussels to sertraline revealed a concentration-dependent accumulation with higher concentrations significantly increasing the sertraline body burden in both genders (maximum SER load at SER16: around 14 and 16 µg/g dw for females and males, equivalent to 2.7 and 3.2 µg/g ww) (Table 1). Previous studies have reported SER as one of the most frequently detected SSRIs in bivalves, with higher concentrations in freshwater than in marine mussels, which is assumed to be related to their habitat influenced by wastewater discharges (lower environmental dilution factor) (de Solla et al., 2016; Du et al., 2014; Grabicová et al., 2022a; Maskrey et al., 2024). In freshwater bivalves exposed to SER0.16, SER loads quantified were 440.2 and 302.8 ng/g dw equivalent to 84.7 and 58.2 ng/g ww in females and males respectively (de Solla et al., 2016; Grabicová et al., 2022b). In a stream in Texas, a maximum concentration of 370 ng/g ww SER was also reported in unionid mussels (Du et al., 2014), a concentration not far from that measured in ZMs exposed at SER1.6 in this study. Additionally, body burden reported in freshwater bivalves, fish and decapods also seem to increase with increasing time (de Solla et al., 2016; Passignat et al., 2025).

Sertraline accumulation was also higher than its active metabolite, norsertraline similar to that observed for fluoxetine and its active metabolite in the mussel (Zindler et al., 2020). Contrary to that observed in the Zebra mussels, the decapod *Carcinus maenas* norsertraline body burden was higher than sertraline (Rodrigues et al., 2015). This disparity in results might be due to the higher sertraline concentration used in the decapod study (100 times) than the current study or the differences in organism physiology. Reported sertraline BAFs in the literature vary broadly between studies and bivalve species (>100,000 fold) (de Solla et al., 2016; Boström et al., 2017), but still highlight the bioaccumulative properties of SER, especially in bivalve tissues (BAF ≥2000). Zebra mussels showed the lowest metabolism of sertraline to norsertraline with moderate dosages (SER1.6, SER3.2), but a higher metabolism at the highest concentration (SER16). For the gender-specific differences, metabolism and bioaccumulation factor between females and males were not significant in most treatment groups although higher in females than males, except for SER3.2. At higher concentrations, females had a higher metabolic capacity (converting sertraline to norsertraline), about twice that of males and a lower BAF.

The effective rate of metabolism of sertraline to norsertraline observed in this study might be due to the differences in the total cytochrome P450 enzymes recruited by the various sertraline treatments. Although not investigated in this study, cytochrome P450 enzymes are instrumental in the metabolism of drugs like sertraline with primary enzymes like CYP2B6, CYP2C19, CYP2C9, CYP3A4, and CYP2D6 involved in its metabolism (Lee et al., 2024). Different P450 enzymes are involved in sertraline metabolism and their contribution varies with sertraline concentrations (Obach et al., 2005; Huddart et al., 2020). At lower concentrations, CYP2D6 and CYP2B6 are prominent and

contributes the most to metabolism, while CYP2C9, CYP3A4, and CYP2C19 are recruited as primary drivers at higher concentrations (Obach et al., 2005; Huddart et al., 2020). An inhibition in the functioning of these enzymes could result in the decline of sertraline metabolism to its active metabolite, similar to that observed at moderate sertraline exposure in this study. Beyond the inhibition of enzymes, the higher ratio at moderate doses could be due to the inability or delay of mussels to recruit other P450 enzymes vital to sertraline metabolism at high doses. Additionally, the recruited P450 enzymes might be insufficient as the levels of total cytochrome P450 enzymes found in bivalves are lower than those found in vertebrates (Livingstone, 1998). Cytochrome P450 enzymes are known to exhibit polymorphism which could lead to difference in pharmacokinetics and pharmacodynamic properties of sertraline thereby influencing the sertraline metabolism at different concentrations (Obach et al., 2005; Huddart et al., 2020).

Hormonal influences are known to influence sertraline metabolism (Ma et al., 2019). For instance, in mammals, female sex hormone supplementation with sertraline administration enhanced the efficacy of sertraline in both males and females with a pronounced beneficial effect in females (estrogen modulation of the serotonergic system) (Ma et al., 2019). Estrogen was also found to influence the serotonin synthesis and serotonin receptor binding and activity. This hormonal influence reported could partially explain the observed differences in sertraline metabolism in male and female mussels at higher concentrations in this study. Although lacking, some studies have also reported the ability of mussels to effectively metabolize sertraline (Passignat et al., 2025) and fluoxetine to its active metabolite with increasing time (Silva et al., 2016). Further studies on sertraline metabolism of Zebra mussels are still needed to better understand the risk of such compound in the environment and to aquatic organisms.

## 3.2. Biomarkers response to sertraline concentrations

PERMANOVA, a non-parametric MANOVA used for explaining the environmental variability within communities in ecology or biology was used on the whole dataset. The results revealed evidence of the significant effect of gender on the biochemical biomarker responses (p = 0.001, df = 5, sum of squares = 0.56626, R² = 0.57759, F = 12.033) and not on the individual biomarker responses (p = 0.051, df = 5, sum of squares = 0.55141, R² = 0.18377, F = 1.8011). PLS-DA was further used to capture the most pertinent biomarkers separating experimental groups and also focus on the gender-specific and tissue-specific biomarker response to sertraline. Treatment groups are labeled low (SER0.16), moderate (SER1.6 and SER3.2) and high (SER16). As reproductive state is an important variable influencing bivalve sensitivity to stressors, the assessment of the gonadal status revealed no differences between groups. All individuals, females and males, were in their spawning period, a particularly sensitive phase for bivalves (very energy-intensive). Gender-differences observed hereafter on biomarkers would be related to the response strategies that males and females use to

<caption>**Table 1**</caption>

Tissue concentrations of sertraline and its metabolite norsertraline (ng/g dw) in Zebra mussels (mean ± sd), and sertraline bioaccumulation factor (BAF, L/kg). Different letters mean significant differences between exposure conditions. Significant differences between males and females for a same condition are illustrated by stars.

<table><thead><tr><th rowspan="2">Gender</th><th rowspan="2"></th><th colspan="2">Ctrl</th><th colspan="2">SER0.16</th><th colspan="2">SER1.6</th><th colspan="2">SER3.2</th><th colspan="2">SER16</th></tr><tr><th>D0</th><th>D28</th><th>D0</th><th>D28</th><th>D0</th><th>D28</th><th>D0</th><th>D28</th><th>D0</th><th>D28</th></tr></thead><tbody><tr><td rowspan="3">Females</td><td>Sertraline (SER)</td><td>41.2 ± 5.9<sup>a</sup></td><td>34.4 ± 14.8<sup>a</sup></td><td>440.2 ± 141.4<sup>b</sup></td><td>4003.0 ± 588.4<sup>c</sup></td><td>7552.1 ± 554.4<sup>d</sup></td><td>14049.4 ± 1887.6<sup>e</sup></td><td>8804</td><td>7552</td><td>2810</td><td></td></tr><tr><td>Mean BAF</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Norsertraline (NorSER)</td><td>26.9 ± 4.5<sup>a</sup></td><td>33.3 ± 8.1<sup>ac</sup></td><td>106.0 ± 40.2<sup>b</sup></td><td>383.0 ± 220.6<sup>bc</sup></td><td>1337.6 ± 265.5<sup>d*</sup></td><td>7296.0 ± 2191.1<sup>e</sup></td><td>1.5 ± 1.3</td><td>10.5 ± 2.7</td><td>5.7 ± 2.1</td><td>1.9 ± 0.9</td><td></td></tr><tr><td rowspan="3">Males</td><td>Ratio SER/NorSER</td><td></td><td></td><td>4.2 ± 3.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sertraline (SER)</td><td>49.8 ± 3.3<sup>a</sup></td><td>24.9 ± 1.9<sup>b</sup></td><td>302.8 ± 99.0<sup>c</sup></td><td>3379.0 ± 539.0<sup>d</sup></td><td>6802.1 ± 1156.0<sup>e</sup></td><td>16829.8 ± 2129.1<sup>f</sup></td><td>6056</td><td>6758</td><td>6802</td><td>3366</td><td></td></tr><tr><td>Mean BAF</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2"></td><td>Norsertraline (NorSER)</td><td>34.5 ± 2.6<sup>a</sup></td><td>24.1 ± 8.6<sup>a</sup></td><td>71.6 ± 13.8<sup>b</sup></td><td>567.4 ± 215.5<sup>c</sup></td><td>856.3 ± 202.8<sup>e*</sup></td><td>4662.8 ± 1297.5<sup>d</sup></td><td>1.4 ± 1.3</td><td>6.0 ± 2.5</td><td>7.9 ± 5.7</td><td>3.6 ± 1.6</td><td></td></tr><tr><td>Ratio SER/NorSER</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
cope with the stress from sertraline exposure.

### 3.2.1. Biomarker response at individual level

The PLS-DA analyses conducted indicated that 56 % of the total variance in the female mussel data (Fig. 1A) was explained by the first two components; 41 % by component 1 and 15 % by component 2 with major contribution from total sugars, total lipids but also condition index for component 1, and from total proteins and respiration rate (RR) for component 2 (biomarkers deemed important, VIP scores ≥0.8, Supp. Table S1). Individual evaluation of the biomarkers (Supp. Fig. S1) revealed no significant difference in most biomarker between the Ctrl and the exposure groups, consistent with the clear segregation between all groups on the PLS-DA plot. The sole evidence of sertraline effect was moderate, between SER3.2 and SER16 treatments (p = 0.012; hotelling pairwise comparisons). The horizontal axis separated SER16 group to the others, which displayed higher condition index but lower total sugar and lipid contents than SER16. The SER3.2 exposed females also showed a significant increase in energy reserves. However, they increased lipid storage while utilizing more proteins and sugars for maintenance and metabolic function which could be a survival strategy and an adaptive mechanism to aid stress response and meet reproductive demands with lipid metabolism. For the male mussel data (Figs. 1B), 73 % of the total variance was explained by the first two components; 55 % explained by component 1 and 18 % by component 2 with major contribution from total sugars, total proteins, SfG, clearance rate (CR) for both experimental groups but also condition index for component 1 and RR and

condition index for component 2 (biomarkers considered important, VIP scores ≥0.8, Supp. Table S1). The control group showed high inter-individual variability with the treatment groups. However, moderate evidences of effects were highlighted by hotelling pairwise comparisons between Ctrl and SER0.16 (p = 0.022), and SER3.2 (p = 0.014), and between SER1.6 and SER3.2 (p = 0.024). Additionally, strong evidence for sertraline effect between SER0.16 and SER3.2 (p = 0.0015), and SER0.16 and SER16 (p = 0.0091) and very strong evidence between SER0.16 and SER1.6 (p = 5.24 E-05) was observed, suggesting measurable concentration-dependent effects of sertraline on male mussels. Exposed male mussels showed a lower condition index than Ctrl (significant decrease only for SER3.2 group). The clearance rate and scope for growth showed a bimodal pattern with a decrease compared to Ctrl when exposed to SER0.16, and an increase at SER1.6 and SER3.2 exposure (Supp. Fig. S1). In bivalves like mussels, accumulation of SSRIs has been reported to impair physiological and metabolic functions as well as alter many biomarkers (Passignat et al., 2025; Chan et al., 2024). Filter feeding, an essential ecosystem function was altered in mussels exposed to fluoxetine (Peters and Granek, 2016), evidenced by increased water turbidity and reduced clearance rate (Gilroy et al., 2017). In this study, clearance rate was significantly reduced in the low sertraline exposed group, suggesting a potential impairment in gill function (Peters and Granek, 2016). These changes further resulted in the alterations of various growth parameters integrated into the scope for growth (SfG), consistent with that reported by other studies (Scarlet et al., 2015). A direct relationship has also been reported to exist between clearance rate, and SfG in bivalves exposed to chemical pollutants hence, a reduction in clearance rate concomitantly results in decreased feeding, energy uptake, and growth (Falfushynska et al., 2024). Our findings hereby suggest a concentration-dependent change in energy uptake and biomass production of male mussels and an overall sensitivity of filter feeding ability to sertraline exposures. Knowing that filter-feeding bivalves can have profound influence on plankton community and water quality, the disturbances in filtration rates observed in exposed bivalves could then have indirect effects on other interacting organisms. Additionally, these changes might alter the energy available for growth and reproduction and for storage in the energy reserves. A measure of the condition index (biomarker of bivalve health and energy reserves) however, revealed that at low sertraline concentration, male mussels reduce their metabolic rate so that the energy reserves, dry and fresh weight are preserved despite low energy uptake. Contrary to mussels exposed to the low dosage, mussels exposed to the moderate dose (1.6 nM) had an enhanced energy uptake but no change in condition index suggesting an increase in metabolic cost for maintenance and metabolic activity with the increased energy uptake compensating for this cost while maintaining reserves of the mussel which was confirmed from the evaluation of the energy reserves (Supp. Fig. S1). Doubling the sertraline concentration (3.2 nM) further increased the energy uptake but reduced the condition index implying an increased metabolic cost for survival, maintenance and detoxification beyond energy uptake thereby relying on the reserves for compensation. Increased energy for detoxification after sertraline exposure was also recorded in adult worms *Tubifex tubifex* thereby reducing available energy for growth and reproduction (Chan et al., 2024). Contrary to the observation in this study, other studies reported a direct correlation between clearance rate and energy reserves (Peters and Granek, 2016). This observation could be associated with tradeoffs between survival, metabolic functions and tissue growth as this concentration might induce immerse stress hence requiring more energy in maintaining ecological functions and stress response. Interestingly, a significant increase in total proteins was observed with little change in total lipids and sugar in the exposed group implying the reliance of male mussels on lipids and sugars for survival and stress response with a shift towards increased protein synthesis during moderate sertraline exposure. At high sertraline exposure (16 nM) however, there was no significant change in any of these biomarkers. A measure of basal metabolism as respiration rate at the

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Partial Least Squares - Discriminant analyses (PLS-DA) showing the separation of experimental groups (Ctrl, SER0.16, SER1.6, SER3.2, SER16) based on their two first component scores, using biomarker responses at the individual scale for female (A, ♀) and male (B, ♂) Zebra mussels. For biomarker abbreviation meaning: see section 2.4.</figcaption></figure>
organismal level revealed a concentration-dependent inhibitory effect of sertraline on metabolic activity and a potential stress-induced reduction in energy budget at intermediate and high concentrations. Conversely, basal metabolism was enhanced at the low dose possible as a first line of response to sertraline stress and to maintain homeostasis but declined in the moderate and high sertraline exposed mussels. Interestingly, the condition index recorded in this study generally had a negative correlation to the individual biomarkers, energy reserves and respiration rate in both males and females implying that the mussel's health and fitness after sertraline exposure comes at a metabolic cost (lower feeding, energy reserves and respiration rate) to the whole organism.

Sertraline exposure also induced a significant decrease in the hemolymph osmolality of both females and males suggesting that bivalves had too much water and/or less electrolytes in their hemolymph than Ctrl individuals. The females showed moderate evidence for sertraline effects (p = 0.04) and the males showed strong to very strong evidence of sertraline effects (p < 0.002). Overall, the hemolymph osmolality was the only biomarker affected in both females and males (Fig. 2). Mollusk hemolymph plays a crucial role in physiological adaptations, maintaining internal balance and providing immune protection. Zebra mussels have the lowest recorded basal hemolymph osmolality among metazoans (i.e., around 47 mOsm/kg H₂O), and leaky external epithelium that makes it very permeable to solutes and water (Byrne and Dietz, 1997). As hyperosmoregulators, freshwater organisms show higher osmolality of their internal fluid (hemolymph) compared to the water in those they live, conditioning strong water gain and high ion losses through their tegument which they need to compensate via osmoregulation to survive. One way is to actively capture ions (using energy) via extrarenal osmoregulatory organs (gills). For example, any oxidative alteration of gills, inducing permeability increase after pollutant exposure (metals and surface acidification) is known to induce decrease in osmolality in several freshwater invertebrates (Garaud et al., 2015). Moreover, several medical publications report SSRI-induced syndrome of inappropriate antidiuretic hormone (siadh), which is an osmoregulatory disorder, characterized especially by excessive release of antidiuretic hormone (vasopressin) and low plasma osmolality leading to hyponatremia (de Picker et al., 2014). Although, to our knowledge, no study on *D. polymorpha* has investigated the presence and functions of vasopressin-like hormones, its phylogenetic conservation (Odekunle et al., 2019) suggests that a siadh-like effect cannot be ruled out here for sertraline-exposed bivalves. What is known is that serotoninergic pathways regulate ion (e.g., Na⁺) and fluid transports, and cardiac

contraction in freshwater bivalves (Canesi et al., 2022; Dietz et al., 2011). Thus, a part of hemolymph hypoosmolality observed in sertraline-treated groups could also be explained by an effect on these pathways. This osmotic stress come with an increased energy expenditure to acclimate to the stressor and ensure cellular protection (Sokolova et al., 2012). As any osmotic imbalance might affect cell function, further studies are needed to assess the effects of sertraline on hemocyte activities of Zebra mussels.

### 3.2.2. Biomarker response at subcellular level

At the subcellular level, the PLS-DA analysis revealed that 47 % of the total variation in the digestive gland data of the female was explained by 37 % of component 1 and 10 % of component 2 (Fig. 3A). All treatment groups overlapped considerably with the control except for SER1.6 (only one data point) and SER16. There was a distinct biomarker profile between SER0.16 and SER16 (hotelling test: strong evidence for sertraline effect p = 0.004) (Fig. 3A). For the gonad, 34 % of the total variation was explained by 18 % of component 1 and 16 % of component 2 (Fig. 3B). A pronounced segregation was observed in the PLS-DA plot of the female gonad especially at higher concentrations (hotelling test: from very strong evidence of differences between SER0.16 and SER16: p = 0.0005; moderate evidence between SER3.2 and SER16: p = 0.016) (Fig. 3B).

In the male mussels, 37 % of the total variation in the digestive gland data was explained by 25 % of component 1 and 13 % of component 2 (Fig. 3C). There was a notable overlap in the PLS-DA plot between the various treatment groups and the control. SER1.6 and SER3.2 (Hotelling test: p = 0.01) revealed a distinct differentiation in biomarker profile and SER16 treatment group showed a clearer separation from other doses (Hotelling test: with SER0.16: p = 0.01, SER1.6: p = 0.0003, SER3.2: p = 0.001) (Fig. 3C), suggesting a dose-dependent response of male digestive gland to sertraline. In the gonad data, 49 % of the total variation was explained by 33 % of component 1 and 16 % of component 2 (Fig. 3D). The clusters of the various treatment groups from the PLS-DA plots were less differentiated as compared to the digestive gland but SER3.2 showed a slight distinction (Hotelling test: with SER0.16: p = 0.02) and SER1.6: p = 0.048) (Fig. 3D).

Biomarkers (biomarkers deemed important, VIP scores ≥0.8, Supp. Table S2) contributing to the total variance observed in the various data were gender and tissue specific while others were common between both gender and tissues analyzed (Fig. 3A, B, 3C, 3D). Total antioxidant capacity (TAC), the total amount of enzymatic and non-enzymatic antioxidants to neutralize reactive oxygen species (ROS) was sensitive in both males and females (Supp. Table 1) suggesting sertraline-induced regulation of the antioxidant system. In the digestive gland, TAC was altered at all exposed doses in mussels. An observed decline in TAC activity in the female gonad at all doses (significantly at the highest dose) and an increase (SER0.16 & SER3.2) and decrease (SER1.6 & SER16) in the male gonad was evident. Reduced TAC suggests depleted antioxidant which may be due to detoxification and defense against increased ROS production while enhanced TAC suggests an enhanced stress response to ROS production or lower ROS (Pham et al., 2025).

Csp3, an activated cell death enzyme was regulated in both gender and tissue except the female gonad (Fig. 3A, B, 3C, 3D). In the digestive gland, Csp3 activity increased in both genders as compared to the control with a significant activity at high doses. In the male gonad however, Csp3 activity declined significantly at intermediate doses (SER1.6) suggesting the propensity of sertraline-induced cell death at intermediate doses in the male mussel and at high doses in the female mussel. Overall, the results suggests that intermediate to higher sertraline dosage compromises the Zebra mussel's immune system and induces neurotoxic effects and antioxidant response to cell death and oxidative stress. Interestingly, LOOH, a biomarker of the oxidative stress and lipid peroxidation contributed only to the total variance observed in the male digestive gland (Fig. 3C) but significantly declined in the female gonad at moderate doses (Supp. Fig. S3).

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** The hemolymph osmolality (mOsm/kg) (mean ± sd) measured in female and male Zebra mussels. Different letters indicate significant differences between experimental groups.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Partial Least Squares - Discriminant analyses (PLS-DA) showing the separation of experimental groups (Ctrl, SER0.16, SER1.6, SER3.2, SER16) based on their two first component scores, using biochemical biomarker responses in the digestive gland and the gonad for females (♀, A and B) and males (♂, C and D) Zebra mussels. For biomarker abbreviation meaning: see section 2.4.</figcaption></figure>

Serotonin, an essential monoanime neurotransmitter is a major contributor to the serotonergic system. In bivalves, serotonin is associated with the neuroendocrine, physiological and immune activity (Canesi et al., 2022; Gagné et al., 2007). Antidepressants, especially SSRIs like sertraline are major contributors to the serotogenic system and have been reported to affect reproduction in bivalves (gamete formation, oocyte maturation, sperm mobility) (Canesi et al., 2022; Gagné et al., 2007). Serotonin acts on bivalve's gonads, with females appearing less sensitive than males to the stimulation (Canesi et al., 2022). The functioning of serotonin requires a rapid uptake and enzymatic degradation which is served by the mitochondrial-bound enzyme monoaminase oxidase (MAO) (Bortolato et al., 2010). MAO breaks down serotonin and inhibiting MAO increases serotonin levels (as well as SSRIs) (Bortolato et al., 2010). In this study, MAO contributed to the total variance in the data of both gender and tissues except the female gonad (Fig. 3A, B, 3C, 3D). A dose-dependent decline in MAO activity was observed especially at moderate (3.2 nM) to high doses (16 nM) indicative of an enhancement in the reuptake of monoamine (serotonin and dopamine), thus generating neurotoxic effects and inhibiting ROS production and oxidative stress (Maranho et al., 2015; Gagné et al., 2007). Contrary to the finding from this study, several studies have reported an increase in oxidative stress in aquatic organisms exposed to various pharmaceuticals including SSRIs as well as inflammation (cyclooxy activity) (Maranho et al., 2015; Gagné et al., 2007). In this study, the findings revealed a gender specific regulation of cyclooxy activity, specifically in the female gonad with an increase at moderate doses and a decline to baseline levels at high doses (Supp. Fig. S3). As cyclooxygenase is not only associated with inflammatory properties (Aguirre-Martínez et al., 2018), but also with the spawning phase (Maranho et al., 2015), its increase is associated with serotonin increase which aid to finalize gamete maturation and initiate egg release (Gagné et al., 2011). As the mussels used in this study were at the final stage of

the reproductive cycle (emission stage), the higher activity could be attributed to the spawning phase and not inflammation.

All other biomarkers contributing to the total variance in the data of both gender and tissues were related to energy metabolism. Sertraline exposure regulated mitochondria biomarkers like ETS, CS, COX, and LDH with these biomarkers being more sensitive in the male mussel than the females and regulated in one or both tissues of the male mussel (Supp. Table 1, VIP score >8). In the male mussel however, mitochondria response to sertraline appears to be more complex (Supp. Figs. S2 and S3). Although all biomarkers were sensitive to sertraline exposure, only the digestive gland seems to experience a significant metabolic shift from aerobic to anaerobic respiration at intermediate sertraline exposure (SER3.2). In the gonad, there was a decline in ETS, CS and LDH activity but an increase in the activity level of COX in the 3.2 nM sertraline exposed group. Overall, this finding suggests a possible impairment of mitochondrial oxidative capacity and ATP generation especially at moderate doses and a partial reliance on lactate and glycolysis to meet energy demands (Rodrigues et al., 2015). Previous studies have reported an alteration in metabolic rate and bioenergetics after exposure to different SSRIs like fluoxetine (Rezende et al., 2021). In mussels, these pharmaceuticals have also been reported to increase the rate of electron transport in mitochondria (Rodrigues et al., 2015). However, these studies in aquatic organisms are generally limiting but vastly available in mammals (de Oliveira, 2016; Li et al., 2012). For instance, fluoxetine, an SSRI like sertraline was found to trigger mitochondrial apoptosis and inhibit the ETS (de Oliveira, 2016). Sertraline was also found to initiate mitochondria dysfunction and inhibit ATP production (Li et al., 2012) with the presence of female sex hormones like estradiol exhibiting a protective role in the mitochondria and alleviating this dysfunction (Shimamoto and Rappeneau, 2017).

In this study, only biomarkers related to aerobic respiration (CS, COX) were sensitive in the female mussels. Although information linking
mitochondrial activity to sex and sertraline administration is limiting, females are known to respond better to sertraline treatments due to sex hormones (Ma et al., 2019; Kornstein et al., 2000) and estrogen was found to be expressed in the mammalian mitochondria (Shimamoto and Rappeneau, 2017) suggesting the potential for a better mitochondrial activity in females than males. A dose-dependent decline in COX activity (an enzyme complex of the ETS involved in aerobic generation of ATP) in the female predominantly in gonad and an increase in LDH activity was also observed. LDH is a key enzyme in ATP generation via glycolysis hence the observation from this study suggests a shift from aerobic respiration towards anaerobic respiration during sertraline exposure in the females as well. This further indicates an impairment of mitochondrial functions like oxidative phosphorylation and signal metabolic stress even at low concentrations prompting the reliance on glycolysis to compensate for energy demands. Similar findings were reported in rat liver and astrocytes where sertraline exposure induce mitochondrial uncoupling, Complex I and V inhibition, ATP depletion, induction of mitochondrial permeability transition and lactate dehydrogenase release (Li et al., 2012). These findings presume the potential for similar effects of sertraline exposure on the mitochondrial of both target and non-target organisms. Albeit, the results further revealed an increase in CS (key TCA cycle enzyme vital to aerobic respiration) activity at the highest sertraline exposure suggesting the initiation of a potential compensatory mechanism by the mitochondria to aid mitochondrial function, the return of aerobic respiration and enhance ATP production. Taken together, our finding suggests that sertraline induces significant changes in energy metabolism with a clear transition from aerobic to anaerobic respiration in both genders. However, the female mussels might have a compensatory mechanism in place to restore aerobic respiration at higher concentration.

### 3.3. Integrated biomarker response (IBR) and effect size

Computing the Hedges effect size revealed multiple biomarkers with diverse effect sizes (small, moderate, large and very large) among the various sertraline treatment groups (Fig. 4). In the female mussel (Fig. 4A, ♂), despite large to very large effect size (1.3 ≤ g) in both tissues for some biomarkers, statistically significance of sertraline treatment was only observed in gonad tissues (i.e., lower CS activity in SER0.16 group, lower Cox activity and LOOH content in SER3.2 group, and lower TAC in SER16 group, compared to Ctrl group). Most individual biomarkers showed medium effects with no statistical

significance. In the male mussels (Fig. 4B, ♂), only Csp3 showed very large effect size in the digestive gland (significant higher activity for SER16 group compared to Ctrl) and in the gonad (significant lower activity for SER1.6 group compared to Ctrl). LDH showed statistically significant effect of sertraline exposure in the digestive gland with large effect size in SER3.2 exposure group (higher activity). For the hemo-lymph osmolality, the findings revealed large effects but no statistical significance for females across all sertraline exposure (except SER1.6, had only 1 sample) and very large effect size for males across all sertraline exposure. Only intermediate and high sertraline concentrations in the male mussel were statistically significant (lower osmolality compared to Ctrl). Condition index, total proteins and total sugars showed very large effect size with significance in at least one of the sertraline exposed groups. Based on these findings, we speculate that sertraline exposure had the most significant effects on subcellular biomarkers, specifically those related to energy metabolism at the mitochondrial level and oxidative stress suggesting an alteration in bioenergetics and immunocompetence of the female gonad and not digestive gland. In the male however, sertraline exposure had significant effect on both individual and subcellular biomarkers (for both tissues). Energy metabolism was altered both at the whole organism and mitochondrial level in the male mussel suggesting energy metabolism biomarkers as crucial targets for sertraline exposure in the Zebra mussel.

A common issue in multi-biomarker studies is the need to transcend independent interpretation of each biomarker, and to assess the overall response pattern they jointly produce. This global assessment simplifies their interpretation in biomonitoring programs (Devin et al., 2014). Herein, integrating all the VIP biomarkers (IBR) at the various levels revealed concentration-dependent effects (Fig. 5). The IBR calculated with VIP biomarkers obtained from the digestive gland showed a global similar pattern between males (♂) and females (♀), with an increased value for SER0.16 group compared to control followed by a similar value as Ctrl at SER1.6 followed by an increase of IBR with increasing sertraline concentrations. For the gonad, differences were observed between males and females. IBR values according to SER concentrations showed a bell-shape with a maximum value for SER1.6 group, whereas for females IBR values decreased with increasing concentrations. A bimodal shape was observed for males with IBR values calculated with “whole organism” VIPs, SER0.16 group showing the lowest IBR value and SER3.2 the highest. For females, the pattern followed a U-shape, with the minimum value observed for SER1.6 group, and similar IBR value between Ctrl and SER16. Regardless, the scale of biological

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Effect size estimates in Hedge's g (with 95 % CI) of biomarkers between sertraline-exposed and unexposed Zebra mussels, after 28d of exposure. Significant effects of sertraline compared to unexposed group (Ctrl) are shown by *. For biomarker abbreviation meaning: see section 2.4.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Integrated biomarker response (IBR) of Zebra mussels at the end of the experiment (day 28) using only the VIP biomarkers from PLS-DA analyses (cut-off = 0.8). Female (♀) and male (♂) responses are separated. Box plots show median values, quartiles (hinges), and whiskers (1.5 × interquartile range). Different letters indicate significant differences between experimental groups. For biomarker abbreviation meaning: see section 2.4.</figcaption></figure>

organization (subcellular or individual), IBR allows to highlight the common non-monotonic concentration-response relationships for endocrine disrupting compounds (EDCs). Another phenomenon related to EDCs, called “low concentration effects”, was also visible with IBR. Significant physiological changes, compared to control, appeared already at the environmental concentrations (SER0.16), implying that data at high concentrations cannot be used to predict effects at lower environmental concentrations. Additionally, these findings further affirm that the response to sertraline exposure is not universal but varies between gender, tissues and the whole organism, hence the various scales should be considered in ecotoxicological studies.

## 4. Conclusion

The present study revealed that increasing sertraline concentration in water increases the sertraline body burden of exposed mussels with mussels exhibiting high sensitivity and low metabolism at moderate concentrations. An increment in the sertraline body burden alters mussel cellular and physiological functions with environmentally relevant dosage (low dose), inhibiting organismal health and fitness and initiating physiological adjustments to maintain homeostasis and organismal function. At moderate concentrations, the results from this study revealed further inhibitory effects with a compromise in mussels' metabolic rate and immune and antioxidant system generating neurotoxic effects while at high concentration, little to no change in cellular and physiological function was identified as levels of sertraline accumulation are the highest and metabolism similar to baseline levels with energy metabolism and immune functions similar to baseline level. We therefore speculate that at this concentration, exposed mussels are unable to significantly mount (either improve or degrade) physiological response to sertraline exposure. Conclusively. Zebra mussels are most sensitive to moderate concentrations with a prominent alteration in cellular and subcellular functions. These alterations are gender and tissue-specific with males showing higher sensitivity to sertraline exposure than females. We therefore speculate that as observed in mammals where female hormones aids a better response to sertraline, a similar mechanism might be at play in mussels too. These findings further emphasis the need to consider gender-specific responses in ecotoxicological studies as the variations could have potential ecological

outcomes for mussel population dynamics. Overall, the findings from this study reveal the need for toxicity assessment even at low environmental concentrations (SER0.16) as significant physiological changes are observed in comparison to the control, and high concentrations cannot exactly predict effects at lower environmental concentrations. Additionally, the findings suggest energy metabolism and bioenergetics as crucial targets for sertraline exposure and possibly other pharmaceuticals, prompting the need for more studies in this area.

### CRediT authorship contribution statement

**Linda Lumor:** Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation. **Anne Vignet:** Writing – original draft, Visualization, Formal analysis, Data curation. **Céline Passignat:** Writing – original draft, Validation, Supervision, Methodology, Formal analysis, Data curation, Conceptualization. **Vincent Felten:** Writing – original draft, Methodology, Formal analysis. **Céline Simon:** Methodology, Formal analysis. **Marie Zaffino:** Methodology, Formal analysis. **Guillaume Grosjean:** Methodology, Formal analysis. **Laure Giambérini:** Writing – original draft, Methodology, Formal analysis. **Laetitia Minguez:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### 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

This work was supported by the French National Research Agency through the Pharma CARE project (ANR-21-CE34-0009). Linda Lumor received a financial support from the Grand-Est Region for her post-doctoral fellowship. This work was partly done with resources from the Pôle de compétences en biologie environmental, ANATELo, LIEC laboratory, UMR 7360 CNRS – Université de Lorraine, and the LiecOscope,
LIEC laboratory, UMR 7360 CNRS – Université de Lorraine.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envres.2025.123422.

## Data availability

Data will be made available on request.

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