# Health check-up of a freshwater bivalve exposed to lithium

Nicolas Lachaux, Carole Cossu-Leguille, Marie Zaffino, Céline Simon, Maximilien Beuret, Laure Giamberini, Laetitia Minguez

► To cite this version:

Nicolas Lachaux, Carole Cossu-Leguille, Marie Zaffino, Céline Simon, Maximilien Beuret, et al. Health check-up of a freshwater bivalve exposed to lithium. Environmental Pollution, 2025, 367, pp.125573. 10.1016/j.envpol.2024.125573. hal-05194636

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Health check-up of a freshwater bivalve exposed to lithium

Lachaux N.<fnref n="1" />*, Cossu-Leguille C. <fnref n="1" />, Zaffino M. <fnref n="1" />,<fnref n="2" />, Simon C. <fnref n="1" />,<fnref n="2" />, Beuret M. <fnref n="1" />,<fnref n="3" />, Giamberini L. <fnref n="1" />,
Minguez L. <fnref n="1" />*

<footnote n="1">¹ Université de Lorraine, LIEC, CNRS, F-57000, Metz, France</footnote>

<footnote n="2">² Pôle de compétences Biologie environnementale, ANATELO, Université de Lorraine, F-57000, Metz, France</footnote>

<footnote n="3">³ Pôle de compétences Chimie analytique environnementale, ANATELO, Université de Lorraine, F-57000, Metz, France</footnote>

<footnote>*Corresponding authors : lachaux.n@gmail.com; laetitia.minguez@univ-lorraine.fr</footnote>
# 1 Abstract

Lithium (Li) has become essential for energy and digital transitions, especially as a component of rechargeable batteries. Its growing uses worldwide lead to increasing anthropogenic releases of Li into the environment, which is making Li as an emerging contaminant. It is thus critical to evaluate the ecotoxicological impact of Li, which has been poorly studied unlike its human toxicology. The objectives of this work were to assess the potential adverse effects of Li on aquatic ecosystems. Bioaccumulation and potential sublethal effects (at individual and cellular levels) of Li have been investigated in the widespread and ecologically relevant freshwater bivalve *Dreissena polymorpha*. To tackle these issues, mussels were exposed to several spiking of 40, 100 and 250 µg Li L⁻¹ for 28 days to reproduce anthropogenic contamination scenario. Results demonstrated that bivalves significantly accumulated Li in a dose-dependent manner, from 2 to 10 µg g⁻¹ dry weight (bioaccumulation factor, BAF ≈ 19 L kg⁻¹). Bioaccumulation of Li reached a steady-state from seven days of exposure and BAF values were constant regardless the exposure concentration indicating a tight regulation of Li body burden. Lithium exposure leads to increased energy demand associated with a higher lactate dehydrogenase activity and the decrease of protein concentrations. The observed weight gain, increased cellular metabolism, decreased apoptosis, induction of antioxidant defenses and cation content modification in *D. polymorpha* were also reported in previous studies on humans. The observed effects intensified with exposure concentration and duration, which implies an increased risk for aquatic organisms exposed to Li chronic contamination. Overall, the present study provides new knowledge concerning the impact of Li on non-targeted species, which have implications for the environmental risk assessment of Li in freshwater ecosystems. It also open new perspectives for the understanding of Li toxicokinetics and toxicodynamics on freshwater organisms.

**Keywords:** Emerging contaminant; Ecotoxicity; Sublethal effects; Bioaccumulation; *Dreissena polymorpha*; Homeostasis; Energy demand

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# 1. Introduction

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The international commitment to achieving a climate-neutral society requires fundamental changes in the way we generate and use energy. The transition towards more climate-neutral countries has led to a significant growth of the markets for electric vehicles and their lithium-ion batteries (European Commission, 2019). The energy transition relies on lithium-ion technologies. Since 2017, lithium (Li) demand has tripled, and could grow tenfold under the International Energy Agency's (IEA) Net Zero Emissions by 2050 Scenario (International Energy Agency, 2023). Despite this increased demand for Li, the waste collection and recycling sectors are still in development. For instance, Li-ion batteries tend to be landfilled with municipal solid waste, due to lack of recycling plants (Thibon et al., 2021a). Moreover, the high mobility of Li makes its removal by wastewater treatment plants challenging (Choi et al., 2019). The increasing extraction and use of Li, combined with inefficient purification protocols, result in greater environmental release. Li is transported over long distances from its sources, leading to heightened environmental accumulation (Barbosa et al., 2023b; Thibon et al., 2021c). Mining activities, chemical industry, sludge-amended agricultural soil, car lubricating grease from road leaching, deficiency in e-waste (e.g. rechargeable batteries) management and recycling are the main sources of anthropogenic Li in aquatic systems (Aral and Vecchio-Sadus, 2011; Bolan et al., 2021; Choi et al., 2019; Kaunda, 2020; Mrozik et al., 2021; Shakoor et al., 2023). Freshwater ecosystems at the vicinity of Li naturally-enriched regions, urbanized and industrial areas or e-waste dumping sites could be particularly vulnerable to Li contamination.

Concentrations of Li in freshwater systems range from 0.07 to 40 µg L⁻¹ (Aral and Vecchio-Sadus, 2011), but can reach 1.6, 3.1 and 13.7 mg L⁻¹ in contaminated rivers in South Korea (Choi et al., 2019), France (EauFrance, 2023) and Ukraine (Sobolev et al., 2019). The increasing use of Li and the resulting environmental contamination have triggered new concerns about its potential impact on aquatic organisms. Several recent studies have shown that Li bioaccumulates and has high biological activity on marine organisms (Aral and Vecchio-Sadus, 2008; Barbosa et al., 2023c, 2023b; Bolan et al., 2021; Perošević et al., 2018; Santos et al., 2023; Thibon et al., 2021a; Viana et al., 2020), knowledge on freshwater species are more limited (Aral and Vecchio-Sadus, 2008; Kszos et al., 2003; Martins et al., 2023). These studies reported Li concentrations in bivalve tissues ranging from 0.4 to 7.1 µg g⁻¹ (bioaccumulation factor = 1.4-10.3 L kg⁻¹) associated with toxic effects. Among all marine species studied, bivalves were among the strongest Li-accumulators (Thibon et al., 2021c) and the most sensitive to Li toxicity (Barbosa et al., 2023b). Those works also showed the importance of
assessing sublethal effects of Li. Toxicological effects of Li on humans and mammal models are more documented, Li being the mainstay of bipolar disorder treatments, but its mode of action is still not yet completely elucidated. Li could affect energy metabolism and reserves, ionoregulation but also induce oxidative stress, neurological alterations, kidney and thyroid disturbances (Adida et al., 2014; Aral and Vecchio-Sadus, 2011; McKnight et al., 2012; Medić et al., 2020). As the biological targets of Li have been conserved through the evolution, its potential impact on freshwater species should be assessed.

Zebra mussels, *Dreissena polymorpha* can provide knowledge about the fate and effects of lithium in fresh waters. As filter-feeding species, bivalve mollusks fullfil ecological functions by contributing to phytoplankton density regulation and water depuration. Their worldwide distribution, their sessile lifestyle and their ability to accumulate large amounts of contaminants (including metals) make bivalves, including *D. polymorpha*, key organisms for biomonitoring programs across the world (Multisanti et al., 2022; Pain-Devin et al., 2014; Palais et al., 2011). Their responsiveness to contaminants can be assessed by measuring biomarkers at different levels of the biological organization (Garaud et al., 2016; Multisanti et al., 2024; Potet et al., 2016), which represents an “early warning” of the potential environmental impact of contaminants. Zebra mussel is considered as model organism in ecotoxicology and it is described as the freshwater counterpart of the blue mussel (Binelli et al., 2015; Hodkovicova et al., 2022). To our knowledge, there is only one study having investigated Li biological effects on a freshwater bivalve (Aral and Vecchio-Sadus, 2008). The authors reported that 24 h of exposure at 185-232 mg L⁻¹ of Li induced 50% of mortality (LC₅₀) in *D. polymorpha* but sublethal effects were not investigated. Under the current scenario of the global demand for Li, studies investigating the ecotoxicological risk of Li are needed to ensure that green technologies will not contribute to the decline in biodiversity.

In this context, we aim to assess bioaccumulation and sublethal effects of Li as a contaminant of emerging concern on the widespread freshwater mussel, *D. polymorpha*. Bivalves were exposed during 28 days to three environmentally relevant Li concentrations. Biochemical and physiological endpoints were assessed for depicting Li-sublethal effects. Bioaccumulation and biological effects of Li were monitored each week for 28 days. The kinetic approach used in this study provides new insights into how *D. polymorpha* handle Li-exposure over time.

## 2. Material and methods

### 2.1. Organism sampling and acclimation
*Dreissena polymorpha* individuals were hand-collected in the Meuse River at St-Mihiel, France (48.887210, 5.534515) in September 2020. They were transferred to the laboratory in a cooler with field water. The day after sampling, soft tissues of five mussels were collected and stored at -80°C until to be processed for Li quantification. This data provides information on lithium impregnation in the field (*in situ* bioaccumulation) for these organisms. The remaining mussels were acclimated for 15 days at field temperature (19°C) in two separate tanks continuously aerated, filled with 12 L of field water. The water was progressively replaced by mineral water, Volvic® (1/3 every two day the first week and complete replacement the second week). Mortality was checked daily and mussels were fed with microalgae, *Chlorella vulgaris*, from our own lab culture (2x10⁶ cell/mussel/day).

## 2.2. Exposure conditions

A stock solution of LiOH (Sigma-Aldrich, reagent grade >98%) at a concentration of 1 g L⁻¹ was prepared in demineralised water and used for all experiment duration. Desired Li concentrations were obtained by dilution of Li stock solution in Volvic® mineral water (12, 15, 8, 9, 12, 74, 6, 0.5 mg L⁻¹ of calcium, chloride, magnesium, sulfate, sodium, hydrogen carbonate, potassium, phosphate, respectively) constituting the exposure medium. Mussels were exposed for 28 days to several spiking of 40, 100 and 250 µg L⁻¹ of Li. The exposure medium was entirely renewed once a week while Li exposure concentration was spiked every two days (four times a week) in order to reproduce a diffuse contamination and to compensate the potential loss of Li due to mussel filtration. For each condition (Li exposure concentration and duration), 25 mussels of close size (23±3 mm) were exposed in a glass tank filled with 5 L of exposure medium. Experimental controls consisted in organisms kept in non-contaminated water under the same food, light and temperature conditions as Li exposed mussels. All mussels were fed daily with *C. vulgaris* (2x10⁶ cell/mussel/day) (Figure 1). Temperature, pH, dissolved oxygen (O₂) and conductivity were monitored daily in each tank the first week of exposure, then once a week the rest of the time.

## 2.3. Lithium concentration measurement and analysis

Lithium quantification in water and mussel tissues was performed by inductively coupled plasma mass spectrometry (Thermo iCap TQ, Thermo Fisher, Bremen, Germany). After 0 (t₀), 7 (t₇), 14 (t₁₄), 21 (t₂₁) and 28 (t₂₈) days of exposure, the whole soft tissue of five individuals per condition were collected and stored at -80°C for bioaccumulation analysis (Figure 1). Previously to elemental quantification, freeze-dried tissues were weighed to the nearest 0.1 mg
with an analytical balance in 15 ml disposable glass vials, then suspended in 2 ml of 69 % nitric acid (NORMATOM, VWR) and digested in batches of 15 using a microwave-assisted system (Milestone Ultrawave) at 220°C and 110 bars for 40 min. Digested samples were diluted with ultrapure water to 50 mL (% Total Dissolved Solid <0.1; %H⁺ (v/v) = 4) and centrifugated prior to analysis. In each batch, at least two blanks and one reference material (ERM-CE278k; Mussel Tissue) were included in the mussel sample. Water (i.e. exposure medium) samples were collected before and after each medium renewal and before and after the first Li spiking. Then, water samples were acidified with HNO₃ 1 % v/v and stored at 4°C for analysis of Li total concentrations. For element quantification in water and mussel tissues, ICP-MS instrument was tuned daily for oxide and doubly charged ions and mass calibration accuracy was checked. The SQ-No gas mode was used only for lithium as it has no polyatomic interference. For all elements SQ-KED (Single Quadrupole—Kinetic Energy Discrimination) and TQ-O2 on-mass or mass-shift modes (Triple Quadrupole Oxygen Reaction) were used to limit potential polyatomic or isobaric interferences (Table S1). External calibration was performed using synthetic multi-elemental solutions (AccuStandard and SPEX Certiprep) acidified with 2% HNO₃ (v/v) (NORMATOM, VWR) (Table S2). The accuracy of the calibration was checked with certified reference surface water (SPS-SW1 Batch 112) and an interlaboratory surface water (AGLAE). The instrumental drift was monitored by running SPS-SW1 and AGLAE surface water every 10–20 samples and by adding Rh and Ir to the samples as internal standards. The measurements were performed three times and averaged for each solution. The reference material ERM-CE278k (Mussel Tissue) was used to check the accuracy of the digestion method, and procedural blanks were measured to assess contamination. For lithium in ERM-CE278k, we found a value of 0.25 ± 0.05 µg g⁻¹ which is in line with (Thibon et al., 2021b) who found 0.17 ± 0.05 µg g⁻¹. Sodium borosilicate glass vials used in digestion of tissue samples induced a non negligible sodium and potassium contamination which is why detection limit of these element are much higher in digested sample than in water (Tables S3 and S4).

Effective Li exposure concentration along the experiment was determined for each experimental condition by calculating the time weighted mean concentration (TWMC) according to OECD (2012):

$$TWMC = \frac{\left( \frac{\text{conc } 0 - \text{conc } 1}{\text{Ln}(\text{conc } 0) - \text{Ln}(\text{conc } 1)} \times \text{renewal duration} \right)}{\text{exposure duration}} \quad (1),$$

with conc 0 and conc 1 being Li concentration measured at the beginning and at the end of the renewal period, respectively; renewal duration = 7 days and exposure duration = 28 days.
Moreover, chemical speciation of Li was estimated by modelling using Visual Minteq 3.1 in order to better understand Li fate in the tested exposure medium.

Bioaccumulation factor (BAF) was calculated as the ratio between Li concentration measured in bivalve tissues and the one measured in the exposure medium (TWMC).

In addition, Li concentrations were measured in algae to quantify the potential accumulation of Li in mussels via the dietary route. Algae (Chlorella vulgaris) were cultivated (initial concentration : 5 x 10⁵ cells/mL) in 2 L-flasks containing 1 L of LC medium (Jouany et al., 1982) (pH 7.0) with lithium at the same concentrations as for mussels (triplicate cultures per exposure concentration), over 7 days. Under continuous illumination (≈ 90 µmol m⁻² sec⁻¹), controlled temperature (24 ± 1 °C) and aeration, algae are still in their exponentially growing phase after 7 days. The water surplus was removed by centrifugation (1200 g, 15 min) and algal pellets quickly frozen. Samples were lyophilized (freeze-dried cell mass per sample ≈ 0.04 g) before further similar processing for Li quantification as for bivalves.

## 2.4. Biomarkers measured at individual level

Eight mussels were sampled at t₀, t₇, t₁₄, t₂₁ and t₂₈ for biomarker analyses at individual level (Figure 1). Length (l), height (h), width (w) of shell (in mm) and fresh weight (fw) of the whole soft tissues (in g) were measured. Condition index (CI) (in g mm⁻³), a proxy of mussel general health (Filgueira et al., 2013) was calculated according to the following formula:

$$CI = \frac{fw}{l \times h \times w} \qquad (2),$$

Scope for growth (SfG) is an index of the whole-animal physiological state (i.e. energy status) to sublethal stress induced by contaminants (Widdows and Staff, 2006). Mussel SfG was measured according to the protocol adapted from Widdows and Johnson (1988) modified by Mubiana and Blust (2007). Briefly, bivalves were placed individually in glass beaker with 180 mL of exposure media or Volvic water for controls, and fed with 1 mL of a suspension of C. vulgaris (6 x 10⁷ cells mL⁻¹) of known caloric content. Initial algae concentration was measured in each beaker by flow cytometry. Mussels were monitored in closed beakers during 100 min in the dark for their oxygen consumption 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, a beaker filled with 180 mL Volvic water and 1 mL of algae but without bivalve was also processed to assess the effect of algae on the measured oxygen concentrations. At the end of
each run, exposure media were sampled for NH₄ concentration analysis by spectrophotometry and final algal concentration was measured. SfG was calculated according to the following formula:

$$192 \quad SfG = AE - (RE + EE) \quad (3),$$

where AE = absorbed energy (associated with feeding rate measurement), RE = respired energy (associated with respiration rate measurement) and EE = excreted energy (associated with ammonia excretion).

Results were expressed as J h⁻¹ g⁻¹ dry weight. Additional information concerning SfG measurement and calculation can be found in Louis et al. (2019).

For carbohydrate and lipid content determination were performed following the method developed by Van Handel (1985a, 1985b) and adapted by Plaistow et al., (2001). A chloroform/methanol (1/2 v:v) – sodium sulfate (Na₂SO₄, 2%) mixture was added to tissue homogenate. After precipitation (30 min at 4 °C), samples were centrifuged at 3000 g for 10 min (4 °C). The resulting supernatant (100 µL) was transferred in culture tubes and placed into a dry bath at 95 °C for 5 min to evaporate the solvent. Then, sulphuric acid (200 µL) was added and the mixture was boiled during 10 min at 95 °C. The tubes were cooled before adding 5 mL of phosphovanillin reagent, mixed and left 10 min before reading the optical density at 535 nm. Phosphovanillin reagent is a mixture of 120 mg of vanillin, 20 mL of ultrapure ethanol (95%) and 80 mL of phosphoric acid (85%). Olive oil was used as standard for lipid assay. Pellets are resuspended in demineralized water (400 µL) and let 15 min on ice. Resuspended pellets are crushed to obtain a homogenous solution: 50 µL of this solution are mixed to 2.45 mL of anthrone. After 17 min at 95°C, the reaction between anthrone and sugars gives a green color to the solution. After 5 min on ice, the absorbance of solutions was read at 630 nm. Glucose was used as standard for carbohydrate assay. Protein content was quantified according to Bradford (1976) and Bovine Serum Albumin was used as standard.

Lithium effects on mineral element composition and regulation were assessed by measuring Na, K, Mg, Ca, P, S, Cu concentrations in the whole soft tissues of mussels used for Li bioaccumulation determination, also by ICP-MS.

## 2.5. Biomarkers measured at subcellular level

At t₀, t₇, t₁₄, t₂₁ and t₂₈, eight mussels were dissected to collect digestive glands and gills, which were stored at -80°C until biomarker analyses (Figure 1). Biological effects of Li at
subcellular level were assessed by analysing energy reserves and metabolism, composition and regulation of elements, oxidative stress and cellular damages. In digestive glands, energy reserves and metabolism were measured at cellular level with triglyceride (TG) content, cytochrome c oxidase (COx) and citrate synthase (CS) activities but also at individual level with protein (PROT), lipid, carbohydrate contents and electron transport system (ETS) activity. COx, CS and ETS are enzymes involved in aerobic metabolism while lactate dehydrogenase (LDH) is a proxy of anaerobic metabolism. Antioxidant capacity was studied as total antioxidant capacity (TAC) and a more specific enzymatic activity (superoxide dismutase, SOD). The induction of potential cellular damages was assessed through lipid peroxidation (LOOH) measurement. Caspase-3 (CSP-3) is an enzyme indicating cellular damage and more especially cell death (apoptosis).

Analyses of TG, PROT, ETS, LDH, CS, SOD, TAC and LOOH were performed using an
automate spectrometer (Konelab 20-XTi, ThermoScientific) while COx and CSP-3 were
measured using a spectrofluorimeter (SAFAS, Xenius). Briefly, digestive glands were weighted
and crushed in 50 mM phosphate buffer supplemented with protease inhibitors (1mM
phenylmethylsulfonyl fluoride (PMSF) and 1mM L-serine borate) at a 4:1 (v/w) ratio, using a
bead beater. After a first centrifugation for 5 min at 250 g, a part of the supernatant was collected
for PROT, TG, LOOH concentrations, CS, ETS and COx activity measurement. The remaining
part of the supernatant was centrifugated 50 min at 20,000 g and used for quantification of
PROT and TAC concentrations, and measurement of LDH, SOD and CSP-3 activities. PROT
and TG concentrations were measured using Thermo-Scientific Konelab ready-to-use reagents.
Briefly, PROT were quantified according to Pyrogallol method that consists in measuring, at
600 nm, the absorbance of the coloured complex Pyrogallol-molybdate with amino groups from
proteins. TG were quantified by measuring the absorbance, at 510 nm, of quinoimine resulting
from the reaction between 4-aminoantipyrine, 4-chlorophenol and H₂O₂ after triglycerides
hydrolysis. ETS activity was determined by monitoring formazan production at 480 nm
resulting to the reduction of p-iodonitrotetrazolium as final electron acceptor in mitochondrial
respiratory chain instead of O₂ (Owens and King, 1975). COx activity was assessed by
measuring cytochrome c oxidation by complex IV of the mitochondrial respiratory chain at 550
nm. LDH was determined by monitoring lactate to pyruvate oxidation by LDH, which reduces
NAD to NADH, the latter being measured at 340 nm. CS activity was determined by measuring
condensation reaction of acetate residue from acetyl coenzyme A and oxaloacetate to form
citrate at 405 nm. SOD activity was determined by monitoring, at 450 nm for 324 sec, formazan
production (inversely proportional to SOD activity) resulting from tetrazolium salt cleaving by
superoxide radical. TAC activity was measured by monitoring AzBTS⁺ reduction by all
antioxidant species including enzymes (GSH reductase, catalase, peroxidase), small molecules
(ascorbate, uric acid, GSH, vitamin E...) and proteins (e.g. albumin, transferrin) (Erel, 2003).
Lipid peroxidation (LOOH) was measured by monitoring Fe²⁺ oxidation by lipid
hydroperoxides (Arab and Steghens, 2004). Then, Fe³⁺ complexation with xylenol orange is
measured at 620 nm. CSP-3 activity was determined by measuring the fluorescence (emission
400 nm; excitation 505 nm) of 7-amino-4-trifluoromethylcoumarin (AFC) cleaved from Asp-
Glu-Val-Asp (DEVD) by CSP-3. Additional information concerning methods of biomarker
measurement can be found in Garaud et al. (2016) and Lachaux et al. (2023).

Total Ca²⁺-ATPase activity was measured on the automated chemistry analyser Konelab 20
Xti in the homogenate of the gills of controls and exposed bivalves. Briefly, gills were
suspended in 6 mL g⁻¹ fw in HEPES buffer (10 mM HEPES pH 7.2, 250 mM sucrose, 1 mM
EDTA, 1 mM PMSF) and crushed with metallic beads in a bead beater (30s). The homogenate
was then centrifuged for 5 min at 250 g. Supernatants containing total Ca²⁺-ATPase were
estimated straight away by quantifying Pi released from Ca²⁺-ATPase activity (Santini et al.,
2011).

Sex of each mussel used for bioaccumulation and biomarker (individual and subcellular
levels) analyses was determined by observation of a gonad smear under optical microscope.

## 2.6. Data analysis

Statistical analyses were conducted with R software (version 3.6.1). The results between
the different experimental conditions were compared performing ANOVA (when normality and
homoscedasticity conditions are respected) otherwise Kruskal-Wallis (KW) test. Two factors
were tested: Li concentration and exposure duration (time). Data normality and
homoscedasticity were checked with Shapiro-Wilk and Bartlett tests respectively. Results were
considered statistically significant in case of p-values < 0.05. In addition, Hedge's g statistic
was calculated based on the mean and standard deviation of each variable (biomarker) with the
package "metafor" (Viechtbauer, 2010). Effect size represents the magnitude of difference
between experimental conditions and was thus used as a complement to ANOVA and KW tests
(Sullivan and Feinn, 2012). Effect sizes were calculated between Li exposed and experimental
control groups to analyse Li effects respect to the control. The effect size is significant when
confidence interval of g does not include 0. A given effect size is considered as very small if 0
285 ≤ g < 0.2; small if 0.2 ≤ g < 0.5; moderate if 0.5 ≤ g < 0.8; large if 0.8 ≤ g < 1.3; very large if
286 1.3 ≤ g (Sullivan and Feinn, 2012). Influence of sex on the results of different analyses was
287 checked by performing ANOVA. Relationship between two variables was evaluated by
288 performing a linear regression test of Pearson (when data normality is respected) otherwise
289 Spearman using the whole data set for each variable. A difference was considered as statistically
290 significant in case of p-value <0.05. A principal component analysis (PCA) was performed to
291 compare biological effects observed in the different exposure conditions (Li concentration and
292 time). The PCA was based on the mean of biomarkers exhibiting significant differences
293 between experimental conditions (in AOV and KW tests) in order to enhance the variance
294 explained by PCA factors (axis) (Lachaux et al., 2023).
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## 3. Results

296 No significant differences were observed between bivalve sex concerning both Li
297 bioaccumulation and biological effects. Consequently, males and females will not be
298 distinguished in the rest of the manuscript.
299

### 3.1. Lithium concentrations in the water and mussels

300 Physicochemical conditions were close between the different tanks and relatively stable
301 along the experiment with an average temperature, pH, conductivity and O₂ concentrations of
302 18.7±0.4 °C, 8.2±0.2, 237±10 µS cm⁻¹ and 9.1±0.2 mg L⁻¹, respectively (Figure S1).
303

303 Concentrations of Li measured show that 100±20 % of Li introduced in the tanks were
304 recovered in the water. Lithium spiking led to an increase of Li concentrations over seven days
305 and was followed by a concentration decrease after medium renewal. This pattern of Li in water
306 was repeated four times along the experiment (between each medium renewal). Average
307 concentrations of Li measured in the water along the experiment (TWMC) were 109, 252 and
308 620 µg L⁻¹ in exposed conditions and 3 µg L⁻¹ in controls. In the rest of the manuscript we will
309 use these TWMC for exposures to 40, 100 and 250 µg L⁻¹ spiking, respectively. Modelling,
310 using Visual Minteq 3.1, predicted that 99.6 % of Li in the water was in the dissolved free ion
311 form for all the treatment concentrations.
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312 Li concentrations measured in control mussels (0.4 ± 0.3 µg g⁻¹) were close to those found
313 in mussels on the field (0.3 ± 0.1 µg g⁻¹). Exposed mussels accumulated significant
314 concentrations of Li compared to controls, in a dose-dependent manner (Figure 2). After 7 days
315 of exposure, Li concentrations in bivalves reached a steady-state, at 2.2, 4.7 and 10.5 µg g⁻¹ dw
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when exposed to 109, 252 and 620 µg L⁻¹ of Li, respectively. Bioaccumulation factors (BAF) at the steady-state were similar regardless Li exposure concentration and was on average 19 L kg⁻¹ (Figure 2). After 7 days of exposure, Li concentrations in algae were 0.5 ± 0.01, 0.8 ± 0.1 and 1.4 ± 0.3 µg g⁻¹ dw when exposed to the same concentrations than bivalves.

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## 3.2. Lithium biological effects

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### 3.2.1. Individual level

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Several effects at individual and subcellular levels have been observed in *D. polymorpha* exposed to Li (Figure 3). At individual level, mussel condition index significantly increased (large effect size) after 14 days of exposure at 109 µg L⁻¹ of Li. Scope for growth (SfG) values were negative for all the experimental groups, but Li exposure induced an increase of SfG values. This shift towards higher SfG in exposed mussels compared to control intensified over time. A significant effect size (very large) on mussels exposed at 109 and 620 µg L⁻¹ Li was highlighted after 28 days of exposure (Figure 3). Total carbohydrate content tends to have a bell-shaped curve characterized by a decrease at t₇ (moderate effect size), then an increase at t₁₄ and t₂₁ (moderate to large effect size) followed by a decrease at t₂₈. Regarding element composition and regulation, Na, Mg and P concentrations were positively correlated to Li concentrations measured in the whole soft tissues of mussels, especially at t₂₁ and t₂₈. On the contrary, Ca, K, S, Cu concentrations in mussel tissues were not significantly affected by Li treatments (Tables S5 and S6).

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### 3.2.2. Subcellular level

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Concerning cellular energy reserves and mitochondrial metabolism, triglyceride contents tend to decrease over time from small to moderate effect size. At t₁₄, protein concentration significantly decreased (very large effect size) while LDH activity increased (large effect size) in the digestive gland of *D. polymorpha* exposed to the higher Li concentration (Figure 3). LDH activity was positively correlated to Li bioaccumulated concentrations. Biomarkers of aerobic metabolism, namely ETS and CS (but not COx), tend to increase over time until reaching a significant effect size (very large) for CS after 28 days of exposure at 109 µg L⁻¹.

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Antioxidant defenses, SOD and TAC, increased after 21 days of exposure at 252 and 109 µg L⁻¹, respectively. TAC was higher at t₂₈ in mussels exposed to 109 µg L⁻¹ (Figure 3). LDH, TAC and SOD levels were positively correlated to Li concentrations in the water and ETS activity and were negatively correlated to PROT and TRIGLY concentrations. Concerning

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cellular damages, CSP-3 activity was significantly lower (very large effect size) in mussels
exposed for seven days to 252 and 620 µg L⁻¹ of Li. Lipid peroxidation in mussels exposed to
the higher Li concentration fluctuated over time, with an increase at t₇ and t₂₈ (moderate effect
sizes) and a decrease at t₁₄ (large effect size). Lithium treatments did not significantly affect
(very small or small effect sizes) lipid content in the whole organism, nor Ca²⁺ATPase and COx
activities (Table S5).

354

3.3.3. Multivariate analysis

355

356

357

358

359

360

361

362

363

364

365

The PCA gathering significant biomarkers illustrates differences in biological effects
between Li exposed and experimental control groups (Figure 4). This distinction appears
mainly along the horizontal axis (representing most of data variance, 37 %) driven by the
subcellular biomarkers TAC and LDH (which are positively correlated to bioaccumulated
concentrations of Li) and in a lesser extent by CSP-3. Differences between exposed and control
groups are visible in a lesser extent along the vertical axis (22 % of data variance) driven by the
individual biomarkers SfG and CI. PCA also shows the increasing difference over time between
exposed and control groups, which is highlighted along the horizontal axis by the subindividual
biomarkers TAC, LDH and in a lesser extent along the vertical axis by SfG. The diagonal from
upper-left to bottom-right shows increasing ETS activity and decreasing CI and PROT
concentrations over time regardless exposure condition (Figure 4).

366

4. Discussion

367

368

369

370

The present work proposes the first assessment of sublethal responses of a freshwater
bivalve exposed to environmental concentrations of Li. Biomarker analysis was associated to
Li quantification in the water and mussel tissues in order to better characterize and understand
Li biological effects.

371

4.1. Lithium concentrations in the water and mussels

372

373

374

375

376

377

378

Lithium concentrations measured in the water corresponded to the amounts added through
Li spiking, which shows the high stability of Li in the tested exposure media. This corroborates
the exclusive presence of Li in the dissolved phase of the water predicted by modelling. Lithium
concentrations measured in the water of exposed conditions (109 – 620 ug L⁻¹) are in line with
those that can be found in rivers under different scenarii of anthropogenic Li contamination
(Choi et al., 2019; EauFrance, 2023; Sobolev et al., 2019). The measured control Li
concentration (3 ug L⁻¹) was close to Li average concentration of French (EauFrance, 2023) and
379
380

US (Kszos and Stewart, 2003) rivers and can thus be considered representative of average natural occurrence of Li in fresh waters.

381
382
383
384
385
386
387
388
389
390
391

Lithium concentrations measured in exposed mussels demonstrate that Li was significantly bioaccumulated (with respect to controls) and thus that Li was bioavailable. This is in line with the predicted occurrence of Li in the free ion form, which is considered as the most bioavailable chemical form of metals (Di Toro et al., 2001). The stability of Li concentration in the water over time suggests that mussels accumulated a small proportion of Li added in tanks and/or that mussels have taken up Li but eliminated it rapidly (fast cycle). Indeed, pharmacokinetic studies conducted in mammal models show that Li is rapidly taken up, distributed among tissues and finally eliminated via kidney with no metabolization nor interaction (Wen et al., 2019). The low concentrations of Li measured in algae indicate that the potential amount of Li accumulated by mussels *via* dietary route was negligible. Consequently, we can assume that Li was mainly accumulated in *D. polymorpha* *via* waterborne exposure.

392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409

To our knowledge, there is only one study that has reported Li concentration in a freshwater bivalve (Pereira et al., 2012). The authors measured 1.41 µg g⁻¹ of Li in the tissues of *Limnoperna fortunei*, collected in a Brazilian river, which is relatively close to the concentrations that we have measured in *D. polymorpha* from control conditions (0.2-0.6 µg g⁻¹). In the present study, Li concentrations accumulated by exposed freshwater mussels (2.2-10.5 µg g⁻¹; BAF = 17-20 L kg⁻¹) were higher than those reported in marine mussels exposed to similar Li concentrations in both laboratory and natural environment (0.4-7.1 µg g⁻¹; BAF = 1.4-10.3 L kg⁻¹) (Barbosa et al., 2023c, 2023a; Perošević et al., 2018; Santos et al., 2023; Thibon et al., 2021a, 2021c; Viana et al., 2020). Such difference can be explained by the lower content of sodium (Na) in fresh waters compared to seawaters, which would lead to a lower competition between Li and Na and thus to a higher bioavailability and bioaccumulation of Li in freshwater systems. Indeed, lithium's proposed mechanism of action involves competing with Na+. It was demonstrated that Li and Na can interact with common transporters (e.g. Na+K+-ATPase pump, Na+/H+ and Ca2+/Na+ exchangers) because of their very close chemical properties including monovalency and electronic configuration (Hesketh et al., 1978; Jakobsson et al., 2017; Thibon et al., 2021a; Ward et al., 1994). Lithium, by entering the cytosol via sodium channels, would accumulate inside the cell lowering the intracellular concentration of Na⁺ (Dudev et al., 2018).

410
411

The concentration-dependent bioaccumulation of Li we observed is a common behaviour of contaminants including Li (Barbosa et al., 2023c; Thibon et al., 2021a). This reflects the
412

strong influence of exposure medium composition on metal bioaccumulation (Allen et al., 1980; de Paiva Magalhães et al., 2015; Lachaux et al., 2022). Lithium bioaccumulation depended on exposure concentration but not on time. Indeed, the Li tissue concentration in exposed mussels appeared to have reached steady-state at or before the first sampling timepoint at seven days. The concentration measured in an organism is a dynamic factor resulting from uptake and loss processes. The chemical properties of lithium make it easier to achieve a steady state (uptake rate equals loss rate) in a maximum of 7 days, as also highlighted in *M. edulis* (Thibon et al., 2021a). These results indicate that *D. polymorpha* regulates Li accumulation depending on Li concentration in the water, and thus suggest the existence of Li homeostasis processes in bivalves. One reason that steady-state conditions were reached in a few days would be the rapid physiological turnover of Li in the organism. Kidneys should be a key organ in the homeostasis of Li (Thibon et al., 2021c) and especially in its elimination (Hedya et al., 2022).

413

414

415

416

417

418

419

420

421

422

423

424

## 4.2. Lithium biological effects

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

During the first 14 days of the experiment, some biomarkers indicated apparently beneficial effects of Li on mussels. The lower CSP-3 activity illustrates a decreased apoptosis in mussels exposed to the highest Li concentrations. The protective effect of Li against cellular death is well described in bipolar patients receiving Li-based treatment as it helps limit neurodegenerative alterations. The antiapoptotic activity of Li is explained by the inhibition of glycogen synthase kinase-3beta by Li, which is an enzyme involved in CSP-3 activation and thus apoptosis induction (Chen et al., 2006; King et al., 2001; Mora et al., 2001). The increased CI is due to a higher mass of mussels exposed to the lower Li concentration compared to controls. It may be linked to the capacity of Li to stimulate weight gain, which is also well documented in bipolar patients treated with Li (Adida et al., 2014; McKnight et al., 2012). An increase of body weight was also reported in the fish *Micropterus salmoides* after Li exposure (Yuan et al., 2022). In the present study, the increased biomass is probably linked to the enhanced filtration rate and food intake (algae) observed in mussels exposed to Li. These effects of Li on CSP-3 activity and CI were transient, diminishing over time and not observable any more after 21 days of exposure.

440

441

442

443

444

All mussels displayed negative SfG values, meaning that available energy for growth and reproduction is low. This can be related to mussel sampling in September, which represents the end of reproduction period when mussels have already used all their energy for gamete production and spawning. However, Li-exposed mussels showed higher SfG than non-exposed mussels. In that case, the energetic balance (i.e. the difference between the energy absorbed
from the food and the energy lost in respiration and excretion) being less negative, exposed
mussels had more energy available for growth and reproduction that is manifest as an increase
in body weight. Indeed, exposed mussels filtered more but lost less energy by respiration. The
trend of increasing mitochondrial respiration (CS, ETS) and carbohydrate content over time
suggest an higher energy demand in bivalves exposed to Li to repare potential tissue damages.
Indeed, an elevation of LDH can also be related to tissue damages (Chan et al., 2013), which
would be in line with the decreased protein concentrations we observed in the digestive gland
of mussels exposed to the higher Li concentration. These results corroborate protein
concentration decrease and protein damages (protein carbonylation) observed in the marine
mollusks, Tritia neritea (Marín Rodríguez et al., 2022) and Venerupis corrugata (Barbosa et
al., 2023a) respectively, exposed to 200-800 µg L⁻¹ of Li. In addition, our results are in line
with protein synthesis inhibition reported in sea urchins embryos after Li exposure (Wolcott,
1982, 1981). Tissue alterations, such as atrophy of digestive alveoli epithelium and digestive
gland degeneration, were reported in M. galloprovincialis exposed to 10 mg L⁻¹ of Li for 21
days (Fraga et al., 2022). Prevalence and intensity of these histopathological effects increased
over time similarly to LDH activity in our study. Although we did not measure LDH activity in
the gills, it is likely that tissue damages occurred also in the gills because it is one of the organ
which most accumulates Li in aquatic animals (Thibon et al., 2023). Alterations of gill tissues
may lead to a deficiency of respiratory functions (e.g. gas fluxes), which could explain the
decreased O₂ consumption observed in Li-exposed mussels. The increased aerobic (CS, ETS)
and anaerobic (LDH) metabolism at cell level would be a way to compensate respiration
deficiency at tissue level.

An elevation of cellular metabolism can have diverse implications on mussel physiology (Impellitteri et al., 2023; Shiry et al., 2023). Firstly, the increased mitochondrial respiration should increase energy (ATP) production, which is in line with the higher P content measured in the tissues of mussels exposed to Li. The increased Na and Mg concentrations in exposed mussels may be explained by an impairment of kidney functions by Li, such as a reduction of glomerular filtration and urine concentration (Adida et al., 2014; McKnight et al., 2012), which would limit Na and Mg elimination. Competitive interactions might also occur between Li,Na and Mg (Aral and Vecchio-Sadus, 2011; Hesketh et al., 1978; Ward et al., 1994) on efflux channels, which could enhance Na and Mg retention in bivalve tissues. Secondly, higher mitochondrial activity may also lead to an increased production of reactive oxygen species (ROS) (Liemburg-Apers et al., 2015; Martyniuk et al., 2024; Nohl et al., 2003). Lithium
exposure can also generate ROS production (Eskandari et al., 2012; Ommati et al., 2021). This
would explain the increasing levels over time of the antioxidant enzymes TAC and SOD in
exposed mussels. It seems that the induction of antioxidant defenses limited cellular damages
except in mussels exposed to 620 µg L⁻¹ for 28 days where lipid peroxidation tend to increase.
Our results are in line with the modulation of antioxidant defenses SOD, catalase, glutathione
peroxidase activities and glutathione contents (GSH/GSSG) reported in marine bivalves
exposed to 100-250 µg L⁻¹ of Li (Barbosa et al., 2023c, 2023a; Viana et al., 2020) and in
freshwater fish after Li exposure (Chandra et al., 2020; El-Tekreti and Yeltekin, 2023).
Moreover, lipid peroxidation was observed in *M. galloprovincialis* exposed from 250 (Santos
et al., 2023) to 750 µg L⁻¹ of Li (Viana et al., 2020) for 28 days.

## 5. Conclusion

The present study revealed that the addition of Li in water, similar to anthropogenic releases, led to increased Li body burden in mussels, reaching higher tissue concentrations than those quantified in marine bivalves exposed to similar Li concentrations. The observed toxicokinetics suggests a tight regulation of Li tissue concentrations by the mussels. The relatively fast and waterborne concentration dependent bioaccumulation of Li by *D. polymorpha* make this species suitable for Li biomonitoring in fresh waters. Its implementation close to potential sources of Li releases such as Li extraction and production plants, electronic waste storage/recycling sites or industrial activities would be useful for Li contamination monitoring and environmental risk assessment. Zebra mussels responses at individual level and subcellular levels revealed that environmentally relevant concentrations of Li caused increased energy demand associated to potential tissue damages illustrated by the higher LDH activity and the decrease of PROT concentrations. Changes of SfG values indicated a modification of energy allocation in Li exposed mussels, which may have repercussions on their ecological functions and affect ecosystem health in a longer-term (> 28 days). Exposed mussels also experienced similar metabolic changes as those reported in bipolar patients receiving Li-based treatment (e.g. weight gain, increased cellular metabolism, decreased apoptosis, induction of antioxidant defenses and cation content modification). Given that patients treated with Li are monitored, since Li can quickly become toxic (narrow therapeutic window), the same care should be taken for the environment. Sublethal responses of *D. polymorpha* were expressed differently throughout the exposure period, which tend to intensify over time. More refined kinetic studies, combining also toxicokinetic (absorption, tissular distribution, cellular fractionation, and elimination) and toxicodynamic (effects) approaches are further needed. A more precise
knowledge of Li uptake and elimination rates in this species would be useful for the
implementation of this species for Li contamination biomonitoring. It would also help also to
decipher Li homeostasis mechanisms and biological targets in freshwater bivalves.

514 Acknowledgements

This work has been supported by the French National Research Agency through the national program "Investissements d'avenir" with the reference ANR-10-LABX-21-01 / LABEX RESSOURCES21. This work was partly done with resources from the Pôle de compétences Chimie Analytique Environnementale and from the Pôle Vivant, ANATELo, LIEC laboratory, UMR 7360 CNRS - Université de Lorraine. We would like to thank also Jean-François Poinsaint from the Pôle de compétences Terrain-Instrumentation (LIEC - ANATELo - Université de Lorraine - CNRS - http://liec.univ-lorraine.fr) for their technical assistance in the field.
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<figure><img src="image_2.png" /><figcaption>Figure 1: Timeline with Li spiking, quantification in the water and collection of bivalves for bioaccumulation and biomarkers measurements.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2:** Bioaccumulation of lithium in *D. polymorpha* as a function of time. Bars represent the mean value of five replicates and vertical bars correspond to standard deviation. Different numbers indicate significant differences between exposure concentrations and different letters show significant differences between exposure times.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3: Effect sizes of the different exposure Li concentrations and duration respectively to control condition.** Symbols and lines represent the mean and 95% confidence interval of effect size. One asterisk indicates a significant effect size compared to control condition (i.e. confidence interval does not include 0). Biomarkers were measured in mussel whole soft tissues, digestive gland (dg) and gills (gi). SfG = Scope for Growth; PROT = protein; TRIGLY = triglycerides; ETS = electron transport system; COx = cytochrome c oxidase; CS = citrate synthase; LDH = lactate dehydrogenase; CaATPase = Ca<sup>++</sup>-ATPase pump; SOD = superoxide dismutase; TAC = total antioxidant capacity; LOOH = lipid hydroperoxides; CSP-3 = caspase-3.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4: Principal component analysis based on biomarkers measured in *D. polymorpha* controls or exposed to three concentrations of lithium for different exposure duration.** Principal component analysis (PCA) was performed on the mean of statistically (ANOVA and/or Kruskal-Wallis tests) significant biomarkers measured in bivalves. Subpanel A corresponds to the distribution of the different experimental groups as a function of biomarker responses and subpanel B represents the correlation circle of variables (biomarkers). Numbers in brackets refer to the percentage of data variance explained by each PCA dimension (axis). PROT = protein; ETS = electron transport system; LDH = lactate dehydrogenase; CI = condition index; SfG = scope for growth; CSP-3 = caspase-3; TAC = total antioxidant capacity; Li<sub>bioacc</sub> = bioaccumulated concentrations of lithium; Biomarkers were measured in bivalve whole tissues, digestive gland (dg), and gills (gi).</figcaption></figure>
<figure><img src="image_6.png" /></figure>

Biological effects:

CSP-3 = caspase-3

PROT = protein content

LDH = lactate dehydrogenase

CI = condition index

SOD = superoxide dismutase

TAC = total antioxidant capacity

SfG = scope for growth

CS = citrate synthase

SfG = scope for growth

Bioaccumulation: [Li]/BAF

µg g⁻¹/L kg⁻¹

CSP-3

PROT

LDH

SfG

10.6/17

CSP-3

SOD

4.7/19

CI

TAC

SfG

CS

TAC

28 days

0

7

14

21

Exposure:

[Li] µg L⁻¹

620

252

109

<figure><img src="image_7.png" /></figure>
**Highlights**

- *D. polymorpha* accumulated Li in a dose-dependent manner (from 2 to 10 ug g⁻¹ dw)

- Bioaccumulation steady-state and similar BAF suggest Li bioaccumulation regulation

- Li increased energy demand, LDH and TAC levels and decreased PROT and CSP levels

- Bivalve responses to Li were comparable to those reported in Li-treated patients

- Sublethal bivalve responses to Li were variable over the 28-days exposure period
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure S1: Temperature (A), dissolved oxygen concentration (B), pH (C) and conductivity (D) evolution along the experiment</figcaption></figure>
<table><caption>**Table S1:** Measurement modes of inductively coupled plasma mass spectrometry (ICP-MS) and most abundant possible interferences</caption><thead><tr><th>Element</th><th>Mass</th><th>Mode(s)</th><th>Most abundant possible polyatomic interferences</th><th>Most abundant possible isobaric interferences</th></tr></thead><tbody><tr><td>Li</td><td>7,016</td><td>SQ-No Gas; SQ-KED; TQ-O2 (On Mass)</td><td>1H + 6Li</td><td>14N++; 15N++</td></tr><tr><td>Na</td><td>22,9898</td><td>SQ-KED; TQ-O2 (On Mass)</td><td>16O + 7Li; 12C + 11B</td><td>46Ti++; 47Ti++</td></tr><tr><td>Mg</td><td>23,985</td><td>SQ-KED; TQ-O2 (On Mass)</td><td>1H + 23Na; 16O + 1H + 7Li</td><td>48Ti++</td></tr><tr><td>P</td><td>30,9738</td><td>TQ-O2 (Mass Shift)</td><td>16O + 1H + 14N</td><td>63Cu++; 62Ni++</td></tr><tr><td>S</td><td>31,9721</td><td>TQ-O2 (Mass Shift)</td><td>16O + 16O; 1H + 31P</td><td>64Zn++; 65Cu++</td></tr><tr><td>K</td><td>38,9637</td><td>SQ-KED; TQ-O2 (On Mass)</td><td>16O + 23Na; 12C + 27Al</td><td>78Se++</td></tr><tr><td>Ca</td><td>43,9555</td><td>TQ-O2 (Mass Shift)</td><td>16O + 1H + 27Al; 12C + 32S; 16O + 28Si</td><td>89Y++; 88Sr++</td></tr><tr><td>Cu</td><td>62,9296</td><td>SQ-KED</td><td>40Ar + 23Na; 12C + 51V</td><td>126Te++</td></tr><tr><td>Rh</td><td>102,9055</td><td>SQ-No Gas; SQ-KED; TQ-O2 (On Mass)</td><td>40Ar + 63Cu</td><td>205Ti ++; 206Pb++</td></tr></tbody></table>

<table><caption>**Table S2:** External calibration inductively coupled plasma mass spectrometry (ICP-MS) quantification with synthetic multi-elemental solutions</caption><thead><tr><th></th><th>Li<br>(µg/L)</th><th>Na<br>(mg/L)</th><th>Mg<br>(mg/L)</th><th>P<br>(mg/L)</th><th>S<br>(mg/L)</th><th>K<br>(mg/L)</th><th>Ca<br>(mg/L)</th><th>Cu<br>(µg/L)</th></tr></thead><tbody><tr><td>Blank</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Standard1</td><td>0,1</td><td>0,01</td><td>0,01</td><td>0,01</td><td>0,01</td><td>0,01</td><td>0,01</td><td>0,1</td></tr><tr><td>Standard2</td><td>1</td><td>0,1</td><td>0,1</td><td>0,1</td><td>0,1</td><td>0,1</td><td>0,1</td><td>1</td></tr><tr><td>Standard3</td><td>10</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>10</td></tr><tr><td>Standard4</td><td>100</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>100</td></tr><tr><td>Standard5</td><td>1000</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>1000</td></tr></tbody></table>

<table><caption>**Table S3:** Accuracy in % (n=4, RSD) estimated for standard reference materials SPS-SW1 and AGLAE interlaboratory surface water. Detection limit estimated with ultrapure water acidified with 2% (v/v) HNO3 (n=10). LD = Mean (blank) + 3 sd (blank)</caption><thead><tr><th>Element</th><th>Detection Limit (µg/L)</th><th>Accuracy ± RSD (AGLAE)</th><th>Accuracy ± RSD (SPS-SW1)</th></tr></thead><tbody><tr><td>Li</td><td>0,0</td><td>104 ± 8,5*</td><td>-</td></tr><tr><td>Na</td><td>15,0</td><td>-</td><td>94,9 ± 8,2</td></tr><tr><td>Mg</td><td>2,0</td><td>-</td><td>98,8 ± 8,7</td></tr><tr><td>P</td><td>10,3</td><td>-</td><td>113,8 ± 10,9</td></tr><tr><td>S</td><td>12,2</td><td>-</td><td>113,6 ± 8,3</td></tr><tr><td>K</td><td>4,7</td><td>-</td><td>109,9 ± 7,9</td></tr><tr><td>Ca</td><td>13,6</td><td>-</td><td>113,8 ± 7,9</td></tr><tr><td>Cu</td><td>0,1</td><td>-</td><td>97,1 ± 8,3</td></tr></tbody></table>

<footnote>* Indicative value only</footnote>
<table><caption>**Table S4: Accuracy in % (n=46 RSD) estimated for standard reference material ERM-CE278k. Detection limit estimated with procedural blanks (n=12). LD = Mean (blank) + 3 sd (blank)**</caption><thead><tr><th>Element</th><th>Detection Limit (µg/g)</th><th>Accuracy ± RSD (ERM-CE278k)</th></tr></thead><tbody><tr><td>Li</td><td>0,1</td><td>-</td></tr><tr><td>Na</td><td>1060,6</td><td>96,2 ± 3,6*</td></tr><tr><td>Mg</td><td>8,9</td><td>108,7 ± 3,8*</td></tr><tr><td>P</td><td>0,7</td><td>-</td></tr><tr><td>S</td><td>2,1</td><td>-</td></tr><tr><td>K</td><td>280,0</td><td>110,3 ± 2,3*</td></tr><tr><td>Ca</td><td>35,0</td><td>118,9 ± 6,4*</td></tr><tr><td>Cu</td><td>0,4</td><td>98,4 ± 2,9</td></tr></tbody></table>

<footnote>*Indicative value only from additional material information</footnote>
<table><caption>**Table S5: Results of biomarker measurements and statistical tests.** The results obtained for the different experimental groups were compared performing analysis of variance (AOV) when normality and homoscedasticity conditions were respected otherwise by performing Kruskal-Wallis (KW) test. Tukey HSD and multiple comparisons tests (for parametric and non-parametric tests, respectively) were conducted as post-hoc tests to identify exactly which groups differ from each other. F and X² values refer to statistical coefficient from parametric and non-parametric tests respectively. A test was considered significant in case of p-value < 0.05. *, ** and *** mean that p-value is lower than 0.05, 0.01 and 0.001 respectively. df = degree of freedom. SD = standard deviation. BCF = bioconcentration factor; Biomarkers were measured in mussel whole soft tissues, digestive gland (dg) and gills (gi). SfG = scope for growth; PROT = protein; TRIGLY = triglycerides; ETS = electron transport system; COx = cytochrome c oxidase; CS = citrate synthase; LDH = lactate dehydrogenase; CaATPase = plasma membrane Ca++-ATPase; SOD = superoxide dismutase; TAC = total antioxidant capacity; LOOH = lipid hydroperoxides; CSP-3 = caspase-3.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">TIME (j)</th><th colspan="10">CONC (µg/L)</th><th colspan="2">CONC (df=3)</th><th colspan="2">TIME (df=4or3)</th><th colspan="2">CONC*TIME (df=8)</th><th rowspan="2">Significant (pvalue&lt;0.05) Post-hoc tests (TukeyHSD or Kruskalmc)</th><th rowspan="2">Pairwise comparison (pairwise t.test or kruskal)</th></tr><tr><th>0</th><th>40</th><th>100</th><th>250</th><th>TOT</th><th>F or KW value</th><th>P-value</th><th>F or KW value</th><th>P-value</th><th>F or KW value</th><th>P-value</th><th>F or KW value</th><th>P-value</th></tr></thead><tbody><tr><td rowspan="5">PROT<sub>gi</sub> (mg/gFW)</td><td>0</td><td>34,2</td><td>8,1</td><td></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>7</td><td>29,9</td><td>8,2</td><td>34,7</td><td>14,7</td><td>28,2</td><td>7,8</td><td>36,0</td><td>5,0</td><td>32,3</td><td>9,4</td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>30,8</td><td>10,2</td><td>31,1</td><td>8,0</td><td>26,9</td><td>8,1</td><td>24,8</td><td>4,7</td><td>28,4</td><td>8,0</td><td>7.5094</td><td>0.1854</td><td>13.922</td><td>0.003013</td><td>t7&gt;t28</td><td>250t7 &gt; 250t28*, 250t7*</td></tr><tr><td>21</td><td>29,3</td><td>6,8</td><td>27,1</td><td>10,7</td><td>34,5</td><td>16,1</td><td>27,9</td><td>9,2</td><td>29,6</td><td>10,9</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>22,5</td><td>7,6</td><td>26,1</td><td>12,2</td><td>22,5</td><td>5,9</td><td>24,1</td><td>6,5</td><td>23,8</td><td>8,1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">PROT<sub>cyte</sub> (mg/gFW)</td><td>TOT</td><td>28,1</td><td>8,5</td><td>29,6</td><td>11,3</td><td>28,0</td><td>10,7</td><td>28,2</td><td>7,9</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>25,9</td><td>7,2</td><td></td><td></td><td></td><td></td><td></td><td></td><td>25,9</td><td>7,2</td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td>23,0</td><td>9,8</td><td>22,1</td><td>9,9</td><td>29,7</td><td>4,9</td><td>23,6</td><td>5,6</td><td>24,5</td><td>8,0</td><td>5.6159</td><td>0.3454</td><td>4.9438</td><td>0.176</td><td>100t28 &lt; 100t7*</td></tr><tr><td>14</td><td>26,4</td><td>2,5</td><td>26,0</td><td>3,9</td><td>24,0</td><td>4,4</td><td>21,7</td><td>2,748</td><td>24,5</td><td>3,8</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>24,2</td><td>2,1</td><td>24,0</td><td>5,1</td><td>26,1</td><td>5,0</td><td>21,2</td><td>3,8</td><td>23,8</td><td>4,3</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">CaATPase (mmoles Pi/gPROT/h)</td><td>TOT</td><td>24,0</td><td>6,8</td><td>21,8</td><td>5,3</td><td>18,0</td><td>7,0</td><td>23,9</td><td>4,4</td><td>21,9</td><td>6,2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>24,4</td><td>6,0</td><td>23,5</td><td>6,2</td><td>24,3</td><td>6,7</td><td>22,6</td><td>4,2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>4,610</td><td>1,227</td><td></td><td></td><td></td><td></td><td></td><td></td><td>4,610</td><td>1,227</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td>4,488</td><td>2,032</td><td>4,868</td><td>1,689</td><td>4,190</td><td>1,213</td><td>4,842</td><td>1,590</td><td>4,602</td><td>1,594</td><td>0.1346</td><td>0.9392</td><td>1.2512</td><td>0.2957</td><td>0.9269</td><td></td></tr><tr><td>14</td><td>4,797</td><td>1,482</td><td>4,598</td><td>0,950</td><td>5,156</td><td>1,823</td><td>4,568</td><td>0,803</td><td>4,780</td><td>1,276</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">TRIGLY (mg/gFW)</td><td>TOT</td><td>4,520</td><td>1,100</td><td>4,019</td><td>1,092</td><td>4,482</td><td>1,311</td><td>3,747</td><td>1,624</td><td>4,176</td><td>1,286</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>4,988</td><td>1,206</td><td>4,509</td><td>1,222</td><td>4,706</td><td>1,811</td><td>5,102</td><td>0,922</td><td>4,826</td><td>1,276</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>4,698</td><td>1,429</td><td>4,485</td><td>1,214</td><td>4,650</td><td>1,526</td><td>4,537</td><td>1,343</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,827</td><td>2,828</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1,827</td><td>2,828</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td>1,840</td><td>3,544</td><td>0,952</td><td>1,191</td><td>0,789</td><td>0,503</td><td>0,836</td><td>0,553</td><td>1,122</td><td>1,903</td><td>5.1345</td><td>0.3997</td><td>6.2964</td><td>0.09805</td><td></td><td></td></tr><tr><td rowspan="5">LOOH (µmol/gPROT)</td><td>TOT</td><td>1,014</td><td>1,466</td><td>0,738</td><td>0,604</td><td>0,670</td><td>0,233</td><td>0,696</td><td>0,775</td><td>0,777</td><td>0,848</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,646</td><td>2,650</td><td>0,860</td><td>0,818</td><td>0,505</td><td>0,861</td><td>0,369</td><td>0,213</td><td>0,845</td><td>1,463</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,632</td><td>2,524</td><td>1,008</td><td>0,974</td><td>0,868</td><td>1,217</td><td>0,880</td><td>1,384</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>8,09</td><td>5,28</td><td></td><td></td><td></td><td></td><td></td><td></td><td>8,09</td><td>5,28</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td>7,45</td><td>4,39</td><td>9,30</td><td>5,87</td><td>8,42</td><td>1,13</td><td>9,59</td><td>2,90</td><td>8,68</td><td>3,82</td><td>3.9972</td><td>0.5498</td><td>1.8287</td><td>0.6087</td><td></td><td></td></tr><tr><td rowspan="5">ETS (mmol O2/gPROT/h)</td><td>TOT</td><td>11,94</td><td>5,13</td><td>10,71</td><td>4,69</td><td>8,80</td><td>1,34</td><td>11,51</td><td>2,00</td><td>9,80</td><td>3,92</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>9,00</td><td>4,50</td><td>9,60</td><td>3,35</td><td>8,66</td><td>2,07</td><td>8,92</td><td>3,83</td><td>9,04</td><td>3,39</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>12,27</td><td>12,54</td><td>7,36</td><td>2,68</td><td>7,66</td><td>1,08</td><td>8,83</td><td>1,74</td><td>9,03</td><td>6,44</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>10,17</td><td>7,34</td><td>9,24</td><td>4,19</td><td>8,38</td><td>1,46</td><td>8,74</td><td>2,77</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,461</td><td>0,603</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1,461</td><td>0,603</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">ETS (mmol O2/gPROT/h)</td><td>TOT</td><td>1,796</td><td>0,738</td><td>1,783</td><td>0,692</td><td>1,336</td><td>0,225</td><td>1,861</td><td>0,892</td><td>1,704</td><td>0,686</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,569</td><td>0,270</td><td>1,418</td><td>0,324</td><td>1,680</td><td>0,347</td><td>1,763</td><td>0,203</td><td>1,607</td><td>0,304</td><td>15.51</td><td>0.00839</td><td>5.7022</td><td>0.127</td><td>250&gt;Field</td><td>100t7 &lt; 100t28*</td></tr><tr><td>TOT</td><td>1,398</td><td>0,147</td><td>1,613</td><td>0,284</td><td>1,522</td><td>0,241</td><td>1,558</td><td>0,274</td><td>1,521</td><td>0,242</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,626</td><td>0,487</td><td>1,802</td><td>0,541</td><td>2,112</td><td>0,629</td><td>1,942</td><td>0,541</td><td>1,871</td><td>0,551</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,597</td><td>0,464</td><td>1,651</td><td>0,483</td><td>1,675</td><td>0,476</td><td>1,773</td><td>0,532</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="3"></th><th rowspan="3">TIME (j)</th><th colspan="12">CONC (µg/L)</th><th colspan="2">CONC (df=3)</th><th colspan="2">TIME (df=4or3)</th><th colspan="2">CONC*TIME (df=8)</th><th rowspan="3">Significant (pvalue&lt;0.05) Post-hoc tests (TukeyHSD or Kruskalmc)</th><th rowspan="3">Pairwise comparison (pairwise t.test or kruskal)</th></tr><tr><th colspan="2">0</th><th colspan="2">40</th><th colspan="2">100</th><th colspan="2">250</th><th colspan="2">TOT</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td rowspan="5">LDH (µmolNADH/gP ROT/h)</td><td>0</td><td>254</td><td>55</td><td></td><td></td><td></td><td></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>7</td><td>238</td><td>62</td><td>216</td><td>24</td><td>231</td><td>25</td><td>233</td><td>69</td><td>230</td><td>49</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>224</td><td>48</td><td>242</td><td>38</td><td>248</td><td>28</td><td>291</td><td>52</td><td>251</td><td>47</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>245</td><td>30</td><td>289</td><td>54</td><td>266</td><td>71</td><td>258</td><td>44</td><td>264</td><td>51</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>239</td><td>50</td><td>281</td><td>67</td><td>276</td><td>43</td><td>281</td><td>62</td><td>269</td><td>56</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>237</td><td>47</td><td>258</td><td>55</td><td>256</td><td>47</td><td>265</td><td>58</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">TAC (µmolTEq/gPR OT/min)</td><td>0</td><td>36,8</td><td>3.8</td><td></td><td></td><td></td><td></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>7</td><td>40,1</td><td>6.2</td><td>35,3</td><td>4.7</td><td>40,7</td><td>9.5</td><td>38,8</td><td>5.4</td><td>38,8</td><td>1.9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>35,4</td><td>4.8</td><td>36,9</td><td>6.7</td><td>38,3</td><td>7.7</td><td>40,1</td><td>8.6</td><td>37,7</td><td>1.0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>34,9</td><td>1.8</td><td>40,1</td><td>3.9</td><td>39,8</td><td>6.9</td><td>43,0</td><td>9.8</td><td>39,7</td><td>6.9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>36,8</td><td>6.0</td><td>46,9</td><td>8.1</td><td>44,5</td><td>7.3</td><td>46,5</td><td>13.4</td><td>43,9</td><td>9.6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>36,9</td><td>5.2</td><td>40,0</td><td>7.7</td><td>40,8</td><td>7.3</td><td>42,1</td><td>9.7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">COx (U/gPROT/min)</td><td>0</td><td>10,2</td><td>2.2</td><td></td><td></td><td></td><td></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>7</td><td>10,4</td><td>7.6</td><td>7,5</td><td>5.1</td><td>7,5</td><td>5.1</td><td>11,2</td><td>7.7</td><td>9,3</td><td>6.4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>7,2</td><td>1.7</td><td>10,1</td><td>3.2</td><td>9,0</td><td>3.7</td><td>8,6</td><td>4.0</td><td>8,7</td><td>3.3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>9,2</td><td>3.2</td><td>9,9</td><td>3.2</td><td>8,5</td><td>2.1</td><td>9,9</td><td>3.0</td><td>9,4</td><td>2.8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>9,4</td><td>5.8</td><td>7,2</td><td>2.3</td><td>9,5</td><td>5.3</td><td>8,2</td><td>2.9</td><td>8,6</td><td>4.2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>9,0</td><td>5.0</td><td>8,7</td><td>3.6</td><td>8,7</td><td>4.0</td><td>9,5</td><td>4.7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">CS (U/gPROT/min)</td><td>0</td><td>138,2</td><td>29.3</td><td></td><td></td><td></td><td></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>7</td><td>154,2</td><td>50.9</td><td>127,0</td><td>23.4</td><td>127,2</td><td>22.0</td><td>137,1</td><td>31.8</td><td>137,1</td><td>34.6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>141,5</td><td>25.3</td><td>139,6</td><td>30.0</td><td>144,6</td><td>39.0</td><td>128,2</td><td>45.2</td><td>138,5</td><td>34.3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>135,3</td><td>19.9</td><td>144,6</td><td>26.9</td><td>126,1</td><td>30.4</td><td>139,4</td><td>28.0</td><td>136,5</td><td>26.1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>133,9</td><td>20.8</td><td>161,4</td><td>18.4</td><td>137,3</td><td>37.4</td><td>167,5</td><td>50.9</td><td>150,0</td><td>35.8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>141,2</td><td>31.1</td><td>143,8</td><td>26.7</td><td>134,0</td><td>32.3</td><td>142,9</td><td>40.3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">SOD (nomEqSOD/g PROT/min)</td><td>0</td><td>61,4</td><td>18.4</td><td></td><td></td><td></td><td></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>7</td><td>66,3</td><td>17.0</td><td>66,6</td><td>22.5</td><td>51,7</td><td>5.7</td><td>62,5</td><td>15.2</td><td>62,0</td><td>16.4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>59,5</td><td>20.6</td><td>51,4</td><td>6.8</td><td>68,1</td><td>23.5</td><td>57,6</td><td>15.4</td><td>59,2</td><td>17.8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>53,0</td><td>9.9</td><td>68,7</td><td>16.3</td><td>76,5</td><td>20.1</td><td>64,2</td><td>13.7</td><td>65,6</td><td>16.8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>58,4</td><td>15.9</td><td>72,1</td><td>25.5</td><td>61,1</td><td>26.0</td><td>74,0</td><td>22.3</td><td>66,4</td><td>22.6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>59,3</td><td>16.1</td><td>64,6</td><td>19.7</td><td>64,8</td><td>21.6</td><td>64,6</td><td>17.0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">CSP3 (µmolpNa/gPR OT/h)</td><td>0</td><td>1,561</td><td>0.440</td><td></td><td></td><td></td><td></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>7</td><td>7,872</td><td>3.932</td><td>7,216</td><td>3.094</td><td>3,339</td><td>1,826</td><td>1,875</td><td>0.772</td><td>4,722</td><td>3.417</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>1,783</td><td>1.082</td><td>4,074</td><td>6.203</td><td>2,250</td><td>1,185</td><td>0,799</td><td>0.172</td><td>2,340</td><td>1.690</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>0,943</td><td>0.592</td><td>3,026</td><td>2.668</td><td>1,280</td><td>0.886</td><td>1,637</td><td>1.353</td><td>1,719</td><td>1.690</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>2,331</td><td>2.617</td><td>1,645</td><td>1.550</td><td>2,674</td><td>2.059</td><td>1,900</td><td>1.970</td><td>2,138</td><td>2.006</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>2,875</td><td>3.307</td><td>3,742</td><td>4.107</td><td>2,354</td><td>1,656</td><td>1,612</td><td>1.304</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">SfG (U/g/h)</td><td>0</td><td>-64</td><td>112</td><td></td><td></td><td></td><td></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>7</td><td>-100</td><td>110</td><td>-88</td><td>95</td><td>-56</td><td>38</td><td>-49</td><td>35</td><td>-74</td><td>77</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>-113</td><td>113</td><td>-86</td><td>45</td><td>-85</td><td>55</td><td>-60</td><td>81</td><td>-84</td><td>75</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>-149</td><td>99</td><td>-72</td><td>85</td><td>-39</td><td>26</td><td>-87</td><td>66</td><td>-87</td><td>81</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>-154</td><td>114</td><td>-42</td><td>39</td><td>-140</td><td>66</td><td>-24</td><td>37</td><td>-88</td><td>90</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>-130</td><td>106</td><td>-71</td><td>70</td><td>-79</td><td>60</td><td>-56</td><td>61</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">Carbohydrate s</td><td>0</td><td>8,0</td><td>3.7</td><td></td><td></td><td></td><td></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>7</td><td>11,6</td><td>6.8</td><td>8,2</td><td>3.7</td><td>8,6</td><td>3.9</td><td>6,9</td><td>4.1</td><td>8,8</td><td>4.8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>6,0</td><td>4.1</td><td>9,1</td><td>6.9</td><td>10,1</td><td>4.1</td><td>6,5</td><td>3.5</td><td>7,9</td><td>4.9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>5,3</td><td>1.7</td><td>11,7</td><td>7.9</td><td>8,5</td><td>7.1</td><td>9,0</td><td>5.3</td><td>8,8</td><td>6.2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>9,8</td><td>7.4</td><td>7,8</td><td>3.8</td><td>5,4</td><td>2.7</td><td>8,6</td><td>3.4</td><td>8,0</td><td>4.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>TOT</td><td>8,2</td><td>5.7</td><td>9,2</td><td>5.8</td><td>8,5</td><td>4.9</td><td>7,8</td><td>4.1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="3"></th><th rowspan="3">TIME (j)</th><th colspan="12">CONC (µg/L)</th><th colspan="2">CONC (df=3)</th><th colspan="2">TIME (df=4or3)</th><th colspan="2">CONC*TIME (df=8)</th><th rowspan="3">Significant (pvalue&lt;0.05) Post-hoc tests (TukeyHSD or Kruskalmc)</th><th rowspan="3">Pairwise comparison (pairwise t.test or kruskal)</th></tr><tr><th colspan="2">0</th><th colspan="2">40</th><th colspan="2">100</th><th colspan="2">250</th><th colspan="2">TOT</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th><th rowspan="2">F or KW value</th><th rowspan="2">P-value</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td rowspan="6">Lipids</td><td>0</td><td>2,2</td><td>0,4</td><td></td><td></td><td></td><td></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>7</td><td>1,8</td><td>0,4</td><td>1,9</td><td>0,4</td><td>2,0</td><td>0,4</td><td>1,8</td><td>0,5</td><td>1,9</td><td>0,4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>2,2</td><td>1,0</td><td>1,9</td><td>0,4</td><td>2,2</td><td>0,4</td><td>2,1</td><td>0,3</td><td>2,1</td><td>0,6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>2,1</td><td>0,8</td><td>2,5</td><td>0,8</td><td>2,4</td><td>0,7</td><td>2,1</td><td>0,6</td><td>2,3</td><td>0,7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>2,0</td><td>0,9</td><td>2,2</td><td>0,4</td><td>2,0</td><td>0,6</td><td>2,3</td><td>0,6</td><td>2,1</td><td>0,6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>2,0</td><td>0,8</td><td>2,1</td><td>0,6</td><td>2,2</td><td>0,5</td><td>2,1</td><td>0,5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">IC (mg/mm³)</td><td>0</td><td>0,087</td><td>0,022</td><td></td><td></td><td></td><td></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>7</td><td>0,080</td><td>0,016</td><td>0,082</td><td>0,013</td><td>0,074</td><td>0,012</td><td>0,079</td><td>0,017</td><td>0,079</td><td>0,015</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>0,070</td><td>0,022</td><td>0,087</td><td>0,012</td><td>0,081</td><td>0,012</td><td>0,075</td><td>0,016</td><td>0,078</td><td>0,017</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>0,072</td><td>0,013</td><td>0,082</td><td>0,014</td><td>0,076</td><td>0,015</td><td>0,078</td><td>0,019</td><td>0,077</td><td>0,016</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>0,071</td><td>0,014</td><td>0,072</td><td>0,013</td><td>0,076</td><td>0,015</td><td>0,073</td><td>0,015</td><td>0,073</td><td>0,014</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>0,073</td><td>0,017</td><td>0,081</td><td>0,014</td><td>0,077</td><td>0,014</td><td>0,076</td><td>0,016</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Na (mg/g)</td><td>7</td><td>1,652</td><td>0,275</td><td>1,844</td><td>1,035</td><td>1,766</td><td>0,193</td><td>2,140</td><td>0,502</td><td>1,851</td><td>0,580</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>1,768</td><td>0,452</td><td>2,322</td><td>1,061</td><td>1,738</td><td>0,241</td><td>1,802</td><td>1,139</td><td>1,908</td><td>0,792</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>1,866</td><td>0,585</td><td>1,532</td><td>0,403</td><td>2,136</td><td>0,810</td><td>2,533</td><td>0,542</td><td>1,989</td><td>0,664</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>2,804</td><td>0,919</td><td>2,476</td><td>0,373</td><td>2,748</td><td>0,920</td><td>2,442</td><td>0,587</td><td>2,618</td><td>0,696</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>2,023</td><td>0,727</td><td>2,044</td><td>0,822</td><td>2,097</td><td>0,715</td><td>2,213</td><td>0,746</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Mg (mg/g)</td><td>7</td><td>1,166</td><td>0,043</td><td>1,082</td><td>0,160</td><td>1,152</td><td>0,128</td><td>1,152</td><td>0,102</td><td>1,138</td><td>0,112</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>1,284</td><td>0,074</td><td>1,348</td><td>0,136</td><td>1,306</td><td>0,142</td><td>1,314</td><td>0,270</td><td>1,313</td><td>0,159</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>1,160</td><td>0,155</td><td>1,250</td><td>0,119</td><td>1,310</td><td>0,176</td><td>1,438</td><td>0,082</td><td>1,282</td><td>0,163</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>1,384</td><td>0,108</td><td>1,364</td><td>0,067</td><td>1,406</td><td>0,144</td><td>1,328</td><td>0,098</td><td>1,371</td><td>0,103</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>1,249</td><td>0,134</td><td>1,261</td><td>0,163</td><td>1,294</td><td>0,165</td><td>1,301</td><td>0,180</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">P (mg/g)</td><td>7</td><td>9,874</td><td>2,946</td><td>9,118</td><td>1,816</td><td>8,720</td><td>3,785</td><td>9,380</td><td>3,320</td><td>9,273</td><td>2,836</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>12,908</td><td>0,595</td><td>12,620</td><td>1,433</td><td>12,560</td><td>0,790</td><td>12,916</td><td>1,024</td><td>12,751</td><td>0,942</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>10,282</td><td>3,210</td><td>10,248</td><td>2,974</td><td>10,884</td><td>4,166</td><td>11,968</td><td>4,607</td><td>10,786</td><td>3,480</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>13,486</td><td>0,772</td><td>12,366</td><td>1,026</td><td>14,746</td><td>1,839</td><td>14,314</td><td>0,732</td><td>13,728</td><td>1,428</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>11,638</td><td>2,612</td><td>11,088</td><td>2,338</td><td>11,728</td><td>3,558</td><td>12,154</td><td>3,153</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">S (mg/g)</td><td>7</td><td>10,916</td><td>1,860</td><td>10,212</td><td>1,873</td><td>10,164</td><td>2,051</td><td>9,960</td><td>1,976</td><td>10,313</td><td>1,820</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>9,896</td><td>0,395</td><td>10,184</td><td>0,979</td><td>9,774</td><td>0,588</td><td>10,290</td><td>1,305</td><td>10,036</td><td>0,844</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>11,296</td><td>2,438</td><td>11,276</td><td>2,940</td><td>11,680</td><td>1,687</td><td>10,935</td><td>0,584</td><td>11,316</td><td>2,000</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>10,142</td><td>1,418</td><td>10,206</td><td>1,361</td><td>10,946</td><td>0,715</td><td>10,860</td><td>0,712</td><td>10,539</td><td>1,081</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>10,563</td><td>1,665</td><td>10,470</td><td>1,838</td><td>10,641</td><td>1,494</td><td>10,489</td><td>1,260</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">K (mg/g)</td><td>7</td><td>2,944</td><td>0,407</td><td>2,564</td><td>0,747</td><td>2,792</td><td>0,843</td><td>3,220</td><td>0,219</td><td>2,880</td><td>0,610</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>3,122</td><td>0,429</td><td>2,360</td><td>1,347</td><td>3,108</td><td>0,050</td><td>3,030</td><td>0,636</td><td>2,905</td><td>0,782</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>2,788</td><td>0,942</td><td>3,006</td><td>0,334</td><td>2,816</td><td>0,736</td><td>3,548</td><td>0,589</td><td>3,013</td><td>0,699</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>3,460</td><td>0,519</td><td>3,334</td><td>0,261</td><td>3,762</td><td>0,581</td><td>3,576</td><td>0,782</td><td>3,533</td><td>0,545</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>3,079</td><td>0,619</td><td>2,816</td><td>0,830</td><td>3,120</td><td>0,705</td><td>3,333</td><td>0,591</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Ca (mg/g)</td><td>7</td><td>13,718</td><td>10,186</td><td>10,326</td><td>11,541</td><td>4,234</td><td>1,357</td><td>7,058</td><td>7,014</td><td>8,834</td><td>8,596</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>5,410</td><td>3,689</td><td>8,120</td><td>4,525</td><td>5,448</td><td>6,502</td><td>5,338</td><td>3,283</td><td>6,079</td><td>4,451</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>4,782</td><td>3,004</td><td>4,508</td><td>3,137</td><td>11,640</td><td>4,819</td><td>4,878</td><td>2,450</td><td>6,535</td><td>4,494</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>6,646</td><td>3,553</td><td>11,742</td><td>17,456</td><td>11,666</td><td>11,289</td><td>7,008</td><td>5,904</td><td>9,266</td><td>10,356</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>7,639</td><td>6,535</td><td>8,674</td><td>10,316</td><td>8,247</td><td>7,308</td><td>6,133</td><td>4,801</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="6">Cu (µg/g)</td><td>7</td><td>18,929</td><td>6,701</td><td>12,340</td><td>2,644</td><td>10,892</td><td>6,001</td><td>11,838</td><td>6,147</td><td>13,500</td><td>6,090</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td>9,838</td><td>1,572</td><td>7,048</td><td>1,170</td><td>17,072</td><td>9,794</td><td>13,988</td><td>8,737</td><td>11,987</td><td>7,250</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td>10,234</td><td>4,402</td><td>12,812</td><td>4,612</td><td>10,198</td><td>3,793</td><td>11,843</td><td>3,170</td><td>11,242</td><td>3,906</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>28</td><td>13,084</td><td>8,376</td><td>14,564</td><td>5,653</td><td>8,688</td><td>1,480</td><td>8,046</td><td>0,616</td><td>11,096</td><td>5,497</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>TOT</td><td>13,021</td><td>6,536</td><td>11,691</td><td>4,610</td><td>11,713</td><td>6,482</td><td>11,407</td><td>5,675</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S6: Correlation between Li and element concentrations measured in the whole soft tissues of *D. polymorpha*.** Values correspond to the correlation coefficient (r) and the associated p-value determined with Pearson or Spearman correlation tests. For each time (t<sub>x</sub>), the data from all tested concentrations (0, 40, 100, 250 µg L<sup>-1</sup>) was used to run/perform correlation test.</caption><thead><tr><th rowspan="3"></th><th colspan="12">Li</th></tr><tr><th colspan="2">t<sub>7</sub></th><th colspan="2">t<sub>14</sub></th><th colspan="2">t<sub>21</sub></th><th colspan="2">t<sub>28</sub></th><th colspan="2">total (t<sub>7-28</sub>)</th></tr><tr><th>r</th><th>pvalue</th><th>r</th><th>pvalue</th><th>r</th><th>pvalue</th><th>r</th><th>pvalue</th><th>r</th><th>pvalue</th></tr></thead><tbody><tr><td>Na</td><td>0.39</td><td>0.090</td><td>-0.03</td><td>0.898</td><td><strong>0.47</strong></td><td><strong>0.041</strong></td><td>0.14</td><td>0.569</td><td>0.22</td><td>0.053</td></tr><tr><td>Mg</td><td>0.19</td><td>0.429</td><td>0.11</td><td>0.654</td><td><strong>0.49</strong></td><td><strong>0.032</strong></td><td>0.04</td><td>0.870</td><td><strong>0.24</strong></td><td><strong>0.031</strong></td></tr><tr><td>P</td><td>-0.05</td><td>0.826</td><td>-0.13</td><td>0.578</td><td>0.27</td><td>0.270</td><td><strong>0.48</strong></td><td><strong>0.032</strong></td><td>0.20</td><td>0.067</td></tr><tr><td>S</td><td>-0.21</td><td>0.369</td><td>0.27</td><td>0.253</td><td>0.25</td><td>0.304</td><td>0.32</td><td>0.174</td><td>0.07</td><td>0.528</td></tr><tr><td>K</td><td>0.29</td><td>0.219</td><td>-0.13</td><td>0.587</td><td>0.12</td><td>0.618</td><td>0.26</td><td>0.267</td><td>0.16</td><td>0.150</td></tr><tr><td>Ca</td><td>-0.40</td><td>0.081</td><td>-0.22</td><td>0.362</td><td>0.34</td><td>0.154</td><td>-0.10</td><td>0.682</td><td>0.01</td><td>0.912</td></tr><tr><td>Cu</td><td>-0.39</td><td>0.092</td><td>0.13</td><td>0.578</td><td>-0.10</td><td>0.680</td><td>-0.37</td><td>0.111</td><td>-0.17</td><td>0.140</td></tr></tbody></table>