# Internal handling, shell accumulation and effects of cobalt on the gastropod Ampullaceana balthica

Maëva Marimoutou, Gilles Bareille, Mathieu Milhe-Poutingon, Clémentine

Gelber, Patrick Baldoni-Andrey, Nicholas Bagger Gurieff, Hélène Tabouret,

Sandra Mounicou, Séverine Le Faucheur

► To cite this version:

Maëva Marimoutou, Gilles Bareille, Mathieu Milhe-Poutingon, Clémentine Gelber, Patrick Baldoni-Andrey, et al.. Internal handling, shell accumulation and effects of cobalt on the gastropod Ampullaceana balthica. Aquatic Toxicology, 2026, 291, pp.107688. 10.1016/j.aquatox.2025.107688. hal-05440243

HAL Id: hal-05440243

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# Internal handling, shell accumulation and effects of cobalt on the gastropod
## *Ampullaceana balthica*

Maëva Marimoutou<sup>a,*</sup>, Gilles Bareille<sup>a</sup>, Mathieu Milhe-Poutingon<sup>a</sup>, Clémentine Gelber<sup>b</sup>, Patrick Baldoni-Andrey<sup>b</sup>, Nicholas Bagger Gurieff<sup>c</sup>, Hélène Tabouret<sup>a</sup>, Sandra Mounicou<sup>a</sup>, Séverine Le Faucheur<sup>a</sup>

ARTICLE INFO

Keywords:

Metal

Molluscs

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Subcellular distribution

A B S T R A C T

Cobalt (Co) is central to the development of new low-carbon energies, becoming an element of emerging concern for the environment. Gastropods are widely distributed in aquatic ecosystems and represent the largest group of the phylum Mollusca. However very few studies have focused on Co impacts on gastropods. We thus aimed to examine the internal handling of Co in gastropods and its excretion into the shell in order to better understand its toxic actions and how these organisms cope with Co stress. To that end, *Ampullaceana balthica* was exposed to increasing Co concentrations (6, 30 and 60 µg·L⁻¹) using outdoor flow-through mesocosms for 28 days in June and October. They were analyzed for their growth, reproduction and Co accumulation in the soft bodies and the outer shell part every week. Cobalt distribution in soft tissues and its subcellular distribution were further examined.

Cobalt induced a decrease in the egg mass number and a delay in their hatching. Its accumulation in the soft tissue and the outer shell exhibited distinct seasonal patterns. Linear relationships with the ambient Co concentrations were only evident in the soft tissues in June and in the shell in October. For both periods, Co was mainly distributed in the hepatopancreas and the mantle, particularly in their mitochondria and granules fractions. We thus discussed the possible pathways of Co from the soft tissues to the shell involving granules. The present results demonstrate that using shells as bioindicators of metal exposure has certain limitations, depending on the organism life cycle.

1. Introduction

Cobalt (Co) is a critical mineral for industries, contributing to the energy transition towards low carbon energy sources and enhancing the performance of magnetic alloys and rechargeable batteries (Stubblefield et al., 2020). Its demand and extraction have thus risen by 95 % between 2008 and 2018 (U.S Geological Survey, 2018). In aquatic environments, dissolved background Co concentrations have been estimated to be between <0.02 to 0.43 µg·L⁻¹ in major rivers (Stubblefield et al., 2020). Nevertheless, in surface waters contaminated by mining activities such as in North America and Africa, Co concentrations can reach up to 2028 µg·L⁻¹ (Banza Lubaba Nkulu et al., 2018; Environment Canada, 2017; Mebane et al., 2015). No environmental quality criteria currently exist

for aquatic environment in Europe but a Predicted No Effect Concentration (PNEC) for fresh water has been established at 1.06 µg·L⁻¹ while a provisional concentration at 1.0 µg·L⁻¹ is available in Canada (Environment Canada, 2017; European Chemical Agency, 2023). To establish these protective environmental criteria, data on Co effects on aquatic organisms, including invertebrates, are still required whereas biomonitoring tools to assess its impacts on aquatic ecosystems would be of interest to protect them.

Among freshwaters organisms, gastropods represent the largest group of the phylum Mollusca. They have an important ecological role as primary consumers and as sources of food for secondary and tertiary consumers such as fish. Molluscs are widely known to accumulate metals to which they are sensitive (De Schamphelaere et al., 2008; Grosell et al.,

<footnote><sup>a</sup> Université de Pau et des Pays de l'Adour, E2S-UPPA, CNRS, IPREM, Pau, France</footnote>

<footnote><sup>b</sup> TotalEnergies OneTech Pole d'Études et de Recherche, Lacq, France</footnote>

<footnote><sup>c</sup> Rio Tinto Closure, France</footnote>

<footnote>* Corresponding author at: Technopôle Helioparc, 2 Av., 411 du Président Pierre Angot, 64053 Pau Cedex 9, Séverine Le Faucheur - Université de Pau et des Pays de l'Adour, IPREM, France.</footnote>

<footnote>E-mail addresses: mae.marimoutou@gmail.com (M. Marimoutou), severine.le-faucheur@univ-pau.fr (S.L. Faucheur).</footnote>
2006; Schlekat et al., 2010). For example, *Lymnea stagnalis* has been demonstrated to be one of the most sensitive species to metals such as Co, lead (Pb) and nickel (Ni) (*De Schamphelaere et al.*, 2008; Grosell et al., 2006; Mattsson and Crémazy, 2023). Effects on its growth were observed at 79 µg·L⁻¹ Co when exposed for 28 days. Nevertheless, very few studies have investigated the toxicity of Co on molluscs, and even fewer on native species from freshwater environment and at Co environmental concentrations.

Metal accumulation in organisms most commonly results into toxicity, which are highly dependent on several factors, such as metal bioavailability, the physico-chemical parameters of ambient water as well as biological processes (Ardestani et al., 2014; Crémazy et al., 2019; Rainbow, 2002). The free metal concentration, as well as the pH, cations (including hardness) and dissolved organic carbon (DOC) of ambient water are well-known parameters to control metal bioaccumulation (Ardestani et al., 2014; Niyogi and Wood, 2004). The nature of metals (essential or not) has also been reported to influence their accumulation and regulation processes (Marigomez et al., 2002; Rainbow, 2002). Cobalt is an essential metal as a major constituent of the vitamin B12, which plays a role in growth and development mechanisms, but at some concentration levels, could lead to toxic effects on organisms (De Schamphelaere et al., 2008; Maxwell, 1951; Vogeler et al., 2022). Metal distribution among tissues varies greatly on the metal and species. In molluscs, they are mainly stored in the gills, mantle and foot when exposed to the dissolved metal forms, and in the digestive glands when exposed to metals under their particulate forms (Marigomez et al., 2002). Nevertheless, oral ingestion of water is also a possible metal uptake route in gastropods, so that metal can also be found in the digestive glands. As such, higher amounts of Cd and Zn were observed in the digestive tract compared to the other organs (foot, mantle and the remaining tissues) in *L. stagnalis* exposed to dissolved metal forms for a short period of time (2–3 h) (Crémazy et al., 2019). To our knowledge, there are no data on the distribution of Co in soft tissues of gastropods that would enable us to assess its regulation and effects on these organisms.

Once accumulated in these organs, at the subcellular level, metals can be distributed into two categories as a function of their physiological roles, i.e., the biologically detoxified metals (BDM) and metal sensitive fractions (MSF). Metallothioneins and metal-rich granules (MRG) both belong to the BDM fractions as mechanisms used by organisms to cope with metal stress (Ma and Wang, 2020; Rosabal et al., 2014; Wallace et al., 2003). Metallothioneins have been demonstrated to regulate metals in aquatic invertebrates (Amiard et al., 2006). Metal-rich granules are mainly located in the outer mantle epithelium and are an important source of rapidly mobilizable Ca to be used for shell formation (Marin et al., 2012; Widad, 2018). In the freshwater bivalve *Anodonta woodiana* exposed to Cd, MRG were the major fraction for Cd accumulation (Chen et al., 2021b). Once metals are complexed to these intracellular ligands, the shell could be used as a potential storage pathway (Marin et al., 2012; Widad, 2018). Indeed, metals accumulated in MRG of the mantle could be released by exocytosis and incorporated into the shell by substituting Ca through pumps during shell formation (Marigomez et al., 2002; Marin et al., 2012; Schöne and Krause, 2016). However, little is known about Co bioaccumulation, compartment exchanges, especially between soft tissue and shell, and processes developed by molluscs to limit metal toxicity.

The present study aimed to examine (1) the internal handling of Co in adult gastropods *A. balthica* and (2) the role of shell in Co sequestration in order to better understand Co toxicity towards gastropods and to assess the potential use of shells as biomonitoring tool. Our working hypotheses were as follows: (1) Co would be distributed among several compartments, as the organisms were exposed to Co through both food and water. The subcellular distribution would differ according to the concentrations of Co exposure and, consequently, toxicity. (2) An increase in Co concentrations in soft tissue would lead to an increase in Co in the shell, as it would be used as a sequestration pathway. We

anticipated that the concentration and duration of Co exposure would control Co accumulation in the shell rather than environmental conditions such as temperature. To verify these hypotheses, the gastropod *A. balthica* was exposed in outdoor mesocosms (PERL, TotalEnergies, France) to increasing Co concentrations (6, 30 and 60 µg·L⁻¹) during 28 days in June and October 2021 and Co partitioning in soft tissues and shell were investigated.

## 2. Material and methods

### 2.1. Experimental conditions

Two experiments in mesocosms were conducted during 28 days in June (spring) and October (autumn) 2021 at the Pilots Rivers platform of PERL (Pole d'Étude et de Recherche de Lacq, TotalEnergies, Lacq, France). These outdoor mesocosms includes twelve artificial streams (40 m long, 50 cm wide and deep, 7.5 m³·h⁻¹ flow rate) continuously filled with natural water from the Gave de Pau River. *Ampullaceana balthica*, the species of interest, is a native gastropod living naturally on site. It is representative of freshwater lentic environments such as lakes, ponds, streams and slow-flowing rivers. It is sessile, ubiquitous and present all over Europe with lifespan of one year and known to accumulate metals (Chen et al., 2021b; Lebreton, 2020).

The day before the exposure experiments, a total of 360 adult organisms were collected by hand in the water distribution structure (Fig. 1). Their average shell size was 8.1 ± 0.5 mm length and 5.3 ± 0.3 mm width in June, and 7.9 ± 0.9 mm length and 5.1 ± 0.5 mm width in October. Thirty of them were then placed in plastic boxes (39 × 29 × 26 cm), which were pierced lengthwise on both sides and closed with fishing net to allow water circulation without losing individuals. A total of twelve plastic boxes were prepared in order to have 3 boxes containing 30 organisms per exposure condition. Three exposure concentrations (nominal concentrations of 6, 30 and 60 µg·L⁻¹ Co) and one condition without added Co (control) were tested; they are referred as C6, C30, C60 and C0 respectively in the following sections. Three solutions of Co (cobalt(II) chloride hexahydrate, 98 %, Thermo Scientific Chemicals) at 1.09 × 10⁻², 5.43 × 10⁻² and 1.09 × 10⁻¹ M were prepared and injected using a piston pump while dispersed in the water using shearing valves (Netzsch, Nemo, Germany) one day before the experiment. The injections were continuous (71 mL·h⁻¹) during the experiment in order to maintain the Co concentrations (C6, C30, C60). In June, only 1 stream per condition (4 streams in total) was used with 3 boxes per stream due to experimental limitations. In October, the experiment was repeated with 3 streams per condition (12 streams in total) with 1 box per stream was used. Every 7, 14, 21 and 28 days of exposure (later referred as D7, D14, D21 and D28), 12 organisms (three gastropods per exposure day per box) were collected for each condition to be sacrificed for both soft tissues and shell analyses.

### 2.2. Water analysis

Water physicochemical parameters (temperature, conductivity, pH and dissolved oxygen) were monitored at each sampling day using a HACH HQ40D multiparameter field probe (HACH, Düsseldorf, Germany). Water was sampled (10 mL) using 0.45 µm polyethersulfone filter (Minisart® NML Syringue Filter, Sartorius) and collected in metal-free tubes (MetalFree®15 mL, Labcon North America) at each exposure time in triplicate for each channel (n = 3) for dissolved Co, cations and anions concentration analyses. Aliquot of water was also sampled (120 mL) in triplicate in amber borosilicate glass vials previously burned at 450 °C and filtered on 0.7 µm GF/F filters (Whatman) to measure DOC concentrations. Note that the probe analyses and water sampling were performed at three different points along the artificial streams, i.e. about 3 and 1 m before the plastic boxes containing the gastropods (while also respecting the 15 m distance after the Co injection) and 3 m behind the boxes. The samples for dissolved Co and cations analyses were acidified
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Experimental design diagram for cobalt exposure experiments on the gastropod *A. balthica* conducted in outdoor mesocosms in June (spring) and October (autumn) 2021 at the Pilots Rivers (PRs) platform of PERL (Pole d'Étude et de Recherche de Lacq, TotalEnergies, Lacq, France). The PRs includes 16 artificial streams (40 m long, 50 cm wide and deep, 7.5 m³·h⁻¹ flow rate) fed continuously by the *Gave de Pau River*. Three Co exposure concentrations (nominal concentrations of 6, 30 and 60 µg·L⁻¹ Co) and one condition without added Co (control) were applied; they are referred as C6, C30, C60 and C0. The streams were selected randomly.</figcaption></figure>

at 2 % with 70 % ultrapure nitric acid (HNO₃; J.T. Baker, Ultrex II). Cobalt concentrations was determined using ICP-MS (ICP-MS-Agilent 7500, Agilent Technologies, Santa Clara (CA), USA) methodology. The certified reference water SLRS-6 (National Research Council of Canada, Ottawa, Canada) was used to ensure the accuracy and precision of Co analyses in the water. The detection limit for Co was 0.01 µg·L⁻¹. The average percentage recovery was 103 ± 5 %. Cations analysis (Ca, Mg, Na and K) was performed using an inductively coupled plasma atomic emission spectrometer (ICP-AES - Thermo iCAP 6000 series, Thermo Fisher Scientific, Waltham (MA), USA). Anions (F, Cl, NO₃ and SO₄) were quantified using a Dionex Aquion® with a Dionex IonPac AS9-HC column and an AERS 500 Carbonate suppressor. Dissolved Organic Carbon concentrations were measured using a Shimadzu TOC-L (module CSH, Marne La Vallée, France) calibrated with potassium phthalate monobasic solutions (Merck, BioUltra > 99.5 %) while a certified solution of sucrose at 10 mg/L C (Reagecon, Fisher Scientific, Vantaa, Finland) was used as reference material. Finally, the free Co²⁺ concentrations were calculated using Windermere Humic Aqueous Model (WHAM) with its default thermodynamic database. Dissolved total Co concentrations and the physico-chemical parameters of the ambient water (pH and the cations, anions and DOC concentrations) were used as input variables.

## 2.3. Cobalt effects on gastropods

Cobalt effects on mortality, growth and reproduction were monitored each week at D7, D14, D21 and D28 for all Co concentrations studied. Dead organisms were counted in order to determine the percentage of mortality for each exposure Co concentration. The average mortality was determined for the three replicates per exposure concentrations. The dead organisms were removed out of the box at the end of each sampling day. To assess the impact of Co on growth, the length and width of the organisms (n = 4 per box) were monitored using a caliper with an accuracy of 0.01 mm. The effects on the reproduction were assessed by counting both the number of egg masses spawn between each date and the number of neonates per box (June) and the number of eggs per mass (October). In October, the size (length and width) of the masses (n = 4 per box) and the size of the eggs (n = 4 per masses) were also examined.

## 2.4. Cobalt accumulation in gastropods

### 2.4.1. Soft tissues analysis

**Total Co analysis** - After collection, organisms were depurated during 2 days in water collected in the control mesocosms and stored at

-20 °C. Soft tissues were separated from the shell using two tweezers and then freeze-dried during one day. Their weights were further measured to convert Co concentrations in µg·g⁻¹ dry weight (dw). The soft tissues were digested in 3 mL of ultrapure HNO₃ (70 %; J.T. Baker, Ultrex II) and 2 mL of hydrogen peroxide (H₂O₂; Ultratrace analysis 30 %, Merck) during 25 min at 220 °C using an UltraWAVE (Milestone, Sorisole, Italy). These conditions were optimal for obtaining a clear digestate. For each exposure concentration and time, triplicates of soft tissues were analysed (96 organisms in total for June and October). Cobalt concentration was measured in the digestates (2 % nitric acid) using ICP-MS (ICP-MS-Agilent 7500, Agilent Technologies, Santa Clara (CA), USA). The certified reference material IAEA-461 (Clam; International Atomic Energy Agency, Vienna, Austria) was used to ensure the accuracy of the measurements. The detection limit was 0.02 µg·g⁻¹ and the recovery rate was 95 ± 4 %.

**Cobalt distribution mapping** - Cobalt distribution was performed directly in the soft tissue by elemental imaging using Laser Ablation coupled to ICP-MS. The methodology was adapted from our previous studies, and sampling and detection parameters were adjusted for Co localization in gastropod soft tissues sections (Arnaudguilhem et al., 2021; Labeyrie et al., 2023). Two organisms collected at D28 at all exposure conditions in October and June were analyzed. After each depuration period, the soft tissues were separated from the shell using two tweezers. The soft tissues were unrolled and placed flat in aluminum foil for storage at -80 °C. Samples were embedded in the optimal cutting temperature compound (4583, Tissue-Tek, Torrance (CA), USA) in the cryotome chamber (Leica CM1950, Leica Biosystems, Wetzlar, Germany) at -17 °C. Sections of 20 µm thickness were made to obtain sagittal cross-sections of entire organisms. Sections were recovered on a microscope slide and dried for 1 day under a laboratory hood before LA-ICP-MS analysis. Laser ablation analysis was performed using a nanosecond at 213 nm laser wavelength (NWR 213 model, ESI, San Jose (CA), USA) coupled to an ICPMS (Agilent 7700 cx, Agilent Technologies, Santa Clara (CA), USA). Further details concerning device settings are given in the supplementary text S1. For sample analysis, the laser beam was set to 30 × 30 µm, the scan speed to 30 µm·s⁻¹ with the maximum 20 Hz laser pulse frequency. The fluency at the sample surface was 4.5–5 J·cm². The entire section was completely ablated by parallel transects. For each ablated line of the section, a csv file was recorded and 2D images were produced using an in-house computer software under Python® developed by Dr G. Salvato Vallverdu. Cobalt mapping in the soft tissue was expressed by the Co intensity (counts per second, cps) for each pixel represented with a color code.

**Cobalt subcellular fractionation** - The subcellular fractionation of Co was analyzed using the a optimized method adapted from Rosabal
et al., 2024, detailed in supplementary Text S2 for all experimental conditions at D28 in October. Once separated from the shell (n = 3 per condition), the soft tissues were dried with filter paper, weighed and transferred into a Tris buffer solution (1:10). A succession of differential centrifugation steps was then performed to obtain the following fractions: the cellular debris (DE), metal-rich granules or NaOH resistant (MRG or GR), organelles (LY), metallothionein (MT), and heat-sensitive protein (HSP) and mitochondria (MI). The Co concentration was determined in each fraction after nitric acid digestion (HNO3; J.T. Baker, Ultrex II), dilution to 2 % nitric acid and measurement using ICP-MS (ICP-MS-Agilent 7500, Agilent Technologies, Santa Clara (CA), USA).

### 2.4.2. Shell analysis

First, a cross-section mapping of Co was performed using fs-LA-ICP-MS on the samples collected at D28 for each Co condition in order to obtain the Co distribution within the shell layers. For that purpose, additional shells were washed with H₂O₂, dried and embedded in an epoxy resin (Araldite 2020; Huntsmann Corporation). They were then sanded and polished with microabrasive discs to obtain a fine cross-section from the apex to the aperture. They were finally carefully rinsed with ultrapure water (Milli-Q water, 18.2 MΩ cm). Cobalt 2D-mappings were generated thanks to successive 20 µm-wide parallel laser ablations applied at the surface of each cross-section. For this strategy as the next one, an IR 1030 nm femtosecond laser ablation system (Alfamet-Novalase, France) coupled to an ICP-MS (Agilent 8900 ICP-MS Triple Quad) was used. Parallel ablations were done with a 20 µm laser beam diameter, a 60 µm·s⁻¹ scan speed at 50 Hz repetition rate (Marimoutou et al., 2023). Subsequently, based on the results obtained from shell cross-section analysis, Co concentrations were determined on three individuals per condition and per sampling day on the outer surface in the last growth stria of the cleaned shell. To this end, a flat piece of the last growth stria (approximatively 0.8 × 0.3 mm) of each replicate were cut using a wire cutter. This part represented the most recent part of the shell close to the peristome. One transect of 400 µm long and 100 µm wide, parallel to the last growth striae, was made per replicate at a speed of 5 µm·s⁻¹. For both strategies, the certified reference materials NIST614, NIST612 and NIST610 (National Institute of Standards and Technology, Gaithersburg (MD), USA) were used to calibrate the Fs-LA-ICPMS. Otolith reference materials FEBS-1 (National Research Council of Canada, Ottawa, Canada) and NIES22 (National Institute for Environmental Studies, Tsukuba, Japan) were analysed as reference materials to check the analytical accuracy. Recoveries for the certified elements, in particular Sr and Ba, were above 90 %. No reference or certified values were available for Co in these materials but the average concentration on repetitive analyses (n = 25) of these certified samples was 0.02 ± 0.01 µg·g⁻¹ (NIES22) and 0.06 ± 0.04 µg·g⁻¹ (FEBS-1), and they remained stable along the different calibration analyses. The limit of detection for Co was 0.20 µg·g⁻¹ for the 2D-mappings analyses and 0.01 µg·g⁻¹ for the transect analyses. The elemental Co raw data were reduced using an Excel VBA Macro (Focal; internal Macro developed by Dr C. Pécheyran, IPREM, Pau, France) and then processed on ImageJ to represent the concentration by a gradient color map.

### 2.4.3. Data analysis and modelling

All data representation and statistical analysis were made with Rstudio (RStudio, 2022). Statistical analyses were limited due to operational differences between the two seasons (1 stream per condition in June vs 3 streams per condition in October) and the number of replicates used for each analysis (n = 3 per condition and per day of sampling). Firstly, analysis of variance (ANOVA) was performed with a significance level below 0.05 to evaluate the effect of the exposure concentration and time on the organisms. However, the conditions of normality and homogeneity were never respected and the Kruskal-Wallis non-parametric test had to be performed.

Accumulation data in the various biological compartments were modelled using a linear equation when possible, using Rstudio software

with the Lm function. The obtained R-Squared value (r²) was used to assess the relationship between the water and accumulated Co. When a linear relationship could not be established, a Michaelis-Menten equation was used to represent Co accumulation using Rstudio software and the drm function. The Michaelis-Menten equation was defined as follows:

$$ \{Co\} = \frac{\{Co\}_{max} \times [Co^{2+}]}{K_m + [Co^{2+}]} $$

With:

- {Co}: Co concentration in the studied compartment (soft tissue or shell) (µg·g⁻¹)

- [Co²⁺]: Free Co concentration in water (µg·L⁻¹)

- Vmax: Co maximal concentration (µg·g⁻¹)

- Km: Michaelis-Menten constant, the substrate concentration at which the reaction rate is half of Vmax (µg·g⁻¹)

Several conditions were tested, including the assumption of the linear regressions, residual homogeneity, residual distribution following a normal distribution and residual independence with Lm test, the Breusch-Pagan test, Shapiro-Wilk test and Durbin-Watson test. The p-value was set at 0.05 for the significance of the different models.

## 3. Results

### 3.1. Exposure conditions

In June, at the start of the exposure experiment, the Gave de Pau water filling the mesocosms had an average Ca, Mg, Na concentrations of 30,498 ± 566 µg·L⁻¹, 2344 ± 36 µg·L⁻¹ and 2794 ± 35 µg·L⁻¹, respectively. Its average pH was 8.07 ± 0.01, its temperature was 17.38 ± 0.14 °C and DOC concentration was 0.52 ± 0.14 mg·L⁻¹ (Table 1). The background dissolved Co concentration was 0.36 ± 0.02 µg·L⁻¹ with a predicted free Co²⁺ fraction of 0.13 ± 0.01 µg·L⁻¹, representing 36 % of the total dissolved Co concentration (Table 1). During the 28 days of exposure, Ca, Mg and Na concentrations slightly increased by 1.1-, 1.3- and 1.2-fold, respectively. Cobalt addition into the mesocosms increased its total dissolved concentrations to 2.39 ± 0.75 µg·L⁻¹, 22.84 ± 0.99 µg·L⁻¹ and 51.60 ± 10.42 µg·L⁻¹ for C6, C30 and C60, respectively. The free Co²⁺ concentrations were calculated to be 0.67 ± 0.32 µg·L⁻¹, 8.34 ± 1.82 µg·L⁻¹ and 16.90 ± 6.51 µg·L⁻¹ for C0, C6, C30 and C60, respectively (Fig. 1). These concentrations remained constant over the course of the experiment (Table 1). The pH ranged between 7.93 ± 0.12 and 8.32 ± 0.07, and was not affected by Co addition. The temperature fluctuated between 16.5 ± 0.5 and 19.39 ± 0.04 °C. The concentrations of DOC varied between 0.31 ± 0.11 and 2.37 ± 0.62 mg·L⁻¹ during the experiment (Table 1).

In October, cation concentrations and pH were similar to those measured at D28 in June (Table 2). They remained stable over the course of the experiment. The temperature values were lower than in June, and decreased over the 28 days of exposure, from 14.20 ± 0.01 °C on D7 to 10.27 ± 0.06 °C at D28. The average DOC concentrations were in similar ranges than those measured in June and remained comparable between control and exposed mesocosms. The total dissolved Co concentrations were 0.30 ± 0.13 µg·L⁻¹, 5.18 ± 0.20 µg·L⁻¹, 33.20 ± 3.23 µg·L⁻¹ and 65.72 ± 2.61 µg·L⁻¹ for C0, C6, C30 and C60, respectively. The predicted free Co concentrations were 0.095 ± 0.059 µg·L⁻¹, 1.557 ± 0.144 µg·L⁻¹, 10.103 ± 1.676 µg·L⁻¹ and 19.800 ± 1.821 µg·L⁻¹ for C0, C6, C30 and C60, respectively (Figure S3). The Co exposure concentrations were similar to those measured in June and remained unchanged over time (Figure S3; Table 1 and 2). The different exposure conditions are further expressed as C0, C6, C30 and C60 in the following sections to simplify the reading, but the precise values are presented in Tables 1 and 2.
<caption>**Table 1**</caption>

Average (and standard deviations; *n* = 3) of the physicochemical parameters measured in the mesocosm exposure media during June experiment. The letters a,b,c and d represent the significant difference between the exposure concentration for a same sampling day. The letters A, B, C, D represent the difference between the different sampling day for a same exposure concentration. Significant differences were assessed using Dunn's post-hoc tests following Kruskal-Wallis non-parametric analysis.

<table><thead><tr><th>Day</th><th>Treatment</th><th>pH</th><th>Temperature (°C)</th><th>Ca<sup>2+</sup>(ug.L<sup>-1</sup>)</th><th>Mg<sup>2+</sup>(ug.L<sup>-1</sup>)</th><th>Na<sup>+</sup>(ug.L<sup>-1</sup>)</th><th>DOC<sup>+</sup>(ug.L<sup>-1</sup>)</th><th>Total Co (ug.L<sup>-1</sup>)</th><th>Free Co<sup>2+</sup>(ug.L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>7</td><td>C0</td><td>8.07 ± 0.01 <sup>A</sup></td><td>17.38 ± 0.14 <sup>a,A</sup></td><td>30,498 ± 566<sup>A</sup></td><td>2344 ± 36 <sup>A</sup></td><td>2794 ± 35 <sup>A</sup></td><td>0.524 ± 0.142<sup>a,A</sup></td><td>0.36 ± 0.02 <sup>a</sup></td><td>0.13 ± 0.01 <sup>a</sup></td></tr><tr><td>7</td><td>C6</td><td>8.26 ± 0.02 <sup>A</sup></td><td>17.37 ± 0.11 <sup>a,A</sup></td><td>30,523 ± 325<sup>A</sup></td><td>2345 ± 26 <sup>A</sup></td><td>2731 ± 32 <sup>A</sup></td><td>0.369 ± 0.001<sup>b,A</sup></td><td>2.12 ± 0.12 <sup>b</sup></td><td>0.51 ± 0.05<sup>b</sup></td></tr><tr><td>7</td><td>C30</td><td>8.18 ± 0.01 <sup>A</sup></td><td>17.54 ± 0.11 <sup>b,A</sup></td><td>30,418 ± 304<sup>A</sup></td><td>2349 ± 55 <sup>A</sup></td><td>2652 ± 27 <sup>A</sup></td><td>0.713 ± 0.179 <sup>a,A</sup></td><td>22.14 ± 0.27<sup>c</sup></td><td>6.35 ± 0.28<sup>c,A</sup></td></tr><tr><td>7</td><td>C60</td><td>8.12 ± 0.08 <sup>A</sup></td><td>17.43 ± 0.13 <sup>b,A</sup></td><td>30,331 ± 155<sup>A</sup></td><td>2324 ± 8 <sup>A</sup></td><td>2622 ± 31 <sup>A</sup></td><td>0.357 ± 0.072 <sup>b,A</sup></td><td>42.30 ± 0.34<sup>d</sup></td><td>14.56 ± 2.61<sup>d,A</sup></td></tr><tr><td>14</td><td>C0</td><td>8.12 ± 0.08 <sup>A</sup></td><td>16.47 ± 0.49 <sup>B</sup></td><td>32,110 ± 364<sup>B</sup></td><td>2781 ± 28 <sup>B</sup></td><td>2576 ± 41 <sup>B</sup></td><td>0.903 ± 0.063 <sup>B</sup></td><td>0.47 ± 0.05 <sup>a</sup></td><td>0.16 ± 0.04 <sup>a</sup></td></tr><tr><td>14</td><td>C6</td><td>8.32 ± 0.07 <sup>A</sup></td><td>16.55 ± 0.49 <sup>B</sup></td><td>31,692 ± 221<sup>B</sup></td><td>2738 ± 36 <sup>B</sup></td><td>2508 ± 71 <sup>B</sup></td><td>0.905 ± 0.034 <sup>B</sup></td><td>2.10 ± 0.06 <sup>b</sup></td><td>0.42 ± 0.10 <sup>b</sup></td></tr><tr><td>14</td><td>C30</td><td>8.17 ± 0.07 <sup>A</sup></td><td>16.69 ± 0.51 <sup>B</sup></td><td>31,498 ± 802<sup>B</sup></td><td>2739 ± 71 <sup>B</sup></td><td>2441 ± 104 <sup>B</sup></td><td>0.913 ± 0.061 <sup>B</sup></td><td>24.33 ± 3.05<sup>c</sup></td><td>7.26 ± 2.32 <sup>c,A</sup></td></tr><tr><td>14</td><td>C60</td><td>8.29 ± 0.08 <sup>A</sup></td><td>16.72 ± 0.46 <sup>B</sup></td><td>31,291 ± 222<sup>B</sup></td><td>2736 ± 25 <sup>B</sup></td><td>2435 ± 76 <sup>B</sup></td><td>0.833 ± 0.062 <sup>B</sup></td><td>45.39 ± 1.40<sup>d</sup></td><td>9.86 ± 2.71 <sup>d,A</sup></td></tr><tr><td>21</td><td>C0</td><td>7.98 ± 0.08 <sup>B</sup></td><td>19.21 ± 0.03 <sup>a,C</sup></td><td>32,892 ± 490<sup>C</sup></td><td>2739 ± 40 <sup>B</sup></td><td>3048 ± 157 <sup>C</sup></td><td>0.472 ± 0.039 <sup>a</sup></td><td>0.74 ± 0.28 <sup>a</sup></td><td>0.33 ± 0.16 <sup>a</sup></td></tr><tr><td>21</td><td>C6</td><td>8.16 ± 0.08 <sup>B</sup></td><td>19.17 ± 0.05 <sup>a,C</sup></td><td>32,650 ± 82<sup>C</sup></td><td>2713 ± 24 <sup>B</sup></td><td>2931 ± 33 <sup>C</sup></td><td>2.372 ± 0.617 <sup>b,C</sup></td><td>3.66 ± 1.93 <sup>b</sup></td><td>1.13 ± 0.68 <sup>b</sup></td></tr><tr><td>21</td><td>C30</td><td>7.96 ± 0.05 <sup>B</sup></td><td>19.35 ± 0.03 <sup>b,C</sup></td><td>32,891 ± 467<sup>C</sup></td><td>2734 ± 44 <sup>B</sup></td><td>2955 ± 54 <sup>C</sup></td><td>2.167 ± 0.601 <sup>b,C</sup></td><td>23.13 ± 0.18<sup>c</sup></td><td>10.10 ± 0.53 <sup>c,B</sup></td></tr><tr><td>21</td><td>C60</td><td>8.08 ± 0.06 <sup>B</sup></td><td>19.39 ± 0.04 <sup>b,C</sup></td><td>32,814 ± 66<sup>C</sup></td><td>2726 ± 24 <sup>B</sup></td><td>2959 ± 23 <sup>C</sup></td><td>0.376 ± 0.095 <sup>a</sup></td><td>49.60 ± 2.95<sup>d</sup></td><td>17.86 ± 1.99 <sup>d,B</sup></td></tr><tr><td>28</td><td>C0</td><td>7.93 ± 0.12 <sup>B</sup></td><td>16.90 ± 0.02 <sup>a,B</sup></td><td>34,906 ± 906<sup>D</sup></td><td>2961 ± 85 <sup>C</sup></td><td>3315 ± 92 <sup>D</sup></td><td>0.306 ± 0.106 <sup>a</sup></td><td>0.43 ± 0.03 <sup>a</sup></td><td>0.02 ± 0.01 <sup>a</sup></td></tr><tr><td>28</td><td>C6</td><td>8.11 ± 0.11 <sup>B</sup></td><td>16.87 ± 0.02 <sup>a,B</sup></td><td>34,826 ± 224<sup>D</sup></td><td>2665 ± 12 <sup>C</sup></td><td>3298 ± 22 <sup>D</sup></td><td>1.373 ± 0.228 <sup>b</sup></td><td>1.69 ± 1.30 <sup>b</sup></td><td>0.61 ± 0.49 <sup>b</sup></td></tr><tr><td>28</td><td>C30</td><td>7.96 ± 0.11 <sup>B</sup></td><td>16.99 ± 0.02 <sup>b,B</sup></td><td>34,583 ± 289<sup>D</sup></td><td>2911 ± 21 <sup>C</sup></td><td>3259 ± 24 <sup>D</sup></td><td>0.411 ± 0.060 <sup>a</sup></td><td>21.77 ± 1.01 <sup>c</sup></td><td>9.64 ± 1.38 <sup>c,B</sup></td></tr><tr><td>28</td><td>C60</td><td>8.07 ± 0.11 <sup>B</sup></td><td>17.08 ± 0.01 <sup>b,B</sup></td><td>34,994 ± 775<sup>D</sup></td><td>2962 ± 78 <sup>C</sup></td><td>3331 ± 87 <sup>D</sup></td><td>0.842 ± 0.084 <sup>b</sup></td><td>69.07 ± 8.30<sup>d</sup></td><td>25.33 ± 8.06 <sup>d,B</sup></td></tr></tbody></table>

<caption>**Table 2**</caption>

Average (and standard deviations; *n* = 3) of the physicochemical parameters of mesocosm exposure media during October experiment. The letters a,b,c and d represent the significant difference between the exposure concentration for a same sampling day. The letters A, B, C, D represent the difference between the different sampling day for a same exposure concentration. Significant differences were assessed using Dunn's post-hoc test following Kruskal-Wallis non-parametric analysis.

<table><thead><tr><th>Day</th><th>Treatment</th><th>pH</th><th>Temp (°C)</th><th>Ca<sup>2+</sup>(ug.L<sup>-1</sup>)</th><th>Mg<sup>2+</sup>(ug.L<sup>-1</sup>)</th><th>Na<sup>+</sup>(ug.L<sup>-1</sup>)</th><th>DOC<sup>+</sup>(ug.L<sup>-1</sup>)</th><th>Total Co (ug.L<sup>-1</sup>)</th><th>Free Co<sup>2+</sup>(ug.L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>7</td><td>C0</td><td>8.23 ± 0.01 <sup>A</sup></td><td>14.20 ± 0.01 <sup>A</sup></td><td>35,734 ± 255 <sup>A</sup></td><td>3697 ± 115 <sup>A</sup></td><td>4531 ± 83 <sup>A</sup></td><td>0.549 ± 0.066 <sup>A</sup></td><td>0.57 ± 0.25 <sup>a</sup></td><td>0.18 ± 0.08 <sup>a</sup></td></tr><tr><td>7</td><td>C6</td><td>8.25 ± 0.02 <sup>A</sup></td><td>14.20 ± 0.10 <sup>A</sup></td><td>35,977 ± 114 <sup>A</sup></td><td>3677 ± 3 <sup>A</sup></td><td>4524 ± 15 <sup>A</sup></td><td>0.541 ± 0.054 <sup>A</sup></td><td>5.42 ± 0.42 <sup>b</sup></td><td>1.61 ± 0.01 <sup>b</sup></td></tr><tr><td>7</td><td>C30</td><td>8.27 ± 0.03 <sup>A</sup></td><td>14.40 ± 0.10 <sup>A</sup></td><td>36,220 ± 279 <sup>A</sup></td><td>3715 ± 26 <sup>A</sup></td><td>4549 ± 45 <sup>A</sup></td><td>0.576 ± 0.059 <sup>A</sup></td><td>34.17 ± 1.95 <sup>c</sup></td><td>9.86 ± 0.66 <sup>c</sup></td></tr><tr><td>7</td><td>C60</td><td>8.28 ± 0.02 <sup>A</sup></td><td>14.4 ± 0.01 <sup>A</sup></td><td>36,043 ± 126 <sup>A</sup></td><td>3671 ± 29 <sup>A</sup></td><td>4487 ± 53 <sup>A</sup></td><td>0.560 ± 0.053 <sup>A</sup></td><td>68.40 ± 0.43 <sup>d</sup></td><td>19.40 ± 0.46 <sup>d</sup></td></tr><tr><td>14</td><td>C0</td><td>8.23 ± 0.01 <sup>A</sup></td><td>12.90 ± 0.01 <sup>B</sup></td><td>36,414 ± 205 <sup>A</sup></td><td>3848 ± 41 <sup>B</sup></td><td>4859 ± 40 <sup>B</sup></td><td>0.689 ± 0.198 <sup>A</sup></td><td>0.23 ± 0.08 <sup>a</sup></td><td>0.07 ± 0.03 <sup>a</sup></td></tr><tr><td>14</td><td>C6</td><td>8.23 ± 0.01 <sup>A</sup></td><td>12.90 ± 0.01 <sup>B</sup></td><td>36,638 ± 238 <sup>A</sup></td><td>3721 ± 195 <sup>B</sup></td><td>4667 ± 156 <sup>B</sup></td><td>0.581 ± 0.064 <sup>A</sup></td><td>5.34 ± 0.61 <sup>b</sup></td><td>1.66 ± 0.21 <sup>b</sup></td></tr><tr><td>14</td><td>C30</td><td>8.23 ± 0.01 <sup>A</sup></td><td>13.00 ± 0.01 <sup>B</sup></td><td>37,191 ± 381 <sup>A</sup></td><td>3476 ± 21 <sup>B</sup></td><td>5092 ± 51 <sup>B</sup></td><td>0.538 ± 0.053 <sup>A</sup></td><td>32.73 ± 2.48 <sup>c</sup></td><td>10.30 ± 0.89 <sup>c</sup></td></tr><tr><td>14</td><td>C60</td><td>8.21 ± 0.01 <sup>A</sup></td><td>12.90 ± 0.01 <sup>B</sup></td><td>36,848 ± 541 <sup>A</sup></td><td>3473 ± 79 <sup>B</sup></td><td>4943 ± 56 <sup>B</sup></td><td>0.552 ± 0.161 <sup>A</sup></td><td>61.80 ± 2.24 <sup>d</sup></td><td>19.73 ± 0.93 <sup>d</sup></td></tr><tr><td>21</td><td>C0</td><td>8.20 ± 0.05 <sup>A</sup></td><td>12.67 ± 0.06 <sup>B</sup></td><td>34,443 ± 125 <sup>B</sup></td><td>4090 ± 33 <sup>C</sup></td><td>3454 ± 227 <sup>C</sup></td><td>0.982 ± 0.061 <sup>B</sup></td><td>0.19 ± 0.17 <sup>a</sup></td><td>0.06 ± 0.06 <sup>a</sup></td></tr><tr><td>21</td><td>C6</td><td>8.20 ± 0.05 <sup>A</sup></td><td>12.70 ± 0.17 <sup>B</sup></td><td>34,545 ± 227 <sup>B</sup></td><td>4114 ± 47 <sup>C</sup></td><td>3750 ± 26 <sup>C</sup></td><td>0.906 ± 0.066 <sup>B</sup></td><td>4.97 ± 0.29 <sup>b</sup></td><td>1.61 ± 0.08 <sup>b</sup></td></tr><tr><td>21</td><td>C30</td><td>8.22 ± 0.08 <sup>A</sup></td><td>12.80 ± 0.10 <sup>B</sup></td><td>34,284 ± 19 <sup>B</sup></td><td>4094 ± 29 <sup>C</sup></td><td>3694 ± 10 <sup>C</sup></td><td>1.101 ± 0.129 <sup>B</sup></td><td>38.69 ± 0.27 <sup>c</sup></td><td>12.17 ± 1.19 <sup>c</sup></td></tr><tr><td>21</td><td>C60</td><td>8.19 ± 0.02 <sup>A</sup></td><td>12.80 ± 0.01 <sup>B</sup></td><td>34,178 ± 27 <sup>B</sup></td><td>4087 ± 19 <sup>C</sup></td><td>3663 ± 4 <sup>C</sup></td><td>0.948 ± 0.042 <sup>B</sup></td><td>68.26 ± 2.05 <sup>d</sup></td><td>22.23 ± 0.31 <sup>d</sup></td></tr><tr><td>28</td><td>C0</td><td>8.35 ± 0.01 <sup>B</sup></td><td>10.30 ± 0.01 <sup>C</sup></td><td>36,638 ± 134 <sup>A</sup></td><td>4200 ± 15 <sup>D</sup></td><td>4004 ± 24 <sup>D</sup></td><td>0.754 ± 0.014 <sup>a,B</sup></td><td>0.22 ± 0.15 <sup>a</sup></td><td>0.06 ± 0.05 <sup>a</sup></td></tr><tr><td>28</td><td>C6</td><td>8.37 ± 0.02 <sup>B</sup></td><td>10.27 ± 0.06 <sup>C</sup></td><td>36,886 ± 272 <sup>A</sup></td><td>4214 ± 22 <sup>D</sup></td><td>4006 ± 37 <sup>D</sup></td><td>0.749 ± 0.197 <sup>a,B</sup></td><td>4.98 ± 0.26 <sup>b</sup></td><td>1.34 ± 0.04 <sup>b</sup></td></tr><tr><td>28</td><td>C30</td><td>8.35 ± 0.05 <sup>B</sup></td><td>10.27 ± 0.06 <sup>C</sup></td><td>36,931 ± 247 <sup>A</sup></td><td>4175 ± 20 <sup>D</sup></td><td>4010 ± 75 <sup>D</sup></td><td>1.158 ± 0.010 <sup>b,B</sup></td><td>27.20 ± 3.71 <sup>c</sup></td><td>8.09 ± 1.65 <sup>c</sup></td></tr><tr><td>28</td><td>C60</td><td>8.34 ± 0.05 <sup>B</sup></td><td>10.27 ± 0.06 <sup>C</sup></td><td>36,857 ± 408 <sup>A</sup></td><td>4160 ± 26 <sup>D</sup></td><td>3965 ± 59 <sup>D</sup></td><td>0.927 ± 0.020 <sup>a,B</sup></td><td>64.42 ± 2.49 <sup>d</sup></td><td>17.83 ± 1.22 <sup>d</sup></td></tr></tbody></table>

## 3.2. Effects of Co on organisms

### 3.2.1. Growth

At the start of the exposure experiments, the studied organisms were adults with an average shell size of 8.1 ± 0.5 mm length and 5.3 ± 0.3 mm width in June, and 7.9 ± 0.9 mm length and 5.1 ± 0.5 mm width in October, considering that growth below 0.01 mm could not be measured. In June, a shell growth was measured during the first 7 days of exposure only for the control gastropods and those exposed at C30, with an average increase of 1.65 and 0.65 mm in shell length, respectively (Fig. 2a). For C6 and C60, the growth was observed later at D14 with an increase of 1.17 and 1.22 mm, respectively. No significant growth was further observed for the rest of the experiment up to D28 (Fig. 2a). In October, a slight but significant growth of 2.12 mm in shell length was observed at D21 and for all exposure concentrations (Fig. 2e).

### 3.2.2. Mortality

In June, mortality increased consistently over time, from 1–3 % to 15–25 % at all Co concentrations (Fig. 2b). No significant differences were found between the control and Co exposures, although a slight increasing trend could be noticed depending on the level of exposure on D21 and D28 sampling days. In October, mortality was slightly opposite, decreasing from the start of the experiment and remaining relatively

constant around 10 % from D21 to the end. Again, no significant differences were observed between the different exposure Co concentrations (Fig. 2f).

### 3.2.3. Reproduction

Reproduction was assessed by counting the number of masses spawned by the organisms. The highest number of masses was observed in the control with a value of 67 ± 4 at D14 in June (Fig. 2c). This number was significantly higher than those measured for gastropods exposed to the three added Co concentrations. In contrast, the number of masses increased significantly with Co exposure concentrations at D21. That trend was also observed at D28, with × 1.4, × 2.1 and × 2.7 for C6, C30 and C60 as compared to the control, respectively. At D28, neonates were counted at all exposure concentrations (Fig. 2d). The highest number was found in the control (195 ± 118), and this number decreased significantly with exposure Co concentrations.

In October and at D7, control gastropods had spawn lower number of masses (2 ± 2) than in June (17 ± 11; Fig. 2g). This number remained stable over the course of the exposure experiment, except for the slight significant increase (× 3.5) at D21. Higher number of masses were observed for C6 and C60 at all exposure time (except at D7 for C60) as compared to the control with a further increase at C6 (× 1.7) and C60 (× 4) between D7 and D14. The numbers of masses for gastropods exposed

<footnote>* DOC: Dissolved organic carbon.</footnote>

<footnote>* DOC: Dissolved organic carbon.</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Impact of Co on *A. balthica* growth (a,e), mortality (b,f) and reproduction (c,d,g,h) in June (top figures) and October (bottom figures). The barplots represent the mean values and the standard deviation as error bars (n = 3). The lower-case letters a,b,c and d represent the significant difference (p < 0.05) between Co exposure concentrations for a same sampling day. Asterisks correspond to the significant differences (p < 0.05) between exposure times. Significant differences were defined using Dunn's post-hoc test following Kruskal-Wallis non-parametric analysis. At the start of the experiment, the shell length was 8.1 ± 0.5 mm in June and 7.9 ± 0.9 mm in October.</figcaption></figure>

to C30 were similar to those of the control, except at D21 at which a higher number (× 2.3) was measured for the control (Fig. 2g). Based on the highest mass numbers observed during the two experiments, the amount was 2.5 × higher in June than in October. Moreover, in contrast with June, no neonates were observed. The effect of Co on masses was assessed using additional parameters with the counting of the number of eggs per mass and egg size (Fig. 2h; Figure S4). The number of eggs per mass was higher for exposure concentrations C6 (× 2) and C60 (× 1.8) than for control (9 ± 2) and C30 (12 ± 2), which presented similar numbers.

## 3.3. Cobalt accumulation in gastropods

### 3.3.1. Whole soft tissues

Cobalt accumulation was examined in the soft tissues of *A. balthica* exposed to free Co²⁺ at D7, D14, D21 and D28 in both June and October. In June, the Co concentration in the control gastropods was 0.40 ± 0.36 µg·g⁻¹ at D7 and remained stable throughout exposure time (Figure S5). Cobalt accumulation increased with the increase in Co concentrations in ambient water. As such, bioaccumulated Co concentrations were 1.96 ± 1.91 µg·g⁻¹, 3.13 ± 0.45 µg·g⁻¹ and 5.75 ± 2.53 µg·g⁻¹ for C6, C30 and C60, respectively. Similarly, to the control gastropods, these concentrations were constant throughout the exposure time (Figure S5). In October, Co concentrations in the soft tissues at D7 were 2.32 ± 1.59 µg·g⁻¹, 5.73 ± 1.93 µg·g⁻¹ and 4.06 ± 0.86 µg·g⁻¹ for the gastropods exposed at C6, C30 and C60, respectively (Figure S5). In control gastropods, Co concentrations were lower than in June (0.08 ± 0.03 µg·g⁻¹; D7). These Co concentrations remained stable for all exposure conditions throughout the experiment. Higher Co concentrations in soft tissues were observed in June for organisms exposed to C60 (average for all sampling day: 9.69 ± 2.95 µg·g⁻¹) compared to October (average for all

sampling day: 4.74 ± 1.33 µg·g⁻¹).

As no effect of exposure time on Co accumulation in soft bodies was observed in the two experiments, data were aggregated to establish a relationship between Co bioaccumulation and free Co exposure concentrations (Fig. 3). In June, a positive correlation was observed between both compartments (r²=0.55; Fig. 3a), whereas in October, a Michaelis Menten curve was the best model to represent the bio-accumulation as a function of ambient free Co²⁺ concentration with a plateau reached at C30 (Fig. 3c).

### 3.3.2. Cobalt distribution in soft tissues

The hepatopancreas, gut, mouth and mantle were identified in the cross-section of the *A. balthica* soft tissues (Fig. 4a). The Co distribution in the gastropod organs were examined in organisms collected at D28 in June and October (Fig. 4b and 4c). As the maximum of Co concentration in the total soft tissues was observed at day 28 in June and remained stable after day 7 in October, only gastropods exposed for 28 days were analysed for all conditions. At all studied concentrations, Co was mainly found in the hepatopancreas and mantle, with the exception of C30, for which a low Co signal intensity was detected in June and a wider distribution throughout the soft tissues was observed in October (Fig. 4).

### 3.3.3. Subcellular Co distribution

Subcellular Co accumulation was further examined in control (C0) and gastropods exposed to Co (C6, C30 and C60) at D28 in June and October (Fig. 5). The average recovery was 81 ± 12 % in June and 88 ± 6 % in October at day 28. In June, Co was mainly found in the granules (GR) in the control gastropods (80 ± 1 %) and in the gastropods exposed at C30 (71 ± 12 %) and at C60 (61 ± 13 %) (Fig. 5a). The remaining Co was found in the mitochondria (MI) with 18 ± 16 % at C0, 15 ± 10 % at C30 and 29 ± 27 % at C60. The gastropods exposed at C6 presented a
<figure><img src="image_4.png" /><figcaption>Fig. 3. Relationship between the Co concentration in soft tissues (a,c) and at the outer-shell layer (b,d) and the free Co concentration in water during the experiment in June (a,b) and October (c,d). In June, a linear regression relationship was found to be the best data fit, whereas a Michaelis-Menten non-linear relationship was used in October for the soft tissues compartment. While in the outer shell layer in June, data were fitted using a Michaelis-Menten non-linear curve, whereas a linear regression best represented October data.</figcaption></figure>

different trend with a higher percentage in MI (85 ± 50 %) than granules (6 ± 1 %). When expressed as accumulated Co concentrations, high Co concentrations are observed at C30 (2.14 ± 0.40 µg·g⁻¹) and at C60 (6.01 ± 1.03 µg·g⁻¹) in GR and in MI at C6 (2.74 ± 1.04 µg·g⁻¹) and C60 (2.45 ± 1.32 µg·g⁻¹) (Fig. 5b).

In October, for the control gastropods, Co was mainly found in the granules (66 ± 22 %) and in the HSP (15 ± 12 %) fractions (Fig. 5c). Upon Co exposure, more than 50 % of Co accumulated in the mitochondria fraction at all exposure Co concentrations (Fig. 5c). The remaining Co distributed in the HSP (20 ± 10 %) at C6, the granules (34 ± 21 %) at C30 and LY (15 ± 9 %), HSP (9 ± 3 %) and granules (6 ± 4 %) at C60. When reported as accumulated Co concentrations, increase of Co in the mitochondria fractions represented 1.33 ± 0.47 µg·g⁻¹ at C6 to a highest value of 3.22 ± 2.75 µg·g⁻¹ at C30 and 2.29 ± 1.75 µg·g⁻¹ at C60 (Fig. 5d). A higher concentration of Co was also observed in the granule fraction (1.36 ± 1.27 µg·g⁻¹) at C30. Cobalt was mainly found in the detoxification fraction (granules) in June while in October, Co was mostly detected in the sensitive fraction (mitochondria).

### 3.3.4. Outer shell surface

Cobalt was mainly found in the outer shell layer for the organisms exposed to Co in June and October (Fig. 6). In June, Co average concentrations in exposed gastropods were significantly higher (C6: 1.05 ± 0.30 µg·g⁻¹; C30: 2.28 ± 0.80 µg·g⁻¹; C60: 2.18 ± 0.45 µg·g⁻¹) than the

control (0.03 ± 0.01 µg·g⁻¹) (Figure S6a). The Co concentrations remained stable for all conditions during the exposure time. In October, similar Co concentrations as June were obtained for control gastropods (0.08 ± 0.06 µg·g⁻¹) and those exposed C6 (0.66 ± 0.26 µg·g⁻¹), C30 (0.95 ± 0.43 µg·g⁻¹) and C60 (1.78 ± 0.17 µg·g⁻¹) (Figure S6c). As for June, no significant difference in Co accumulation was observed between the different exposure days. Similar average Co concentration was found for all organisms exposed to Co over the whole experiment in June (1.39 ± 0.46 µg·g⁻¹, *n* = 36) and in October (0.87 ± 0.50 µg·g⁻¹, *n* = 36).

These concentrations remained constant over the exposure experiment in June and October. Therefore, all accumulation data were used to establish the relationship between free Co concentrations in ambient water and the Co concentration in the last growth stria of the outer shell. In June, the relation was described by a Michaelis-Menten curve, with a plateau reached at C30 (Fig. 3b). However, in contrast in October, a positive correlation was obtained between the Co concentration in the outer shell and the exposure concentrations (r²=0.49; Fig. 3d).

## 4. Discussion

### 4.1. Cobalt toxicity

In both experiments carried out in June and October, exposure to Co
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Microscopic observation of a sagittal section of *A. balthica* soft tissue (a). The imaging of Co distribution in soft tissue section of *A. balthica* analysed by LA-ICP-MS (c) at day 28 (D28) for all exposure conditions (C0, C6, C30 and C60) in June (b) and October (c).</figcaption></figure>

did not induce significant mortality among organisms. The natural mortality ranged from 1 to 30 %, with comparable values observed in the control and exposed gastropods. In laboratory experiments, no mortality was also observed for the gastropod *L. stagnalis* exposed to total dissolved Co ranging from 0.02 to 1000 µg·L⁻¹ for 28 days (De

Schamphelaere et al., 2008). Furthermore, a short-term LC50 value of 61,600 µg·L⁻¹ total dissolved Co has been reported for *L. stagnalis* (Stubblefield et al., 2020), which is much higher than our highest tested concentrations (56 ± 17 µg·L⁻¹ in June and 64 ± 4 µg·L⁻¹ in October). The low mortality is also supported by the low level of Co found in the
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Average percentage of Co distribution between the different subcellular fractions in *A. balthica* soft tissues (a) on day 28 for experiments of June and October. The Co concentration (µg·g⁻¹) in the different fractions (b) as function of the exposure conditions (C0, C6, C30 and C60). The different fractions obtained were the granules (GR), the metallothionein (MT), the lysosomes (LY), the heat-sensitive-proteins (HSP), the cellular debris (DE) and the mitochondria (MI). The barplots represent the mean values and the standard deviation as error bars (n = 3). The letters a, b, c and d represent the significant difference (*p* < 0.05) between the exposure concentrations performed by Dunn's post-hoc test following Kruskal-Wallis non-parametric analysis for each fraction.</figcaption></figure>

BDM fractions. Indeed, the MT fraction are known to be involved in the regulation of essential metals (Amiard et al., 2006; Marigomez et al., 2002) whereas granules are involved in the intracellular complexation of non-essential metals such as Cd and Ni (Chen et al., 2021b; Ma and Wang, 2020). In our study, most Co was mostly accumulated in the mitochondria at all exposure Co concentrations, notably in October, suggesting that gastropods did not induce regulating processes upon Co exposure. No effect of Co on growth was observed in both experiments and similar range of growth rate was observed in summer for the gastropod *Radix* with 2.41-2.86 mm/month (Chen et al., 2021a). Again, these results are in good agreement with those reported on *L. stagnalis* growth with a decrease only occurring at 79 µg·L⁻¹ total dissolved Co concentration (De Schamphelaere et al., 2008).

Although no effect was observed on mortality and growth, impact on

reproduction was found. In June, control gastropods had a higher number of masses, which spawn earlier than gastropods exposed to Co. Their number of mass then decreased over time, in accordance with a hatching period of 10–15 days reported for *A. balthica* (Lebreton, 2020). For exposed Co gastropods, the mass numbers increased with Co concentrations and with exposure time, but neonates decreased as a function of Co concentrations. This result could be explained by a negative impact of Co on the hatching stage as observed in the laboratory at comparable concentrations with *L. stagnalis* exposed to 40 µg·L⁻¹ Co for 28 days whereas the EC50 for the reproduction of *L. stagnalis* was 60 µg·L⁻¹ Co (De Schamphelaere et al., 2008). Cadmium and Zn have also been shown to disrupt mollusc reproduction by reducing embryonic hatching rates (Coeurdassier et al., 2004; De Schamphelaere et al., 2008). These metals could induce embryonic malformations, delay on
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Imaging of Co distribution of *A. balthica* shell cross-section analysed by Fs-LA-ICP-MS for all exposure conditions (C0, C6, C30 and C60) in June and October. The upper part of the shell represents the outer surface and the bottom part the inner surface of the shell.</figcaption></figure>

the embryogenesis, mainly in the veliger stage, and lead to the death of the embryo (Coeurdassier et al., 2004). Moreover, hatching involves chemical, osmotic and mechanical processes. Copper was reported to inhibit the hatching enzyme chorionase and impacted the osmotic regularity, by reducing enzymatic activity and muscle movement involved in hatching in fish (Dave and Xiu, 1991). Therefore, it is possible that Co might impact these different processes in similar way (Dave and Xiu, 1991). In October, fewer egg masses were found than in June and no neonate was observed in the control conditions. This is in accordance with the reported optimal temperature of 22 °C for spawning, mass development and hatching for *A. balthica* (Lebreton, 2020). The average temperature was 17.38 ± 0.14 °C in June and 14.20 ± 0.01 °C in October, declining over time. Environmental conditions were thus more favorable for reproduction in June than in October (Kuroda and Abe, 2020; Lebreton, 2020). Cobalt effect on the gastropod reproduction could thus not be assessed at this time of the year. These results suggest that very low levels of Co contamination in natural fresh water, i.e. a five-fold increase in dissolved and free Co concentrations as well as in Co accumulation, compared to natural conditions, could affect the maintenance of gastropod populations during the mollusc reproductive season. This could therefore indirectly destabilise communities that are ecologically linked to them.

## 4.2. Cobalt accumulation in soft tissues

Cobalt accumulated in soft bodies ranged between 0.08 – 18.04 µg·g⁻¹, in good agreement with the literature (Gawad and Soad, 2018; Meguid et al., 2017). Cobalt is naturally found in mollusc soft tissues as an essential metal found in vitamin B12 responsible for metabolic processes such as DNA synthesis and with a functional role in the nervous system (Maxwell, 1951; Nurjanah et al., 2015; Vogeler et al., 2022). A

quick equilibrium was established between its concentration in the soft tissues and the ambient environment, within 7 days of exposure for both seasons, but the relationships between the two differs according to the seasons. This last point will be discussed further in the Section 4.4. Both the hepatopancreas and mantle zone were the targeted organs, suggesting Co exposure through food and water, respectively (Flessas et al., 2000; Croteau et al., 2007; Bonnet, 2020). Food sources were most likely biofilms growing on the rocks placed in the plastic box and on the plastic box walls, which also absorbed Co from the ambient environment. Subcellular analysis further revealed that, in June, gastropods exposed to Co initiated mainly a detoxification response with storage in granules. Granules are well-known to play a fundamental role in metal detoxification in molluscs in which excess of metals precipitate to form insoluble concretion (Marin et al., 2012). In the outer epithelium of mantle, they additionally represent an internal source of Ca for molluscs, which could re-dissolve them (Widad, 2018). Released ions could then be transported into the extrapallial space of the mantle and be used for the shell formation (Marin et al., 2012).

In October, Co was mainly found in the mitochondria fractions for all exposure concentrations. Mitochondria play a key role in oxidative phosphorylation, which generates ATP, used as the primary energy source for most biochemical and physiological processes such as growth, movement and homeostasis (Ghiselli et al., 2021; Wallace et al., 2003). As such, the mitochondria fraction is considered as a sensitive fraction and the increase of Co binding to this pool could have negative impacts on organisms. These high concentrations in mitochondria observed in October may explain the Co concentration. Nevertheless, detoxification systems were not initiated with low concentrations in the GR and MT fractions in October. The low Co concentrations in the MT fraction may suggest that detoxification systems were not initiated. Nevertheless, the MT may be induced in the first period exposure (<7 days) and incorporated in the
lysosomes to be degraded (Amiard et al., 2006). Low percentages were also observed with the essential metal Zn on bivalves with 4 % found in MT and 2 % in GR (Wallace et al., 2003). Nevertheless, high concentrations of Co were found in both mitochondria and granules fractions in gastropods exposed to C30.

### 4.3. Cobalt accumulation in shell

The general process of shell mineralization consists of the reaction of Ca, bicarbonate ions and organic compounds in the extrapallial space in the mantle, forming the biomineral CaCO₃ (Marin et al., 2012; Widad, 2018). Metals can then accumulate into the shell by substituting Ca through Ca pumps, binding to the organic phase of the shell or being stored in the intracrystal of the shell (Marin et al., 2012; Schöne and Krause, 2016). Analyses performed with laser ablation on shell cross-section in the area of the last growth stria revealed that Co was mainly distributed on the outer shell surface, as demonstrated for other metals such as Cu, Ni, Pb and Zn (Marimoutou et al., 2023). This layer in *A. balthica* represents the periostracum, mainly composed of organic compounds and the upper part of the aragonitic prismatic layer. Periostracum is secreted by the periostracal groove as a soluble liquid film, which becomes insoluble and sclerotized by a tanning process involving quinone (Marin et al., 2012). This suggests that one of the ways in which metals could be transported to the shell may be by the binding to organic components involved in the formation of the periostracum. The ranges of Co concentrations in the outer layer measured using LA-ICP-MS were 0.01- 4.13 µg·g⁻¹. Data on trace elements using LA-ICP-MS on gastropods in freshwater are limited (Stringer and Prendergast, 2023). Moreover, Co concentrations in the outer shell was found to be already at equilibrium with exposure media at D7 with similar concentrations over time. Furthermore, no difference was observed between the exposure concentrations and the Co accumulated in the outer shell layer in June. In October, however, outer shell Co concentrations were higher for C30 and C60. Differences in Co storage in the shell according to seasons will be discussed in the next section.

### 4.4. Relationship between Co concentration in water, soft tissue and shell

Different patterns and levels of Co accumulation were observed in soft bodies and shells depending on the month of exposure. Several parameters could influence metal accumulation by organisms, such as metal bioavailability and water parameters (pH, hardness) as well as the organism physiology. Exposure periods were mainly differentiated by cation concentrations in the water, with lower Ca concentrations in June and therefore representing potentially less competition with Co for uptake. This period was also characterized by higher temperatures than October. This parameter influences growth and reproduction in *A. balthica* such that June represents more suitable environmental conditions for reproduction than October.

In June, Co accumulation in soft tissues was characterized by a positive correlation with free Co concentration (Fig. 2). Moreover, higher concentrations were observed in June compared to October. A study on *Mytilus edulis* in different sea sites demonstrated higher Co accumulation in soft tissues during spring (Knopf et al., 2020). This is the reproductive state period of *A. balthica* with a higher number masses produced in June than in October. In fact, several studies have highlighted the influence of the reproductive cycle on metal accumulation in molluscs, with a significant accumulation of metals (Cu, Cd and Pb) during this period (Azizi et al., 2018; Mbandzi et al., 2022; Yoloğlu et al., 2018). During gametogenesis, the gonads increase considerably and can constitute a trap for metals incorporation in the organism. Moreover, Co is an essential metal found in the vitamin B12, which is naturally involved in the biological processes of the organism such growth, development and respiration processes (Maxwell, 1951; Nurjanah et al., 2015). Unlike Co in soft tissue, shell Co accumulation rapidly reached a plateau of about 2 µg·g⁻¹ in average (Fig. 3) during the spring season.

This result would suggest that gastropods could use additional Co for important metabolic processes and limit its detoxification and storage in the shell.

In October, Co accumulation in soft tissues were comparable over Co exposure concentrations and reach a plateau of about 4 µg·g⁻¹ in average. On the contrary, Co accumulation in shells increased linearly with exposure concentrations. Cobalt could then be more efficiently regulated in the soft tissues and stored in the shell. The shell would be thus a more important detoxification pathway in October than in June. Similar results were observed in the gastropod *Lanistes carinatus* and *Lymnea palustris* exposed to Co (Amiard and Amiard-Triquet, 1979; Gawad and Soad, 2018). They suggest that the shell is a longer storage compartment for Co, and that the shell is a better compartment to link to Co exposure from water. Cobalt was additionally found to be stored in the mantle and at the subcellular level in granules and mitochondria during both experiments. Granules are known as detoxification response in metal exposure and could re-dissolve and disperse their ions in the extrapallial fluid, and could be involved in shell formation (Marin et al., 2012; Schöne and Krause, 2016).

In summary, our results clearly emphasized contrasting behavior between the two seasons with regard to the accumulation of Co in the two compartments (soft tissue and shell), which is probably in line with the organism's requirements. Gastropods exposed to Co initiated a detoxification response more significant in June than October while no linear relation between Co concentration in water and in shell was observed, a reverse behavior being found in October.

Regarding the transfer of Co to the shell, we could suggest that granules could constitute a significant pathway for shell Co transfer, either directly or indirectly or both. Indeed, Co was found mainly in the outer part of the shell of *A. balthica* at the interface between the periostracum, composed of organic molecules, and the prismatic layer composed of calcium carbonate. The granules present in the mantle could contribute to the formation of Co dissolved pool in the mantle during their redissolution, although a dissolved fraction of Co could also be already present. Part of this reservoir of dissolved Co may be complexed to organic molecules constitutive of the periostracum during its formation process, and thus be incorporated into the shell directly in the periostracum (Fig. 7: pathway 1). Another part could substitute Ca through pumps (Ca²⁺-ATPase) during shell formation and may be absorbed between the inner surface of the periostracum (Fig. 6: pathway 2) and the carbonate layer or complexed to the inner periostracum surface (Marin et al., 2012; Schöne and Krause, 2016). Finally, the granules undissolved containing Co could be (Fig. 6: pathway 3) directly trapped at the interface between the periostracum and the calcium carbonate prismatic layer.

## 5. Conclusion

In the present study, we demonstrated that Co impacted gastropod reproduction at low Co concentrations. We highlighted that Co accumulation in *A. balthica* was influenced by its life-cycle stage. During active reproductive phase, gastropods linearly accumulated Co in the soft tissues, while showing limited incorporation into the shell. In contrast, during less active periods, Co was mainly handled through storage in the shell. These results suggest a shift in metal-storage strategy depending on the gastropod physiological status. Tissue- and subcellular-level analyses provide further insight into the mechanisms of Co accumulation and storage in the gastropod. Our results indicated a key role of granules, and potentially shell storage, in Co sequestration processes. Cobalt may be stored as granules in the mantle and subsequently transferred to the shell during periostracum formation. The present study also demonstrates that the use of shell of *A. balthica* as bioindicators may be limited by the life stage (physiological activity) of the individual and Co fate in soft tissues. Further research into the life cycle of the species as well as the processes of shell growth and formation is required before Co accumulation in the shell of *A. balthica* can be
<figure><img src="image_8.png" /><figcaption>Fig. 7. Possible pathways for Co accumulation in the gastropod shell.</figcaption></figure>

used as an indicator of metal contamination. Additionally, further studies on the fate of Co in soft tissues of gastropods are needed to improve our understanding of the relationship between Co accumulation in soft tissue and shell incorporation.

### CRediT authorship contribution statement

**Maëva Marimoutou:** Methodology, Software, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization.
**Gilles Bareille:** Conceptualization, Writing – review & editing, Supervision.
**Mathieu Milhe-Poutingon:** Formal analysis, Writing – review & editing.
**Clémentine Gelber:** Writing – review & editing.
**Patrick Baldoni-Andrey:** Writing – review & editing, Funding acquisition.
**Nicholas Bagger Gurieff:** Writing – review & editing, Funding acquisition.
**Hélène Tabouret:** Methodology, Writing – review & editing.
**Sandra Mounicou:** Methodology, Writing – review & editing.
**Séverine Le Faucheur:** Conceptualization, Methodology, Writing – review & editing, Supervision, Project administration, Funding acquisition.

### 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.

### Acknowledgment

The authors would like to thank all the technicians and the environmental service team at PERL for their valuable support during the experiments conducted on the pilot rivers. We are also grateful to Mathieu Milhe-Poutingon for his technical support in the laboratory, particularly with sample analysis. Funding for this research was provided by the Research Partnership Chair E2S-UPPA–TotalEnergies–Rio Tinto (ANR-16-IDEX-0002).

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.aquatox.2025.107688.

### Data availability

Data will be made available on request.

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