# Unveiling the hidden threat: Molecular, cellular and behavioral effects of dietborne inorganic mercury and methylmercury in Dreissena polymorpha

Clarisse Seguin, Alice Marant, Séverine Palacios-Paris, Isabelle Bonnard, Jean-Luc Loizeau, Elise David, Damien Rioult, Claudia Cosio

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Clarisse Seguin, Alice Marant, Séverine Palacios-Paris, Isabelle Bonnard, Jean-Luc Loizeau, et al. Unveiling the hidden threat: Molecular, cellular and behavioral effects of dietborne inorganic mercury and methylmercury in Dreissena polymorpha. Chemosphere, 2025, 376, pp.144306. 10.1016/j.chemosphere.2025.144306. hal-05007905

HAL Id: hal-05007905

https://hal.science/hal-05007905v1

Submitted on 27 Mar 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Unveiling the hidden threat: Molecular, cellular and behavioral effects of dietborne inorganic mercury and methylmercury in *Dreissena polymorpha*

<figure><img src="image_3.png" /></figure>

Check for
updates

Clarisse Seguin<sup>a</sup>, Alice Marant<sup>a</sup>, Séverine Palacios-Paris<sup>a</sup>, Isabelle Bonnard<sup>a</sup>,
Jean-Luc Loizeau<sup>c</sup>, Elise David<sup>a, d</sup>, Damien Rioult<sup>a, b, d</sup>, Claudia Cosio<sup>a, *, d</sup>

## HIGHLIGHTS

* The effects of dietary Hg exposure were investigated in *Dreissena polymorpha*.

* The bioaccumulation factor was higher for methyl-Hg (MeHg) than for inorganic Hg (IHg).

* MeHg exposure reduced filtration rates at 1h compared to IHg.

* Histopathological damage was observed at the highest concentrations.

* Hg in diet caused oxidative stress.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

Handling editor: Lena Q. Ma

**Keywords:**

Bioaccumulation

Energy metabolism

Filtration

Histopathology

Microalgae

Oxidative stress

## ABSTRACT

Methylmercury (MeHg) bioaccumulation in food webs has been recognized as a significant health risk for over 50 years, yet most studies focus on high concentrations of waterborne inorganic mercury (IHg). This study investigates the effects of dietborne mercury (Hg) exposure at environmentally realistic IHg and MeHg levels on a freshwater food chain.

Freshwater mussels, *Dreissena polymorpha*, were fed with microalgae previously contaminated with 2 and 20 fg IHg or MeHg per cell for 4 d. Filtration behavior, Hg bioaccumulation, histopathology, antioxidant enzyme activity, and gene expression related to defense and energy metabolism were measured across gills, digestive glands, and other soft tissues (rests) for 1, 2, and 4 d. While all microalgae were filtered at the end of feeding, only MeHg exposure led to a reduced filtration at the beginning of feeding. Bioaccumulation factors were higher for MeHg than IHg, particularly in gills. Dietborne MeHg also caused more fibrosis and structural changes in gills than IHg, in line with bioaccumulation. Necrosis, tubular atrophy, and hemocyte infiltration were observed in

<footnote><sup>a</sup> Université de Reims Champagne-Ardenne, Université Le Havre Normandie, INERIS, Normandie Univ, UMR-I 02 SEBIO, Reims, France</footnote>

<footnote><sup>b</sup> Université de Reims Champagne-Ardenne, URCATech, MOBICYTE, Reims, France</footnote>

<footnote><sup>c</sup> Department F.-A. Forel for Environmental and Aquatic Sciences, Institute for Environmental Sciences, University of Geneva, Boulevard Carl-Vogt 66, Geneva 4, 1211, Switzerland</footnote>

<footnote>This article is part of a special issue entitled: Mercury in the Environment published in Chemosphere.</footnote>

<footnote>* Corresponding author.</footnote>
the digestive gland. Both IHg and MeHg triggered oxidative stress, as evidenced by significant changes in antioxidant enzyme activities and increased lipid peroxidation levels. MeHg exposure significantly upregulated the sod gene in rests and modulated cs and aox genes involved in energy metabolism in gills and digestive gland, depending on exposure duration. The findings aligned with established Hg toxicity targets but demonstrated notable differences in response depending on Hg forms and tissue type, emphasizing the importance of Hg speciation and tissue type in assessing toxicity.

# 1. Introduction

Mercury (Hg) is a global priority pollutant in aquatic ecosystems due to its threat to plants, animals, and humans. Methylmercury (MeHg) is recognized as a significant public health risk, particularly for humans exposed through the consumption of contaminated fish and seafood. As a result, Hg is targeted by the European Stockholm Convention and the global Minamata Convention, both of which aim to protect human health and the environment by reducing Hg's harmful effects and limiting anthropogenic emissions worldwide.

Atmospheric Hg contamination contributes to the contamination of soils and aquatic environments through both wet and dry deposits of inorganic Hg (IHg) (Driscoll et al., 2013). This deposited IHg can subsequently undergo biotic methylation in sediment and suboxic micro-environments through microbial activity, producing MeHg (Driscoll et al., 2013; Yu et al., 2018). MeHg then bioaccumulates in primary producers and biomagnifies in food webs, resulting in increasing tissue concentrations that correlate with organismal age and trophic level (Carrasco et al., 2008; Bravo et al., 2014). Consequently, dietary exposure to MeHg is the primary route of contamination for animals (Wang et al., 2012; Mao et al., 2021; van Rooyen et al., 2023). In this context, the burden and the toxicity of dietary MeHg in aquatic biota are expected to rise in the future (Pack et al., 2014; Schaefer et al., 2020).

Chronic exposure to dietary Hg for humans can lead to neurological and psychological effects including tremors, personality changes, anxiety, sleep disorders and depression (Järup, 2003; Yang et al., 2020). Similar toxic effects of Hg were observed in vertebrates and invertebrates, manifesting as inhibited growth and feeding, reduced embryo survival, developmental abnormalities, impaired locomotion, and decreased ability to capture active prey (Yang et al., 2020). Other effects such as behavioral changes, histological and DNA damages or oxidative stress were observed in aquatic invertebrates exposed to waterborne IHg (Table 1).

In freshwater fish, dietary exposures to high concentration of MeHg caused mortality, lipid peroxidation, brain damage, altered male reproductive behavior, impaired locomotion and feeding, with symptom severity correlating with Hg bioaccumulation (reviewed in Depew et al.,

2012). In *Crassostrea angulata* dietary exposure to high IHg concentrations (mg/L) led to reprotoxicity, oxidative stress and histological lesions to testicular and ovarian tissues (Zhang et al., 2013). In zebrafish, dietary MeHg and IHg exposure for 2 months (50 ng Hg/g, 80 % as MeHg; 10 µg Hg/g, 95 % as MeHg; 10 µg Hg/g, 99 % as IHg) caused more damages in muscle and liver tissues following MeHg exposure than IHg (Gentès et al., 2015). Additionally, Hg form influenced responses at the molecular level, as seen in the differential expression of 18 genes related to defense, oxidative stress, apoptosis, nervous system signaling, and mitochondrial metabolism, suggesting distinct toxicity pathways for IHg and MeHg (Gentès et al., 2015).

The plant-animal interface plays an essential ecological and ecotoxicological role in aquatic ecosystems, driving the transfer of energy, nutrients, and contaminants to higher trophic levels. Studies on the dietary impact of Hg on lower trophic level animals, such as invertebrate primary consumers that play a crucial role in introducing Hg into the food chain, remain limited. Notably, freshwater biota remains significantly less studied than marine biota in the context of Hg fate and toxicity. Moreover, many studies on waterborne Hg have used concentrations $10^3$ to $10^6$ × higher than those observed in natural environments and have often excluded MeHg, significantly reducing their environmental relevance. Consequently, the effects of MeHg biomagnification within food webs are not well understood and therefore difficult to accurately predict. Despite advances in understanding global Hg fluxes due to decades of research on the Hg cycle, key questions remain unanswered. Notably, the link between Hg bioaccumulation, molecular responses, and behavioral changes are underexplored (Evers et al., 2024; Morcillo et al., 2017).

Mussels belong to the 2nd largest animal phylum, Mollusca, which comprises over $10^6$ species (Fernández Robledo et al., 2019). Among these, bivalves are particularly important for benthic-pelagic coupling; they filter large volumes of water, cycle particulate matter and phytoplankton, alter community composition in their surroundings, and contribute to higher trophic levels (Newell, 2004; Smith et al., 2018). Mussels feed on phytoplankton, a group crucial to the cycling of MeHg, and they can also directly accumulate contaminants from the water column, making them ideal representatives of macrobenthic biota for

<table><caption>**Table 1**
Studies on effects of waterborne Hg in aquatic vertebrates and invertebrates.</caption><thead><tr><th>Organism</th><th>Hg</th><th>Concentration (µg/L)</th><th>Duration</th><th>Effects</th><th>Reference</th></tr></thead><tbody><tr><td>Ictalurus melas</td><td>IHg</td><td>35, 70, 140</td><td>10 d</td><td>Alteration in enzyme activities involved in defense against reactive oxygen species</td><td>Elia et al. (2003)</td></tr><tr><td>Pelteobagrus fulvidraco</td><td>IHg</td><td>2, 10</td><td>6 weeks</td><td>Histological damages, oxidative stress, immunotoxicity</td><td>Sun et al. (2018)</td></tr><tr><td>Cyprinus carpio</td><td>IHg</td><td>10</td><td>4 d</td><td>Oxidative stress, genotoxicity DNA damage</td><td>García-Medina et al., 2017</td></tr><tr><td>Bathymodiolus azoricus</td><td>IHg</td><td>20</td><td>21 d</td><td>Valve opening and shell gaping</td><td>Kádár et al. (2005)</td></tr><tr><td>Cerastoderma edule</td><td>IHg</td><td>1, 10, 100</td><td>6 d</td><td>Alteration in the estimated desaturases and elongases' activities, increase in enzymatic and non-enzymatic antioxidants, histological damages</td><td>Bejaoui et al. (2020)</td></tr><tr><td>Corbicula fluminea</td><td>IHg</td><td>30</td><td>8 d</td><td>Decrease in filtration rate, oxidative stress</td><td>Oliveira et al. (2018)</td></tr><tr><td>Ruditapes philippinarum</td><td>IHg</td><td>10</td><td>4 d</td><td>Decrease in filtration rate, immunomodulation, histological damages</td><td>Sikdokur et al., 2020</td></tr><tr><td>Dreissena polymorpha</td><td>MeHg</td><td>0.2 to 0.5</td><td>1-7 d</td><td>Alterations in cell and organelle structure, enzyme and gene expression related to oxidative stress defense, modulation of proteins involved in stress response, development, cytoskeleton, energy and amino acid metabolism</td><td>Baratange et al. (2022), 2023</td></tr><tr><td>Tigriopus japonicus</td><td>MeHg</td><td>0 to 1</td><td>4 d</td><td>Adverse effects on growth rate and reproduction, increase of antioxidant enzymatic activities, activation of MAPK pathways</td><td>Lee et al. (2017)</td></tr></tbody></table>
studying Hg uptake and effects (Bravo et al., 2014; Roditi et al., 2000; Zuykov et al., 2013). A field study along the Gambia River (Senegal), in areas recently impacted by artisanal gold mining, showed that bivalves accumulated more Hg than other animals, highlighting the significant role of these lower-trophic-level species play in Hg transfer from sediments to higher trophic levels (Niane et al., 2015). In freshwater ecosystems, *D. polymorpha* is highly tolerant to environmental changes (Palos-Ladeiro et al., 2017), widely distributed throughout the Northern Hemisphere and provides valuable ecosystem services, such as supporting drinking water production, and reducing eutrophication through filtration (5–400 ml/bivalve/h; Baldwin et al., 2002) and phytoplankton consumption valued at 12 k€/10⁶ m³ (Wang et al., 2021; Ludovisi et al., 2022). Furthermore, *D. polymorpha* serves as a food source for crustaceans, fish, birds and mammals (Molloy et al., 1997; Kobak and Kakareko, 2011), impacting population dynamics across freshwater ecosystems. Presence of *D. polymorpha* was linked to increased Hg in fish, likely due to biomagnification of MeHg in freshwater environments (Blinick et al., 2024). Nonetheless, the bio-accumulation and impact of dietary IHg and MeHg in *D. polymorpha* remain unknown.

Here, we aimed to investigate the bioaccumulation and effects of dietary IHg and MeHg in *D. polymorpha* to test whether MeHg causes more effects than IHg. We analyzed enzyme activities and transcript levels involved in antioxidant defense, lipid peroxidation as an indicator of oxidative stress, and histological damage. Analyses were conducted on the gills, digestive gland, and other soft tissues (rests): gills were chosen because they are involved in filtration and are the first tissues exposed to microalgae; the digestive gland as it plays a detoxification role and facilitates microalgae digestion; and the rests as they represent indirect dietary exposure after digestion. Filtration behavior in *D. polymorpha* was also assessed as a proxy for feeding activity.

## 2. Material and methods

### 2.1. Consumables and glasswares

Consumables and glassware were cleaned with three consecutive acid baths, each lasting one week: 10 % (v/v) HNO₃ (1st bath) and 10 % (v/v) HCl (2nd and 3rd baths). After each bath, items were rinsed three times with demineralized water and dried under a fume hood.

### 2.2. Biological models

*D. polymorpha* specimens were collected in April 2021 from Der Lake (Grand Est, France 48°36'22.02" N, 4°46'34.0" E), cleaned, and size-selected (18–20 mm). To avoid any sex bias, females were identified by gonadal puncture and distributed across 3 tanks filled with natural spring water (spring Aurèle, Cristaline®). Mussels underwent a 20 d depuration period at the lake's collection temperature (10.5 °C) and were fed twice weekly with 4 × 10⁶ cells/individual/d of *Nannochloropsis* (Nanno 3600 1L, Reed Mariculture, Planktovie). Tanks were continuously aerated, and water was refreshed before feeding. One week prior to exposure, 60 mussels were randomly placed in exposure tanks with 45 ml of water per individual to acclimate and establish byssal attachment.

### 2.3. Hg exposure of *Nannochloropsis* sp

Exposures were designed to simulate a microalgal bloom of 3.5 × 10⁴ cell/ml exposed to 70 ng/L THg, which is the Environmental Quality Standards (EQS) in Europe, corresponding to 2 fg/cell. We also used a concentration 10 × higher to represent a site contaminated with Hg. Microalgae cell concentration was measured using flow cytometry (BD Accuri C6). Initially, 2.9 ± 0.27 × 10¹⁰ *Nannochloropsis* cells in spring water (spring Aurèle, Cristaline) were exposed to five conditions: a control without Hg, 2 fg/cell of IHg (IHg2), 2 fg/cell of MeHg (MeHg2),

20 fg/cell of IHg (IHg20) and 20 fg/cell of MeHg (MeHg20) using IHg standard solution (Hg(NO₃)₂ 1 g/L, Sigma-Aldrich Supelco) and MeHg standard solution (CH₃HgCl 1 g/L, Alfa Aesar). Following exposure, microalgae were placed on an orbital shaker for 2h and then centrifuged for 10 min at 1300 g. The supernatant was discarded, and the microalgae were resuspended in spring water before being again centrifuged for 10 min at 1300 g to remove any unbound Hg.

### 2.4. Dietary exposure of *D. polymorpha*

Water was renewed daily prior to feeding mussels with a nominal concentration of 1 × 10⁸ microalgae cells/individual/d, while the effective concentration reached 0.42 ± 0.12 × 10⁸ cells/individual/d. Before each water renewal, water sample from each tank were collected, filtered (Sterivex, 0.45 µm), acidified to 0.5 % with HCl (Suprapur), and stored in Teflon tubes at 4 °C until Hg analysis. Eighteen mussels per tank were sampled at 24, 48, and 96 h for various analyses: bio-accumulation (n = 6), gene expression level (n = 6), protein activity and abundance (n = 6). Gills, the digestive gland, and other soft tissues (rests) were dissected, frozen in liquid nitrogen, and stored at -80 °C until analysis. The mussels that remained in the tanks at 96 h (n = 6) were used for histopathological analyses. No mortality of *D. polymorpha* was observed during the experiment.

### 2.5. Hg analysis

For bioaccumulation analysis, soft tissues of mussels were rinsed before dissection 5 min with 1 mM EDTA and 1 mM cysteine to remove loosely bound Hg. For total Hg (THg = IHg + MeHg) measurements, mussels and microalgae samples were freeze-dried and analyzed by atomic absorption spectrometry using the Advanced Hg Analyzer AMA 254 (Altec s.r.l., Czech Republic). For MeHg analysis, mussels and microalgae samples were freeze-dried, grinded, and digested in 30 % (v/v) HNO₃ at 60 °C for 12 h. THg concentrations in water were analyzed using MERX® T System (Brooks Rand Instruments, Seattle, WA, USA), while MeHg concentration in water, microalgae, and mussels were analyzed using MERX® M System (Brooks Rand Instruments, Seattle, WA, USA). Analytical quality was verified using certified reference material (DORM-4; NRC - CNRC) and blanks. THg and MeHg recovery was determined at 93 ± 6 and 100 ± 12 %, respectively. The bio-accumulation factor (BAF, g⁻¹) was calculated at 96 h as: (THg effective concentration in mussels (µg/g))/(THg effective amount in 10⁸ cells of microalgae (µg) × 4 d of exposure) (Table 2). Before exposure, IHg concentration in the whole body of *D. polymorpha* was 0.07 ± 0.01 µg/g, with MeHg accounting for 46 % of the total Hg. These values were subtracted from final bioaccumulation data.

### 2.6. Hg effects in *D. polymorpha*

#### 2.6.1. Filtration rate

To observe the filtration behavior of *D. polymorpha*, we adapted the system previously described by Géba et al. (2021). Each mussel was placed in a tube with 45 ml of spring water continuously bubbled with air to both oxygenate the medium and prevent microalgae sedimentation. Four tubes, each housing one mussel, were prepared for each

<table><caption>**Table 2**
Concentration of THg and MeHg (fg/cell) in *Nannochloropsis* at the beginning of exposure.</caption><thead><tr><td>Condition</td><td>THg</td><td>MeHg</td></tr></thead><tbody><tr><td>Control</td><td>0.00 ± 0.00</td><td>0.00</td></tr><tr><td>IHg2</td><td>0.59 ± 0.01</td><td>0.00</td></tr><tr><td>MeHg2</td><td>1.10 ± 0.02</td><td>0.01</td></tr><tr><td>IHg20</td><td>10.07 ± 0.08</td><td>0.00</td></tr><tr><td>MeHg20</td><td>6.52 ± 0.23</td><td>3.87</td></tr></tbody></table>
condition. After one day of acclimation, mussels were fed with $1 \times 10^8$ microalgae cells/individual/d prepared as described in section 2.4. For each condition, a tube without mussel was prepared, as negative control, which also received microalgae to ensure that microalgae concentration was stable in the filtration device. The number of unconsumed microalgae cells was measured daily in each tube 1 h (beginning) and 22 h (end) after feeding by flow cytometry (NovoCyte Advanteon) using cell complexity (SSC) and cell size (FSC) at a flow rate of 35 µL/min, using 50 µL of sample. The filtration rate (FR) was expressed as the percentage of filtered cells in 45 mL per hour and calculated using the formula: $FR = ((N_{initial} - N_{final})/N_{initial}) \times 100$ where N = number of microalgae in 45 mL.

### 2.6.2. Histopathology

The protocol for histopathology was previously described in Baratange et al. (2022). Entire soft tissues were immediately fixed in Bouin's aqueous solution for 24 h. Tissues were then dehydrated and embedded in paraffin following the protocol detailed in Paris-Palacios et al. (2000). Five µm-sections were obtained, stained with nuclear fast red (NR) and picro-indigo-carmin (PIC), and mounted on glass slides for microscopic analysis. Mussels exhibiting parasite(s) were excluded from the study. To evaluate the frequency and the intensity of potential pathologies in gills or the digestive gland, three sections from each mussel were examined and scored (adapted from Jaffal et al., 2015; Jacquin et al., 2019). A global score of histological perturbations was calculated as the median value of all scores.

### 2.6.3. Antioxidant enzymes and lipid peroxidation

Protocols were previously detailed in Baratange et al. (2022). Briefly, samples were homogenized using a 400 MM homogenizer (Restch®, Eragny, Luxembourg) with a single stainless-steel bead (8 mm) for 2 × 30 s intervals at 30 Hz in 800 µL of 50 mM phosphate buffer (pH 7.4) containing 1 mM Phenylmethylsulfonyl fluoride (PMSF) and 1 mM L-Serine borate per 100 mg of tissues. Protein concentration, glutathione-S-transferase (GST) activity, and lipid peroxidation were measured using a Gallery® spectrometer (ThermoFisher Scientific). Protein concentration was determined at 600 nm using the red pyrogallol method. GST activity was assessed at 340 nm with 1-chloro-2, 4-dinitrobenzene (0.9 mM) and reduced glutathione (1 mM) in phosphate buffer (0.1 M, pH 6.5). Lipid hydroperoxide levels (LOOH), indicative of lipid peroxidation damage, were measured at 620 nm using the Fox method. Catalase (CAT) activity was evaluated at 240 nm with a Cary 50 spectrophotometer (Agilent) using hydrogen peroxide (14 mM) in phosphate buffer (50 mM, pH 7.4). SuperOxide Dismutase (SOD) activity was measured at 340 nm with a Tecan® spectrophotometer (Spark™ 10 M) using β-mercaptoethanol (10 mM) and NADH (7.5 mM) in phosphate buffer (50 mM, pH 7.4).

### 2.6.4. Gene relative expression level

Total RNA was extracted from tissues using TRI Reagent™ according to manufacturer instructions (Euromedex). RNA purity was assessed by measuring the absorbance ratio at 260 and 280 nm with a micro-volume spectrophotometer (GenovaNano, Jenway). RNA integrity was evaluated using electrophoresis on 1 % agarose gel in TBE (Tris Borate EDTA 0.5 %) buffer. Reverse transcription (RT) was performed using 400 ng of RNA with the Thermo Scientific Verso cDNA kit employing a mixture of oligo-dT and random primers as specified in manufacturer instructions. Quantitative PCR (qPCR) analysis was conducted using a BioRad CFX 96 system with Absolute Blue SYBR Green ROX Mix (Thermo Scientific) on cDNA samples diluted (1 : 10) in a total volume of 15 µL. Cycling conditions consisted of 95 °C for 2 min, 95 °C for 5 s and 60 °C for 45 s, repeated 40 × . Target genes included catalase (cat), glutathione-s-transferase (gst), superoxide dismutase (sod) and metallothionein (mt) involved in detoxication mechanisms and atp synthase (atp), cytochrome c oxidase A (cox), lactate deshydrogenase (ldh), amp kinase subunit α (ampk), citrate synthase (cs) and alternative oxidase (aox)

involved in energy metabolism. The relative expression levels of target genes were calculated using the $2^{-\Delta\Delta Ct}$ method with actin and s3 protein serving as reference genes (Table S1). Thereafter, to distinguish gene expression level from enzyme activities, the name of the former is written in lower case and the later in upper case.

### 2.7. Statistics

Statistical analyses were conducted using R software version 4.0.3. We compared the effect of all the exposure conditions (ctrl, MeHg2, MeHg20, IHg2 and IHg20) independently for each time point (24 h, 48 h, 96 h). For each endpoint, the normality of residuals (Shapiro-Wilk's test, α = 0.01) and homoscedasticity (Brown Forsythe's test, α = 0.05) were evaluated prior to interpreting the ANOVA results. A one-way ANOVA was then performed followed by the Tukey post-doc test (α = 0.05). When necessary, a log transformation was applied to meet these criteria. If only the homoscedasticity criterion was not met, one-way Welch's ANOVA (α = 0.05) was performed followed by the Games Howell post-hoc test (α = 0.05). In case where neither criterion was met, a nonparametric one-way Kruskall-Wallis test (α = 0.05) was conducted, followed by the Wilcoxon's post-hoc for independent samples test with the Hochberg p-value adjustment (α = 0.05) for multiple comparisons.

## 3. Results

### 3.1. Filtration rate

Filtration activity was used as a proxy for feeding. The highest FR at the beginning of feeding was observed in mussels exposed to microalgae contaminated with IHg2 (93.3 ± 8.0 %) and IHg20 (87.5 ± 14.7 %) at 24 h; however, these values were not significantly different from the control (Fig. 1). Notably, FR of mussels exposed to IHg was significantly higher than FR measured without mussel, which was not observed for control, MeHg2 and MeHg20 exposures. Additionally, filtration activity appeared to be reduced in mussels exposed to MeHg compared to those exposed to IHg, with a significant difference observed between mussels exposed to IHg2 and MeHg20. Nonetheless these differences in FR at the beginning of feeding were not significant on following days (Fig. 1), although MeHg2 exposure reduced strongly FR in 38 % of the individuals. Nonetheless, at the end of feeding, FR reached 98.80 % ± 0.39 for control, 97.69 % ± 3.46 for IHg2, 85.38 % ± 15.79 for MeHg2, 95.48 % ± 5.19 for IHg20, and 99.21 % ± 0.63 for MeHg20. These results supported that all mussels demonstrated active filtration over the whole exposure under our experimental conditions. By the end of feeding, all microalgae had been filtered, confirming trophic Hg uptake.

### 3.2. Hg bioaccumulation

Both IHg and MeHg were bioaccumulated in tissues of *D. polymorpha*, exhibiting high variability among individuals but higher values in gills and digestive gland than rests (Fig. 2, Table S2). Overall, the mean of bioaccumulation increased over time, with MeHg bioaccumulation being higher than IHg bioaccumulation. In gills, THg concentrations with MeHg20 were 13 × , 12 × and 6 × higher at 24, 48 and 96 h, respectively, compared to MeHg2. Similarly, THg concentrations with IHg20 were respectively 12 × , 16 × and 8 × higher at 24, 48 and 96 h than those for IHg2. These trends were consistent in the digestive gland and rests. Exposure to MeHg20 in gills resulted in BAF 17 × higher than IHg20. Conversely, exposure to IHg2 led to a BAF 10 × and 3 × higher than IHg20 and MeHg20 respectively. As anticipated, % MeHg was higher during MeHg exposure. Moreover, % MeHg in IHg20 exposure also increased compared to control and IHg2 in all tissues at 48h and 96h, reaching values approximately 200 × higher.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Filtration rate (%) of *D. polymorpha* and negative control (no mussel) at the beginning of feeding at 24 h (A), 48 h (B) and 96 h (B). Different letters indicate statistically different groups (n = 4; *p* ≤ 0.05).</figcaption></figure>

### 3.3. Histopathology

In the gills of both control and exposed mussels, we observed collagen deposits indicative of fibrosis, alterations in the shape of the gill filaments, and erosion of the ciliary border (Fig. 3). These changes were more frequent and severe in mussels exposed to MeHg. The gut, stomach, and style bag of mussels displayed very few histopathological alterations under control and IHg conditions, with only moderate fibrosis and some cellular alterations noted in the gut (Table 3).

In the digestive gland, collagen deposits around digestive tubules were thin in control but larger in mussels exposed to IHg and MeHg. Some collagen deposited in interstitial areas indicated higher levels of fibrosis. Abnormal shape and staining of digestive cells suggested both plasmolysis (large round and clear cell) and necrosis (small irregular dark cell) in the most severely affected tubules.

Mussels fed with algae contaminated with both IHg and MeHg, exhibited histological damages consistent with a typical inflammatory response, including important amount of hemolymphatic fluid leading to some edema in the digestive gland, a high number of circulating cells concentrated around the more severely altered digestive tubules, and obvious inter-tubule fibrosis. These alterations were accompanied by a high frequency of abnormal cells with perturbed nuclei, as well as tubular breakage, atresia, or complete destruction. Alterations were more frequent and intense in gills and guts of mussels exposed to MeHg compared to those exposed to IHg. However, nuclear alterations were more pronounced in digestive glands of mussels exposed to IHg. Notably, fibrosis in gut were higher in mussels exposed to IHg20 and MeHg20.

### 3.4. Antioxidant enzyme activities and lipid peroxidation

Both MeHg and IHg in the diet impacted enzyme activities and LOOH abundance (Table 4). In gills, CAT activity was generally lower across all the Hg exposures compared to the control, with a significant reduction (2.9 ×) in MeHg20 exposure. In gills in IHg2 at 24 h, LOOH concentration increased. In the digestive gland, CAT activity increased by 4.6 × in IHg2 and 3.5 × in MeHg2 at 24 h compared to control. At 24 h, LOOH concentration increased in IHg2 (1.7 ×), MeHg2 (1.8 ×) and IHg20 (1.7 ×) exposures. In rests, SOD activity decreased in MeHg20 exposure at 24 h (2.1 ×), 48 h (1.8 ×) and 96 h (2.8 ×), while it increased in MeHg2 exposure at 24 h (1.5 ×), 48 h (1.4 ×) and 96 h (1.4 ×). In rests, LOOH concentration rose significantly in MeHg2 at 24 h (2.4 ×), 48 h (3.2 ×) and 96 h (2.6 ×), as well as in MeHg20 at 24 h (1.6 ×), 48 h (2.8 ×) and 96 h (2.2 ×) compared to control. In IHg2, LOOH concentration was higher at 48 h (2.3 ×) and 96 h (2.1 ×), though no significant difference was observed in IHg20 exposure compared to the control.

### 3.5. Gene expression level

At the transcript level, the expression level of the *sod* gene involved in antioxidant defenses was significantly upregulated (2.74 ×) in the rests of mussels exposed 96 h to MeHg20 compared to the control (Table 5). Genes related to energy metabolism also showed significant changes of expression compared to the control: in the digestive gland at 96 h, *cs* transcripts were downregulated 1.92 × and 1.56 × in MeHg2 and IHg20, respectively; in gills at 48 h, *aox* transcripts were downregulated by 1.79 × and 1.70 × in IHg2 and MeHg20 respectively; in the rests at 24 h, *aox* gene expression level was upregulated 2.13 × in IHg20. Further differences between Hg exposures were observed: at 24 h in rests, *mt* transcripts were more upregulated in MeHg2 than MeHg20, and *atp* transcript levels were higher in rests at 24 h in IHg20 than IHg2. No significant changes were detected in the expression of *cat*, *gst*, *cox*, *ldh* and *ampk* gene expressions under these experimental conditions.
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Concentrations of THg (µg/g; bar chart), % of IHg and MeHg (pie chart) and the bioaccumulation factor (BAF g⁻¹) in gills (A), digestive gland (B) and rests (C) of *D. polymorpha* exposed to IHg and MeHg by diet (mean ± SD; n = 3, p < 0.05).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Histopathological alterations of the digestive gland (A–C) and gills (D–F) of *D. polymorpha* exposed by diet to control (A, D), IHg20 (B, E) and MeHg20 (C, F); \*: alteration of gill filament shape, ⋅: erosion of the ciliary border, a: algae observed in the mucus surrounding gills, fc: fine digestive tubules, Lc: large digestive tubules, F: fibrosis, ⚪: altered tubules, ⚪: enlargement of interstitial areas, accumulation of coelomocytes and fibrosis; tubules are stained blue with indigo.</figcaption></figure>

## 4. Discussion

Comparing the bioaccumulation and effects of dietary IHg and MeHg in *D. polymorpha* revealed differences in both their bioaccumulation patterns and subsequent impacts. In MeHg exposure, mussels showed a reduced FR at the beginning of feeding at 24 h and in 38 % of individuals in longer exposures. However, *D. polymorpha* bioaccumulated more MeHg than IHg in the three tissues, with highest concentrations in gills in MeHg20 exposure. In congruence, there was more histological damages observed in MeHg exposures. In contrast, effects on antioxidant enzymes were mainly observed in rests, that were not directly exposed to diet and few modulations were observed in gene expression.

*D. polymorpha* accumulated both IHg and MeHg over the 96 h-

exposure period with higher BAFs for MeHg2 and MeHg20 in the gills and higher BAF for IHg2 in the digestive gland. This is in line with a higher THg concentration in the digestive gland at 96 h than in other tissues. In previous studies, *D. polymorpha* exposed to 280 ng/L MeHg in water for 1 and 7 d had BAF of 755 and 3973 respectively (Baratange et al., 2022). Our findings suggested that dietary exposure to MeHg led to lower BAF values than water exposure in *D. polymorpha*, although comparison remains limited because of quite different concentrations. However, data on IHg bioaccumulation in *D. polymorpha* remain unavailable. For comparison, in *Perna perna* exposed to 50 µg/L IHg in water over 2 and 4 d, whole soft tissues accumulated 3 and 7 µg THg/g respectively (Anandraj et al., 2002). This is 5.8 × and 3.4 × lower than for IHg2 in the digestive gland of *D. polymorpha*, and comparable to those found in the rests after 2 and 4 d (3.44 and 5.36 µg/g), in our study. This suggests comparable Hg bioaccumulation in *D. polymorpha* when exposed via diet, underscoring the relevance of dietary exposure in realistic environmental conditions. Nonetheless, these comparisons between whole tissue and specific organs emphasize a notable gap in research on Hg organotropism and fate in bivalves.

Consistent with the higher BAF for MeHg than IHg in gills, MeHg exposure appeared to reduce filtration activity during the first hour compared to IHg, though this effect did not persist with longer exposures. By the end of feeding, all microalgae had been filtered, confirming trophic Hg uptake and indicating no correlation between early filtration behavior and final bioaccumulation levels. There is a lack of studies examining the effects of dietary Hg on bivalve filtration rates, limiting direct comparisons with our experimental conditions and underscoring the need for more research concerning bivalves. For instance, a study exposing *D. polymorpha* to 40 µg/L of waterborne MeHg and IHg for 2 h reported non-significant difference in FR compared to control (Faria et al., 2009), though the concentration used was approximately 500 × the Hg EQS. In *C. fluminea*, a 1 h exposure to 30 µg/L waterborne IHg reduced filtration activity by 78 % (Oliveira et al., 2018). Similarly, in *P. perna*, exposure to 50 µg/L IHg for 2, 4, 8, 16, 24 d significantly reduced FR by 42 and 61 % by 2 and 16 d, respectively, compared to the control (Anandraj et al., 2002). However, these studies were conducted at concentrations 400-700 × above the EQS, raising questions about their environmental relevance and limiting comparability with our exposure levels. Our findings suggest that MeHg exposure may influence FR of *D. polymorpha* in the first hour, a response not observed with IHg and during prolonged exposures. Together with other studies, these results suggest Hg may alter filtration behavior in bivalves. Gills of *Mytilus edulis* and *Crassostrea virginica* were shown to contain serotonergic and dopaminergic fibers which function both as peripheral and ganglionic neurotransmitters that regulate cilia beating rate (Sauvey et al., 2021). In *D. polymorpha* flow rate was shown to be under the control of gills muscles that responded to neurotransmitters (Medler and Silverman, 1997). Effects on motor functions related to valve opening and shell gaping have been documented, for example in *B. azoricus* exposed to 20 µg/L IHg over 21 d under controlled laboratory conditions (Kádár et al., 2005), consistent with its expected neurotoxicity (Baatrup, 1991). Similarly, shell gaping in *Corbicula fluminea* in response to IHg exposure was observed for exposures ≥ 5.1 µg/L and duration ≥ 5 h (Tran et al., 2007). Thus, a reduction in filtration could indicate a neurotoxic effect of MeHg in our experimental conditions. Despite these findings, further

<caption>**Table 3**</caption>

Histopathological scores in *D. polymorpha* exposed by diet to control, IHg2, IHg20, MeHg2 and MeHg20.

<table><thead><tr><th>Organ</th><th>Pathology</th><th>Control</th><th>IHg2</th><th>IHg20</th><th>MeHg2</th><th>MeHg20</th></tr></thead><tbody><tr><td rowspan="2">Gills</td><td>Fibrosis</td><td>15 ± 8</td><td>17.5 ± 5</td><td>14 ± 3</td><td>24 ± 8</td><td>22 ± 8</td></tr><tr><td>Alteration</td><td>11 ± 3</td><td>28.5 ± 24</td><td>20 ± 6</td><td>50 ± 18</td><td>40 ± 15</td></tr><tr><td>Gut</td><td>Destructuration and alteration</td><td>0</td><td>5 ± 7</td><td>0</td><td>50 ± 11</td><td>45 ± 10</td></tr><tr><td rowspan="4">Digestive gland</td><td>Inflammation</td><td>0.3 ± 0.8</td><td>2.8 ± 1.0</td><td>2.2 ± 1.0</td><td>1.0 ± 0.5</td><td>1.2 ± 0.7</td></tr><tr><td>Fibrosis</td><td>0.4 ± 0.5</td><td>0.8 ± 0.4</td><td>1.8 ± 0.4</td><td>0.6 ± 0.3</td><td>1.1 ± 0.6</td></tr><tr><td>Tubule alteration (%)</td><td>15 ± 5</td><td>31 ± 8</td><td>34 ± 6</td><td>32 ± 8</td><td>34 ± 9</td></tr><tr><td>Nuclear alteration (%)</td><td>22 ± 7</td><td>44 ± 10</td><td>45 ± 6</td><td>32 ± 5</td><td>33 ± 6</td></tr></tbody></table>
<caption>**Table 4**</caption>

Enzyme activities of CAT (U/g protein), GST (µmol CDNB/g protein · min), SOD (U/g protein) and LOOH (µmol TBH/g protein) in gills, digestive gland and rests of *D. polymorpha* exposed by diet 24 h, 48 h and 96 h to IHg and MeHg (mean ± SD, n = 4 to 6 animals; different letters show statistical significance p < 0.05).

<table><thead><tr><th>Tissue</th><th>Time</th><th>Exposure</th><th>CAT</th><th>GST</th><th>SOD</th><th>LOOH</th></tr></thead><tbody><tr><td rowspan="12">Gills</td><td rowspan="5">24 h</td><td>Control</td><td>102.57 ± 70.67 <sup>a</sup></td><td>125.35 ± 34.15 <sup>a</sup></td><td>4.14 ± 1.20 <sup>a</sup></td><td>18.76 ± 2.28 <sup>a</sup></td></tr><tr><td>IHg2</td><td>49.93 ± 31.08 <sup>a</sup></td><td>118.26 ± 27.01 <sup>a</sup></td><td>5.06 ± 1.52 <sup>a</sup></td><td>44.34 ± 6.55 <sup>b</sup></td></tr><tr><td>MeHg2</td><td>43.85 ± 20.38 <sup>a</sup></td><td>108.39 ± 21.07 <sup>a</sup></td><td>4.36 ± 1.10 <sup>a</sup></td><td>21.48 ± 11.94 <sup>a</sup></td></tr><tr><td>IHg20</td><td>47.84 ± 21.47 <sup>a</sup></td><td>104.53 ± 32.30 <sup>a</sup></td><td>4.41 ± 0.38 <sup>a</sup></td><td>15.53 ± 0.71 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>34.48 ± 13.75 <sup>b</sup></td><td>176.02 ± 73.83 <sup>a</sup></td><td>4.84 ± 1.55 <sup>a</sup></td><td>22.44 ± 13.21 <sup>ab</sup></td></tr><tr><td rowspan="5">48 h</td><td>Control</td><td>44.74 ± 9.65 <sup>a</sup></td><td>133.19 ± 25.19 <sup>a</sup></td><td>4.72 ± 0.74 <sup>a</sup></td><td>49.12 ± 13.88 <sup>a</sup></td></tr><tr><td>IHg2</td><td>87.62 ± 64.65 <sup>a</sup></td><td>94.23 ± 22.39 <sup>a</sup></td><td>4.84 ± 0.96 <sup>a</sup></td><td>26.74 ± 16.71 <sup>a</sup></td></tr><tr><td>MeHg2</td><td>34.80 ± 20.38 <sup>a</sup></td><td>127.22 ± 29.84 <sup>a</sup></td><td>5.18 ± 1.77 <sup>a</sup></td><td>25.73 ± 14.78 <sup>a</sup></td></tr><tr><td>IHg20</td><td>61.89 ± 38.45 <sup>a</sup></td><td>138.63 ± 27.14 <sup>a</sup></td><td>4.79 ± 1.49 <sup>a</sup></td><td>28.65 ± 12.81 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>39.04 ± 38.28 <sup>a</sup></td><td>121.57 ± 34.40 <sup>a</sup></td><td>4.51 ± 1.05 <sup>a</sup></td><td>43.32 ± 20.57 <sup>a</sup></td></tr><tr><td rowspan="12">Digestive gland</td><td rowspan="5">96 h</td><td>Control</td><td>66.23 ± 47.02 <sup>a</sup></td><td>144.65 ± 43.93 <sup>a</sup></td><td>4.94 ± 2.61 <sup>a</sup></td><td>34.35 ± 15.70 <sup>a</sup></td></tr><tr><td>IHg2</td><td>51.06 ± 13.02 <sup>a</sup></td><td>116.13 ± 26.76 <sup>a</sup></td><td>3.54 ± 0.65 <sup>a</sup></td><td>26.14 ± 2.00 <sup>a</sup></td></tr><tr><td>MeHg2</td><td>33.52 ± 15.54 <sup>a</sup></td><td>118.64 ± 33.42 <sup>a</sup></td><td>4.91 ± 1.44 <sup>a</sup></td><td>24.86 ± 17.29 <sup>a</sup></td></tr><tr><td>IHg20</td><td>54.56 ± 9.81 <sup>a</sup></td><td>110.04 ± 18.25 <sup>a</sup></td><td>4.38 ± 0.74 <sup>a</sup></td><td>19.46 ± 9.40 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>65.85 ± 30.33 <sup>a</sup></td><td>137.03 ± 13.28 <sup>a</sup></td><td>3.77 ± 0.58 <sup>a</sup></td><td>20.50 ± 6.27 <sup>a</sup></td></tr><tr><td rowspan="5">24 h</td><td>Control</td><td>46.32 ± 23.65 <sup>a</sup></td><td>245.07 ± 68.30 <sup>a</sup></td><td>5.97 ± 2.30 <sup>a</sup></td><td>38.77 ± 23.61 <sup>a</sup></td></tr><tr><td>IHg2</td><td>212.41 ± 89.52 <sup>b</sup></td><td>239.84 ± 47.80 <sup>a</sup></td><td>6.46 ± 1.41 <sup>a</sup></td><td>34.26 ± 19.86 <sup>a</sup></td></tr><tr><td>MeHg2</td><td>164.15 ± 37.96 <sup>b</sup></td><td>256.13 ± 69.47 <sup>a</sup></td><td>5.86 ± 1.09 <sup>a</sup></td><td>36.71 ± 18.20 <sup>a</sup></td></tr><tr><td>IHg20</td><td>96.35 ± 62.30 <sup>ab</sup></td><td>237.60 ± 100.51 <sup>a</sup></td><td>7.18 ± 3.17 <sup>a</sup></td><td>31.07 ± 15.76 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>123.93 ± 80.21 <sup>ab</sup></td><td>237.36 ± 69.54 <sup>a</sup></td><td>6.56 ± 1.58 <sup>a</sup></td><td>36.55 ± 18.97 <sup>a</sup></td></tr><tr><td rowspan="12">Rests</td><td rowspan="5">48 h</td><td>Control</td><td>93.48 ± 63.56 <sup>a</sup></td><td>236.28 ± 46.86 <sup>a</sup></td><td>4.82 ± 1.44 <sup>a</sup></td><td>55.54 ± 7.84 <sup>a</sup></td></tr><tr><td>IHg2</td><td>173.46 ± 43.06 <sup>a</sup></td><td>227.04 ± 58.03 <sup>a</sup></td><td>5.39 ± 0.26 <sup>a</sup></td><td>43.92 ± 12.01 <sup>a</sup></td></tr><tr><td>MeHg2</td><td>108.86 ± 67.05 <sup>a</sup></td><td>263.92 ± 112.80 <sup>a</sup></td><td>5.99 ± 1.71 <sup>a</sup></td><td>41.16 ± 19.86 <sup>a</sup></td></tr><tr><td>IHg20</td><td>135.49 ± 22.46 <sup>a</sup></td><td>320.98 ± 81.76 <sup>a</sup></td><td>5.06 ± 0.87 <sup>a</sup></td><td>33.79 ± 15.99 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>95.41 ± 62.15 <sup>a</sup></td><td>247.94 ± 102.08 <sup>a</sup></td><td>6.66 ± 2.07 <sup>a</sup></td><td>36.60 ± 16.46 <sup>a</sup></td></tr><tr><td rowspan="5">96 h</td><td>Control</td><td>210.42 ± 35.15 <sup>a</sup></td><td>258.30 ± 5.08 <sup>a</sup></td><td>5.03 ± 1.21 <sup>a</sup></td><td>65.51 ± 9.87 <sup>a</sup></td></tr><tr><td>IHg2</td><td>137.41 ± 73.50 <sup>a</sup></td><td>295.58 ± 80.64 <sup>a</sup></td><td>4.95 ± 0.58 <sup>a</sup></td><td>39.57 ± 8.95 <sup>a</sup></td></tr><tr><td>MeHg2</td><td>114.95 ± 27.10 <sup>a</sup></td><td>231.50 ± 93.63 <sup>a</sup></td><td>4.60 ± 1.53 <sup>a</sup></td><td>37.26 ± 11.20 <sup>a</sup></td></tr><tr><td>IHg20</td><td>145.56 ± 42.35 <sup>a</sup></td><td>307.05 ± 62.55 <sup>a</sup></td><td>6.02 ± 0.64 <sup>a</sup></td><td>39.46 ± 8.91 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>114.79 ± 53.54 <sup>a</sup></td><td>312.61 ± 106.77 <sup>a</sup></td><td>4.21 ± 0.45 <sup>a</sup></td><td>50.59 ± 13.31 <sup>a</sup></td></tr><tr><td rowspan="12">48 h</td><td>Control</td><td>29.97 ± 11.50 <sup>a</sup></td><td>140.36 ± 24.30 <sup>a</sup></td><td>4.86 ± 0.40 <sup>a</sup></td><td>34.50 ± 7.84 <sup>a</sup></td></tr><tr><td>IHg2</td><td>30.78 ± 5.30 <sup>a</sup></td><td>204.17 ± 24.05 <sup>b</sup></td><td>8.51 ± 1.41 <sup>b</sup></td><td>48.26 ± 16.02 <sup>ab</sup></td></tr><tr><td>MeHg2</td><td>37.98 ± 14.40 <sup>a</sup></td><td>143.43 ± 26.37 <sup>a</sup></td><td>7.49 ± 0.98 <sup>b</sup></td><td>81.94 ± 10.13 <sup>c</sup></td></tr><tr><td>IHg20</td><td>32.51 ± 16.56 <sup>a</sup></td><td>137.43 ± 36.84 <sup>a</sup></td><td>5.14 ± 0.33 <sup>a</sup></td><td>26.49 ± 75.08 <sup>a</sup></td></tr><tr><td>MeHg20</td><td>24.90 ± 6.44 <sup>a</sup></td><td>155.64 ± 40.86 <sup>ab</sup></td><td>2.33 ± 1.11 <sup>c</sup></td><td>56.82 ± 4.72 <sup>b</sup></td></tr><tr><td>Control</td><td>30.07 ± 6.35 <sup>a</sup></td><td>150.13 ± 19.44 <sup>a</sup></td><td>4.87 ± 0.58 <sup>a</sup></td><td>23.65 ± 4.17 <sup>a</sup></td></tr><tr><td>IHg2</td><td>40.64 ± 13.28 <sup>a</sup></td><td>167.08 ± 43.65 <sup>ab</sup></td><td>6.60 ± 1.28 <sup>ab</sup></td><td>54.93 ± 10.98 <sup>bc</sup></td></tr><tr><td>MeHg2</td><td>41.82 ± 17.74 <sup>a</sup></td><td>155.62 ± 32.46 <sup>ab</sup></td><td>6.88 ± 0.78 <sup>b</sup></td><td>76.07 ± 11.64 <sup>b</sup></td></tr><tr><td>IHg20</td><td>47.09 ± 15.63 <sup>a</sup></td><td>209.31 ± 30.19 <sup>b</sup></td><td>9.47 ± 0.78 <sup>c</sup></td><td>49.89 ± 13.52 <sup>ab</sup></td></tr><tr><td>MeHg20</td><td>49.00 ± 19.59 <sup>a</sup></td><td>144.04 ± 36.67 <sup>a</sup></td><td>2.75 ± 0.53 <sup>d</sup></td><td>65.73 ± 6.17 <sup>c</sup></td></tr><tr><td rowspan="5">96 h</td><td>Control</td><td>45.30 ± 28.25 <sup>a</sup></td><td>165.93 ± 33.36 <sup>a</sup></td><td>5.08 ± 0.57 <sup>a</sup></td><td>29.13 ± 11.48 <sup>a</sup></td></tr><tr><td>IHg2</td><td>25.85 ± 4.92 <sup>a</sup></td><td>148.87 ± 20.44 <sup>a</sup></td><td>10.08 ± 2.10 <sup>b</sup></td><td>60.86 ± 7.65 <sup>bc</sup></td></tr><tr><td>MeHg2</td><td>39.69 ± 6.40 <sup>a</sup></td><td>150.17 ± 38.09 <sup>a</sup></td><td>7.17 ± 0.43 <sup>b</sup></td><td>76.44 ± 10.00 <sup>b</sup></td></tr><tr><td>IHg20</td><td>44.59 ± 23.80 <sup>a</sup></td><td>208.19 ± 49.39 <sup>a</sup></td><td>8.70 ± 1.38 <sup>b</sup></td><td>41.68 ± 14.77 <sup>ac</sup></td></tr><tr><td>MeHg20</td><td>35.18 ± 6.54 <sup>a</sup></td><td>153.82 ± 29.08 <sup>a</sup></td><td>1.82 ± 0.47 <sup>c</sup></td><td>63.14 ± 3.56 <sup>bc</sup></td></tr></tbody></table>

investigation is necessary to confirm whether such neurotoxic effects impact feeding. In bivalves, the neural regulation of filtration for feeding remains a debated topic, as filtration rates appeared mainly influenced by the physical characteristics of microalgae and chemosensory detection (Rosa et al., 2018).

Histopathological analyses have shown that waterborne Hg bio-accumulation can significantly impact mussel tissues: IHg exposure has been reported to damage gills and disrupt the structure of digestive gland tissues, leading to necrosis, tubular atrophy, and hemocyte infiltration (Chalghmi et al., 2016; Sikkokur et al., 2020) aligning with our observations. For example, *Mytilus edulis* exposed to 50 µg/L IHg for 14 d exhibited gill injuries, including cilia erosion and severe degeneration of digestive tubules (Amachree et al., 2014). At lower IHg concentrations, *Cerastoderma edule* exposed to 10 µg/L IHg for 6 d developed fibrosis in interstitial tubules and extensive hemocytes infiltration, which we also observed here (Bejaoui et al., 2020). In the present study, MeHg exposure led to 22–50 % fibrosis and gill alterations, while 32–34 % of digestive gland tubules were affected. These effects are consistent with those seen in *D. polymorpha* exposed for 7 d to 430 ng/L of waterborne MeHg (Baratange et al., 2022). Comparing our findings with the reported effects of waterborne Hg, dietary Hg exposure appears to produce similar tissue damages in mussels. Notably, MeHg caused more

pronounced pathologies than IHg, likely due to its higher bio-accumulation, except for nuclear alterations in digestive glands. However, the scarcity of histopathological studies on MeHg in bivalves limits direct comparisons. Cellular ultrastructure, a key indicator of cellular function, has proven valuable in ecotoxicology (Segner and Braunbeck, 1990; Biagianti-Risbourg, 1997). Here, findings indicated that dietary MeHg bioaccumulation in gills leads to cellular alterations, which may compromise tissue functionality.

A selection of biomarkers was used to evaluate defense responses (Oliveira et al., 2015). We observed significant increase in CAT, GST and SOD activity in *D. polymorpha* exposed to dietary IHg2, IHg20, consistent with responses observed in other species exposed to waterborne IHg. For instance, CAT, GST, and SOD activities increased in *C. edule* exposed for 6 d to 1, 10 and 100 µg/L IHg (Bejaoui et al., 2020). Similarly, CAT and GST activities rose in *C. fluminea* exposed 8 d to 30 µg/L IHg in water (Oliveira et al., 2018). In addition, MeHg2 and MeHg20 exposures elevated SOD activity in rests, aligning with findings in *D. polymorpha* exposed for 7 d to 280 ng/L MeHg in water, which showed 1.6 × increased in SOD activity in gills and 1.2 × increase in the digestive gland (Baratange et al., 2022). This comparison reveals distinct cellular responses, although similar Hg bioaccumulation was reported in the latter study. Increase in LOOH levels, which indicate oxidative damage,
<table><caption>**Table 5**
Relative gene expression level in gills, digestive gland and rests of *cat*, *sod*, *gst*, *mt* involved in defense and *atp*, *cox*, *ldh*, *ampk*, *cs*, and *aox*, involved in energy metabolism of *D. polymorpha* exposed 24 h, 48 h and 96 h by diet to IHg and MeHg (mean ± SD, n = 3 to 6 animals; different letters show statistical significance, p < 0.05).</caption><thead><tr><th>Tissue</th><th>Time</th><th>Condition</th><th>cat</th><th>sod</th><th>gst</th><th>mt</th><th>atp</th><th>cox</th><th>ldh</th><th>ampk</th><th>cs</th><th>aox</th></tr></thead><tbody><tr><td rowspan="10">Gills</td><td rowspan="5">24 h</td><td>Ctrl</td><td>1.00 ± 0.30<sup>a</sup></td><td>1.00 ± 0.37<sup>a</sup></td><td>1.00 ± 0.36<sup>a</sup></td><td>1.00 ± 0.90<sup>a</sup></td><td>1.00 ± 0.56<sup>a</sup></td><td>1.00 ± 0.37<sup>a</sup></td><td>1.00 ± 0.42<sup>a</sup></td><td>1.00 ± 0.38<sup>a</sup></td><td>1.00 ± 0.28<sup>a</sup></td><td>1.00 ± 0.44<sup>a</sup></td></tr><tr><td>IHg2</td><td>1.38 ± 0.90<sup>a</sup></td><td>1.02 ± 0.90<sup>a</sup></td><td>1.08 ± 0.43<sup>a</sup></td><td>0.76 ± 0.55<sup>a</sup></td><td>1.37 ± 0.49<sup>a</sup></td><td>1.02 ± 0.26<sup>a</sup></td><td>1.02 ± 0.56<sup>a</sup></td><td>1.04 ± 0.81<sup>a</sup></td><td>1.08 ± 0.38<sup>a</sup></td><td>0.70 ± 0.39<sup>a</sup></td></tr><tr><td>MeHg2</td><td>0.82 ± 0.22<sup>a</sup></td><td>1.12 ± 0.53<sup>a</sup></td><td>0.85 ± 0.30<sup>a</sup></td><td>1.11 ± 0.93<sup>a</sup></td><td>1.31 ± 0.80<sup>a</sup></td><td>1.24 ± 0.74<sup>a</sup></td><td>0.85 ± 0.32<sup>a</sup></td><td>1.73 ± 1.37<sup>a</sup></td><td>1.18 ± 0.72<sup>a</sup></td><td>0.53 ± 0.47<sup>a</sup></td></tr><tr><td>IHg20</td><td>0.67 ± 0.27<sup>a</sup></td><td>1.16 ± 0.51<sup>a</sup></td><td>1.07 ± 0.39<sup>a</sup></td><td>0.73 ± 0.37<sup>a</sup></td><td>1.60 ± 0.57<sup>a</sup></td><td>1.25 ± 0.37<sup>a</sup></td><td>0.70 ± 0.34<sup>a</sup></td><td>1.49 ± 0.23<sup>a</sup></td><td>0.83 ± 0.27<sup>a</sup></td><td>0.86 ± 0.26<sup>a</sup></td></tr><tr><td>MeHg20</td><td>1.46 ± 0.64<sup>a</sup></td><td>1.82 ± 0.70<sup>a</sup></td><td>0.65 ± 0.45<sup>a</sup></td><td>1.07 ± 0.74<sup>a</sup></td><td>1.78 ± 0.67<sup>a</sup></td><td>1.29 ± 0.49<sup>a</sup></td><td>0.97 ± 0.32<sup>a</sup></td><td>0.93 ± 0.37<sup>a</sup></td><td>1.01 ± 0.36<sup>a</sup></td><td>1.34 ± 1.12<sup>a</sup></td></tr><tr><td rowspan="5">48 h</td><td>Ctrl</td><td>1.00 ± 0.26<sup>a</sup></td><td>1.00 ± 1.27<sup>a</sup></td><td>1.00 ± 0.28<sup>a</sup></td><td>1.00 ± 0.36<sup>a</sup></td><td>1.00 ± 0.48<sup>a</sup></td><td>1.00 ± 0.43<sup>a</sup></td><td>1.00 ± 0.74<sup>a</sup></td><td>1.00 ± 0.95<sup>a</sup></td><td>1.00 ± 0.42<sup>a</sup></td><td>1.00 ± 0.27<sup>a</sup></td></tr><tr><td>IHg2</td><td>0.81 ± 0.57<sup>a</sup></td><td>0.90 ± 0.24<sup>a</sup></td><td>0.83 ± 0.37<sup>a</sup></td><td>0.64 ± 0.42<sup>a</sup></td><td>1.29 ± 0.35<sup>a</sup></td><td>1.13 ± 0.49<sup>a</sup></td><td>0.62 ± 0.13<sup>a</sup></td><td>2.21 ± 0.90<sup>a</sup></td><td>1.11 ± 0.32<sup>a</sup></td><td>0.56 ± 0.10<sup>b</sup></td></tr><tr><td>MeHg2</td><td>0.61 ± 0.53<sup>a</sup></td><td>0.70 ± 0.45<sup>a</sup></td><td>0.69 ± 0.36<sup>a</sup></td><td>0.54 ± 0.21<sup>a</sup></td><td>1.40 ± 1.40<sup>a</sup></td><td>1.30 ± 1.03<sup>a</sup></td><td>0.85 ± 0.25<sup>a</sup></td><td>2.43 ± 2.17<sup>a</sup></td><td>1.25 ± 0.22<sup>a</sup></td><td>1.19 ± 0.66<sup>ab</sup></td></tr><tr><td>IHg20</td><td>1.64 ± 1.46<sup>a</sup></td><td>0.91 ± 0.39<sup>a</sup></td><td>0.95 ± 1.03<sup>a</sup></td><td>0.73 ± 0.37<sup>a</sup></td><td>1.27 ± 0.93<sup>a</sup></td><td>0.83 ± 0.42<sup>a</sup></td><td>0.59 ± 0.07<sup>a</sup></td><td>1.43 ± 0.59<sup>a</sup></td><td>1.36 ± 1.62<sup>a</sup></td><td>0.23 ± 0.34<sup>ab</sup></td></tr><tr><td>MeHg20</td><td>1.53 ± 0.98<sup>a</sup></td><td>1.14 ± 0.81<sup>a</sup></td><td>0.99 ± 0.59<sup>a</sup></td><td>1.06 ± 1.19<sup>a</sup></td><td>2.16 ± 1.23<sup>a</sup></td><td>0.83 ± 0.23<sup>a</sup></td><td>0.68 ± 0.38<sup>a</sup></td><td>1.06 ± 0.61<sup>a</sup></td><td>1.55 ± 2.30<sup>a</sup></td><td>0.59 ± 0.08<sup>b</sup></td></tr><tr><td rowspan="10">Digestive gland</td><td rowspan="5">96 h</td><td>Ctrl</td><td>1.00 ± 0.21<sup>a</sup></td><td>1.00 ± 0.64<sup>a</sup></td><td>1.00 ± 0.68<sup>a</sup></td><td>1.00 ± 0.64<sup>a</sup></td><td>1.00 ± 0.43<sup>a</sup></td><td>1.00 ± 0.33<sup>a</sup></td><td>1.00 ± 0.51<sup>a</sup></td><td>1.00 ± 0.59<sup>a</sup></td><td>1.00 ± 0.37<sup>a</sup></td><td>1.00 ± 0.50<sup>a</sup></td></tr><tr><td>IHg2</td><td>0.87 ± 0.32<sup>a</sup></td><td>0.96 ± 0.33<sup>a</sup></td><td>1.33 ± 0.21<sup>a</sup></td><td>0.49 ± 0.22<sup>a</sup></td><td>1.56 ± 1.14<sup>a</sup></td><td>0.74 ± 0.41<sup>a</sup></td><td>0.55 ± 0.11<sup>a</sup></td><td>0.92 ± 0.45<sup>a</sup></td><td>0.82 ± 0.38<sup>a</sup></td><td>0.80 ± 0.89<sup>a</sup></td></tr><tr><td>MeHg2</td><td>1.10 ± 0.40<sup>a</sup></td><td>1.25 ± 0.74<sup>a</sup></td><td>0.89 ± 0.11<sup>a</sup></td><td>0.73 ± 0.57<sup>a</sup></td><td>1.96 ± 0.63<sup>a</sup></td><td>1.01 ± 0.78<sup>a</sup></td><td>0.69 ± 0.31<sup>a</sup></td><td>1.08 ± 0.59<sup>a</sup></td><td>0.95 ± 0.45<sup>a</sup></td><td>1.71 ± 0.39<sup>a</sup></td></tr><tr><td>IHg20</td><td>1.33 ± 0.32<sup>a</sup></td><td>1.76 ± 0.86<sup>a</sup></td><td>0.96 ± 0.75<sup>a</sup></td><td>0.76 ± 0.18<sup>a</sup></td><td>1.52 ± 1.24<sup>a</sup></td><td>0.69 ± 0.53<sup>a</sup></td><td>0.86 ± 1.23<sup>a</sup></td><td>1.17 ± 1.24<sup>a</sup></td><td>1.41 ± 1.91<sup>a</sup></td><td>0.86 ± 1.28<sup>a</sup></td></tr><tr><td>MeHg20</td><td>1.04 ± 0.38<sup>a</sup></td><td>1.57 ± 0.79<sup>a</sup></td><td>0.82 ± 0.47<sup>a</sup></td><td>0.82 ± 0.36<sup>a</sup></td><td>2.12 ± 1.45<sup>a</sup></td><td>0.76 ± 0.40<sup>a</sup></td><td>0.53 ± 0.24<sup>a</sup></td><td>0.46 ± 0.24<sup>a</sup></td><td>1.16 ± 0.21<sup>a</sup></td><td>1.85 ± 1.40<sup>a</sup></td></tr><tr><td rowspan="5">24 h</td><td>Ctrl</td><td>1.00 ± 0.44<sup>a</sup></td><td>1.00 ± 0.41<sup>a</sup></td><td>1.00 ± 0.91<sup>a</sup></td><td>1.00 ± 1.04<sup>a</sup></td><td>1.00 ± 1.95<sup>a</sup></td><td>1.00 ± 1.07<sup>a</sup></td><td>1.00 ± 1.58<sup>a</sup></td><td>1.00 ± 0.68<sup>a</sup></td><td>1.00 ± 1.90<sup>a</sup></td><td>1.00 ± 0.66<sup>a</sup></td></tr><tr><td>IHg2</td><td>1.05 ± 1.47<sup>a</sup></td><td>1.35 ± 1.08<sup>a</sup></td><td>0.90 ± 0.65<sup>a</sup></td><td>0.43 ± 0.79<sup>a</sup></td><td>0.70 ± 1.08<sup>a</sup></td><td>1.29 ± 0.81<sup>a</sup></td><td>0.72 ± 0.58<sup>a</sup></td><td>1.01 ± 0.59<sup>a</sup></td><td>0.60 ± 0.22<sup>a</sup></td><td>0.65 ± 0.52<sup>a</sup></td></tr><tr><td>MeHg2</td><td>0.57 ± 0.32<sup>a</sup></td><td>1.03 ± 0.60<sup>a</sup></td><td>0.54 ± 0.24<sup>a</sup></td><td>0.64 ± 0.44<sup>a</sup></td><td>0.49 ± 0.18<sup>a</sup></td><td>0.90 ± 0.43<sup>a</sup></td><td>0.60 ± 0.32<sup>a</sup></td><td>0.59 ± 0.34<sup>a</sup></td><td>0.48 ± 0.46<sup>a</sup></td><td>0.59 ± 0.26<sup>a</sup></td></tr><tr><td>IHg20</td><td>0.68 ± 0.38<sup>a</sup></td><td>1.82 ± 0.63<sup>a</sup></td><td>0.88 ± 0.49<sup>a</sup></td><td>1.20 ± 0.40<sup>a</sup></td><td>0.51 ± 0.18<sup>a</sup></td><td>0.95 ± 0.23<sup>a</sup></td><td>0.41 ± 0.34<sup>a</sup></td><td>0.65 ± 0.44<sup>a</sup></td><td>0.54 ± 0.17<sup>a</sup></td><td>0.59 ± 0.29<sup>a</sup></td></tr><tr><td>MeHg20</td><td>1.00 ± 0.66<sup>a</sup></td><td>1.33 ± 0.31<sup>a</sup></td><td>0.67 ± 0.37<sup>a</sup></td><td>1.01 ± 0.95<sup>a</sup></td><td>0.74 ± 0.73<sup>a</sup></td><td>1.71 ± 1.85<sup>a</sup></td><td>0.47 ± 0.35<sup>a</sup></td><td>1.13 ± 0.51<sup>a</sup></td><td>0.82 ± 0.38<sup>a</sup></td><td>1.17 ± 0.44<sup>a</sup></td></tr><tr><td rowspan="5">48 h</td><td>Ctrl</td><td>1.00 ± 0.50<sup>a</sup></td><td>1.00 ± 0.54<sup>a</sup></td><td>1.00 ± 1.25<sup>a</sup></td><td>1.00 ± 1.10<sup>a</sup></td><td>1.00 ± 0.97<sup>a</sup></td><td>1.00 ± 0.36<sup>a</sup></td><td>1.00 ± 0.80<sup>a</sup></td><td>1.00 ± 0.29<sup>a</sup></td><td>1.00 ± 0.42<sup>a</sup></td><td>1.00 ± 0.52<sup>ab</sup></td></tr><tr><td>IHg2</td><td>0.63 ± 0.20<sup>a</sup></td><td>1.09 ± 0.45<sup>a</sup></td><td>0.80 ± 1.38<sup>a</sup></td><td>0.86 ± 0.61<sup>a</sup></td><td>1.03 ± 0.72<sup>a</sup></td><td>0.75 ± 0.44<sup>a</sup></td><td>0.57 ± 0.16<sup>a</sup></td><td>1.06 ± 0.28<sup>a</sup></td><td>0.82 ± 0.34<sup>a</sup></td><td>0.60 ± 0.17<sup>a</sup></td></tr><tr><td>MeHg2</td><td>0.59 ± 0.34<sup>a</sup></td><td>0.78 ± 0.44<sup>a</sup></td><td>0.71 ± 0.23<sup>a</sup></td><td>0.69 ± 0.44<sup>a</sup></td><td>1.24 ± 0.67<sup>a</sup></td><td>0.65 ± 0.43<sup>a</sup></td><td>0.47 ± 0.26<sup>a</sup></td><td>0.87 ± 0.41<sup>a</sup></td><td>0.71 ± 0.72<sup>a</sup></td><td>0.98 ± 0.59<sup>ab</sup></td></tr><tr><td>IHg20</td><td>0.60 ± 0.65<sup>a</sup></td><td>1.02 ± 1.15<sup>a</sup></td><td>0.69 ± 0.30<sup>a</sup></td><td>0.86 ± 0.25<sup>a</sup></td><td>1.02 ± 0.08<sup>a</sup></td><td>0.63 ± 0.41<sup>a</sup></td><td>0.60 ± 0.06<sup>a</sup></td><td>0.79 ± 0.48<sup>a</sup></td><td>0.61 ± 0.15<sup>a</sup></td><td>0.62 ± 0.20<sup>a</sup></td></tr><tr><td>MeHg20</td><td>0.47 ± 0.35<sup>a</sup></td><td>1.10 ± 0.44<sup>a</sup></td><td>0.62 ± 0.20<sup>a</sup></td><td>0.99 ± 0.73<sup>a</sup></td><td>1.53 ± 0.54<sup>a</sup></td><td>0.75 ± 0.34<sup>a</sup></td><td>0.69 ± 0.44<sup>a</sup></td><td>1.10 ± 0.80<sup>a</sup></td><td>1.09 ± 0.33<sup>a</sup></td><td>1.99 ± 0.24<sup>b</sup></td></tr><tr><td rowspan="10">Rests</td><td rowspan="5">96 h</td><td>Ctrl</td><td>1.00 ± 0.80<sup>a</sup></td><td>1.00 ± 0.35<sup>a</sup></td><td>1.00 ± 1.23<sup>a</sup></td><td>1.00 ± 0.70<sup>ab</sup></td><td>1.00 ± 0.57<sup>a</sup></td><td>1.00 ± 0.70<sup>a</sup></td><td>1.00 ± 0.34<sup>a</sup></td><td>1.00 ± 0.36<sup>a</sup></td><td>1.00 ± 0.29<sup>a</sup></td><td>1.00 ± 0.60<sup>a</sup></td></tr><tr><td>IHg2</td><td>1.04 ± 0.53<sup>a</sup></td><td>1.53 ± 0.58<sup>a</sup></td><td>1.31 ± 0.80<sup>a</sup></td><td>1.61 ± 1.05<sup>ab</sup></td><td>1.11 ± 0.26<sup>a</sup></td><td>1.08 ± 0.80<sup>a</sup></td><td>0.57 ± 0.51<sup>a</sup></td><td>1.24 ± 0.27<sup>a</sup></td><td>0.78 ± 0.17<sup>ab</sup></td><td>0.79 ± 0.29<sup>a</sup></td></tr><tr><td>MeHg2</td><td>0.69 ± 0.49<sup>a</sup></td><td>0.95 ± 0.61<sup>a</sup></td><td>0.48 ± 0.34<sup>a</sup></td><td>0.52 ± 0.24<sup>a</sup></td><td>0.87 ± 0.33<sup>a</sup></td><td>0.80 ± 0.30<sup>a</sup></td><td>0.50 ± 0.34<sup>a</sup></td><td>0.67 ± 0.80<sup>a</sup></td><td>0.52 ± 0.13<sup>b</sup></td><td>0.98 ± 0.35<sup>a</sup></td></tr><tr><td>IHg20</td><td>0.92 ± 0.34<sup>a</sup></td><td>1.64 ± 0.90<sup>a</sup></td><td>1.13 ± 0.83<sup>a</sup></td><td>1.44 ± 0.95<sup>b</sup></td><td>0.67 ± 0.52<sup>a</sup></td><td>0.72 ± 0.23<sup>a</sup></td><td>0.76 ± 0.26<sup>a</sup></td><td>0.95 ± 0.22<sup>a</sup></td><td>0.64 ± 0.13<sup>ab</sup></td><td>0.74 ± 0.39<sup>a</sup></td></tr><tr><td>MeHg20</td><td>0.82 ± 0.46<sup>a</sup></td><td>1.51 ± 0.83<sup>a</sup></td><td>0.83 ± 0.33<sup>a</sup></td><td>0.95 ± 0.23<sup>ab</sup></td><td>1.21 ± 0.88<sup>a</sup></td><td>0.77 ± 0.25<sup>a</sup></td><td>0.65 ± 0.29<sup>a</sup></td><td>0.79 ± 0.75<sup>a</sup></td><td>0.74 ± 0.30<sup>ab</sup></td><td>1.41 ± 1.28<sup>a</sup></td></tr><tr><td rowspan="5">24 h</td><td>Ctrl</td><td>1.00 ± 0.31<sup>a</sup></td><td>1.00 ± 0.69<sup>a</sup></td><td>1.00 ± 0.56<sup>a</sup></td><td>1.00 ± 0.67<sup>a</sup></td><td>1.00 ± 0.19<sup>ab</sup></td><td>1.00 ± 0.55<sup>a</sup></td><td>1.00 ± 0.62<sup>a</sup></td><td>1.00 ± 0.75<sup>a</sup></td><td>1.00 ± 0.52<sup>a</sup></td><td>1.00 ± 0.35<sup>a</sup></td></tr><tr><td>IHg2</td><td>0.87 ± 0.65<sup>a</sup></td><td>0.73 ± 0.46<sup>a</sup></td><td>0.73 ± 0.33<sup>a</sup></td><td>0.79 ± 0.71<sup>a</sup></td><td>0.66 ± 0.20<sup>a</sup></td><td>1.80 ± 0.74<sup>a</sup></td><td>1.07 ± 0.36<sup>a</sup></td><td>1.01 ± 0.58<sup>a</sup></td><td>0.69 ± 0.18<sup>a</sup></td><td>0.77 ± 0.41<sup>ab</sup></td></tr><tr><td>MeHg2</td><td>0.79 ± 0.53<sup>a</sup></td><td>0.80 ± 0.40<sup>a</sup></td><td>0.69 ± 0.49<sup>a</sup></td><td>0.68 ± 0.50<sup>a</sup></td><td>0.62 ± 0.41<sup>ab</sup></td><td>1.14 ± 0.82<sup>a</sup></td><td>1.02 ± 0.30<sup>a</sup></td><td>0.89 ± 0.90<sup>a</sup></td><td>1.15 ± 0.90<sup>a</sup></td><td>1.14 ± 0.56<sup>ab</sup></td></tr><tr><td>IHg20</td><td>0.74 ± 0.29<sup>a</sup></td><td>1.17 ± 0.71<sup>a</sup></td><td>1.49 ± 1.03<sup>a</sup></td><td>1.04 ± 0.41<sup>a</sup></td><td>1.54 ± 1.13<sup>b</sup></td><td>2.46 ± 1.37<sup>a</sup></td><td>1.44 ± 0.53<sup>a</sup></td><td>1.59 ± 1.41<sup>a</sup></td><td>1.11 ± 0.50<sup>a</sup></td><td>2.13 ± 0.72<sup>b</sup></td></tr><tr><td>MeHg20</td><td>1.12 ± 0.67<sup>a</sup></td><td>0.76 ± 0.38<sup>a</sup></td><td>0.76 ± 0.28<sup>a</sup></td><td>1.05 ± 1.22<sup>a</sup></td><td>0.88 ± 1.53<sup>ab</sup></td><td>1.73 ± 1.55<sup>a</sup></td><td>1.22 ± 0.31<sup>a</sup></td><td>1.02 ± 0.87<sup>a</sup></td><td>1.50 ± 0.53<sup>a</sup></td><td>1.63 ± 0.50<sup>ab</sup></td></tr><tr><td rowspan="5">48 h</td><td>Ctrl</td><td>1.00 ± 0.23<sup>a</sup></td><td>1.00 ± 0.99<sup>a</sup></td><td>1.00 ± 0.39<sup>a</sup></td><td>1.00 ± 0.56<sup>a</sup></td><td>1.00 ± 0.27<sup>a</sup></td><td>1.00 ± 0.26<sup>a</sup></td><td>1.00 ± 0.41<sup>a</sup></td><td>1.00 ± 0.30<sup>a</sup></td><td>1.00 ± 0.34<sup>a</sup></td><td>1.00 ± 0.37<sup>a</sup></td></tr><tr><td>IHg2</td><td>0.77 ± 0.22<sup>a</sup></td><td>1.77 ± 1.43<sup>a</sup></td><td>0.94 ± 0.34<sup>a</sup></td><td>0.91 ± 0.14<sup>a</sup></td><td>0.91 ± 0.52<sup>a</sup></td><td>0.78 ± 0.45<sup>a</sup></td><td>0.98 ± 0.41<sup>a</sup></td><td>1.82 ± 2.85<sup>a</sup></td><td>0.92 ± 0.68<sup>a</sup></td><td>1.12 ± 0.65<sup>a</sup></td></tr><tr><td>MeHg2</td><td>0.79 ± 0.21<sup>a</sup></td><td>1.25 ± 0.40<sup>a</sup></td><td>0.93 ± 0.34<sup>a</sup></td><td>0.95 ± 0.70<sup>a</sup></td><td>1.03 ± 0.80<sup>a</sup></td><td>0.64 ± 0.30<sup>a</sup></td><td>0.89 ± 0.27<sup>a</sup></td><td>1.10 ± 0.75<sup>a</sup></td><td>1.07 ± 0.70<sup>a</sup></td><td>1.46 ± 0.89<sup>a</sup></td></tr><tr><td>IHg20</td><td>0.63 ± 0.24<sup>a</sup></td><td>1.65 ± 1.17<sup>a</sup></td><td>0.91 ± 0.54<sup>a</sup></td><td>1.12 ± 0.83<sup>a</sup></td><td>1.01 ± 0.34<sup>a</sup></td><td>0.77 ± 0.28<sup>a</sup></td><td>0.90 ± 0.22<sup>a</sup></td><td>1.22 ± 0.73<sup>a</sup></td><td>0.95 ± 0.39<sup>a</sup></td><td>1.08 ± 0.43<sup>a</sup></td></tr><tr><td>MeHg20</td><td>1.07 ± 0.27<sup>a</sup></td><td>1.46 ± 1.85<sup>a</sup></td><td>0.76 ± 0.32<sup>a</sup></td><td>0.84 ± 0.89<sup>a</sup></td><td>1.07 ± 0.92<sup>a</sup></td><td>0.81 ± 0.55<sup>a</sup></td><td>1.02 ± 0.54<sup>a</sup></td><td>1.31 ± 1.19<sup>a</sup></td><td>1.40 ± 0.70<sup>a</sup></td><td>1.21 ± 0.57<sup>a</sup></td></tr><tr><td rowspan="5">96 h</td><td>Ctrl</td><td>1.00 ± 0.46<sup>a</sup></td><td>1.00 ± 0.68<sup>a</sup></td><td>1.00 ± 0.57<sup>a</sup></td><td>1.00 ± 0.38<sup>ab</sup></td><td>1.00 ± 0.34<sup>a</sup></td><td>1.00 ± 0.16<sup>a</sup></td><td>1.00 ± 0.35<sup>a</sup></td><td>1.00 ± 0.28<sup>a</sup></td><td>1.00 ± 0.34<sup>a</sup></td><td>1.00 ± 0.20<sup>a</sup></td></tr><tr><td>IHg2</td><td>1.24 ± 0.71<sup>a</sup></td><td>1.92 ± 1.01<sup>ab</sup></td><td>2.02 ± 0.41<sup>a</sup></td><td>0.55 ± 0.79<sup>ab</sup></td><td>1.81 ± 0.68<sup>a</sup></td><td>0.26 ± 0.82<sup>a</sup></td><td>1.19 ± 0.09<sup>a</sup></td><td>1.16 ± 0.59<sup>a</sup></td><td>1.16 ± 0.54<sup>a</sup></td><td>0.89 ± 0.40<sup>a</sup></td></tr><tr><td>MeHg2</td><td>1.07 ± 0.65<sup>a</sup></td><td>1.29 ± 0.65<sup>ab</sup></td><td>1.15 ± 1.01<sup>a</sup></td><td>1.96 ± 1.30<sup>a</sup></td><td>1.29 ± 0.17<sup>a</sup></td><td>1.12 ± 0.43<sup>a</sup></td><td>1.23 ± 0.27<sup>a</sup></td><td>1.07 ± 1.07<sup>a</sup></td><td>1.30 ± 0.37<sup>a</sup></td><td>0.99 ± 0.46<sup>a</sup></td></tr><tr><td>IHg20</td><td>1.16 ± 0.21<sup>a</sup></td><td>2.25 ± 1.00<sup>ab</sup></td><td>1.50 ± 1.27<sup>a</sup></td><td>1.35 ± 0.76<sup>ab</sup></td><td>1.22 ± 0.35<sup>a</sup></td><td>0.94 ± 0.48<sup>a</sup></td><td>1.47 ± 0.46<sup>a</sup></td><td>1.11 ± 0.34<sup>a</sup></td><td>1.44 ± 0.48<sup>a</sup></td><td>1.03 ± 0.54<sup>a</sup></td></tr><tr><td>MeHg20</td><td>1.13 ± 0.08<sup>a</sup></td><td>2.74 ± 0.50<sup>b</sup></td><td>1.90 ± 0.97<sup>a</sup></td><td>0.33 ± 1.04<sup>b</sup></td><td>1.31 ± 0.47<sup>a</sup></td><td>1.02 ± 0.62<sup>a</sup></td><td>1.28 ± 0.25<sup>a</sup></td><td>1.25 ± 0.81<sup>a</sup></td><td>1.16 ± 0.51<sup>a</sup></td><td>1.36 ± 0.71<sup>a</sup></td></tr></tbody></table>
were also observed 2.5 × higher in *C. fluminea* compared to control (Oliveira et al., 2018), aligning with our observations in rests of *D. polymorpha* exposed to IHg and MeHg at 24, 48 and 96 h. Among the three tissues examined, rests showed the most rapid and highest lipid peroxidation levels, while gills showed no oxidative stress. Our study demonstrated that Hg ingested through contaminated microalgae influenced antioxidant enzyme activity and caused lipid peroxidation in *D. polymorpha* tissues, both through direct exposure and after digestion. This suggested that dietborne Hg can impair or even overwhelm the repair and protective mechanisms against oxidative damage. Conversely, Hg exposure through water and diet elicited broadly similar overall responses in *D. polymorpha*, likely due to inherent chemical properties of Hg. However, the specific exposure pathway and the form of Hg led to distinct, nuanced responses when examined in detail.

In our experimental conditions, we observed changes in the expression of genes related to defense responses. Specifically, *sod* transcripts were upregulated in rests of *D. polymorpha* exposed to MeHg20 compared to control. This upregulation of *sod* transcripts may suggest an increase in transcription to counter the observed decrease in SOD activity when *D. polymorpha* is exposed to MeHg20. Similar upregulation of *sod* and *cat* transcripts was previously reported in gills of *D. polymorpha* exposed 7 d to 430 ng/L waterborne MeHg (Baratange et al., 2022), consistent with the increased ROS production often associated with Hg exposure and its impact on antioxidant defenses. In addition, we observed only one significant gene response in MeHg20 at 96h within rests, which are indirectly exposed by microalgal digestion. Mitochondria and energy metabolism are additional key toxicity targets of Hg. MeHg exposure impacted mitochondria (Ferreira et al., 2018; Corrêa et al., 2020), causing mitochondrial membrane depolarization in synaptosomes (Hare and Atchison, 1992), as well as mitochondrial bodies and membrane damage that impair metabolic capacities (Baratange et al., 2022). Previous studies showed that proteins involved in energy metabolism were modulated, as observed via shotgun proteomics in *D. polymorpha* exposed to 430 ng/L waterborne MeHg (Baratange et al., 2023). Here, transcripts associated with energy metabolism were downregulated in gills and digestive gland: *cs* is involved in the first phase of the Krebs cycle (Siu et al., 2003) and *aox* in the integration of carbon metabolism and electron transport (Vanlerberghe and Ordog, 2002). Their downregulation suggested an impact of Hg on the energy metabolism of *D. polymorpha*, particularly in organs directly exposed to contaminated food. Overall, the distinct response patterns observed between IHg and MeHg exposure supported that each Hg form had different molecular toxicity targets.

## 5. Conclusion

Data presented here demonstrated that dietborne Hg led to molecular and cellular damage in *D. polymorpha* across all three studied tissues. The most pronounced histological effects occurred at higher concentrations of Hg, with greater severity observed for MeHg compared to IHg exposures, aligning with the differences in BAF. Additionally, the filtration behavior of *D. polymorpha* indicated varying toxicity between dietary MeHg and IHg, though both forms led to significant bioaccumulation within *D. polymorpha* tissues. The use of enriched stable isotopes of Hg would be useful to characterize its speciation and bioconversion in microalgae and zebra mussels to further characterize the dietary transfer of Hg between the two species. Overall, these data suggested that exposure to dietborne IHg and MeHg caused toxicity symptoms similar to those observed with waterborne Hg exposure. However, the effects varied between the gills, digestive gland, and rests. This variability likely arises from the different modes of exposure: gills are affected by direct contact, the digestive gland is impacted through both contact and digestion, while rests are influenced exclusively after digestion. This highlights the importance of understanding the fate of Hg in relation to the observed responses. For example, genes involved in antioxidant responses were activated in rests, whereas genes

involved in energy metabolism were affected in gills and the digestive gland. In addition, differences in effects between IHg and MeHg also plaid for different toxicity pathways. These differences are not fully elucidated by endpoints used here. Non-targeted high throughput approaches, such as metabolomic and proteomic could provide valuable insights into the molecular toxicity pathways of IHg and MeHg.

Due to their ecological significance, abundance, and potential for biomonitoring, it is crucial to study the fate and cellular toxicity pathways of dietary Hg at environmental concentrations in representative bivalves. Aquatic ecosystems are vulnerable and interconnected. Hg contamination can affect the health of various species within these ecosystems, from plankton, low trophic level invertebrates to fish and mammals, including humans. Governments and environmental organizations require comprehensive data on Hg risks to develop more effective regulations and conservation strategies. Understanding the dietary pathways of Hg and the associated toxicity will help assess its overall impact on biodiversity and ecosystem stability.

## CRediT authorship contribution statement

Clarisse Seguin: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. Alice Marant: Methodology, Investigation, Formal analysis. Séverine Palacios-Paris: Writing – review & editing, Validation, Resources, Investigation. Isabelle Bonnard: Investigation. Jean-Luc Loizeau: Writing – review & editing, Validation, Resources. Elise David: Writing – review & editing, Validation, Resources. Damien Rioult: Writing – review & editing, Supervision, Methodology, Investigation. Claudia Cosio: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Claudia Cosio reports financial support was provided by Greater Reims Urban Community. Claudia Cosio reports financial support was provided by MeSeine Innovation. Claudia Cosio reports financial support was provided by French National Research Agency (ANR-23-CE34-0019 Mercur-Food). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We are grateful to the URCA Tech Mobicycle Platform at URCA for access to cytometry, qPCR facilities and technical support. Authors would like to thank Dr Iris Barjhoux for her guidance and support in navigating the statistical analyses, as well as Laurence Delahaut, Arthur Marchand, Nicolas Borie and Benjamin Bertaux for technical help. Authors also benefited from the French GDR “Aquatic Ecotoxicology” framework which aims at fostering stimulating scientific discussions and collaborations for more integrative approaches. The designs used for the graphical abstract come from www.freepik.com.

## Appendix A Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2025.144306.

## Data availability

Data will be made available on request.
## References

Amachree, D., Moody, A.J., Handy, R.D., 2014. Comparison of intermittent and continuous exposures to inorganic mercury in the mussel *Mytilus edulis*: accumulation and sub-lethal physiological effects. Ecotoxicol. Environ. Saf. 109, 133-142. https://doi.org/10.1016/j.ecoenv.2014.07.025.

Anandraj, A., Marshall, D., Gregory, M., McClurg, T., 2002. Metal accumulation, filtration and O2 uptake rates in the mussel *Perna perna* (Mollusca: Bivalvia) exposed to Hg2+, Cu2+ and Zn2+. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 132, 355-363. https://doi.org/10.1016/S1532-0456(02)00081-9.

Baatrup, E., 1991. Structural and functional effects of heavy metals on the nervous system, including sense organs, of fish. Comp. Biochem. Physiol., C: Compar. Pharmacol. 100, 253-257. https://doi.org/10.1016/0742-8413(91)09103-6.

Baldwin, B.S., Mayer, M.S., Dayton, J., Pau, N., Mendilla, J., Sullivan, M., Moore, A., Ma, A., Mills, E.L., 2002. Comparative growth and feeding in zebra and quagga mussels (*Dreissena polymorpha* and *Dreissena bugensis*): Implications for North American lakes. Can. J. Fish. Aquat. Sci. 59, 680-694. https://doi.org/10.1139/f02-043.

Baratange, C., Baali, H., Gaillet, V., Bonnard, I., Delahaut, L., Gaillard, J.-C., Grandjean, D., Sayen, S., Gallorini, A., Le Bris, N., Renault, D., Breider, F., Loizeau, J.-L., Armengaud, J., Cosio, C., 2023. Bioaccumulation and molecular effects of carbamazepine and methylmercury co-exposure in males of *Dreissena polymorpha*. Sci. Total Environ. 897, 165379. https://doi.org/10.1016/j.scitotenv.2023.165379.

Baratange, C., Paris-Palacios, S., Bonnard, I., Delahaut, L., Grandjean, J., Wortham, L., Sayen, S., Gallorini, A., Michel, J., Renault, D., Breider, F., Loizeau, J.-L., Cosio, C., 2022. Metabolic, cellular and defense responses to single and co-exposure to carbamazepine and methylmercury in *Dreissena polymorpha*. Environ. Pollut. 300, 118933. https://doi.org/10.1016/j.envpol.2022.118933.

Bejaoui, S., Chaabane, F., Fouzai, C., Chetoui, I., Chalbi, E., Nechi, S., Chalghaf, M., El Cafi, M., Soudani, N., 2020. Exploring the impacts of mercury chloride exposure on fatty acids profile, oxidative stress response and histomorphological aspect of *Cerastoderma edule* detoxifying organs. Ecol. Indic. 118, 106798. https://doi.org/10.1016/j.ecolind.2020.106798.

Biagianti-Risbourg, S., 1997. Les perturbations (ultra)structurales du foie des poissons utilisées comme biomarqueurs de la qualité sanitaire des milieux aquatiques. In: Lagadic Amiard, J.C., Caquet, T., Ramande, F. (Eds.), Utilisation de biomarqueurs en écotoxicologie, aspects fondamentaux. Masson Pub., Paris, pp. 355-391.

Blinick, N.S., Link, D., Ahrenstorff, T.D., Bethke, B.J., Fleishman, A.B., Janssen, S.E., Krabbenhoft, D.P., Nelson, J.K.R., Rantalai, H.M., Rude, C.L., Hansen, G.J.A., 2024. Increased mercury concentrations in walleye and yellow perch in lakes invaded by zebra mussels. Sci. Total Environ. 957, 177515. https://doi.org/10.1016/j.scitotenv.2024.177515.

Bravo, A.G., Cosio, C., Amouroux, D., Zopfi, J., Chevalley, P.-A., Spangenberg, J.E., Ungureanu, V.-G., Dominik, J., 2014. Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chlor-alkali plant. Water Res. 49, 391-405. https://doi.org/10.1016/j.watres.2013.10.024.

Carrasco, L., Díez, S., Soto, D.X., Catalan, J., Bayona, J.M., 2008. Assessment of mercury and methylmercury pollution with zebra mussel (*Dreissena polymorpha*) in the Ebro River (NE Spain) impacted by industrial hazardous dumps. Sci. Total Environ. 407, 178-184. https://doi.org/10.1016/j.scitotenv.2008.07.031.

Chalghmi, H., Bourdineaud, J.-P., Haouas, Z., Gourves, P.-Y., Zrafi, I., Saidane-Mosbahi, D., 2016. Transcriptomic, biochemical and histopathological responses of the clam *Ruditapes decussatus* from a metal-contaminated Tunis Lagoon. Environ. Contam. Toxicol. 70, 241-256. https://doi.org/10.1007/s00244-015-0185-0.

Corrèa, M.G., Bittencourt, L.O., Nascimento, P.C., Ferreira, R.O., Aragão, W.A.B., Silva, M.C.F., Gomes-Leal, W., Fernandes, M.S., Dionízio, A., Buzalaf, M.R., Crespo-Lopez, M.E., Lima, R.R., 2020. Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: ultrastructural, proteomic and biochemical damages associated with reduced neuronal density. Ecotoxicol. Environ. Saf. 191, 110159. https://doi.org/10.1016/j.ecoenv.2019.110159.

Depew, D.C., Basu, N., Burgess, N.M., Campbell, L.M., Devlin, E.W., Drevnick, P.E., Hammerschmidt, C.R., Murphy, C.A., Sandheinrich, M.B., Wiener, J.G., 2012. Toxicity of dietary methylmercury to fish: derivation of ecologically meaningful threshold concentrations. Environ. Toxicol. Chem. 31, 1536-1547. https://doi.org/10.1002/etc.1859.

Driscoll, C.T., Mason, R.P., Chan, H.M., Jacob, D.J., Pirrone, N., 2013. Mercury as a global pollutant: sources, pathways, and effects. Environ. Sci. Technol. 47, 4967-4983. https://doi.org/10.1021/es305071v.

Elia, A.C., Galarini, R., Taticchi, M.I., Dörr, A.J.M., Mantilacci, L., 2003. Antioxidant responses and bioaccumulation in *Ictalurus melas* under mercury exposure. Ecotoxicol. Environ. Saf. 55, 162-167. https://doi.org/10.1016/S0147-6513(02)00123-9.

Evers, D.C., Ackerman, J.T., Åkerblom, S., Bally, D., Basu, N., Bishop, K., Bodin, N., Braaten, H.F.V., Burton, M.E.H., Bustamante, P., Chen, C., Chételat, J., Christian, L., Dietz, R., Drevnick, P., Eagles-Smith, C., Fernandez, L.E., Hammerschlag, N., Harnelin-Vivien, M., Harte, A., Krümmel, E.M., Brito, J.L., Medina, G., Barrios Rodriguez, C.A., Stenhouse, I., Sunderland, E., Takeuchi, A., Tear, T., Vega, C., Wilson, S., Wu, P., 2024. Global mercury concentrations in biota: their use as a basis for a global biomonitoring framework. Ecotoxicology 33, 325-396. https://doi.org/10.1007/s10646-024-02747-x.

Faria, M., Carrasco, L., Díez, S., Riva, M.C., Bayona, J.M., Barata, C., 2009. Multi-biomarker responses in the freshwater mussel *Dreissena polymorpha* exposed to

polychlorobiphenyls and metals. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149, 281-288. https://doi.org/10.1016/j.cbpbc.2008.07.012.

Fernández Robledo, J.A., Yadavalli, R., Allam, B., Pales Espinosa, E., Gerdol, M., Greco, S., Stevick, R.J., Gómez-Chiarri, M., Zhang, Y., Heil, C.A., Tracy, A.N., Bishop-Bailey, D., Metzger, M.J., 2019. From the raw bar to the bench: bivalves as models for human health. Dev. Comp. Immunol. 92, 260-282. https://doi.org/10.1016/j.dci.2018.11.020.

Ferreira, F.F., Nazari, E.M., Müller, Y.M.R., 2018. MeHg causes ultrastructural changes in mitochondria and autophagy in the spinal cord cells of chicken embryo. J. Toxicol. 2018, 8460490. https://doi.org/10.1155/2018/8460490.

García-Medina, S., Galar-Martínez, M., Gómez-Oliván, L.M., Ruiz-Lara, K., Islas-Flores, H., Gasca-Pérez, E., 2017. Relationship between genotoxicity and oxidative stress induced by mercury on common carp (*Cyprinus carpio*) tissues. Aquat. Toxicol. 192, 207-215. https://doi.org/10.1016/j.aquatox.2017.09.019.

Géba, E., Rioult, D., Palluel, O., Dedourge-Geffard, O., Betouille, S., Aubert, D., Bigot-Clivot, A., 2021. Resilience of *Dreissena polymorpha* in wastewater effluent: use as a bioremediation tool? J. Environ. Manag. 278, 111513. https://doi.org/10.1016/j.jenvman.2021.111513.

Gentès, S., Maury-Brachet, R., Feng, C., Pedrero, Z., Tessier, E., Legeay, A., Mesmer-Dudons, N., Baudrimont, M., Maurice, L., Amouroux, D., Gonzalez, P., 2015. Specific effects of dietary methylmercury and inorganic mercury in zebrafish (*Danio rerio*) determined by genetic, histological, and metallothionein responses. Environ. Sci. Technol. 49, 14560-14569. https://doi.org/10.1021/acs.est.5b03586.

Hare, M.F., Atchison, W.D., 1992. Comparative action of methylmercury and divalent inorganic mercury on nerve terminal and intraterritorial mitochondrial membrane potentials. J. Pharmacol. Exp. Therapeut. 261, 166-172.

Jacuin, L., Gandar, A., Aguirre-Smith, M., Perrault, A., Hénaff, M.L., Jong, L.D., Paris-Palacios, S., Laffaille, P., Jean, S., 2019. High temperature aggravates the effects of pesticides in goldfish. Ecotoxicol. Environ. Saf. 172, 255-264. https://doi.org/10.1016/j.ecoenv.2019.01.085.

Jaffal, S., Betouille, S., Biagianti-Risbourg, S., Terreau, A., Sanchez, W., Paris-Palacios, S., 2015. Heavy metal contamination and hepatic toxicological responses in brown trout (*Salmo trutta*) from the Kerguelen Islands. Polar Res. https://doi.org/10.3402/polar.v34.22784.

Järup, L., 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68, 167-182. https://doi.org/10.1093/bmb/lg032.

Kádár, E., Costa, V., Santos, R.S., Lopes, H., 2005. Behavioural response to the bioavailability of inorganic mercury in the hydrothermal mussel *Thymopylioides azoricus*. J. Exp. Biol. 208, 505-513. https://doi.org/10.1242/jeb.01415.

Kobak, J., Kakareko, T., 2011. The effectiveness of the induced anti-predator behaviour of zebra mussel *Dreissena polymorpha* in the presence of molluscivorous roach *Rutilus rutilus*. Aquat. Ecol. 45, 357-366. https://doi.org/10.1007/s10452-011-9359-7.

Lee, Y.H., Kang, H.-M., Kim, D.-H., Wang, M., Jeong, C.-B., Lee, J.-S., 2017. Adverse effects of methylmercury (MeHg) on life stages, antioxidant systems, and MAPK signaling pathways in the copepod *Tigrionus japonicus*. Aquat. Toxicol. 184, 133-141. https://doi.org/10.1016/j.aquatox.2017.01.010.

Ludovisi, A., Goretti, E., Pallottini, M., Lucentini, L., Pizzirani, C., Vizzini, S., Mancinelli, G., 2022. Stable isotope analysis reveals trophic segregation between the invasive zebra mussel *Dreissena polymorpha* and the native duck mussel *Anodonta anatina* in Lake Trasimeno (Italy). Hydrobiologia 849, 2091-2108. https://doi.org/10.1007/s10750-022-04846-4.

Mao, L., Liu, X., Wang, Z., Wang, B., Lin, C., Xin, M., Zhang, B.-T., Wu, T., He, M., Ouyang, W., 2021. Trophic transfer and dietary exposure risk of mercury in aquatic organisms from urbanized coastal ecosystems. Chemosphere 281, 130836. https://doi.org/10.1016/j.chemosphere.2021.130836.

Medler, S., Silverman, H., 1997. Functional organization of intrinsic gill muscles in zebra mussels, *Dreissena polymorpha* (Mollusca: Bivalvia), and response to transmitters in vitro. Invertebr. Biol. 116, 200-212. https://doi.org/10.2307/3226897.

Molloy, D.P., Karatayev, A.Y., Burlakova, L.E., Kurandina, D.P., Luriele, F., 1997. Natural enemies of zebra mussels: predators, parasites, and ecological competitors. Rev. Fish. Sci. Aquac. 5, 27-97. https://doi.org/10.1080/10641269709388593.

Morcillo, P., Esteban, M.A., Cuesta, A., Morcillo, P., Esteban, M.A., Cuesta, A., 2017. Mercury and its toxic effects on fish. AIMS Environ. Sci. 4, 386-402. https://doi.org/10.3934/envIRON.2017.3.386.

Newell, R.I.E., 2004. Ecosystem influences of natural and cultivated populations of suspension-feeding bivalve molluscs: a review. J. Shellfish Res. 23, 51-62.

Niane, B., Guédron, S., Moritz, R., Cosio, C., Ngom, P.M., Deverajan, N., Pfeifer, H.R., Poté, J., 2015. Human exposure to mercury in artisanal small-scale gold mining areas of Kedougon region, Senegal, as a function of occupational activity and fish consumption. Environ. Sci. Pollut. Res. 22, 7101-7111. https://doi.org/10.1007/s11356-014-3913-5.

Oliveira, P., Barboza, L.G.A., Branco, V., Figueiredo, N., Carvalho, C., Guilhermino, L., 2018. Effects of microplastics and mercury in the freshwater bivalve *Corbicula fluminea* (Müller, 1774): filtration rate, biochemical biomarkers and mercury bioconcentration. Ecotoxicol. Environ. Saf. 164, 155-163. https://doi.org/10.1016/j.ecoenv.2018.07.062.

Oliveira, P., Lopes-Lima, M., Machado, J., Guilhermino, L., 2015. Comparative sensitivity of European native (*Anodonta anatina*) and exotic (*Corbicula fluminea*) bivalves to mercury. Estuar. Coast Shelf Sci. 167, 191-198. https://doi.org/10.1016/j.ecss.2015.06.014.

Pack, E. chul, Lee, S., Kim, C., Lim, C., Sung, D., Kim, M., Park, K., Lim, K., Choi, D., Kim, S., 2014. Effects of environmental temperature change on mercury absorption in aquatic organisms with respect to climate warming. J. Toxicol. Environ. Health, Part A 77, 1477-1490. https://doi.org/10.1080/15287394.2014.955892.

Palos-Ladeiro, M., Barjhoux, I., Bigot-Clivot, A., Bonnard, M., David, E., Dedourge-Geffard, O., Geba, E., Lance, E., Lepretre, M., Magniez, G., Rioult, D., Aubert, D.,
Villena, I., Daniele, G., Salvador, A., Vulliet, E., Armengaud, J., Geffard, A., Ladeiro, M.P., Barjhoux, I., Bigot-Clivot, A., Bonnard, M., David, E., Dedourge-Geffard, O., Geba, E., Lance, E., Lepretre, M., Magniez, G., Rioult, D., Aubert, D., Villena, I., Daniele, G., Salvador, A., Vulliet, E., Armengaud, J., Geffard, A., 2017. Mussel as a tool to define continental watershed quality. In: Organismal and Molecular Malacology. IntechOpen. https://doi.org/10.5772/67995.

Paris-Palacios, S., Biagjanti-Risbourg, S., Vernet, G., 2000. Biochemical and (ultra) structural hepatic perturbations of *Brachydanio rerio* (Teleostei, Cyprinidae) exposed to two sublethal concentrations of copper sulfate. Aquat. Toxicol. 50, 109–124. https://doi.org/10.1016/S0166-445X(99)00090-9.

Roditi, H.A., Fisher, N.S., Sãoudo-Wilhelmy, S.A., 2000. Uptake of dissolved organic carbon and trace elements by zebra mussels. Nature 407, 78–80. https://doi.org/10.1038/35024069.

Rosa, M., Ward, J.E., Shumway, S.E., 2018. Selective capture and ingestion of particles by suspension-feeding bivalve molluscs: a review. J. Shellfish Res. 37, 727–746. https://doi.org/10.2983/035.037.0405.

Sauvey, A., Denis, F., Hégaret, H., Le Roy, B., Lelong, C., Jolly, O., Pavie, M., Fauchot, J., 2021. Interactions between filter-feeding bivalves and toxic diatoms: influence on the feeding behavior of *Crassostrea gigas* and *Pecten maximus* and on toxin production by *Pseudo-nitzschia*. Toxins 13, 577. https://doi.org/10.3390/toxins13080577.

Schaefer, K., Elshorbany, Y., Jafarov, E., Schuster, P.F., Striegl, R.G., Wickland, K.P., Sunderland, E.M., 2020. Potential impacts of mercury released from thawing permafrost. Nat. Commun. 11, 4650. https://doi.org/10.1038/s41467-020-18398-5.

Segner, H., Braunbeck, T., 1990. Qualitative and quantitative assessment of the response of milkfish, *Chanos chanos*, fry to low-level copper exposure. In: Perkins, F.O., Cheng, T.C. (Eds.), Pathology in Marine Science. Academic Press, San Diego, pp. 347–368. https://doi.org/10.1016/B978-0-12-550755-4.50040-9.

Siu, P.M., Donley, D.A., Bryner, R.W., Alway, S.E., 2003. Citrate synthase expression and enzyme activity after endurance training in cardiac and skeletal muscles. J. Appl. Physiol. 94, 555–560. https://doi.org/10.1152/japplphysiol.00821.2002.

Sikdokur, E., Belivermiş, M., Sezer, N., Pekmez, M., Bulan, Ö.K., Kılıç, Ö., 2020. Effects of microplastics and mercury on manila clam *Ruditapes philippinarum*: feeding rate, immunomodulation, histopathology and oxidative stress. Environ. Pollut. 262, 114247. https://doi.org/10.1016/j.envpol.2020.114247.

Smith, C.S., Ito, M., Namba, M., Nakaoka, M., 2018. Oyster aquaculture impacts *Zostera marina* epibiont community composition in Akkeshi-ko estuary, Japan. PLoS One 13, e0197753. https://doi.org/10.1371/journal.pone.0197753.

Sun, Y., Li, Y., Rao, J., Liu, Z., Chen, Q., 2018. Effects of inorganic mercury exposure on histological structure, antioxidant status and immune response of immune organs in yellow catfish (*Pelteobagrus fulvidraco*). J. Appl. Toxicol. 38, 843–854. https://doi.org/10.1002/jat.3592.

Tran, D., Fournier, E., Durrieu, G., Massabuau, J.-C., 2007. Inorganic mercury detection by valve closure response in the freshwater clam *Corbicula fluminea*: integration of time and water metal concentration changes. Environ. Toxicol. Chem. 26 (1), 1545–1551. https://doi.org/10.1897/06-390R1.

Vanlerberghe, G.C., Ordog, S.H., 2002. Alternative oxidase: integrating carbon metabolism and electron transport in plant respiration. In: Foyer, C.H., Noctor, G. (Eds.), Photosynthetic Nitrogen Assimilation and Associated Carbon and Respiratory Metabolism. Springer, Netherlands, Dordrecht, pp. 173–191. https://doi.org/10.1007/0-306-48138-3_11.

Van Rooyen, D., Erasmus, J.H., Gerber, R., Nachev, M., Sures, B., Wepener, V., Smit, N.J., 2023. Bioaccumulation and trophic transfer of total mercury through the aquatic food webs of an African sub-tropical wetland system. Sci. Total Environ. 889, 164210. https://doi.org/10.1016/j.scitotenv.2023.164210.

Wang, S., Li, B., Zhang, M., Xing, D., Jia, Y., Wei, C., 2012. Bioaccumulation and trophic transfer of mercury in a food web from a large, shallow, hypereutrophic lake (Lake Taihu) in China. Environ. Sci. Pollut. Res. 19, 2820–2831. https://doi.org/10.1007/s11356-012-0787-2.

Wang, J., Koopman, K.R., Collas, F.P.L., Posthuma, L., de Nijs, T., Leuven, R.S.E.W., Hendriks, A.J., 2021. Towards an ecosystem service-based method to quantify the filtration services of mussels under chemical exposure. Sci. Total Environ. 763, 144196. https://doi.org/10.1016/j.scitotenv.2020.144196.

Yang, L., Zhang, Y., Wang, F., Luo, Z., Guo, S., Strähle, U., 2020. Toxicity of mercury: molecular evidence. Chemosphere 245, 125586. https://doi.org/10.1016/j.chemosphere.2019.125586.

Yu, R.-Q., Reinfelder, J.R., Hines, M.E., Barkay, T., 2018. Syntrophic pathways for microbial mercury methylation. ISME J. 12, 1826–1835. https://doi.org/10.1038/s41396-018-0106-0.

Zhang, Q.-H., Huang, L., Zhang, Y., Ke, C.-H., Huang, H.-Q., 2013. Proteomic approach for identifying gonad differential proteins in the oyster (*Crassostrea angulata*) following food-chain contamination with HgCl2. J. Proteomics 94, 37–53. https://doi.org/10.1016/j.jprot.2013.08.018.