# Transformations of aquatic mercury species by the diatom Cyclotella meneghiniana

João Santos, Javier Garcia-Calleja, Emmanuel Tessier, Séverine Le Faucheur, Zoyne Pedrero, David Amouroux, Vera Slaveykova

► To cite this version:

João Santos, Javier Garcia-Calleja, Emmanuel Tessier, Séverine Le Faucheur, Zoyne Pedrero, et al. "Transformations of aquatic mercury species by the diatom Cyclotella meneghiniana." Environmental Pollution, 2025, 374, pp.126248. 10.1016/j.envpol.2025.126248. hal-05044701

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# Transformations of aquatic mercury species by the diatom
*Cyclotella meneghiniana*☆

João P. Santos<sup>a</sup>, Javier Garcia-Calleja<sup>b D</sup>, Emmanuel Tessier<sup>b</sup>, Séverine Le Faucheur<sup>b</sup>,
Zoyne Pedrero<sup>b</sup>, David Amouroux<sup>b D</sup>, Vera I. Slaveykova<sup>a, * D</sup>

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

## ARTICLE INFO

### Keywords:

- Methylmercury
- Inorganic mercury
- Accumulation
- Demethylation
- Hg(II) reduction

## ABSTRACT

Phytoplankton are traditionally viewed as simple bioaccumulators and key entry points for mercury (Hg) into aquatic trophic chain. However the more recent findings suggest that they can function as dynamic biological systems capable of enhancing Hg cycling reactivity and altering its speciation. Nevertheless, the role of phytoplankton species in mercury transformations remains largely overlooked. The present study examined inorganic mercury (Hg(II)) methylation, monomethylmercury (MeHg) demethylation, and the production of dissolved gaseous mercury (Hg(0)) following exposure to sub-nanomolar concentrations Hg(II) or MeHg, representative of contaminated environments. Diatom *Cyclotella meneghiniana* was selected as a representative phytoplankton species due to its widespread presence in diverse aquatic ecosystems. To track transformation pathways, isotopically labeled Hg species were used to distinguish between methylation and demethylation processes. The results demonstrated rapid accumulation of both Hg(II) and MeHg in the diatom cells. A cellular demethylation of MeHg into Hg(II), primarily occurring within the cell debris fraction, but no detectable Hg(II) methylation was observed. The reduction of Hg(II) to Hg(0) was found to be biologically mediated and independent of the photosynthetic system. No significant production of Hg(0) after MeHg exposure was determined. Overall, these findings imply that phytoplankton species could actively contribute to mercury cycling in aquatic environments through cellular transformation processes, including MeHg demethylation and Hg(II) reduction.

## 1. Introduction

Mercury is a ubiquitous global pollutant (Pavithra et al., 2023). In freshwater systems, mercury can be found in different forms, with inorganic mercury (Hg(II)) being the most abundant species, followed by monomethylmercury (CH₃Hg⁺ or MeHg) and gaseous elemental mercury (Hg(0)) (Branfireun et al., 2020). Different biotic and abiotic transformation processes control mercury speciation in aquatic environment, including methylation/demethylation, reduction/oxidation and sulfidation (Gao et al., 2022). The understanding of these transformations and speciation is central for improved understanding of the toxicity and trophic transfer efficiency of bioaccumulated Hg(II) and MeHg (Le Faucheur et al., 2014). Extensive systematic work and recent advances in microbial mercury methylation (Peng et al., 2024),

demethylation (Barkay and Gu, 2022) and photochemical reduction (Oladoye et al., 2024) have been comprehensively reviewed.

Phytoplankton, traditionally regarded as simple bioaccumulators and primary entry points of mercury into the trophic chain (Le Faucheur et al., 2014), have received comparatively little attention for their potential role in actively transforming mercury at the cellular level. However, evidences suggest that these phytoplankton species can influence mercury cycling through biochemical processes, such as MeHg demethylation and the reduction of Hg(II). Indeed, MeHg demethylation has been observed in multiple algal and cyanobacterial species (Bravo et al., 2014; Franco et al., 2018; Li et al., 2022; Yin et al., 2022). Reduction and demethylation were also demonstrated in the diatom *Thalasiosira weissflogii* (Devars et al., 2000; Morelli et al., 2009) and green alga *Chlamydomonas reinhardtii* (Bravo et al., 2014). However,

<footnote><sup>a</sup> University of Geneva, Faculty of Sciences, Earth and Environment Sciences, Department F.-A. Forel for Environmental and Aquatic Sciences, Environmental Biogeochemistry and Ecotoxicology, 66 Blvd Carl-Vogt, CH, 1205, Geneva, Switzerland</footnote>

<footnote><sup>b</sup> Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les matériaux, Pau, 64000, France</footnote>

<footnote>* This paper has been recommended for acceptance by Prof. Wen-Xiong Wang.</footnote>

<footnote>* Corresponding author.
E-mail address: vera.slaveykova@unige.ch (V.I. Slaveykova).</footnote>
transformation yields and demethylation rate constants remain to be quantified. A comparative study on 15 different algal species found that 6 species (dinoflagellates, chrysophytes, and diatoms) could demethylate MeHg (Li et al., 2022). More recently, a light-independent pathway of MeHg demethylation by the green alga *Chlorella vulgaris*, the marine diatom *Chaetoceros gracilis* and two cyanobacteria with the singlet oxygen identified as the main driver was reported (Liang et al., 2023). Demethylation was also observed in natural pico-nanoplankton from a eutrophic lake (Cossart et al., 2021). By contrast, no direct evidence of Hg(II) methylation by phytoplankton species has been found. Nevertheless, studies reported a positive correlation between phytoplankton productivity and MeHg levels (Lazaro et al., 2019; Lázaro et al., 2013; Tsui et al., 2010; Xing et al., 2018) or methylation rates in open oceans (Heimbürger et al., 2010).

The reduction of Hg(II) to gaseous Hg(0) by different phytoplankton species has been also reported (Gregoire and Poulain, 2014; Grégoire and Poulain, 2016; Kelly et al., 2006; Mason et al., 1995; Morelli et al., 2009; Oh et al., 2011; Poulain et al., 2004; Poulain et al., 2007). For example, diatom *Thalassiosira weissflogii* produced dissolved gaseous mercury under both light and dark conditions, suggesting biological rather than photochemical transformation (Morelli et al., 2009). Green alga *C. autotrophica*, flagellate *Isochrysis galbana* and diatom *T. weissflogii* were found to convert Hg(II) into dissolved gaseous mercury, phytochelatin (PC) complexes and metacinnabar in algal species-specific manner (Wu and Wang, 2014). The reduction of Hg(II) by *C. vulgaris* was also demonstrated, but the organic matter released or obtained after cell degradation was shown to reduce more Hg(II) than living algal cells (Liang et al., 2023).

Potential drivers for Hg transformations in organisms, such as plants, algae and animals have been discussed and challenges related with the study of *in vivo* Hg transformations have been recently discussed (Li et al., 2024). The above examples highlight significant progress in understanding the role of phototrophic organisms in Hg species transformation within aquatic environments, revealing them as important yet often overlooked drivers of Hg cycling, as reviewed (Cossart et al., 2022; Li et al., 2024). Based on the existing literature, we can hypothesize that the phytoplankton species play a more important role in mercury cycling. Rather than acting solely as bioaccumulators, these microorganisms could actively mediate mercury transformation through a range of cellular processes.

This study aims to systematically investigate the transformation of mercury species by diatoms providing new insights into the relative significance of these processes. We examine Hg(II) methylation, MeHg demethylation, and the production of dissolved gaseous Hg(0) following exposure to Hg(II) or MeHg. For the first time, we explore the specific cellular fractions where these transformations occur, advancing our understanding of the mechanisms driving diatom-mediated mercury transformation. Diatom *Cyclotella meneghiniana* was chosen as a representative photosynthetic organism commonly found in diverse aquatic ecosystems (Wehr et al., 2015).

# 2. Materials and methods

## 2.1. Materials and reagents

All glassware underwent trace metal cleaning procedures as described elsewhere (Garcia-Calleja et al., 2021). Trace solutions of ¹⁹⁹Hg-enriched inorganic mercury and ²⁰¹Hg-enriched methylmercury (ISC-Science, Oviedo, Spain) were used as spikes in bioassays with diatoms. Standard solutions of ¹⁹⁸Hg-enriched inorganic mercury and ²⁰²Hg-enriched methylmercury (ISC-Science) were used to quantify the endogenous Hg species (¹⁹⁹Hg(II) and CH₃²⁰¹Hg, hereafter written as ²⁰¹MeHg). Hg(II) (mercury ICP standard, CarlRoth, Arlesheim, Switzerland) and MeHg (standard solution, Alfa Aesar, Kandel, Germany) solutions were used to determine Hg(II) transformation to Hg(0). Working solutions were prepared daily by appropriately diluting the

stock standard solutions in 1 % hydrochloric acid (30 % HCl, Suprapur® solution, Merck) and stored at 4 °C until use.

## 2.2. Algal culture conditions and exposure

The diatom *Cyclotella meneghiniana* (CCAC 0036 strain Central Collection of Algal Cultures, Cologne Centre, Germany) was axenically cultivated in a synthetic freshwater medium (SFM) under visible light with a light intensity of 100 µEm⁻² s⁻¹, a light regime of 14:10 h (light: dark), at 20 °C (Santos et al., 2024). The air was filtered by two gold-coated quartz sand traps (Brooks-Rand Ltd., Seattle, WA) and a capsule filter (0.2 µm Aervent®) to remove possible mercury and biological contamination and purged through the system with a flow rate of 20 mL min⁻¹ (Fig. S1). Cultures of *C. meneghiniana* were collected at the mid-exponential phase (120 h post-inoculum) by centrifugation at 1300 g for 15 min at 20 °C. Subsequently, the cells were rinsed and resuspended in Hg-free exposure medium to reach a final cell density of 1.0 × 10⁶ cells mL⁻¹.

## 2.3. Mercury accumulation and transformation by microalgae

Two different experimental series were performed to investigate the potential capacity of *C. meneghiniana* to transform Hg species, including Hg(II) methylation to MeHg and MeHg demethylation to Hg(II) and Hg(0) formation after Hg(II) or MeHg exposure. As biotic transformations of Hg are considered primarily intracellular processes (Hsu-Kim et al., 2013), the uptake of Hg(II) and MeHg by *C. meneghiniana* was first assessed.

### 2.3.1. Uptake, Hg methylation and MeHg demethylation assay

To assess the potential methylation (¹⁹⁹Hg(II) to ¹⁹⁹MeHg) and demethylation (²⁰¹MeHg to ²⁰¹Hg(II)) by *C. meneghiniana*, cells were exposed to a mixture of 3 nM ¹⁹⁹Hg(II) and 0.3 nM ²⁰¹MeHg during 72 h. Incubations were carried out in the incubator at a light/dark cycle of 14h/10h, constant light intensity of 100 µEm⁻² s⁻¹ and a constant temperature of 20 °C. Although these concentrations are higher than those found in the natural environment, they are among the lowest reported for the incubation of mercury species with photosynthetic unicellular organisms (Gojkovic et al., 2022). Furthermore, the selected concentrations were not toxic, as they induced growth inhibition in less than 5 % of the cells (Santos et al., 2024). Abiotic exposure in the absence of diatom cells, as well as respective unexposed controls were run. All tests were performed with three independent replicates. To identify where these transformations occur in cell suspensions, we analyzed the cellular fraction (CellF), extracellular fraction (ExtraF), whole cell system fraction (SystemF, combining CellF and ExtraF), and two subcellular fractions (see SI for details): the debris fraction (DebrisF), containing membranes, cell walls, and nuclei, and the cytosolic fraction (CytoF), which includes organelles, cytosol, and associated proteins (Lavoie et al. (2009). The subcellular fractions were obtained from diatom cells that had been collected and flash-frozen in liquid nitrogen to stop the metabolic activity following a procedure previously applied (Millet et al., 2024). A good mass balance for both Hg(II) and MeHg was obtained in the cellular fractionation procedure (Fig. S2).

### 2.3.2. Reduction assay

The potential reduction of Hg(II) or MeHg to Hg(0) by *C. meneghiniana* was examined after exposing cells to 1.8 nM Hg(II) or 0.32 nM MeHg under light and dark conditions. Abiotic controls were performed to assess abiotic contributions. The experimental setup (Fig. S3) was designed to capture gaseous Hg(0) during exposure experiments (details in SI). Briefly, 50 mL of Hg-free medium or resuspended cells were added to each sealed flask. Dark-condition flasks were covered with foil and pre-incubated for 2 h, while light-condition flasks remained exposed. All flasks were purged with Hg-free air before incubation to remove potential Hg(0) contamination. Gaseous Hg(0) was
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Dynamic changes of ¹⁹⁹Hg(II) (a) and ²⁰¹MeHg (b) concentrations in the SystemF for biotic and abiotic exposure. ¹⁹⁹Hg(II) (c) and ²⁰¹MeHg (d) concentrations along the exposure period in the three different fractions. Significant differences for each fraction are expressed with letters and were determined using Two-way ANOVA, followed by Tukey's multiple comparisons test. Data are means ± SD (n = 3).</figcaption></figure>

sampled continuously at 77 ± 4 mL min⁻¹, and after ~3 h, gold-coated traps were sealed and analyzed within the same day. Following trap collection, samples were taken to determine total mercury in different fractions (SystemF, CellF, ExtraF). Solid matrices (SystemF, CellF) were digested in ultrapure previously digested in ultrapure HNO₃ (65 % w/w, for analysis (max. 0.005 ppm Hg) EMSURE®) at 85 °C during 12 h as described elsewhere (Santos et al., 2022), before proceeding with the THg analysis.

## 2.4. Analytical methods for Hg determination

A ThermoFisher Scientific Gas chromatograph (Trace GC) coupled to a ThermoFisher Scientific ICP-MS (X-Series 2) was used for the quantification of natural and isotopically enriched Hg(II) and MeHg (GC-ICPMS). All sample fractions were analyzed by species-specific isotope dilution and GC-ICPMS, as described elsewhere (Garcia-Calleja et al., 2021). The detection limit (LOD) of Hg compounds for GC-ICP-MS analytical method was 0.25 pM and 0.14 pM for Hg(II) and MeHg, respectively. The mathematical approach for quantification of the samples by quadruple isotope dilution analysis can be found in previous studies (Bouchet et al., 2018; Garcia-Calleja et al., 2021).

Gaseous elemental Hg(0) production was measured using gold-coated silica (Amasil™ with a trapping capacity of 80 µg of Hg, a recovery >97 % between temperatures of 30 °C and 210 °C), followed by their subsequent analysis. After collection, each trap was sealed with polypropylene Teflon-lined caps and stored in double PE Zip-lock bags for analysis later on the same day. Mercury collected in the traps was thermally desorbed at 500 °C and determined by double amalgamation

on gold-AFS (DA/Au-AFS) (Tekran 2500), using pure argon as a carrier gas, as previously described elsewhere (Bouchet et al., 2011; Duval et al., 2023). The calibration curve for the method presented a high linearity of 99 % confidence level (R² > 0.99). Hg(0) was collected using a glass chamber equipped with a capillary tube, liquid mercury, a thermometer, and an inlet for micro-syringe insertion, by applying the Dumarey equations (Dumarey et al., 2010). The concentration of Hg in each sample was determined by measuring the peak of Hg and subtracting the subsequent blank peak of the same trap. The method's detection limit was estimated to be 0.03 pM, which is well below the observed concentration range.

The total mercury concentration in the samples collected in Hg(II) reduction assay was analyzed in accordance with the EPA method 1631 guideline (Environmental Protection Agency U.S., 2002) and the MERX-T manufacturer's guidelines using a MERX® Automated Total Mercury Analytical System (Brooks Rand Instruments, Seattle, WA, USA). The LOD of the method is 6 × 10⁻¹⁴ mol g⁻¹. The accuracy of the digestion and measurements was checked by analyzing certified reference material of unicellular microalga *Scenedesmus obliquus* (IAEA-450) with a recovery efficiency of 109 ± 2 % (n = 3).

## 2.5. Statistical analysis

Graphical and statistical analyses were performed using GraphPad Prism 10 Software. The Shapiro-Wilk test was used to assess the normal distribution of data. One-way or two-way ANOVA was utilized to determine statistically significant differences between groups, depending on one or two independent variables, respectively. Significant
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Concentrations of spiked ¹⁹⁹Hg(II) (a) and ²⁰¹MeHg (d) and produced ¹⁹⁹MeHg (b) and ²⁰¹Hg(II) (e) in the cellular fraction and the two subcellular fractions (cytosolic and debris fractions) during the three exposure times (5 min, 24 h and 72 h). Percentage of methylation (c) and demethylation (f) in cellular and subcellular fractions (cytosolic and debris fractions), over three exposure durations (5 min, 24 h, and 72 h) considering the initial Hg concentration (see Eqs. S1-S2). Two-way ANOVA was performed to determine, followed by Tukey's multiple comparisons tests, where significance within each fraction is presented by different letters.</figcaption></figure>

differences between and within results from different exposure times
were determined using Tukey's multiple comparisons test.

**3. Results and discussion**

**3.1. Hg(II) and MeHg accumulation in diatoms**

The concentration of Hg(II) in diatom suspensions (SystemF), consistently decreased over the exposure time, reaching ~20 % reduction after 72 h (Fig. 1a). This indicates that diatom cells help retain Hg (II) in the system, reducing its loss compared to abiotic conditions. In contrast, abiotic exposure resulted in a significantly greater decrease of 62–79 % over the same period suggesting very low retention of Hg(II) in the solution and possible reduction to Hg(0) followed by volatilization.

The Hg(II) measurements in CellF and ExtraF provided further insights (Fig. 1c and 2a). ¹⁹⁹Hg(II) in the CellF and DebrisF significantly increased from 5 min to 24 h exposure with a subsequent significant decrease at 72 h. This decline may result from Hg(II) desorption/efflux (Quevedo-Ospina et al., 2023) or Hg(II) transformations to MeHg or Hg (0). However, the constant Hg(II) concentration in ExtraF as well as CytoF over time suggests no significant efflux from diatoms. In addition, Hg(II) in DebrisF was much higher than Hg(II) in CytoF, which is consistent with the previously observed high level of accumulation (84.5 %) in the cellular debris and 15.5 % in the CytoF for *C. meneghiniana* when exposed to similar concentrations of Hg(II) (Pickhardt and Fisher, 2007).

A significant decrease of MeHg in diatoms suspensions was also
observed over 24 h followed by an increase at 72h (Fig. 1b). However,
no similar changes in MeHg concentrations were seen in the abiotic
exposure which was rather constant over time. The MeHg concentration
in the CellF increased significantly from 5 min to 24 h, followed by a
decrease toward 72 h, which corresponded to an inverse trend in ExtraF
(Fig. 1d). This observation would suggest an initial rapid uptake of

MeHg followed by a significant efflux/desorption of MeHg by the cells
and corroborates with a literature study reporting a decrease in MeHg
concentration in the CellF from 24 to 72 h and suggesting that the
metabolic activity of the cells can affect the MeHg accumulation (Tada
and Marumoto, 2020). In addition, the differing pattern in the CellF
upon exposure to Hg(II) and MeHg could also be due to the difference in
the uptake mechanism of these two species. Indeed as discussed in our
previous study (Santos et al., 2024), Hg(II) predominantly forms the
neutral complex Hg(OH)₂ in SFM, which can cross cell membranes via
passive diffusion. This aligns with previous findings by (Mason et al.,
1995) who demonstrated that neutral mercury complexes readily diffuse
across lipid bilayers. Conversely, MeHg primarily forms positively
charged complexes, such as (CH₃Hg)₂OH⁺, which may enter cells via
specific transport mechanisms (Beauvais-Flück et al., 2017).

**3.2. Methylation and demethylation of mercury species**

Concerning the contribution of the transformation process to a
decrease of the Hg(II) in the diatoms suspensions, no measurable
methylation of Hg(II) was found as newly formed ¹⁹⁹MeHg as a result of
¹⁹⁹Hg(II) methylation by *C. meneghiniana* cells was not detected under
the studied conditions (Fig. 2c) ¹⁹⁹MeHg concentrations obtained were
comparable at different times (Fig. 2b) and close to or below the method
detection limit of 0.14 pM MeHg. The absence of methylation in the
present study is expected since diatoms lack the *hgcAB* gene responsible
for Hg(II) methylation in different microorganisms (Parks et al., 2013;
Vigneron et al., 2021). Moreover, such a transformation would not be
strategically advantageous for microalgae species, due to the higher
toxicity of MeHg (Gojkovic et al., 2022). Our results are in agreement
with previous studies on phototrophs including cyanobacterium *Nostoc
paladosu* and *Synechocystis* sp. PCC 6803, green algae *C. reinhardtii* and *C.
vulgaris* which also showed no detectable methylation of Hg(II) (Bravo
et al., 2014; Franco et al., 2018; Garcia-Calleja et al., 2021; Li et al.,
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Determination of recovered Hg(0) measured after adding Hg(II) (a) or MeHg (b) in light and dark conditions. Biotic treatment: presence of 9.05 × 10⁵ cells mL⁻¹, abiotic treatment no cells. Data means ± SD (n = 3). Significant differences between treatments are represented by different letters (p < 0.05).</figcaption></figure>

2022; Yin et al., 2022).

²⁰¹MeHg was demethylated by diatom cells as evidenced by the significant increase in concentration of the newly formed ²⁰¹Hg(II) from 5 min to 24 h in the CellF, followed by a significant decrease to 72 h (Fig. 2e). Similar results were observed in the DebrisF, with no differences found in CytoF, suggesting that demethylation might occur in the DebrisF. Our results indicate that biological demethylation (system yield, SI, Eq. S(2))occurred, reaching 0.6 ± 0.3 % in CellF and 1.0 ± 0.4 % in DebrisF after 72 h of exposure (Fig. 2f).

Additionally, the cellular demethylation yield, calculated based on the assumption that demethylation occurred within the diatom cells, was compared with the actual accumulated ²⁰¹MeHg in the CellF. Consequently, a significant cellular demethylation yield was observed in the CellF and DebrisF with demethylation reaching respectively 4.2 ± 2.8 % and 7.1 ± 2.8 % after 72 h. No newly formed ²⁰¹Hg(II) was found in the abiotic exposure. The latter result is in agreement with the lack of abiotic demethylation for light-visible range under oxic conditions similar to our study (Lotfi-Kalahroodi et al., 2023).

Our findings regarding biological demethylation by diatoms align with recent literature reporting that four phytoplankton species, including green algae *Chlorella vulgaris* and the diatom *Chaetoceros gracilis*, demethylate MeHg after exposure at 25 nM (Liang et al., 2023). However, the demethylation rates observed in our study are significantly lower than those reported by Liang et al., (2023), likely due to the approximately 100-fold lower MeHg concentrations used here, differences in algal species, or variations in cell density.

The mechanisms underlying MeHg demethylation in diatoms remain uncertain, but insights from other organisms suggest potential pathways. Dark demethylation, in which endogenous ROS, particularly singlet oxygen generated in green algae and diatoms, mediate MeHg breakdown, has been reported (Liang et al., 2023). Additionally, intracellular redox reactions mediated by glutathione or other thiol-containing compounds may contribute to the transformation of MeHg into Hg(II) (Garcia-Calleja et al., 2021; Liang et al., 2023). Unlike bacteria, diatoms lack the *mer* gene system, ruling out enzymatic demethylation via the *merB* gene (Barkay and Gu, 2022). Similarly, *mer*-independent oxidative demethylation (Barkay and Gu, 2022) appears unlikely in diatoms given their metabolic constraints. However a recent detection of Hg(0) formation from MeHg in coastal seawaters suggests the potential for reductive demethylation (Kleindienst et al., 2025). Further research is needed to elucidate whether diatoms possess distinct biochemical or photochemical mechanisms driving Hg demethylation by ohytoplankton species such as diatoms.

### 3.3. Reduction of mercury species

The ability of diatom *C. meneghiniana* to produce Hg(0) during Hg(II) exposure, which could be volatilized was explored. Hg(II) spiking (Fig. 3a) resulted in similar production yields of Hg(0) (system yield, see

SI) of 2.86 ± 0.25 % and 2.32 ± 0.38 % for light and dark biotic exposure conditions, respectively after ~3 h incubation. When assuming that only the cellular Hg was transformed, cellular yields of 5.9 ± 1.3 % and 8.6 ± 1.5 % for light and dark incubation were found (Fig. S4). Abiotic conditions presented much lower production of Hg(0) in both light and dark conditions, with no significant differences between them. Although we initially expected higher Hg(0) production under light conditions, given the use of fluorescent lamps emitting only visible light, no significant Hg(II) reduction was observed. Indeed, only ultraviolet light has been shown to play a critical role in Hg photoreduction (Luo et al., 2020). Overall, in both dark and light conditions, the production of Hg(0) was significantly higher under biotic conditions than under abiotic conditions, indicating biotic Hg(II) reduction.

The ability of diatom *C. meneghiniana* to produce Hg(0) after MeHg exposure (i.e. Hg-reductive demethylation) was very low. Contrary to Hg(II), MeHg exposure showed no differences across the different treatments including visible light (Fig. 3b), which could be linked to the low demethylation rates presented by *C. meneghiniana* as discussed above. The abiotic findings are consistent with the literature, which shows that no MeHg photodemethylation followed by photoreduction was observed in an oxic system exposed to only visible light (Lotfi-Kalahroodi et al., 2023). The present results demonstrate that Hg (II) reduction to Hg(0) is biotically-mediated, but neither by photosynthetic metabolism, nor other light mediated processes. Indeed, such a light-independent process was further established in other studies where direct measurements of gaseous Hg were assessed under light and dark conditions. For instance, it was shown that the capacity of *Euglena gracilis* to produce Hg(0) was light-independent (Devars et al., 2000). Exposure of marine diatom *Thalassiosira weissflogii* to 5 nM Hg(II) (comparable to the present study: 1.76 nM Hg(II)), led to similar results of Hg(0) production between light and dark conditions (Morelli et al., 2009). Furthermore, the percentage of produced Hg(0) after 1 h exposure was around 1 % which corroborates with our findings (system yield, 1.06 ± 0.09 % h⁻¹ and 0.88 ± 0.15 % h⁻¹ for light and dark biotic incubation of Hg(II)). In addition, comparable Hg(0) production was observed after comparing the effects of both light and dark incubation on *Chattonella marina* (Zhang et al., 2021). Interestingly, when comparing the reducing capacity of coastal surface water with communities above 3 µm (where diatom species are included), the authors observed no differences between light and dark incubation in terms of Hg(0) production (Zhang et al., 2021). The results in this previous study exhibit 20–40 % purgeable Hg(0) after 24 h, which is close to our findings estimating a daily reduction yield of ca. 24 %. More recently, various microalgae (e.g., the flagellate *Chattonella marina*, the diatom *Phaeodactylum tricornutum*, and the green alga *Dunaliella salina*, etc.) were shown to promote the reduction of Hg(II) to Hg(0) (Zhang et al., 2021). The different tested microalgae species exhibited different reducing Hg(II) yields as previously observed (Wu and Wang, 2014), emphasizing the species specificity for this transformation. The rates of
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Summary of the overall transformation detected in the current study after exposing *C. meneghiniana* to Hg(II) and MeHg. Hg(II) and MeHg is accumulated inside the cells of *C. meneghiniana*. MeHg was subject to oxidative demethylation to Hg(II). While no Hg(II) methylation to MeHg was observed. *C. meneghiniana* reduced Hg(II) to Hg(0) with Hg(II). **The influence of ROS on oxidative MeHg demethylation was based on literature** (Liang et al., 2023). Created in BioRender. Santos, J. (2025) https://BioRender.com/w58a587.</figcaption></figure>

Hg(0) production measured in our study (0.25 ± 0.02 amol cell⁻¹ day⁻¹ or 9.3 ± 0.7 pM h⁻¹) were in the same order as in other studies that investigated diatom *Thalassiosira weissflogii* where Hg(0) rates were 0.29 amol cell⁻¹ day⁻¹ (Mason et al., 1995), 2.6 amol cell⁻¹ day⁻¹ (Morelli et al., 2009) and 36.2 ± 4.8 pM h⁻¹ (Wu and Wang, 2014). The variations in the Hg(II) reduction potential within and between species should be carefully addressed since factors such as species, cell densities, incubation protocols, and Hg concentrations can influence experimental outcomes (Zhang et al., 2015).

Understanding the fate of Hg species in the aquatic environment is essential to determine the potential Hg impact in the aquatic environment. Our findings have demonstrated that photosynthetic microorganisms, particularly diatoms, which are ubiquitous primary producers found in aquatic environments, play a significant role in Hg bioaccumulation and transformations. More precisely, diatoms exhibit a notable capacity for bioaccumulation/sequestration of mercury species, serving as the primary entry point into the trophic food web. The results of the present study demonstrated that the cellular burden of the Hg(II) and MeHg is dependent not only on the uptake and efflux processes but also on potential transformation processes including MeHg demethylation and gaseous Hg(0) formation. Compiling our results we propose a conceptual framework for the prevailing transformations in *C. meneghiniana* exposed to sub-nanomolar concentrations of Hg(II) and MeHg (Fig. 4), found in contaminated environment (Caballero-Gallardo et al., 2022). Diatoms are able to demethylate MeHg into less toxic Hg (II), decreasing the amount of MeHg present in the aquatic system and thus reducing mercury biomagnification potential to top predators. They also contribute to the reduction of Hg(II) to Hg(0) and thus could facilitate its transfer from the water to the atmosphere, reducing Hg concentration in the aquatic system and at the same time reducing the potential Hg availability for methylmercury (MeHg) formation in the aquatic environment (Nerentorp Mastromonaco et al., 2017). These transformations significantly impact Hg cycling in aquatic systems by altering the abundance of different Hg species, thus influencing its entry and fate into the trophic food chain. For instance, the increased activity of methylators promotes higher MeHg production, resulting in greater accumulation up to the trophic chain. MeHg is of particular concern because it does not only bioaccumulate but also biomagnifies along the trophic chain (Gentes et al., 2021; Gojkovic et al., 2022; Lavoie et al., 2013), causing a serious health threat for humans and top predators. Overall, the findings of the present study, along with recent research, imply that phytoplankton species (e.g., diatoms) are not just simple

bioaccumulators of mercury but actively contribute to its biogeochemical cycling. Through these transformation processes, they could enhance mercury reactivity, influencing its speciation, mobility, and potential bioavailability in aquatic ecosystems. This challenges the conventional view of phytoplankton as simple bioaccumulators and highlights their role as important players in the biogeochemical cycling of mercury.

## 4. Conclusions

Understanding the bioaccumulation and transformation of Hg species once in the aquatic environment is essential to understand their toxicity and trophic transfer potential of the aquatic environment. Our findings have demonstrated that diatoms rapidly accumulated both Hg (II) and MeHg. MeHg demethylation occurred primarily within the cell debris fraction at a rate of 0.06 ± 0.03 % h⁻¹ over 72 h, with cell debris contributing 0.1 ± 0.04 % h⁻¹. The reduction of Hg(II) to elemental mercury (Hg(0)) was biologically mediated and independent of light, as evidenced by comparable rates in light (2.2 ± 0.5 % h⁻¹) and dark (3.3 ± 0.6 % h⁻¹). These findings suggest that phytoplankton organisms, such as diatoms, could play an important role in mercury species demethylation and reduction to volatile Hg(0) facilitating mercury transfer from water to the atmosphere and reducing its availability for MeHg formation in aquatic environments.

### CRediT authorship contribution statement

**João P. Santos:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

**Javier Garcia-Calleja:** Formal analysis, Data curation. **Emmanuel Tessier:** Formal analysis, Data curation. **Séverine Le Faucheur:** Writing – review & editing, Resources. **Zoyne Pedrero:** Writing – review & editing, Supervision. **David Amouroux:** Writing – review & editing, Validation, Resources, Conceptualization. **Vera I. Slaveykova:** Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgments

The present study was financially supported by the Swiss National Science Foundation grants № 175721 and French Agence National de la Recherche grant No17-C34-0014. Figures S1, S3 and 4, as well as Graphical abstract were created with BioRender.com.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2025.126248.

## Data availability

Data will be made available on request.

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