# Mercury compound-specific stable isotope fractionation in high-altitude lake ecosystems of the Bolivian Altiplano

Jérémy Masbou, David Point, Sylvain Bouchet, Jeroen Sonke, Carlos Molina, Carla Ibanez, Anne Lorrain, Julio Pinto, Dario Acha, David Amouroux

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► To cite this version:

Jérémy Masbou, David Point, Sylvain Bouchet, Jeroen Sonke, Carlos Molina, et al. Mercury compound-specific stable isotope fractionation in high-altitude lake ecosystems of the Bolivian Altiplano. Science of the Total Environment, 2025, 983, pp.179630. 10.1016/j.scitotenv.2025.179630. hal-05085532

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Submitted on 26 May 2025

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# Mercury compound-specific stable isotope fractionation in high-altitude lake ecosystems of the Bolivian Altiplano

Jérémy Masbou<sup>a,b,*</sup>, David Point<sup>b,**</sup>, Sylvain Bouchet<sup>c,d</sup>, Jeroen E. Sonke<sup>b</sup>, Carlos Molina<sup>e</sup>, Carla Ibanez<sup>e</sup>, Anne Lorrain<sup>f</sup>, Julio Pinto<sup>e</sup>, Dario Acha<sup>e</sup>, David Amouroux<sup>c</sup>

## HIGHLIGHTS

* Significant biomagnification of mono-methylmercury (MMHg) was observed in high-altitude food webs

* Extensive Hg-CSIA dataset indicate that the sediment and/or the epibenthic environment is the primary MMHg source

* The Hg mass-independent fractionation pattern in food webs helps identify multiple MMHg entry points

* Atmospheric Hg(II) sources dominate MMHg bio-accumulated in high-altitude food webs

## GRAPHICAL ABSTRACT

<figure><img src="image_5.png" /></figure>

## ARTICLE INFO

Editor: Xinbin Feng

Keywords:

Mercury

Isotopes

CSIA

FOODWEB

Lake

ALTITUDE

## ABSTRACT

A combination of mercury (Hg) speciation and compound-specific stable isotope analyses was employed to trace the origin and fate of monomethylmercury (MMHg) in the high-altitude food webs of Lakes Titicaca (TTKK) and Uru Uru (UU). Significant MMHg biomagnification was observed, with concentrations reaching up to 2 µg.g⁻¹ Hg in top predators. Hg isotopes lake-specific trends were identified in relation to trophic position (δ¹⁵N) and MMHg fractions. In particular, Δ¹⁹⁹MMHg increased from 0 ‰ in UU epiphytic biofilm to ≈2 ‰ and ≈4 ‰ in UU and TTKK piscivorous fish, respectively. Both δ²⁰²MMHg and Δ¹⁹⁹MMHg signatures indicate that the sediment and/or the epibenthic environment is the primary MMHg source in both food webs. However, an additional MMHg pool, associated with photodegraded MMHg, was identified entering the two food webs at a different trophic level. Photodemethylation was estimated to account for 21 % of MMHg degradation before it entered TTKK Lake

<footnote><sup>a</sup> Institut Terre et Environnement de Strasbourg (ITES), University of Strasbourg/ENGEES, CNRS UMR 7063, France</footnote>

<footnote><sup>b</sup> Géosciences Environnement Toulouse, CNRS/IRD/Université Toulouse III - Paul Sabatier, 31400 Toulouse, France</footnote>

<footnote><sup>c</sup> Université de Pau et des Pays de l'Adour, CNRS, IPREM, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les Matériaux, Pau, France</footnote>

<footnote><sup>d</sup> Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, ETH Zurich, Universitaetstrasse 16, CHN, CH-8092 Zurich, Switzerland</footnote>

<footnote><sup>e</sup> Instituto de Ecología, Unidad de Calidad Ambiental (UCA), Carrera de Biología, Universidad Mayor de San Andrés, Campus Universitario de Cota Cota, casilla 3161, La Paz, Bolivia.</footnote>

<footnote><sup>f</sup> IRD, Univ Brest, CNRS, Ifremer, LEMAR, IUEM, F-29280 Plouzané, France</footnote>

<footnote>* Correspondence to: J. Masbou, Institut Terre et Environnement de Strasbourg (ITES), University of Strasbourg/ENGEES, CNRS UMR 7063, France.</footnote>

<footnote>** Corresponding author.
E-mail addresses: masbou@unistra.fr (J. Masbou), david.point@ird.fr (D. Point).</footnote>
food web at the fish level, and 16 % before reaching UU Lake food web at the invertebrate level. Even-Hg MIF (Δ²⁰⁰Hg) shows that both Hg(0) atmospheric deposition and geogenic inputs contributed to Hg accumulation in sediments, while the food web adds significant atmospheric Hg(II) signatures, with up to 94 % of Hg(II) contributing to Lake TTKK fish MMHg. These findings underscore the high potential of Hg-CSIA revealing the dominant role of atmospheric Hg(II) deposition and distinct MMHg pools in driving MMHg bioaccumulation in high-altitude lake food webs.

# 1. Introduction

Mercury (Hg) is a global pollutant impacting terrestrial and aquatic environments worldwide due to the long range transport of its predominant atmospheric species, elemental gaseous Hg(0). It is emitted by both natural and anthropogenic sources and can reach remote regions (Fitzgerald et al., 1998), such as high-altitude lake ecosystems (Guédron et al., 2017; Loewen et al., 2007; Marusczak et al., 2011; Zhang et al., 2014a). Once released into the atmosphere, elemental mercury (Hg(0)) undergoes oxidation within 6–18 months (Horowitz et al., 2017), transforming into divalent mercury (Hg(II)) (Gworek et al., 2020). Both Hg(0) and Hg(II) deposit on continental and aquatic surfaces via wet and dry deposition mechanisms (Jiskra et al., 2021). Once deposited, a fraction of the Hg pool can be methylated primarily through biotic pathways (Ma et al., 2019), although abiotic processes can also contribute (Wang et al., 2022). This methylation leads to the formation of monomethylmercury (MMHg), a neurotoxic and bioaccumulative organic Hg compound (Ullrich et al., 2001). Methylation has been mainly documented in anoxic and/or suboxic environments such as i) the sediment-water interface (Merritt and Amirbahman, 2009; Warner et al., 2003), ii) oxygen depleted water columns (Eckley and Hintelmann, 2006; Soerensen et al., 2018), iii) or in microniches such as particulate flocs and biofilms (Alanoca et al., 2016a; Bouchet et al., 2018b; Cleckner et al., 1999; Gascón Díez et al., 2016; Guimaraes et al., 2000; Hamelin et al., 2011). In aphotic and photic zones of aquatic ecosystems, MMHg can be degraded through various dark and light induced pathways, while in surface waters exposed to UV radiations, photodemethylation remains the most important mechanism (Amyot et al., 1997; Barkay and Gu, 2021; Hammerschmidt and Fitzgerald, 2006b; Klapstein and O'Driscoll, 2018; Seller et al., 1996). The residual pool of MMHg, governed by the balance between methylation and demethylation processes, represents the net baseline production of MMHg that can be assimilated and biomagnified in the food webs (Masbou et al., 2018; Perrot et al., 2012).

For the past two decades, Hg stable isotopes have been used efficiently to trace Hg sources and identify key transformation processes in aquatic ecosystems (Tsui et al., 2020). Most Hg transfer processes, as well as abiotic and biotic transformation pathways, induce mass-dependent fractionation (MDF), thereby complicating the interpretation of δ²⁰²Hg signatures—particularly in biological matrices, where distinct isotopic compositions are often observed between inorganic Hg (II) and MMHg (Masbou et al., 2018; Perrot et al., 2016; Zhang et al., 2022). On the contrary, photochemical reactions such as Hg(II) photo-reduction and MMHg photodegradation result in unique mass independent fractionation affecting odd isotopes (odd-MIF, expressed as Δ¹⁹⁹Hg or Δ²⁰¹Hg) which allow to trace the extent of these major pathways in aquatic ecosystems (Bergquist and Blum, 2007; Chandan et al., 2014; Kwon et al., 2012; Masbou et al., 2015). MIF signatures are preserved along the food webs (Kwon et al., 2012; Laffont et al., 2009) and several studies have used the MMHg odd-MIF recorded in freshwater or marine biota to identify Hg sources and to assess the magnitude of MMHg photodegradation in the water column and its controlling factor before incorporation in the food webs (Bergquist and Blum, 2007; Blum et al., 2013; Lepak et al., 2018; Motta et al., 2020; Point et al., 2011; Renedo et al., 2020; Sherman and Blum, 2013). On the other hand, the more recent discovery of even-Hg MIF (Cai and Chen, 2016; Gratz et al., 2010), which is likely driven by photochemical reactions in

the upper atmosphere offers a new angle to track the relative importance of Hg(II) and Hg(0) deposition on terrestrial (Enrico et al., 2017; Enrico et al., 2016; Jiskra et al., 2022; Obrist et al., 2017; Zheng et al., 2016) or oceanic systems (Jiskra et al., 2021).

While most Hg stable isotopes measurements have been performed on the total Hg resulting in so-called Hg bulk stable isotope analysis (Hg-BSIA), the recent development of Hg compound specific isotope analysis (Hg-CSIA) allows to determine MMHg or inorganic Hg specific isotopic composition (Epov et al., 2010; Masbou et al., 2013; Rosera et al., 2020; Zhang et al., 2022). Recent studies have thus demonstrated that Hg species generally exhibit distinct isotopic signatures, with MMHg typically showing enrichment in δ²⁰²Hg relative to Hg(II) in both sediment and biological matrices (Perrot et al., 2016; Rosera et al., 2020). The use of Hg-CSIA, particularly when considering both even and odd MIF, offers significant potential for improving our understanding of MMHg sources and transformations across different spatial scales. At the ecosystem scale, analyses of sediments and fish muscle can provide broad insights into MMHg dynamics. Investigating additional sample types, such as zooplankton, plankton, and biofilms, allows a more detailed examination of MMHg sources to food webs and spatial heterogeneity at a more localized scale. Furthermore, while different tissues—such as muscle, liver, or kidney—may not directly help in discriminating sources and transformations, their analysis provides valuable information on species-specific depuration pathways and Hg redistribution within organisms, contributing to a more comprehensive understanding of Hg cycling (Bouchet et al., 2018a; Gao et al., 2022; Rosera et al., 2020).

The Bolivian Altiplano is one of the largest high-altitude region (3800 m) in the world, characterized by a unique endoreic basin made of a succession of water bodies: Lakes Titicaca (TTKK, the most important freshwater reservoir in the Andes), Uru Uru (UU), Poopó down to the Copaisa salt flat. Climate change, expanding urban demography, mining activities, and agriculture have disturbed the hydrological, chemical and ecological equilibrium of this fragile ecosystem (Zubieta et al., 2021). High-altitude lakes from this region are worth studying because they are sensitive indicators of surrounding pollutions, but also represent unique extreme environments with intense diurnal and seasonal gradients of biological, chemical and physical parameters that are known to enhance the transformations governing the Hg cycle (Ahonen et al., 2018; Alanoca et al., 2016b; Guédron et al., 2017; Masbou et al., 2018; Point et al., 2011; Xu et al., 2016). Mercury even MIF (Δ²⁰⁰Hg) records in TTKK sediments pointed out a significant shift from a main geogenic source during the early Holocene (fgeog = 79 %) to a predominant atmospheric Hg source nowadays (fgeog = 20 %) (Jiskra et al., 2022). In Lake UU, the combination of shallow waters, acid mine drainage, and urban effluents has been shown to create eutrophic conditions that enhance Hg methylation in the benthic environment and its potential release into the water column, with sedimentary environments representing a primary MMHg source in these shallow ecosystems (Alanoca et al., 2016a; Alanoca et al., 2016b; Guédron et al., 2020a). More specifically, new potential sources such as organic aggregates, epibenthic and epiphytic biofilms have been identified to either produce or accumulate MMHg, though their respective contributions to the MMHg budget and trophic transfer remain unclear (Alanoca et al., 2016a; Alanoca et al., 2016b; Guédron et al., 2020a). Diurnal MMHg cycles of high amplitude in Lake UU, and to a lower extent in TTKK, were thus attributed to differences in the depth of the water column and MMHg photodegradation extent balancing the sediment benthic efflux of MMHg to the water column
(Alanoca et al., 2016a; Guédron et al., 2020b). Despite growing evidence of Hg methylation and transformation processes in freshwater ecosystems, there remain significant knowledge gaps regarding the factors influencing the proportions of MMHg across different lakes. Limited understanding exists about how environmental and ecological variables, such as water chemistry and microbial activity, shape the distribution and production of MMHg in various systems. These gaps impede our capacity to predict MMHg bioaccumulation in extreme aquatic food webs and its associated risks to both ecosystems and human health.

In this study, we conducted an extensive sampling effort, analyzing over 400 samples across multiple food web compartments in freshwater ecosystems. Using both Hg-BSIA and CSIA, we examined baseline components including sediment, epiphytic biofilm, aquatic plants, and invertebrates, along with diverse biota samples representing various trophic levels in the food webs of Lakes TTKK and UU. In a top-down approach, the objectives of this study were to (i) assess the bioamplification patterns of MMHg in contrasting high-altitude food webs; (ii) identify the sources of MMHg pools and their assimilation within these food webs; and (iii) examine the sources of Hg(II) and Hg(0) that contribute to the formation of bioavailable MMHg in lakes within complex and extreme environments.

## 2. Materials and methods

### 2.1. Study sites and sample collections

Lake UU is located in the Oruro Department of Bolivia at 3686 m a.s.l. and its surface and depth vary between 120 and 350 km² and 0.25–1 m, respectively (Guédron et al., 2017). Lake TTKK is the largest freshwater reservoir in the Andes region located between Bolivia and Peru at an altitude of 3809 m. It is composed of two nearly separate basins (Fig. S-1): the great lake called “Lago Mayor” (7131 km²; mean depth = 100 m; max depth = 285 m) and the small lake called “Lago Menor” (1428 km²; mean depth = 9 m; max depth = 40 m) (Dejoux, 2012). Lake TTKK has a single outlet, the Río Desaguadero, flowing south towards Lakes UU and Poopó down to Uyuni salt flats where it progressively evaporates (Cross et al., 2000; Dejoux, 2012). This endoreic system forms the Titicaca-Desaguadero-Poopó-Coipasa Salt Lake (TDPS) assemblage. Lake TTKK water is slightly alkaline with a salinity of 1 g.L⁻¹ (Dejoux, 2012), while Lake UU displays brackish waters (up to 5 g.L⁻¹) resulting from the increased evaporation towards the south (Guédron et al., 2017).

Samples collections and locations are presented in Table S-1 and Fig. S-1 respectively. Overall, a total of 398 samples originating from both lakes were collected during different field campaigns between 2005 and 2014. Lake TTKK samples were collected at the Huatajata reference station in the shallow and eutrophicated Lago Menor part presented in Fig. S-1 and previously described (Guédron et al., 2017; Heredia et al., 2022). Samples encompass sediments (UU: n = 33; TTKK: n = 10), aquatic plants (UU: n = 34; TTKK: n = 0; Myriophyllum sp., Ruppia spp. and Schoenoplectus californicus subsp. tatora), epiphytic biofilm (UU: n = 7; TTKK: n = 2), invertebrates (UU: n = 34; TTKK: n = 11), as well as various tissues and organs from fish (UU: n = 70; TTKK: n = 87; muscle, fins, viscera, head and scales) and waterbirds (UU: n = 27; TTKK: n = 0; blood, muscle, liver and feathers), as detailed in the SI. As they are the most represented, muscle tissues were used for inter-species comparison and statistical tests unless specified. After collection, samples were stored at –18 °C and freeze-dried within a week.

### 2.2. HgT and MMHg concentrations, δ¹³C and δ¹⁵N isotope ratios measurements

Total Hg concentrations (expressed on a dry weight basis) of freeze-dried samples were determined using a DMA-80 analyzer (Milestone, USA) at the Geosciences Environnement Toulouse (GET) laboratory. Accuracy was checked against the following reference materials: NRCC-DOLT-4 (dry Dogfish liver), NRCC-TORT-2 (dry Lobster

Hepatopancreas) and JRC-BCR-414 (dry Plankton). Hg concentrations of reference materials were not statistically different from the certified values (Table S-2).

A MMHg selective extraction method (SEM) was used in this work to determine MMHg concentrations on biological samples when a sufficient sample mass was available (i.e. > 250 mg). Details of the method, measurement accuracy and reference materials analysed are given in Table S-3 and previous work (Masbou et al., 2013). Once extracted, MMHg concentrations were determined by CV-AFS following the EPA1631 protocol using a custom-made gold amalgamation system connected to a Brooks Rand (Seattle, USA) AFS detector.

Carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios were measured on defatted (organic tissues) or decarbonated (sediments) sample fractions (preparation detailed previously (Bligh and Dyer, 1959; Masbou et al., 2015)) by Elemental Analysis - Isotope Ratio Mass Spectrometry at the Institut du Littoral et de l'Environnement (La Rochelle, France) and at the Institut Terre et Environnement de Strasbourg (Strasbourg, France). External instrument reproducibility for δ¹³C and δ¹⁵N is typically ±0.1 ‰.

### 2.3. Analytical procedure HgT-BSIA and MMHg-CSIA

Depending on the mass availability and concentrations, selected samples were processed for subsequent HgT-BSIA and/or MMHg-CSIA analysis. The details of the isotopic dataset are presented in Table S-1. Fish and birds are well represented due to their high MMHg concentrations, whereas epiphytic biofilms, with their lower HgT concentrations, were limited to 3 isotopic analyses (UU: n = 1; TTKK: n = 2).

Mercury bulk stable isotopic measurements on total Hg (HgT-BSIA) and on MMHg fraction (MMHg-CSIA) were determined by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS; Neptune, Thermo Finnigan Neptune) with continuous flow cold vapour (CV) generation using Sn(II) reduction (CETAC HGX-200) using the same methodology as previous studies (Enrico et al., 2017; Masbou et al., 2018). All samples were analysed at the Géosciences Environnement Toulouse laboratory using this setup, except for the sediment samples, which were analysed at the IPREM laboratory (Pau, France) using CV-MC-ICP-MS (Nu Instruments).

HgT-BSIA were performed by CV-MC-ICPMS on total Hg acidic extracts obtained according to a previously detailed procedure (see (Masbou et al., 2018) and in the SI). MMHg-CSIA was performed by CV-MC-ICPMS applied to the MMHg purified fraction obtained by the SEM, described above, on selected biological samples when a minimum sample mass of 0.25 g was obtained. Briefly, about 0.1 to 0.3 ml of acidic BrCl were added to a 20-fold diluted subsample of the purified MMHg-thiosulfate and analysed similarly as the other samples for BSIA by CV-MC-ICP-MS. The detailed methodology is available elsewhere (see (Masbou et al., 2013; Masbou et al., 2015) for details)) and in the SI. Briefly, solutions containing between 0.5 and 5 ngHg.g⁻¹ were analysed by CV-MC-ICP-MS and bracketed with NIST 3133 standards of concentrations matching within 15 %. Depending on the sample mass availability and concentrations, sample measurements were replicated between 1 and 4 times.

Mercury isotopic compositions are expressed in δ notation and reported in parts per thousand (‰) deviation from the NIST SRM 3133 standard according to the following equation:

$$ \delta^{\text{xxx}}\text{Hg}(\text{‰}) = \left[ \left( \frac{\left( \frac{\text{xxx Hg}}{\text{198 Hg}} \right)_{\text{sample}}}{\left( \frac{\text{xxx Hg}}{\text{198 Hg}} \right)_{\text{NIST3133}}} \right) - 1 \right] \times 1000 \quad (1) $$

where xxx represents the mass of each Hg isotope.

δ²⁰²Hg characterizes the mass dependent isotope fractionation (MDF) while mass independent isotope fractionation (MIF) is expressed by the Δ notation:
$$ \Delta^{199}\text{Hg}(\text{\textperthousand}) = \delta^{199}\text{Hg} - (\delta^{202}\text{Hg} \times 0.2520) \quad (2) $$

$$ \Delta^{200}\text{Hg}(\text{\textperthousand}) = \delta^{200}\text{Hg} - (\delta^{202}\text{Hg} \times 0.5024) \quad (3) $$

$$ \Delta^{201}\text{Hg}(\text{\textperthousand}) = \delta^{201}\text{Hg} - (\delta^{202}\text{Hg} \times 0.7520) \quad (4) $$

Long term reproducibility during the different series of experiments, was evaluated with NRCC-DOLT-4 (fish and birds, HgT-BSIA and MMHg-CSIA) and JRC-BCR-414 (aquatic plants, epiphytic biofilm and invertebrates, HgT-BSIA and MMHg-CSIA) and NIST-1944 (sediment samples, HgT-BSIA) in-house control materials (Foucher and Hintelmann, 2006; Masbou et al., 2013). Measurements performed at different concentration levels were highly consistent in terms of precision and accuracy (Table S-4). Similar long-term reproducibilities were observed for HgT-BSIA and MMHg-CSIA, reaching 0.13 ‰ and 0.08 ‰ (2SD, n = 31) for NRCC-DOLT-4 δ²⁰²Hg and Δ¹⁹⁹Hg respectively. Secondary reference material NIST 8610 (UM-Almaden) was also analysed, with δ²⁰²Hg values of -0.53 ± 0.10 ‰ (2SD, n = 46) consistent with certified δ²⁰²Hg = -0.56 ± 0.03 ‰ (Standards and Technology, 2017). Uncertainties applied to samples were the larger of either the 2SD on the NRCC-DOLT-4 control material or the 2SD on sample replicates.

## 2.4. Statistics

Statistical analyses were performed using JMP® software (SAS Institute, USA). Unless indicated, all the given results are mean ± 95 % CI. One-way ANOVA tests were performed when comparing two datasets. Homogeneity of variances was tested using the Brown-Forsythe test. In the case of unequal variances, a Welch ANOVA test was used. For slope comparison, Real Statistics Resource Pack for Microsoft Excel® (http://www.real-statistics.com) was used.

# 3. Results and discussion

## 3.1. Characterization of lakes' food webs

The trophic structures of Lakes UU and TTKK (Lago Menor, mesotrophic) and the corresponding δ¹⁵N vs. δ¹³C signatures are described in Fig. S-2 and S-3 respectively. In Lake UU, δ¹⁵N showed a continuous enrichment from epiphytic biofilm presenting an average value of 1.9 ± 0.7 ‰ to 4.6 ± 0.8 ‰ in invertebrates, 7.3 ± 0.6 ‰ in piscivorous birds and 8.9 ± 0.3 ‰ in piscivorous fish (n = 4, 20, 7 and 11, respectively). This trend is consistent with δ¹⁵N enrichments of 3–4 ‰ previously observed between trophic levels (Post, 2002). However, aquatic plants and sediments presented higher δ¹⁵N values (3.9 ± 0.9 ‰ and 3.6 ± 0.7 ‰, n = 17 and 28 respectively) compared to epiphytic biofilm, which differ from typical sediment-based food web structures. Sediments and plants δ¹⁵N values are however consistent with values observed in similar samples from high-altitude Yarmouk Tibetan lake (Xu et al., 2016). Despite a more limited set of taxa in Lake TTKK, δ¹⁵N showed a similar pattern with average values increasing from -0.2 ± 1.1 ‰ in sediments to 7.0 ± 0.5 ‰ in piscivorous fish (n = 6 and 7, respectively). These observations are consistent with baseline δ¹⁵N values of 1.0 ± 1.2 ‰ and -1.3 ± 2.4 ‰ determined in Lake TTKK suspended particulate matter and sediments respectively (Heredia et al., 2022). These variations in baseline δ¹⁵N between the two lakes are consistent with previous observations, which demonstrate elevated δ¹⁵N levels in nitrogen-enriched, shallow ecosystems of TTKK impacted by anthropogenic activities (e.g., ammonia input) (Heredia et al., 2022). In contrast, nitrogen-limited open-lake environments rely predominantly on biological nitrogen fixation as a key source of bioavailable nitrogen (NH₃), contributing to the nitrogen pool with δ¹⁵N values ranging from -1 to -3 ‰, which closely approximate those of atmospheric N₂ (ca. 0 ‰) (Chappuis et al., 2017; Zhang et al., 2014b).

In Lake UU, δ¹³C values ranged from -18.5 ± 1.8 ‰ in epiphytic biofilm to -26.1 ± 0.3 ‰ in Totoras (semi-aerial plants) (Fig. S-3). This

is coherent with a previous study on the lake's vegetation (Heredia et al., 2022), showing that the predominance of emerged macrophytes led to isotopic values within the range of terrestrial C3 plants (-32 to -22 ‰), with most carbon being captured from the atmosphere (i.e., δ¹³CₚCO₂ = -8 ‰). On the contrary, Lake TTKK food web displayed more positive δ¹³C values within a narrower range, from -16.4 ± 0.2 ‰ in sediments to -18.6 ± 0.2 ‰ in epiphytic biofilm. This is in line with δ¹³C values found in terrestrial C4 plants, i.e. -23 to -9 ‰ (Chappuis et al., 2017) and dissolved inorganic carbon (DIC) being the main carbon source (Heredia et al., 2022). Overall, these results suggest that, despite the similar lengths and structures of the food webs, organisms in each lake may rely on slightly different carbon sources due to varying proportions of C3 versus C4 plants.

## 3.2. Large and consistent Hg biomagnification in biota from Lakes UU and TTKK

As expected, both HgT concentrations and proportion of MMHg (% MMHg) were found to increase with trophic level (Fig. 1, (Lavoie et al., 2013)). Primary producer organisms, i.e. aquatic plants and associated epiphytic biofilm, exhibited the lowest HgT concentrations (17 ± 3 and 29 ± 18 ng.g⁻¹, n = 23 and 6, respectively) whereas piscivorous fish and water birds at the top of the food webs exhibited the highest HgT (706 ± 245 and 2972 ± 2407 ng.g⁻¹, n = 11 and 7, respectively). Invertebrates, including mainly zooplankton, showed intermediate HgT concentrations of 111 ± 25 ng.g⁻¹ (n = 29). The %MMHg was the lowest in the sediments (3 ± 5 ‰, n = 5) and increased progressively along the UU food web, up to 76 ± 8 ‰ and 92 ± 4 ‰ (n = 45) in waterbirds and fish respectively. In fish from Lake TTKK, HgT concentrations reached 240 ± 54 and 629 ± 193 ng.g⁻¹ (muscle, n = 79) in non-piscivorous and piscivorous species, respectively. These concentrations are slightly but significantly lower (p < 0.05) than those found in the same fish species from Lake UU.

<figure><img src="image_6.png" /><figcaption>Fig. 1. a) log₁₀ [HgT] vs δ¹⁵N and b) % MMHg/HgT vs δ¹⁵N observed in Lake UU and TTKK food webs.</figcaption></figure>
MMHg trophic magnification slopes (TMS), calculated according to $Log_{10}[MMHg] = TMS \times \delta^{15}N + b$, reached values of $0.45 \pm 0.23$ ($r^2 = 0.84, p < 0.005$) and $0.37 \pm 0.88$ ($r^2 = 0.97, p = 0.11$) for the Lake UU and TTKK, respectively. Therefore, the biomagnification trends seem to be similar between the two lakes, even if the smaller dataset for TTKK makes the value less precise and despite different MMHg levels previously reported in sediments and surface waters (Guédron et al., 2017). These biomagnification slopes are similar to those observed in the Ti-betan Nam Co and Yamdrok high-altitude Lakes, exhibiting $Log_{10}[MMHg] = 1.2$ [TL] (TMS $\approx$ 0.36) and $Log_{10}[MMHg] = 1.34$ [TL] (TMS $\approx$ 0.40), respectively (Xu et al., 2016). They are significantly higher than the mean TMS value of $0.24 \pm 0.08$ found in worldwide freshwaters food-webs (Lavoie et al., 2013). Assuming a trophic level of 1 for epiphytic biofilm and a $\delta^{15}N = +3.4$ ‰ enrichment per TL (Post, 2002), the lengths of both Lake TTKK and UU food webs are equal, both reaching $\Delta$TL (trophic level of top predator – trophic level of baseline) of 2.3. They are also similar to the mean $\Delta$TL of $1.8 \pm 0.8$ observed in the worldwide meta-analysis (Lavoie et al., 2013), thus excluding such a bias. The high biomagnification observed in the Lake UU and TTKK systems could be explained by three main hypotheses. First, the efficient utilization of food and the high assimilation of MMHg by zooplankton, as previously reported (Xu et al., 2016; Zhang et al., 2014a), may enhance the transfer of Hg through the food web. Second, elevated baseline Hg concentrations at the base of these lake systems could contribute to higher Hg levels in aquatic organisms and a steeper biomagnification slope, as suggested in other studies (Hammerschmidt and Fitzgerald, 2006a; Harris et al., 2007). Third, slow growth rates of organisms in these systems may lead to reduced growth dilution of Hg,

resulting in greater bioaccumulation and biomagnification, a mechanism supported in previous research (Chételat et al., 2021; Ward et al., 2010). However, the applicability of the latter two hypotheses to the TTKK system remains uncertain. A comparative analysis with other lakes did not reveal any distinctive HgT concentrations in Lake TTKK relative to Lake Victoria or Lake Baikal, for instance (Guédron et al., 2017). Previous studies in Lake TTKK and UU (Alanoca et al., 2016a; Bouchet et al., 2018b) have clearly demonstrated that sulfate reduction activity and subsequent Hg methylation is largely stimulated in both sediment and epibenthic biofilms as a consequence of benthic eutrophication in both lakes. These processes generate a high proportion of MMHg in aquatic compartments of both lakes making bioaccumulation of Hg at the base of the foodweb to be very efficient (Alanoca et al., 2016b; Bouchet et al., 2018b; Guédron et al., 2020a; Guédron et al., 2020b; Guédron et al., 2017).

### 3.3. Tracing MMHg transfer in lake food webs via Hg-BSIA and CSIA

Hg-BSIA and CSIA data are presented in Fig. 2 and Fig. S-5. Overall, the results showed exclusively positive $\Delta^{199}$Hg mean values, ranging from $0.02 \pm 0.10$ ‰ in epiphytic biofilm from UU ($n = 1$), up to $4.2 \pm 0.7$ ‰ in fish from TTKK ($n = 21$). Hg MDF ($\delta^{202}$Hg), showed both negative and positive values, ranging from $\delta^{202}$HgT = -0.61 ± 0.10 ‰ ($n = 1$) up to $\delta^{202}$MMHg = 0.92 ± 0.17 ‰ ($n = 7$) for TTKK and UU invertebrates, respectively. In Lake UU, Hg-BSIA and MMHg CSIA showed relatively restrained values (Fig. 2) but two groups of samples could be identified: i) samples associated with food web baseline such as sediment and epiphytic biofilm displaying $\delta^{202}$Hg $\approx$ -0.5 ‰ and

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Influence of $\delta^{15}N$ (left) and MMHg / HgT ratio (right) on $\delta^{202}$Hg (MDF, up) or $\Delta^{199}$Hg (MIF, down) tracers. Isotope data are MMHg-CSIA except those indicated with *, which are Hg-BSIA. Due to the occasional unavailability of $\delta^{15}N$ and MMHg/HgT data for some samples, the number of samples may vary between the left and right panels. For the detailed database, please refer to the SI.</figcaption></figure>
$\Delta^{199}$Hg ≈ 0 ‰ values, and ii) invertebrates, aquatic plants, fish and birds showing enriched Hg signatures with $\delta^{202}$Hg ranging between 0.5 and 1 ‰ and $\Delta^{199}$Hg between 1 and 2.5 ‰. In Lake TTKK, Hg-BSIA and CSIA signatures were more variable with more negative $\delta^{202}$HgT ≈ − 0.6 ‰ and positive $\Delta^{199}$HgT ≈ 0.4–0.7 ‰ values in both sediment and invertebrate compartments, rising up to $\delta^{202}$MMHg ≈ 0.8 ‰ and $\Delta^{199}$MMHg ≈ 4.2 ‰ in fish.

We observe an overall increase of $\delta^{202}$Hg with $\delta^{15}$N and MMHg/HgT (p < 0.05, Fig. 2c), which can be explained by the progressively higher proportion of MMHg, that is isotopically heavier than inorganic Hg(II) (Bouchet et al., 2023; Laffont et al., 2011; Masbou et al., 2018; Meng et al., 2020; Perrot et al., 2010; Xu et al., 2016). Similar $\delta^{202}$MMHg values (p = 0.17) are observed between piscivorous fish (0.72 ± 0.13 ‰, n = 8) and birds (0.66 ± 0.25 ‰, n = 7), respectively, at the top of the Lake UU food web due to their similar foraging behaviors (see SI for details). Non-piscivorous fish and birds however differ (p < 0.05) with $\delta^{202}$MMHg = 0.59 ± 0.09 ‰ (n = 8) and 0.42 ± 0.19 ‰ (n = 7) respectively, suggesting more diversified feeding strategies, potentially including migratory behavior. Interestingly, invertebrates and aquatic plants, which are the main preys of benthic species, presented significantly lower $\delta^{15}$N values (4.6 ± 0.8 ‰ (n = 20, p < 0.05) and 3.9 ± 0.8 ‰ (n = 17, p < 0.05), respectively) but slightly higher $\delta^{202}$MMHg values (0.92 ± 0.17 ‰ (n = 7, p < 0.001) and 0.85 ± 0.17 ‰ (n = 8, p < 0.05), respectively) compared to their predators (benthic fish). These elevated $\delta^{202}$MMHg values in prey may reflect the potential influence of microbial demethylation processes occurring within the gastrointestinal tracts of benthic fish at the basis of kinetic isotope effect and MMHg light isotope preferential demethylation (Li et al., 2022; Poulin et al., 2021). As already shown, $\delta^{13}$C is slightly correlated with $\delta^{15}$N (r² = 0.4, Fig. S-3) in Lake UU food web with sediment and low trophic level organisms showing higher $\delta^{13}$C values. This is in agreement with several studies showing that organisms with higher $\delta^{13}$C values preferentially feed benthically instead of pelagically (Fry, 1988; Newman et al., 2011). Most previous studies found that $\Delta^{199}$Hg values were not influenced by trophic processes (Kwon et al., 2012; Masbou et al., 2018; Tsui et al., 2020) while a few observed an "apparent" increase in $\Delta^{199}$Hg from invertebrates to top predators in both freshwaters and seawater food webs (Das et al., 2009; Day et al., 2012; Perrot et al., 2012; Senn et al., 2010). It suggests that the positive relationships we observed between $\Delta^{199}$Hg and $\delta^{15}$N could be caused by a mixing of sources in the diet of upper organisms from benthic to more pelagic or piscivorous organisms or by different integration time and biological turnover of in vivo Hg between preys and predators. This remains however limited in Lake UU (< 1 m water depth) where the benthic compartment is the dominant food source for whole the food web. Despite similar $\delta^{13}$C between benthic and piscivorous fish, the latter exhibit significantly higher values for $\Delta^{199}$HgT (2.58 ± 0.11 ‰, n = 8) compared to benthic fish (1.61 ± 0.26 ‰, n = 10, p < 0.01), suggesting more pelagic MMHg uptake as influenced by photochemical degradation (e.g. odd MIF of Hg isotopes).

In summary, biomagnification patterns in the two lakes are similar, but Hg-CSIA reveals shifts in dietary sources for top predators in Lakes UU and TTKK. At lower trophic levels, MMHg sources are questioned by the relatively high $\delta^{15}$N values (3.6 ± 0.7 ‰, n = 28) observed in Lake UU sediments which place them outside the MMHg/T/HgT vs. $\delta^{15}$N trend (Fig. 1b). Similar $\delta^{15}$N values (3.8 ± 0.9 ‰, n = 17) are found in widely distributed Totoras semi-aerial plants, suggesting an atmospheric N source to UU sediments via Totoras degradation. Additionally, epiphytic biofilm may significantly contribute to the bioavailable MMHg pool in Lake UU due to i) $\delta^{15}$N and $\delta^{13}$C alignment between epiphytic biofilms and higher organisms (Fig. S-3 and S-4), and ii) their notable MMHg fraction (15 ± 12 ‰ (n = 4)). Since benthic environments have been previously identified as a primary source of MMHg to the surface waters of Lake UU (Guédron et al., 2020a), the use of MMHg CSIA is essential to fully elucidate the complex origins of MMHg within the food webs.

### 3.4. Tracing MMHg sources and fate in lake food webs using CSIA

MMHg-CSIA data in food web baseline ($\delta^{15}$N < 3.5 ‰) offered further insights into the potential source(s) of MMHg entering the food webs. In Lake TTKK, $\delta^{202}$Hg and $\Delta^{199}$Hg vs. $\delta^{15}$N (or %MMHg) patterns are observed (Fig. 2c and d) from sediment $\delta^{202}$HgT = −0.60 ± 0.27 ‰ and $\Delta^{199}$HgT = 0.38 ± 0.19 ‰ (n = 10) to fish $\delta^{202}$MMHg = 0.83 ± 0.11 ‰ and $\Delta^{199}$MMHg = 4.21 ± 0.69 ‰ (n = 21). The main shift in $\Delta^{199}$HgT values from low to high trophic organisms seems to occur between invertebrates ($\Delta^{199}$HgT = 0.72 ± 0.10 ‰, n = 1) and benthic fish ($\Delta^{199}$HgT = 4.29 ± 0.80 ‰, n = 15) even if caution must be exerted due to the limited number of invertebrate samples and the shorter integration time of this compartment compared to fish and sediments.

With homogenous $\delta^{202}$Hg and $\Delta^{199}$Hg ~ 0–0.2 ‰ (HgT and MMHg) at the UU food web baseline (sediment and epiphytic biofilm), a common Hg source for bioavailable Hg(II) and MMHg is suggested. In particular, sediment $\delta^{202}$HgT = 0.21 ± 0.19 ‰ (n = 5) is similar to epiphytic biofilm $\delta^{202}$MMHg = 0.17 ± 0.10 ‰ (n = 1), but significantly lower than high trophic level organisms which exhibit homogeneous and higher $\delta^{202}$MMHg = 0.6–0.7 ‰ (Fig. 2a). Moreover, $\Delta^{199}$Hg is homogeneous between HgT and MMHg fractions in all fish and bird tissue samples (all tissues, p > 0.05). Epiphytic biofilm $\Delta^{199}$MMHg = 0.0 ± 0.1 ‰ (n = 1) is very similar to sediment $\Delta^{199}$HgT = 0.2 ± 0.2 ‰ (n = 5) suggesting that sedimentary MMHg is readily incorporated into epiphytic biofilm without being photodemethylated. More samples are however needed to further support this hypothesis. Aquatic plants show variable and intermediate values of $\Delta^{199}$MMHg = 1.4 ± 0.6 ‰ (n = 8). At the upper trophic level, i.e. invertebrates where MMHg/HgT ≈ 50 ‰, the $\Delta^{199}$MMHg values are already comparable to those found in high trophic level organisms MIF ($\Delta^{199}$HgT or $\Delta^{199}$MMHg = 1.6–2.1 ‰). This similarity suggests that, at this trophic level, the MMHg incorporated originates from a source that has undergone additional photo-demethylation prior to assimilation. While epiphytic biofilm has been considered a potential dietary source for invertebrates, it may not be the sole or direct contributor. Instead, free-floating phytoplankton and/or dissolved MMHg—particularly in environments like UU, where dissolved MMHg concentrations are high (up to 1.8 ng.L⁻¹, Guédron et al., 2020a) could also play a role. Further investigations are needed to clarify the relative contributions of these potential MMHg sources within the food web.

Recently, Rosera et al. showed significantly different $\delta^{202}$Hg in HgT and MMHg fractions in both sediment and plankton from two different US lakes (Rosera et al., 2020). They also detected $\delta^{202}$Hg and $\Delta^{199}$Hg shifts of ~ +0.4 ‰ and ~ +1.5 ‰ respectively, between sediments and plankton and proposed a correction to estimate the initial $\delta^{202}$Hg before photochemical fractionation. Applying this correction to Lake UU high trophic level samples led to $\delta^{202}$MMHg$_{corr}$ ~ 0.2–0.3 ‰ estimations for fish and birds which is similar to the $\delta^{202}$MMHg = 0.17 ± 0.10 ‰ (epiphytic biofilm, n = 1) and $\delta^{202}$HgT = 0.21 ± 0.19 ‰ (sediments, n = 5) baseline values. MMHg in higher organisms thus appear to originate from sediment and/or epiphytic biofilm with no other processes than photochemical reactions in Lake UU. Shallow waters (< 1 m) observed in Lake UU likely promote benthic processes and the incorporation of sediment-derived Hg into the food web (Alanoca et al., 2016b). This results in low $\Delta^{199}$HgT magnitudes (~ 1.5–2 ‰) between the base and the apex of the food web.

Applying $\delta^{202}$Hg correction on Lake TTKK epiphytic biofilm and fish results in $\delta^{202}$Hg$_{corr}$ ~ -0.9 ‰ and $\delta^{202}$MMHg$_{corr}$ ~ 0 ‰ respectively, which is still slightly different from the $\delta^{202}$HgT = −0.6 ± 0.3 ‰ (n = 10) observed in sediments. Other processes, such as microbial activity or various sources and turnover times of MMHg, are anticipated to differ between high and low trophic level samples in Lake TTKK. Both metabolic processes and microbial transformation have been shown to eventually affect $\delta^{202}$Hg but not $\Delta^{199}$Hg signatures (Li et al., 2022). Metabolic isotope fractionation is however limited in fish and the incorporation of different MMHg sources is privileged for explaining the
shift of δ²⁰²Hg in Lake TTKK high level organisms. This hypothesis is however balanced by the similar δ²⁰²MMHg and Δ¹⁹⁹MMHg (p > 0.05) values observed between piscivorous and benthic fish, suggesting that the source of MMHg is comparable regardless of the different sediment-related dietary sources. Previous work observed significant Hg methylation in epibenthic biofilms and bottom Characea's periphyton in addition to surface sediments, while it remains negligible and Totora's epiphytic biofilm from the same area in Lake TTKK (Bouchet et al., 2018a, 2018b). Bottom sediments environments are thus considered as a net source of MMHg to the water column (Guédron et al., 2020b).

Overall, MMHg CSIA results confirm that sediment is a major and direct MMHg source to the lake UU food web but an additional MMHg pool, corresponding to photodegraded MMHg likely originating from sediment environments (surface sediments, benthic biofilms), contributes to the bioavailable pool of MMHg for the food webs in lake TTKK. These results are consistent with previous observations of high proportions of MMHg in water, rising up from <1 to ~50 % of THg from Lake TTKK to Lake UU (Guédron et al., 2017). Such high %MMHg were explained by a high in situ water column MMHg production in relation to the sulfate rich substrate, the low oxygen levels of the water column, and the stabilization of MMHg due to abundant ligands present in UU alkaline waters (Bouchet et al., 2018b). In addition to Hg photochemical reaction tracing by odd-Hg MIF, even-Hg MIF were applied to address Hg(II) sources (Bouchet et al., 2022).

### 3.5. Extent of photochemical reactions revealed by MIF signatures along lakes' foodwebs

The coupled information between MIF amplitude and Δ¹⁹⁹Hg/Δ²⁰¹Hg slopes has proved relevant to decipher and assess Hg(II) photo-reduction and MMHg photodegradation processes (Bergquist and Blum, 2007). When considering Hg-BSIA and MMHg-CSIA in the sediment and foodweb samples, the Δ¹⁹⁹Hg/Δ²⁰¹Hg slopes are 1.29 ± 0.03 (n = 156) and 1.25 ± 0.03 (n = 34) for Lake UU and TTKK, respectively (Fig. 3a). It confirms the variations previously observed in the slope imprinted during MMHg photodegradation resulting from the different MMHg/Sred-DOM ratios of each lake (Sred-DOM = reduced organic sulfur, (Bouchet et al., 2022)). Using in-situ incubation experiments, we previously found significantly different Δ¹⁹⁹Hg/Δ²⁰¹Hg slopes of 1.34 ± 0.02 (DOC = 16 mg.L⁻¹, depth = 0.25-2 m, lake UU), 1.33 ± 0.02 (DOC = 6 mg.L⁻¹, depth = 0.25-2 m, lake TTKK) and 1.24 ± 0.03 (DOC = 3 mg.L⁻¹, depth = 5 m, lake TTKK) during MMHg photodegradation in

representative waters from Lakes UU and TTKK (Bouchet et al., 2022).

With significantly different average values for Δ¹⁹⁹MMHg observed in fish from Lake TTKK and UU (p < 0.001), i.e. 4.3 ± 0.9 (n = 15) and 1.7 ± 0.2 ‰ (n = 28), respectively, the two food webs record MMHg that have undergone distinct photodegradation extents prior to biota incorporation. These extents were estimated by using the Rayleigh equation and the isotope fractionation factors ε<sub>Δ199</sub> = -12.4 ± 1.1 ‰ and ε<sub>Δ199</sub> = -18.5 ± 3.9 ‰ previously determined for lake UU and lake TTKK (depth = 5 m) (Bouchet et al., 2022). MMHg photodemethylation was found to range between 11 ± 4 % (aquatic plants, n = 8) and 19 ± 1 % (piscivorous fish, n = 8) in UU biota, except in epiphytic biofilm where it is close to zero. In Lake TTKK, it is 5 ± 1 % in invertebrate (n = 1) and 21 ± 4 % in benthic fish (muscle, n = 15), respectively (Fig. 3b). These results indicate that the differences in % photodegradation between Lake TTKK and Lake UU are less pronounced than expected. This suggests that factors beyond the overall photic zone and residence time play a role in controlling MMHg photodegradation. Notably, the extent of MMHg photodegradation varies across different compartments, likely due to local conditions such as OM quality/quantity and organism density, which influence MMHg residence time before incorporation and subsequent photodegradation. This highlights an important advancement from our previous work: Hg-CSIA can be used to evaluate localized Hg transformations more precisely, shedding light on the extent of photochemical reactions occurring in microenvironments such as biofilms and zooplankton.

Overall calculated demethylation yields are relatively low compared to those of 65 %, 68 % or 76 % estimated in Tibetan Lakes (Xu et al., 2016), Lake Michigan (Bergquist and Blum, 2007) or Lake Baikal (Perrot et al., 2012) respectively but closer to 27 % observed in Norwegian lakes (Poste et al., 2015). This discrepancy is mainly due to lower isotope fractionation factors (ε) considered here rather than MIF magnitudes that are consistent across the different lakes. With strong ultraviolet intensity at high-altitudes, higher demethylation rates were expected at the Bolivan Altiplano lakes and these results confirm that demethylation efficiency is not only influenced by UV light intensity but rather by a complex combination of MMHg residence time, UV light attenuation in the water column as influenced by particle and dissolved organic matter (DOM) concentration and type in surface waters.

With higher Hg(II) proportions, the Δ¹⁹⁹Hg/Δ²⁰¹ slopes for HgT in sediments are 1.06 ± 0.9 (n = 5) and 1.08 ± 0.25 (n = 9) for lake UU and TTKK respectively (Fig. 3a and S-9). These values align with the photoreduction of Hg(II) prior to its incorporation. Bergquist and Blum

<figure><img src="image_8.png" /><figcaption>**Fig. 3.** a) Δ¹⁹⁹Hg vs. Δ²⁰¹Hg (all data including Hg-BSIA and MMHg-CSIA) observed in Lake UU and TTKK foodwebs. b) MMHg demethylation yields determined in Lake UU and TTKK foodwebs. * For sediment, as Δ¹⁹⁹Hg vs. Δ²⁰¹Hg is consistent with Hg(II) photo-reduction, yield represents photo-reduced Hg(II).</figcaption></figure>
(2007) reported a slope of approximately 1 for Hg photoreduction at high Hg/DOC ratios, whereas Zheng and Hintelmann observed values around 1.2 under lower Hg/DOC conditions. However, the large uncertainties associated with the slope estimates preclude any definitive interpretation regarding an intermediate Hg/DOC ratio or the potential coexistence of two Hg pools, one subjected to photoreduction and the other remaining unaffected. The isotope fractionation factor $\epsilon_{\Delta199} = -2.7 \text{ ‰}$ determined by (Zheng and Hintelmann, 2009) at low Hg/DOC ratios was used to calculate the extent of Hg(II) photoreduction. Sediments exhibited low Hg(II) photoreduction, reaching 8 ± 4 % in Lake UU and 13 ± 6 % in Lake TTKK. These results are consistent with the iHg photoreduction rates previously reported, which showed generally higher photoreduction in TTKK, except at the BC station, where eutrophication is most pronounced (Bouchet et al., 2022). This pattern likely reflects the longer residence time and enhanced light penetration in TTKK, attributed to its lower concentrations of dissolved and particulate organic matter compared to UU. Given the scarcity of comparable estimates in the literature, these findings warrant further discussion as they provide unique insights into Hg cycling in lake sediments.

To summarize, Hg-CSIA approach allowed to confirm different MMHg assimilation from low to high trophic level organisms in Lake TTKK and UU. The very shallow (<1 m) Lake UU exhibits a more homogeneous MMHg source dominated by sediments whereas the deeper Lake TTKK (<1–40 m) foodweb has MMHg contributions both from sediments and from photic zone MMHg.

### 3.6. Tracking Hg(0) vs. Hg(II) sources: insights from even-Hg MIF

Hg even-Hg MIF ($\Delta^{200}$Hg and $\Delta^{204}$Hg, Table S-10b) is thought to be generated exclusively by upper atmospheric photochemical reactions and is therefore considered as a conservative tracer for lower atmospheric Hg deposition pathways. Being only 300 km far from each other, the two lakes probably record similar Hg atmospheric deposition signatures coming from the two main air masses clusters identified as the “Madre de Dios” watershed (Amazonian origin) and Central Volcanic Zone (Pacific Ocean) (Koenig et al., 2021). However, the high seasonal variability linked to local sources, such as biomass burning, may lead to slight differences in atmospheric Hg deposition between the two lakes. A recent study suggested that $\Delta^{200}$Hg values of 0.14 ‰ and - 0.05 ‰ are reasonable estimates, in this context, for Hg(II) (both wet and dry) and atmospheric Hg(0) deposition, respectively (Jiskra et al., 2021). On the other hand, Hg(0) used for amalgamation and found in tailings deposits from Bolivian gold mining exhibit $\Delta^{200}$Hg from - 0.01 to 0.03 ‰ (Laffont et al., 2011; Schudel et al., 2019). With $\Delta^{200}$Hg = -0.05 ± 0.04 ‰ observed atmospheric Hg during biomass burning (Richter et al., 2023), long-range atmospheric $\Delta^{200}$Hg (Hg(0)) signature is not significantly different from local $\Delta^{200}$Hg (Hg(0)) sources, both were pooled and named Hg(0). Except aquatic plants where mixed atmospheric/water carbon sources are likely (see previous part), a homogenous $\Delta^{200}$Hg value of 0.04 ± 0.02 ‰ was found within the food web of Lake UU (Fig. 4a) whereas sediments exhibit a significantly lower average value of -0.01 ± 0.03 ‰ (n = 5, p* < 0.05).

In Lake UU, sediments appear dominated by Hg(0) signatures while the rest of the food web presents a mixed signature between Hg(0) and Hg(II). Through isotopic mass balance calculations, Hg geogenic sources were found to contribute 12 % of UU sediments whereas Hg(0) sources accounted for 53 and 78 % in sediment and fish respectively. Hg(0) contribution to the UU biota ranges between 46 and 52 % except aquatic plants where Hg(0) accounts for 22 %. This high Hg(0) source contribution to the Lake UU food web is in line with the correlation observed between THg and tracers of exploited ores, and the high concentration of trace metal(oid)s in sediments downstream of the Oruro and Huanuni mining areas (Tapia et al., 2012). Enhanced leaching and erosion of both dissolved and particulate Hg from mining waste deposits were suspected in Lake UU during the wet season (Alanoca et al., 2016b). Altogether these results could explain the moderate MMHg concentrations

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** a) $\Delta^{200}$Hg values measured in the various compartments of Lake UU and TTKK. b) Apportionment of Hg(0) and Hg(II) (wet and dry) sources to the various compartments of Lake UU and TTKK. Hg(geogenic) source was added to sediment samples. Isotope data are MMHg-CSIA except those indicated with *, which are HgT-BSIA.</figcaption></figure>

observed in lake UU fish (649 ± 98 ng.g$^{-1}$, n = 47) that do not reflect high HgT concentrations found in the surrounding sediments (182 ± 142 ng.g$^{-1}$, n = 5 and elsewhere (Alanoca et al., 2016b)).

In Lake TTKK, a similar shift is observed between the $\Delta^{200}$Hg values found in sediments and the food web, i.e. 0.05 ± 0.02 and 0.14 ± 0.02 ‰, respectively. The overall range of $\Delta^{200}$Hg values is however significantly higher than in Lake UU, clearly indicating that Hg(II) deposition act as a dominant source to the food web (up to 94 % in fish) whereas sediments record a mix of sources (20 % geogenic Hg /37 % Hg(II) /42 % Hg(0)). These results support the hypothesis of very dynamic exchanges between atmospheric, aquatic and benthic Hg cycling and that MMHg source in the Lake TTKK originate mostly from atmospherically deposited Hg, before methylation and foodweb uptake in this aquatic system. Hg(II) contributions from 51 % to 95 % in sediment and fish respectively (Fig. 4b) are consistent with recent estimations of Hg(II) contribution being up to 82 % in TTKK sediments dated from the late Holocene to present (Jiskra et al., 2022). With a surface of 8000 km$^2$ and a volume of 900 km$^3$, Lake TTKK can collect extensive Hg(II) dry or wet depositions, which would largely dilute local anthropogenic Hg(0) inputs. This finding aligns with prior observations indicating i) negligible local anthropogenic contributions in Lake TTKK outside the area impacted by the main tributary outlet discharging waters from the suburbs of La Paz (Heredia et al., 2022) and ii) the relatively low HgT concentrations (66.1 ± 10.4 ng.g$^{-1}$) in the sediments of Lake TTK (Guédon et al., 2020b) compared to those reported for contaminated sites affected by mining activities in the Altiplano region (Alanoca et al., 2016b). Consequently, this may explain why Hg concentrations in the TTKK food web are not significantly different from those in relatively more pristine environments.
CRediT authorship contribution statement

**Jérémy Masbou:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. **David Point:** Writing – review & editing, Investigation, Funding acquisition, Conceptualization. **Sylvain Bouchet:** Writing – review & editing, Investigation, Conceptualization. **Jeroen E. Sonke:** Writing – review & editing, Resources. **Carlos Molina:** Investigation. **Carla Ibanez:** Investigation. **Anne Lorrain:** Writing – review & editing, Investigation. **Julio Pinto:** Investigation. **Dario Acha:** Writing – review & editing, Investigation. **David Amouroux:** Writing – review & editing, Investigation, 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.

Acknowledgements

This work is a contribution to LA PACHAMAMA (ANR CESA program, no. ANR-13-CESA-0015-01) and ECCO CYTRIX COMIBOL projects. We thank Jérôme Chmeleff and Laure Laffont for expert management of the OMP mass spectrometry and mercury lab facilities.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2025.179630.

Data availability

Data will be made available on request.

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