# Temporal change of arsenic in leachates from Sargassum spp. biomass deposit: mass balance, speciation and microbial biotransformation pathways

Hugues Thouin, Mohamed Fikrat, Fabienne Battaglia-Brunet, Catherine Joulian, Mickael Charron, Adrien Mestrot, Véronique Vacchina, Fabienne Séby, Patrick Ollivier

► To cite this version:

Hugues Thouin, Mohamed Fikrat, Fabienne Battaglia-Brunet, Catherine Joulian, Mickael Charron, et al. Temporal change of arsenic in leachates from Sargassum spp. biomass deposit: mass balance, speciation and microbial biotransformation pathways. Science of the Total Environment, 2025, 1005, pp.180840. 10.1016/j.scitotenv.2025.180840. insu-05352602

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# Temporal change of arsenic in leachates from *Sargassum* spp. biomass deposit: mass balance, speciation and microbial biotransformation pathways

Hugues Thouin<sup>a,∗</sup>, Mohamed Fikrat<sup>a</sup>, Fabienne Battaglia-Brunet<sup>a,b</sup>, Catherine Joulian<sup>a</sup>, Mickael Charron<sup>a</sup>, Adrien Mestrot<sup>c</sup>, Véronique Vacchina<sup>d</sup>, Fabienne Séby<sup>d</sup>, Patrick Ollivier<sup>a</sup>

## HIGHLIGHTS

* *Sargassum* biomass decay was studied in a closed controlled experiment.

* As concentration in *Sargassum* leachates reached 10 mg/L.

* After 120 days, 50 % of the initial *Sargassum* As content was released in leachate.

* High levels of volatile As species were released during the first weeks.

* Bacterial diversity changes during *Sargassum* decomposition impact the As fate.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /><figcaption>Arsenic in leachate</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Microbial community</figcaption></figure>

<figure><img src="image_6.png" /></figure>

## ARTICLE INFO

### Keywords:

* Sargassum decomposition
* Arsenic content and speciation
* Bacterial community
* Environmental risks

## ABSTRACT

The decomposition of washed ashore *Sargassum* biomass is a major health and environmental concern due to arsenic (As) release. A 120-day mesocosm experiment without replication, simulating natural conditions at stockpiled *Sargassum* sites (temperature, sunlight, rain), was conducted to study the fate of As. In fresh *Sargassum*, total As concentrations were 85 mg/kg (dw). As<sup>III</sup> was the most abundant species, followed by TMAsO, As<sup>V</sup>, AsB and DMAs<sup>V</sup>. In leachates, the As concentration was initially 10 mg/L and then decreased to 1.5 mg/L after 90 days of experimental monitoring. As<sup>V</sup> was more abundant than As<sup>III</sup>, and varying amounts of AsB and DMAs<sup>V</sup> were also detected. High arsine content, i.e. mainly trimethylarsine ((CH<sub>3</sub>)<sub>3</sub>As), was released during the first weeks. After 120 days, 50 % of the initial total *Sargassum* As content was released in the leachate, while 51 µg volatilised in the atmosphere (0.06 ‰). As remaining in this biomass was still high (55 mg/kg dw) due to the substantial mass loss during its degradation. Bacterial diversity increased over time in the leachate and decomposed biomass, suggesting that bacteria had a major role in the As fate and speciation. According to the

<footnote><sup>a</sup> BRGM, F-45060, Orléans, France</footnote>

<footnote><sup>b</sup> Université d'Orléans, CNRS, BRGM, ISTO, UMR 7327, 45071, Orléans, France</footnote>

<footnote><sup>c</sup> Institute of Geography, University of Bern, Hallerstrasse 12, 3012, Bern, Switzerland</footnote>

<footnote><sup>d</sup> UT2A, 2, avenue du Président Angot, 64053, Pau, France</footnote>

<footnote>* Corresponding author.
E-mail address: h.thouin@brgm.fr (H. Thouin).</footnote>
French legislation, the discharge limits in terms of As concentration in leachates of the decaying *Sargassum* biomass from hazardous waste treatment facilities would often be exceeded.

# 1. Introduction

Since 2011, the Caribbean and Gulf of Mexico coasts have experienced massive seasonal arrivals of holopelagic *Sargassum* (Van Tussenbroek et al., 2017; Oviatt et al., 2019). Satellite imagery has highlighted the presence of a Great Atlantic *Sargassum* Belt (GASB) extending from West Africa to the Gulf of Mexico (Brooks et al., 2018; Wang et al., 2019). These blooms are driven by factors such as Inter-Tropical Convergence Zone (ITCZ) winds, ocean circulation and nutrient inputs (Oviatt et al., 2019; Wang et al., 2019). Three holopelagic *Sargassum* morphotypes have been reported in these blooms: *S. natans* VIII, *S. natans* I and *S. fluitans* III (Schell et al., 2015; Amaral-Zettler et al., 2017; Dibner et al., 2022).

These phenomena cause beach deterioration, while threatening local biodiversity, fisheries and tourism (Ofori and Rouleau, 2020; Robledo et al., 2021). The seaweeds begin to rot shortly after reaching shallow waters and beaches, thereby reducing light and oxygen levels in near-shore water and resulting in the mortality of seagrasses and their associated fauna (Van Tussenbroek et al., 2017). *Sargassum* is also a human health concerns due to the large amounts of toxic gases, including hydrogen sulphide and ammonia, that are produced when the seaweeds start decomposing on the seashore (Resiere et al., 2021).

*Sargassum* is able to absorb metals and metalloids from the pelagic environment (Rodríguez-Martínez et al., 2020; Ortega-Flores et al., 2022). The accumulation of both essential and non-essential metals and arsenic (As) in different seaweed thallus parts has been documented in several studies (Bonanno and Orlando-Bonaca, 2018; García-Seoane et al., 2018). Among these accumulated elements, the toxic metalloid As has been detected in algal food and other *Sargassum*-derived products (Devault et al., 2022a; Magura et al., 2019; Milledge et al., 2020; Rodríguez-Martínez et al., 2020, Cipolloni et al., 2023). High concentrations of metals and metalloids such as iron (Fe), copper (Cu), zinc (Zn), cadmium (Cd) and As in seaweed biomass can lead to environmental issues and may hamper their use as fertiliser, livestock feed and foods for human consumption (Rodríguez-Martínez et al., 2020). Before washing ashore, the As concentration in *Sargassum* biomass may vary according to: (1) spatiotemporal scales, which depend on the previous trajectory of the floating *Sargassum* rafts, (2) phosphate, copper and iron concentrations in the biomass (Mamun et al., 2019, Cipolloni et al., 2023, Ortega-Flores et al., 2022), and (3) the concentration of uronic acids and sulphate from fucoidan (Ortega-Flores et al., 2022). As speciation in *Sargassum* spp. from the French West Indies has been studied by Devault et al. (2022b), who reported that AsV is the dominant form of As in offshore *Sargassum*, whereas AsIII, DMAsV (dimethylarsinic acid), MMAsV (monomethylarsonic acid) and AsB (arsenobetaine) were also detected. *Sargassum* beaching induces a modification in the As content and speciation. Total As, AsV, MMAsV and AsB contents are significantly higher in dried beached samples than in floating algae (Devault et al., 2022b).

Metals and As primarily accumulated in living *Sargassum* are released in the liquid phase generated by seaweed cell lysis and rain-water leaching of dead biomass. The resulting aqueous phases represent pollution sources for coastal sea water, and for surface or underground freshwater (García-Sánchez et al., 2020). In 1.25 L reactors, Olguin-Maciel et al. (2022) studied leachate produced by *Sargassum* biomass decomposition in natural conditions along the Mexican Caribbean coast. Their leachate production calculations indicated that each ton of fresh *Sargassum* that decomposes at a controlled temperature of 27 °C could produce 316 L of leachate. This leachate in turn could contain 5.67 g of As and other potentially toxic metals (e.g. B, Al, Cu).

Arsenic can occur in the form of diverse inorganic and organic

chemical species that can undergo microbial transformation. Oxidation of AsIII (Battaglia-Brunet et al., 2002) may correspond to resistance or energy production mechanisms (Bryan et al., 2009). Reduction of AsV can be achieved via the *ars* system, whose main function is detoxification with AsV reduced by an arsenate reductase ArsC (Martin et al., 2001), or by the *arr* dissimilatory reduction system associated with anaerobic respiration (Oremland and Stoltz, 2003). Methylation of As may either play a role in resistance or microbial chemical warfare (Viacava et al., 2020) and can induce the generation of volatile As species (Mestrot et al., 2013a). In addition, some micro-organisms are involved in the degradation (Pengprecha et al., 2005) or the production of organoarsenic compounds (Xue et al., 2018). The complexity of decaying *Sargassum* biomass, involving solid phases and leachates, with many available organic compounds, a range of salinities and diverse microflora, probably induces many simultaneous As speciation transformations. Moreover, the As speciation pattern governs the mobility and toxicity of this element in leachates and water phases. Another issue could be linked to As volatilisation during algal biomass degradation as microorganisms can produce volatile As species, which are collectively referred to as ‘arsines’. The most common species are inorganic arsine (AsH₃) as well as mono-, di- and trimethylarsine ((CH₃)AsH₂, (CH₃)₂AsH and (CH₃)₃As, respectively). These can be produced during anaerobic (Michalke et al., 2000; Mestrot et al., 2013b) or aerobic (Diaz-Bone et al., 2011; Zhai et al., 2021) biodegradation of wastes with a high organic matter content.

In this context, we aimed to study variations in the concentration and speciation of As during the natural degradation process of *Sargassum* biomass from the French West Indies. The study was conducted in a mesocosm under controlled conditions close to those prevailing at stockpiled *Sargassum* sites, thereby enabling precise assessment of As species over time. Regarded as a case study due to the lack of replication and the experimental scale, this setup allowed examination of the different compartments likely to contain As were thus examined, i.e. the liquid phase (leachate), gas phase, and solid algal biomass, while assessing the impacts of microbial processes.

# 2. Material and methods

## 2.1. *Sargassum* sampling and characteristics

*Sargassum* seaweed was collected at Anse du Belley, Sainte-Anne, Guadeloupe (France) (16.2320, -61.3686) and then directly transported by plane to the BRGM laboratory (Orléans, France) in three 110 L drums after draining the sea water. The corresponding 42 kg (9.66 kg dry weight equivalent) of fresh *Sargassum* biomass was visually estimated to be composed of a mixture of about 20 % *S. natans* VIII and 80 % *S. fluitans* III.

## 2.2. Mesocosm experiment

Immediately after receipt, the *Sargassum* samples were deposited in an empty 220 L plastic tank equipped with bottom pipe-connected outlet. This tank was placed in a stainless-steel column allowing atmosphere control (Supplementary material SM1). The mesocosm water jacket temperature was set at 25 °C, in line with the average temperature prevailing in the West Indies over the study period. The day-night cycle was simulated by LED lighting with a cycle of 12 h light and 12 h darkness.

Based on precipitation time series data for the French West Indies (Martinique) in recent years, we designed a time series reproducing the monthly cumulative precipitation pattern for the May–August period
and tailored to the *Sargassum* surface inside the tank. The equivalent precipitation levels were 52.5 mm for May (days 4–34), 115 mm for June (days 35–62) and 540.5 mm for July and August (days 63–118). The watering time series is presented in Supplementary material SM2. Rainfall was simulated using pipes equipped with sprinklers. Three simulated rainfall volumes were applied (1 L, 5 L and 10 L), with a frequency of two watering events per week. Slightly mineralised Mont Roucous water (pH 5.85, 3.1 mg.L⁻¹ Na⁺, 2.4 mg.L⁻¹ Ca²⁺, 0.5 mg.L⁻¹ Mg²⁺, 2.0 mg.L⁻¹ SO₄²⁻, 6.3 mg.L⁻¹ HCO₃⁻, 3.0 mg.L⁻¹ NO₃⁻) was used as laboratory rain water (Thouin et al., 2018). The first rainfall event was applied 10 days after the *Sargassum* biomass had been placed in the monitored column.

Time domain reflectometry probes (TDR) (TRIME-PICO32, IMKO) were inserted in the *Sargassum* bed (middle layer) and in the column atmosphere to measure moisture and temperature. Specific chemotrips for volatile As species (Mestrot et al., 2009) were placed in the air inlet and outlet and replaced weekly. Air was injected into the column at a flow-rate of 1.5 L.min⁻¹.

The leachate was collected at the column outlet and weighed after each watering event in glass bottles (2 L or 5 L) or cans (20 L) continuously flushed with N₂ to avoid oxidation. The physicochemical parameters (Eh, pH, conductivity and temperature) were immediately measured and the leachate was sampled for chemical analysis and microbial community characterisation, and then stored in a N₂ atmosphere at 5 °C.

At the end of the experiment, the final *Sargassum* biomass was removed from the plastic tank in three layers of equivalent thicknesses (top, middle and bottom), weighted and sampled for As content, As speciation and biomolecular analysis. The samples were frozen at -80 °C for microbial community characterisation and freeze dried for As analysis.

## 2.3. Arsenic characterisation of Sargassum biomass

A mass of 0.2 g of dried algae was mineralised in 4 mL of nitric acid (70 %) and 2 mL of hydrogen peroxide (30 %) on a hot block at 90 °C for 3 h. After dilution with ultrapure water, the total As content was measured by inductively coupled plasma mass spectrometry (ICP-MS 7800, Agilent Technologies). The ⁷⁵As isotope was used for quantification. IAEA-423 Certified Reference Material (Vienna, Austria) was analysed together with the samples. Recoveries calculated from the certified concentration values (127 ± 7 mg/kg) were in the 91–101 % range.

Two different extraction protocols were used for As speciation in freeze dried algae. For the first one, 0.2 g of dried algae were extracted with 10 mL of a mixture of nitric acid (1 %) and hydrogen peroxide (2 %) on a hot block at 95 °C for 90 min and then supplemented with ultrapure water to 50 mL volume. For the second one, 0.2 g of dried algae were sequentially extracted with 10 mL of a water/methanol mixture (1/1, v/v and then 1/9, v/v) for 1 h in an ultrasonic bath. The As³⁺, As⁵⁺, MMA⁵⁺, DMAs⁵⁺, arsenocholine (AsC) and AsB species in extracts, regardless the protocol, were analysed by high performance liquid chromatography (UHPLC 1290, Agilent Technologies) coupled with ICP-MS (ICP-MS 7800, Agilent Technologies). The present study was focused on volatile and water soluble As species and no lipid As species were investigated. As no certified reference material was available, the analysis accuracy was verified by spiking the matrix with As³⁺, As⁵⁺, MMA⁵⁺, DMAs⁵⁺, AsC and AsB species prior to extraction. The recoveries obtained ranged from 89.1 to 108.6 %, depending on the species. The TMAO species was also investigated but its quantification was only estimated in the absence of certified standard solution.

## 2.4. Chemical leachate analysis

A 1 mL volume of filtered (0.45 µm) and acidified (HNO₃) leachate was mineralised in 2 mL of nitric acid (70 %) and 1 mL of hydrogen

peroxide (30 %) on a hot block at 95 °C for 1 h. After dilution with ultrapure water, the total As content was measured by ICP-MS (ICP-MS 7800, Agilent Technologies) using the ⁷⁵As isotope; and Ca, Mg, K, S, Na, Fe and Mn were measured by ICP-AES (Arcos II, Spectro) using 393,366 nm, 279,553 nm, 769,896 nm, 180,731 nm, 588,995 nm, 238,204 nm and 257,611 nm wavelengths, respectively. In each case, element quantification was carried out by external calibration.

The filtered (0.2 µm) leachates conditioned in sterile N₂-filled gas tight glass tubes were 10-fold diluted and then analysed for As speciation by HPLC-ICP-MS, using the same procedure as that described above for the *Sargassum* extract. As³⁺, As⁵⁺, MMA⁵⁺, DMAs⁵⁺, AsC and AsB species quantification was carried out by external calibration.

Anion (sulphite, sulphate, thiosulphate, nitrate and phosphate) concentrations were determined in the filtered (0.2 µm) leachates by ion chromatography equipped with conductivity detector (Model 930, Metrohm). For this, 20 µL of leachates were injected in a Metrosep A Supp 5 column (150 × 4 mm) equipped with a guard column. The mobile phase was 1 mM NaHCO₃ and 3.2 mM Na₂CO₃ in isocratic mode. The flow rate was set at 0.7 mL/min. Quantification was carried out by external calibration.

The total organic carbon (TOC) and dissolved organic carbon (DOC) concentrations were respectively measured in unfiltered and filtered (0.45 µm) leachate, acidified with H₂SO₄, using a TOC (5050/SSM 5000-A, Shimadzu) elemental analyser.

## 2.5. Volatile As species analysis

Chemotrips specifically designed and validated to quantitatively measure As volatilisation were placed in the inlet and outlet of the traps to monitor the production and temporal variations in volatile As species (Mestrot et al., 2009, Müller et al., 2022). To account for possible trap saturation, two traps were set up in series in the outlet to catch any carry-over. The flow rate was ~1.5 L/min. To monitor temporal variations, the chemotrips were replaced on a weekly basis and stored in individual plastic bags.

Traps were opened and extracted by microwave (MARS 6, CEM) using a 5 mL solution of 1 % HNO₃ and 1.2 % H₂O₂ at 95 °C and then filtered at 0.22 µm and stored at 4 °C using the procedure detailed in Müller et al. (2022). Post-extraction, all volatile As species were converted to their oxy-anion counterparts (e.g. AsH₃ as AsV and (CH₃)₃As as TMAsO) and could thus be separated, identified and quantified by HPLC-ICP-MS (HPLC 1260, ICP-MS 7700×, Agilent Technologies). An anion exchange chromatographic column (PRP-X100, 10 µm, 4.1 × 150 mm, Hamilton) with a 6 mM ammonium nitrate and 6 mM ammonium phosphate mobile phase (pH 5.6) was used. Isocratic elution was performed for 12 min at a 1 mL/min flow rate. A standard containing TMAsO, DMAsV, MMAV and AsV at 10 µg/L was used for species identification. A solution containing rhodium in 1 % (v/v) HNO₃ was introduced from an external line as internal standard. The amount of As in the traps was blank-subtracted (unused chemotrips as blanks as well as outlet-inlet to account for possible external contamination). The sum of the two outlet traps was calculated if any carry-over was detected. The detection limit, calculated as 3.3 times the standard deviation of the blanks (n = 7), was 11.9 ng.

## 2.6. DNA extraction, functional gene detection and next generation sequencing

For microbial community characterisation in the sampled leachate, 50 mL of leachate were centrifuged at 10,000g for 20 min and the resulting pellets were frozen at -20 °C until DNA extraction. For microbial community characterisation in the final *Sargassum* biomass, the frozen samples were milled in sterile mortars and ~ 0.5 g of sub-sample was stored at -20 °C until DNA extraction. Genomic DNA was extracted in triplicate from the stored samples with the FastDNA® SPIN kit for soil (MP Biomedicals, Illkirch, France) according to the manufacturer's
instructions, using a FastPrep-24™ instrument at a speed of 5 m/s for 30 s.

The presence of the following functional genes involved in inorganic As biotransformation was determined by PCR: *aioA*, encoding the catalytic subunit of arsenite oxidase; *arsB*, encoding As<sup>III</sup>-efflux pump of Ars resistance system *arrA*, encoding respiratory ('dissimilatory') arsenate reductase; and *arsM*, encoding arsenite methyltransferase. Primers and PCR conditions are outlined in Supplementary material SM3.

To determine the taxonomic composition of microbial communities, the V4-V5 region of the gene coding 16S rRNA (Bacteria and Archaea domains) was amplified using the barcoded, universal primer set 515WF/918WR. PCR reactions were performed using the AccuStart II PCR ToughMix kit and cleaned (HighPrep PCR beads, Mokascience). Pooled triplicates were submitted for sequencing on an Illumina MiSeq instrument at GeT-PlaGe (Auzeville, France). These steps were performed by INRAE Transfert Methys (Narbonne, France).

The sequencing datasets were processed using the Find, Rapidly, OTUs with Galaxy Solution (FROGS) bioinformatics pipeline (Escudière et al., 2018). Fastq paired reads were merged with the Vsearch software package, and clustered into operational taxonomic units (OTUs) using Swarm with an aggregation distance clustering of 1. Chimera and OTUs with a proportion < 0.0005 % of all sequences were removed. Taxonomic affiliation was conducted using BLAST and the Silva 138.1 database for 16S rRNA gene sequences. The raw datasets are available on the European Nucleotide Archive system (project accession number: PRJEB80819).

## 2.7. Statistical analysis

Statistical analyses were performed using R (http://www.r-project.org/; version 4.3.3).

A generalised additive model (GAM) was used to fit variations in pH, Eh, EC, majors and As species concentrations during the experiment using the mgcv package (version 1.9-1). A thin plate regression spline was used. The model smoothness was controlled by the number of knots and the associated effective number of degrees of freedom. The optimal number of knots was estimated through cross-validation. QQ plots (sample quantiles plotted against theoretical quantiles) and Shapiro tests were used to assess the normality of the model residuals. We examined autocorrelations of the model residuals. The model validity was also assessed through Adj.R2. An analysis of deviance was used to assess the significance of the null hypothesis for the smooth function. For the smooth function, the finite difference method was used to calculate the first derivative of the function and the associated confidence interval. This first derivative was used to estimate the rate of change to identify periods of statistically significant change.

Alpha and beta diversity were determined for each sample using the microeco 1.2.0 package (Liu et al., 2021). The ggalluvial package (version 0.12.5) was used to draw the stack histogram of the bacterial relative abundances in the leachate. PCoA analysis of the bacterial community based on UniFrac distances (Lozupone and Knight, 2005) was performed on leachates and on the different decayed *Sargassum* biomass layers at the end of the experiment. A Pearson correlation matrix was calculated to determined relationships between the relative abundance of the 20 most abundant genera and environmental factors in leachates (n = 20). P values were adjusted by the false discovery rate (FDR). Linear discriminant analysis (LDA) effect size (LEfSe, Segata et al., 2011) was performed from phylum to genus level in different depth layers of decayed *Sargassum* biomass at the end of the experiment.

# 3. Results

## 3.1. Arsenic in Sargassum biomass

Arsenic content and speciation were analysed upon reception of the *Sargassum* sample (composed of *S. natans* VIII and *S. fluitans* III) and at

the end of the experiment (Table 1). The initial *Sargassum* sample, contained 85 mg of As per kg of dry *Sargassum*, with a total As content of 821 mg in all *Sargassum* biomass used in the experiment. Arsenic speciation, using extraction by diluted HNO₃/H₂O₂ for full As recovery without distinguishing between As<sup>III</sup> and As<sup>V</sup>, revealed that about two thirds of the water soluble As content was inorganic and that the organic species were shared between DMAs<sup>V</sup>, TMAsO and AsB (Table 1). Extraction by a water/methanol mixture to determine if the inorganic As was As<sup>III</sup> or As<sup>V</sup>, did not enable full As recovery, but revealed that As<sup>III</sup> was the main inorganic As form in the initial *Sargassum* biomass.

At the end of the experiment, the As content was measured in the bulk sample and in three layers: top surface layer (0–2 cm) composed of dry barely degraded *Sargassum*, middle (2–7 cm) and bottom (7–12 cm) layers composed of very degraded *Sargassum*. The As content decreased from 103 to 52 mg/kg with depth, and the bulk sample contained 55 mg/kg. This As was mainly inorganic, while DMAs<sup>V</sup> was the sole organic As species remaining in *Sargassum* biomass at the end of the experiment.

## 3.2. Variations in the leachate chemical parameters

### 3.2.1. Physicochemical parameters organic carbon and major ion

The leachate pH ranged from 6 to 9, increasing with time from days 0 to 50–60 (Fig. 1). A significant increase in pH was observed during the first 40 days of the experiment. The redox potential was low (around –300 mV ref. Ag/AgCl) during the first 30 days of the experiment, then it significantly increased from days 30 to 110, reaching values close to +200 mV at the end of the experiment.

Electric conductivity (EC) variations were characterised by two phases of significant decrease, i.e. the first from days 0 to 10, and the second from days 50 to 100. Between these two phases, the EC slightly increased between days 30 and 50. The temperature within the *Sargassum* biomass reached its maximum value of ~40 °C during the first days of the experiment (SM4). It tended to diminish to reach the water jacket value (25 °C) after day 60. However, punctual temperature peaks were observed around days 14, 31, 47 and 82.

At the outset of the experiment, the leachate contained high total organic carbon (TOC) and dissolved organic carbon (DOC) concentrations, with initial values of 4.6 g/L and 3.9 g/L, respectively (Supplementary material SM7). These concentrations sharply decreased and reached a steady concentration of 170–180 mg/L between days 21 and 42. A second decrease was then observed, with concentrations gradually dropping to ~100 mg/L until day 60, after which they remained stable until the end of the experiment. The TOC and DOC concentrations were nearly identical, except during the first days of the experiment, where a small fraction of organic carbon was present in particulate or colloidal form. Throughout the rest of the experiment, dissolved organic carbon molecules were predominantly present in the leachate.

The main soluble elements analysed in the leachate were potassium (K) and sodium (Na), whose concentrations followed the EC pattern. An initial decrease was observed from days 0 to 26, followed by an increase between days 27 and 40, and a final decreasing phase from day 41 until the end of the experiment (Fig. 2A). Nitrate (NO₃⁻) and phosphate (PO₄³⁻) were not detected in the early stages of the experiment but started emerging in the leachate around day 90, reaching maximum concentrations of 250 mg/L and 25 mg/L, respectively. The analysis of sulphur species in the leachate highlighted a significant peak sulphate (SO₄²⁻) concentration that began around day 25, just before the increased rainfall flow, and reached its maximum value (700 mg/L) between days 45 and 50 (Fig. 2B). Thiosulphate (S₂O₃²⁻) was present in relatively high but variable concentrations during the first 80 days of the experiment, whereas sulphite (SO₃²⁻) was only detected (at low concentration) at the beginning of the experiment.

### 3.2.2. Arsenic concentrations

The total leachate As concentration was maximal (~10 mg/L) at the beginning of the experiment (Fig. 3A). This initial high concentration
<table><caption>**Table 1**
Arsenic concentration and speciation in initial and final *Sargassum* biomass samples.</caption><thead><tr><th></th><th>Total As (mg/kg)<sup>(1)</sup></th><th>As<sup>V</sup> (mg/kg)<sup>(2)</sup></th><th>As<sup>III</sup> (mg/kg)<sup>(2)</sup></th><th>Inorg. As (mg/kg)<sup>(3)</sup></th><th>DMAs<sup>V</sup> (mg/kg)<sup>(3)</sup></th><th>MMAs<sup>V</sup> (mg/kg)<sup>(3)</sup></th><th>AsB<sup>a</sup> (mg/kg)<sup>(3)</sup></th><th>AsC<sup>b</sup> (mg/kg)<sup>(3)</sup></th><th>TMAsO (mg/kg)<sup>(3)</sup></th></tr></thead><tbody><tr><td>Initial bulk</td><td>85 ± 7</td><td>8.6 ± 0.8</td><td>52 ± 2</td><td>56 ± 4</td><td>11 ± 2</td><td>&lt;0.1</td><td>9 ± 2</td><td>&lt;0.2</td><td>12.3 ± 0.3</td></tr><tr><td>End bulk</td><td>55 ± 8</td><td>-</td><td>-</td><td>50 ± 8</td><td>5.7 ± 0.2</td><td>&lt;0.1</td><td>&lt;0.1</td><td>&lt;0.2</td><td>&lt;0.2</td></tr><tr><td>End, surface layer (0–2 cm)</td><td>103 ± 6</td><td>-</td><td>-</td><td>90 ± 3</td><td>3.9 ± 0.4</td><td>&lt;0.1</td><td>&lt;0.1</td><td>&lt;0.2</td><td>&lt;0.2</td></tr><tr><td>End, middle layer (2–7 cm)</td><td>58 ± 9</td><td>-</td><td>-</td><td>36 ± 4</td><td>4.4 ± 0.4</td><td>&lt;0.1</td><td>&lt;0.1</td><td>&lt;0.2</td><td>&lt;0.2</td></tr><tr><td>End, bottom layer (7–12 cm)</td><td>52 ± 4</td><td>-</td><td>-</td><td>36 ± 5</td><td>5 ± 1</td><td>&lt;0.1</td><td>&lt;0.1</td><td>&lt;0.2</td><td>&lt;0.2</td></tr></tbody></table>

(1) Mineralization and ICP-MS quantification, (2) MeOH-H₂O extraction and HPLC-ICP-MS quantification and (3) diluted HNO₃-H₂O₂ extraction and HPLC-ICP-MS quantification.

(-) not determined.

a Arsenobetaine.

b Arsenocholine.

<figure><img src="image_7.png" /><figcaption>**Fig. 1.** Variations in pH, redox potential (Eh ref. Ag/AgCl) and electric conductivity (EC) of the leachates. GAM is represented by solid line and 95 % confidence intervals is between the dashed lines. Blue lines indicate times periods with a significant increase, while red lines reflect periods with a significant decrease, based on the first derivative of the GAM. Background: white corresponds to a low precipitation regime, light grey to a medium precipitation regime and darker grey to a high precipitation regime (see SM2 for details).</figcaption></figure>

rapidly decreased over the first 26 days, when it reached a minimum of 3 mg/L. An increase was observed between days 27 and 39, with the total As concentration rising to 5.7 mg/L, followed by a continuously decreasing final phase. By the end of the experiment, the total As concentration was ~0.5 mg/L As. Total As, AsV, AsB and DMAsV concentrations were plotted and fitted with GAM, with significant concentration variations noted in SM5.

The main As species detected in the leachate were AsV, AsIII, DMAsV and AsB (Fig. 3A). Moreover, two unidentified As species were present at the beginning of the experiment, which accounted for 20 % of the total As. The oxidised inorganic form (AsV) was the dominant species throughout the experiment. Other As species were present in significant amounts until day 90, after which AsV represented more than 90 % of the total As. Prior to day 90, AsIII presented transient peaks, particularly
<figure><img src="image_8.png" /><figcaption>**Fig. 2.** Variations in the concentrations of major ionic species, anions (A) and sulphur compounds (B) in the leachate. Background: white corresponds to a low precipitation regime, light grey to a medium precipitation regime and darker grey to a high precipitation regime (see SM2 for details). Data are given in Supplementary material SM6.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Variations in concentrations of the main As species in the leachate and quantities of volatile As analysed in the mesocosm atmosphere (A) and cumulated As and As species in the leachate during the experiment (B). Background: white corresponds to a low precipitation regime, light grey to a medium precipitation regime and darker grey to high precipitation regime (see SM2 for details). Data are given in Supplementary material SM6.</figcaption></figure>

between days 14 and 26, where it became the dominant species. AsB was present as a minor As species from the beginning of the experiment to day 70, with its concentration increasing significantly from days 30 to 60, followed by a decrease. DMAs<sup>V</sup> was present in the leachate from days 0 to 30, with a significant increase between days 7 and 14 (SM5).

In addition to dissolved As species, volatile As compounds were

produced throughout the experiment, with their atmospheric concen-
tration peaking between days 9 and 30. Volatile As was composed of
(CH3)3As and (CH3)2AsH, respectively oxidised and analysed as TMAsO
and DMAsV. The proportions of these species remained stable over time,
with 92.7 ± 2.3 % of TMAsO (mean for all samples, with standard de-
viation; n = 16) and 6.6 ± 2.4 % DMAsV.
The cumulated quantity of As leached from the *Sargassum* biomass (9.66 kg dry weight) throughout the experiment was ~400 mg (Fig. 3B). The majority of this leached As (60 %) was in the form of AsV, while AsIII accounted for 12.5 %. Organic As species, including DMAsV and AsB, each represented 4 % of the total As release. The highest As release rate was noted between days 40 and 90, reaching a maximum of 4.55 mg/d. The total quantity of volatile As produced reached 50 µg, with the highest emission rate of 2.23 µg/d occurring between days 4 and 26 (Fig. 3B).

### 3.3. Variations in microbial parameters

#### 3.3.1. Microbial community in leachate

After sequencing and treatment steps, a total of 558,540 16S rRNA gene sequences were obtained from the 11 leachate samples. Only three OTUs affiliated with the Archaea domain were detected (2824 sequences). The richness and diversity of the microbial community in the leachate continuously increased over the time course of the experiment (Fig. 4A). Numbers of OTUs detected in the leachate increased from 201 in the initial leachate to 420 after 119 days of *Sargassum* decomposition. This increase in richness also led to an increase in Shannon and InvSimpson diversity indices. Beta diversity analysis showed continuous changes in the microbial community structure in the leachate, with a marked shift between the microbial composition in the first leachate

(day 4), that monitored at day 30, and the community at the end of the experiment (Fig. 4B). The identity and proportion of the 20 most abundant genera, all affiliated with the Bacteria domain (Fig. 4C), also indicated this pattern. The community always contained OTUs affiliated with aerobic or facultative aerobic genera: *Celeribacter* (Lee et al., 2012), *Winogradskyella* (Nedashkovskaya et al., 2005), *Balneola* (Urios et al., 2008), SM1A02 (Rambo et al., 2020), *Martelella* (Rivas et al., 2005), *Cerasicoccus* (Yoon et al., 2007), *Puneiceicoccus* (Choo et al., 2007), *Hydrogenovibrio* (Nishihara et al., 1991), *Amorphus* (Zeevi Ben Yosef et al., 2008), *Edaphobaculum* (Cao et al., 2017), *Terasakiella* (Satomi et al., 2002) and *Melioribacter* (Podosokorskaya et al., 2013). The microbial community also included OTUs affiliated with anaerobic bacteria: *Sphaerochaeta* (anaerobic fermentative bacterium found in marine environments; Miyazaki et al., 2014), *Desulfovibrio* (sulphate-reducing bacteria), and *Thiomicrospira* (anaerobic thiosulphate-oxidising and nitrate-reducing bacterium; Timmer-Ten Hoor, 1975). Moreover, *Denitrovibrio*, a strict anaerobe that uses nitrate as electron acceptor (Myhr and Torsvik, 2000), emerged as a major genus from days 26 to 61. An OTU affiliated with *Francisella*, i.e. a genus often described as being an intracellular pathogen of vertebrate and invertebrate hosts found in marine environments (Duodu et al., 2012), was also present, with a peak proportion found on day 11.

A Pearson correlation matrix between the relative abundance of the 20 most abundant genera and environmental variables highlighted

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Variations in leachate bacterial communities and the effects of geochemical parameters on bacterial communities according to the time of the experiment. A. Alpha diversity variations (Observed OTUs, Chao1, InvSimpson and Shannon index). B. Principal coordinate analysis (PCoA) of UniFrac distances C. Relative abundance of the 20 dominant genera in leachates. D. Pearson correlation heatmap of the 20 most abundant genera and environmental factors (n = 11; FDR adjusted p-value; ***p = 0.001, **p = 0.01, *p = 0.05).</figcaption></figure>
interactions between changes in the bacterial community structure and
significant changes in the geochemical composition of leachates and
environmental parameters (Fig. 4D). Distinct leachate bacterial/envi-
ronmental signatures were identified in this matrix, corresponding to
different phases of the experiment. The first leachates were charac-
terised by high concentrations of organic carbon, total As and As<sup>III</sup>, a
high saline signature and the presence of *Balneola*, *Celeribacter*, *Winog-
radskyella* and SM1A02. The leachates collected between days 10 and 30
were characterised by high DMAs<sup>V</sup> and thiosulphate concentrations, an
increased *Sargassum* temperature, a low redox potential and air moist-
ture, and the presence of *Martelella*, *Francisella* and MSLB3. Volatile
arsine production was also highest during this period (Fig. 3B). Leach-
ates collected between days 30 and 60 corresponded to high AsB and

SO₄²⁻ concentrations and the presence of *Legionella* and *Magnetovibrio*, while *Edaphobaculum* and *Melioribacter* in the last leachates were correlated with high NO₃⁻ and PO₄³⁻ concentrations.

The functional genes *aioA* and *arsM*, which were respectively involved in As<sup>III</sup> oxidation and As methylation, were detected in most of the leachates (Supplementary material SM3). The *arrA* gene involved in dissimilatory As<sup>V</sup> reduction was not detected in the leachates collected in the latter phases of the experiment (between days 73 and 122). The presence *arsB* gene involved in the As<sup>III</sup>-efflux pump of the *ars* resistance system, could not be determined due to non-specific amplification.

3.3.2. *Microbial community in residual decayed Sargassum biomass*

At the end of the experiment, clear microbial community

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Bacterial community structure of the residual decayed *Sargassum* biomass clustered by depth layer. A. Boxplots of alpha diversity (Observed OTUs, Chao1, InvSimpson and Shannon index; n = 4–5; different lowercase letters denote significant differences among different groups in the variance analysis with ANOVA, p < 0.05). B. Principal coordinate analysis (PCoA) of UniFrac distances (95 % confidence ellipse). C. Pie plot of the relative abundance at the phylum level. D. Linear discriminant analysis (LDA) Effect size (LEfSe) rank plot of differentially abundant bacterial taxa between layers, expressed as LDA logarithmic scores. E. Bar charts of the relative abundance of taxa determined by LEfSe analysis. TOP: surface layer (0–2 cm) of residual *Sargassum* biomass, MID: middle layer (2–7 cm), BOT: bottom layer (7–12 cm).</figcaption></figure>
stratification was observed in residual decayed *Sargassum* biomass (Fig. 5). A total of 495,643 16S rRNA gene sequences were obtained from the 14 residual *Sargassum* samples. Only four OTUs affiliated with the Archaea domain were detected (792 sequences). Richness and alpha diversity indices significantly increased with depth (Fig. 5A). The beta diversity analysis highlighted a distinct microbial community structure across the three depth layers, thus highlighting vertical differentiation in the microbial composition (Fig. 5B). The relative abundance of phyla varied according to depth (Fig. 5C). In the surface layer (TOP, 0–2 cm), the bacterial community was dominated by Bacteroidota, Proteobacteria, Actinobacteria and Planctomycetota, which represented 85 % of the total abundance. The middle level (MID, 2–7 cm) was characterised by a 50 % relative abundance of Proteobacteria, and by a substantial proportion of Myxococcota. The bottom layer (BOT, 7–12 cm) had the highest microbial diversity, with the presence of additional phyla such as Spirochaetota, Verrucomicrobiota, Desulfobacterota and Deferribacterota.

LEFSe (Linear discriminant analyses Effect Size; Segata et al., 2011) revealed significant taxa most likely to explain differences between the three depth layers. The LDA scores and relative abundances of significant taxa are represented in Fig. 5D and E. This analysis highlighted that TOP was significantly characterised by *Streptomyces*, *Muricauda* and *Nocardiopsis* (Bennur et al., 2016) genera; MID by *Pelagibius* (Choi et al., 2009), and BOT by *Spirochaeta* 2 and *Denitrovibrio* genera and the Desulfobacterota phylum. Significant taxa thus hosted in the decomposed algae biomass included aerobic bacteria in the surface and middle layers and anaerobic bacteria in the bottom layer.

Comparison of the main phyla found in this biomass and in the leachate (Supplementary material SM8 and Fig. 5C) indicated closer similarity between the bacterial communities of the BOT biomass and leachate (common phyla: Verrumicrobiota, Spirochaetota, Deferribacterota and Desulfobacterota) than between leachate and MID (Proteobacteria in common) and TOP layers (Actinobacteriota and Bacteroidota in common).

## 4. Discussion

Changes in the composition of both the decaying *Sargassum* biomass and its leachate were monitored for 120 days in conditions close to those that prevail at stockpiled *Sargassum* sites. The progressive increase in pH observed could have been related to the release of ammonia species and the dissolution of carbonate concretions found at the surface of floating seaweeds (Salter et al., 2020). The high EC and major ion concentrations (K, Na, sulphate) reflected the marine origin of the biomass. Although these levels decreased after 40 days, driven by increased watering, *Sargassum* leachate release into the environment presents a risk of soil and groundwater salinisation (Devault et al., 2021).

The leachate bacterial communities continuously changed during the experiment. This phenomenon could be attributed to salinity variations and the sequential availability of energy sources (electron donors and acceptors) within the system. Notably, on day 4, typical marine bacteria such as *Balneola*, *Celeribacter*, *Winogradskyella* (Urios et al., 2008; Lee et al., 2012; Nedashkovskaya et al., 2005) were detected in the leachate. These taxa were rapidly replaced by other bacteria, including halophilic aerobic heterotrophs such as *Martelella* (Rivas et al., 2005) and halotolerant anaerobic fermentative bacteria such as *Sphaerochaeta* (Bidzheva et al., 2020). Two taxa, *Maliorobacter* and *Edaphobaculum* (already observed in soil; Cao et al., 2017) were negatively correlated with salinity related parameters (EC, Na and K) suggesting that their development may have been favored by the progressive decrease in salinity. Beyond the changes in bacterial composition, these communities always included both aerobic and anaerobic microorganisms. This could be explained by the heterogeneous nature and stratification of the decaying *Sargassum* biomass. This stratification was clearly illustrated by the distinct bacterial community structuring from the surface to the bottom of the *Sargassum* bed at the end of the experiment (Fig. 5).

Several OTUs associated with the *Sargassum* leachate were phylogenetically related to potential pathogens. In particular, the *Francisella*-related OTU was one of the three most abundant OTUs on day 11, and the *Legionella*-related OTU was found to be one of the most abundant on days 42 and 49. *Francisella* has been reported to be associated with diseases of fish and other aquatic animals (Duodu et al., 2012) and is able to form ex vivo biofilms (Schaudinn et al., 2023). The presence of *Legionella* in saline coastal environments may be linked to seawater contamination by coastal polluted streams (Sinigalliano et al., 2007). Besides the presence of these two potential pathogens among the 20 most abundant OTUs observed, several minor OTUs detected in our seaweed samples were affiliated with potential pathogens, i.e. *Coxiella*, *Mycobacterium*, *Rhodococcus* and *Leptospira* genera. The presence of potential pathogens in *Sargassum* biomass—floating or in stockpile sites—has been reported, including Legionellales, *Coxiella* and *Rickettsiales* (Hervé et al., 2021), along with OTUs affiliated with the *Vibrio* genus. The presence of potentially pathogenic *Vibrio* strains on the surface of floating *Sargassum* collected in their marine environment was reported to be a potential human health issue associated with the handling of *Sargassum* biomass (Mincer et al., 2023). In our study, the relative abundance of potential pathogen OTUs decreased between 50 days and the end of the experiment. This decline coincided with an increase in redox potential and a decrease of EC observed after 40 days, suggesting that changes in environmental conditions within the piles may contribute to the disappearance of potential pathogens over time, thus reducing the health risk associated with handling aged *Sargassum* biomass.

Changes observed in the leachate chemical composition suggested a succession of reactions associated with the degradation of the seaweed biomass, which is composed of ~25 % DW of carbohydrates (Olguín-Maciel et al., 2022). These sugars could have been consumed through aerobic and anaerobic respiration, but also fuelled fermentation, as suggested by the detected presence of *Sphaerochaeta* throughout the experiment. This bacterium was reported to grow chemoorganotrophically with mono-, di- and polysaccharides, with the major end products of glucose fermentation being carbon dioxide, in addition to acetate, ethanol and $H_2$ (Miyazaki et al., 2014) that could feed the retrieved *Hydrogenovibrio*- and/or *Desulfovibrio*-affiliated bacteria. Sulphate-reduction producing toxic $H_2S$ gas is one of the main nuisances linked to inland *Sargassum* stockpiling. Here, the sulphate-reducing *Desulfobacterota*-related OTUs were overrepresented in the decayed *Sargassum* biomass bottom layer and the *Desulfovibrio*-affiliated OTU was among the 20 most abundant OTUs in the leachate. *Sargassum* leachates produced by Domínguez-Maldonado et al. (2024) also contained microorganisms metabolising sulphur (oxidation or reduction) or $H_2$, while chemoheterotrophy, sulphur compound respiration and nitrate reduction were among the main functions predicted from the 16S rRNA gene-based prokaryotic diversity in the solid phase of *Sargassum* storage sites (Hervé et al., 2021).

Microbial assemblies of heterotrophic organisms that contribute to the degradation of seaweed cell structures participated in As release in the aqueous phase. The detection of As-transformation related genes in the leachates, including the *arrA* genes linked to dissimilatory $As^V$ reduction and *arsM* genes linked to As methylation, also suggested that the bacterial compartment contributed to the transformation of As species during the *Sargassum* biomass degradation process. During the early phase of *Sargassum* decay (days 0 to 30), which could correspond to the release of the most soluble algal biomass fraction (Kristensen, 1994), the leachate contained a significant proportion of $As^{III}$, i.e. the major As species found in the initial biomass. Despite the bacterial $As^{III}$ oxidation potential (presence of *aioA* genes), this reaction might have been slowed down because of the low redox conditions and the high dissolved organic matter concentration (Lescure et al., 2016). During this period, sequential peaks of $DMAs^V$ in the leachate and $(CH_3)_3As$ in the atmosphere were observed, suggesting that active As methylation was under way. This process could have been associated with the high
dissolved organic carbon concentration, while the production of volatile As species may also be promoted by the relatively high temperature (Huang et al., 2012; Müller et al., 2022). Sulphate-reducing procaryotes, which were detected in both the *Sargassum* leachates (Fig. 4C) and the final biosolids (bottom, Fig. 5), are frequently cited as being producers of methylated As species (Hu et al., 2024). In addition, the upper biosolids zone was characterised by the presence of *Streptomyces*-related organisms (top, Fig. 5), i.e. a group of bacterial genera that includes aerobic As-methylating species (Kuramata et al., 2015). These findings indicated that the *Sargassum* decaying biomass produced volatile As species, driven by microbial activity and facilitated by available OM and relatively high temperature (SM. 4B).

A second phase (days 30 to 85) was characterised by the release of AsB and major ions (Ca, K, Mg, Ca; Fig. 3A) and a peak in As concentration (Fig. 2A), which may have resulted from the degradation of slowly biodegradable polymeric molecules constituting the seaweed cell walls. This phase corresponded to the emergence of *Denitrovibrio* in the leachate, i.e. an anaerobic nitrate- and As<sup>V</sup>-reducing bacterium (Denton et al., 2013). During this phase, the degraded biomass compaction that occurred at the bottom of the *Sargassum* bed could have generated suitable conditions for anaerobic metabolisms. Among the 20 most abundant OTUs found in the leachate, two were affiliated with genera that could perform respiratory As<sup>V</sup> reduction, i.e. *Denitrovibrio* (Denton et al., 2013) that remained one of the five most abundant OTUs during the second month of the experiment and *Melioribacter* (*Podosokorskaya et al., 2013*) which were abundant OTUs at day 119. *Melioribacter*, which is reportedly able to use arsenate or nitrite as electron acceptor, is a facultative anaerobe able to consume various sugars, including polysaccharides (*Podosokorskaya et al., 2013*). These As<sup>V</sup>-reducing bacteria may have contributed to the production of As<sup>III</sup>, whose concentration fluctuated, with a last peak noted between days 75 and 85.

Finally, after day 85, concentrations of most of the chemical species decreased, primarily driven by the high watering regime and leading to elution and dilution phenomena. Moreover, a freshwater supply, combined with a possible decrease in the readily biodegradable organic molecule concentration, promoted leachate reoxygenation. This was reflected by the rise in the redox potential, the emergence of nitrate and phosphate and the proportion of As<sup>V</sup> in the As species balance.

Regarding the overall fate of As during *Sargassum* biomass decay, the As concentration in the leachate peaked at 10 mg/L and was still >100 µg/L (~500 µg/L) at the end of the experiment. The total amount of As released in the leachate represented ~50 % (408.8 mg) of the fresh *Sargassum* biomass As content (821.1 mg). On average, 1 kg of fresh *Sargassum* released a total of 9.7 mg As in 120 days, which was consistent with the levels reported by Olguin-Maciel et al. (2022) in their study on *Sargassum* from the Mexican Caribbean coast—they estimated that 1 t of seaweed biomass could release 5.67 g of As in 180 days through leachate generation under controlled temperature conditions. This As release represented 52.71 % of the initial stock of As contained in dry tissue. In our experiment, *Sargassum* biomass was watered to mimic climatic conditions prevailing in the French West Indies, thereby enabling us to monitor variations in leached As fluxes over time. Under French regulation, the maximum As concentration allowed in industrial plant discharge water is 25 µg/L, and 0.2 mg/L for hazardous waste treatment facilities if the discharge exceeds 0.5 g/day (Order of 2 February 1998 related to water withdrawals and consumption, as well as a range of diverse emissions from installations classified for environmental protection [Journal Officiel n°52, 3 March 1998]). After 4 months of leaching, a dump of 19.5 t of fresh *Sargassum* biomass would exceed this discharge limit in terms of annual discharged As mass. Yet the quantities of seaweed biomass dumped at stockpile sites is substantially higher, e.g. the volume of *Sargassum* dumped at the Casperre-De-Marie-Galante stockpile site is estimated to be 6000 m³/year (Le Hoher et al., 2019), i.e. ~3000 t of seaweed (225 kg of As). Moreover, the flux of As released in the leachate was not linear during *Sargassum* biomass degradation and depended on the amount of water passing through the

heap. The maximum flux of As released in the leachate, ~0.1 mg/day/kg of fresh *Sargassum*, occurred between 40 and 90 days when the watering had been increased (Fig. 2B). The results of the present study showed that the discharge limits in terms of As concentration were often exceeded in decaying *Sargassum* biomass leachate, with potential associated impacts on the receiving environment. Concerning As volatilisation, according to our quantification of TMAsO and DMAs in the gas phase, the same site receiving 3000 t/year of fresh *Sargassum* could diffuse up to 3.7 g As/year in the atmosphere. This is one order of magnitude higher than the flux recorded for 1 ha of paddy soil in Bangladesh (240 mg/ha/year; Mestrot et al., 2011), and might contribute, together with H₂S and NH₃ emission, to the poor air quality that prevails close to *Sargassum* stockpiled sites.

## 5. Conclusion

*Sargassum* biomass decay on stockpiled sites is known for the resulting nuisances associated with the release of toxic H₂S and NH₃ gases. In addition to these emissions, biodegradation of this seaweed biomass induces the release of both volatile and dissolved As. The leachate contains a broad range of different As chemical species with diverse toxicity levels and geochemical properties that may drive their transport and fate in surface freshwater, sea water, soil and groundwater. The fate of As during *Sargassum* biomass decay was qualitatively and quantitatively determined through an original mesocosm experiment. The results highlighted active production of volatile As species during the early decay phase, the presence of substantial dissolved As in the leachate for over 100 days, and the persistence of 50 % of the initial As in the *Sargassum* biomass after more than 3 months. The simultaneous characterisation of physicochemical and microbial processes during this biomass degradation and characterisation of As speciation and the mass balance generated essential data for assessment of the risks for humans and the environment, and for the design of suitable stockpile sites and leachate treatment processes.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2025.180840.

### CRediT authorship contribution statement

**Hugues Thouin:** Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. **Mohamed Fikrat:** Investigation. **Fabienne Battaglia-Brunet:** Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. **Catherine Joulian:** Writing – review & editing, Formal analysis. **Mickael Charron:** Investigation. **Adrien Mestrot:** Writing – review & editing, Investigation, Conceptualization. **Véronique Vacchina:** Writing – review & editing, Methodology, Investigation. **Fabienne Séby:** Writing – review & editing, Methodology, Investigation. **Patrick Ollivier:** Writing – review & editing, Supervision, Project administration, Funding acquisition.

### Funding

This research was conducted within the framework of the Sarg As&CLD project funded by the French National Research Agency (ANR), contract No. ANR-19-SARG-0003-01.

### 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

The authors thank Christophe Mouvet (BRGM) for the acquisition of financial support for the project leading to this publication. Enzo
Civallero and Simon Chapron (BRGM) are thanked for their kind help in sampling and sending the fresh *Sargassum* samples from the West Indies; Teresa Chávez-Capilla (GIUB) for As volatile speciation analyses; Nicolas Devau (BRGM) for GAM application support. David Manley edited the English manuscript.

## Data availability

Data will be made available on request.

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