Mercury (Hg) is a globally significant environmental contaminant, primarily entering ecosystems in its inorganic form. In aquatic environments, inorganic Hg transforms into methylmercury (MMHg), a potent neurotoxin that bioaccumulates and biomagnifies through food webs, posing risks to human and ecosystem health. The ocean plays a crucial role in global Hg cycling, receiving Hg from various sources, including atmospheric deposition, riverine input, and hydrothermal vents. MMHg is notably enriched in the subsurface ocean, a major exposure pathway for humans through consumption of deep-sea organisms. However, the deep ocean's capacity for Hg methylation and demethylation remains poorly understood. Seafloor cold seeps, found along tectonic plate margins, are characterized by unique microbial communities driven by chemosynthesis. These microbial communities, including sulfate-reducing bacteria, methanogens, and anaerobic methane-oxidizing archaea, are known Hg methylators. This suggests cold seeps could be significant, yet unaccounted for, hotspots for Hg methylation and MMHg demethylation in global Hg models. This research aimed to investigate this hypothesis by analyzing Hg and MMHg concentrations, Hg isotopic composition, and microbial communities in sediments from the Haima cold seep in the South China Sea.

Previous studies have highlighted the importance of the ocean in global mercury cycling, noting the significant enrichment of methylmercury in the subsurface ocean. However, understanding of the deep ocean's role in mercury methylation and demethylation remained limited. Existing global mercury models largely ignore the potential impact of cold seeps, despite the known presence of mercury-methylating microbes in similar environments. Studies have shown that organic matter significantly impacts mercury distribution and speciation in marine sediments, with reduced sulfur compounds binding Hg. Further, the use of mercury stable isotopes has emerged as a powerful tool for tracing sources and processes of mercury in the environment, differentiating mass-dependent fractionation (MDF) from mass-independent fractionation (MIF).

This study utilized sediment cores and bivalve samples ( *Gigantidas haimaensis* and *Calyptogena marissinica*) collected from three environments within the Haima cold seep: an active seep area, an inactive seep area, and a reference area. Sediment cores were sectioned into 2 cm slices for geochemical and molecular analyses. Porewater was extracted to analyze dissolved inorganic carbon (DIC), δ¹³C-DIC, and sulfate concentrations. Total Hg and MMHg were measured using a Direct Mercury Analyzer (DMA 80) and gas chromatography-cold vapor atomic fluorescence spectrometry (GC-CVAFS), respectively. Hg isotopic analysis, including δ²⁰²Hg and mass-independent fractionation (MIF) values (Δ¹⁹⁹Hg, Δ²⁰⁰Hg, Δ²⁰¹Hg), was performed using a Nu-Plasma II MC-ICP-MS. Metagenomic sequencing was conducted on DNA extracted from sediment samples to identify microbial communities and associated Hg-related functional genes (hgcA, merA, merB). Statistical analysis evaluated differences in Hg and MMHg concentrations between the different seep environments. Phylogenetic analysis of Hg-metabolizing genes was performed using maximum likelihood tree construction.

The study revealed significantly higher Hg and MMHg concentrations in the upper sediment column (0–12 cm) of the active seep area compared to the inactive and reference areas. Hg concentrations were 2.4 times higher and MMHg concentrations were 10.5 times higher in the active seep area. The MMHg and MMHg/Hg maxima coincided in both active and inactive seep areas, with the active seep showing much higher values. In the active area, the MMHg maximum correlated with the depth of Hg/TOC maximum and occurred within the sulfate-methane transition zone (SMTZ). The inactive seep area showed MMHg and MMHg/Hg peaks at the core-top layer, potentially linked to the decomposition of dead clams. Bivalves from the active seep area had higher total Hg concentrations than those from the inactive seep area. Total Hg in sediments was significantly correlated with total organic carbon (TOC) content, indicating organic matter's role in Hg binding. Hg/TOC ratios were higher in the active seep area, suggesting either increased Hg binding to organic matter or preferential degradation of organic matter not strongly binding Hg. Analysis of Hg isotopic composition showed distinct differences between the active seep area and the inactive/reference areas. The active area showed a slope of Δ¹⁹⁹Hg/Δ²⁰¹Hg of 1.23 ± 0.10, suggesting abiotic dark redox reactions. The inactive and reference areas had a slope closer to 1, indicating Hg(II) photoreduction before deposition. In the active area, δ²⁰²Hg and odd-MIF values showed a decreasing trend with depth, potentially due to a combination of Hg deposition from the upper ocean and dark redox reactions. Metagenomic analysis revealed abundant Hg-related genes (hgcA, merA, merB) in all sediment cores. Diverse bacterial and archaeal lineages were identified as potential Hg methylators, including several previously unknown clades. The study also identified microbes potentially involved in Hg demethylation and reduction.

The findings demonstrate the significant role of cold seeps as hotspots for Hg accumulation and MMHg production in the deep ocean. The enrichment of Hg and MMHg in active seep areas, supported by Hg isotopic signatures and the presence of Hg-metabolizing microbes, confirms the hypothesis that these environments are not just sinks for Hg but active sites of transformation and potentially sources of MMHg to the deep-sea ecosystem. The distinct Hg isotopic signatures in active versus inactive and reference areas highlight the influence of abiotic dark redox reactions occurring at active seeps. The identification of previously unknown microbial lineages associated with Hg cycling expands our understanding of deep-sea microbial ecology and the biogeochemical cycles in extreme environments. This research provides compelling evidence of a previously unaccounted-for mechanism in global mercury cycling, urging the revision of global Hg budgets and models to include the contribution of cold seeps.

This study conclusively demonstrates that deep-sea cold seeps serve as a previously unrecognized significant sink for inorganic mercury and a source of methylmercury in the deep ocean. The enrichment of Hg and MMHg in active seep areas, along with distinct Hg isotopic signatures and the presence of diverse Hg-metabolizing microbial communities, underscores the importance of incorporating cold seeps into global Hg cycling models. Future research should focus on quantifying Hg and MMHg fluxes from cold seeps to better understand their contribution to global Hg cycling. Further investigation into the specific microbial mechanisms driving Hg methylation and demethylation in these unique ecosystems is also warranted.

The study focuses on a single cold seep site in the South China Sea. While the global extrapolation provides an estimate of the potential global impact, the findings need further validation across diverse cold seep systems worldwide. The global estimate relies on assumptions regarding the consistency of Hg and MMHg concentrations across different seeps and their active areas. Future research should conduct similar analyses in a wider range of cold seep environments to confirm the generality of the findings. More detailed investigation into the processes controlling Hg and MMHg fluxes from cold seeps is needed to refine the global estimations and quantify their contribution to global mercury budgets.