Water column acoustic data were collected with a dedicated acquisition protocol by vessel-hull mounted multibeam echosounders during the MAYOBS51 cruises in 2019, 2020 and 2021 (Kongsberg™ EM122 12 kHz), and during the GEOFLAMME52 cruise in 2021 (RESON™ 7150 24 kHz)., Data were post-processed with SonarScope (https://doi.org/10.17882/87777) and GLOBE (https://doi.org/10.17882/70460) software for the identification and location of the acoustic plumes53.
A Sea-Bird Electronics™ CTD (SBE911plus) was used together with a rosette water sampler (SBE-32) equipped with 24 OTE 10L bottles for hydrographic measurements. For optical backscatter measurements a SeaPoint™ nephelometer were attached to the CTD probe. Upon CTD/rosette recovery, bottles were immediately sampled for gas, pH, silicates and total alkalinity analysis on board. pH was measured over 10ml samples using a Metrohm pH-meter, while total alkalinity measurements were performed over 10–30 ml water samples using Methrom titrimeter.
Gases analysis from hydrocast operations were performed onboard directly after sampling from unpoisoned samples. Hydrogen and Carbon dioxide concentrations were determined by headspace technique with HID detection54 while Methane concentrations were determined by purge and trap technique coupled with GC-FID detection55.
During the GEOFLAMME cruise, gas seeping from the vents (consisting mainly of liquid CO2) was collected using the PEGAZ gas-bubble sampler25. In order to estimate seep fluid discharge and associated fluxes, 3D reconstruction of the seafloor based on ROV images, along with seep fluid counting and sampling were performed over zone B, specifically on site B0, one of the eldest and most vigorous sites. Immediately after recovery, the cylinder (50ml) of the PEGAZ sampler was positioned on a titanium cell equipped with a high pressure sensor and connected to a gas extractor for subsampling26. Once the vacuum has been achieved throughout the system, the Pegaz cylinder was first opened to the cell to evaluate the initial pressure. It was afterwards gradually opened to the gas extractor in order to expand, dry and subsample gases into vacuumed stainless-steel canisters of 50 to 1000 ml capacity equipped with gas-tight valves. Note that at ambient atmospheric pressure and temperature, liquid CO2 decompresses quickly in the gas extractor and changes phase to a gas. The residual gas remaining in the extraction line was injected on board into an SRA Instruments ® micro-chromatograph for gas analysis26. Aliquots of extracted gases were also recovered in copper tubes for onshore analysis of helium and δ13CCO2 isotopes at INGV30,33. To allow for a reasonable (and safe) CO2 decompression in the extraction line, only one or two droplets of gases were collected with the PEGAZ sampler.
Radiocarbon analyzes were carried out on CO2 droplets collected from the seeps around the hydrate field. The gas was collected, purified then converted to graphite56 before being measured at the Artemis LMC14 AMS facility57.
In situ Raman spectroscopy on hydrates mounds.
Visual recognition and identification of the white mounds was performed using the ROV video transects GFL-PL783-14 and GFL-PL785-16. Raman spectra were recorded using a “custom-made” spectrometer for in situ measurements named Ramses and mounted on the ROV58,59 It is equipped with a Horiba Jobin Yvon axial spectrometer and can perform real-time Raman spectroscopy on liquids, gases, and solids at depths up to 4800 m. Housed in Titanium, it features a 600 gr/mm grating for a spectral resolution of 10 cm− 1 and is equipped with two lasers (532 nm and 691 nm) for different sample types. It is coupled to an Andor DU440 CCD sensor.
Fluid flow rates estimates
Three ROV dives (GFL-ROV-PL776-07, GFL-ROV-PL778-09; GFL-ROV-PL785-16) were visually inspected to map, count and classify seep outlets of every active site in the entire Horseshoe area. A dedicated survey was performed in site B0 to quantify flow rates: a small funnel (530ml total volume) with volumetric graduation marks was used to measure flow rates on each event noted. The funnel was also deployed on site C1, D1, E0 and G0. Observed seeps were then classified into groups as a function of hydrate presence or absence and of liquid CO2 flow rates low flow rate < 10 ml s− 1, medium flow rates between 10 and 40 ml s− 1 and high flow rates > 40 ml s− 1. We calculated the number of seeps per category, average and standard deviation of the flow rate for each category. Density of seeps on site B0 was then calculated for each flow rate category and extrapolated over the entire Horseshoe area by assuming the same density repartition at all seeping sites than in site B0. Error estimate was set to 50% to account for site disparities. All annotated events and associated description and flux information are provided as supplementary material.
Megafauna mapping
Recognition and identification of megafauna was performed using the ROV videos acquired during GFL-PL07, GFL-PL16, and transects dedicated to the 3D reconstruction of site B. The Victor6000 has multiple cameras but because video acquisition was not set up for habitat mapping, the downward-looking camera could not be exploited properly. For this study we thus used the main ROV camera, and both the downward-looking and main cameras for site B. Images were analyzed using the ADELIE video annotation software using 3 criteria “sessile fauna category” (including dead Anthozoa), “organisms’ density” and “substrata type”. Because of the uncertainty in the identification of gorgonian- and coral-like morphotypes, these observations were grouped under the ‘undetermined Anthozoa’ modality (Extended Data Table 2). The definition of dead Anthozoa was based on the absence of color but also on the presence of living organisms around. For example, white corals observed among coloured corals or gorgonians were not considered as dead. Conversely, isolated colourless corals surrounded by broken sessile organisms covered by bacterial mats were annotated as ‘dead’. During the annotation process, associated metadata (timecode, image name, geographical coordinates) were recorded with ADELIE software and compilation was performed on ArcGIS software. The opportunistic nature of this dataset did not allow for the acquisition of quantitative data and each faunal record was thus processed as one observation. To assess the impact of liquid CO2 on faunal distribution, the mean proportion of each taxa, and ‘dead’ vs living corals, between active sites and background areas was compared using a Kruskall-Wallis test.