Toffin Laurent

Le Floch

Measuring the biological impact of drilling waste on the deep seafloor: an experimental challenge

Keywords: drill cutting, drilling waste, ecotoxicology, bioassay, hydrostatic pressure, deep-sea, environmental assessment -Introduction

The depletion of traditional oil fields is driving the oil & gas industry to explore new exploitation sites previously considered as unprofitable. Deep-sea oil fields represent one of these new areas of exploitation. Well drilling during exploration and production operations generate large quantities of drilling waste whose biological impact on the deep-sea floor remains largely unknown. Because of the harsh abiotic factors characterizing this environment, the evaluation of this impact remains challenging. High hydrostatic pressure is the prominent factor which will affect in-situ biological processes. This review will examine the feedback on the various strategies used to evaluate the biological impact of deep-sea drilling waste deposition as well as the current technological limitations. Given the complexity of this issue, a good perspective strategy would be to trend towards the research and development of more relevant bioassays, especially considering the crucial factor of hydrostatic pressure.

Drilling waste comprises 2 categories: drill cuttings and spendt drilling muds (Veil JA., 2002;[START_REF] Ellis | Discharged drilling waste from oil and gas platforms and its effects on benthic communities[END_REF][START_REF] Cochrane | Detection of deposited drill cuttings on the sea floor -A comparison between underwater hyperspectral imagery and the human eye[END_REF].

Drilling muds are the compounds and fluids that are injected into the borehole to facilitate the drilling process, for example to lubricate and cool the drill bit, assist the upward transport of drill cuttings and also for hole stabilization. Three main types of drilling muds exist:

-Oil-based mud (OBM) mainly composed of oil or diesel.

-Synthetic-based mud (SBM) whose composition is based, for example, on ester, ether, acetyl or olefin.

-Water-based mud (WBM) also called water-based fluid (WBF) using in situ water (fresh or seawater).

Other compounds typically added to the mud for weighting and other enhancing properties include clay (e.g. bentonite), ilmenite (FeTiO3), barite (BaSO4), organic polymers, inorganic salts and various organic additives [START_REF] Breuer | Drill cutting accumulations in the Northern and Central North Sea: a review of environmental interactions and chemical fate[END_REF].

The release of drilling waste has a number of consequences from both practical and financial viewpoints as well as in terms of environmental issues. The type and amount of discharges vary according to the type of drilling operation (e.g. exploratory or production) and the site-specific characteristics such as geographical properties and water depths as well as reservoir location and structure. In addition, local legislations will dictate how the waste is to be handled; whether it will be deposited at source-point, translocated on the sea floor, dispersed at the sea surfaceor whether at least some of it must be taken to land. Furthermore, the environmental and socio-economic considerations are constantly evolving, especially as the industry is moving into new areas such as the Arctic and the deep sea. As a result, both the industry and the environmental regulatory authorities face a myriad of challenges to which there currently is no single, unified answer.

The present study will present a brief review of the different aspects related to offshore drilling waste from the oil and gas industry, with particular emphasis on deep-sea operations (> 750 m, sensu ISO 16665). We will examine the various tools currently used to study environmental impacts on the sea-floor and assess their relevance to the particular challenges arising as drilling operations inevitably move into increasingly deeper locations, also within the context of the prevailing environmental legislations.

J o u r n a l P r e -p r o o f -Legislative framework

The Montego Bay Convention (CMB) and the Marpol convention are historical and globally recognized agreements on the protection of the oceans. They nevertheless do not provide a fully clear, complete and functional legislative framework to regulate offshore oil & gas exploration/production activities [START_REF] Durand | Dispersed oil decreases the ability of a model fish (Dicentrarchus labrax) to cope with hydrostatic pressure[END_REF]. These activities generally take place on the continental shelf or within the 200 nautical mile limit from the margin (exclusive economic zone: EEZ). The administration of the continental shelf and EEZ comes under the responsibility of the corresponding coastal state. De facto, national or regional laws prevail for the legislation of such offshore activities. To limit negative impacts on the marine environment, drilling waste releases are increasingly under strict regulation. Nevertheless, without unifying international agreement legislation, tolerance variations between countries hosting exploitation sites are common (IOGP report, 2016). Existing regulations mainly target releases of chemicals like metals, additives not derived from hydrocarbon and hydrocarbons.

In the following section, we will examine concrete examples of the most advanced laws and regulatory systems around the world (national and regional) to depict the major environmental management philosophies. These examples are also summarized in the table 1.

The US legislative framework follows the United States Environmental Protection Agency (USEPA) regulations. This is a proteiform approach with no real constancy between oil and gas exploration/production cases/regions/states. It can be considered as pragmatic exploitation of local legislative specificities. For example, regulatory approaches will be dramatically different between oil and gas development areas of the US part of the Gulf of Mexico (bordered by the states of Florida, Alabama, Mississippi, Louisiana and Texas) and the Alaskan part of the Chukchi Sea (IOGP report, 2016). Thus, USEPA regulations, designed to avoid a "compliance burden" for industries, can be considered as a variable geometry legislative framework (Menzie CA, 1982;[START_REF] Rana | Environmental Regulations, Technology, and Cost of Compliance for Oil & Gas Operations[END_REF]. Canada also follows such a system through the involvement of institutions like the C-NLOPB (Canada-Newfoundland and Labrador Offshore Petroleum Board). A similar system exists in Australia where states' and territories' law agreements prevail over federal and Commonwealth legislations (Filder & Noble, 2012;[START_REF] Fraser | An international comparison of governmental disclosure of hydrocarbon spills from offshore oil and gas installations[END_REF].

In northern Europe, the vast majority of offshore infrastructures are located in the North Sea (NS) (Purser & Thomsen., 2011), with increasing activity in the Norwegian Sea and the south-western J o u r n a l P r e -p r o o f Barents Sea. In more southern regions, some minor fields exist in the Adriatic Sea, off the Sicilian coasts and in the Balearic Sea [START_REF] Pinder | Offshore oil and gas: global resource knowledge and technological change[END_REF]. European legislation has the highest level of requirement in comparison with the rest of the world [START_REF] Durand | Dispersed oil decreases the ability of a model fish (Dicentrarchus labrax) to cope with hydrostatic pressure[END_REF]. For example, in Norway, since 2001 OBM and subsequent drill cuttings are no longer permitted to be released at sea. If the concentration of hydrocarbons (HC) in sand, mud and cuttings exceed 10 g.kg -1 , they are systematically recovered and shipped to shore for land disposal [START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF]. This kind of approach is mainly based on the "precautionary principle" and the "polluter pays principle" that were initiated with Helcom (Helsinki Commission) in the 1980s for the Baltic Sea (despite its low available hydrocarbon reserves) as well as with the OSPAR (Oslo-Paris) convention in the early 1990s for the North-Eastern Atlantic [START_REF] Ekins | Zero emissions of oil in water from offshore oil and gas installations: economic and environmental implications[END_REF]. They clearly aim at a generalization of the aim of zero harmful discharges to the sea by 2020 [START_REF] Knol | The uncertainties of precaution: Zero discharges in the Barents Sea[END_REF], which is also in line with the European Union's Marine Strategy Framework Directive (MSFD), which states that all the European seas should attain Good Environmental Status (GES) by 2020. It has to be highlighted that this legislation considers only hazardous discharges without concern for conventional discharges. Similar systems with a comparable degree of requirement are also operating for the Mediterranean Sea, with the Barcelona Convention and its offshore protocol, as well as for the Persian Gulf via the Kuwait Regional Convention for Co-operation on the Protection of the Marine Environment from Pollution [START_REF] Durand | Dispersed oil decreases the ability of a model fish (Dicentrarchus labrax) to cope with hydrostatic pressure[END_REF]. It is worth noting that the most advanced law agreements on offshore operations all concern very limited geographical areas. In the future, it is reasonable to expect a worldwide trend toward standardization of such procedures. Therefore, in a perspective of ecological and economical ongoing relevance, in accordance with the Porter hypothesis (commercial competitiveness is positively driven by environmental regulations through innovations) [START_REF] Ambec | The Porter hypothesis at 20: can environmental regulation enhance innovation and competitiveness?[END_REF], current and new processes used for exploration/production in the oil & gas industry must overtake these probable evolutions.

-General ecological impacts

The ecological impact of drilling waste is mainly linked to its spatial behavior after emission at sea (if so). This behavior is determined by its physicochemical properties (composition, density, size of particular material, solubility), the total depth of the water column as well as the speed of the dominant stream (or local energy regime). Finally, the total volume of discharged waste will also deeply influence the deposition phenomenon, especially with deep well mining. During emission, drilling waste will split into two plumes:

J o u r n a l P r e -p r o o f -An upper plume containing low density, small sized and rapidly dissolving material (mainly drilling fluids) whose patch diffusion speed is a function of the surface stream and upper water column.

-A lower plume based on drill cuttings which will settle to, and accumulate on, the sea bottom.

From an ecological perspective, the lower plume will have a stronger impact on the ecosystem through the durable formation of a drill cuttings pill on the sea floor. The deposition of this waste pill can be simulated according to environmental and the drill cuttings' physicochemical parameters. By convenience, the physicochemical properties of drill cuttings can be condensed to their overall lift in the water column. The water column depth and the dominant stream are the main two environmental parameters. The morphology of drill cutting deposition will be the resulting barycenter of these 3 forcing parameters as a function of their respective intensities (Figure 1). The accumulation of drill cuttings will durably alter the physicochemical properties of the sea floor [START_REF] Breuer | Drill cutting accumulations in the Northern and Central North Sea: a review of environmental interactions and chemical fate[END_REF]. The waste pill will mainly generate a rapid microbial-driven anoxia of the sediment followed by decreased redox potential and release of exogenous compounds (e.g. heavy metals, hydrocarbon). These changes will consequently impact the seafloor biocenosis from the microbiota to the fauna.

-Known impacts on the benthos

The impact assessment of contaminated or uncontaminated drill cuttings on benthic ecosystems is the result of three approaches: a physico-chemical analysis of the sediment (redox parameters, Size Exclusion Chromatography for polymers, Gas chromatography for hydrocarbons…), an ecological analysis of benthos (both meiofauna and macrofauna) and an ecotoxicological analysis using key species of macrofauna (annelids, nematodes, molluscs and crustaceans) [START_REF] Jorissen | Impact of oil-based drill mud disposal on benthic foraminiferal assemblages on the continental margin off Angola[END_REF].

Due to the strong biological impact observed with oil-based muds (OBM), oil-based fluids (OBF) is now not currently applied and requires special agreement to be used [START_REF] Hurley | Environmental effects of ex plo-ratory drilling offshore Canada: environmental effects monitoring data and literature review[END_REF]. Indeed, OBM impact assessment demonstrated large-scale release with an increase in barium and total hydrocarbon concentrations in sediment located 2000 to 6000 m from the platforms. The major problem is the persistent impacts on benthic communities [START_REF] Olsgard | A comprehensive analysis of effects of offshore oil and gas exploration and production on the benthic communities of the Norwegian continental shelf[END_REF][START_REF] Daan | Environmental effects of a discharge of drill cuttings contaminated with ester-based drilling muds in the North Sea[END_REF], with composition change such as the loss of rare species and an increase in pollution-tolerant species [START_REF] Gray | Detection of initial effects of pollution on marine benthos: an example from the Ekofisk and Eldfisk oil fields, North Sea[END_REF][START_REF] Olsgard | A comprehensive analysis of effects of offshore oil and gas exploration and production on the benthic communities of the Norwegian continental shelf[END_REF] and/or opportunistic species [START_REF] Davies | Environmental effects of the use of oil-based drilling muds in the North Sea[END_REF]. to 20 km and 0.2 to 2 km, respectively for WBF and SBF. Benthic ecosystems are impacted by both WBF and SBF at a distance of 100 to 1000 m from the well. As for OBFM, alterations in diversity, abundance and structure are reported in benthic community (Netto et al., 2009;Eliss et al., 2012).

Evidence of sediment impact on benthic communities is highlighted by the loss of suspension feeders, and an increase in deposit feeders and polychaetes. This visual evolution is particularly striking and demonstrates one of the major environmental impacts of drilling activities is the increase in suspended sediments in the deep-sea bottom and its relation to physical changes in substrate (Netto et al., 2009).

Bottom current is a natural conveyor of suspended sediment. In the Norwegian shelf (White et al., 2012), an important area for oil and gas operations in the North-East Atlantic, drillings lead to an increase in sediment suspension and drift toward suspensivorous-specific ecosystems like deep-water coral reefs. Deep-water coral reefs are considered as biodiversity hotspots [START_REF] Roberts | Reefs of the deep: the biology and geology of coldwater coral ecosystems[END_REF] sheltering hundreds of associated organisms which cover almost every phylum [START_REF] Rogers | The biology of Lophelia pertusa (Linnaeus 1758) and other deepwater reef-forming corals and impacts from human activities[END_REF].

Consequently, they have a crucial role in deep benthic ecosystems. Anthropic activities have reported effects on deep-water coral reefs (Roger, 1999). For example, a direct destructive effect is caused by bottom trawling by fisheries [START_REF] Althaus | Impacts of bottom trawling on deep-water ecosystems of seamounts are long-lasting[END_REF]. Nevertheless, most anthropic effects on deep-water scleractinian coral reefs are indirect and the consequence of an increase in suspended sediment (Pilskaln et al., 1998;[START_REF] Lepland | Barite and barium in sediments and coral skeletons around the hydrocarbon exploration drilling site in the Traena Deep, Norwegian Sea[END_REF][START_REF] Trannum | Effects of sedimentation from water-based drill cuttings and natural sediment on benthic macrofaunal community structure and ecosystem processes[END_REF]. Impact assessments of drilling activities on deep-water corals have long studied this issue, with different levels of coral organization and timescales (see below). One of the major species of framework-forming deep-water coral builder is Lophelia pertusa [START_REF] Freiwald | Reef-forming cold-water corals[END_REF][START_REF] Roberts | Cold-water Corals: The Biology and Geology of Deep-sea Coral Habitats[END_REF]. L. pertusa can be relatively tolerant, in lab conditions, to short term exposure to drill cuttings and natural sediment [START_REF] Larsson | Sedimentation on the cold-water coral Lophelia pertusa: cleaning efficiency from natural sediments and drill cuttings[END_REF]. Bausant et al. (2018) determined a threshold drill cutting concentration of 10 mg.L -1 (with particles smaller than 63 μm for 97% of them), above which, changes in coral polyp behaviors were observed.

Under high sedimentation, L. pertusa exhibited resilience to hypoxic, and even anoxic, episodes [START_REF] Allers | Resistance of Lophelia pertusa to coverage by sediment and petroleum drill cuttings[END_REF]. Due to its ability to endure anoxia only for a short time and the slow recovery after this stress episode, [START_REF] Allers | Resistance of Lophelia pertusa to coverage by sediment and petroleum drill cuttings[END_REF] suggest an anoxia tolerance rather than an anaerobic metabolism.

So, we can assume that a part of the L. pertusa mechanism to cope with hypoxia and anoxia is probably present in their mitochondria.

J o u r n a l P r e -p r o o f

Research efforts should now point out these mechanisms and analyze the respiratory chain of the coral model, like L. pertusa, after exposure to sediment or polluted sediment. In particular, they should examine the possibility that mitochondrial alterations may not have any effect at a higher level of integration (e.g. polyp behavior). This compensation phenomenon is recognized and probably hides some fitness changes. Indeed, the compensation phenomenon has an energetic cost and could have a major effect of organism ontogenesis, as described by [START_REF] Hsing | Evidence of lasting impact of the Deepwater Horizon oil spill on a deep Gulf of Mexico coral community[END_REF] during a 17-month study following the DeepWater Horizon oil spill. Using remoted operational vehicles (ROV), the authors performed environmental monitoring on deep-water corals contaminated by oil-polluted flocs. They demonstrated the long-term impact of oil contamination on corals by observing the modification of coral colonies which were more and more colonized by hydroid fouling. The short-term impacts seemed to be relatively low. However, the effects which continued for months led to the reduction of coenosarc tissue and the onset of opportunistic species on the newly apparent coenosteum. [START_REF] Godø | Real time observation system for monitoring environmental impact on marine ecosystems from oil drilling operations[END_REF] tested a methodology to obtain real time observation system for monitoring the potential environmental impact of drilling operations. The system is a coupling sensor platform (optical and acoustic) with a surface communication buoy. As mentioned by authors, the experimental system requires some improvements, but could offer in the future a relevant way to assess the environmental impact during all phases of drilling operation and also between several drill sites. In the same purpose, Purser (2015) performed a relevant monitoring studies of potential impact of drilling activities on small size L. pertusa reefs in the Morvin field (350m depth) located in the Norwegian margin. Using several ROVs, nine coral reefs were video surveyed before, during, immediately after and one year after drilling activities. Modeling of drill cutting transport following releases allowed the author to generate three levels of reef exposure: negligible, occasional exposures (>5 ppm) or repeated exposures (<5 ppm). Author reported no observation of modification of the reef structure and associate megafauna abundance over time whatever the modelled exposure concentration. However, this study only focused his survey on adult fauna and the question of larvae behavior in drilling activities context is worrying. Indeed, [START_REF] Järnegren | Effects of drill cuttings on larvae of the cold-water coral Lophelia pertusa. Deep Sea Research Part II[END_REF] have demonstrated than adult L. pertusa can survive with high sediment load. However if the same high sediment load occurs during larval time, it will kill all or the major part of the larval cohort.

This study demonstrated the deleterious effect that a major discharge of sediment in deep-sea ecosystem could have during coral spawning time.

J o u r n a l P r e -p r o o f -Reported impact on microflora

Similarly to benthos, deep seafloor microflora will be dramatically impacted by drilling waste release. Nevertheless, comparatively to benthic zoocenosis, very few extensive studies have been conducted on sediment microbiota. Most of the in situ studies focus on the biogeochemical modification of the sediment as well as the biodegradation of hydrocarbon and/or drilling fluids. Notably, metagenomic approaches on drill cutting are still scarce compared to more classic studies e.g. hydrocarbon impacted shore. Reported studies point out a seafloor bacterial community shift in cases of drilling waste deposition [START_REF] Main | Hydrocarbon contamination affects deep-sea benthic oxygen uptake and microbial community composition[END_REF][START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF]. This shift is accompanied by biogeochemical perturbation affecting the S and N-cycle [START_REF] King | Microbial responses to the Deepwater Horizon oil spill: from coastal wetlands to the deep sea[END_REF] and a rapid decrease of dissolved oxygen (36h, [START_REF] Main | Hydrocarbon contamination affects deep-sea benthic oxygen uptake and microbial community composition[END_REF] leading to localized anaerobic spots [START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF] and a potentially strong redoxcline [START_REF] Schaanning | Biodegradation of Esters and Olefins in Drilling Mud Deposited on Arctic soft-bottom communities in a low-temperature Mesocosm[END_REF][START_REF] Trannum | Biological and biogeochemical effects of organic matter and drilling discharges in two sediment communities[END_REF]. Microbial community restructuration is also marked by a functional shift toward an enrichment of the hydrocarbon anaerobic metabolism repertoire [START_REF] King | Microbial responses to the Deepwater Horizon oil spill: from coastal wetlands to the deep sea[END_REF]. Interestingly, this means that some phylogenetic groups can be used as microbial bioindicators [START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF] like proteobacteria clade [START_REF] Napp | Comparative metagenomics reveals different hydrocarbon degradative abilities from enriched oil-drilling waste[END_REF].

The real impact of deep-sea abiotic factors (hydrostatic pressure, low temperature, anaerobicity) is not clear but at least they seem not to inhibit drilling waste biodegradation by indigenous microbiota [START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF]. As mentioned above, exhaustive studies into the impact of drilling waste on the seafloor microbiota, especially on community structuring, remain anecdotal. To fill this gap in knowledge, some parallels can be drawn with studies about the impact of hydrocarbon pollution on deep-sea floor microbiota. Initiated after the Macondo blowout (MBO) in 2010, numerous results from the Gulf of Mexico Research Initiative (GOMRI) perfectly illustrate this (http://gulfresearchinitiative.org/). During this major environmental crisis, the authorities authorized the intensive use of Corexit 9500 to disperse and stimulate the microbial biodegradation of the accidentally released crude oil [START_REF] Azwell | The Macondo blowout environmental report[END_REF].

Biodegradation of hydrocarbons always starts by the simplest compounds, e.g. short chain alkanes (Heads et al., 2006). In the case of the MBO, the biodegradation of small compounds in previously dispersed oil led to oil sedimentation promoted by marine oil snow formation and flocculants accumulation (MOSSFA) [START_REF] Ramseur | Deepwater Horizon oil spill: the fate of the oil[END_REF][START_REF] Passow | Marine snow formation in the aftermath of the Deepwater Horizon oil spill in the Gulf of Mexico[END_REF][START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF][START_REF] Brooks | Sedimentation pulse in the NE Gulf of Mexico following the 2010 DWH blowout[END_REF]. This "dirty blizzard hypothesis" was simulated by ex situ lab experiments and proven to be a microbially driven phenomenon [START_REF] Passow | Marine snow formation in the aftermath of the Deepwater Horizon oil spill in the Gulf of Mexico[END_REF]. In many ways, this contamination of the J o u r n a l P r e -p r o o f environment by MOSSFA can be related to pollution with drilling waste. In both cases, a dual emission plume is observed (IOGP report 2016, [START_REF] Daly | Assessing the impacts of oil-associated marine snow formation and sedimentation during and after the Deepwater Horizon oil spill[END_REF] (Figure 2).

That is particularly interesting considering the vast range of bathymetric profiles impacted which can be used to virtually assess the potential effect of drilling waste according various technical scenarii. Like for drilling waste, MOSSFA deposition enriches the seafloor with organic or inorganic pollutants but also generates microbial seeding with strains from the water column and deep-biosphere [START_REF] Joye | Microbial dynamics following the Macondo oil well blowout across Gulf of Mexico environments[END_REF][START_REF] Arnosti | Oil-derived marine aggregates-hot spots of polysaccharide degradation by specialized bacterial communities[END_REF][START_REF] Mason | Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill[END_REF][START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF]. Due to the intense and focused nature of the MBO, effects on the microbiota were different from natural hydrocarbon seepage, characteristic of a prolific hydrocarbon basin such as the Gulf of Mexico (Joye et al., 2016). These seeps are natural biodiversity hotspots with well-adapted interdependent biological communities from microbial to deepsea animals (Cordes et al., 2009). MBO dramatically impacted deep-sea and shoreline communities [START_REF] White | Cold-water coral ecosystem (Tisler Reef, Norwegian Shelf) may be a hotspot for carbon cycling[END_REF]Fisher et al., 2014a;Fisher et al., 2014b[START_REF] Silva | Coral injuries observed at mesophotic reefs after the Deepwater Horizon oil discharge[END_REF]. Similarly to drilling waste, widespread deposition of MOSSFA generated sedimentary biogeochemistry changes. Perturbations of the N and S-cycles were reported [START_REF] Joye | Microbial dynamics following the Macondo oil well blowout across Gulf of Mexico environments[END_REF]. [START_REF] Hastings | Changes in sediment redox conditions following the BP DWH blowout event[END_REF] also reported changes in sediment redox conditions following the MBO event with a decrease in the pore water oxygen concentration and a shoaled redoxcline for 3 years before a gradual return to pre-impact values. A metagenomic and metabolic survey of the sedimentary microbial response to MBO pointed out temporal and spatial biodegradation patterns (both in term of localization in the gulf and in the sedimentary column). These patterns are mainly linked to the nature of the hydrocarbon contaminants and oxygen bioavailability. Briefly, Deltaproteobacteria (e.g. Desulfatibacillum sp) were involved in anaerobic biodegradation of aliphatic and aromatic hydrocarbons in the first months after the spill and subsequent MOSSFA deposition [START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF]. At the same time Gammaproteobacteria (e.g.

Oceanospirillales such as Alcanivorax sp) were involved in the aerobic biodegradation of n-alkanes, aliphatic and simple aromatic hydrocarbons [START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF]. Alphaproteobacteria (e.g. Roseovarius spp) were able to aerobically degrade more recalcitrant aromatic oil compounds such as PAHs (Kotska et al., 2011;[START_REF] Kimes | Metagenomic analysis and metabolite profiling of deep-sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill[END_REF]. Interestingly, these trends, although perfectible, seem independent of hydrostatic pressure. For experimental and economic reasons, ongoing research should assess this point using long time scale pressurized microcosm experiments. Indeed, enrichment culture with native piezophile and piezotolerant communities on a simulated and stabilized sea bottom could more accurately highlight communities and functional shift during drilling waste/MOSSFA biodegradation. The industrial exploitation of the deep-sea bottom can lead to waste production and consequently to potential environmental risks. As required by the legislation (e.g. OSPAR convention in the North-East Atlantic), these risks, or in the worst case the pollution, must be assessed using several methods including ecotoxicological tests, also known as bioassays. Since bioassays never reflect the full reality of the field, their ecological relevance is questionable; they nevertheless still offer an elegant way to assess the relative toxicity of chemicals in the environment. To be ecologically coherent, bioassays must consider several abiotic parameters, such as temperature, salinity or pH, variable parameters of water ecosystems.

In aquatic environments, hydrostatic pressure depends on the height of the water column and hence increases linearly with depth. At 2000 m under the ocean surface the pressure is close to 200 tons per square meter (202 bars). About 70% of the ocean [START_REF] Parkes | Culturable prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure, anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)[END_REF] is made up of deep-sea environments with an average pressure of 380 bars and up to 1100 bars in the sediment layer of the Mariana Trench. Hydrostatic pressure is, like temperature, a thermodynamic parameter of the environment. Increasing hydrostatic pressure favors the state of smaller volumes and hydrostatic pressure is liable to influence a huge number of fundamental biological structures and processes including macromolecular properties [START_REF] Johnson | The theory of rate processes in biology and medicine[END_REF]Kunugi,1992), biochemical reaction rates and equilibrium [START_REF] Johnson | The kinetic basis of pressure effects in biology and chemistry[END_REF][START_REF] Somero | Hydrostatic pressure and adaptation to the deep sea[END_REF] or biological membrane fluidity [START_REF] Macdonald | The effects of pressure on the molecular structure and physiological functions of cell membranes[END_REF]. Furthermore, hydrostatic pressure interacts with other fundamental factors of the environment and in particular with temperature and osmotic pressure [START_REF] Kornblatt | The effects of osmotic and hydrostatic pressures on macromolecular systems[END_REF][START_REF] Sébert | Pressure and temperature interactions on cellular respiration; e review[END_REF]Hochachka, 2005[START_REF] Yancey | Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses[END_REF]. For these reasons, the effects of hydrostatic pressure on biological systems are too important to be set aside or, worst, extrapolated from atmospheric pressure results.

Hydrostatic pressure is one of the most difficult abiotic parameters to control in the laboratory.

It requires specialized equipment and expensive maintenance. Consequently, literature on bioassays at simulated depths is scarce. Furthermore, bioassays under pressure must meet a number of criteria to be relevant. In addition to being able to control the hydrostatic pressure, the bioassays should be easily applicable within a short timeframe. An ideal test would be suitable to respond to a routine request for environmental monitoring as well as in an emergency context in case of deep-water pollution. To be J o u r n a l P r e -p r o o f economically viable, the experimental device should require limited maintenance between trials. Finally, a comparison with a standardized bioassay usually used at atmospheric pressure would be a plus in order to monitor continuity in different ecosystems ranging from bathyal to coastal zone.

-Animals assay

Deep-sea ecotoxicological studies (hereafter referred as eco-piezotoxicological studies) are a new field for the assessment of anthropic effects. Data are still scarce and several approaches are used to explore this new issue [START_REF] Lemaire | Hydrostatic pressure and the experimental toxicology of marine fishes: The elephant in the room[END_REF]. One of these approaches uses piezotolerant species brought from the deep-sea and exposed to chemicals at atmospheric pressure. This was performed on several taxa of different trophic levels, such as deep-sea coral (see Benthos report impact section), a giant deep amphipod Eurythenes gryllus (Olsen et al., 2015;Camus & Gulliksen, 2005) and sablefish Anoplopoma fimbria [START_REF] Mcconville | The sensitivity of a deep-sea fish species (Anoplopoma fimbria) to oil-associated aromatic compounds, dispersant, and Alaskan North Slope crude oil[END_REF]. [START_REF] Boschen | Mining of deep-sea seafloor massive sulfides: a review of the deposits, their benthic communities, impacts from mining, regulatory frameworks and management strategies[END_REF] highlighted, in a review about the mining of deep-sea seafloor massive sulphides (SMS), the need of relevant bioassays to assess the potential impact of these SMS mining on the benthic fauna. Such bioassays should avoid using shallow water species that do not naturally face "extreme" abiotic parameters such high hydrostatic pressure, total darkness and/or cold water.

Nevertheless, the use of piezotolerant shallow-water species could offer a relevant compromise for the chemical or anthropic impact assessments in deep-sea environments. Indeed, several ecopiezotoxicological studies have been performed under high hydrostatic pressure using hyperbaric chambers: on an intertidal and sub-littoral polychaete Pomatoceros lamarcki exposed at 101 and 304 bars [START_REF] Vevers | The role of hydrostatic pressure on developmental stages of Pomatoceros lamarcki (Polychaeta: Serpulidae) exposed to water accomodated fractions of crude oil and positive genotoxins at simulated depths of 1000-3000 m[END_REF], on an European marine shrimp Palaemon varians at a range from 100 to 400 bars [START_REF] Domingues | Hydrostatic pressure on cadmium toxicity in Palaemon varians[END_REF], on a demersal fish with seabass Dicentrarchus labrax at 138 bars (Dussauze et al., 2017) and at 50 and 150 bars [START_REF] Lemaire | High hydrostatic pressure influences the in vitro response to xenobiotics in Dicentrarchus labrax liver[END_REF] and a benthic fish, turbot Scophthalmus maximus at 101 bars [START_REF] Dussauze | In vitro Diving Simulation: A New Approach to Assess Biological Impact of Hydrocarbons at Depth[END_REF]Dussauze et al., in prep). Kopecka-Pilarczyk & Coimbra (2010a;2010b) investigated the effect of pressure on relevant ecotoxicological biomarkers on the liver (EROD, GST, CAT, SOD, Lipid peroxidation...) of a shallow water fish Pagellus bogaraveo (Kopecka-Pilarczyk & Coimbra, 2010a) and on European eel Anguilla anguilla (Kopecka-Pilarczyk & Coimbra, 2010b). This last study is interesting due to the choice of the model species. The European eel has a life history containing a deep-sea phase. This led to the final and more relevant approach, but which is also more difficult, consisting in analyzing deep-sea species under simulated depth. Lemaire et al.

J o u r n a l P r e -p r o o f

(2012) pressurized liver slices of rock grenadier Coryphaenoides rupestris fished at 530 meters depth at 1, 50 and 150 bars. To date, in vitro tools coupled with a hyperbaric chamber [START_REF] J O U R N A L P R E -P R O O F Lemaire | Precisioncut liver slices to investigate responsiveness of deep-sea fish to contaminants at high pressure[END_REF][START_REF] Dussauze | In vitro Diving Simulation: A New Approach to Assess Biological Impact of Hydrocarbons at Depth[END_REF] seem to offer the best compromise between ecological relevance and cost of experimentation in eco-piezotoxicology.

Depressurization and pressurization remain the major issue in eco-piezotoxicology. Repetitive pressure changes have been shown to alter the oxygen consumption of deep-living fish [START_REF] Roer | Effects of pressure on oxygen consumption in cottid fish from lake Baikal[END_REF]. The effects of pressure changes were also investigated by [START_REF] Theron | Hydrostatic pressure and cellular respiration: are the values observed postdecompression representative of the reality under pressure?[END_REF] on mitochondrial and cellular respiration on the European eel acclimatized at atmospheric pressure or at 101 bars, showing that pressure changes strongly affect aerobic metabolism. Nevertheless, the effects of depressurization and pressurization (300 bars) were studied on the hydrothermal vent shrimp Rimicaris exoculata after copper exposure [START_REF] Auguste | Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata[END_REF]. The results of this study suggest that the biomarkers' responses to metal exposure in shrimps were not affected by several depressurizationpressurization cycles in comparison with in situ results on the same species.

Finally, The use of species performing Diurnal Vertical Migration could avoid the problem of depressurization-pressurization effects due to their adaptive capacities to living at several bathymetries. Zooplankton species such Calanoid copepods could fit for such a pressure bioassay. Their life histories are known for many species and can be easily controlled in a lab. Given their size, they can be used in mini hyperbaric chamber as developed by [START_REF] Dussauze | In vitro Diving Simulation: A New Approach to Assess Biological Impact of Hydrocarbons at Depth[END_REF] which requires little maintenance.

Furthermore, a correlation with a standardized bioassay could be made with the copepod Acartia tonsa, a model species of an ISO protocol and recommended for the OSPAR protocol.

Finally, as described by [START_REF] Boschen | Mining of deep-sea seafloor massive sulfides: a review of the deposits, their benthic communities, impacts from mining, regulatory frameworks and management strategies[END_REF], the adaptation of bioassays under pressure focused on several trophic levels or performed them in situ using ROV could generate relevant biological and ecological data on the chronic and accumulative potential impacts of an anthropic activities in deepsea and therefore how to mitigate them.

-Microbiological assay

The majority of sub-seafloor microorganisms live under high hydrostatic pressure, and are likely to be well suited to high pressure considering its dramatic effects on microbial viability and growth [START_REF] Yanagibayashi | Changes in the microbial community in Japan Trench sediment from a depth of 6292 m during cultivation without decompression[END_REF]. Moreover, deep-sea sediments are unique environments in which J o u r n a l P r e -p r o o f hydrostatic pressure has an impact on the concentration of gases (e.g. CH4, CO2, other short hydrocarbon gases, H2S). Cell membranes are often the first barrier affected by hydrostatic pressure which will decrease membrane fluidity concomitantly with pressure increase. Ecophysiological adaptation to high hydrostatic pressure drives microorganisms to increase the proportion of unsaturated fatty acids in their cell membrane to maintain sufficient fluidity [START_REF] Mangelsdorf | A quantitative assessment of pressure dependent adaptive changes in the membrane lipids of a piezosensitive deep sub-seafloor bacterium[END_REF]. Water content is also an important factor in the inactivation of microbes under high pressure. Vegetative cells are more sensitive to high pressure than spores. High pressure affects the physiology and biochemistry of cells by decreasing the stability of protein folding and hindering the ability of enzymes for metabolic activities.

In response to high pressure, microorganisms growing optimally at pressures above 100 bars (corresponding to a depth of 1000 m) are piezophilic or those tolerating elevated pressures are piezotolerant. Obligatory piezophilic microorganisms grow only at elevated pressures. These pressureadapted microorganisms are unable to survive decompression within hours. During sampling at depth, potential damage and/or inactivation of cells due to the depressurization of samples can occur when atmospheric pressure is reached. These effects increase for microorganism of the deep-biosphere where microorganisms are subjected to both hydrostatic and lithostatic pressure. That is probably one of the reasons why strict pieziophilic microorganisms have not yet been isolated from deep-sea sediments in comparison with other deep environments.

Early in the infancy of marine microbiology, deep-sea microbial diversity was explored during series of oceanographic cruises known as the "Travailleur" and "Talisman" expeditions with a French "marine" microbiologist Adolphe-Adrien Certes who first provided protocols for the collection of microbial samples and conducted cultivation of microbial populations from waters and sediments collected at depths ranging from 927 to 5100 m (Certes, 1884). Later, the pioneering work of two American microbiologists Claude Zobel and Richard Morita in the 1950s [START_REF] Zobell | Bacterial life at the bottom of the Philippine Trench[END_REF]) demonstrated using avant garde sophisticated samplers that microbial activity in deep-sea sediments was very low at in situ conditions (i.e. low temperature, high pressure and low nutrient levels). All the techniques used (in situ incubation, aboard ex situ incubation with or without decompression of samples) led to the same observation: organic matter degradation was slower at hydrostatic than atmospheric pressure, suggesting that the microbes probably originated from the surface instead of the deep sea or, at least, that only the piezotolerant species survived the depressurization. This kind of results was again recently J o u r n a l P r e -p r o o f confirmed by a study on depressurized deep-sea GoM bacterial communities recovered from Niskin Bottles and subjected to an onboard pressurized bioassay with oil from the MBO [START_REF] Marietou | The effect of hydrostatic pressure on enrichments of hydrocarbon degrading microbes from the Gulf of Mexico following the Deepwater Horizon oil spill[END_REF]. This pioneering work pointed out that to obtain a representative overview of deep-sea microbial communities, pressure is a crucial factor. An isobaric approach with samples from the deep-sea environments is essential and yet challenging because changes in pressure and temperature can result in substantial out-gassing that destroys the structural integrity of the sample as well as altering the composition and the activity of its microbial communities. This has been supported by contemporary studies systematically showing that microorganisms from enrichment cultures of depressurized samples are different to those detected by molecular approaches directly from pristine samples [START_REF] Yanagibayashi | Changes in the microbial community in Japan Trench sediment from a depth of 6292 m during cultivation without decompression[END_REF][START_REF] Edgcomb | Comparison of Niskin vs. in situ approaches for analysis of gene expression in deep Mediterranean Sea water samples[END_REF]. In the case of a hypothetical study about the consequences of severe anthropogenic perturbation like drilling waste deposition, there is no reason to expect a different outcome. Consequently, deep-sea microbiological bioassays thrive with a recurrent issue, namely: the incubation of piezophilic microorganisms in relevant and uninterrupted deep-sea environmental conditions (temperature, pH, hydrostatic pressure, and oxygen partial pressure -PaO2). To overcome these subsequent technical constraints, pressurized microbiological bioassays evolved following 3 strategies:

-Ex situ incubation with an indigenous consortium -In situ incubation with an indigenous consortium -Cultivation of pure piezotolerant strains In the second half of the 20 th century, sophisticated devices or prototypes were developed to collect water, fluids, sediments and animals as well as to cultivate microbes inhabiting such ecosystems (Gundersen & Mountain, 1972;Jannasch et al., 1982;[START_REF] Yayanos | Recovery and maintenance of live amphipods at a pressure of 580 bars from an ocean depth of 5700 meters[END_REF][START_REF] Yayanos | Evolutional and ecological implications of the properties of deep-sea barophilic bacteria[END_REF][START_REF] Yayanos | Isolation of a deep-sea barophilic bacterium and some of its growth characteristics[END_REF]1981;Yayanos & Dietz, 1983). Pressurized microbiological bioassays are directly derived from these technologies.

Ex situ incubation with a local consortium

Derived from the first deep-sea bioprospection tools, ex situ incubation protocols were developed before in-situ protocols. The reasons are diverse and include the need for direct observation J o u r n a l P r e -p r o o f driven by naturalistic curiosity, technological limitations and initial ignorance as well as incomprehension of ecophysiologically piezosensitive microbial communities. [START_REF] Bianchi | A high-pressure serial sampler to measure microbial activity in the deep sea[END_REF] expanded the pioneer concept of Jannasch and colleagues (Jannasch et al., 1973;[START_REF] Jannasch | Retrieval of concentrated and undecompressed microbial populations from the deep sea[END_REF][START_REF] Macdonald | The effects of pressure on the molecular structure and physiological functions of cell membranes[END_REF] into a multi-chamber setup that allows up to eight pressurized samples to be taken together during a single deployment. The first pressurized deep-sea sediment cores were obtained in 1995 at the Black Ridge and Carolina Rise during the Ocean Drilling Program (ODP) cruise (Leg 164) using the Pressure Core Sampler (PCS), a device that is capable of maintaining in situ pressures of up to 690 bars (depth of around 6900 m) [START_REF] Francis | Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[END_REF]. In 2003, the Multi-Autoclave-Corer (MAC) and the Dynamic Autoclave Piston Corer (DAPC) were deployed in relatively shallow gas hydrate-bearing sediments in the northern Gulf of Mexico. These devices retrieved, for the first time, near-surface sediment cores under pressure. The systems were designed to recover sediment cores at in situ pressures of up to 142 bars and 304 bars respectively [START_REF] J O U R N A L P R E -P R O O F Heeschen | In situ hydrocarbon concentrations from pressurized cores in surface sediments, Northern Gulf of Mexico[END_REF]. In addition, deepsea shallow sediment samples (around 5 mL) obtained by the manned submersible Shinkai 6500 with a scoop have been successfully brought to the surface in a pressure-and temperature-retaining device [START_REF] Kato | 31 Handling of Piezophilic Microorganisms[END_REF]. Subsequently, enrichment cultures were obtained without decompression [START_REF] Yanagibayashi | Changes in the microbial community in Japan Trench sediment from a depth of 6292 m during cultivation without decompression[END_REF] in a large laboratory high-pressure culture device called the "DeepBath system" [START_REF] Kato | 31 Handling of Piezophilic Microorganisms[END_REF]. The importance of obtaining uncontaminated microbial samples led to the development of the Autonomous Microbial Sampler (AMS), which guarantees that uncontaminated fluid samples can be obtained for microbial and nucleic acid analyses from a variety of environments, including deep-sea hydrothermal vents characterized by steep biogeochemical gradients [START_REF] Taylor | Autonomous Microbial Sampler (AMS), a device for the uncontaminated collection of multiple microbial samples from submarine hydrothermal vents and other aquatic environments[END_REF]. The AMS protects samples against temperature changes, but does not maintain the in-situ pressure. Parkes et al.

(2009) developed a system known as Deep-isoBUG which maintains sediment samples under elevated pressure without decompression (up to 250 bars) for enrichment and isolation of microorganisms at pressures of up to 1000 bars. Deep-isoBUG is a subsampling transfer system, which could be coupled with the pressure-retaining core drilling system HYACINTH [START_REF] Schultheiss | Pressure coring, logging and subsampling with the HYACINTH system[END_REF][START_REF] Alain | Microbial Life of the Deep Biosphere[END_REF] and the PRESS core-cutting system [START_REF] Parkes | Culturable prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure, anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)[END_REF]. These systems allow sub-seafloor sediment samples to be sub-sampled and transferred into an isolation culture chamber of a design similar to the device described by Jannasch et al., (1982) or a chemostat for further processing and bioassays. WHOI (Woods Hole Oceanographic Institute) developed a stand-alone sediment core sampling device [START_REF] Sheryll | New technology for uncontaminated and pressure controlled deep-sea sampling:" Deep Ocean Benthic Sampler[END_REF]) also designed to be interfaced with existing high-pressure tools for isolation of pure J o u r n a l P r e -p r o o f cultures of microorganisms in the absence of decompression (Jannasch et al., 1982;[START_REF] Jannasch | Deep-sea microbiology[END_REF]Taylor, 1978;[START_REF] Jannasch | Retrieval of concentrated and undecompressed microbial populations from the deep sea[END_REF][START_REF] Wirsen | A study of deep-sea natural microbial populations and barophilic pure cultures using a high-pressure chemostat[END_REF]. The more recently developed lander DOBS (Deep Ocean Benthic Sampler) is able to obtain core sediment samples from the deep ocean without microbial contamination and under in-situ pressure and temperature conditions until retrieval by the ship. Another system called the MAC-EXP system was designed by [START_REF] Jackson | A system for retrieval and incubation of benthic sediment cores at in situ ambient pressure and under controlled or manipulated environmental conditions[END_REF] for the retrieval and incubation of benthic sediments under in-situ pressure and temperature conditions and in controlled or manipulated environmental conditions. This system is able to sample down to 3500 m and to incubate sediments on-board without decompression. Moreover, during incubation, the core chambers can be connected to a laboratory incubation system in order to control enrichment culture, but also oxygen and temperature. The MAC-EXP was successfully used to make a laboratory bioassay related to the effect of pressure and temperature on deep-sea hydrocarbonoclastic microbial communities in artificially oil-polluted subarctic sediments [START_REF] Perez Calderon | Pressure and temperature effects on deep-sea hydrocarbon-degrading microbial communities in subarctic sediments[END_REF].

-In situ incubation with a local consortium

This second approach consists in directly using local abiotic specificities to perform incubation or enrichment using a local microbial population. An underwater vehicle can be used for a short-term experiment of up to 24h [START_REF] J O U R N A L P R E -P R O O F Cahet | In situ experimentation at the water/sediment interface in the deep sea: 2. Biotransformation of dissolved organic substrates by microbial communities at 2000 m depth in the Bay of Biscay[END_REF]. A cheaper, safer and more convenient possibility is the use of specific deep-sea landers. According to [START_REF] Tengberg | Benthic chamber and profiling landers in oceanography-a review of design, technical solutions and functioning[END_REF]: "Lander is a general term for any autonomous, unmanned oceanographic research vehicle that free-falls to the sea-floor unattached to any cable, and then operates independently on the sea-floor". Once the experiment is over, the lander float backs to the surface for recovery by a ship. Experiments can be conducted from a few days up to 1 year [START_REF] Kanzog | Microbial colonisation of artificial and deep-sea sediments in the Arctic Ocean[END_REF]. Using deep-sea benthic chambers, this kind of device can work under in-situ deep-sea conditions for the study of microbial response to enrichment with various sources of organic carbon [START_REF] Witte | Rapid response of a deep-sea benthic community to POM enrichment: an in situ experimental study[END_REF]Bürhing et al., 2006). After the MBO, bioassays with oil enrichment were performed in Gulf of Mexico sediment at 1226 m depth [START_REF] Orcutt | Microbial response to oil enrichment in Gulf of Mexico sediment measured using a novel long-term benthic lander system[END_REF]. This 5-month experiment, using the MIcrobial Methane Observatory for Seafloor Analysis (MIMOSA) lander, was the first to describe the response of deep-sea microbiota to oil release with such a degree of accuracy in terms of the relevance of abiotic factors. Their results clearly pointed out strong differences in comparison with ex-situ experiments (e.g. impact on microbial metabolism and composition). Despite their relative simplicity, landers can be quite efficient and sophisticated. The MIMOSA lander and the "Autonomous Sediment Sampler and Injection Ultra-Lander System" are good examples. The latter J o u r n a l P r e -p r o o f device, developed by a team of international deep-sea scientists and engineers led by R. Glud (University of Southern Denmark), enables the collection of in situ hadal sediment cores using separate motors to drive four core liners with core catchers into the sediment, where they can perform incubations or fixation of samples at the seafloor (Coley K., 2015). Due to their in situ location, landers also present technical limits including the reduced spectrum of analytical possibilities compared with lab experiments, the obligatory simplest experimental matrix as well as the difficulty to perform time course experiments.

-Cultivation of pure piezotolerant strains Cultivation of axenic microbial strains is a convenient, versatile and reproducible way to deepen ecophysiological mechanisms at play during a bioassay (e.g. detailed metabolism of pollutants, gene expression). Cultivation of axenic strictly piezophilic microorganisms is still very challenging even for routine maintenance. Deep-sea piezotolerant strains appear to be a better compromise for pressurized bioassays. Piezotolerant strains can be routinely cultivated under atmospheric pressure before being progressively acclimated to deep-sea hydrostatic pressure for bioassays. Anaerobic bioassays up to 100 bars are relatively easy to process with a simple stainless-steel chamber and HPLC pump to generate the pressure. Using such a system, [START_REF] Scoma | Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?[END_REF] demonstrated that piezosensitive hydrocarbonoclastic bacterial strains presented lower oil biodegradation capacities under pressure.

Preprocessing of culture manipulations for pressurized bioassays beyond 100 bars could be performed in a specific anaerobic chamber with a controlled atmosphere when anaerobic culture conditions are required. In general, two types of pressure equipment are available [START_REF] J O U R N A L P R E -P R O O F Marteinsson | Physiological responses to stress conditions and barophilic behavior of the hyperthermophilic vent archaeon Pyrococcus abyssi[END_REF]. One consists in a manual pump and several independent reactors to be used at room or cold temperature. These are routinely used for preservation of deep-sea samples collected under presumed in-situ pressure. A pressure vessel is also used for incubating enrichment cultures and monitoring microbial growth. In the laboratory, the second system is composed of several reactors associated with tubular ovens connected to a pump, a hydraulic accumulator, and an electronic control device. Each reactor can be used at selected temperatures and pressures independently of the others. Experiments can be carried out at up to 600 bars and 300°C. Microbial cultures are not directly performed in reactors.

Instead, incubation of pure cultures and sediment slurry enrichments at elevated pressures is conducted in serum vials or glass and/or plastic syringes completely filled with media and placed in the reactor, which is then fully filled with water and sealed with a lid secured. The gas atmosphere in the syringes and vials is completely removed. The vessel is pressurized using a gas-powered pump or gas tank and J o u r n a l P r e -p r o o f the pressure is transferred to the samples via the flexible butyl rubber stopper or the piston of the syringe (Figure 3).

Another anaerobic or microaerobic system commonly used for bioassays substitutes the pump with a screw-piston mechanism to generate the pressure and a steel ball or a magnetic stirring bar to mix the medium at up to 400 bars for the more efficient systems [START_REF] Schedler | Microbial degradation of crude oil at high pressure[END_REF]. This kind of device allows subsampling during incubation directly from the reactor or glass vial depending on the effective volume thanks to specific valves (Figure 4). The loss of pressure is compensated by screwing the piston. Screwpiston reactors can be autoclaved with medium after residual oxygen is replaced with nitrogen to avoid the emergence of reactive oxygen species at high oxygen partial pressure [START_REF] Bean | Effects of oxygen at increased pressure[END_REF][START_REF] Cabiscol Català | Oxidative stress in bacteria and protein damage by reactive oxygen species[END_REF]. In the case of microaerobic cultivation, the amount of oxygen cannot be adjusted in the time and gradually decreases during the bioassay.

Screw-piston reactors were successfully used to demonstrate that pressure cannot be discounted to estimate the biodegradation rate of aromatic hydrocarbons after the MBO. A bioassay was carried out with 2 model hydrocarbonoclastic microbial strains in presence of n-hexadecane and naphthalene and showed that some metabolic pathways were more impacted by pressure than previously estimated [START_REF] Schedler | Effect of high pressure on hydrocarbon-degrading bacteria[END_REF]. Finally, a Particle Sinking Simulator (PASS) was developed by Tamburini and colleagues [START_REF] J O U R N A L P R E -P R O O F Tamburini | Effects of hydrostatic pressure on microbial alteration of sinking fecal pellets[END_REF]. This promising device can be used to study the behavior of the microbiota inhabiting sinking particles (herein, fecal pellets) in a simulated sink by progressively increasing the hydrostatic pressure in a rotating incubator at up to 40 MPa under controlled temperature (but not O2 partial pressure). It would be interesting to test this device with a piezotolerant strain and simulated MOSSFA [START_REF] Ramseur | Deepwater Horizon oil spill: the fate of the oil[END_REF][START_REF] Passow | Marine snow formation in the aftermath of the Deepwater Horizon oil spill in the Gulf of Mexico[END_REF][START_REF] Nguyen | Perturbation of seafloor bacterial community structure by drilling waste discharge[END_REF][START_REF] Brooks | Sedimentation pulse in the NE Gulf of Mexico following the 2010 DWH blowout[END_REF].

In spite of all these interesting prototypes, a technological gap has still to be bridged to develop an efficient, cost-effective and convenient high-pressure laboratory tool and method. The 3 strategies reviewed above all present different interests and disadvantages from a practical view which are summarized in the table 2. These highlighted criteria should pave the way to the conception of the next generation approach. Amongst all these criteria a clear trend emerges. The ideal next generation process should be ideally able to avoid decompression during transfer of subsamples as well subsequent incubation of piezophilic or piezotolerant microbes in switchable aerobic/anaerobic J o u r n a l P r e -p r o o f controlled conditions over a long period of time (several months). Alternatively, a proper bioprospection of piezotolerant bacterial strains whose the deep sea origin is well assessed (e.g. piezotolerance and psychrophily of strains isolated with an axenic device from deep sea sediment) could serve to build a reference biocollection for simplified microbiological bioassays (Strategy 3.). These types of approach are necessary to properly evaluate the environmental impact of deep-sea pollution such as drilling waste depositions.

-Conclusion and Prospects

In the upcoming decades, the worldwide increase in demand for resources will put the deepsea under pressure [START_REF] Dussauze | Extreme Environments: The New Exploration/Production Oil Area Problem[END_REF]. The oil & gas industry is no exception to this trend. Despite the absence of unified, global legislation, local regulations tend to harmonize and be more and more restrictive on the Exploration and Production conditions of offshore exploitation. Oil & gas industry stakeholders will have to adapt to these changes to maintain their competitiveness. The fact that reported impacts are generally site-specific points out the need for a systematic risk assessment study coupled with in situ measurements as well numerical simulation for each new drilling area. It is highly expectable that a global consensus will emerge in this direction. To date, while the numerical tools and in situ exploration/monitoring tools (e.g. ROV, landers) present a high degree of operability, a functional, versatile and cost-effective tool, and associated protocol, must be developed for high pressure bioassays. Ideally, this technological solution would be switchable between microbiological and animal assay, anoxic and oxic conditions and suitable with a broad spectrum of abiotic factors (T°,pH,PaO2).

It should be designed to be simple, exploiting existing proven technological subsolutions and needs to be suitable for on-board experiments in terms of robustness and dimensions. Such a tool could easily become the reference for high pressure bioassays.

Declaration of interest statement

All the authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have

J

  o u r n a l P r e -p r o o f According to the review of Eliss et al. (2012) on sediment barium concentrations, water-based drilling fluids have a higher chemical impact than synthetic-based fluids, with an influence zone from 2

  J o u r n a l P r e -p r o o f -Pressurized biological assay: experimental background and technical challenges.

J

  o u r n a l P r e -p r o o f influenced its outcome.

Figure 1 :

 1 Figure 1: Waste distribution as is a barycenter of waste lift, depth and stream speed as a function of their respective intensities. The concentration of waste is represented by a heatmap from important (hot) to low (purple).

JFigure 2 :Figure 3 :

 23 Figure 2: Schematic representation of the similarities between idealized plumes and deposition between deepwater offshore drilling operation and DWH hydrocarbon propagation post blowout. J o u r n a l P r e -p r o o f

Figure 4 :

 4 Figure 4: Schematic view of a screw-piston reactor (adapted from Schedler, 2016).

J

  o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Regulation for the Mediterranean Sea is defined by the Barcelona Convention and its offshore protocol

The regulation goes for a 0 discharges.

But a list of authorized substances is implemented and if their concentrations are below a limit, then discharges are also authorized.

European Union's Marine Strategy Framework Directive (MSFD), which states that all the European seas should attain Good Environmental Status (GES) by 2020.

For French Guyana, the Guyana decree was implemented in 2012 by the French government.

French Guyana decree say that the amount of drilling fluid must be lower than 5% of the total waste volume


E Aprile

J Aalbers

F Agostini

M Alfonsi

L Althueser

F D Amaro

M Anthony

V C Antochi

F Arneodo

L Baudis

B Bauermeister

M L Benabderrahmane

T Berger

P A Breur
email: s.breur@nikhef.nl

A Brown

E Brown

S Bruenner

G Bruno

R Budnik

C Capelli

J M R Cardoso

D Cichon

D Coderre

A P Colijn

J Conrad

J P Cussonneau

M P Decowski

P De Perio

P Di Gangi

A Di Giovanni

S Diglio

A Elykov

G Eurin

J Fei

A D Ferella

A Fieguth

W Fulgione

A Gallo Rosso

M Galloway

F Gao

M Garbini

L Grandi

Z Greene

C Hasterok

E Hogenbirk

J Howlett

M Iacovacci

R Itay

F Joerg

S Kazama

A Kish

G Koltman

A Kopec

H Landsman

R F Lang

L Levinson

Q Lin

S Lindemann

M Lindner

F Lombardi

J A M Lopes

E López Fune

C Macolino

J Mahlstedt

A Manfredini

F Marignetti

T Marrodán Undagoitia

J Masbou

D Masson

S Mastroianni

M Messina

K Micheneau

K Miller

A Molinario

K Morå

Y Mosbacher

M Murra

J Naganoma

K Ni

U Oberlack

K Odgers

B Pelssers

F Piastra

J Pienaar

V Pizzella

G Plante

R Podviianiuk

N Priel

H Qiu

D Ramírez García

S Reichard

B Riedel

A Rizzo

A Rocchetti

N Rupp

J M F Dos Santos

G Sartorelli

N Šarčević

M Scheibelhut

S Schindler

J Schreiner

D Schulte

M Schumann

L Scotto

M Selvi

P Shagin

E Shockley

M Silva

H Simgen

C Therreau

D Thers

F Toschi

G Trinchero

C Tunnell

N Upole

M Vargas

O Wack

H Wang

Z Wang

Y Wei

C Weinheimer

D Wenz

C Wittweg

J Wulf

Z Xu

J Ye

Y Zhang

T Zhu

J P Zopounidis

Constraining the spin-dependent WIMP-nucleon cross sections with XENON1T

Keywords: numbers: 95.35.+d, 14.80.Ly, 61.25.Bi, 95.55.Vj

We report the first experimental results on spin-dependent elastic WIMP-nucleon scattering from the XENON1T dark matter search experiment. The analysis uses the full tonne-year exposure of XENON1T to constrain the spin-dependent proton-only and neutron-only cases. No significant signal excess is observed, and a profile likelihood ratio analysis is used to set exclusion limits on the WIMP-nucleon interactions. This includes the most stringent constraint to date on the WIMPneutron cross section, with a minimum of 6.3 × 10 -42 cm 2 at 30 GeV/c 2 and 90% confidence level. The results are compared with those from collider searches and used to exclude new parameter space in an isoscalar theory with an axial-vector mediator.

Introduction.-There is a preponderance of astrophysical evidence that a non-luminous, massive component known as dark matter (DM) comprises about 26.5% of the total energy density of the Universe [1,2]. Still, the particle nature of this component remains unknown. One attractive DM candidate is the weakly interacting massive particle (WIMP), which arises naturally in several extensions of the Standard Model of particle physics [3,4]. This has motivated many experimental searches for both the decay and self-annihilation products of WIMPs (indirect detection) [5], for WIMP production at particle accelerators [6], and for WIMPs scattering off atomic nuclei on Earth (direct detection) [7]. One leading direct detection technique uses liquid xenon (LXe) time projection chambers (TPCs), placed underground to reduce backgrounds induced by cosmic rays [8][9][10][11]. XENON1T, the largest and most sensitive of these experiments to date, is a dual-phase (liquid and gas) xenon TPC located at a depth of 3600 m water-equivalent at the INFN Laboratori Nazionali del Gran Sasso in L'Aquila, Italy [12].

XENON1T contains 3.2 t of ultra-pure LXe, with 2 t in the active detector volume, and is outfitted with two arrays of 127 and 121 Hamamatsu R11410-21 3" photomultiplier tubes (PMTs) [13,14] facing the LXe from above and below, respectively. Energy depositions in the active volume produce scintillation photons as well as ionization electrons. The scintillation light is promptly detected by the PMTs (S1), while the electrons are drifted upward through the LXe by a uniform electric drift field. At the liquid-gas interface, the electrons are extracted by an electric field towards the anode, producing proportional scintillation light (S2) via electroluminescence, which is also detected by the PMTs. The time difference between the S1 and the S2 is proportional to the depth of the original interaction. Combined with the PMT hit-pattern of the S2, this allows for 3D position reconstruction of the event. The reconstructed position helps eliminate background events due to radioactivity from materials in and around the TPC, which largely occur near its edge. The position is also used to apply corrections for variations in signal collection efficiencies. The ratio between S2 and S1 signal sizes is used to discriminate between nuclear recoils (NRs) due to WIMPs or neutron backgrounds, and electronic recoils (ERs) due to β or γ backgrounds.

Since WIMPs are non-relativistic [3], the WIMPnucleus interaction cross section can be written as the sum of a part which increases with the mass of the target nucleus (spin-independent, or SI), and an axial-vector part which couples to the nuclear spin (spin-dependent, or SD) [4,15]. Recently, XENON1T reported SI results from a tonne-year exposure, which achieved the lowest ever background in a direct detection experiment and set the most stringent 90% C.L. upper limit to date on the SI cross section for WIMP masses above 6 GeV/c 2 [8].

We also reported the first direct detection constraints on the WIMP-nucleus interaction involving pion exchange currents, an additional scalar component that can dominate if the standard SI interaction is absent or strongly suppressed [16]. In this Letter, we present the first limits on the SD WIMP-nucleon cross sections from the XENON1T experiment, using data from the full tonneyear exposure.

Spin-Dependent Theory.-The SD interaction of WIMPs with nuclei is described by the WIMP-quark axial-vector-axial-vector Lagrangian [17]. At low momentum transfer q, the Lagrangian can be evaluated using chiral effective field theory (EFT) [18,19], and the differential cross section can be written as

dσ SD dq 2 = 8G 2 F (2J + 1)v 2 S A (q), (1)
where G F is the Fermi constant, v is the WIMP velocity in the rest frame of the detector, J is the initial groundstate angular momentum of the nucleus, and S A (q) is the axial-vector structure factor (all equations shown in natural units). The structure factor is conveniently expressed in terms of the isoscalar (a 0 ) and isovector (a 1 ) WIMP-nucleon couplings as

S A (q) = a 2 0 S 00 (q) + a 0 a 1 S 01 (q) + a 2 1 S 11 (q). (2)
The interaction strength is described by these couplings, while the nuclear structure information is absorbed by the S ij factors. The two unknown couplings (a 0 , a 1 ) yield a two-dimensional plane of parameter space to search, unlike in the SI case, where the WIMP is typically assumed to have an equal (purely isoscalar) coupling to protons and neutrons.

In the limit of zero momentum transfer, the structure factor simplifies to

S A (0) = (2J + 1)(J + 1) 4πJ × |(a 0 + a 1 ) S p + (a 0 -a 1 ) S n | 2 , (3)
where S p and S n are the expectation values of the total proton and neutron spin operators in the nucleus, and a 1 contains a correction to the isovector coupling a 1 due to chiral two-body currents involving the exchange of a pion. Experimental SD searches constrain the theory using the special cases a 0 = a 1 = 1 ("proton-only") and a 0 = -a 1 = 1 ("neutron-only"). These cases are convenient because at the one-body (a 1 → a 1 ) level, S A (0) depends only on the total spin expectation values of the protons and neutrons in the nucleus, respectively [19]. Two naturally-occurring isotopes of xenon have nonzero nuclear spin, 129 Xe (spin-1/2) and 131 Xe (spin-3/2), with natural abundances of 26.4% and 21.2%, respectively [20,[START_REF] Rumble | CRC Handbook of Chemistry and Physics[END_REF]. Residual gas analyzer measurements show consistency with these natural abundances in XENON1T within the 1% precision of the measurement device. The remaining 52.4% of xenon has negligible sensitivity to the SD interaction. As both xenon isotopes have an odd number of neutrons, it follows that | S n | | S p |. Specifically, in 129 Xe S n = 0.329 and S p = 0.010, while in 131 Xe S n = -0.272 and S p = -0.009 [19]. Consequently, XENON1T is more sensitive to the neutron-only case, but also has non-zero sensitivity to the proton-only case since a 0a 1 = 0 in Eq. ( 3) due to the aforementioned two-body contribution.

The total expected NR spectrum dR/dE r can be written as

dR dE r = 2ρ χ m χ dσ SD dq 2 vf ( v)d 3 v, (4)
where m χ is the WIMP mass, ρ χ is the local WIMP density and f ( v) is the WIMP velocity distribution in the rest frame of the detector. q = √ 2E r m Xe , with m Xe the mass of a xenon nucleus. A standard isothermal WIMP halo, as in [8], is assumed, with v 0 = 220 km/s, ρ χ = 0.3 GeV/(c 2 × cm 3 ), v esc = 544 km/s, and Earth velocity v E = 232 km/s [START_REF] Lewin | [END_REF]. In the neutron-or protononly case, the differential scattering cross section can be rewritten as

dσ SD dq 2 = σ SD χN 3µ 2 N v 2 π 2J + 1 S N (q), (5)
where µ N is the reduced mass of the WIMP-nucleon system, S N (q) is the axial-vector structure factor for a proton or neutron (N = {n, p}) in xenon (from using the corresponding couplings in Eq. ( 2)), and σ SD χN is the scattering cross section between a WIMP and a single proton or neutron, at zero momentum transfer [4,16]. Using Eq. ( 5), the recoil spectrum in Eq. ( 4) becomes proportional to σ SD χN . This unknown parameter is used to set limits as a function of WIMP mass.

Detailed calculations of S N have been carried out in [19] for many isotopes relevant to experimental searches, including 129 Xe and 131 Xe. These calculations use a detailed nuclear shell model to represent the nuclear states, reproducing the ground-state energies and ordering of energy levels from spectroscopic measurements. Ref. [19] expands on [23], which was extensively compared with alternative calculations in [24].

The theoretical uncertainties reported in [19] mainly come from the two-body current contribution, specifically the density of the nuclear states and the low-energy constants in chiral EFT. These uncertainties have a larger effect on σ SD χp than on σ SD χn , since the SD WIMP-proton sensitivity in a xenon target chiefly relies on two-body interactions with neutrons. Since it is difficult to characterize the distribution of these uncertainties, it is conventional [24,32,33] to take the mean of the range of structure factors given, rather than including the uncertainty on the scattering rate in the statistical inference.

Example recoil spectra for the neutron-and proton-only cases are shown in Fig. 1, along with the standard SI spectrum (scaled by 10 -4 ) [8] for reference. The resulting SD rates are much lower than the SI rates. This is mostly explained by the SD structure factor in Eq. ( 3), which is O(1), while the analogous SI form factor scales with the square of the number of nucleons, due to the coherence of the interaction over the nucleus.

Analysis method.-SI and SD WIMP-nucleus scattering produce similar recoil spectra in XENON1T, and both interactions produce observables through the same NR process. We therefore use signal corrections and event selection criteria identical to [8]. The dark matter search is limited to events within an inner 1.30 ± 0.01 t LXe fiducial mass, with corrected S1s between (3, 70) photoelectrons, accepting NRs of about 5 -41 keV nuclear recoil energy on average. The livetime analyzed is 278.8 days, consisting of a 32.1 day run [25] and a 246.7 day run, resulting in a total exposure of 1.0 t × yr. Background models, also retained from [8], include data-driven models for accidental coincidence of lone S1s and S2s, and events with reduced charge signal due to interactions at the detector surfaces. The ER (β and γ) and NR (radiogenic neutrons and coherent elastic neutrino-nucleus scattering) backgrounds are modeled using energy depositions from GEANT4 simulations, passed to a Monte-Carlo (MC) of ER and NR response in LXe, XENON1T detector physics, and detection efficiency [28]. The parameters in the MC are determined from a simultaneous fit to calibration data using ER [26] ) cases in LXe for a 10 GeV/c 2 (solid) and 100 GeV/c 2 (dashed) WIMP and a WIMP-nucleon cross section of 10 -45 cm 2 . The bands on the SD spectra come from uncertainties in the contribution to SD scattering from interactions involving the exchange of a pion between two nucleons (two-body currents). The WIMP search region in XENON1T is depicted by the total efficiency curve (gray dotted).

and NR [27] sources taken periodically throughout the exposure. The signal region in the DM search data was blinded prior to the determination of the event selection and background models [8]. For each WIMP mass, the SD signal recoil spectrum calculated from Eq. ( 4) is propagated through the same MC to generate the expected distribution of S1s and S2s from corresponding WIMPnucleon interactions. Statistical inference is done using a three-dimensional (corrected S1, corrected S2 in the bottom PMT array, and radius) unbinned extended likelihood, profiled over nuisance parameters [28]. In addition to these three dimensions, the likelihood distinguishes between events in an inner 0.65 t core and those in an outer section of the fiducial mass to incorporate the difference in the expected neutron background rate, as in [8]. Nuisance parameters are included to account for uncertainties in ER response, detection and selection efficiencies, and background rates. To safeguard against interpreting an under-prediction of ERs as a signal excess, an additional WIMP-like component is added to the background model and constrained by ER calibration data [28,29]. Upper limits and two-sided intervals are computed using a Feldman-Cousins-based method [30], with a Neyman band constructed from a profiled likelihood ratio test statistic [31]. Background-only simulations are performed to calculate the range of possible upper limits under many repetitions of the XENON1T exposure.

Results.-For all WIMP masses considered, and for both the neutron-and proton-only cases, the data are XENON1T 90% C.L. upper limit on the spindependent WIMP-neutron cross section from a 1 tonne-year exposure. The range of expected sensitivity is indicated by the green (1σ) and yellow (2σ) bands. Also shown are the experimental results from XENON100 [24], LUX [32] and PandaX-II [33]. We use the 'chiral EFT' limit from PandaX-II, since it is based on the same SD interaction model as all other shown results.

consistent with the background-only hypothesis. The local discovery p-values at WIMP masses of 6, 50, and 200 GeV/c 2 in the neutron-only (proton-only) case are 0.6, 0.4, and 0.2 (0.6, 0.3, and 0.1), respectively. Fig. 2 and Fig. 3 show the 90% C.L. upper limits, as well as the 1σ and 2σ sensitivity bands, on the SD WIMP-neutron and WIMP-proton cross sections, respectively. Differences between the limit and the median sensitivity due to fluctuation of the background are within the 2σ statistical uncertainty.

The mean values of the structure factors are used both for the observed limits and the sensitivity distributions. To estimate the impact of the theoretical uncertainty on the result, a cross-check was performed by taking the minimum and maximum values of the structure factors, and using the asymptotic distribution of the test statistic to set limits for each case [34]. At 50 GeV/c 2 , the upper limit on the WIMP-neutron cross section shifts downwards (upwards) by a factor of 1.1 (1.1) when taking the minimum (maximum) structure factor values. Similarly, the upper limit on the WIMP-proton cross section shifts downwards (upwards) by a factor of 1.6 (2.2) due to the larger dependence of the proton-only sensitivity on the uncertain two-body component.

The neutron-only limit (Fig. 2) is the most stringent constraint from a direct detection experiment for WIMP masses above 6 GeV/c 2 , with a minimum of 6.3 × 10 -42 cm 2 for a 30 GeV/c 2 WIMP. The protononly limit (Fig. 3) is the most stringent constraint from a LXe direct detection experiment, though fluorine-based superheated liquid experiments such as PICASSO [35], XENON1T 90% C.L. upper limit on the spindependent WIMP-proton cross section from a 1 tonne-year exposure. The range of expected sensitivity is indicated by the green (1σ) and yellow (2σ) bands. Selected experimental results are shown for XENON100 [24], LUX [32], PandaX-II [33] and PICO-60 [38]. We use the 'chiral EFT' limit from PandaX-II, since it is based on the same SD interaction model as all other shown results.

SIMPLE [36], and PICO-60 [37,38] have consistently led the field in directly constraining the WIMP-proton cross section.

Collider experiments are sensitive to WIMPs through searches for final states of pp collisions with missing transverse energy, which can be attributed to the production of escaping DM particles. These searches are complementary to those carried out by direct detection experiments, but direct comparison of the resulting limits requires that a model of DM interactions with Standard Model particles is specified. Following the approach of [37], we use a model recommended by the LHC Dark Matter Working Group [39], and frequently used by AT-LAS and CMS, to compare results with direct detection SD searches [41,42]. In this model, the WIMP is a Dirac fermion of mass m χ , and has an s-channel interaction with quarks, mediated by a spin-1 particle of mass m med with an axial-vector coupling to both the WIMP and the quarks. Additionally, the mediator couples equally to all quark flavors, so the WIMP-nucleon interaction is isoscalar.

Since the two-body pion exchange currents are purely isovector [19], the corresponding correction terms vanish in the isoscalar case. Consequently, the structure factor, and thus the recoil spectra, differ in both shape and rate from the neutron-only and proton-only cases, so this model should be treated as a third, distinct case.

The model contains four free parameters: the mediator mass m med , m χ , the mediator-quark coupling g q , and the mediator-WIMP coupling g χ . Following [39], the cross section can now be written as

σ SD χn = σ SD χp = 0.31 π g 2 q g 2 χ µ 2 N m 4 med . (6)
New signal models are generated, and statistical inference is performed via the same method as for the neutrononly and proton-only cases, resulting in a 95% C.L. upper limit on the WIMP-nucleon cross section σ SD χN for the isoscalar case. After substituting the conventional values of the couplings (g q = 0.25, g χ = 1), we can use Eq. 6 to transform this upper limit into the m med -m χ plane, and compare it directly with collider experiments for this particular simplified model. As shown in Fig. 4, the constraint from XENON1T data excludes new parameter space in this theory, and represents the most stringent constraint from a direct detection experiment. We note that in a complete treatment of the comparison between the LHC and direct detection experiments, one should include isospin-violating corrections to the couplings due to the difference in energy scales [40]. This cannot be simply included because the structure factor becomes dependent on the mediator mass. As this effect would enhance the rate of WIMP-neutron scattering relative to WIMP-proton scattering, our limit can be viewed as conservative. XENON1T 95% C.L. upper limit in the m medmχ plane for a simplified isoscalar model with an axial-vector mediator and a Dirac WIMP, where the mediator-quark (gq) and mediator-WIMP (gχ) couplings are fixed to 0.25 and 1.0, respectively. Shown for comparison are 95% C.L. limits from PICO-60 [37], ATLAS [41], and CMS [42]. The shaded regions are excluded by the correspondingly colored limits.

Conclusion.-We have analyzed the data from a tonne-year exposure of XENON1T, which is currently the leading dual-phase liquid xenon direct detection experiment. The SD WIMP-neutron and WIMP-proton spectra were calculated from the work of Ref. [19]. For the data selection, background models and statistical interference, the same methods have been used as for the XENON1T SI analysis [8]. The data are consistent with the background-only hypothesis and the resulting limits were computed. These results are the first constraints on the SD interaction from XENON1T and improve upon previous constraints from LXe experiments. Additionally, the XENON1T 95% C.L. upper limit in the m med -m χ plane for a simplified isoscalar model has been compared with constraints from collider searches and excludes new parameter space. This work is part of a program to constrain a large set of theoretical parameters using XENON1T, including the SI [8] and WIMP-pion [16] interactions, along with future work to constrain a broader set of WIMP-quark couplings using EFT methods.

Israeli Centers Of Research Excellence (I-CORE), Pazy-Vatat, Initial Training Network Invisibles (Marie Curie Actions, PITNGA-2011-289442), Fundacao para a Ciencia e a Tecnologia, Region des Pays de la Loire, Knut and Alice Wallenberg Foundation, Kavli Foundation, and Istituto Nazionale di Fisica Nucleare. Data processing is performed using infrastructures from the Open Science Grid and European Grid Initiative. We are grateful to Laboratori Nazionali del Gran Sasso for hosting and supporting the XENON project.

FIG. 1 .

 1 FIG.1. Comparison of the WIMP-nucleus recoil spectra in the SD neutron-only (green), SD proton-only (blue), and SI (orange, scaled by 10 -4 ) cases in LXe for a 10 GeV/c 2 (solid) and 100 GeV/c 2 (dashed) WIMP and a WIMP-nucleon cross section of 10 -45 cm 2 . The bands on the SD spectra come from uncertainties in the contribution to SD scattering from interactions involving the exchange of a pion between two nucleons (two-body currents). The WIMP search region in XENON1T is depicted by the total efficiency curve (gray dotted).

  FIG. 2.XENON1T 90% C.L. upper limit on the spindependent WIMP-neutron cross section from a 1 tonne-year exposure. The range of expected sensitivity is indicated by the green (1σ) and yellow (2σ) bands. Also shown are the experimental results from XENON100[24], LUX[32] and PandaX-II[33]. We use the 'chiral EFT' limit from PandaX-II, since it is based on the same SD interaction model as all other shown results.

  FIG. 3.XENON1T 90% C.L. upper limit on the spindependent WIMP-proton cross section from a 1 tonne-year exposure. The range of expected sensitivity is indicated by the green (1σ) and yellow (2σ) bands. Selected experimental results are shown for XENON100[24], LUX[32], PandaX-II[33] and PICO-60[38]. We use the 'chiral EFT' limit from PandaX-II, since it is based on the same SD interaction model as all other shown results.

  FIG. 4.XENON1T 95% C.L. upper limit in the m medmχ plane for a simplified isoscalar model with an axial-vector mediator and a Dirac WIMP, where the mediator-quark (gq) and mediator-WIMP (gχ) couplings are fixed to 0.25 and 1.0, respectively. Shown for comparison are 95% C.L. limits from PICO-60[37], ATLAS[41], and CMS[42]. The shaded regions are excluded by the correspondingly colored limits.

Acknowledgements.-The authors would like to thank A. Schwenk, M. Hoferichter, and J. Menéndez for useful discussions on SD theory and nuclear structure. We gratefully acknowledge support from the National Science Foundation, Swiss National Science Foundation, German Ministry for Education and Research, Max Planck Gesellschaft, Deutsche Forschungsgemeinschaft, Netherlands Organisation for Scientific Research (NWO), Netherlands eScience Center (NLeSC) with the support of the SURF Cooperative, Weizmann Institute of Science,