José Seco
email: jseco@ua.pt

José C Xavier

Paco Bustamante

João P Coelho

Ryan A Saunders

Nicole Ferreira

Sophie Fielding

Miguel A Pardal

Gabriele Stowasser

Thainara Viana

Geraint A Tarling

Eduarda Pereira

Andrew S Brierley

Main drivers of mercury levels in Southern Ocean Lantern fish Myctophidae

Keywords: Trace element, metal, bioaccumulation, mesopelagic fish, Antarctic

Myctophids are the most abundant fish group in the Southern Ocean pelagic ecosystem and are an important link in the Antarctic marine food web. Due to their major ecological role, evaluating the level of mercury (Hg) contamination in myctophids is important as a step towards understanding the trophic pathway of this contaminant. The concentrations of total Hg were determined in muscle, gill, heart and liver tissue of 9 myctophid species to quantify tissue partitioning variability between species. Organic Hg concentration and proportion in muscle was also determined. Hg concentrations were higher in the liver and heart than in muscle and gills, but the proportion of organic Hg was almost 100% in muscle, indicating that the main uptake route for Hg is through the diet. Most of the species analysed have similar vertical and horizontal distributions, and similar feeding modes and prey.

Geographical and temporal variability of Hg concentrations was examined using samples from 3 different years (2007/08, 2015/16 and 2016/17) and 2 locations (South Georgia and South Orkneys Islands). Our results appear to indicate a decreasing trend in Hg contamination over the last decade, particularly gill tissue, which is in agreement with a previous study on squid from the same region. There was no significant variability in Hg concentration between the different sampling locations. Hg levels were consistent with values reported previously for myctophids around the world, indicating low global-scale geographic variability. A positive relationship between fish size and Hg concentration was found for most species, with the exception of Electrona antarctica females, which may be explained through Hg elimination by egg laying. We estimate that myctophids collectively comprise a Southern Ocean mercury 'reserve' of  1.82 metric tonnes.

Capsule: Myctophids are a key group in the Southern Ocean food web, and have shown a possible decreasing trend in mercury contamination over the decade.

Introduction

Mercury (Hg) is one of the most known hazardous elements and it has a global dispersion [START_REF] Selin | Global Biogeochemical Cycling of Mercury: A Review[END_REF]UNEP, 2013). Hg has a long-range dispersion capacity [START_REF] Streets | Global and regional trends in mercury emissions and concentrations, 2010-2015[END_REF] and reaches remote areas such as Antarctica where relatively high Hg concentrations have been reported in seawater [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Once into the ocean, Hg is methylated by microorganisms enhancing its bioavailability to biota and allowing its bioaccumulation and biomagnification in food webs (Eagles-Smith et al;2018). Different oceanographic features such as strong upwelling currents in the Southern Ocean, and seasonal sea ice cover at high latitudes, create conditions that favour Hg methylation leading to increased methylHg (MeHg) concentrations [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. MeHg detrimental effects include changing biochemical processes, damaging cells and tissues, and reducing reproductive success in fish [START_REF] Sandheinrich | Methylmercury in fish: Recent advances in assessing toxicity of environmentally relevant exposures[END_REF][START_REF] Scheuhammer | Recent progress on our understanding of the biological effects of mercury in fish and wildlife in the Canadian Arctic[END_REF]. As MeHg biomagnifies along food webs, longlived apex predatory species are at high toxic risk. As prey from mesopelagic habitats are enriched in MeHg as a result of enhanced methylation rates in deep waters (Monteiro et al. 1995[START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF], predators feeding on mesopelagic prey are likely to be at higher risk than epipelagic feeders.

Knowledge of deep sea organisms and communities remains fragmentary [START_REF] Cvitanovic | Improving knowledge exchange among scientists and decision-makers to facilitate the adaptive governance of marine resources: A review of knowledge and research needs[END_REF]. Mesopelagic fish are a particularly understudied group [START_REF] Catul | A review on mesopelagic fishes belonging to family Myctophidae[END_REF], especially lanternfish (Family Myctophidae; hereafter myctophids), which are the most abundant and diverse Family, with ~250 species globally and a biomass of at least 550-660 million tonnes [START_REF] Bone | Biology of fishes[END_REF][START_REF] Gjøsaeter | A review of the world resources of mesopelagic fish[END_REF]. As a consequence of their high biomass, myctophids are important components of oceanic ecosystems and global biogeochemical cycles [START_REF] Irigoien | Large mesopelagic fishes biomass and trophic efficiency in the open ocean[END_REF]. They are crucial in the transfer of energy and contaminants such as Hg through oceanic food webs, linking primary consumers and macrozooplankton to higher predators [START_REF] Saunders | Myctophid Fish (Family Myctophidae) Are Central Consumers in the Food Web of the Scotia Sea (Southern Ocean)[END_REF]. However, major uncertainties still remain regarding the extent of Hg bioaccumulation and Hg speciation in myctophids, particularly in the Southern Ocean [START_REF] St John | A Dark Hole in Our Understanding of Marine Ecosystems and Their Services: Perspectives from the Mesopelagic Community[END_REF].

As elsewhere, myctophids are an important part of the Southern Ocean, food web [START_REF] Murphy | Spatial and temporal operation of the Scotia Sea ecosystem: a review of large-scale links in a krill centred food web[END_REF]. There they consume mainly planktic crustaceans [START_REF] Lourenço | Life cycle, distribution and trophodynamics of the lanternfish Krefftichthys anderssoni (Lonnberg, 1905) in the Scotia Sea[END_REF][START_REF] Pakhomov | Prey composition and daily rations of myctophid fishes in the Southern Ocean[END_REF][START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF], and in turn a major food source for a range of marine predators, including large predatory fish [START_REF] Fenaughty | Diet of the Antarctic toothfish (Dissostichus mawsoni) from the Ross Sea, Antarctica (subarea 88.1)[END_REF][START_REF] Stevens | Diet of Antarctic toothfish (Dissostichus mawsoni) from the Ross Sea region, Antarctica[END_REF], cephalopods [START_REF] Cherel | Diet of the squid Moroteuthis ingens (Teuthoidea: Onychoteuthidae) in the upper slope waters of the Kerguelen Islands[END_REF][START_REF] Olson | The role of squid in open ocean ecosystems[END_REF]; K. L. [START_REF] Phillips | Predation on myctophids by the squid Moroteuthis ingens around Macquarie and Heard Islands: stomach contents and fatty acid analyses[END_REF][START_REF] Rodhouse | Trophic relations of the cephalopod Martialia hyadesi (Teuthoidea: Ommastrephidae) at the Antarctic Polar Front, Scotia Sea[END_REF], marine mammals [START_REF] Newland | Diet of juvenile southern elephant seals reappraised by stable isotopes in whiskers[END_REF] and seabirds [START_REF] Xavier | Interannual variation in the diets of two albatross species breeding at South Georgia: implications for breeding performance[END_REF]. Since most myctophids are opportunistic feeders with a broad dietary range, preying on the most available food resources at their disposal [START_REF] Cherel | Isotopic niches and trophic levels of myctophid fishes and their predators in the Southern Ocean[END_REF][START_REF] Pakhomov | Prey composition and daily rations of myctophid fishes in the Southern Ocean[END_REF][START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF][START_REF] Stowasser | Food web dynamics in the Scotia Sea in summer A stable isotope study[END_REF], they are considered good bioindicators of contamination levels in the local marine environment.

In mid-trophic level fish such as myctophids, Hg can be accumulated biologically via two main pathways: absorption from the environment through the surfaces involved in respiration (e.g. gills and skin), and absorption through ingestion of contaminated prey. Whilst waterborne uptake can be a significant pathway, particularly under high environmental exposure levels, the majority of Hg is absorbed through food intake (G. R. [START_REF] Phillips | The Relative Contributions of Methylmercury from Food or Water to Rainbow Trout (Salmo gairdneri) in a Controlled Laboratory Environment[END_REF]. Although many studies have examined the general diets and trophodynamics of myctophids across the globe [START_REF] Hudson | Myctophid feeding ecology and carbon transport along the northern Mid-Atlantic Ridge[END_REF][START_REF] Olivar | Trophic position of lanternfishes (Pisces: Myctophidae) of the tropical and equatorial Atlantic estimated using stable isotopes[END_REF][START_REF] Van Noord | Oceanographic influences on the diet of 3 surface-migrating myctophids in the eastern tropical Pacific Ocean[END_REF], only a few studies globally have investigated Hg bioaccumulation in these fish [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF][START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF][START_REF] Gibbs | Heavy Metal Concentrations in Museum Fish Specimens: Effects of Preservatives and Time[END_REF][START_REF] Lahaye | New insights from age determination on toxic element accumulation in striped and bottlenose dolphins from Atlantic and Mediterranean waters[END_REF][START_REF] Martins | Temporal and spatial changes in mercury concentrations in the North Atlantic as indicated by museum specimens of glacier lanternfish Benthosema glaciale (Pisces: Myctophidae)[END_REF][START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF][START_REF] Windom | Arsenic, cadmium, copper, mercury, and zinc in some species of[END_REF], and only two studies have focussed on the Southern Ocean [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]Cipro et al., 2018b). Due to their central role in the Southern Ocean pelagic food web, evaluating the level of Hg contamination in myctophids is important as a step towards understanding the trophic pathway of this contaminant. Such studies are also essential for establishing robust baselines for future environmental monitoring that will inform potential ecosystem management strategies in the context of the Minamata convention objectives (Gustin et al. 2016).

In the present study, Hg concentrations were measured in myctophids collected across the Scotia Sea, one of the most productive regions of the Southern Ocean [START_REF] Holm-Hansen | Temporal and spatial distribution of chlorophyll-a in surface waters of the Scotia Sea as determined by both shipboard measurements and satellite data[END_REF], and spatial, temporal, inter-specific and ontogenetic patterns were examined. The Scotia Sea is home to globally important populations of higher predator species such as penguins, flying birds, seals and whales [START_REF] Murphy | Spatial and temporal operation of the Scotia Sea ecosystem: a review of large-scale links in a krill centred food web[END_REF], and important commercial fisheries [START_REF] Constable | Managing fisheries to conserve the Antarctic marine ecosystem: practical implementation of the Convention on the Conservation of Antarctic Marine Living Resources (CCAMLR)[END_REF], so understanding Hg pathways is important. Specifically, we assessed the total Hg (T-Hg) concentrations in four tissues (muscle, gills, heart and liver) and organic Hg as a proxy of MeHg (O-Hg) in the muscle of the nine biomass-dominant myctophids (Electrona antarctica, Electrona carlsbergi, Gymnoscopelus braueri, Gymnoscopelus nicholsi, Gymnoscopelus opisthopterus, Gymnoscopelus fraseri, Protomyctophum bolini, Krefftichthys anderssoni and Nannobrachium achirus) from two regionally distinct food webs in the Scotia Sea (South Georgia and South Orkneys Islands) in different years (2007/08, 2015/16 and 2016/17). These regions are characterised by different environmental conditions (South Orkney Islands, an Antarctic island group which experiences winter sea ice (Murphy et al., 1995); South Georgia, a sub-Antarctic island free of sea ice (Rogers et al., 2015)), zooplankton population dynamics and myctophid community composition/structure, as well as different higher predator-prey dynamics [START_REF] Murphy | Comparison of the structure and function of Southern Ocean regional ecosystems: The Antarctic Peninsula and South Georgia[END_REF], so together facilitate an interesting comparison into how Hg may affect different components of the overall Southern Ocean ecosystem. This study therefore provides important insights on T-Hg and O-Hg accumulation in Southern Ocean myctophids that are essential for monitoring the health of the Southern Ocean food web in a resource management context. All samples were caught during at night, between 0 and 1000 m, using either 8 m 2 or 25 m 2 mouth-opening Rectangular Midwater Trawl nets (RMT8 or RMT25; [START_REF] Piatkowski | Nekton community of the Scotia Sea as sampled by the RMT 25 during austral summer[END_REF][START_REF] Roe | A new multiple rectangular midwater trawl (RMT 1+8M) and some modifications to the institute of oceanographic sciences' RMT 1+8[END_REF]. Myctophids were identified onboard to species level and standard length (SL) measured to the nearest mm. Individuals were then frozen at -20ºC for subsequent laboratory analyses, with each fish species preserved separately in different plastic bags.

Material and methods

Sampling

Laboratory procedures

In the laboratory, all 200 specimens were re-measured and weighed to the nearest 0.01g before dissection. Sex and maturity of post-juveniles was determined [START_REF] Hulley | Myctophidae[END_REF]. For most specimens, the gills, heart and liver were extracted, as well as a sample of muscle (without the skin). For the 2 smallest species, Krefftichthys anderssoni and Protomyctophum bolini, organ dissection was not possible and only muscle was collected.

Samples were frozen in sterile containers and lyophilized during two days and ground to a fine powder for further analyses of Hg. T-Hg was determined by atomic absorption spectrometry (AAS) with thermal decomposition and gold amalgamation, using an Advanced Mercury Analyser LECO AMA-254. Quantification of O-Hg was performed through a chemical digestion described in [START_REF] Válega | Determination of Organic Mercury in Biota, Plants and Contaminated Sediments Using a Thermal Atomic Absorption Spectrometry Technique[END_REF]. Briefly, biological tissues were digested with a mixture of 18% KBr in 5% H2SO4, followed by extraction of organic mercury into toluene. The aqueous fraction resulting from the addition of a Na2S2O3 solution was then analyzed for mercury by thermal decomposition atomic absorption spectrometry with gold amalgamation (all reagents used were Hg-free, p.a. grade). The method does not differentiate different forms of mercury-Hg or other mercury-Hg compounds, such as dimethylHg or ethylHg, and all can be contained in the extract. However, organic forms are only present at very low levels at depth in the world ocean [START_REF] Hintelmann | Organomercurials. Their formation and pathways in the environment[END_REF]) so virtually all the OHg quantified here would be 100% MethylHg. Where there was low individual mass (less than 200 mg), samples for O-Hg analyses were obtained by combining multiple individuals of the same species of similar sizes collected from the same location. Analytical precision and accuracy were determined several (>4) times a day for the following certified reference materials: DORM-4 (n = 59, 96 ± 13%) fish protein and ERM-BB422 (n = 108, 100 ± 4%).

DORM-4 was used to certify O-Hg analyses, with an efficiency of 99 ± 8% (n = 24). All analyses were repeated 2-3 times until a relative standard deviation <10% was achieved.

Detection limits for thermal decomposition atomic absorption spectrometry is 0.01 ng of T-Hg and 0.004 µg g -1 for O-Hg. Hg concentrations are given as µg g -1 dry weight (dw).

Statistical analysis

Relationships between Hg concentration and myctophid standard length were examined by correlation. Shappiro-Wilk and Bartlett's test were used to test the normality and homogeneity of the data respectively. For the comparison of Hg in tissues (muscle, gills, heart and liver), Friedman tests were. To evaluate differences among years, locations and sexes Wilcoxon rank and Kruskal-Wallis tests were performed.

Results

Spatial and temporal variation in T-Hg concentrations in myctophids

T-Hg concentrations in the muscle of the 8 analysed myctophid species sampled during 2007/08 varied between 0.026 µg g -1 (in K. anderssoni) and 0.418 µg g -1 (in G. opisthopterus; Fig. 2; Table 1). In 2015/16, only 3 species were caught and T-Hg concentrations in the muscle ranged from 0.072 to 0.441 µg g -1 , with the highest and lowest values observed in E. antarctica (Fig. 2; Table 1). During 2016/17, T-Hg concentrations varied across the 6 species from 0.022 µg g -1 (K. anderssoni) to 0.424 µg g -1 (Gymnoscopelus fraseri; Fig. 2; Table 1). Of the sampled myctophids, only 3 species (E. antarctica, G. braueri and G. nicholsi) were caught repeatedly each year. There were no significant differences in muscle T-Hg concentrations between years for G. braueri or G. nicholsi (Kruskal-Wallis test, H = 5.366, p = 0.068; Kruskal-Wallis test, H = 4.859, p = 0.0852, respectively) while significantly lower T-Hg concentrations were observed for E. antarctica in 2016/17 than in 2007/08 and 2015/16 (Kruskal-Wallis test, H = 13.81, p = 0.001; Table 1). There were statistically significant differences in gill T-Hg concentration between years in E. antarctica, G. braueri and G. nicholsi (Kruskal-Wallis test, H = 33.51, p < 0.001; H = 17.84, p < 0.001; H = 14.38, p < 0.001). By contrast, in Krefftichthys anderssoni and Protomyctophum bolini, which were caught both in 2007/08 and 2016/17, no significant differences in T-Hg muscle concentrations between years was evident (Mann-Whitney test, U = 43, p = 0.426; Mann-Whitney test, U = 66, p = 0.359, respectively).

To minimize the effect of temporal variation on the spatial analysis, comparisons were only performed within species between the 2 consecutive sampling years (2015/16 and 2016/17). During these surveys, only 3 species (E. antarctica, G. braueri and G. nicholsi) were caught consistently year on year at both South Georgia (north of the Southern boundary of the Antarctic Circumpolar Current Front; SACCF) and the South Orkneys Islands (south of the SACCF). There was no significant difference in T-Hg muscle concentrations in the two Gymnoscopelus species between the two locations (G. braueri, Kruskal-Wallis test, H = 5.366, p = 0.068; G. nicholsi, Kruskal-Wallis test, H = 4.859, p = 0.085). However, the concentrations of T-Hg in E. antarctica were significantly lower at the South Orkney Islands (0.126 ± 0.068 µg g -1 ) than at South Georgia (0.216 ± 0.082 µg g -1 ; Fig. 3).

Inter-specific variations in T-Hg concentrations in myctophids

Inter-specific variations in T-Hg concentrations in muscle were apparent for samples pooled by year and location. During 2008/09, the 3 species of Southern Ocean Gymnoscopelus (G. braueri, G. nicholsi and G. opisthopterus) and the two species of Electrona (E. antarctica and E. carlsbergi) had higher T-Hg concentrations than K. anderssoni and P. bolini (Kruskal-Wallis test, H = 48.66, p < 0.0001). However, the levels of T-Hg in Nannobrachium achirus were not significantly different from the other species caught during this time (Fig. 2; Table 1). No statistical differences were observed between T-Hg concentrations in any of the 3 fish species (E. antarctica, G. braueri and G. nicholsi) caught in 2015/16 (Kruskal-Wallis test, H = 4.432, p = 0.109). However, for fish caught in 2016/2017, E. antarctica, G. fraseri, G.

nicholsi each had higher T-Hg concentrations than K. anderssoni, whilst no significant interspecific differences were observed for either G. braueri and P. bolini (Kruskal-Wallis test, H = 28.7, p < 0.0001; Fig. 2; Table 1).

Gender-specific and ontogenetical variations in T-Hg concentrations

To increase the robustness of our analysis on gender-and size-related effects on T-Hg accumulation in myctophids, only samples with no significant differences across years were pooled for each species. Thus, samples were pooled across all years for every species, except E. antarctica, for which only samples from 2007/08 and 2015/16 were pooled.

No significant gender-related differences in T-Hg muscle concentrations were observed for any species other than G. nicholsi and K. anderssoni where, in both, males had higher T-Hg concentrations than females (Mann-Whitney test, U = 41, p = 0.005 and U = 56, p = 0.017, respectively), Size-(and hence age-) related patterns in T-Hg concentration were evaluated for species where there was a sufficient sample size (n > 5) across the full expected size ranges (Fig. 3). 3) and a decrease of T-Hg with size, was only found in females of E. antarctica.

Variations in T-Hg concentrations in the different tissues of Southern Ocean myctophids

Four different tissue types, muscle, gills, heart and liver, were analysed for T-Hg levels in each species, except K. anderssoni and P. bolini that were too small for us to achieve adequate extraction of these tissues (Table 1).

Overall, T-Hg concentrations varied between tissues, with different patterns of variation between species and years (Table 1). However, in the majority of the species analysed from 2007/08 (χ 2 = 92.74, p < 0.001) and 2015/16 (χ 2 = 135.8, p < 0.001) samples, the heart and liver had consistently higher T-Hg concentrations than the muscle and gills, which contained the lowest overall concentrations. In 2016/17 samples (χ 2 = 70.47, p < 0.001), gills had always lower concentration than muscle. Also, liver from G. fraseri was the only tissue to have an average T-Hg concentration greater than 1 µg g -1 (1.11 ± 0.644 µg g -1 ).

O-Hg concentrations in Southern Ocean myctophids

Concentrations of O-Hg in muscle of the analysed species ranged between 0.051 and 0.493 µg g -1 . There were no significant differences in O-Hg concentrations between species (Kruskal-Wallis test, H = 6.428, p = 0.491; Table 2). The overall percentage of muscle O-Hg relative to muscle T-Hg was consistently greater than 75%, and close to 100% in most species (Table 2), indicating that the T-Hg found in myctophid muscle is predominantly the organic form.

Discussion

Prior to this study, knowledge of Hg in Southern Ocean myctophids was quite limited, despite their major role in the Southern Ocean food webs. In this study, we identified the main intrinsic (e.g., sex, size) and extrinsic (e.g., year, sampling location) drivers that influence Hg levels in myctophids. Furthermore, our results showed that myctophids represent an important reservoir of bioavailable Hg across the Southern Ocean.

Spatial and temporal trends in muscle T-Hg concentration

In this study we examined, for the first time, short-term changes in T-Hg concentrations in the biomass-dominant myctophid community at South Georgia and the South Orkney Islands.

We found no differences in T-Hg concentrations in muscle between sampling years for most species, although the high standard deviation may have masked the decreasing trend shown in those species that were caught in all sampling years. The high SD arose possibly due to the low sample size in some species that was in part due to logistical constraints for sampling in remote environments like the Southern Ocean. However, Electrona antarctica had statistically lower concentrations in 2016/17 than in the other years (see Fig. 2). Furthermore, the significantly lower T-Hg concentrations in gills in 2016/17 than in 2007/08 indicates a possible decreasing trend of Hg bioavailability in the water. Such a decreasing pattern over time has been observed for squid from the same region at the same time scale ( [START_REF] Seco | Mercury levels in Southern Ocean squid: Variability over the last decade[END_REF]. The pattern of decline in T-Hg tissue concentrations in both squid and myctophids over this 10-year period suggests a decrease in the bioavailability of Hg around South Georgia in the last decade. However, due to the comparatively shorter life spans of squid than to myctophids (around 1 to 2 years in squid [START_REF] Arkhipkin | Diversity in growth and longevity in short-lived animals: squid of the suborder Oegopsina[END_REF][START_REF] Boyle | Cephalopods ecology and fisheries[END_REF][START_REF] Xavier | A review on the biodiversity, distribution and trophic role of cephalopods in the Arctic and Antarctic marine ecosystems under a changing ocean[END_REF] versus ~ 3 to 8 years in myctophids (Linkowski, 1987;[START_REF] Linkowski | Population biology of the myctophid fish Gymnoscopelus nicholsi (Gillber, 1911) from the western South Atlantic[END_REF][START_REF] Saunders | Age and growth of Brauer's lanternfish Gymnoscopelus braueriand rhombic lanternfish Krefftichthys anderssoni(Family Myctophidae) in the Scotia Sea, Southern Ocean[END_REF]), acute changes in environmental pollutants are more likely to be reflected in squid due to a greater turnover in individuals within their populations. In contrast, myctophids are likely to retain and integrate Hg contamination from the environment into their muscle over longer periods of exposure, making it more appropriate to look for patterns of T-Hg decrease in gill tissue as gills will more immediately reflect a difference in bioavailability of Hg in the water.

Generally, longer-lived animals take longer to reflect any alteration in habitat contaminant levels [START_REF] Fränzle | Complex bioindication and environmental stress assessment[END_REF].

Habitat use has a major effect on Hg accumulation, since longer exposure in more contaminated areas will result in higher concentrations of this element in tissues [START_REF] Desta | Habitat use and trophic position determine mercury concentration in the straight fin barb Barbus paludinosus, a small fish species in Lake Awassa, Ethiopia[END_REF][START_REF] Bourg | Effect of body length, trophic position and habitat use on mercury concentrations of sharks from contrasted ecosystems in the southwestern Indian Ocean[END_REF]. In this study, little evidence was found of regional variation in T-Hg concentrations from the 3 species that were caught concurrently at South Georgia and the South Orkneys (E. antarctica, G. braueri and G. nicholsi), with regional differences only apparent for E. antarctica. Electrona antarctica specimens from South Georgia had lower concentrations of T-Hg than those from the South Orkneys, but samples from the South Orkneys Islands were, on average, 20 mm smaller, suggesting that the observed spatial pattern could reflect differences due to body size (i.e., bioaccumulation level) rather than differences in environmental factors. Indeed, other studies have shown that body size is typically positively correlated with T-Hg concentration in fish [START_REF] Barghigiani | Size Related Mercury Accumulations in Edible Marine Species from an Area of the Northern Tyrrhenian Sea[END_REF][START_REF] Bosch | Mercury accumulation in Yellowfin tuna (Thunnus albacares) with regards to muscle type, muscle position and fish size[END_REF][START_REF] Somers | Adjusting mercury concentration for fish-size covariation: a multivariate alternative to bivariate regression[END_REF]; present study). However, G. braueri and G. nicholsi had similar T-Hg concentrations in both locations, regardless of body size, whilst an opposite trend was apparent for the pelagic euphausiid Antarctic krill (Euphausia superba), collected in the same surveys [START_REF] Seco | Spatial variability in total and organic mercury levels in Antarctic krill Euphausia superba across the Scotia Sea[END_REF]. In this last study, Antarctic krill collected around the South Orkney Islands had higher T-Hg concentrations than those caught at South Georgia, a pattern that was attribute to the presence of sea ice around the South Orkney Islands, as ice formation may act as a trap for contaminants precipitating from the atmosphere. Both G. braueri and G. nicholsi feed on Antarctic krill at South Georgia and the South Orkneys [G. braueri 10% and G. nicholsi 25% of Index of relative importance [START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF]], which suggests that the regional differences in Hg levels of this prey species should also be reflected in these myctophids if Hg accumulation by ingestion was the predominant pathway in the Scotia Sea [e.g. [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF][START_REF] Paiva | The Influence of Diet on Mercury Intake by Little Tern Chicks[END_REF]]. However, the intake of T-Hg from other shorter-lived prey, such as copepods, small euphausiids and amphipods, and the long-term incorporation of Hg in the myctophid tissues might mask this krill-myctophid interaction on small spatial and temporal scales, such as those in the study of [START_REF] Seco | Spatial variability in total and organic mercury levels in Antarctic krill Euphausia superba across the Scotia Sea[END_REF]. Myctophids migrate across the Scotia Sea [START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF] and variability in time across this spatially extensive habitat may also mask any regional difference in Hg bioavailability. Such a question deserves further investigation to clarify the importance of spatial variation of Hg in myctophids and the role of their different prey in Hg bioaccumulation.

At a global scale, the concentrations of T-Hg found in the literature for myctophids studied globally were consistent with those reported here (Table 3). The approximately uniform concentrations are unexpected given the large range of habitats and species sampled, each with different ecology, depth distribution, growth, and diet. It seems that myctophids, from a global perspective, have similar T-Hg concentrations, despite their ostensible differences in physiology, biology and ecology. Comparisons among these studies have to be done with caution, however, as methodological differences such as sample preservation, analytical approaches and sample size may invalidate direct comparisons between studies.

To the best of our knowledge, there are only two other studies on Hg in Southern Ocean myctophids [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]Cipro et al., 2018b), both of which are on samples from the sub-Antarctic Kerguelen Islands (Indian Ocean sector). E. antarctica from the Kerguelen Islands had T-Hg concentrations between 2 and 4 times lower (0.066 ± 0.015 µg g -1 ; (Cipro et al., 2018b)) than in all of our sampled years/locations, even though there was a high degree of overlap in sizes of fish analysed between studies. In contrast, the Gymnoscopelus species that occurred in our and previous studies, G. fraseri and G. nicholsi, had similar values for equivalent sized individuals [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]Cipro et al., 2018a). Although there are differences in diet and prey field between the two regions (South Georgia and Kerguelen), E. antarctica from Kerguelen Islands appear to feed more frequently on Thysanoessa macrura [START_REF] Clarke | DNA-based diet analysis of mesopelagic fish from the southern Kerguelen Axis[END_REF], while individuals from South Georgia feed mainly on Euphausia superba or Themisto gaudichaudii [START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF]. This difference in diet alone seems unlikely to explain the regional differences in T-Hg, as levels of mercury in T. macrura from Kerguelen Islands were similar to those in E. superba from the Scotia Sea (Cipro et al., 2018b;[START_REF] Seco | Spatial variability in total and organic mercury levels in Antarctic krill Euphausia superba across the Scotia Sea[END_REF]. Furthermore, species-specific estimates of Hg levels across the whole prey field of myctophids from both Kerguelen Islands and the Scotia Sea are unknown, and the role of these taxa in the transfer of Hg to myctophids and upper trophic levels remains unclear. A possible lower fraction of O-Hg in the prey species from the Kerguelen Islands compared to their counterparts in the Scotia Sea [START_REF] Seco | Spatial variability in total and organic mercury levels in Antarctic krill Euphausia superba across the Scotia Sea[END_REF] could lead to a lower rate of Hg accumulation in myctophids in this region, given the shorter turnover of MeHg in fish tissue (~400 days [START_REF] Downs | Mercuty in Precipitation and Its Relation to Bioaccumulation in Fish: A Literature Review[END_REF]), and the more limited trophic transfer potential of inorganic Hg compare to O-Hg. These diverse results reinforce the importance of species-specific analyses of Hg levels, when trying to understand spatial patterns in myctophid Hg accumulation patterns through regional predator-prey interactions.

Gender-based and ontogenetic patterns in T-Hg concentration in Southern Ocean myctophids

Physiological and biological factors, such as sex and size, are known to influence Hg concentration in fish [START_REF] Bastos | Sex-related mercury bioaccumulation in fish from the Madeira River, Amazon[END_REF][START_REF] Dang | Why mercury concentration increases with fish size? Biokinetic explanation[END_REF][START_REF] Gewurtz | Influence of fish size and sex on mercury/PCB concentration: Importance for fish consumption advisories[END_REF][START_REF] Bourg | Effect of body length, trophic position and habitat use on mercury concentrations of sharks from contrasted ecosystems in the southwestern Indian Ocean[END_REF]. When assessing the effect of body size in T-Hg concentration in the muscle of Southern Ocean myctophids, we observed a general positive trend of increasing T-Hg with increasing size, except for E. antarctica females, for which this relationship was negative. In other species, such as E. carlsbergi, G. fraseri, and G. opisthopterus, the correlations were not significant (see Fig. 3). The positive relationship between T-Hg and size in fish is well established [START_REF] Dang | Why mercury concentration increases with fish size? Biokinetic explanation[END_REF][START_REF] Gewurtz | Influence of fish size and sex on mercury/PCB concentration: Importance for fish consumption advisories[END_REF][START_REF] Somers | Adjusting mercury concentration for fish-size covariation: a multivariate alternative to bivariate regression[END_REF], with a tendency for increased Hg bioaccumulation with age, mainly in muscle tissue. Also, larger fish tend to feed on larger prey that usually bioaccumulates greater Hg concentrations [START_REF] Chouvelon | Species-and size-related patterns in stable isotopes and mercury concentrations in fish help refine marine ecosystem indicators and provide evidence for distinct management units for hake in the Northeast Atlantic[END_REF] which, coupled with the tendency for lower Hg excretion rates in larger fish, [START_REF] Trudel | Modeling the elimination of mercury by fish[END_REF] results in higher concentrations in larger (older) individuals. This is also true for our results, where T-Hg increases with size in males, however, females generally show a trend for lower T-Hg accumulation rates with size than males. Thus, females in some myctophid species may have a Hg excretion system that does not occur in males. Egg laying is a well-known Hg elimination mechanism for most oviparous animals [e.g., arthropods [START_REF] Bakker | Maternal transfer of trace elements in the Atlantic horseshoe crab (Limulus polyphemus)[END_REF][START_REF] Saxton | Maternal transfer of inorganic mercury and methylmercury in aquatic and terrestrial arthropods[END_REF], crustaceans [START_REF] Seco | Spatial variability in total and organic mercury levels in Antarctic krill Euphausia superba across the Scotia Sea[END_REF], amphibians [START_REF] Bergeron | Bioaccumulation and maternal transfer of mercury and selenium in amphibians[END_REF], fish [START_REF] Khadra | The fish or the egg: Maternal transfer and subcellular partitioning of mercury and selenium in Yellow Perch (Perca flavescens)[END_REF][START_REF] Sackett | Maternally transferred mercury in wild largemouth bass, Micropterus salmoides[END_REF] and in seabirds [START_REF] Brasso | Penguin eggshell membranes reflect homogeneity of mercury in the marine food web surrounding the Antarctic Peninsula[END_REF][START_REF] Pedro | Mercury accumulation in gentoo penguins Pygoscelis papua: spatial, temporal and sexual intraspecific variations[END_REF]], and this mechanism might explain the different bioaccumulation patterns between sexes.

Inter-specific variations in T-Hg concentrations in muscle of Southern Ocean myctophids

Species-specific traits like feeding ecology, vertical and horizontal distribution, metabolism or physiology play an important role in Hg accumulation. In the present study, however, size and sex seemed to more important drivers for Hg accumulation in myctophids, as the smallest species had the lowest Hg concentrations in muscle (K. anderssoni and P. bolini), whilst concentrations were broadly congruent within the larger species. The lack of an inter-specific signal may be due to overlap in the distribution and diet patterns in the studied community. Most of the analysed species feed upon the same zooplankton prey species, such as the copepods Metridia spp., Rhincalanus gigas, Pleuromamma robusta and Calanoides acutus and the euphausiid Thysanoessa spp. [START_REF] Lourenço | Life cycle, distribution and trophodynamics of the lanternfish Krefftichthys anderssoni (Lonnberg, 1905) in the Scotia Sea[END_REF][START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF], with the exception of E. antarctica that feeds mostly on E. superba and on the hyperiid amphipod T. gaudichaudii [START_REF] Saunders | Seasonal variation in the predatory impact of myctophids on zooplankton in the Scotia Sea (Southern Ocean)[END_REF]. Most species were found across the Scotia Sea, with all species co-occurring in the northern Scotia Sea region (see Fig. 1). The vertical distribution patterns were also broadly similar among most species with the greatest concentrations of fish occurring above 400 m, particular at night [START_REF] Collins | Latitudinal and bathymetric patterns in the distribution and abundance of mesopelagic fish in the Scotia[END_REF][START_REF] Saunders | Myctophid Fish (Family Myctophidae) Are Central Consumers in the Food Web of the Scotia Sea (Southern Ocean)[END_REF]2018). Specific details of metabolic and physiological characteristics of myctophids are still unknown but, due to the phylogenetic proximity of the analysed species, one would assume that there should not be significant differences on Hg accumulation or excretion mechanisms among these species.

Tissue allocation of Hg in the Southern Ocean myctophids

Significant differences in T-Hg levels were observed in muscle, heart, liver and gill tissues in all species examined. Heart and liver tissues consistently showed higher concentrations than muscle and gill tissues. Large variations in Hg concentrations in heart tissue was consistently observed between individuals, probably due to differences in blood volume inside the heart chambers. The presence of blood fluid in the heart would decrease the overall Hg content in this organ, as Hg in fish blood was between 3 and 15 times lower than muscle in different fish species [START_REF] Eilser | Compendium of trace metals and marine biota 2: Vertebrates[END_REF][START_REF] Hamada | On mercury and selenium in tuna fish tissues III. Mercury distribution in yellowfin tuna[END_REF][START_REF] Shultz | The distribution of total and organic mercury in seven tissues of the Pacific blue marlin, Makaira nigricans[END_REF].

The liver, as an organ responsible for detoxification and transformation of toxins [START_REF] Maršálek | The content of total mercury and methylmercury in common carp from selected Czech ponds[END_REF][START_REF] Yamashita | Total mercury and methylmercury levels in commercially important fishes in Japan[END_REF], was expected to have high concentrations of T-Hg. Indeed, higher T-Hg levels occur in the liver of fish than in muscle tissue (liver/muscle index), such that a high liver: muscle contamination ratio is regarded to be an appropriate bioindicator for highly contaminated habitats [START_REF] Evans | Trace element concentrations in fish livers: implications of variations with fish size in pollution monitoring[END_REF][START_REF] Gonzalez | Comparative Effects of Dietary Methylmercury on Gene Expression in Liver, Skeletal Muscle, and Brain of the Zebrafish (Danio rerio)[END_REF][START_REF] Havelková | Comparison of Mercury Distribution Between Liver and Muscle -A Biomonitoring of Fish from Lightly and Heavily Contaminated Localities[END_REF]. Although the Southern Ocean is thought of as a fairly pristine environment, it is already known that, due to the Hg atmospheric cycle and special conditions for Hg methylation processes, greater than expected O-Hg concentrations occur in the region [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. It is likely that this bioavailability of O-Hg can be reflected on the high liver/muscle index. Present results are in agreement with a previous study in the Southern Ocean on the bald rockcod Pagothenia borchgrevinki, which also showed Hg concentrations in the liver to be twice that in muscle [START_REF] Honda | Organ and Tissue Distribution of Heavy Metals, and Their Growth-related Changes in Antarctic Fish, Pagothenia borchgrevinki[END_REF].

The main uptake route of T-Hg in fish is through the diet, with only low percentages (~10%) of the whole body burden T-Hg originating from waterborne Hg absorbed by the gills (G. R. [START_REF] Phillips | The Relative Contributions of Methylmercury from Food or Water to Rainbow Trout (Salmo gairdneri) in a Controlled Laboratory Environment[END_REF]. Nevertheless, muscle comprises the majority of individual biomass and therefore is the tissue with the highest ecological relevance, as it will be the carrier for most Hg to the next trophic level. Myctophids are an important prey to several Southern Ocean predators [START_REF] Sabourenkov | Myctophids in the diet of Antarctic predators[END_REF] and are also the most abundant mesopelagic fish family in the Southern Ocean (70-200 Mt) [START_REF] Catul | A review on mesopelagic fishes belonging to family Myctophidae[END_REF]. Assuming the conservative lowest muscle concentration found in the present study, for all species, and the lowest biomass estimation, this would mean that  1.82t of Hg are potentially bioavailable to Southern Ocean predators, in this family of mesopelagic fish alone. It is therefore likely that myctophids may be viewed as "Hg light bulbs" in the Southern Ocean, given their significant role in Hg bioaccumulation and trophic transfer processes in this remote environment.

Conclusions

Our study indicates a decreasing trend in Hg levels in myctophids over the last decade, especially in the gills, suggesting a decreasing bioavailability of this element in the water over this period. Further monitoring is required to confirm this pattern. There was little evidence of any regional variability in Hg contamination in the Southern Ocean, and indeed myctophid species appeared to have the same Hg range globally. Hg concentration generally increased with fish body size, which is a proxy for age, with the exception of E. antarctica females, that had a decreasing pattern, most likely a result of Hg elimination through egg laying. Higher concentrations of Hg were found in the liver and heart followed by muscle and gills. At a species level, Hg accumulation is mainly driven by species size, gender and developmental stage, as well as temporal variation in Hg availability in the environment.

  Myctophids were caught on three multidisciplinary research cruises aboard the RRS James Clark Ross around South Georgia (north of the Southern boundary of the Antarctic Circumpolar Current Front [SACC ]) and the South Orkney Islands (south of the SACC) during austral summer between 2007/08 and 2016/17 (Figure 1). The surveys around South Georgia were undertaken between December 2007 and February 2008 and December 2016 and January 2017, whilst the survey around the South Orkneys islands was undertaken between December 2015 and January 2016.

  Three patterns were noted between the different species/sexes: 1) No influence of size in T-Hg concentrations [in females of G. braueri, in males of G. braueri, G. nicholsi, K. anderssoni and juveniles of P. bolini]; 2) an increase of concentration of T-Hg with size [in males of G. nicholsi, K anderssoni and P. bolini, E. antarctica and P. bolini and in juveniles of G. braueri];

Figure 1 .

 1 Figure 1. Sampling sites and distributions of species captured around the Scotia Sea across all sampling years. SAF -Sub Antarctic Front; SACCF -Southern boundary of the Antarctic Circumpolar Current Front.

Figure 2 .

 2 Figure 2. Total mercury concentrations (Mean ± 1SD, µg g -1 dw) in the muscles of Southern Ocean myctophids, collected in the Scotia Sea in the austral summers of 2007/08, 2015/16 and 2016/17.

  Total mercury concentrations (Mean ± 1SD, µg g -1 dw) versus standard length (mm) in the muscles of Southern Ocean myctophids and significant regression lines, collected in the Scotia Sea in the austral summers of 2007/08, 2015/16 and 2016/17.

	2007/08 2015/16 2016/17 s t G . o p i s t h o p t t . E a n a r c i c a h . G n i c o l s i u r l c E . a r s b e g i G . b r a e r i e r u . n r n K . a d e s s o i P . b o l i n i N a c h i r u s G . f r a s e r i Tissues 2007/08 n Standard Length Weight Muscle Gills Electrona antarctica 16 73 ± 16 46 -98 5.5 ± 3.4 1.1 -12.7 0.21 ± 0.07 0.09 -0.33 0.28 ± 0.05 0.19 -0.37 0.59 ± 0.16 Heart 0.35 -0.99 Electrona carlsbergi 15 75 ± 4 67 -81 6.0 ± 0.9 4.7 -7.7 0.15 ± 0.03 0.11 -0.18 0.31 ± 0.06 0.26 -0.43 0.57 ± 0.12 0.42 -0.80 Gymnoscopelus braueri 5 102 ± 40 63 -164 14 ± 16 2.0 -38.8 0.16 ± 0.03 0.11 -0.18 0.40 ± 0.12 0.24 -0.56 0.77 ± 0.28 0.61 -1.29 Gymnoscopelus nicholsi 5 137 ± 11 124 -153 28 ± 6.3 21 -36 0.29 ± 0.12 0.18 -0.47 0.25 ± 0.04 0.18 -0.29 0.50 ± 0.08 0.42 -0.62 Gymnoscopelus opisthopterus 7 131 ± 20 109 -164 24 ± 10 13 -39 0.23 ± 0.12 0.11 -0.34 0.14 ± 0.05 0.08 -0.23 0.52 ± 0.08 0.41 -0.62 Nannobrachium achirus 6 129 ± 11 111 -142 18 ± 5.4 11 -25 0.11 ± 0.03 0.07 -0.16 0.15 ± 0.041 0.10 -0.20 0.24 ± 0.05 0.19 -0.32 Krefftichthys anderssoni 10 38 ± 4 34 -47 1.0 ± 1.8 0.3 -6.0 0.04 ± 0.01 0.03 -0.04 n.a. n.a. Protomyctophum bolini 10 42 ± 8 31 -56 1.2 ± 0.7 0.5 -2.6 0.09 ± 0.03 0.07 -0.16 n.a. n.a. 2015/16 Electrona antarctica 36 80 ± 8 63 -96 6.9 ± 2.6 3.1 -14 0.22 ± 0.08 0.07 -0.44 0.22 ± 0.06 0.11 -0.37 0.49 ± 0.17 0.27 -0.91 Gymnoscopelus braueri 18 114 ± 9 94 -127 12 ± 3.3 5.7 -16 0.18 ± 0.03 0.13 -0.22 0.17 ± 0.07 0.09 -0.41 0.57 ± 0.11 0.43 -0.82 Gymnoscopelus nicholsi 9 142 ± 11 125 -154 30 ± 5.8 20 -39 0.17 ± 0.04 0.11 -0.24 0.16 ± 0.02 0.14 -0.19 0.31 ± 0.04 0.27 -0.36 2016/17 Electrona antarctica 15 61 ± 16 39 -83 3.3 ± 2.5 0.7 -7.7 0.13 ± 0.07 0.06 -0.30 0.07 ± 0.04 0.03 -0.15 0.19 ± 0.13 0.07 -0.48 Gymnoscopelus braueri 7 92 ± 34 57 -134 9.0 ± 9.2 1.1 -25 0.12 ± 0.06 0.08 -0.24 0.08 ± 0.03 0.05 -0.14 0.41 ± 0.43 0.15 -1.28 Gymnoscopelus fraseri 8 75 ± 10 56 -86 4.1 ± 1.7 1.5 -6.5 0.22 ± 0.11 0.12 -0.42 0.15 ± 0.06 0.10 -0.26 0.39 ± 0.36 0.12 -1.03 Gymnoscopelus nicholsi 5 132 ± 3 129 -137 21 ± 1.4 20 -23 0.15 ± 0.02 0.12 -0.18 0.10 ± 0.01 0.09 -0.11 0.29 ± 0.07 0.20 -0.38 Figure 3. Species Krefftichthys anderssoni 11 52 ± 12 40 -70 1.87 ± 1.35 0.7 -3.9 0.05 ± 0.04 0.02 -0.14 n.a. n.a.	Liver 0.36 ± 0.16 0.18 -0.80 0.29 ± 0.12 0.19 -0.56 0.51 ± 0.12 0.37 -0.62 0.59 ± 0.09 0.52 -0.73 0.42 ± 0.19 0.20 -0.69 0.65 ± 0.23 0.34 -0.94 n.a. n.a. 0.36 ± 0.14 0.09 -0.79 0.58 ± 0.16 0 .24 -0.94 0.33 ± 0.05 0.26 -0.38 0.15 ± 0.09 0.05 -0.33 0.50 ± 0.30 0.20 -0.99 1.11 ± 0.64 0.25 -2.11 0.39 ± 0.12 0.19 -0.48 n.a.	Friedman test X2 30.5 25.6 14.0 14.0 16.0 17.0 79.6 19.5 41.7 22.9 17.0 22.9 14.0	p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
	Protomyctophum bolini	17	43 ± 10 28 -57	1.24 ± 0.76 0.2 -2.6	0.11 ± 0.03 0.07 -0.20	n.a.	n.a.	n.a.

Table 1 .

 1 Number of analyzed individuals, standard length (mm), weight (g) and total mercury concentrations (Mean ± 1SD, min -max, µg g -1 dw) in different tissues of Southern Ocean myctophids collected in the Scotia sea in the austral summer of 2007/08, 2015/16 and 2016/17. n.a.: not analysed.

Table 2 .

 2 Total mercury (T-Hg, ± 1SD) and organic mercury (O-Hg, ± 1SD) concentrations (µg g -1 dw) and percentage (± 1SD ) of O-Hg in in the muscles of Southern Ocean myctophid, collected in the Scotia Sea in the austral summers of 2007/08, 2015/16 and 2016/17.

	Species	T-Hg	O-Hg	%O-Hg
	Electrona antartica	0.17 ± 0.07	0.141 ± 0.07	79 ± 9
	Electrona carlsbergi	0.12 ± 0.01	0.107 ± 0.02	88 ± 8
	Gymnoscopelus braueri	0.17 ± 0.06	0.114 ± 0.03	80 ± 11
	Gymnoscopelus nicholsi	0.33 ± 0.14	0.331 ± 0.18	95 ± 17
	Gymnoscopelus opisthopterus	0.12 ± 0.02	0.099 ± 0.02	79 ± 4
	Gymnoscopelus fraseri	0.16 ± 0.004	0.157 ± 0.03	97 ± 18
	Krefftichthys anderssoni	0.10 ± 0.04	0.096 ± 0.06	88 ± 13
	Protomyctophum bolini	0.13 ± 0.02	0.123 ± 0.02	96 ± 2

Table 3 .

 3 Standard length (SL; average or range; in mm), total mercury (T-Hg) average and/or range concentrations (µg g -1 dw), sampling location and year and preservation method of global myctophids from published data

	Species	SL	T-Hg	Range	Location	Year	Preservation	Reference
							method
							Formaldehyde /
	Benthosema glaciale	39 -53	0.11	-	Gulf Stream	1993	Ethanol	Martins et al. 2006
							Formaldehyde /
	Benthosema glaciale	39 -53	0.14	-	Gulf Stream	1952	Ethanol	Martins et al. 2006
							Formaldehyde /
	Benthosema glaciale	39 -53	0.15	-	Gulf Stream	1971	Ethanol	Martins et al. 2006
							Formaldehyde /
	Benthosema glaciale	39 -53	0.17	-	Gulf Stream	1976	Ethanol	Martins et al. 2006
							Formaldehyde /
	Benthosema glaciale	39 -53	0.2	-	Gulf Stream	1936	Ethanol	Martins et al. 2006
							Formaldehyde /
	Benthosema glaciale	39 -53	0.22	-	Gulf Stream	1963	Ethanol	Martins et al. 2006

Acknowledgments

Thanks go to the Masters, officers and crew of RSS James Clark Ross for the help provided during all the sampled years. We also thank Giulia Pompeo for her help with the mercury analysis. We acknowledge the Portuguese Foundation for the Science and Technology (FCT) for the PhD grant to JS (SRFH/PD/BD/113487). JPC is funded by CESAM (UID/AMB/50017/2019) and the Integrated Program of SR&TD 'Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate' (Centro-01-0145-FEDER-000018), co-funded by Centro 2020 program, Portugal 2020 and the European Regional Development Fund. The Institut Universitaire de France (IUF) is acknowledged for its support to PB as a Senior Member. We also thank MARE (MARE -UID/MAR/04292/2019) for the support to JX. Ecosystems Programme at the British Antarctic Survey supported GAT, GS, RAS and SF.


M Sandoval-Reyes

Monica M Eskander

Joao Pires
email: joao.m.pires@tecnico.ulisboa.pt

Bruno Lacarrière
email: bruno.lacarriere@mines-nantes.fr

Joao Pires

João M Pires

Carlos A Silva
email: carlos.santos.silva@tecnico.ulisboa.pt

Feasibility analysis of

Keywords: Combined heat, cooling and power, Distributed generation, Electricity, cooling and heating microgrid, University campus

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction and Background

Energy consumption is closely related to climate change issues, but also it is directly correlated with life quality of the population. To maintain a suitable life standard while reducing environmental impact, it is necessary to support energy efficiency technologies.

When the need of thermal and electrical energy is coupled, a feasible option is Combined Heat and Power (CHP), also known as cogeneration; this process allows the simultaneously generation of useful thermal and electrical energy from a single fuel or energy source -either fossil or non-fossil-.

According to CODE2 reports [START_REF]CODE2 -Cogeneration Observatory and Dissemination Europe[END_REF], CHP saves upwards 25% of the primary energy compared to separate production of both outputs. Nowadays [START_REF]Cogeneration and District Energy[END_REF], 10% of the electricity consumed worldwide is produced with CHP; however, the usage of this technology is mainly concentrated in countries with focused governmental policies such as Denmark, Finland, Russia, Germany and Italy.

With the addition of other technologies such as heat pumps or absorption cooling systems, it is possible to produce cooling from the heat and power generated at the CHP, and then the whole system receives the name of Combined Heat, Cooling and Power (CHCP), also known as trigeneration.

The economic viability for a CHP or CHCP in a specific building, industry or district, strongly depends on the heat and power demand, as well as the price of electricity and the fuel (nowadays, natural gas is the most spread fuel across CHP technology).

Due to the capital investment required for a CHP, to make it economically feasible, it is important to have the unit or plant operating as many hours as possible [START_REF]Combined heat and power for buildings. Selecting, installing and operating CHP in buildings -A guide for building services engineers[END_REF]. As a general rule, applications which have a simultaneous demand for heat and power for more than 5000 hours are good candidates for this technology.

One of the advantages of CHP and CHCP systems is that they contribute greatly to the development of Distributed Generation Microgrids, because while electrical energy can be delivered to consumers over very long distances, cogenerated heat must be used at close proximity of the production place [START_REF] Pagliarini | Modeling of a thermal energy storage system coupled with combined heat and power generation for the heating requirements of a University Campus[END_REF]. The capacity of CHCP systems ranges from less than 1 kW in domestic dwellings (microcogeneration) to more than 10MW in hospitals, airports [START_REF] Cardona | Energy saving in airports by trigeneration. Part I: Assessing economic and technical potential[END_REF] or university campus, and as much as 300MW to supply energy to an urban district [START_REF] Wu | Combined cooling, heating and power: A review[END_REF].

-CHP usage in Universities

Universities are good cases for CHCP implementation because they are equipped with heating and air-conditioning systems, lighting, electric equipment and energy requests from many facilities such as labs and sporting units, throughout the year.

In 2002, Wu and Wang [START_REF] Wu | Combined cooling, heating and power: A review[END_REF] made a literature review of CHCP system and they mentioned three specific university examples. As microsystem, the Shanghai Jiao Tong University with a gas-fired reciprocating engine of 12 kWe and a 10 kW adsorption chiller. As medium size system, the Chesapeake Building of the University of Maryland having a micro-turbine that generates 60kWe and a 65 kW single effect absorption chiller. As large-scale system, the University of Illinois in Chicago with a 20.2MWe CHCP plant coupled with two-stage absorption chillers and electrical centrifugal chillers.

In 2002 also, Oliveira et al. [START_REF] Oliveira | A combined heat and power system for buildings driven by solar energy and gas[END_REF] in the University of Porto, ran a numerical simulation and prototyped an absorption cooling unit running with a hybrid solar and gas turbine system; the latter also generates electricity while running. They achieved cooling capacities up to 5 kW and electrical output up to 1.5 kW and concluded that the system competitiveness is higher in hotter climates.

In 2010, Pagliarini et al. [START_REF] Pagliarini | Modeling of a thermal energy storage system coupled with combined heat and power generation for the heating requirements of a University Campus[END_REF] evaluated technically and economically a cogeneration facility coupled with a thermal energy storage system, using as case a buildings belonging to University of Parma Campus. The results showed the optimal heat storage volume and how does it affect the auxiliary boiler contribution (reducing CO2 emissions), the waste heat and the annual income.

In 2013, Bracco et al. [START_REF] Bracco | The University of Genoa smart polygeneration microgrid test-bed facility: The overall system, the technologies and the research challenges[END_REF] described the Savona Campus Smart Polygeneration Microgrid (SPM) in the University of Genoa, Italy. The system includes three CHP gas turbines with combined ratings of 160kW electrical and 284kW thermal outputs, an H2O-LiBr (Water -Lithium Bromide) absorption chiller that is used for cooling the student library during summer, among other technologies. Today, they have even implemented a mathematical model to optimally manage the SPM and reduce even more the CO2 emissions and primary energy consumption [START_REF] Bracco | A mathematical model for the optimal operation of the University of Genoa Smart Polygeneration Microgrid: Evaluation of technical, economic and environmental performance indicators[END_REF].

The list of cases where CHP or CHCP are installed in Universities is long [START_REF] Environmental | Combined Heat and Power. A guide to developing and Implementing Greenhouse Gas Reduction Programs[END_REF][START_REF]Combined heat and power for buildings. Selecting, installing and operating CHP in buildings -a guide for building services engineers[END_REF][START_REF]Combined heat and power (CHP) in Universities[END_REF] and includes among others the Massachusetts Institute of Technology, which has a CHP installed capacity of 20MWe; the Queens Medical Centre-University Hospital in Nottingham, UK with a 4.9MWe CHP; the University of Florida's Shandis Cancer Hospital with a 4.3MWe installed capacity; the Liverpool University, UK with 3.6MWe; the York University, UK with 1.03MWe (supplying 60% of its electricity demand and 20% of the heat); the Coventry University, UK with 600kWe as installed capacity; the Harvard University's Hotel that supplies from 10 to 15% of its electricity demand with a 75kWe CHP and the Bangor University, UK also with 75kWe installed capacity.

From the literature review, it is evident that there are two types of applications for CHCP in the Universities, and the classification depends on the scale of the system installed. The first group includes the CHCP systems with capacities up to 1MW, which are used to supply energy to small Campus or even individual buildings; the second group comprises capacities from 3.6 to 20MW, which provide service for the entire Campus.

-Portugal current status and legal framework

According to the CODE2 Report, in 2014, the CHP installed capacity was 1.3 GWe -around 14% of the electricity supplied in Portugal-, with applications embedded in major industrial activities where heat at suitable conditions is required [START_REF]D5.1 Final Cogeneration Roadmap non pilot Member State: Portugal[END_REF].

There are few applications in buildings and in district heating; regarding the latter, there is one in Parque das Nações, Lisbon, where the company ClimaEspaço has an installed capacity of 79 MW of cooling energy, 81 MW of heat and 5MW of electricity [START_REF] Climaespaço | A Origem de ClimaEspaço[END_REF].

Starting in 1996, aimed by EU policies regarding energy efficiency, Portugal established a transparent and stable legal framework for CHP, allowing CHP installed capacity to reach 1.45GWe in 2012, mostly based on natural gas and with an overall equivalent efficiency of 65%. This installed capacity share was achieved mainly among industries such as pulp and paper, textile, chemicals and refined products, which use the additional income of selling the surplus of power produced by CHP to the grid, to increase businesses' competitiveness, especially for the exporting industry. However, over the period 2010-2012 the Government made changes in existing legislation which impacted negatively on CHP promotion and development. Following the transposition of the Cogeneration Directive 2004/8/CE, the main change in the legal framework for CHP is the cogeneration facilities have to be excluded from the regulated electricity tariff after 20 years.

Then, since 2012, the CHP installed capacity in Portugal has decreased due to the financial economic crisis coupled with a less favorable regulation; even though, the latter still promotes the installation of cogeneration units with a regulated tariff.

Case Study and Methodology

The objective of this paper is to present a technical and economic feasibility analysis for the deployment of an electricity, cooling and heating microgrid developed for a University Campus located in Lisbon, Portugal.

As a case study, the IST -Alameda Campus was selected and the idea is to analyze if the use of a CHCP system is feasible under the climate, and regulatory and operational framework available.

The IST -Alameda Campus has a compound area of 107,137.00m2 and comprises of 26 buildings including classrooms, labs, multiple cafeterias with cooking installations, sport facilities and a day nursery. The campus serves more than 10,000 users every day (students, faculty members, researchers and general staff). It has a large demand of electricity, over 10 GWh per year, and a significant share of it is being use for heating and cooling services; while natural gas is used only for the campus pool and the heating system in two of the newest and largest buildings.

EnergyPLAN as a modelling tool

To model the case study, Energy Plan [START_REF] Energyplan | [END_REF] will be used. This is a bottom-up free modelling software that assists the design of energy planning strategies by running mid-term scenarios for the entire energy system, based on hourly basis, for a region (nation, municipality or district, building). The output allows to compare different scenarios technically and economically through energy balance of annual productions, fuel consumption, import and export of electricity and the total costs related to the system.

The structure of EnergyPLAN allows to consider the heat, cooling and electricity demand from individual households or districts. On the other hand, the supply could come from a centralized or distributed generation, including the industrial sector as a producer and consumer. Finally, different renewable energy sources, including CHCP, could be combined among them to comply with the energy system demand. Some of the outputs that are used as discrimination factors among different scenarios are the total energy required within the energy system, the amount of CO2 emitted, the annual costs and the amount of electricity imported or exported.

The software has been widely used in Denmark [START_REF] Mathiesen | 100% Renewable energy systems, climate mitigation and economic growth[END_REF] and UK [START_REF] Connolly | The first step towards a 100% renewable energy-system for Ireland[END_REF] for different academic and nonacademic projects.

For this work, the results will be presented in terms of Total Annual Cost and amount of CO2 emitted.

IST -Alameda Campus Reference Data

The total IST Alameda Campus energy demand and distribution profiles are as follows [START_REF]Data provided by the Department "Campus Sustentável[END_REF]. The data shown in Table 1 includes the energy required for all the activities inside the Campus throughout the year and it is important to highlight two aspects; first, that the electricity demand is bigger than the heat and cooling demand; and second, that 71.4% of the heat demand is already supplied with a Natural Gas Boiler, however, the cooling demand is completely supplied with electric units, which leaves room for improvement of the system through Absorption Cooling Units implementation.

The Absorption Cooling System uses heat, instead of mechanical energy, to provide cooling. Compared to mechanical chillers, this has lower COP (Coefficient of Performance), but it can be energized by lower-grade heat. On the other hand, there are two types of Absorption Cooling Systems, the Single-effect and the Double-effect; basically the difference is that the latter could reach up to 40% higher COP than the first one, but it also needs higher grade of thermal input (more pressurized steam) [START_REF]Use Low-Grade Waste Steam to Power Absorption Chillers -Energy Tips: STEAM[END_REF].

The pattern in Fig. 1 reflects the electricity demand distribution throughout the year in hourly basis.

From here, it is important to notice that the electricity profile increases up to 20% during winter, when more electric heat is required, and up to 15% during summer, when electric cooling needs reach their maximum. Also, there is a drop in electric demand during August and is basically due to the fact that the campus is closed. Same, but in lower scale, happens during Christmas, New Year and weekends. On the other hand, the information used for the model regarding energy market in Portugal is provided in the Table 2. [START_REF]Enerdata -Odyssee, Portugal -Energy efficiency report[END_REF] 295 grams CO2 / kWh Electricity Price [START_REF]Data provided by the Department "Campus Sustentável[END_REF] 0.137 € / kWh Natural Gas price [START_REF]Data provided by the Department "Campus Sustentável[END_REF] 26.66 € / GJ

General description of the different scenarios explored and the costs considered

Using EnergyPLAN as a tool, the different Scenarios that will be explored and compared are:

REFERENCE SCENARIO:

It considers that all the electricity is bought from the national electrical grid and reflects that all the cooling demand is provided through electrical cooling systems and the heat demand is covered by a mix of Natural Gas boilers and electric heaters (as shown in Table 1). Among all mentioned until now, the Scenario 2, which is the better from the last group, is selected (see following section for more detail) and then it is combined with PV (Photovoltaic) modules to construct the next scenarios.

SCENARIO 1 -100kW CHP, 3MW Boiler, 211kW Absorption Cooling

SCENARIO 4 -300kW PV, 100kW CHP, 3MW Boiler, 211kW Absorption Cooling System:

It considers the installation of a 300kW PV system inside the Campus.

SCENARIO 5 -544kW PV, 100kW CHP, 3MW Boiler, 211kW Absorption Cooling System:

It considers the installation of a 544kW PV system inside the Campus and in the neighboring buildings.

SCENARIO 6 -800kW PV, 100kW CHP, 3MW Boiler, 211kW Absorption Cooling System:

It considers the installation of an 800kW PV system inside the Campus and in the neighboring buildings.

Detailed description of the costs considered in each scenario and results

At the REFERENCE SCENARIO, the costs included are shown in Table 3. The results from the Reference Scenario, which will be useful to assess the feasibility of the other scenarios proposed, are shown in Table 4.

Table 4. EnergyPLAN output from the reference scenario.

Scenario Total Annual Cost [k€]

CO2 annual emissions [kt]

Electricity used from the national grid [GWh/y] REFERENCE 1,423.30 3.05 10.06

In SCENARIOS 1, 2 AND 3, the use of a Single effect H2O-LiBr (Water -Lithium Bromide) Absorption Cooling System is assumed. This systems was selected based on the annual cooling demand for the IST Alameda Campus, and as long as the cooling demand is fixed, the implementation of this Absorption Cooling System will be permanent throughout all the scenarios, allowing the substitution of the electric cooling system in the Reference scenario. The objective is to use heat energy and a small amount of electricity to produce cooling. The characteristics of Absorption System are displayed in Table 5 [START_REF]Midwest CHP Application Center, Combined Heat and Power (CHP) Resource Guide[END_REF]. The scenarios vary depending on the size of the CHP unit and the back-up Boiler. Table 6 shows the characteristics of each selection [START_REF]Catalog of CHP Technologies -Section 2. Technology Characterization -Reciprocating Internal Combustion Engines[END_REF]. The importance of the inclusion of a backup Boiler is to produce only heat whenever two situations arise; first, when it is not economically feasible to run the CHP and produce both electricity and heat; or second, when the installed capacity of the CHP is not enough to cover the heat demand.

The characteristics of the backup boiler are shown in Table 7 [START_REF]Energy Sources, Production Costs and Performance of Technologies for Power Generation, Heating and Transport (Unofficial Version)[END_REF]. As a result of running these three scenarios with the characteristics described above, the Table 8 was generated. It shows clearly that the best option in term of Total Annual Cost is the 2 nd Scenario using a 211 kW Single effect H2O-LiBr Absorption Cooling Unit coupled with a 633kW CHP and a 2MW Boiler; however, the CO2 emissions are higher than the Reference. This is mainly because electricity in Portugal has in general a share of renewable resources higher than 50%. In order to find out the amount of PV modules that could be installed at the IST Alameda Campus, the team performed a measurement of the area available at the roofs inside the Campus (4 th Scenario) and in the neighboring buildings (5 th and 6 th scenario). The latest two scenarios are conceived as an option to create an electricity, cooling and heating microgrid which extends further the University Campus. From Table 10, it is evident that as larger the PV system, the larger the savings and lowest the CO2 emissions, but then the limit is the available space to install the PV system.

Discussion and future work

This work intends to be a preliminary technical and economical assessment of an electricity, cooling and heating microgrid located in the Alameda Campus of the Instituto Superior Técnico. The different scenarios analyze the feasibility of using a CHCP system and PV modules in different scales.

Evaluating the Total Annual Cost and the CO2 emissions results, it can be concluded that the optimal combination is the 6 th scenario with a 211 kW Single effect H2O-LiBr Absorption Cooling Unit coupled with a 633kW CHP, 2MW Boiler and 800kW PV.

Still, there are some further interesting discussions regarding this case study.

First, if the 2 nd scenario is analyzed carefully, it is evident that the usage of the CHCP is economically feasible; this is because the electricity demand is bigger than the thermal demand, which allows to use the CHCP at its maximum capacity supplying the heat demand as much as possible and on the other hand, the electricity produced is used in situ.

Second, the CO2 emissions in the 1 st , 2 nd and 3 rd scenarios, when using the CHCP system, increases compared with the Reference scenario. This is because the share of renewables in the Portuguese electricity production system is around 50%, which makes it relatively clean electricity.

Third, in a case study like this, which has a large electricity only demand, the installation of PV helps to reduce the Total Annual Cost and CO2 emissions, but the constrain factor is the space to install the system.

Fourth, scenarios 5 th and 6 th are considering the integration of neighboring areas to install PV panels, which in fact could lead to the creation an electricity, cooling and heating microgrid which extends further the University Campus to the neighboring buildings.

As future work, two tasks are evident. For the energy system suggested inside the University Campus, a proper lay-out and management strategy must be assessed. On the other hand, regarding the creation of an electricity, cooling and heating microgrid which extends further the University Campus to neighboring buildings, a careful feasibility analysis must be done.

Fig. 1 .

 1 Fig. 1. Electricity demand distributions through the year for the IST Alameda campus in hourly basis.

Fig. 2

 2 Fig.2 and 3show the heat and cooling demand patterns throughout the year in hourly basis. These profiles are representative of the Atlantic climate and characterizes Lisbon; with large heat demands from November to February, large cooling demand from June to August, and a mixed heat and cooling demand in mid-seasons (from March to May and from September to October).

Fig. 2 .

 2 Fig. 2. Heat demand distributions through the year for the IST Alameda campus in hourly basis.

Fig. 3 .

 3 Fig. 3. Cooling demand distributions through the year for the IST Alameda campus in hourly basis.

  System: It considers the full substitution of electrical cooling systems by absorption cooling systems, as well as the installation of a 100kW CHP and a 3MW back-up Boiler.SCENARIO 2 -633kW CHP, 2MW Boiler, 211kW Absorption Cooling System:It considers the full substitution of electrical cooling systems by absorption cooling systems, as well as the installation of a 633kW CHP and a 2MW back-up Boiler.SCENARIO 3 -1,121kW CHP, 2MW Boiler, 211kW Absorption Cooling System:It considers the full substitution of electrical cooling systems by absorption cooling systems, as well as the installation of a 1,121kW CHP and a 2MW back-up Boiler.

Table 1 .

 1 IST Alameda campus total energy demand.

		Installed Capacity* [kW] Consumption [GWh/y]
	Electricity	1,673.50	8.82
	Electric Heaters	30.36	0.16
	Heaters through NG Boilers	75.89	0.40
	Electric Coolers	204. 92 kW ≈ 58.27 RT	1.08 GWh/y ≈ 307,079.90 RT-h/y
	*Considering 24hr/day, 366days/year and a 0.6 plant usage factor.

Table 2 .

 2 Other costs included at the model.

	CO2 emissions price [20]

Table 3 .

 3 Equipment cost considered at the reference scenario.

	Period [years]	Operation & [% of investment] Maintenance		Total Investment cost [k€]
	Electrical Chillers	25	5	126,26
	Electrical Heat Pumps	25	5	2.85
	MCC system	25	5	15.18
	Boiler [22, p. 20]	25	1	0.135/MWe* 76kW = 10.3 k€

*The total investment costs include equipment, transportation and installations costs.

Table 5 .

 5 Characteristics of the single effect H2O-LiBr absorption cooling system selected.

	COP

* US GDP Deflator data and currency exchange rate (1 US Dollar = 1.09 Euro) was used to adjust the prices.

Table 6 .

 6 Characteristics of CHP systems selected for scenarios 1, 2 and 3.

		SCENARIO 1	SCENARIO 2	SCENARIO 3
		100kW CHP, 3MW	633kW CHP, 2MW	1,121kW CHP, 2MW
		Boiler, 211kW	Boiler, 211kW	Boiler, 211kW
		Absorption Cooling	Absorption Cooling	Absorption Cooling
		System	System	System
	Base load
	Electric	100	633	1,121
	Capacity [kW]
	Electrical efficiency [%]	27.0%	34.5%	36.8%
	Total Heat recovered [kW]	196	815	1,266
	Thermal efficiency [%]	53.0%	44.4%	41.6%
	Installation Cost* [€/kW]	2,660.54	2,602.74	2,170.64
	O&M Cost [%
	of installation	1.69 %	1.69 %	1.69 %
	cost]
	Back-up Boiler capacity [MW]	3	2	2
	* US GDP deflator data and currency exchange rate (1 US dollar = 1.09 euro) was used to adjust the prices.

Table 7 .

 7 Back-up boiler characteristics and prices.

	Installed Costs per RT*	135 € / kWth
	O&M Costs*	1% of the installation costs
	Life time cycle	25 years

Table 8 .

 8 EnergyPLAN output from the reference, 1 st , 2 nd and 3 rd scenarios. the 2 nd scenario is taken and adjusted by adding PV modules to generate electric energy. The proposal is to use as base a 300W PV module made of Monocrystalline Silicon and an area of 2m 2 . The objective is to make different arrangements of it to reach diverse PV installed capacity at the different scenarios.

	Scenario	Total Annual Cost [k€]	CO2 annual emissions [kt]	Electricity used from the national grid [GWh/y]
	REFERENCE	1,423.30	3.05	10.06
	SCENARIO 1: 100kW CHP, 3MW
	Boiler, 211kW Absorption Cooling	1,323.80	3.15	8.61
	System
	SCENARIO 2: 633kW CHP, 2MW
	Boiler, 211kW Absorption Cooling	1,298.50	3.16	7.66
	System
	SCENARIO 3: 1,121kW CHP, 2MW
	Boiler, 211kW Absorption Cooling	1,304.00	3.16	7.27
	System
	In SCENARIOS 4, 5 AND 6,

Table 9 .

 9 PV system characteristics and prices.

	Installed Costs per W [25-26]	3.34 € / W
	O&M Costs	2% of the installation costs
	Life time cycle	25 years

Table 10 .

 10 EnergyPLAN output from the reference, 2 nd , 4 th , 5 th and 6 th scenarios.

	Scenario	Total Annual Cost [k€]	CO2 annual emissions [kt]	Electricity used from the national grid [GWh/y]
	REFERENCE	1,423.30	3.05	10.06
	SCENARIO 2: 633kW CHP, 2MW Boiler, 211kW Absorption Cooling System	1,298.50	3.16	7.66
	SCENARIO 4 -300kW PV, 100kW CHP, 3MW Boiler, 211kW Absorption Cooling System	1,285.35	3.01	7.14
	SCENARIO 5 -544kW PV, 100kW CHP, 3MW Boiler, 211kW Absorption Cooling System	1,276.20	2.88	6.73
	SCENARIO 4 -800kW PV, 100kW CHP,
	3MW Boiler, 211kW Absorption Cooling	1,270.29	2.76	6.33
	System

Acknowledgments

The authors of this work thank Rui Pereira and João Patricio from the Project "Campus Sustentável" at the Instituto Superior Técnico, as well as, Diana Neves, from IN+, Instituto Superior Técnico, Universidade de Lisboa; for their help to get the data used in this model.

H2O -Water

LiBr -Lithium Bromide NG -Natural Gas O&M -Operation and Maintenance PRE -Special Regime Production


Vincent Rafik
email: vincent.rafik@insa-toulouse.fr

Alain Daidié

Bertrand Combes

Clément Chirol

Simplified Analysis of the Early Stage Self-loosening of a Shearloaded Bolted Joint

Keywords: self-loosening, bolted joints, shear-load, finite elements analysis

The self-loosening of a joint through unscrewing of the bolt is a phenomenon mainly occurring when the assembly is solicited by transverse repeated loads. Previous works highlighted that the transversal sliding of the bearing surfaces, either in the threads or underneath the bearing surfaces of bolts and/or nuts, is its root cause and that this phenomenon begins during the first loading cycles. In order to study the early stage of this self-loosening, a simplified numerical model has been developed. The latter factors the bearing surfaces of the bolt, the preload, the friction coefficient, the amplitude of the shear-load and the fastener's material. Through measurements and interpretation of the results, the shearing of the fastener has been identified as the main deformation leading to the self-loosening of the assembly, while the bending of the fastener shank limits the self-loosening. Moreover, according to the values of preload and shear-load, the behaviors were identified, and an interpretation has been proposed.

I. Introduction

A. The self-loosening in the literature

A few scientists such as Reid, Hattori or Dinger [START_REF] Reid | Detailed modeling of bolted joints with slippage[END_REF][START_REF] Hattori | Loosening and Sliding Behaviour of Bolt-Nut Fastener under Transverse Loading[END_REF][START_REF] Dinger | Design of multi-bolted joints to prevent self-loosening failure[END_REF] highlighted that in some specific conditions, the selfloosening of the fasteners clamping the pieces of a structure together can be noticed. This phenomenon might be due to an excessive cycled shear-loading of the parts. In order to focus on this phenomena, several papers are studying the self-loosening of a single bolted joint to get rid of the additional phenomena implied by the bolting sequence in a multi bolted assembly [START_REF] Junker | New Criteria for Self-Loosening of Fasteners Under Vibration[END_REF][START_REF] Zhou | Anti-loosening performance of coatings on fasteners subjected to dynamic shear load[END_REF][START_REF] Zadoks | An investigation of the self-loosening behavior of bolts under transverse vibration[END_REF][START_REF] Jiang | A Study of Early Stage Self-loosening of Bolted Joints[END_REF].

Junker [START_REF] Junker | New Criteria for Self-Loosening of Fasteners Under Vibration[END_REF] is the first to have studied this phenomenon. Thanks to a specific test bench, he considered the sliding of the bearing surfaces of the fasteners and the bolt's bending as its root cause. Dinger and Zhou [START_REF] Dinger | Design of multi-bolted joints to prevent self-loosening failure[END_REF][START_REF] Zhou | Anti-loosening performance of coatings on fasteners subjected to dynamic shear load[END_REF] studied the influence of the bolt coating on the self-loosening of the joint. They noticed that the lower was the friction coefficient, the most severe was its self-loosening as it can be seen on the Fig. 1. Whichever the coating Zhou considered; the loss of normal strength could be observed. Indeed, the higher was the friction coefficient, the latter and the slower was the phenomena. Zadoks interested himself in the behavior of the sliding surfaces [START_REF] Zadoks | An investigation of the self-loosening behavior of bolts under transverse vibration[END_REF]. He factored the transverse sliding and the plate and bolt deformation in order to propose an explanation to the self-loosening. He described the phenomena thanks to ratios between the relative circumferential and transverse velocities, either in the threads or under the head. According to Jiang [START_REF] Jiang | A Study of Early Stage Self-loosening of Bolted Joints[END_REF], the loosening curves can be split into two parts, Fig. 2. Moreover, he stated that the early loosening "Stage I", is driving the phenomena as the first movement or deformation, breaking the bolt's steadiness, were the most important one.

B. Purpose of the study

Understanding which process implies the early self-loosening might help us to avoid the loss of preload due to the rotation of the fastener. To do so, a simplified numerical model of the contact between the fastener's bearing surfaces and the clamped part will be developed. Once this model has been validated thanks to specific tests, an analysis will be done in order to identify the process and more specifically the deformation, either shearing or bending, leading to the self-loosening of the assembly. Finally, an analytical approach will aim at proposing a definition of the loosening load, the effort implying the loss of preload.

II. The numerical analysis

A. Presentation of the model

We will study an assembly of three plates (Fig. 3.a) tightened together by a bolt, solicited transversally by a sinusoidal shaped load. Our model, developed on Abaqus (Fig. 3.b), only represents the head of the fastener, a part of its shank and its bearing plates. The bolt (diameter 9.52 mm, in red), is in titanium whereas the plate (with bore clearance of 30µm, in light grey) is in aluminum. The simulation is composed of 3 steps. The first one is an axial loading F0 to apply a preload on section Sb. A maximum preload of 28 000 N, chosen according to Airbus standards, corresponds to 60% of the yield strength for the Ti4Al6V. The second one is an axial torque C0 applied to the section Sb, to consider the reaction of the nut's threads on the bolt's threads due to the preload. The last one is the shear-loading of the assembly by a sinusoidal shear-load Ft(t) at a frequency of 5 Hz, applied to the cylinder Ct.

Two contacts with Coulomb friction are modeled (Fig. 3b):

• Contact ①: Between the aluminum coated bolt's head and the primer coated bearing surface of the plate, f = 0.06. • Contact ②: Between the aluminum coated bolt's rod and the uncoated hole of the plate, f = 0.08. The variation of three parameters will be tested. Their values are presented Table 1. The influence of the preload and shear-load will aim at identifying the process leading to the self-loosening whereas the Young modulus of the bolt will highlight which deformation implies the phenomena.

B.

The results

Influence of the shear-load

In a first step, the influence of the amplitude of the shear-load has been investigated. For each preload value, the loosening speed (i.e. the mean rotation of the bolt for one loading cycle, measured in steady state after a stabilization time) has been plotted vs the amplitude of the shear-load, Fig. 4. The curves can be split into several parts from A to E: • Part AB: The head of the fastener sticks to its bearing surface. Therefore, the bolt cannot rotate.

• Part BC: Beyond a certain shear-load, the head starts to slip on the plate. The loosening of the fastener can be noticed. Up to a preload of 15 000 N, this part is vertical, which correspond to the Coulomb theory. The corresponding loading will be qualified as "loosening load". As soon as the solicitation reaches it, the distance of the head's slipping will be the clearance as the bending is negligible. For higher preload, the shear-load necessary for the head to slip will imply the fastener bending. Then, the slipping distance will be lower as well as the loosening speed. • Part CD: For low preload, once the fastener slides, its shank will contact the plate hole. The sliding will be stopped and the loosening speed will be the same whatever the shear-load in the part CD. For higher preload, the fastener will bend more, the slipping distance and the loosening speed will be lower. But when the shearload increases, the contact between the shank and its hole gets closer to the head. So the slipping distance and the loosening speed increases with the shear-load. • Part DE: The shear-load is sufficiently high to cause the plastic deformation of the hole, causing its ovalization, then the possible sliding distance becomes larger and the loosening angle caused by the head slipping is higher.

2. Influence of the preload Then, the influence of the normal stress has been investigated. In order to do so, the preload and the torque applied during the first and second simulation steps has been changed a simulation from another. For each of them, the evolution of the loosening load, defined by points B on Fig. 4, has been plotted on the Fig. 5. The loosening load is proportional to the preload, which matches the Coulomb theory. This result shows that for this level of shear-load and for any preload considered, the plate's deformation is negligible as the fastener will slide on the plate for a specific load which could be calculated thanks to an analytical model with simple hypothesis.

Influence of the Young modulus

Bending and shearing of the shank are caused by the shear-loading of the fastener. In order to define which one implies the loss of preload, the influence of the fastener material has been investigated. The loosening speed for preload of 28 000 N and shear-load of 10 000 N for different Young modulus of the bolt has been plotted Figure 6. We observe that the higher the fastener's Young modulus is, the lower its bending deformation is, and the more severe its self-loosening. So we can conclude that the bending of the bolt prevents the self-loosening. This can be attributed to the contact of the bolt shank and its hole, that happens earlier when the shank bends, and which decreases the amplitude of the sliding of the head on the plate as seen before.

III. Conclusion

The self-loosening is a phenomenon characterized by a loss of preload within the joint coming from unscrewing. Its root cause is the sliding of the bolt's bearing surface caused by an excessive shear-load. It has been proven that the early stage of the phenomena is driving the subsequent rotation. In order to study the behavior of the bolt during the first cycles, a simplified numerical model including only a part of the bolt and a plate has been developed. Thanks to it, the influences of the preload, the amplitude of shear-load and the fastener material have been investigated. The results highlighted that whichever the preload and beyond a certain shear-load, the bolt will self-loosen, matching the Coulomb theory. However, for high value of preload, this simple theory is not sufficient as the bending of the bolt shank induces a contact of the shank on its hole, which reduces the sliding of the bolt head and the unscrewing. Finally, the shearing of the bolt has been identified as the main solicitation implying the selfloosening. Indeed, the higher is the bolt's bending rigidity, the more severe is its self-loosening.

Figure 1 :

 1 Figure 1:Loosening curve for different fastener coatings[START_REF] Zhou | Anti-loosening performance of coatings on fasteners subjected to dynamic shear load[END_REF]

Figure 2 .

 2 Figure2. The self-loosening sequence,[START_REF] Jiang | A Study of Early Stage Self-loosening of Bolted Joints[END_REF]

Figure 3 .

 3 Figure 3. Definition of the complete assembly (a) and the model (b)

Figure 4 .

 4 Figure 4. Comparison of loosening speed vs shear-load, for different preloads.

Figure 5 .

 5 Figure 5. Loosening load vs the preload.

Figure 6 .

 6 Figure 6. Influence of the Young Modulus on the loosening speed.

Table 1 .

 1 Definition of the parameters

	Parameters	Shear-load (N)	Preload (N)	Young modulus (GPa)
	Number of values	50	5	10
	Range of values	30 -25 000	500 -25 000 60 -250