Dragos G Zaharescu

Peter S Hooda
email: p.hooda@kingston.ac.uk

Carmen I Burghelea

Viktor Polyakov
email: viktor.polyakov@ars.usda.gov

Antonio Palanca-Soler

Climate change enhances the mobilization of naturally occurring metals in high altitude environments

Keywords: Climate Change, High Altitude, Trace Elements, Lakebed Sediment Record, Weathering and Transportation, Contaminant Risk

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Introduction

Crossing certain boundary of climate thresholds could have serious consequences for humanity, including major changes in the Earth's other systems, such as the water and geochemical cycles [START_REF] Rockström | A safe operating space for humanity[END_REF]. Similar to Polar Regions, mountain-top environments are generally more sensitive to climatic change and hence are likely to experience the consequential effects first. This sensitivity comes from stronger interactions between the major circulation patterns of the atmosphere and orography, resulting in greater changes in local environment, including cloud formation and precipitation, snow/ice level, surface moisture regime, soil erosion, heat transfer on the vertical and Foehn winds [START_REF] Geiger | The Climate Near the Ground[END_REF][START_REF] Beniston | Climatic change at high elevation sites: an overview[END_REF]) -all these directly can influence the biogeochemical cycles [START_REF] White | Effects of climate on chemical weathering in watersheds[END_REF][START_REF] Camarero | Application of MAGIC to Lake Redó ( Central Pyrenees ): an assessment of the effects of possible climate driven changes in atmospheric precipitation , base cation deposition , and weathering rates on lake water chemistry[END_REF][START_REF] Piao | The impacts of climate change on water resources and agriculture in China[END_REF]. This is supported by the rise in temperature observed in mountain regions in recent decades, which in some places is about five-fold greater than the global warming average [START_REF] Beniston | Climatic change at high elevation sites: an overview[END_REF]Diaz & Bradley, 1997;[START_REF] Nogues-Bravo | Exposure of global mountain systems to climate warming during the 21st Century[END_REF].

In high mountain catchments the biogeochemical cycling of trace elements (TEs) is generally governed by a weathering-limited regime [START_REF] Stallard | Geochemistry of the Amazon 2. The influence of geology and weathering environment on the dissolved load[END_REF]. The geogenic inputs of TEs to mountain lakes may however be enhanced by climate change driven denudation of metal/metalloid-bearing minerals and their subsequent transport and burial in the sediments. Increased mobilization of TEs can have adverse impacts on the wider environment, including water quality [START_REF] Savage | Arsenic speciation in pyrite and secondary weathering phases , Mother Lode Gold District , Tuolumne County , California[END_REF] and human health [START_REF] Charlet | Arsenic in shallow, reducing groundwaters in Southern Asia: an environmental health disaster[END_REF]. There is a considerable literature supporting increased accumulation of TEs and other contaminants in Polar and mountain regions, largely due to their long-range atmospheric transportation from anthropogenic activities [START_REF] Macdonald | Recent climate change in the Arctic and its impact on contaminant pathways and interpretation of temporal trend data[END_REF][START_REF] Outridge | Trace metal profiles in the varved sediment of an Arctic lake[END_REF][START_REF] Michelutti | Temporal trends of pollution Pb and other metals in east-central Baf fi n Island inferred from lake sediment geochemistry[END_REF][START_REF] Bing | Historical trends of anthropogenic metals in Eastern Tibetan Plateau as reconstructed from alpine lake sediments over the last century[END_REF]. Also, sediment core studies have suggested changes in land use and climate during the Holocene being responsible for erosion and geochemical changes seen in lake sediments [START_REF] Augustsson | Effects of land use and climate change on erosion intensity and sediment geochemistry at Lake Lehmilampi, Finland[END_REF]. Despite various attempts to understand the influence of climate change on mountain geochemical processes [START_REF] Psenner | Climate driven pH control of remote alpine lakes and effects of acid deposition[END_REF], its potential impact on TEs release and accumulation in mountain catchments remains a major question, partly due to the complex nature of the processes involved and strong year-toyear variability. Secondly, any direct approach is not likely to provide reliable information on how weathering and transport of weathered material may have changed through time and its coupling with climate change.

Lake sediments are one of the endpoints for TEs emitted from both natural and anthropogenic sources; they can provide an accurate archive of changes in the surrounding landscapes [START_REF] Yang | Geochemistry of Pyramid Lake sediments: influence of anthropogenic activities and climatic variations within the basin[END_REF], including geochemical cycling of TEs. Here we present data from a Pyrenean reservoir (Lake Bubal; Figure 1), and examine the effects of changing climate patterns on TEs accumulation in a sediment profile covering > 3 decades of historical record.

Methods

Hydrology and climate.

Reservoir lakes are generally good sentinels of climate change. They can reflect changes in the catchment environment over known periods of time since their construction, usually by erecting dams [START_REF] Williamson | Lakes and reservoirs as sentinels , integrators , and regulators of climate change[END_REF]. Our study was conducted in Bubal lake catchment (headwaters of the River Gallego), a postglacial valley in the Central Pyrenees Spain;Figure 1). This catchment, of about 305.5 km 2 , follows a north-south orientation and is characterised by a series of interconnected lakes and typical altitude streams that drain water from the surrounding mountain slopes. Some of the lakes were transformed into reservoirs in the 1950s-60s and the main river in the catchment (Gellego) was dammed in 1971 to form Lake Bubal at 1085 m a.s.l., with its catchment peaks extending up to 3058 m a.s.l. The lake has a surface area of 234 ha, a used capacity of 64 hm 3 and annual water output of 382 hm 3 [START_REF] Limnos | Diagnóstico y gestión ambiental de embalses en el ámbito de la cuenca hidrográfica del Ebro[END_REF]. At about 6 water renewals each year the lake experiences a relatively short water residence time and frequent water level oscillations. Average depth of the reservoir is 27 m, which is also the depth at the sampling location. The thermocline depth varies between 5-12 m. All such lakes in the region have been used for both, hydroelectricity and supplying water for agriculture and potable purposes in the valley and the region further downstream.

The long-term mean annual precipitation for the catchment is 1077 mm (averaged over 1972-2005). The dominant air mass direction is from W-NW bringing precipitations mainly from the Atlantic [START_REF] Dessens | A critical examination of the precipitation records at the Pic du Midi observatory, Pyrenees, France[END_REF]. Hence, the long-distance aerial transport of contaminants must be limited in this region. This is also supported by low atmospheric deposition of metals found in the central Pyrenees at the altitude of Bubal catchment, which is considered to be similar to European background levels [START_REF] Bacardit-Penarroya | Trace elements biogeochemistry in high mountain lake catchments: identifying anthropogenic versus natural components from the atmospheric contamination legacy in remote natural areas[END_REF]. Moreover, the bulk precipitation in the Central Pyrenees is more neutral than in other Central European sites (e.g. Alps) mainly due to lower acidic pollutant levels (SO x and NO x ) [START_REF] Camarero | Chemistry of bulk precipitation in the central and Eastern Pyrenees, Northeast Spain[END_REF]. There is also a reported decrease in acid depositions in the high Pyrenees in the last decades following the overall reductions in industrial emissions [START_REF] Mosello | Trends in the water chemistry of high altitude lakes in Europe[END_REF]. Therefore, the main potential source of TEs in core sediments is from the surrounding geology.

Geology.

The geology of Bubal catchment is dominated by Devonian deposits (limestone, sandstone and lutite) shaped by Permian and Carboniferous materials at west and Cretaceous limestone at south (Figure 1). This geology is marked by the extrusion of Cauterets-Panticosa granitic batholith at NE which generated a low-grade contact metamorphism aureole [START_REF] Subias | Hydrothermal events in the Valle de Tena ( Spanish Western Pyrenees ) as evidenced by fluid inclusions and trace-element distribution from fluorite deposits[END_REF]. This has significant presence of W-Au and F-Zn-Pb vein mineralisations formed in the Devonian limestone that contain fluorite (CaF 2 ), sphalerite, galena (PbS), pyrite (FeS 2 ), chalcopyrite (CuFeS 2 ), siderite (FeCO 3 ) and green and white fluorite [START_REF] Subias | Yacimientos Hidrotermales de Pb-Zn-F del Área de Sallent de Gállego -Panticosa, Pirineos Occidentales[END_REF]. Pyrite in mineral deposits has As concentrations reaching 250 ± 40 mg kg -1 , while the hosting rock As content is relatively smaller and more consistent (87.5 ± 0.5 mg kg -1 ) [START_REF] Subias | Yacimientos Hidrotermales de Pb-Zn-F del Área de Sallent de Gállego -Panticosa, Pirineos Occidentales[END_REF]. This suggests that pyritecontaining materials (e.g. shale, schist) are potential geological source of As and other metals. Geogenic inputs of TEs have been reported in the waters and sediments of this zone, particularly in the east side [START_REF] Garrido | The thermalisme of the batholite of Panticosa (Huesca): hydrochemistry characteristics and relationship with the hydrothermal system of Cauterrets-Panticosa granitic massif (Spain-France)[END_REF]Zaharescu et al., 2009b) (Figure 1).

Samples collection and profile characteristics.

The sampling strategy covered the sedimentary record from the major catchment depository: Lake Bubal, 1085 m a.s.l. (Figure 1 and S1a). A 50 cm sediment core was extracted from the lake in bathymetric depression toward the edges of the former river (42°41'49.97"N, 0°18'51.65"W; see Interactive Map File), where the interferences from sediment slumping or turbidity were minimal. The sampling was conducted in August 2005 when the north half of the lake bed was exposed during an unusually extreme drought. A pit was dugout in an area free of potential disturbances to extract the sediment core (Figure 1 and S1a). Therefore, no sediment compression/disturbance occurred during sampling. The cobles and forest soil at the bottom of the core clearly indicated the depth from where the new deposits were formed since 1971 when the dam was erected. The core was therefore expected to comprise a record from the year when this part of the river was transformed into reservoir.

The profile was brownish, indicative of oxic environment. Sediment organic content was low (3.39 ± 1.5 %, as loss-on-ignition). The profile, visually comprised of fine alluvial sediments (silt-fine sand), showed continuous record of sedimentation and no important evidence of remixing or bioturbation during the pit excavation and sampling. It is known that, in the absence of significant changes in core texture, the variation in TEs along the sediment profile can reflect the variation in their historical input [START_REF] Santschi | Accumulation of sediments, trace metals (lead, copper) and total hydrocarbons in Narragansett Bay, Rhode Island[END_REF][START_REF] Mecray | Historical trace metal accumulation in the sediments of an urbanized region of the lake Champlain watershed, Bulington, Vermont[END_REF].

The core was sectioned in the field using a wide stainless-steel spatula at 1.5 cm intervals. This is consistent with the averaged sediment deposition rate of 1.5 cm year -1 for the core extracted from upper Lake Respomuso [START_REF] Lavilla | Depth profile of trace elements in a sediment core of a high-altitude lake deposit at the Pyrenees, Spain[END_REF] (Figure 1), and also with the relatively low depositional rates reported for other Pyrenean reservoirs [START_REF] Valero-Garces | Sediment sources and siltation in mountain reservoirs : a case study from the Central Spanish Pyrenees[END_REF]. It also corresponded to the sedimentation laminae along the profile, likely produced during spring thaw, which implies sediments were not disturbed by mixing. Furthermore, sediment metal content from the present study was compared to surface sediment data collected throughout the catchment (Zaharescu et al., 2009a andZaharescu et al., 2009b).

Climate data were provided by the Spanish National Institute of Meteorology, Madrid (INM) and covered the period 1972-2005.

Particle size distribution.

Sediment samples were estimated for sand, silt and clay contents by sedimentation of a homogeneous suspension following a modification of the hydrometer method for soils [START_REF] Bouyoucos | Hydrometer method improved for making particle size analysis of soils[END_REF]. About 3 g air dried sample was suspended in 45 ml dispersant solution (5% Na-hexametaphosphate in deionised water) and shaken to separate the aggregates. Fractions (pellet) of sand and silt were collected after 40" and 6h 25' of settling, respectively, and the clay fraction after 15 min centrifugation at 3000 rpm (1006 G-force). Sediment pellets were then oven dried at 50 ⁰C for 24h and weighted. Results were expressed in % dry weight. Particle size distribution with depth showed a natural variability in sediment transportation and deposition with silt and clay fractions varying in step with the metals investigated (Figure S1).

Radionuclide dating.

The samples were oven dried at 40 ⁰C for 2 days, gently dispersed using ceramic pestle and mortar, sieved through 0.25 mm mesh size and stored in sealed plastic bags. Radioactive dating of 137 Cs and 210 Pb followed the method described in [START_REF] Walling | Conversion Models for Use in Soil-Erosion, Soil-Redistribution and Sedimentation Investigations[END_REF]. The unsupported 210 Pb activity vs. depth followed an exponential curve, meaning the core has little or no post-depositional disturbance(Figure S1). A peak expected for the 1986 Chernobyl incident in the stratigraphic record of 137 Cs fallout was relatively low. Nonetheless relatively low 137 Cs variability with depth together with 210 Pb profile support the idea that sediment supply rates to this site, although varied in granulometry, was largely constant and thus, the subsampling procedure was appropriate. The observed variability in radionuclide distribution and particle size is expected for such mountain system where climate variability such as winter catchment freezing, and other hydrological processes can slightly modify the otherwise constant input into the lake [START_REF] Appleby | Radiometric dating of sediment records in European mountain lakes[END_REF][START_REF] Kotarba | Pb dating of young holocene sediments in high-mountains lakes of the Tatra mountains[END_REF].

Analysis of trace and major elements, and organic matter.

For TEs analysis, the < 0.25 mm homogenised sediment of each subsampled layer (n = 33) was subjected to semitotal digestion following USEPA Method 3050B for inductively coupled plasma mass spectroscopy, ICP-MS (USEPA, 1999), to extract the biologically-relevant element fraction. As, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn were determined by ICP-MS, using standard operating conditions and following standard QA/QC protocols. This entailed replicated certified reference materials (standard reference sediments SRM 2704, GSD-1 and GSD-4), and reagent blanks, to estimate the accuracy of the analytical method. The TEs analysis in terms of precision and accuracy was highly reliable, except for Cd. Cadmium due to its very low level had a high relative standard deviation (~25 %), which was considered unsatisfactory and removed from further analysis (see Supplementary Information).

Major minerogenic elements Ti, Fe, Al and Li were characterised following USEPA Method 3051A for ICP-OES (USEPA, 2007). A certified reference material from the National Water Research Institute of Canada (CRM WQB3), procedural blank and three samples were analysed in triplicate to optimize the accuracy, reproducibility and recovery rate of the extraction procedure, which were found consistent with the method used (see Supplementary Information).

Sediment organic matter content was estimated gravimetrically as loss on ignition at 550 ⁰C for 4 h [START_REF] Rowell | Soil science. Methods and Applications[END_REF].

Data analysis.

First, a stepwise multiple linear regression model was fit to trace and major elements data (log-transformed) to determine the geochemical origin of TEs. Given the high correlation coefficients between TEs (Spearman rank ρ > 0.5, p < 0.01), and to simplify inter-elemental natural variability in relation to climate change they were summarised as regression factor scores by principal component analysis (PCA). This combined score variable is expected to offer a general response of sediment core metal content to climate variables.

Data representing average monthly temperature and precipitations at Bubal included: maximum and mean temperature, total precipitation (mm), and the frequency of days with rain, snow, storm and freezing days. The climate was reconstructed from the monthly means of variables at Escarilla reference station (1170 m a.s.l.; Figure 1 and (Agustí-Panareda, [START_REF] Agustí-Panareda | Reconstructing temperature variations at high elevation lake sites in Europe during the instrumental period[END_REF]. The freezing level was estimated from the mean monthly air temperature at a standard adiabatic lapse rate of 0.65 ⁰C 100 m -1 [START_REF] Thies | Unexpected Response of High Alpine Lake Waters to Climate Warming[END_REF].

In the few cases where temperature values were missing in reference (Escarilla) dataset, the gaps were completed with data from the nearest neighbouring station after previously correcting for the monthly mean differences between the nearest (previous and subsequent) common years [START_REF] Rosenblüth | Recent temperature variations in southern South America[END_REF][START_REF] Peterson | Homogeneity adjustments of in situ atmospheric climate data: A review[END_REF]. Except for 6 cases (i.e. months, randomly distributed) for which the average temperature needed to be transferred from a lower station (60 km south); the stations used for correction were all in the valley, within 7 km of the main station. The accuracy of the method was tested using various approaches. First, a Pearson product moment correlation of the instrumental data between stations showed coefficients > 0.8 at above 99.9 % significance level. Subsequently, cross-validation test [START_REF] Michaelson | Cross-validation in statistical climate forecast models[END_REF] found reasonably low errors, considering the mountain topography, of < 0.63 ⁰C month -1 between the obtained values and the instrumental records. Furthermore, the interpolated and the measured values were highly correlated (r 2 > 0.97, p < 0.01). This indicated that the method used was reliable. Precipitation variables (amount and frequency) did not require transformation as the station used for correction was within 1 km distance from the reference station. These calculations were performed in PASW 18 (former SPSS) for Windows.

To filter out inter-annual variability, and therefore increase the interpretability of long term behaviour of climatic factors, the variables were smoothed along time using locally estimated scatterplot smoothing (LOESS), which uses a squared polynomial function. A Gaussian smoothing parameter (α) of 1 was used to infer major modifications in temperature and precipitation patterns between the beginning and the end of the record period. A Gaussian filter (α = 0.1) helped to examine the variability of TEs in step with climatic variables. The significance of the long term annual and seasonal trends in climate was tested by the nonparametric Mann-Kendall test.

To unveil the multivariate association between sediment TE contents and predictor climatic variables, we used principal component analysis (PCA). For this, the TEs (As, Co, Cr, Cu, Mn, Ni, Pb and Zn) were summarised as standardised (centred to mean = 0, standard deviation = 1) PCA regression factor scores before using them as response variable in the model. Annual averages/sums of monthly values of climate variables were also standardised prior to using them in PCA.

Temporal variability of the TEs-climate relationships was quantified by crosscorrelation. Since autocorrelation within series prevents significance testing, a permutation test helped infer the null distribution of 95 % of the cross-correlation values under the expectation of no correlation between any two variables of interest [START_REF] Blois | Small mammal diversity loss in response to late-Pleistocene climatic change[END_REF]. This was achieved by holding one variable constant, randomly rearranging the values in the second variable and recalculating the correlation coefficient. We repeated the process 1,000 times and calculated the upper and lower 2.5 % cut-off values for the confidence interval. Coefficients outside the confidence interval were considered significant. Strongest correlations between climate and TEs were r = -0.650 and r = 0.603 at lag = 4 for snow days and spring freezing level, respectively (null intervals -0.320 to 0.334, and -0.331 to 0.309); r = -0.623 at lag = 0 for rain days (null intervals -0.347 to 0.358) and, r = 0.578 and r = -0.599 at lag = 1 for freezing days and storm days, respectively (null intervals -0.338 to 0.320, and -0.347 to 0.343).

Variance partitioning using adjusted r 2 of linear regression [START_REF] Legendre | Numerical ecology[END_REF] helped distinguish the contribution of different sets of variables to the temporal variation of TEs while accounting for lag responses. The variables used for this analysis were standardised. Three sets of explanatory variables potentially important for metal mobilization-climate change relationship were used: (A) spring freezing level and snow days, (B) rain days and record period, and (C) freezing days and storm days. Variables within sets were also similar in their lag effect. The significance of variation portions was tested by redundancy analysis with a 200 permutations test [START_REF] Legendre | Numerical ecology[END_REF]. The contribution of each set of variables was significant at p < 0.01. Both, cross-correlation and variance partitioning were performed using R statistical language (version 2.10.0).

Finally, a conceptual diagram integrating the major results was modelled in ArcInfo 9.3 package for Windows.

Results and discussion

3.1 Trace element source. The sediment core was primarily composed of mobilised terrestrial inorganic detritus, brought into the lake during thaw and rain events. The stepwise multiple linear regression significantly related As, Co, Cr, Cu, Mn, Ni, Pb and Zn with major lithogenic constituents Ti, Fe, Al and/or Li (Figure 2). The metal distribution profiles in the sediment core over time show similar pattern (Figure 3a). This similarity together with TEs relationship with lithogenics clearly demonstrates a common origin, as well as a common mechanism of metal mobilisation i.e. via erosion and burial. This is also supported by a strong relationship between TEs and silt and clay size fractions along the core (Pearson r = 0.34 to 0.9 (p < 0.05), with finer particles inherently hosting more TEs due to a larger surface area. Redox processes in lake' environment can potentially redistribute metals in bottom sediment, influencing their distribution along the sediment profile. Profile distribution of Fe/Mn-two redox sensitive elements, showed no major shift or trend (Figure S1 e-f). This is reflective of natural Fe-Mn oxide inputs, which often host TEs-hence some connection to TEs is expected. Given the high rate of inflow-outflow of water in/from Bubal reservoir, a low nutrient content (UTE RBE, 2009), largely ice-free winter and low measured organic matter content in its sediment, it is not surprising that no evidence of redox-driven TEs mobilisation along the profile was found (Figure 2). This would have separated the elements into two distinct groups, i.e. redox sensitive (As, Co, Cr, Mn, Ni and Fe) and non-sensitive (Cu, Pb, Zn, Al, Li and Ti), which was not the case. This means metals accumulated in the core have not been redistributed by redox processes, and thus the core is intact.

The sediment core TE contents display a relatively moderate variability, with CV of 8.7-22.9 % (Table S1), reflecting the extent of the variation in their input from the catchment. Three clearly visible metal peaks observed were at 27-30 cm, 18-21 cm and a relatively broader increase between 3 and 12 cm depth (Figure 3a). This, points to three major metal input events, corresponding to the estimated periods of 1985-1987, 1991-1993 and 1997-2003, respectively. Except Cr, all other TEs show increasing trends, from lower values in the 1970s and the 1980s to higher accumulation in recent years (Figure 3a), as supported by a significant association of TEs with depth (Pearson r = 0.68, p < 0.001). This could, in theory, result from increased atmospheric metal and proton deposition, change in the land cover, and/or climate change driven increased mobilization.

Rain acidity and airborne pollution inputs (including metals) are relatively low in the Pyrenees compared to other European mountain ranges, and they have significantly decreased with the implementation of industrial emission control technologies since the early 1970s [START_REF] Mosello | Trends in the water chemistry of high altitude lakes in Europe[END_REF][START_REF] Pacyna | Current and future emissions of selected heavy metals to the atmosphere from anthropogenic sources in Europe[END_REF]. This means acidity input is not likely to 1970 1975 1980 1985 1990 1995 2000 1975 1985 1995 1500 2000 2500 3000 Spring freezing leve Inset plot shows the variability in spring freezing level across the record period (in grey) together with its long term trend (in red) as given by LOESS fit line at α = 1. The trend was significant at p < 0.05 (Mann-Kendall test). Horizontal dashed line is at the mean of series.

have increased the mineralisation/ dissolution rates, and significant reductions in atmospheric metal deposition across Europe [START_REF] Pacyna | Current and future emissions of selected heavy metals to the atmosphere from anthropogenic sources in Europe[END_REF] cannot be responsible for increasing sediment metal accumulation trends seen here. Catchment vegetation is also not expected to be responsible for TEs mobilisation, as the land cover has not undergone any major changes during the record period. It is thus quite likely that this is mainly an outcome of increased denudation of catchment surfaces driven by changing climate.

Having discounted significant atmospheric contributions and land cover/use, this leaves climate change as the expected driver of TEs mobilisation. To investigate this, we evaluated the relationship between a number of climatic variables and sediment metal contents for the 1972-2004 sedimentation period.

Change in catchment's climate. The annual temperatures in Bubal lake

catchment showed decadal fluctuations with overall increases between the early 1970s and the mid-2000s of about 1.73 and 1.77 ⁰C for the mean and maximum temperatures, respectively (Figure 4). This is similar to the global temperature rise in mountain regions, and is more than twice compared with the northern hemisphere average [START_REF] Nogues-Bravo | Exposure of global mountain systems to climate warming during the 21st Century[END_REF][START_REF] Piao | The impacts of climate change on water resources and agriculture in China[END_REF]. The precipitation amount did not change significantly over the studied period; however, the frequencies of rain and snow days decreased by 18.20 and 48.25 %, respectively, especially since the mid-1980s (Figure 3b andd and Figure 4). This indicates that precipitation became less frequent but more intense. This obviously means a reduction in permanent stream-flow but increase in temporary runoff and drier soil surfaces. The seasonal changes in the climatic parameters broadly followed the annual trends, with lower frequencies of winter and spring snow days and summer rain days, particularly since the mid-1980s (Figure 4, variability not shown). This reveals a shift in the climate scenario since the mid-1980s to mildly warmer seasons with drier winters, as given by less snow fall/ accumulation, and a slight change in surface freezing days. The rise in temperature also meant an elevation of the freezing line from about 1944 m a.s.l. in 1972 to 2489 m a.s.l. in 2003, with consequential increase in the catchment area remaining uncovered, i.e. below 0 ⁰C isotherm (Figure 4 and Figure 5c).

Climate-enhanced lithogenic trace element mobilization.

The aforementioned changes in temperature, precipitation and freezing line could alter catchment weathering, snow-cover period and surface, water bodies' heat balance and metabolism [START_REF] White | Effects of climate on chemical weathering in watersheds[END_REF]. All these could result in greater transportation of TEs in the catchment either in mineral form or associated to mineral-organic complexes. Spring freezing level, record period, sediment TE contents and freezing days were inversely related to the precipitation variables (Figure 3b-f and Figure 5). Variance partition further revealed that the largest portion of variance in sediment metal accumulation was explained by an interaction between the climatic factors. It unveils a rather complex scenario in which it is not possible for a single factor to account for the variability in sediment metal deposition. Instead a pool of factors work in concert to significantly increase metal denudation from exposed mountain geology over the 30 years period, followed by their transport by turbulent runoff into the streams system during storm events/spring thaw and further deposition into the lake bed. A conceptualised example built upon the results (Figure 5c) illustrates that a sharp elevation of spring freezing level (increasingly unfrozen surface, in yellow) and changes in the other climate patterns in the past three decades have the potential to substantially increase TEs release from the lake catchment. The sets of explanatory variables were: A-exposed surface (spring freezing level and snow days), B-changes in wet-dry period over time (rain days and record period) and C-extreme events (storm days and freezing days) together with their first order (AB and AC) and second order (ABC) interactions. Interaction between climate factors explained most of variation in TEs concentrations in the lake sediments over time. The variances explained by each variable separately were: snow days: 47.5 %, spring freezing level: 45.4 %, record period: 44.3 %, rain days: 36.8 %, freezing days: 33.9 % and storm days: 33.8 %. These portions were significant at p < 0.01. c, A conceptualised example illustrating the increased TEs release in catchment environment (Respomuso lake subcatchment at 2130-3058 m a.s.l., enclosed on a dark grey polygon in Fig. 1) following the sharp elevation of spring freezing level (increasingly unfrozen surface, in yellow) and changes in other climate patterns in the past three decades. The estimates show that great part of the catchment (or above 1944 m a.s.l.) was snow covered before 1972 but it dramatically decreased in coverage in recent years (yellow area).

The complexity of the processes involved is further increased by a lag found in TEs peaks in relationship to climate variability (Figure 3b-f). Given the nature of the weathering processes, a lagged response is naturally expected, as denudation, transport and final burial of sediments are physically time-dependent. Nevertheless, it is implicit that an increase in the number of drier periods (as defined by the decreased frequency of precipitation events) increases weathering and denudation of the catchment slopes (during wet/dry cycles), enhancing the amount of TEs transported and buried into the lakebed sediments. Unexpectedly high metal increment along the core profile is consistent with chemical weathering of catchment's metal sulphides, e.g. pyrites, which during the dry span within the wet-dry cycles oxidise to metal sulphates and oxides. During subsequent wet cycles these metal-rich weathering byproducts can easily be leached [START_REF] Griethuysen | Temporal variation of trace metal geochemistry in floodplain lake sediment subject to dynamic hydrological conditions[END_REF] into the catchment water-bodies, being aided by relatively poorly developed soils on the mountain slopes. The climate change mediated increased mobilization is further supported by the association of major lithogenic elements Ti, Fe, Al and Li with TEs in the sediment (Figure 2).

The reduction in snow-cover, together with the elevation of spring freezing line (increasingly unfrozen surfaces) also appeared to have contributed significantly to enhanced release of TEs, by exposing additional rock/soil surfaces to denudation processes. It has been shown that weathering rates in granitic catchments can increase about one order of magnitude when uncovered mineral surface experiences substantial warming while precipitation volume is constant [START_REF] White | Effects of climate on chemical weathering in watersheds[END_REF].

Such increases in mineral surface temperatures are possible during spring/summer on the south facing slopes of catchments that remain increasingly snow-uncovered especially when precipitation events have also diminished. In Lake Bubal catchment the drainage of mineral deposits from the surrounding metamorphic belt is the major potential source of TEs (Figure 1). These ores have significant presence of TEs in vein-type mineralization with As content as high as 250 ± 40 mg kg -1 [START_REF] Subias | Las rnineralizaciones filonianas de F-Zn-Pb de Las Marmoleras (Pirineo occidental , prov. Huesca): caracterización geológica, mineralógica y geoquímica [in Spanish][END_REF]. These As and/or TEs containing sulphides can oxidise relatively rapidly under neutral pH and, therefore, release As and other elements in the environment at enhanced rates [START_REF] Davis | The nexus between groundwater modelling pit lake chemogenesis and ecological risk from arsenic in the Getchell Main Pit, Nevada[END_REF], being aided by a relatively high diffusion factor of oxygen in drier surfaces and modified climate patterns. Total precipitation, which is usually ascribed to intensify weathering, however showed no particular influence. This would suggest that changes in precipitation patterns are stronger than the total amount of precipitation in mobilising TEs.

Potential environmental risk.

The incremental metal release with changing climatic pattern raises then the question of potential risks to the environment. Arsenic in the surface sediment layers (< 15 cm) exceeded its safe guide concentration of 17 mg kg -1 for the protection of aquatic life (CCME, 1999) (Figure 3a). This is important since As is a priority carcinogenic pollutant ranking first in international hazardous substances lists, and it can be a human safety concern even at background values of 7-18 mg kg -1 [START_REF] Dani | Arsenic for the fool : An exponential connection[END_REF]. Metals in sediments can pose significant and increasing threat to the wider environment if their natural background source is high (CCME, 1999;[START_REF] Dani | Arsenic for the fool : An exponential connection[END_REF]. Nickel also reached its safe threshold of 36 mg kg -1 [START_REF] Dani | Arsenic for the fool : An exponential connection[END_REF] in surface sediments. The elevated As and Ni in the upper sediment layers are consistent with their general distribution in stream sediments and waters in this catchment, with As values in surface (stream and lake) sediments as high as 161.2 (Zaharescu et al., 2009a;Zaharescu et al., 2009b) and 339.5 [START_REF] Lavilla | Depth profile of trace elements in a sediment core of a high-altitude lake deposit at the Pyrenees, Spain[END_REF] mg kg -1 . Although TEs in Lake Bubal may not cause immediate concern, the evidence of their increased mobilisation and high background levels means climate change could trigger the release of metals from their natural stores. This could lead to potentially detrimental consequences for the ecosystems and beyond, particularly in regions where significant natural metal storage sites experience major climate change.

Conclusion.

We acknowledge that the relationship between climate change and TEs mineralisation/mobilisation and their accumulation is complex and controlled by a multitude of factors. Our results nevertheless demonstrate a clear link between increased TEs accumulation in lakebed sediment and changes in key climate drivers. To clearly determine whether the increased mobilisation of natural metal sources seen herein is an extended and ongoing process warrants further large scale surveys, particularly in sensitive regions characterised by metal-rich geologies.

2 concentrated HCl, HNO 3 and perchloric acid (HClO 4 ). The samples were subjected to ultrasound assisted digestion at 60 ⁰C for 15 min, then kept on 125 ⁰C sand bed until nearly complete evaporation. A certified reference material from the National Water Research Institute of Canada (CRM WQB3), procedural blank and three samples were analysed in triplicate to optimize the accuracy, reproducibility and recovery rate of the extraction procedure. After digestion, samples were filtered through Whatman filter 541 and made up to 50 ml with ultrapure water. Overall, analysis of the major elements was highly reliable in terms of precision and accuracy as per the method used. PTF Teflon beakers were used throughout the analyses. All reagents were ultra-pure Aristar grade. Stock standard solutions were Merck Certificate AA standards. Milli-Q water was used in all samples, dilutions and standard solutions, as appropriate.

Figure 1 .

 1 Figure 1. Hydro-geological context. Lake Bubal catchment in the Central Pyrenees with the representation of mineralization areas and sampling sites. Enclosed in a dark grey rectangle is Respomuso catchment exemplified in Fig. 5c. Map was assembled in Adobe Photoshop CS3 (https://www.adobe.com/) after Subias, Moritz & Fernandez-Nieto (1998). Inset topography map is after NASA & JPL (2003).

Figure 2 .

 2 Figure 2. Geogenic control of sediment TEs content. Stepwise multiple linear regression of trace metals (summarised as regression factor scores by principal component analysis) versus their standardised values predicted by major elements Ti, Fe, Al and Li, together with upper and lower 95 % confidence intervals. Significance of the model p < 0.001.

Figure 3 .

 3 Figure 3. Lake sediment trace metal levels have risen and fallen in step with climate changes during the period 1972-2006. a, Observed variation of TMs (mg kg -1) in bottom sediments of Lake Bubal (Central Pyrenees), with major trends represented by dashed black lines. The metals exhibit clearly visible peaks in concentrations at 27-30, 18-21 and 3-12 cm depths. Vertical (dashed) lines in red represent sediment guide values for the protection of aquatic ecosystem(CCME, 1999). b-f, Sediment TMs (As, Co, Cr, Cu, Mn, Ni, Pb and Zn, summarized as regression factor scores of principal component analysis) trend-black dashed line, against b-rain days, c-storm days, d-snow days, e-freezing days and f-spring freezing level, and their LOESS (locally estimated scatterplot smoothing) fit lines (dark grey line). Horizontal lines are set at means of series. 95 % Pearson correlation significance (*) between TEs and climate variables is drawn after 1,000 permutations test. Climate variables are shifted with respect to TMs on their identified lag response.

Figure 4 .

 4 Figure 4. Bubal lake catchment has experienced significant changes in annual and seasonal climate in the period 1972-2006. Long term climate trends are drawn from a locally estimated scatterplot smoothing (LOESS) regression, filter factor α = 1 span. Trend significance (*; p < 0.05) was given by Mann-Kendall test.Inset plot shows the variability in spring freezing level across the record period (in grey) together with its long term trend (in red) as given by LOESS fit line at α = 1. The trend was significant at p < 0.05 (Mann-Kendall test). Horizontal dashed line is at the mean of series.

Figure 5 .

 5 Figure 5. Climate-drivers of increased metal mobilisation. a, Relationship between climate variables (input as LOESS predicted values, 0.1 span), TMs (As, Co, Cr, Cu, Mn, Ni, Pb and Zn, summarised as PCA regression factor scores) and record period shown in the projection of PC1-PC2 of principal component analysis. Figures highlight a tight relationship between sediment TEs input, spring freezing level, precipitation and freezing day frequencies, supporting thus the hypothesis of an increased metal mobilisation with climate change. Symbols key: ■, elements correlating to PC1; and •, element correlating to PC2. b, Variance in sediment trace metal contents over time was partitioned among climate factors.The sets of explanatory variables were: A-exposed surface (spring freezing level and snow days), B-changes in wet-dry period over time (rain days and record period) and C-extreme events (storm days and freezing days) together with their first order (AB and AC) and second order (ABC) interactions. Interaction between climate factors explained most of variation in TEs concentrations in the lake sediments over time. The variances explained by each variable separately were: snow days: 47.5 %, spring freezing level: 45.4 %, record period: 44.3 %, rain days: 36.8 %, freezing days: 33.9 % and storm days: 33.8 %. These portions were significant at p < 0.01. c, A conceptualised example illustrating the increased TEs release in catchment environment (Respomuso lake subcatchment at 2130-3058 m a.s.l., enclosed on a dark grey polygon in Fig.1) following the sharp elevation of spring freezing level (increasingly unfrozen surface, in yellow) and changes in other climate patterns in the past three decades. The estimates show that great part of the catchment (or above 1944 m a.s.l.) was snow covered before 1972 but it dramatically decreased in coverage in recent years (yellow area).

Acknowledgements

This work was financially supported by a Félix de Azara award of Huesca Provincial Council, Spain, as well as by University of Vigo, Spain and Kingston University London, UK. The sponsors were not involved in the research or article preparation. We are deeply grateful to the Spanish National Institute of Meteorology in Madrid, Spain for providing climate data, to Mariano Lastra-Valdor, from the University of Vigo, for his substantial support during project execution, as well as to Craig Rasmussen, Joel Cuello and Cody Brown from the University of Arizona for their help with particle size analysis.

Author contributions

D.G. Zaharescu, C.I. Burghelea and P.Hooda contributed equally to the conceptual development, material analysis and manuscript preparation. V. Polyakov contributed the analysis and interpretation of radiometric samples. A. Palanca-Soler supported sediment analysis and meteorological data acquisition.

Additional Information

Competing financial interests: The authors declare no competing financial interests. 2009). Snow-thaw and rainwater comprise the major water input, and no significant industrial or extensive agricultural activities exist/existed in the area. There may be some localised emissions from tourism activities in the catchment. However, local aerial contribution during peak tourism season is low (Zaharescu et al., 2009). The economy in the valley is mostly tourism dependent. Vegetation cover and land use have not changed markedly over the last decades.

SUPPLEMENTARY INFORMATION

Analysis for trace, major elements and organic matter contents. Quality control protocol for trace metal (As, Cd. Co, Cr, Cu, Mn, Ni, Pb and Zn) analysis included reagent blanks, certified reference materials and sample duplicates. The ICP-MS was highly sensitive for the elements in terms of detection limit and reproducibility. Overall the analysis was highly reliable, with % relative standard deviation (RSD) between replicates and between measurements of the same sample being < 5 %. The RSD for Cd was about 25 %, which is not uncommon at elemental levels close to the detection limit; nonetheless it was not considered satisfactory enough and was therefore removed from further analysis. The percentage recoveries of the analysed elements were generally consistent with that specified for USEPA Method 3050B used (USEPA, 1999). . Major elements (Ti, Fe, Al and Li) were determined following USEPA Method 3051A for ICP-OES (USEPA, 2007). Briefly, this involved ashing a portion (0.1g) of dried sediment at 550 ⁰C for 10 h to remove organic matter before being digested by using a mixture of

Supplementary tables

Table S1. Variability in trace and major (represented on grey background) element contents (mg kg -1 dry weight) in the < 0.25 mm sediment fraction of Lake Bubal core.

Supplementary figures

P<0.05

Supplementary references


Kristian Rauhe Harreby

Aritra Kundu

Bo Geng

Thomas Stieglitz

Pawel Maciejasz

David Guiraud

Tim Boretius

Ken Yoshida

Winnie Jensen

Harreby Kr

& Jensen

Recruitment Selectivity of Single and Pairs of Transverse, Intrafascicular, Multi-channel Electrodes (TIME) in the Pig Median Nerve

Keywords: TIME, pig model, median nerve, stimulation, electrode

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Selective peripheral neural electrodes may be used as means to restore sensory and motor functions. Advanced prosthetic limbs with sensing abilities have been developed for amputees [START_REF] Carrozza | Design of a cybernetic hand for perception and action[END_REF] . However, the lack of reliable, safe and selective interfaces to intuitively link the user to the multiple degrees of freedom and control in these prosthetics device is currently hindering their use.

Designers of peripheral nerve electrodes face a trade-off between selectivity and invasiveness. The result is an array of different neural interfaces which have been under development. These include the multipolar cuff electrode, the thin-film longitudinal intra-fascicular electrode (tfLIFE), transverse intra-fascicular multi-channel electrode (TIME) and the Utah Slanted Electrode Array (USEA). All have different selectivity / invasiveness ratios [START_REF] Boretius | A transverse intrafascicular multichannel electrode (TIME) to interface with the peripheral nerve[END_REF][START_REF] Yoshida | Multichannel intraneural and intramuscular techniques for multiunit recording and use in active prostheses[END_REF][START_REF] Branner | Long-term stimulation and recording with a penetrating microelectrode array in cat sciatic nerve[END_REF] .

The TIME consists of a flexible polyimide substrate loop, which holds 6 iridium oxide coated contact sites on each side of the loop [START_REF] Boretius | A transverse intrafascicular multichannel electrode (TIME) to interface with the peripheral nerve[END_REF] . Being transversely implanted into the nerve, the TIME is considered to be more invasive than the cuff electrode; but since its substrate is flexible and penetrates the nerve only once, it is considered less invasive than the USEA. A recent study in the sciatic nerve of rats showed that the TIME could selectively recruit three muscles innervated by this nerve, thus showing both intra-and inter-fascicular selectivity [START_REF] Badia | Comparative analysis of transverse intrafascicular multichannel, longitudinal intrafascicular and multipolar cuff electrodes for the selective stimulation of nerve fascicles[END_REF] . However, recent studies in the median nerves of pigs have found that a single TIME is capable of selectively reaching only a subset of a large poly-fascicular nerve [START_REF] Kundu | Acute stimulation selectivity of transverse and longitudinal and transverse implantation of intrafascicular electrode in pig animal model[END_REF][START_REF] Harreby | Evaluation of the chronic stimulation performance of the Transverse, Intrafascicular, Multi-channel Electrode (TIME) in the Göttingen mini-pig[END_REF] .

In a poly-fascicular nerve, fascicles adjacent to the TIME are most likely to be selectively recruited. By implanting several TIMEs within the same peripheral nerve, more fascicles will be located adjacent to an electrode and, thus, a larger part of the nerve can potentially be selectively recruited. This study aims to investigate the ability to selectively recruit muscles innervated by the median nerve of the pig, when pairs of TIMEs are inserted in different configurations. Electrical stimulation consisting of rectangular, cathodic current pulses (100 µs duration) of increasing current intensities was presented at 2 Hz to each contact of the individual TIMEs, and the EMG responses from the seven muscles were simultaneously recorded. For Group A: 40 µA to 800 µA (40 µA steps), each current level was repeated five times (STG2008, Multichannel systems, Germany). In Case B, stimulation current was increased from 20 µA to 800 µA (20 µA steps) and each level was repeated three times (Stim'ND, Neuromedics, France [START_REF] Andreu | A distributed architecture for activating the peripheral nervous system[END_REF] ). A stainless steel needle placed subcutaneously on the thorax served as anode.

Material and Methods

Experimental procedures

Offline analysis

The EMG was bandpass filtered (0.1 -2 kHz, Matlab, R2011a, MathWorks, USA). The rootmean-square value of the evoked EMG (time window: 2-12 ms after stim. pulses) was used for assessing nerve recruitment. To obtain the recruitment level of individual muscles (EMG RL ), root-mean-square values obtained for the same stimulation intensity were median filtered and normalized to the maximal evoked amplitude (100 %) obtained in individual muscle of each pig. Recruitment curves were linearly interpolated to 1 µA resolution. The selectivity index of an individual muscle (SI M ), j, was defined as:

Statistics

SI D results from each TIME was regarded as one group (SI D,All ) of single TIMEs, no matter if implanted at an angle or in parallel. To illustrate the separation between SI and RC, results for the different groups were compared using a t-test (p < 0.05 was regarded as statistical significant). No corrections were made for multiple comparisons.

Results

The SI D for single TIMEs were similar no matter whether they were implanted at an angle or in parallel (0.27 ± 0.02 & 0.36 ± 0.04, respectively, p = 0.23, mean ± standard error of mean) and so they were pooled together in SI D,All (0.28 ± 0.02). The SI DD for all three types of paired TIME configurations (SI DD45 = 0.38 ± 0.03 , SI DD90 = 0.41 ± 0.03, SI DD|| = 0.56) show a significant improvement compared to SI D,All , see Fig. 2 a.

There is a weak trend for SI DD90 to be higher than SI DD45 (p = 0.34), whereas, the SI DD|| is higher than both SI DD45 and SI DD90 . However, this does not reach a level of significance (p = 0.06 and p = 0.11). The SI DD|| =0.56 obtained for the single case, was greater than any single SI DD obtained in the two groups of TIMEs paired at an angle (max{SI DD45 }= 0.47 & max{SI DD90 } = 0.52).

The mean RC for TIMEs paired at an angle are close to zero (0.03 ± 0.10 and -0.01 ± 0.14). The RC DD|| seems to be lower than that for RC DD45 and RC DD90 , however, this did neither reach a level of significance (p = 0.10 and p = 0.21). The one pair of TIMEs evaluated in parallel, resulted in a RC DD||| = -0.59, which was lower than any value obtained in the two other groups (min{RC DD45 } = -0.39 & min{RC DD90 } = -0.58).

Discussion

The ability to selectively innervate the median nerve of a pig was significantly improved by using two TIMEs compared to a single TIME. There was a trend for 45º separation to provide the least improvement, 90º separation to provide intermediate improvement and for TIMEs implanted in parallel to show the highest improvement. Thus, as expected, results indicate that the more spatially separated TIMEs were within the nerve, the higher was the gain in selectivity.

A single TIME may be sufficient to selectively recruit the whole sciatic nerve of the rat [START_REF] Badia | Comparative analysis of transverse intrafascicular multichannel, longitudinal intrafascicular and multipolar cuff electrodes for the selective stimulation of nerve fascicles[END_REF] . However, in the poly-fascicular nerve of pig, results indicated that single TIMEs could only selectively recruit a subset of the nerve [START_REF] Kundu | Comparison of acute stimulation selectivity of transverse and longitudinal intrafascicular electrodes in pigs[END_REF][START_REF] Harreby | Evaluation of the stimulation selectivity of transverse intrafascicular multichannel electrodes in the chronic Göttingen mini-pig : preliminary results[END_REF] . These results indicate that implantation of several TIMEs, ideally interfacing different non-overlapping nerve regions may be an approach to interface a whole poly-fascicular human size nerves.

The drawback with implantation of several TIMEs will obviously be an increase in the invasiveness of the combined nerve interface. An alternative to the implantation of several TIMEs might be to implant a single USEA. However, in relation to distributing a number of contact sites inside the nerve, the USEA approach is less effective and more invasive than the TIME, as each USEA contact site entail a needle to penetrate the nerve [START_REF] Branner | Long-term stimulation and recording with a penetrating microelectrode array in cat sciatic nerve[END_REF] . In contrast, the TIME distributes 12 contact sites inside the nerve when penetrating it only once. In addition, the TIME consists of a flexible polyimide substrate as compared to the stiff silicon structure of the USEA. Thus, several, TIMEs might still be preferred over an USEA.

The current study has been limited by having only one pair of TIMEs for the parallel configuration. In this configuration the Stim'ND prototype stimulator was used for the first time in a mammal model. Although, this stimulator produced results consistent with the commercial STG2008 stimulator, more experiments have to be performed to validate the performances of this stimulator under in-vivo conditions. Therefore results from the parallel TIME configurations should be interpreted with care. However, our initial hypothesis is consistently supported by the results, i.e. that less spatial overlap between paired TIMEs inside the nerve (Fig. 1) will result in less redundancy in the selectively recruited muscles (Fig. 2b) and, thus, result in the highest selectivity gain (Fig. 2a).

Conclusions

This study investigated the use of pairs of TIMEs to interface a large poly-fasicular nerve. The ability of the TIME to selectively recruit such nerves was significantly improved by using pairs of TIMEs as compared to using one single TIME. Results indicate that the optimal improvement occur when TIMEs are placed in parallel to each other in such a way that they interfaced non-overlapping nerve regions.

  Acute pig experiments were carried out in 6 domestic pigs (25-40 kg). Under full anaesthesia the pigs were intubated and positioned in the supine position. The median nerve was exposed and two or four TIMEs were implanted either through the middle of the nerve at different implantation angles (Group A, 5 pigs, 16 TIMEs, with 45º between TIMEs) or parallel to each other with different offsets from the midline of the nerve (Case B, 1 pig, 2 TIMEs; see Fig 1 a & b). Seven muscles innervated by the median nerve were exposed and epimysial electromyographic (EMG) patch electrodes were attached. The muscles were: Pronator teres, Palmaris longus, Flexor carpi radialis, Flexor digitalis superficialis, Flexor digitorium profundus, Abductor pollicis brevis and Humeral head of deep digital flexor.

Fig. 1 .

 1 Fig. 1. Illustration of insertion schemes. (a) Example of four TIMEs which are placed in a nerve with a 45º angle in between. Based on the results from individual TIMEs, the SI DD and RC could be calculated for TIMEs implanted at different angles (45º and 90º) relatively to each other. (b) TIMEs were implanted in parallel through the nerve, thus minimizing overlapping regions of recruitment.

(eq. 1 )

 1 SI M,j was set to zero if the EMG RL did not reach 30 %. The device selectivity for each individual TIME devices (SI D ) and for pairs of TIMEs (SI DD ) were defined as: (eq. 2) (eq. 3) SI DD was calculated for TIME devices implanted with a 45º angle (SI DD45 ), a 90º angle (SI DD90 ) or in parallel (SI DD| |, see Fig 1.). In general, SI values near 0 indicate no selectivity or a lack of recruitment, and a value close to 1 indicate good selectivity (muscles can be individually recruited). The recruitment correlation (RC) for pairs of TIMEs was defined using the Pearson correlation coefficient (PCC). (eq. 4) refers to a seven dimensional vector containing the SI M for all muscles monitored. Values close to -1 indicate that each of the TIMEs in a pair selectively recruit different muscles, whereas, values close to 1 indicate that the same muscles are selectively recruited by the two TIME devices. The latter would imply that little is gained by implanting more than one TIME.

Fig. 2 .

 2 Fig. 2. (a) SI results of individual and pairs of TIME devices. (b) Results of RC for the pairs of TIME configurations. Errors bars indicate standard error of mean.

Acknowledgements

The work was funded by European Union under the 7 th Framework program (grant CP-FP-INFSO 224012/TIME). We wish to thank the staff at Aalborg Biomedical Laboratory for their generous help during our experiments.

Author's Address

Kristian Rauhe Harreby, PhD krauhe@hst.aau.dk


Jérôme Lambert

Isabelle Cantat

Renaud Delannay

Rajmund Mokso

James A Glazier

François Graner

Experimental growth law for bubbles in a "wet" 3D liquid foam

Keywords: numbers: 82, 70, Rr, 83, 80, Iz

We used X-ray tomography to characterize the geometry of all bubbles in a liquid foam of average liquid fraction φ l ≈ 17 % and to follow their evolution, measuring the normalized growth rate G = V -1 3 dV dt for 7000 bubbles. While G does not depend only on the number of faces of a bubble, its average over f -faced bubbles scales as G f ∼ f -f 0 for large f s at all times. We discuss the dispersion of G and the influence of V on G.

Liquid foams consist of bubbles of gas separated by a continuous liquid phase occupying a fraction φ l of the foam's volume. Liquid foams coarsen because gas slowly diffuses through the liquid films from high pressure bubbles to low pressure bubbles so the high pressure bubbles eventually disappear. The dynamics of the coarsening differs in the two limiting cases of dry and infinitely wet foams.

In a bubbly liquid, with φ l ∼ 1, bubbles are spherical and well separated. As in emulsions and diphasic materials, the bubbles coarsen via "Oswald ripening" as theoretically described by Lifshitz, Slyozov and Wagner (LSW) [1,2] using a mean-field approach.

In the dry foam limit, φ l ≪ 1, bubbles are polyhedral and touch each other. The gas flux between two neighboring bubbles is proportional to their pressure difference, hence to the mean curvature of the film separating them. From simple dimensional arguments [3], the growth rate of an individual bubble of volume V in a 3D foam must be of the form:

dV dt = V 1 3 G (1)
G depends both on the shape of the bubble rescaled to unit volume and on physico-chemical characteristics of the liquid, gas and of the surfactant through the effective gas diffusivity

D ef f : G = -D ef f S HdS V 1 3 , ( 2
)
where H is the mean curvature of a surface element dS of the bubble. Theoretical treatment of coarsening in dry foams usually make an analogy with 2D foams, for which von Neumann's law (vNM), which is exact, states that the growth rate of the area of a bubble is proportional to n -6, where n is its number of edges. This law has been validated experimentally. In 3D, in contrast, the existence of the extra radius of curvature means that the growth rate of a bubble does not depend only on its number of faces [4,[START_REF] Glazier | Foams, emulsion and their application[END_REF]. Thus deriving a growth law in 3D requires additional assumptions and results are only approximate. Attempts to relate the growth rates of individual bubbles or the average growth rates of classes of bubbles to simple geometrical properties have had a mixed success. Indeed many attempts have been done to find a relation between the right-hand-side (rhs) of eq. 2 and simple geometrical properties. Mullins [START_REF] Mullins | [END_REF] and several groups (see [4] and references therein) derived estimates for this rhs based on ideal regular bubbles and obtained G M ∼ f 1/2 for large f . Numerical simulations of coarsening have computed G for large numbers of bubbles. Potts Model [3,7], vertex model [8] and Surface Evolver [9] simulations all exhibit a strong correlation between bubbles' number of faces and growth rate with a small dispersion. We can then check if Mullins' model holds on average for realistic foams by evaluating the average values

G f =< G > f over sets of f -faced bubbles. The data admit both G f ∼ (f -f 0 ) and G f ∼ (f -f 0 ) 1 2
fits. Experimental measurements of G f using optical tomography [10,11] and NMR [12,13,[START_REF] Prause | Proceedings of the 5th Experimental Chaos Conference Orlando[END_REF][START_REF] Prause | [END_REF] observed too few bubbles to distinguish (f

-f 0 ) from (f -f 0 ) 1 2 .
Here we investigate foam coarsening experimentally in the previously unstudied intermediate wetness regime with φ l ∼ 10 -20 %, which is not known in theory nor in simulations, but which is widely used in most foam applications [START_REF] Weaire | The physics of foams[END_REF]. We determine G and G f for several thousand bubbles and study the cross-over between the LSW and the vNM regimes. Recent progress in X-ray tomography [START_REF] Lambert | [END_REF] permits us to take 3D images of thousands of bubbles in 2 minutes, and allows us to monitor a foam for many hours. We find that, for a given set of f -faced bubbles, the dispersion of G is small. Therefore we focus on G f . While the shape of the distribution of the number of faces per bubble changes during the experiment (implying that our foam has not reached a statistically invariant scaling state), the shape of G f vs. f stays constant. This quantity thus seems to be a robust parameter to characterize foam coarsening. G f depends linearly on f for large f. We then study the dispersion of G as a function of f and V and we finally discuss whether f or V is more relevant to determine the dynamics of a bubble, on the basis of the strong correlation existing between both quantities through Lewis' law.

The X-ray tomography apparatus at the ID19 beamline of the European Synchrotron Radiation facility (ESRF) Grenoble, France, enables us to visualize the coarsening of a wet liquid foam contained in a 1 cm 3 cubic volume. The spatial resolution of the images is 10 µm in each direction, smaller than the smallest bubbles which appear in at least two successive images (i.e. any bubble smaller than 10 µm diameter disappears in less than 2 minutes. It thus never enters our calculations of growth rates). Thus we can image all bubbles which we can analyze (by definition, we do not include in G f bubbles which disappear within one time step). Obtaining adequate image contrast between liquid and gas phases is a major difficulty in this kind of experiment since the liquid phase occupies only a small percentage of the total volume -the liquid fraction φ l [START_REF]To achieve a relatively high contrast, we[END_REF]. In addition, the bubble walls must not move significantly during the time required to acquire an image otherwise topographic reconstruction of the foam will fail. The characteristic evolution time for gas diffusion in ∼ 1 mm 3 air bubbles is several minutes. Our experimental acquisition time of 2 min per image enabled us to clearly identify and follow bubbles during their evolution. To reduce the complicating effects of gravitational drainage of the foam and to maintain a constant and relatively high liquid fraction, we used a high-viscosity liquid composed of 100 mL de-ionized water, 0.1 g sodium dodecyl phosphate, 0.003 g dodecanol and 1 g gelatine (not enough to gelify the liquid). In addition, we used a pump to provide a constant supply of liquid to the top of the foam which maintained the liquid fraction, decreasing from 20% at the top to 10% at the bottom [START_REF] Lambert | [END_REF].

We identified bubbles in an image using a watershed method detailed in [START_REF] Lambert | [END_REF]. We measured the liquid fraction in a region by summing the number of pixels of that volume containing more than a threshold amount of liquid. φ l was thus estimated with an error ∆φ l = 0.5%.

We developed a correlation algorithm using the commercial Aphelion [19] software package to track bubbles from image to image, enabling us to determine their growth rates.

Determining a bubble's number of faces in a wet foam is ambiguously defined [START_REF] Lambert | [END_REF], since neighboring bubbles may be distant. We compared the distance separating two bubbles to the characteristic size of their vertices, i.e. the crossing of the bubbles' edges where most of the liquid accumulates. We assumed that if the distance between two bubbles was less than half this characteristic size, they were neighbors and shared a face. Thus small bubbles located at vertices may have fewer than 4 faces. We automatically discarded from our analysis bubbles which touched the container walls. 48 minutes after production, the foam's liquid fraction varied from 20% at its top to 10% at its bottom as a result of the continuous liquid injection at its top. To test the influence of the liquid fraction on foam dynamics, we defined four subregions corresponding to different heights and thus wetnesses in our column.

for each image, we measured each bubble's number of neighbors f and growth rate G.

figure 1 shows G vs. f for the 3582 bubbles in the topmost horizontal slice of the cell 48 min after foam creation, and the distribution of Gs for f = 12 (inset). Error bars on the measurement of G result of the incertitude on the measurement of V at two successive times.

∆V was determined for each bubble by assuming that a 1 voxel thick sheet could randomly be added or removed from each of its faces. G varies for different bubbles with the same f s to a much greater extent than the experimental uncertainty. Hence it is not an exact function of the unique variable f , although the two quantities are correlated, showing that 3D bubbles do not obey an exact equivalent of von Neumann's law. However for the ensemble of bubbles with a given f , the dispersion of G around its average is not large, as the inset of fig. 1 shows for f=12, since one can clearly see the increase of G with f : The dispersion σ G coincides with the variation of G f for ∆f ≈ 5 (with f > 10). We therefore compute the average value G f to investigate its dependence on φ l and the structure of the foam.

Foam structure changes during coarsening. We characterize the structure by measuring the distribution of bubbles' number of faces ρ(f ). figure 2 Figure 3 shows G f at two different times for different values of φ l . Despite a shift in the value of f at which G f crosses 0, the general dependence of G f vs. f is unchanged: G f goes to 0 as f → 0 for f < 10, and G f is linear for f > 10.

Since in the limit φ l → 1 G should depend on V only , we plot G vs. V in Figure 4. As expected, both quantities correlate.

In order to check if the number of faces describes better a bubble's behavior than its volume as it is expected in the dry foam limit [3], we scatter-plot G vs. V for three different sets of f -faced bubbles in fig. 5. It is clear that, at fixed f , G increases slightly with V .

The shift of the clouds corresponding to different f -set over the V axis can be explained by the strong correlation between < V > f and f , that we plot in the inset of fig. 5, usually known as Lewis' law [7,9,[START_REF] Weaire | The physics of foams[END_REF].

We experimentally observe a power law relation between < V > f and f : We compared the dispersion of the data points for scatter plots of G vs. f (fig. 1) and vs. V (fig. 4). for f = 12 (476 bubbles) we obtained a standard deviation for G of ∆G = 10.5 µm 2 .s -1 . On the basis of the empirical Lewis' law V f ∼ f 2.2 we plotted on fig. 5, we compute a ∆V corresponding to ∆f = 1 and find ∆G = 12.1 µm 2 .s -1 around V 12 . This small difference does not allow to conclude that one parameter is better than the other.

V f =< V > f ∼ f α , with α ≈ 2.
Future experiments using foams of very different structures (bidisperse, monodisperse, scaling state...) and liquid fractions may allow to obtain various relations between f and V , and thus to determine which parameter, V or f , influences G most significantly.

Our experiments studied a moderately wet foam (14% < φ l < 20%), for the dynamics of which we have no theoretical predictions. G does not depend uniquely on f and G f disagrees with mean-field theories for both wet and dry foams. G f increases linearly with f for f > 10 as Glazier numerically observed using Potts' simulations [3]. Thomas et al [7] have confirmed Glazier's results and extended them to additional initial bubble size distributions. As in dry foam simulations, G f increases to zero as f decreases for small f s [3,7]. Let us notice that there are still some spurious bubbles due to the noise in image segmentation. Some of them are artificially correlated, but their number has been evaluated to be very small, and their Inset: < V > f as a function of f for φ l = 14, 17 and 20%.

influence on the results is poor as was checked by introducing a filter on the size of the bubbles taken into account.

In contrast to 2D, the dispersion of G around G f is intrinsic to the foam, as f does not suffice to describe the dynamics of a bubble. In addition we have checked the stability of the linearity of G f for early stages of the evolution of the foam. The foams contains too few bubbles at late stages to compute statistically relevant G f s. It will be of great interest to observe G f in later stages, in particular during a possible scaling regime.

FIG. 1 :

 1 FIG. 1: Scatterplot of G vs. f in the topmost slice (φ l = 20 %) of the experimental cell. Inset: histogram of G for 12-faced bubbles.

  shows ρ(f ) at different times and for different values of φ l . The shape of the distribution varies significantly with time and liquid fraction. The average number of neighbors ranges from 10.7 ± 5.5 (φ l = 20%) to 11.0 ± 4.5 (φ l = 14%).

FIG. 2 :

 2 FIG.2: Solid red and blue lines: ρ(f ) for φ l = 20%, 48 min and 375 min after foam creation. Solid green and black lines: ρ(f ) for φ l = 14% and 17%, 48 min after foam creation.

1 )FIG. 3 :

 13 FIG. 3: Blue and red points G f vs. f at times 48 min and 375 min after the making of the foam for φ l = 20 %. The standard deviation of G around G f for each f is pictured by the bars. Green and black points : G f vs. f at time 48 min for φ l = 14 % and φ l = 17 %.

3 )FIG. 4 :FFIG. 5 :

 345 FIG.4: Growth rate G vs. V , volume of the bubbles inside the topmost slice (φ l = 20 %) in the 48 min old foam.


Caceres

S E A Orrigo
email: sonja.orrigo@ific.uv.es

B Rubio

Y Fujita

B Blank

W Gelletly

J Agramunt

A Algora

P Ascher

B Bilgier

L Cáceres

R B Cakirli

H Fujita

E Ganioglu

M Gerbaux

J Giovinazzo

S Grévy

O Kamalou

H C Kozer

L Kucuk

T Kurtukian-Nieto

F Molina

L Popescu

A M Rogers

G Susoy

C Stodel

T Suzuki

A Tamii

J C Thomas

Competition of β-delayed protons and β-delayed γ rays in 56 Zn and the exotic β-delayed γ-proton decay

Competition of β-delayed protons and β-delayed γ rays in 56

Zn and the exotic β-delayed γ-proton decay

Introduction

Decay spectroscopy is a powerful tool for exploring the structure of nuclei at the drip-lines. β-decay studies, in particular, provide direct access to the absolute values of the Fermi and Gamow-Teller transition strengths, B(F) and B(GT), respectively.

The proton-rich 56 Zn nucleus was observed for the first time at GANIL in 1999 [1]. 56 Zn is a weakly-bound nucleus lying very close to the proton drip-line. It has a quite small proton separation energy, S p = 560(140) keV [2], and third component of the isospin quantum number T z = -2.

The first study of the β decay of 56 Zn was reported in ref . [3]. More recently, some interesting results on 56 Zn decay have been reported in ref . [4]. Among them the discovery of a rare and exotic decay mode, β-delayed γproton decay, which has been seen for the first time in the fp shell. The consequences of this rare decay sequence for the determination of the Gamow-Teller (GT) strength have also been analyzed.

The experiment

The experimental study of 56 Zn decay was performed at GANIL in 2010. The experiment used a primary beam of 58 Ni 26+ to produce 56 Zn. The 58 Ni beam, of 3.7 eμA and accelerated to 74.5 MeV/nucleon, was fragmented on a natural Ni target, 200 μm thick. The fragments were selected by the LISE3 separator and implanted into a Double-Sided Silicon Strip Detector (DSSSD). The detection set-up comprised the aforementioned DSSSD detector, 300 μm thick, a silicon ΔE detector located 28 cm upstream, and four EXOGAM Ge clovers surrounding the DSSSD.

The EXOGAM clovers were used to detect β-delayed γ rays. The purpose of the DSSSD was the detection of both the implanted fragments and the subsequent charged-particle decays, i.e., β particles and β-delayed protons. An implantation event was defined by simultaneous signals in both the ΔE and DSSSD detectors. A decay event was defined by a signal above threshold (50-90 keV) in the DSSSD and no coincident signal in the ΔE.

The implanted ions were identified and selected by putting a gate in a two-dimensional identification matrix, obtained by combining the energy loss signal from the ΔE detector and the Time-of-Flight. The latter was defined as the time difference between the cyclotron radio-frequency and ΔE signal.

Results on the β decay of 56 Zn

The results on the β decay of 56 Zn [4] are summarized in the decay scheme in fig. 1 and in table 1, and discussed below.

A half-life of T 1/2 = 32.9(8) ms was obtained for 56 Zn, in agreement with ref . [3]. To determine T 1/2 , a decay-time spectrum has been constructed from the time correlations between a decay event in a given pixel of the DSSSD (with a total of 256 pixels) and any implantation signal that occurred before and after it in the same pixel, satisfying the identification condition required to select 56 Zn. The analysis of the charged-particle spectrum measured in the DSSSD has provided new spectroscopic information on the energy levels populated in the 56 Cu nucleus, the β-daughter of 56 Zn. These levels are shown in fig. 1. The comparison of this level spectrum with that of the mirror 56 Co, obtained by the 56 Fe( 3 He,t) charge exchange reaction [5], has been very fruitful.

The analysis of the γ spectrum measured in the EXOGAM clovers and γ-proton coincidences have identified three γ rays at 309, 861 and 1835 keV.

Absolute B(F) and B(GT) strengths have been determined (table 1).

Competition of β-delayed protons and β-delayed γ rays

In the first study of the 56 Zn β decay [3], the emission of β-delayed protons was observed but no β-delayed γ rays were seen. This was not a surprise because, in general, in proton-rich nuclei the proton decay is expected to dominate for states well above (>1 MeV) the proton separation energy S p . The consequence is that normally the β feeding is directly inferred from the measured intensities of the proton peaks. However, cases where there is a competition between β-delayed proton emission and β-delayed γ deexcitation have also been observed, e.g., in ref s. [3,6].

In the T z = -2 → -1, β + decay of 56 Zn to 56 Cu, the 56 Zn ground state decays with a Fermi transition to its Isobaric Analogue State (IAS) in 56 Cu. It should be noted that the de-excitation of this T = 2, J π = 0 + IAS via proton decay to the ground state of 55 Ni (T = 1/2, J π = 7/2 -) is isospin forbidden. Therefore the proton emission that we observe can only happen through a T = 1 isospin impurity present in the IAS. Moreover in general, when the proton emission is isospin forbidden, the competitive emission of de-exciting γ rays from the IAS also becomes possible and can be observed even from IAS lying at an excitation energy well above S p [3,6].

The competition between β-delayed protons and γ rays has indeed been observed in 56 Zn. The γ decays represent 56(6)% of the total decays from the 3508 keV IAS. Thus one has to take into account the intensities of both the proton and γ peaks to determine the Fermi strength correctly.

We have also found evidence for the fragmentation of B(F) due to a strong isospin mixing with a 0 + state at 3423 keV [4], which is important in terms of the mass evaluation [7]. The isospin impurity in the 56 Cu IAS, α 2 = 33(10)% (defined as in ref . [5]), and the off-diagonal matrix element of the charge-dependent part of the Hamiltonian, H c = 40(23) keV, which is responsible for the isospin mixing of the 3508 keV IAS (T = 2, J π = 0 + ) and the 0 + part of the 3423 keV level (T = 1), are similar to the values obtained in the mirror 56 Co nucleus [5].

Thus, the proton decay of the IAS proceeds thanks to the T = 1 component. However, considering the quite large isospin mixing in 56 Cu, the much faster proton decay (t 1/2 ∼ 10 -18 s) should dominate on the γ deexcitation (t 1/2 ∼ 10 -14 s in the mirror). This is not the case since we are still observing the γ decay of the IAS in competition with it.

The knowledge on the nuclear structure of the three nuclei involved in the decay, i.e., 56 Zn, 56 Cu and 55 Ni, can provide us with a possible explanation for the hindrance of the proton decay. Shell model calculations are in progress to clarify this point.

The β-delayed γ-proton decay

Besides the competition between β-delayed proton emission and γ decay, the exotic sequence of β-delayed γ-proton decay has been detected. Indeed 56 Zn does β decay to its IAS in 56 Cu and from there we observe the emission of two γ rays of 861 and 1835 keV, populating the 56 Cu levels at 2661 and 1691 keV, respectively. Due to the low S p , these levels are still proton-unbound and thereafter they decay by proton emission. Consequently the rare and exotic β-delayed γ-proton decay has been observed. In addition to these two branches, there is a third case. The 1691 keV level emits a γ ray of 309 keV, going to the level at 1391 keV that is again proton-unbound and then it de-excites by proton emission.

The β-delayed γ-proton decay has been observed here for the first time in the fp shell. This rare decay mode was seen only once before, in the sd shell in 32 Ar [6], but the consequences for the determination of B(GT) were not addressed in ref [6].

The observation of this special decay mode is very important because it does affect the conventional way to determine B(GT) near the proton drip-line. For a proper determination of B(GT), indeed, it is crucial to correct the intensity of the proton transitions for the amount of indirect feeding coming from the γ de-excitation. This finding indicates that it is important to employ γ detectors in such studies. This decay mode is expected to be significant in heavier proton-rich nuclei with T z ≤ -3/2 under study at RIKEN.

Figure 1 :

 1 Figure 1: Scheme of the β decay of 56 Zn. The solid lines indicate observed proton or γ transitions, while the dashed lines correspond to transitions observed in the mirror 56 Co nucleus.

Table 1 :

 1 β feedings, Fermi and Gamow Teller transition strengths to the 56 Cu levels populated in the β + decay of 56 Zn.

	E X (keV)	I β (%) B(F) B(GT)
	3508(140)*	43(5) 2.7(5)
	3423(140)	21(1) 1.3(5) ≤0.32
	2661(140)	14(1)	0.34(6)
	2537(140)	0	0
	1691(140)	22(6)	0.30(9)
	1391(140)	0	0
	*Main component of the IAS.

7

9

Acknowledgements

This work was supported by the Spanish MICINN grants FPA2008-06419-C02-01, FPA2011-24553; Centro de Excelencia Severo Ochoa del IFIC SEV-2014-0398; CPAN Consolider-Ingenio 2010 Programme CSD2007-00042; Junta para la Ampliación de Estudios Programme (CSIC JAE-Doc contract) co-financed by FSE; MEXT, Japan 18540270 and 22540310; Japan-Spain coll. program of JSPS and CSIC; Istanbul University Scientific Research Projects, Num. 5808; UK Science and Technology Facilities Council (STFC) Grant No. ST/F012012/1; Region of Aquitaine. R.B.C. acknowledges support by the Alexander von Humboldt foundation and the Max-Planck-Partner Group. We acknowledge the EXOGAM collaboration for the use of their clover detectors.