Neha Mehta

Karim Benzerara
email: karim.benzerara@sorbonne-universite.fr

Benjamin D Kocar
email: bkocar@exponent.com

Virginie Chapon

Sequestration of Radionuclides Radium-226 and Strontium-90 by Cyanobacteria Forming Intracellular Calcium Carbonates

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Introduction

Cyanobacteria are a phylogenetically and ecologically diverse group of photosynthetic bacteria, playing a vital role in the global cycling of numerous elements such as carbon (C), calcium (Ca) and phosphorus (P) [START_REF] Hutchins | Nutrient Cycles and Marine Microbes in a CO2-Enriched Ocean[END_REF] . In particular, their impact on the global carbon cycle is of significant interest as they sequester atmospheric CO2 into organic carbon and biogenic calcium carbonates (CaCO3) through calcification [START_REF] Riding | Cyanobacterial Calcification, Carbon Dioxide Concentrating Mechanisms, and Proterozoic-Cambrian Changes in Atmospheric Composition[END_REF] . In cyanobacteria, this process has long been considered as extracellular and non-biologically controlled. However, this paradigm was challenged by the recent discovery of several species of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) inclusions in very diverse environments. [START_REF] Benzerara | Intracellular Ca-Carbonate Biomineralization Is Widespread in Cyanobacteria[END_REF] Couradeau et al. first described this intracellular calcification in the cyanobacterium Gloeomargarita lithophora, isolated from an alkaline freshwater lake Alchichica microbialite, Mexico [START_REF] Couradeau | An Early-Branching Microbialite Cyanobacterium Forms Intracellular Carbonates[END_REF] . These ACC inclusions measured several hundreds of nanometers in diameter, were poorly crystalline and were composed of CO3 and Ca-Mg-Sr-Ba, with Ba/Ca and Sr/Ca atomic ratios in inclusions higher by factors of 1370 and 90 respectively relative to the solutions in which cells grew [START_REF] Benzerara | Intracellular Ca-Carbonate Biomineralization Is Widespread in Cyanobacteria[END_REF][START_REF] Couradeau | An Early-Branching Microbialite Cyanobacterium Forms Intracellular Carbonates[END_REF] . Recently, it was confirmed that cyanobacteria capable of forming intracellular ACC contained a much higher content of alkaline earth elements (AEE) than all other cyanobacteria [START_REF] De Wever | Evidence of High Ca Uptake by Cyanobacteria Forming Intracellular CaCO 3 and Impact on Their Growth[END_REF] . In addition to intracellular ACC inclusions, G. lithophora also forms intracellular polyphosphate inclusions (PolyP), polymers of orthophosphate which may serve as reserves of P and/or energy for cells [START_REF] Li | Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses[END_REF] .

Using stable isotopes, Cam et al. showed that G. lithophora preferentially accumulated Ba over Sr and finally Ca within intracellular PolyP and ACC. Currently, the origin of such a surprising selectivity remains unclear. The proven ability of G. lithophora to selectively sequester Sr over Ca within intracellular inclusions provides an intriguing microbial framework to examine if this ability extends to radioactive AEE such as radium ( 226 Ra) and radio-strontium ( 90 Sr). In the environment, 90 Sr and 226 Ra are present at trace concentrations, along with excess dissolved Ca. Whether the observed selective accumulation of AEE is extended to 90 Sr and 226 Ra at such low concentrations and in the presence of high concentrations of Ca remains to be explored.

Radium-226 is a naturally occurring radioactive alkaline earth element (AEE) with a half-life of 1600 y. It is one of the dominant soluble radionuclides present in groundwater [START_REF] Szabo | Occurrence and Geochemistry of Radium in Water from Principal Drinking-Water Aquifer Systems of the United States[END_REF] .

In the United States, 226 Ra activity in drinking water is regulated at 0.2 Bq L -1 8 . Potential exposure to Ra could originate from the disposal and management of large volumes of Rabearing waste streams associated with uranium mining, phosphate mining and milling operations, coal mining and power plants, and conventional and unconventional oil and gas extraction [START_REF]The Environmental Behaviour of Radium: Revised Edition[END_REF] . The Ra content of these waste streams can range from 100-1200 Bq L -1 , which far exceeds the regulatory limit of 2.2 Bq L -1 Ra in effluent waste stream 10 . During disposal and storage of relatively large volumes of these streams, leaching of Ra (and other toxic elements) could result in contamination of terrestrial and subsurface aquatic environments [START_REF] Warner | Impacts of Shale Gas Wastewater Disposal on Water Quality in Western PA[END_REF][START_REF] Chellam | Physical-Chemical Treatment of Groundwater Contaminated by Leachate from Surface Disposal of Uranium Tailings[END_REF] .

In contrast, radio-Strontium ( 90 Sr) is a radionuclide of sole anthropogenic origin produced by nuclear fission reactions. With its long half-life of 29 years, 90 Sr persists sufficiently long in the environment to warrant health concerns. The main sources of 90 Sr in the environment are the atmospheric tests of nuclear weapons conducted from 1945 to 1980's and nuclear accidents, such as Fukushima and Chernobyl [START_REF] Casacuberta | 90Sr and 89Sr in Seawater off Japan as a Consequence of the Fukushima Dai-Ichi Nuclear Accident[END_REF][START_REF] Vakulovsky | Cesium-137 and Strontium-90 Contamination of Water Bodies in the Areas Affected by Releases from the Chernobyl Nuclear Power Plant Accident: An Overview[END_REF] . Measured 90 Sr specific activity in the surface waters adjacent to the Fukushima Daiichi Nuclear Power Plant varied between 0.2 -400 kBq m -3 , which were about 4 orders of magnitude greater than the preaccident level (1 Bq m -3 ) [START_REF] Povinec | Radiostrontium in the Western North Pacific: Characteristics, Behavior, and the Fukushima Impact[END_REF] . Strontium is very soluble, highly mobile and is bioavailable in the environment. Its soluble form, Sr 2+ can be transferred from contaminated soils and water into living organisms through the food chain. A chemical analog of Ca 2+ , 90 Sr 2+ concentrates in bone tissues in the human body, increasing the probability of bone cancer after accumulation and prolonged exposure [START_REF]Selected Radionuclides Important to Low-Level Radioactive Waste Management[END_REF] .

Several physiochemical strategies have been developed for decontamination of 226 Ra and 90 Sr from effluent waste streams. However, in many cases their application is limited due to economic constraints [START_REF] Pagnanelli | Biosorption of Metal Ions on Arthrobacter Sp.: Biomass Characterization and Biosorption Modeling[END_REF] . In addition, the presence of high concentrations of Ca and other co-contaminants often present within waste streams limits the selective removal of 226 Ra and 90 Sr using the traditional schemes [START_REF] Volesky | Detoxification of Metal-Bearing Effluents: Biosorption for the next Century[END_REF][START_REF] Brower | Comparison of Ion-Exchange Resins and Biosorbents for the Removal of Heavy Metals from Plating Factory Wastewater[END_REF] . On the other hand, bioremediation strategies are gaining significant interest owing to their relative cost-effectiveness and ecofriendliness [START_REF] Lloyd | Bioremediation of Radioactive Waste: Radionuclide-Microbe Interactions in Laboratory and Field-Scale Studies[END_REF][START_REF] Gadd | Minerals and Microbes: Geomicrobiology and Bioremediation[END_REF][START_REF] Ruggiero | Actinide and Metal Toxicity to Prospective Bioremediation Bacteria[END_REF] . Various types of organisms including fungi, algae, cyanobacteria, and phytoplankton have been reported to retain 226 Ra and 90 Sr [START_REF]The Environmental Behaviour of Radium: Revised Edition[END_REF][START_REF] Tsezos | Adsorption of Radium-226 by Biological Origin Absorbents[END_REF] . Different pathways of sequestration can be involved. 226 Ra and 90 Sr can adhere to the surface of living and dead cells. Ligands (carboxyl, amine, hydroxyl, phosphate, and sulfhydryl groups) present in the cell membrane of both Gram-positive and Gram-negative bacteria can bind positively charged metals through adsorption [START_REF] Newsome | The Biogeochemistry and Bioremediation of Uranium and Other Priority Radionuclides[END_REF] . Some examples of such microorganisms adsorbing 226 Ra include dead microbial biomass of Pseudomonas aeruginosa, Nostoc carneum, Nostoc insulare, Oscillatoria geminanat, Spirullina laxissima, Pseudomonas fluorescens, Streptomyces nives, and mixed bacterial cultures from activated wastewater sludges [START_REF] Tsezos | Adsorption of Radium-226 by Biological Origin Absorbents[END_REF][START_REF] Pohl | Adsorption of Radionuclides (134Cs, 85Sr, 226Ra, 241Am) by Extracted Biomasses of Cyanobacteria (Nostoc Carneum, N. Insulare, Oscillatoria Geminata and Spirulina Laxis-Sima) and Phaeophyceae (Laminaria Digitata and L. Japonica; Waste Products from Alginat[END_REF] .

The dead biomass of bacterial strains isolated from areas with a high level of natural radiation, such as Citrobacter freundii, Chromobacterium, Chryseobacterium, and Corynebacterium also have been shown to adsorb 226 Ra [START_REF] Satvatmanesh | Biosorption of 226Ra in High Level Natural Radiation Areas of Ramsar, Iran[END_REF] . In the case of 90 Sr, studies have often been performed using stable Sr isotopes to examine the 90 Sr remediation potential of an organism. Some organisms such as the algae Scenedesmus spinosus and Oedogonium sp.

Nak 1001, the cyanobacteria Oscillatoria homogenea and Stigonema ocellatum NIES-2131, and the aquatic plant Egeria densa We2 have been reported to show high stable-Sr adsorption [START_REF] Liu | Biosorption of Strontium from Simulated Nuclear Wastewater by Scenedesmus Spinosus under Culture Conditions: Adsorption and Bioaccumulation Processes and Models[END_REF][START_REF] Fukuda | Global Searches for Microalgae and Aquatic Plants That Can Eliminate Radioactive Cesium, Iodine and Strontium from the Radio-Polluted Aquatic Environment: A Bioremediation Strategy[END_REF] . In addition to adsorption, many types of microorganisms may accumulate 226 Ra and 90 Sr within extracellularly or intracellularly precipitated biogenic minerals. The formation of biominerals occurs in response to localized changes in cellular or extracellular microenvironments. To our knowledge, there is no known micro-organism which has been reported to biomineralize 226 Ra. Several organisms have been screened for their ability to (co)-precipitate Sr. For example, the microalga Chlorella vulgaris, soil bacterium Sporosarcina pasteurii and the bacterium Halomonas sp. induce co-precipitation of strontianite (SrCO3) within extracellularly precipitated CaCO3 mineral phase; the desmid Closterium moniliferum forms celestite (SrSO4) within its vacuole by concentrating preferentially Sr and Ba over Ca [START_REF] Krejci | Selective Sequestration of Strontium in Desmid Green Algae by Biogenic Co-Precipitation with Barite[END_REF][START_REF] Lee | Photosynthetic Biomineralization of Radioactive Sr via Microalgal CO2absorption[END_REF][START_REF] Achal | Bioremediation of Strontium (Sr) Contaminated Aquifer Quartz Sand Based on Carbonate Precipitation Induced by Sr Resistant Halomonas Sp[END_REF][START_REF] Lauchnor | Bacterially Induced Calcium Carbonate Precipitation and Strontium Coprecipitation in a Porous Media Flow System[END_REF] . More recently, Cam et al. showed stable-Sr is selectively accumulated within intracellular ACC and PolyP inclusions in the cyanobacterium G. lithophora [START_REF] Cam | Selective Uptake of Alkaline Earth Metals by Cyanobacteria Forming Intracellular Carbonates[END_REF] . Owing to selectivity for Sr compared to Ca, G. lithophora forms carbonate with higher Sr content in comparison to other bacteria forming extracellular carbonates. This means that the mass of the decontamination by products will therefore be lower which may offer a cost advantage. Some of the non-photosynthetic bacteria which are known to precipitate CaCO3 efficiently, exhibit lower Sr removal efficiency in comparison to G. lithophora. For example, S. pasteurii removes upto 59% of Sr from the solution whereas G. lithophora removes 100% of Sr from the solution [START_REF] Lauchnor | Bacterially Induced Calcium Carbonate Precipitation and Strontium Coprecipitation in a Porous Media Flow System[END_REF][START_REF] Cam | Selective Uptake of Alkaline Earth Metals by Cyanobacteria Forming Intracellular Carbonates[END_REF] .

Despite screening of several organisms for removal of 90 Sr and 226 Ra, these studies have not demonstrated their potential for use in bioremediation. Indeed, research on 90 Sr bioremediation has mostly utilized stable-Sr isotope as an analog of 90 Sr and at relatively high concentrations. Owing to the radiotoxicity of 90 Sr, it is unclear whether the results demonstrated using stable Sr isotopes would be replicated to the same extent in presence of 90 Sr. Further, 226 Ra and 90 Sr removed from solution through adsorption to biomass is prone to rapid desorption upon changes in aqueous chemistry. Moreover, biomass is not selective and other divalent cations (e.g. Ca, Ba), which are often present at relatively high concentrations in 226 Ra and/or 90 Sr contaminated aqueous environments, may outcompete 226 Ra and/or 90 Sr for binding sites, resulting in low removal efficiency [START_REF] Ruggiero | Actinide and Metal Toxicity to Prospective Bioremediation Bacteria[END_REF][START_REF] Newsome | The Biogeochemistry and Bioremediation of Uranium and Other Priority Radionuclides[END_REF] . Thus, effectiveness of 226 Ra and 90 Sr bioremediation strategies will be dictated by (micro)organisms capability to selectively remove the radionuclide at trace concentration in the presence of competing elements while also tolerating ionizing radiation.

Here, the role of the cyanobacterium G. lithophora forming intracellular biominerals on sequestration of 226 Ra and 90 Sr at trace concentrations is evaluated. We selected G.

lithophora to evaluate as a potential bioremediation tool for removing 90 Sr and 226 Ra from solution for several reasons. Because of the photoautotrophic lifestyle of cyanobacteria, G. lithophora may exhibit tolerance to substantial levels of ionizing radiation [START_REF] Bailey | Photoprotection in Cyanobacteria: Regulation of Light Harvesting[END_REF] . Moreover, unlike other (micro)organisms, G. lithophora has been shown to preferentially sequester Sr over Ca within intracellular ACC and PolyP inclusions, despite their chemical similarity. These characteristics make G. lithophora a promising candidate for bioremediation of 226 Ra and 90 Sr. Proving the ability of G. lithophora to sequester 226 Ra and 90 Sr through a novel (co)-precipitation pathway will illustrate its promise for use in bioremediation efforts; also, owing to its widespread ecological distribution [START_REF] Ragon | 16S RDNA-Based Analysis Reveals Cosmopolitan Occurrence but Limited Diversity of Two Cyanobacterial Lineages with Contrasted Patterns of Intracellular Carbonate Mineralization[END_REF] , its cultivability and the availability of its genome sequence [START_REF] Moreira | Description of Gloeomargarita Lithophora Gen. Nov., Sp. Nov., a Thylakoid-Bearing, Basal-Branching Cyanobacterium with Intracellular Carbonates, and Proposal for Gloeomargaritales Ord[END_REF] , G. lithophora serves as an ideal model organism for examining underlying biochemical processes responsible for bioaccumulation of radioactive AEE in microbes. Accordingly, the goals of the current study are 1) to investigate and quantify 226 Ra and 90 Sr uptake by G. lithophora using laboratory batch incubations, and 2) to identify retention mechanism of 226 Ra and 90 Sr by G. lithophora.

EXPERIMENTAL SECTION:

Incubations and Culture Conditions

The cyanobacterial strain G. lithophora was obtained from Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, Paris, France and cultured in the BG-11 medium at 30 o C under continuous light (5-10 µmol m 2 s -1 ) as described by Moreira et al. [START_REF] Moreira | Description of Gloeomargarita Lithophora Gen. Nov., Sp. Nov., a Thylakoid-Bearing, Basal-Branching Cyanobacterium with Intracellular Carbonates, and Proposal for Gloeomargaritales Ord[END_REF] . The BG-11 medium contained 17.6x10 -3 M NaNO3, 0.23x10 -3 M K2HPO4, 0.3x10 -3 M MgSO4, 2.5x10 -4 M CaCl2, 3.1x10 -5 M citric acid, 2x10 -5 M ferric ammonium citrate, 3x10 -6 M ethylenediaminetetraacetic acid (EDTA), 5x10 -4 M NaHCO3, and trace metals [START_REF] Stanier | Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria[END_REF] . The optical density (OD) of the suspensions was measured at 730 nm; based on the previous work by Cam et al. [START_REF] Cam | Selective Uptake of Alkaline Earth Metals by Cyanobacteria Forming Intracellular Carbonates[END_REF] , the relationships between OD, cell density and cell dry mass of G. lithophora, were estimated as 9 x 10 7 cells. mL -1 OD unit -1 and 3.65 x 10 -4 g of dry mass.

OD unit -1 . mL -1 .

226

Ra uptake by G. lithophora 226 Ra uptake was measured during the growth stage of G. lithophora cultures, which were started at low cell density (ODt=0=0.039± 0.01). Pre-cultures used for inoculation were centrifuged at 5000 g for 15 min and the cell pellets were re-suspended in BG-11 amended with 226 Ra and total Ca as chloride salts at concentrations of 7.5 x 10 -10 M (i.e. 6153 Bq L -1 226 Ra) and 6.32 x 10 -4 M, respectively and buffered at pH 8 using HEPES buffer. Triplicate incubations were performed. Three types of controls were included in the experimental design:1) an abiotic control consisting of BG-11 medium amended with 7.5 x 10 -10 M of 226 Ra and 6.32 x 10 -4 M of Ca 2+ . 2) Inactivated cells control consisted of G. lithophora cells cultured in BG-11, killed by gamma irradiation ( 60 Co source, 55 h) and re-suspended at an OD of 1 in fresh BG-11 medium with 5.2 x 10 -10 M (i.e. 5600 Bq L -1 ) of 226 Ra and 2.5 x 10 -4 M of Ca 2+ . This control was used to quantify 226 Ra uptake by dead G. lithophora biomass and adsorption of 226 Ra to walls of the culture vessel. 3) Living cells of Synechocystis sp.

PCC 6803 (obtained from Pasteur Collection of Cyanobacteria) cultured in BG-11 and amended with 5.2 x 10 -10 M (i.e. 5600 Bq L -1 ) of 226 Ra and 2.5 x 10 -4 M of Ca 2+ . This species has been shown to be unable to biomineralize intracellular ACC [START_REF] Benzerara | Intracellular Ca-Carbonate Biomineralization Is Widespread in Cyanobacteria[END_REF] . The OD of Synechocystis sp. PCC 6803 cultures at the start of experiment was equal to 0.02 ± 0.002. Dissolved Ca 2+ concentration and residual 226 Ra activity in solution were measured in all solutions as a function of time. For dissolved Ca 2+ measurements, 100 µl of the culture were mixed with 2 ml of standard BG-11 medium and were filtered on 0.22 µm PES syringe filters. Filtrates were acidified with 3 mL of 3 % HNO3. Dissolved Ca 2+ concentrations were measured using an Agilent Inductively Coupled Optical Emission Spectrometer (ICP-OES).

Concentration of Ca 2+ was also measured in the reagent blank (2 mL BG-11 + 3 mL 3 % HNO3) to eliminate background signal.

For measurement of residual 226 Ra in solution, 500 µl of the culture were mixed with 2 ml of standard BG-11 medium and were filtered on 0.22 µm PES syringe filters. Filtrates were acidified with 3% HNO3 and counted using a Canberra High Purity Germanium Detector (HPGe) Gamma Spectrometer. Energy and efficiency calibration of gamma spectroscopy was performed using a mixed multi-nuclide Eckert & Ziegler™ aqueous gamma standard. 226 Ra was directly measured from its gamma peak at 186.5 keV.

90

Sr uptake by G. lithophora 90 Sr uptake by G. lithophora was studied following a different strategy. In this case, cell suspensions of G. lithophora at high cell density were used. The high cell density of the culture is expected to provide a faster uptake of 90 Sr. Cells previously grown in standard BG-11 to an OD of 1 were centrifuged at 8500 g for 10 min and the cell pellets were resuspended in standard BG-11 at an OD of 0.5 (i.e. 0.1825 g L -1 ). The suspensions were then amended with 8.8 x 10 -11 M of 90 Sr (i.e. 4 x 10 4 Bq L -1 ). Three types of controls were included in the experiment: 1) an abiotic control consisting of BG-11 medium amended with 8.8 x 10 -11 M of 90 Sr. 2) Inactivated cells control consisting in G. lithophora cells grown in BG-11 medium, freeze-dried and killed by autoclaving (121 °C in 20 min). Dried biomass was suspended at 0.2 g L -1 in BG-11 amended with 8.8 x 10 -11 M of 90 Sr. The protocol of cell inactivation was different in this case than for experiments with 226 Ra. However, both approaches were similarly efficient at providing dead cell controls which eventually show low sorption capabilities of 226 Ra and 90 Sr as detailed later in the manuscript 3) Living cells of Synechocystis sp. PCC 6803 grown in BG-11, collected by centrifugation at 8500 g for 10 min, re-suspended in fresh BG-11 at an OD of 0.5 and amended with 8.8 x 10 -11 M of 90 Sr.

Triplicate incubations were performed for all experimental treatments. Total 90 Sr and residual 90 Sr in solution were measured in all solutions after 0.5, 24 and 48 h incubation in the light. Temperature was maintained at 30°C within a MINITRON incubator (INFORS) with shaking at 100 rpm. For residual 90 Sr quantification, 1 mL of cell suspension was centrifuged at 8500 g for 10 min and the supernatant was filtered on a 0.22 m PVDF syringe filter. The volume of the filtered supernatant was measured as 930 ± 2 L. Two milliliters of scintillating solution (Ultima Gold TM , PerkinElmer) were added to the filtrates before counting on a Tri-Carb® 3100TR low activity liquid scintillation analyzer (PerkinElmer). For total 90 Sr content quantification, 930 µL of cell suspension were mixed with 2 mL scintillating solution and counted. 90 Sr standard solutions were prepared with the 90 Sr stock solution (LEA, Pierrelatte, France), diluted in BG-11 medium and counted as described for the other samples. The counts per second (CPS) values were corrected from the background activity, measured with a control made of 930 µL of BG-11 medium and 2 mL scintillation solution. The concentration of 90 Sr (Bq L -1 ) in the samples was derived from the standards.

Calculation of bioaccumulation of 90 Sr and 226 Ra

The mass of 90 Sr and 226 Ra accumulated by the cells at a time t and normalized by the cell mass was determined as follows:

For 226 Ra: and for 90 Sr:

where X denotes radionuclide activity (Bq L -1 ),  is the factor to convert activity of a radionuclide to mass units and t is the sampling time. The value of  for 226 Ra and 90 Sr is equal to 1.2x10 -13 (mol Bq -1 ) and 2.2x10 -15 (mol Bq -1 ).

RESULTS AND DISCUSSION

Kinetics of 90 Sr uptake by G. lithophora

In order to demonstrate the uptake of radionuclides by G. lithophora, we first monitored uptake of 90 Sr at high cell density to avoid any potential toxic effects on its growth by 90 Sr. The residual 90 Sr activity in solution during exposure of G. lithophora cells to 8.8 x 10 -11 M 90 Sr is shown in Figure 1. 90 Sr was rapidly removed from the solution in the presence of G. lithophora: the residual 90 Sr activity in the solution decreased from 46.243 ± 0.604 kBq L -1 to 0.753 ± 0.129 kBq L -1 within 1 h, corresponding to the sequestration of 98 % of the radionuclide by the cells. The presence of divalent ions (e.g. Ca 2+ , Mg 2+ ) in solution did not inhibit 90 Sr uptake by G. lithophora cells, consistent with results obtained with stable Sr [START_REF] Cam | Selective Uptake of Alkaline Earth Metals by Cyanobacteria Forming Intracellular Carbonates[END_REF] . Following the observed rapid decline of the residual 90 Sr activity in the solution, the rate of 90 Sr uptake by G. lithophora decreased. At the end of incubation (48 h), the residual 90 Sr activity reached 0.608 kBq L -1 , corresponding to 99 % of radionuclide removal by the cells. Here, removal of 90 Sr by G. lithophora occurred from solutions containing a high concentration of Ca, with an initial Ca/ 90 Sr molar ratio of 2.7x10 [START_REF] Li | Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses[END_REF] . Thus G. lithophora can efficiently sequester 90 Sr at trace concentrations despite the presence of a large excess of competing cations. Within abiotic controls, only 12% of 90 Sr was removed from solution within 5 hours, and 90 Sr activity remained constant thereafter for the duration of the experiment.

Kinetics of 226 Ra uptake by G. lithophora

Based on the promising uptake of 90 Sr exhibited by G. lithophora cells, the next step was to investigate whether the uptake of radiotoxic AEE could also occur during the growth stage of G. lithophora. For this purpose, we measured 226 Ra uptake upon the growth of G. lithophora cells starting at a low cell density.

The uptake of 226 Ra by G. lithophora was examined within low-cell density cultures for reasons described above, and occurred in two stages (Figure 2, left). In the first stage, 226 Ra was rapidly removed from solution, with 226 Ra activity decreasing from 5964 ± 192.2 Bq L -1 to 1227.68 ± 85.5 Bq L -1 in 144 h. This removal was accompanied by a slight decrease in dissolved Ca 2+ (Figure 2, right). In the second stage, the rate of removal of 226 Ra from the solution slowed down and residual 226 Ra activity in the solution further decreased to 82 Bq L -1 in 1392 h. The initial period of this 2 nd stage was marked with an appreciable

Retention mechanisms of 226 Ra and 90 Sr in G. lithophora

other mechanisms, we compared uptake of 226 Ra and 90 Sr between living and dead cells of G. lithophora. The average uptake of 226 Ra in inactivated cell control, composed of dead cells of G. lithophora, was equal to 0.8 ± 0.1 µg (g of dry mass) -1 ; in contrast the average226 Ra uptake by active G. lithophora cells reached 3.9 ± 0.9 µg (g of dry mass) -1 (Table 1Table 1). Likewise, the average uptake of 90 Sr by dead G. lithophora cells was equal to 8.87 ± 0.2 ng (g of dry mass) -1 ; in contrast the average 90 Sr uptake rate by active cells of G. lithophora measured 47.97 ± 1.5 ng (g of dry mass) -1 (Table 1). Second, the relationship between biomineralization and sequestration of 226 Ra and 90 Sr was tested by comparing uptake of 226 83 ± 14 % of 226 Ra over the same duration (Figure 3). Similarly, incubation over 24 h of the nonbiomineralizing Synechocystis at relatively high cell density (OD=0.5) retained 26 ± 1 % of 90 Sr, while G. lithophora retained 99.5 ± 10 % of the initial 90 Sr (Figure 3). While there could be differences in the cell surface composition between both strains, we argue that these differences are likely not substantial enough to induce 6 times more 226 Ra and 4 times more 90 Sr retention in G. lithophora than in Synechocystis PCC 6803. Altogether, these results suggest specific cellular processes are responsible for 226 Ra and 90 Sr retention by G. lithophora. One of the physicochemical processes that has been used widely for sequestering low levels of contaminants from aqueous solutions is adsorption. Here, based on Figure 3 and The dissolved concentration of Ca 2+ in 226 Ra uptake experiments by active cells of G. lithophora was higher than the concentration of dissolved Ca 2+ in the inactivated cell control and the Synechocystis cultures. Because of competing effects for adsorption between Ca and Ra, this implies that the degree of 226 Ra adsorption measured in the inactivated cell and by Synechocystis controls was relatively overestimated compared to that in the presence of active G. lithophora cells will likely be less in presence of excess Ca 2+ furnished in 226 Ra uptake experiments (Figure 2). Moreover, the cell normalized rate of uptake measured for 90 Sr is consistent with those measured by Cam et al. for stable Sr isotope and shown to be associated with intracellular uptake rather than adsorption [START_REF] Cam | Selective Uptake of Alkaline Earth Metals by Cyanobacteria Forming Intracellular Carbonates[END_REF] . These observations taken altogether suggests that mechanism(s) other than adsorption are involved in 226 Ra and 90 Sr uptake in G. lithophora.

Previously, TEM analyses of G. lithophora cells cultured in BG-11, amended with high initial concentrations of stable Ba and Sr isotopes showed that most of the Sr and Ba were sequestered within intracellular carbonates and to a lesser extent in intracellular polyphosphates. Unfortunately, TEM analysis was not possible here to assess 226 Ra and 90 Sr distribution within the cell because their concentrations were below the detection limits of EDXS [START_REF] Blondeau | Impact of the Cyanobacterium Gloeomargarita Lithophora on the Geochemical Cycles of Sr and Ba[END_REF] . Nevertheless, considering 226 Ra and 90 Sr chemical affinities and similarity to Ca and Ba, we propose that incorporation of 226 Ra and 90 Sr into intracellular carbonate (and polyphosphate) inclusions is likely the primary mechanisms for their sequestration in G.

lithophora. It remains a mystery why G. lithophora would sequester radiotoxic 226 Ra and 90 Sr elements within the cell. Unlike Ca, which is essential as a co-factor for cyanobacteria in the photosystem II complex [START_REF] Debus | The Manganese and Calcium Ions of Photosynthetic Oxygen Evolution[END_REF] , there is no documented biological function of 226 Ra, 90 Sr or Ba. Some studies have argued that trace amounts of non-essential elements can be assimilated together with their corresponding essential analogues [START_REF] Newsome | The Biogeochemistry and Bioremediation of Uranium and Other Priority Radionuclides[END_REF] . In fact, non-selective transport of Sr 2+ through cation channels have already been acknowledged by other studies [START_REF] Gupta | Radiostrontium Transport in Plants and Phytoremediation[END_REF] . Hence, 226 Ra and 90 Sr could be "mistaken" for its chemical analog Ca, an essential element, thereby resulting in their favorable uptake in G. lithophora. Alternatively, while this is not ascertained by the present experiments, selective uptake of 226 lithophora was not inhibited by Ca 2+ , present at approximately 6 orders of magnitude higher concentration than 226 Ra and 90 Sr. These observations favor involvement of another biochemical process at play in G. lithophora. Typically, ion selectivity between similar ions such Sr, Ra, Ba, Ca requires an ion transport pathway to have some specific binding sites over at least part of its length. Future studies characterizing the affinity and specificity of various transporters involved in uptake of AEE in G. lithophora along with measurement of 90 Sr and 226 Ra uptake by G. lithophora at varying Ca: 90 Sr/ 226 Ra ratio may shed some light on molecular processes involved in uptake of 90 Sr and 226 Ra by G. lithophora.

Maximum uptake of 226 Ra and 90 Sr by G. lithophora

Understanding the maximum uptake capacity of 226 Ra and 90 Sr by G. lithophora is an important criterion for its assessment as a potential bioremediation strategy. The maximum radionuclide uptake capacity by a microorganism is an elusive concept as a zero net uptake rate doesn't always signal maximum uptake capacity. For example, net uptake may appear to cease when the amount of radionuclide added to the solution is exhausted before the cells reach their maximum uptake capacity. In the case of 226 Ra, the total 226 Ra accumulated in G. lithophora over the total duration of incubation equals to 133 kBq (g of dry mass) -1 (Figure 2 and Table1). The OD decline after 792 h may possibly indicate cell lysis and/or cell aggregation, settling and/or attachment of cells to the flasks. Visual inspection of the cultures after 792 h did reveal some aggregation and settling of the cells (data no shown).

The decrease in the uptake of 226 Ra during the 2 nd stage may signal that the cells have reached maximum 226 Ra uptake capacity. To test whether this is the case, cultures of G. lithophora were grown in BG-11 containing 226 Ra at a concentration 25 times higher than the experiment here. The high 226 Ra concentration experiments showed that over 30 days 70% of 226 Ra was removed and 962 kBq (g of biomass) -1 226 Ra sequestered by G. lithophora (Figure S4). This implies that the decrease in 226 Ra uptake rate during 2 nd stage is not because cells reached maximum uptake capacity but for some other reasons yet to be discovered. Similar to 226 Ra, total 90 Sr accumulated by G. lithophora in 24 h was equal to 250 kBq (g of biomass) -1 (Figure 1 and Table1). To test whether this amount of 90 Sr sequestered was equal to maximum uptake capacity, culture of G. lithophora at high cell density were successively spiked with 90 Sr and uptake of 90 Sr measured in the supernatant.

The multiple spike experiments showed that 924 kBq (g of biomass) -1 was incorporated within G. lithophora over 24 hr (Figure S5). After 24 h, no further 90 Sr accumulation was measured as the cells exhausted the amount of 90 Sr provided. Together, these observations suggest that in the present study, measured 226 Ra and 90 Sr accumulation by G. lithophora is a lower estimate of its maximum uptake capacity.

Environmental Implications and Bioremediation

Both uptake capacity and selectivity play an important feature for assessment of an organism's effectiveness for 90 Sr and 226 Ra remediation. Tables 2 and3 Another aspect of bioremediation of radionuclides in the environment is the ability of microorganism to survive in the presence of ionizing radiation. The intracellular uptake of radiotoxic 226 Ra and 90 Sr could potentially result in oxidative damage in DNA, proteins and lipids due to the generation of reactive oxygen species. Cyanobacteria have been detected in radioactive areas surrounding Chernobyl nuclear reactor [START_REF] Ragon | Sunlight-Exposed Biofilm Microbial Communities Are Naturally Resistant to Chernobyl Ionizing-Radiation Levels[END_REF] , uranium mining operations [START_REF] Lusa | Microbial Communities in a Former Pilot-Scale Uranium Mine in Eastern Finland -Association with Radium Immobilization[END_REF] and in vicinity of hydrothermal spring caves [START_REF] Enyedi | Radioactive Environment Adapted Bacterial Communities Constituting the Biofilms of Hydrothermal Spring Caves (Budapest, Hungary)[END_REF] , suggesting that they may have some degree of resistance to ionizing radiation. However, little is known about G. lithophora's radiotolerance and response to oxidative stress and future experiments characterizing the viability of G. lithophora cells when exposed to elevated radiation could provide some insights in this regard.

G. lithophora sequesters226 Ra and 90 Sr within intracellular ACC inclusions at very low extracellular Ra/Ca concentration ratios, offering a unique solution to the general problem of remediating 226 Ra and 90 Sr in aqueous environments with naturally high Ca concentrations. Further studies investigating the stability of intracellular inclusions in environmentally relevant matrices and optimization of various process parameters such as maximum 226 Ra and 90 Sr uptake capacity, and tolerance to other metalloids present in the waste streams will be needed to ensure successful development of G. lithophora based bioremediation approach for 90 Sr and 226 Ra.

ASSOCIATED CONTENT

Figure 1 :

 1 Figure 1: Time evolution of residual 90 Sr activity in the solution during exposure of G. lithophora cells to 8.8 x 10 -11 M 90 Sr (▲) and in the abiotic control (Δ), both in BG-11. The dash lines denote the initial total activity of 90 Sr added to the cultures. When not visible, error bars are smaller than the symbols.

Figure 2 :

 2 Figure 2: (Left) Time evolution of residual 226 Ra activity in the solution during growth of G. lithophora with 8 x 10 -10 M 226 Ra ( ) and in abiotic control (o), both in BG-11. Error bars denote the standard deviations of triplicate measurements. The horizontal dash line indicates the activity of 226 Ra added to the BG-11 media used for growing G. lithophora. (Right) Time evolution of dissolved Ca 2+ ( ) concentration and OD (⎐) of cell suspension during growth of G. lithophora in BG-11 amended with 8 x 10 -10 M 226 Ra. When not visible, error bars are smaller than the symbols, except for the abiotic control, for which triplicate measurements were not performed. The vertical dash line indicates the beginning of the second stage of 226 Ra uptake.

  Ra and 90 Sr by living cells of the non-biomineralizing Synechocystis and living cells of G. lithophora. The OD of Synechocystis during the incubation period is shown in Figure S1. Living cells of the nonbiomineralizing Synechocystis retained 14 ± 2 % of 226 Ra in 336 h, while G. lithophora retained

Figure 3 :

 3 Figure 3: (Left) Time evolution of residual 226 Ra activity in the solution during growth of G. lithophora (•) and Synechocystis PCC 6803 (■) in BG-11. (Right) Time evolution of residual 90 Sr activity in high cell density suspensions (OD=0.5) of G. lithophora (▲) and Synechocystis PCC 6803 (Δ), both suspended in BG-11. The activity of 226 Ra added to BG-11 media is shown as dash line for G. lithophora cultures, and solid line for Synechocystis cultures. The activity of 90 Sr added to BG-11 media is shown as dash line for G. lithophora and Synechocystis cultures. Error bar shows ±σ.

  Ra and 90 Sr by G. lithophora should may also be considered because of two reasons: (1) Cam et al. showed that G. lithophora selectively uptakes stable Sr over Ca 33 ; (2) Uptake of 226 Ra and 90 Sr by G.

1 S2Figure S1 :

 1S1 figures. Data on OD of cultures in absence of radionuclide, saturation indices of Ca and Sr solids in the solution,226 Ra uptake by G. lithophora from BG-11 containing 210 kBq L -1

S4

Figure S4 :

 S4 Figure S4: Cell grown in BG-11 amended with 210 kBq L -1 226 Ra, and 6.32x10 -4 M Ca. The squares denote residual 226 Ra activity in the solution and circles denote OD of the cell suspension.

Figure S5 :

 S5 Figure S5: G. lithophora cells suspended at OD=0.5 in BG-11 amended with 90 Sr. The BG-11 was successively spiked with 90 Sr to measure maximum uptake of 90 Sr by G. lithophora.The total amount of 90 Sr uptake measured 160 kBqL -1 (40 kBqL -1 per spike), which considering an OD of 0.5 equals to 924 kBq (g of biomass)-1

Table 1 :

 1 Comparison of 226 Ra and 90 Sr uptake per unit biomass in different incubationsMicrobial processes of226 Ra and 90 Sr sequestration can broadly be categorized into

		Sample ID		226 Ra Retained* µg (g
	three	groups:	extracellular	biomineralization,	adsorption,	and	intracellular
	biomineralization 24 . Microbially induced extracellular biomineralization requires localized
	supersaturation of the solution with respect to Ca-carbonate phases. This results in
	precipitation of carbonate solids and 226 Ra and 90 Sr may co-precipitate within these

of dry mass) -1 90 Sr retained** ng (g of dry mass) -1

  solids and be removed from the solution. Using Visual Minteq[START_REF] Gustafsson | Visual MINTEQ 3.0 User Guide[END_REF] , we calculated the saturation state of the solution with respect to Ca-carbonate phases to verify if this mechanism was operational. Visual MINTEQ predicts that the experimental culture media amended with226 Ra and 90 Sr remained undersaturated with respect to Ca-carbonate phase(s) (FigureS2-S3). Moreover, the state of solution undersaturation with respect to Cacarbonate solids is consistent with the observation that dissolved 226 Ra and 90 Sr activity remained close to their initial activities in the abiotic control (Figure1-2). Therefore, extracellular precipitates of Ca-carbonate phases could not be a plausible sink of 226 Ra and

	Live G. lithophora cells	3.9 ± 0.9	47.9 ± 1.5
	Dead G. lithophora cells	0.8 ± 0.1	8.87 ± 0.2

* mass uptake is average uptake during the first stage of Ra retention. The error bar shows ±σ ** mass uptake is average uptake when cells were exposed to 8.8 x 10 -11 M of 90 Sr for 1 h. extracellular

90

Sr in our study.

Table 1 ,

 1 adsorption of 226 Ra and 90 Sr at walls of culture flasks, at the cell surface and dead G. lithophora biomass, removed 22% 226 Ra and 18% 90 Sr from solution, suggesting that adsorption alone cannot reproduce the degree of 226 Ra and 90 Sr retained by G. lithophora.

  compare90 Sr and226 Ra uptake capacity of G. lithophora with other organisms known to uptake90 Sr or226 Ra.For the screened bioremediation candidates, uptake capacities of 90 Sr and 226 Ra are reported assuming ideal conditions, i.e. in absence of Ca and other divalent ions. Such conditions are not representative of environments contaminated with 90 Sr and 226 Ra, which contain excess Ca and/or other divalent ions. Presence of Ca and other divalent ions in the solution may compromise the uptake of226 Ra and 90 Sr. For example, 40% of 90 Sr was removed by Vetiveria zizanoids from solution containing Ca/ 90 Sr molar ratio of 3.6 x 10 6 , while 94% removal was achieved in absence of Ca[START_REF] Singh | Phytoremediation of 137cesium and 90strontium from Solutions and Low-Level 29 Nuclear Waste by Vetiveria Zizanoides[END_REF] . In another example, Calotropis gigantea exhibited high accumulation of 90 Sr (104 ng g -1 ) but from a solution containing only 90 Sr[START_REF] Eapen | Phytoremediation of Radiostrontium ( 90 Sr) and Radiocesium ( 137 Cs) Using Giant Milky Weed (Calotropis Gigantea R.Br.) Plants[END_REF] .Whether the high 90 Sr removal efficiency of C. gigantea is maintained within an environmentally relevant matrix remains unknown. In the case of 226 Ra, the uptake exhibited by dead biomass was achieved under conditions where226 Ra was the only AEE present and therefore unlikely to be replicated in the environment to the same extent. Therefore, the current bioremediation strategies listed in Table 2-3 are inadequate in efficiently achieving selective removal of 226 Ra and 90 Sr in the presence of Ca.

Table 2 :

 2 Comparison of226 Ra uptake by G. lithophora with previously reported experimental data on226 Ra uptake by organisms.On the other hand, G. lithophora exhibits highest uptake of 90 Sr and226 Ra among all the active and dead microorganisms. Since OD values in experiments using 90 Sr were measured under the same conditions and setup as in Cam et al[START_REF] Moreira | Description of Gloeomargarita Lithophora Gen. Nov., Sp. Nov., a Thylakoid-Bearing, Basal-Branching Cyanobacterium with Intracellular Carbonates, and Proposal for Gloeomargaritales Ord[END_REF] , their conversion to cell numbers and cell mass are expected to be correct. Alternatively, we note that quantity of 22 only because presence of 226 Ra during growth of G. lithophora may induce physiological changes and alter the previously reported relationship between OD, cell density, and cell mass[START_REF] Moreira | Description of Gloeomargarita Lithophora Gen. Nov., Sp. Nov., a Thylakoid-Bearing, Basal-Branching Cyanobacterium with Intracellular Carbonates, and Proposal for Gloeomargaritales Ord[END_REF] .Unlike previously reported uptake studies, G. lithophora uptakes 90 Sr and 226 Ra in presence of excess Ca-initial molar ratio of Ca/ 90 Sr and Ca/ 226 Ra of the cultures equal 2.3x10 6 and 1.2x10 6 respectively. Interestingly,226 Ra accumulation by G. lithophora's overwhelmingly exceeds226 Ra uptake capacity of Dicranopteris linearis, identified as a226 Ra hyper-accumulator[START_REF] Chao | Accumulation of Radium in Relation to Some Chemical Analogues in Dicranopteris Linearis[END_REF] . The remarkable ability of G. lithophora to retain high activities of226 Ra and 90 Sr in comparison with other organisms makes it an attractive candidate for bioremediation applications in decontamination of low salinity radioactive wastewater

	Radium Uptake

226

Ra sequestered per unit biomass of G. lithophora cells might be approximate estimations * based on G. lithophora grown in elevated

226

Ra activity in a preliminary experiment (Figure

S4

) streams. Such a strategy could be envisioned as an ex-situ treatment, integrated (e.g. as a bioreactor) within an existing wastewater treatment plant. Compared to G. lithophora, other

90

Sr/ 226 Ra bioremediation candidates are mostly plants (Table2-3). There are practical considerations of using cyanobacterium G. lithophora vs. plants for bioremediation, such as harvesting of biomass, growth times, duration of decontamination and treatment of radioactive biomass. A complete development of G. lithophora based bioremediation would require determining these aspects in future studies.

Table 3 :

 3 Comparison of Sr uptake in G. lithophora with previously reported experimental data on 90 Sr uptake by

	organisms
		Uptake capacity (ng g -1 )	% removal
	Organism
	V. zizanoides (0mM Ca) 42	-	94%
	V. zizanoides (40 mM Ca) 42	-	40%
	Paspalum notatum 49 **	0.14	20%
	Sorghum halense 49 **	0.14	32%
	Panicum virginatum 49 **	0.15	23%
	Amaranthus retoflexus 50	0.000077	5%
	Calotropis gigantea 43 *	103.37	97%
	G. lithophora (this study) +	177	99%
	* Uptake achieved in 168 h
	** Concentration in plant tissue after 8 weeks of harvest

+

Based on multi-spike experiment (Figure

S5

)

Ra and 90 Sr sequestration by G. lithophora. First, to distinguish adsorption to dead cells from

Ra, and data on uptake of 90 Sr by G. lithophora during successive spike experiment.

ACKNOWLEDGMENTS

We would like to thank Timothy McClure (MIT) for his assistance with ICP-OES, Ryan Samz and Mitchell S Galanek, MIT Environmental Health and Safety, for supplying 226 Ra stock solutions, radiation protection equipment and safety expertise. We thank Fériel Skouri-Panet and Margot Coutaud (IMPMC) for assistance in the Sr experiments. The authors declare no competing financial interest.

REFERENCE


Xochilt Gutierrez

Federica Schiavi

Hans Keppler
email: hans.keppler@uni-bayreuth.de

The adsorption of HCl on volcanic ash

Keywords: Adsorption, ash, hydrogen chloride, volcanic gases, ozone

Understanding the interaction between volcanic gases and ash is important to derive gas compositions from ash leachates and to constrain the environmental impact of eruptions. Volcanic HCl could potentially damage the ozone layer, but it is unclear what fraction of HCl actually reaches the stratosphere. The adsorption of HCl on volcanic ash was therefore studied from -76 to + 150 ˚C to simulate the behavior of HCl in the dilute parts of a volcanic plume. Finely ground synthetic glasses of andesitic, dacitic, and rhyolitic composition as well as a natural obsidian from Vulcano (Italy) served as proxies for fresh natural ash. HCl adsorption is an irreversible process and appears to increase with the total alkali content of the glass.

Adsorption kinetics follow a first order law with rate constants of 2.13 . 10 -6 s -1 to 1.80 . 10 -4 s -1 in the temperature range investigated. For dacitic composition, the temperature and pressure dependence of adsorption can be described by the equation ln c = 1.26 + 0.27 ln p -715.3/T, where c is the surface concentration of adsorbed HCl in mg/m 2 , T is temperature in Kelvin, and p is the partial pressure of HCl in mbar. A comparison of this model with a large data set for the composition of volcanic ash suggests that adsorption of HCl from the gas phase at relatively low temperatures can quantitatively account for the majority of the observed Cl concentrations. The model implies that adsorption of HCl on ash increases with temperature, probably because of the increasing number of accessible adsorption sites. This temperature dependence is opposite to that observed for SO 2 , so that HCl and SO 2 are fractionated by the adsorption process and the fractionation factor changes by four orders of magnitude over a temperature range of 250 K. The assumption of equal adsorption of different species is therefore not appropriate for deriving volcanic gas compositions from analyses of adsorbates on ash. However, with the experimental data provided here, the gas compositions in equilibrium with the ash surfaces can be calculated. In particular, for dacitic composition, the molar ratio of S/Cl adsorbed to the ash surface is related to the molar S/Cl ratio in the gas phase according to the equation ln (S/Cl) adsorbed = 2855 T -1 + 0.28 ln (S/Cl) gas -11.14. Our data also show that adsorption on ash will significantly reduce the fraction of HCl reaching the stratosphere, only if the initial HCl content in the volcanic gas is low (< 1 mole %). For higher initial HCl concentrations, adsorption on ash has only a minor effect. While HCl scavenging by hydrometeors may remove a considerable fraction of HCl from the eruption column, recent models suggest that this process is much less efficient than previously thought.

Our experimental data therefore support the idea that the HCl loading from major explosive eruptions may indeed cause severe depletions of stratospheric ozone.

Introduction

The short-term climatic impact of volcanic eruptions is mostly due to the interaction of sulfur and halogen species in volcanic gases with the atmosphere. The surface cooling observed after major explosive eruptions is caused by the injection of sulfur dioxide into the stratosphere, where it is photochemically oxidized to sulfate aerosols.

These aerosols backscatter sunlight and may remain in the stratosphere for months or even years (e.g. [START_REF] Mccormick | Atmospheric effects of the Mt Pinatubo eruption[END_REF][START_REF] Robock | Volcanic eruptions and climate[END_REF]. Ozone destruction is another potential effect of large explosive eruptions, but it is much less understood than the surface cooling effect. [START_REF] Black | Acid rain and ozone depletion from pulsed Siberian Traps magmatism[END_REF] suggested that the end-Permian mass extinction partially resulted from ozone depletion associated with the Sibirian Trap magmatism. Measurable depletions in stratospheric ozone were observed after the 1991 Mt. Pinatubo eruption [START_REF] Brasseur | Mount Pinatubo aerosols, chlorofluorocarbons, and ozone depletion[END_REF]Granier 1992, Solomon et al. 1998). They were attributed to the catalytic effect of sulfate aerosol surfaces, which may enhance the concentration of active chlorine species involved in ozone destruction [START_REF] Solomon | Increased chlorine dioxide over Antarctica caused by volcanic aerosols from Mount Pinatubo[END_REF][START_REF] Hofmann | Ozone loss in the lower stratosphere over the United States in 1992 -1993: Evidence for heterogeneous chemistry on the Pinatubo aerosol[END_REF]). However, another mechanism could be the direct injection of volcanic hydrogen chloride (HCl) into the stratosphere. A study by [START_REF] Tabazadeh | Stratospheric chlorine injection by volcanic eruptions: HCl scavenging and implications for ozone[END_REF] suggested that this mechanism is inefficient, because hydrogen chloride would be nearly completely washed out of the eruption column by condensing water. More recent models of eruption plumes [START_REF] Textor | Injection of gases into the stratosphere by explosive volcanic eruptions[END_REF], however, show that most of the water in the plume is present as solid ice, which greatly reduces the efficiency of HCl scavenging. Moreover, most of the ice particles are lifted upwards, so that more than 25 % of the HCl originally contained in the plume may reach the stratosphere. This prediction was confirmed for the case of a high-latitude eruption by direct sampling of a volcanic cloud from Hekla volcano [START_REF] Rose | Atmospheric chemistry of a 33-34 hour old volcanic cloud from Hekla Volcano (Iceland): Insights from direct sampling and the application of chemical box modeling[END_REF], Millard et al. 2006). Spectroscopic observations also suggested an increase of 40 % in the total column of HCl above 12 km altitude after the 1982 El Chicón eruption [START_REF] Mankin | Increased stratospheric hydrogen chloride in the El Chicon cloud[END_REF], see also [START_REF] Woods | Halite particles injected into the stratosphere by the 1982 El Chicón eruption[END_REF], while at most a slight increase in stratospheric HCl was observed after the 1991 Mt. Pinatubo eruption (Mankin et al. 1992, Wallace and[START_REF] Wallace | The effect of the Pinatubo cloud on hydrogenchloride and hydrogen-fluoride[END_REF]. [START_REF] Kutterolf | Combined bromine and chlorine release from large explosive volcanic eruptions: A threat to stratospheric ozone?[END_REF] estimated the Cl and Br release from 14 large explosive eruptions in Nicaragua in the last 70 00 years and concluded that most of these eruptions likely caused significant ozone destruction in the stratosphere. On the other hand, elevated HCl concentrations are only rarely detected in ice cores; this may, however, be due to the volatilization of HCl in the presence of H 2 SO 4 [START_REF] Wagnon | Loss of volatile acid species from upper firn layers at Vostok, Antarctica[END_REF][START_REF] Zdanowicz | Mount Mazama eruption: Calendrical age verified and atmospheric impact assessed[END_REF][START_REF] Thamban | Aerosol perturbations related to volcanic eruptions during the past few centuries as recorded in an ice core from the Central Dronning Maud land[END_REF]. The limited data available may imply that the stratospheric HCl yield of eruptions varies considerably, depending on initial gas composition and on the processes occurring in the eruption column.

Hydrogen chloride (HCl) concentrations in volcanic gases typically range from < 0.05 to > 5 mole %, with most data probably in the 0.1 to 1 mole % range [START_REF] Symonds | Volcanic-gas studies: Methods, results, and applications[END_REF][START_REF] Fischer | Fluxes of volatiles (H 2 O, CO 2 , N 2 , Cl, F) from arc volcanoes[END_REF]). Relatively high HCl concentrations and HCl/SO 2 ratios are often found in arc volcanoes. Estimated annual fluxes of HCl vary widely, from 0.4 to 170 Tg/a [START_REF] Symonds | Contributions of Cl-and F-bearing gases to the atmosphere by volcanoes[END_REF][START_REF] Halmer | The annual volcanic gas input into the atmosphere, in particular into the stratosphere: a global data set for the past 100 years[END_REF]. [START_REF] Pyle | Halogens in igneous processes and their fluxes to the atmosphere and oceans from volcanic activity: A review[END_REF] used a compilation of measured HCl/SO 2 ratios and total SO 2 fluxes to estimate the HCl flux from arc volcanoes at 4.3 ± 1 Tg/a, which is believed to dominate the global flux.

However, the flux of HCl reaching the stratosphere is certainly much lower, because only large eruptions penetrate the tropopause and processes in the eruption column may deplete HCl. While the depletion by water and ice has already been investigated in some detail, little is known about the possible effect of HCl adsorption on volcanic ash [START_REF] Rose | Scavenging of volcanic aerosols by ash: Atmospheric and volcanologic implications[END_REF]. [START_REF] Ayris | HCl uptake by volcanic ash in the high temperature eruption plume: Mechanistic insights[END_REF] studied the interaction of HCl with ash under near-vent, high-temperature conditions (200 -800 ˚C). Only for alkali-rich tephrite and phonolite glasses, reaction with HCl to NaCl was observed, but not for dacite and rhyolite. The very short residence time of ash at near-vent conditions during an explosive eruption may further limit the extent of adsorption at high temperature. This suggests that HCl adsorption, if it is an important process at all, must occur in the low-temperature portion of the dilute plume. We therefore studied the adsorption of HCl on ashes of rhyolitic, dacitic, and andesitic composition from -76 to + 150 ˚C at variable HCl partial pressures covering the plausible conditions in a dilute volcanic plume.

Experimental methods

Fe-free synthetic glasses of rhyolitic, dacitic, and andesitic composition were used as proxies for fresh volcanic ash. The glasses were prepared from homogenized, stoichiometric mixtures of analytical grade SiO 2 , Al(OH) 3 , MgO, CaCO 3 , Na 2 CO 3 , and K 2 CO 3 . After decarbonation in a platinum crucible, the charge was melted at 1600 ˚C for 1 hour and quenched in deionized water, yielding a clear glass with some bubbles. Microprobe analyses are given in Table 1. The glass was then dried and ground four times for seven minutes each in a planetary mill. No liquid was added to avoid adsorption on fresh surfaces. The surface area of the powders was determined with a Quantachrome instrument by measuring the BET adsorption isotherm of krypton at 77 K. To constrain the isotherm, seven data points were measured in a range of pressures ranging from 5 to 30 % of the condensation pressure of Kr. 100 -200 mg of sample were outgassed at 200 ˚C for 24 hours before the measurement.

The specific surfaces obtained were in the range of 1.5 -4.9 m 2 /g. These numbers are quite comparable to those obtained for fine (< 100 m) natural ash (mostly 1.1 -2.1 m 2 /g for a sieved fraction < 100 m; Demelle et al. 2005;4.3 m 2 /g for phreatomagmatic ash from Eyafjallajökull; [START_REF] Gislason | Characterization of Eyjafjallajökull volcanic ash particles and a protocol for rapid risk assessment[END_REF].

Adsorption measurements were carried out in a glass apparatus equipped with a Vacuubrand DVR 5 pressure gauge, following the procedure described by [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF]. In brief, 30 -50 g of the sample powder were placed in a sample tube and the entire system was evacuated. HCl gas was then filled into a reservoir flask up to the desired pressure. Once the valve between the sample tube and the reservoir was opened, an instantaneous pressure drop occurred due to the expansion of the gas into the sample tube, followed by a slow decrease in pressure due to the adsorption of HCl on the sample surface. From the known volumes of the system, the measured pressure drop and the temperature, the amount of adsorbed HCl can be calculated. All required formulae are given by [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF]. For calculating the amount of adsorbed HCl, the ideal gas law was used, which is a very good approximation for HCl under the low pressures and the temperatures of interest.

Once the pressure in the system had stabilized, the valve between sample and gas reservoir was closed and the pressure in the reservoir was increased. After opening the valve again, the next point of the adsorption isotherm was measured. This procedure was repeated until the pressure in the system reached nearly one bar.

Desorption experiments started from close to one bar by decreasing the pressure in the reservoir with a vacuum pump. After opening the valve to the sample, pressure first increased, due to expansion of the gas from the sample tube to the reservoir, followed by a slow increase due to desorption of HCl from the sample surface. After pressure stabilized, the valve between reservoir and sample was closed and the procedure was repeated, until the pressure in the reservoir was close to zero mbar. Measuring an entire adsorption and desorption cycle for a sample took several weeks. Experiments at 150 ˚C were carried out by immersing the sample tube in an oil bath; for experiments at -76 ˚C, the sample tube was cooled with a mixture of dry ice and acetone. Main sources of error in the surface concentrations are the specific surfaces of the powders, the pressures, and the volumes in the system.

Wet chemical analyses were carried out in order to confirm the measured HCl surface concentrations. About 10 g of HCl-loaded glass sample were mixed with 100 ml of distilled water and stirred for 24 hours in a closed Erlenmeyer flask. The solution was then filtered off and some NaHCO 3 added to adjust to neutral pH. Chloride was then determined by titrating against a 0.1 N AgNO 3 solution with Ag 2 CrO 4 as endpoint indicator (Mohr method). Surface concentrations (in mg/m 2 ) were then obtained by dividing the mass of measured HCl by the surface area of the sample according to the BET data.

Results

Kinetics of adsorption

Figure 1 shows the experimentally observed decrease of pressure during an adsorption experiment. For every adsorption step, the data can be described by an equation (p -p eq ) = (p 0 -p eq ) e -t

(1)

where p is pressure, p eq is the equilibrium pressure, and p 0 is the initial pressure after opening the valve between gas reservoir and sample;  is the rate constant and t is time. Values of p eq and of  were obtained by fitting equation (1) to the data for every adsorption step. Since in the ideal gas law, pressure is proportional to the number of moles in the gas phase, (p -p eq ) is proportional to the number of gas atoms n that still will be absorbed and (p 0 -p eq ) is proportional to the initial number of gas atoms n 0 that ultimately will end up on the sample surface. The above equation ( 1) is therefore equivalent to a first-order-law of reaction kinetics n = n 0 e -t (2)

with the same rate constant as in equation ( 1).

Experimentally determined rate constants are compiled in Table 2; measured values for  range from 2.13 . 10 -6 s -1 to 1.80 . 10 -4 s -1 . The timescale for reaching equilibrium is in the order of magnitude of the inverse of these rate constants. For dacite at room temperature, a clear decrease of  with the initial gas pressure of the adsorption experiment is observed. This may be related to the fact that in the experiments at higher pressure, the most reactive surface sites had already been saturated with HCl in the previous steps of the adsorption experiment. In other words, with increasing surface coverage of the sample, the rate of adsorption decreases. This effect is not so clearly seen for the other samples. Interestingly, the rate constants for HCl adsorption on dacite at -76 ˚C are in most cases higher than at room temperature. This is unexpected, as reaction rates should generally increase with temperature.

However, as will be discussed below, only the most reactive surface sites seem to be accessible for HCl adsorption at -76 ˚C, and this higher reactivity probably also translates into higher rate constants. Desorption experiments also followed a firstorder-kinetics, with rate constants similar to those for adsorption on the same sample.

However, only very little desorption was generally observed and therefore, desorption kinetics will not be further discussed.

Adsorption isotherms

Adsorption and desorption isotherms of HCl for andesitic, dacitic and rhyolitic glasses are shown in Figure 2. With increasing pressure, the amount of HCl adsorbed on the surface first increases sharply and then levels off and reaches a plateau. This behavior corresponds to a type I adsorption isotherm in the Brunauer classification [START_REF] Brunauer | On a theory of the van der Waals adsorption of gases[END_REF], which is typical for a chemisorption process, where the adsorbed molecule forms strong chemical bonds with the substrate. Above about 500 mbar, there is a slight further increase in surface concentration that may indicate minor multilayer adsorption.

The desorption isotherms show that the adsorption process is nearly irreversible, i. e.

the surface concentration of HCl remains nearly constant when pressure is decreased to zero mbar. This behavior is again consistent with a chemisorption process involving the formation of strong chemical bonds between HCl and the substrate. The HCl surface concentration observed for andesitic and rhyolitic glasses are rather similar, while those for the dacite glass are 2 to 3 times higher. Inspection of Table 1 shows that this effect cannot be due to the SiO 2 , Al 2 O 3 , CaO or MgO contents of the glass, since the percentage of these components in the dacite glass is between that for rhyolite and andesite. However, the total alkali (Na 2 O + K 2 O) content of the dacite is significantly higher than for the other two compositions. Since the alkalis are the strongest bases in the system, it is plausible that they enhance the reaction with the acidic HCl molecule. This is consistent with the enhanced reactivity of alkali-rich glasses with HCl vapor at high-temperature, near vent conditions [START_REF] Ayris | HCl uptake by volcanic ash in the high temperature eruption plume: Mechanistic insights[END_REF].

Note, however, that the alkali contents of the synthetic glasses used for this study are not necessarily representative of a global average of rhyolite or dacite compositions and individual rhyolites may very well have higher total alkalis than some dacites.

The temperature dependence of HCl adsorption was explored for dacite, by carrying out additional experiments at -76 ˚C and at 150 ˚C. The results are shown in Figure 3.

Adsorption increases with temperature, probably because at low temperature adsorption occurs only on some particularly reactive sites, with the number of available sites increasing with temperature. This is a typical behavior sometimes observed for chemisorption, where the formation of chemical bonds requires some activation energy that can only be overcome above a certain temperature (e.g. [START_REF] Batzill | Surface science studies of gas sensing materials: SnO 2[END_REF][START_REF] Duran-Munoz | Analysis of the CO 2 chemisorption reaction mechanism in lithium oxosilicate (Li 8 SiO 6 ): a new option for high-temperature CO 2 capture[END_REF].

The surface concentrations of HCl remaining on the samples after the end of the desorption experiments, as derived from the measurements of the isotherms, were checked by argentometric titration of leachate solutions, with generally good agreement (Supplementary Table S1). Only the dacite sample from the experiment at room temperature may have lost some HCl by handling on humid air before the argentometric analysis. For the comparison, the surface concentrations were converted into wt. % using the specific surfaces given in Table 1.

A regression model for the adsorption of HCl on dacite glass

Schmauss and Keppler (2014) showed that the temperature and pressure dependence of the adsorption of SO 2 on volcanic ash may be described by a modified Freundlich

isotherm ln c = A T -1 + B ln p + C (3)
where c is the surface concentration in mg/m 2 , T is temperature in K and p is the partial pressure of the absorbed gas in mbar. A least squares fit of all the experimental data for HCl adsorption on dacite glass using the same equation yielded A = -715.3 ± 55.3, B = 0.27 ± 0.03, and C = 1.26 ± 0.29, with R 2 = 0.93. A comparison of measured and predicted surface concentrations of HCl is shown in Figure 4.

HCl adsorption on a natural obsidian sample

In order to test whether the adsorption data obtained by using synthetic samples are also applicable to natural, Fe-bearing volcanic glasses, we also measured the adsorption of HCl on a natural alkali-rhyolitic obsidian from the 1739 AD eruption of Vulcano (Aeolian Islands, Italy; see Table1). This obsidian contains a small fraction of feldspar microlites. In adsorption experiments at room temperature, both the kinetics of adsorption and the adsorption isotherm were found to be quite similar to the synthetic glasses (Fig. 5). The HCl surface concentrations observed for the Vulcano obsidian are somewhat higher than for the synthetic rhyolite and resemble more those for synthetic dacite (Fig. 2). This is, however, entirely consistent with the elevated bulk alkali content in this sample, supporting the idea that this is the primary compositional parameter influencing HCl adsorption.

Discussion

Applicability of the experimental data to adsorption in volcanic plumes

The experiments in the present study were carried out in the presence of HCl alone, while in the volcanic plume, H 2 O is usually the most abundant component, together with smaller amounts of SO 2 , HCl and traces of other gases. CO 2 may be an abundant species as well, but since the CO 2 molecule lacks a permanent dipole moment, its absorption on surfaces can likely be ignored. The much more important question is therefore, whether the data obtained in the present study may be applied to the situation in a volcanic plume, where HCl is a minor component compared to H 2 O.

Very likely this is possible, because the glass powders used in the experiments described here were exposed to air of average humidity at ambient conditions during the preparation of the experiments. Both studies on synthetic silicate glasses and of volcanic ashes show that under ambient conditions and also in a volcanic plume, the surface of a glass particle will be covered with a complete layer of adsorbed H 2 O molecules [START_REF] Razouk | The adsorption of water vapor on glass surfaces[END_REF]Salem 1948, Delmelle et al. 2005). Accordingly, there should be no difference in HCl surface concentration between the glass powders studied here in pure HCl alone and the adsorption of HCl on ash in the volcanic plume.

Dissolution of HCl gas in liquid water adhering to ash particles is a separate effect, which will be discussed below.

Figure 6 compares Cl concentrations adsorbed on the surface of volcanic ashes from more than 40 individual eruptions with the experimental data for dacite glass obtained in this study. To convert surface concentrations (in mg/m 2 ) into bulk concentrations (mg Cl / kg ash), the specific surface area of the ash has to be assumed. Figure 6 shows predicted maximum bulk concentrations (vertical lines) for two different specific surfaces of 1 m 2 /g, which is at the low end of this parameter for fine ash (Demelle et al. 2005;[START_REF] Gislason | Characterization of Eyjafjallajökull volcanic ash particles and a protocol for rapid risk assessment[END_REF]) and 0.1 m 2 /g, more characteristic for coarser ash (see supplementary Table S2 for examples). The vertical lines in Figure 6 give maximum concentrations achievable at a given specific surface and temperature at high HCl partial pressures. However, inspection of Figure 3 shows that similar concentrations may already be reached at low HCl partial pressures (10 -100 mbar) realistic for a volcanic plume. Figure 6 shows that adsorption of HCl from the gas phase can account for the vast majority of the observed Cl concentrations. Only the data for Irazu and San Miguel appear anomalously high, but they could still be explained by surface adsorption if the specific surface of these ashes were higher, in the order of 2 -4 m 2 /g, within the range confirmed by measurements on fine ashes In a volcanic plume, ash may interact with aerosols or become coated by films of liquid water or ice. [START_REF] Rose | Scavenging of volcanic aerosols by ash: Atmospheric and volcanologic implications[END_REF] suggested that the accumulation of aerosol particles, particularly of H 2 SO 4 is a main source for adsorbed material on ash. However, H 2 SO 4 aerosols are usually only a minor species, subordinate to SO 2 in volcanic plumes close to the vent (e.g. [START_REF] Mather | Oxygen and sulfur isotopic composition of volcanic sulfate aerosol at the point of emission[END_REF]. [START_REF] Delmelle | Gas/aerosolash interaction in volcanic plumes: New insights from surface analyses of fine ash particles Earth Planet[END_REF] see also [START_REF] Gislason | Characterization of Eyjafjallajökull volcanic ash particles and a protocol for rapid risk assessment[END_REF]) detected Cl and S on the surface of ash particles by X-ray photoelectron spectroscopy, but not by SEM. They concluded that Cl and S form coatings with a thickness of a few nm at most; they further suggested that this observation together with the observed surface depletion in Na, K, Mg, and Ca argues against the deposition of material via aerosols, but is more consistent with precipitation from a liquid film. However, their data are equally consistent with our model of direct adsorption from a gas phase, since the shape of the adsorption isotherms indeed suggests the formation of a mono-molecular layer of HCl on the ash surface. Coating of ash particles by water certainly occurs in some parts of the plume, but this process is reversible. Once the water evaporates, the dissolved HCl and SO 2 will also be lost, unless some surface reaction has occurred. However, if the surface is in equilibrium with the liquid film and the liquid is in equilibrium with a gas phase, then the surface is also in direct equilibrium with the gas. In other words, this process should lead to the same surface concentration as observed in our experiments. The fact that surface reactions, but not the solubility in liquid water determine the origin of leachable material on ash surfaces is also supported by the observation of a systematic offset between S/Cl ratios adsorbed on ash and S/Cl ratios in rainwater [START_REF] Edmonds | Rainwater and ash leachate analysis as proxies for plume chemistry at Soufrière Hills volcano, Montserrat[END_REF]. If the relative solubility of SO 2 and HCl in liquid water somehow determined the S/Cl ratio on the ash surface, this ratio should be the same as in rainwater that had interacted with the plume.

Deriving volcanic gas compositions from adsorbate analyses

The analysis of volatile species adsorbed on fresh ash surfaces is a convenient and frequently used method to infer volcanic gas compositions (e.g. [START_REF] Rose | Scavenging of volcanic aerosols by ash: Atmospheric and volcanologic implications[END_REF][START_REF] Williams | Eruption of the Nevado des Ruiz volcano, Colombia on 13 November 1985: Gas flux and fluid geochemistry[END_REF][START_REF] Armienta | Geochemistry of ash leachates during the 1994-1996 activity of Popocatépetl volcano[END_REF][START_REF] De Hoog | Sulfur and chlorine degassing from primitive arc magmas: temporal changes during the 1982-1983 eruptions of Galunggung (West Java, Indonesia)[END_REF][START_REF] Armienta | Chemistry of ash-leachates to monitor volcanic activity: An application to Popocatépetl volcano, central Mexico[END_REF][START_REF] Bagnato | Leachate analyses of volcanic ashes from Stromboli volcano: A proxy for the volcanic gas plume composition?[END_REF], 2013). Usually, the implicit assumption is made that the concentration ratio of adsorbed S, Cl, and F is somehow comparable to the molar ratio of the corresponding species in the gas phase of the plume. Our experimental data, together with published results for the adsorption of SO 2 on volcanic ash [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF] where S/Cl now refers to the molar ratio in the gas and on the glass surface.

Figure 7 shows the ratio of S/Cl on dacitic ash surfaces, as predicted by equation 7 for a range of temperatures and gas compositions. Obviously, the adsorbed S/Cl ratio is a very strong function of temperature. In fact, the temperature effect is much stronger than the effect of the S/Cl ratio in the gas. For example, near 0 ˚C, increasing the S/Cl ratio in the gas by a factor of 10 has the same effect on the absorption as reducing temperature by less than 20 ˚C. The relatively small effect of gas composition is due to the factor of 0.28 in front of the ln (S/Cl) gas term in equation ( 7); it implies that under otherwise equal conditions, (S/Cl) in the adsorbate only increases by a factor of (S/Cl) gas 0.28 , i.e. a 10-fold increase in this ratio in the gas phase only translates into a less than 2-fold increase in the adsorbate. The 0.28 exponent is related to the effect seen in Figure 2 that the amount of adsorbed HCl first increases sharply at low p HCl , but then levels off at higher p HCl . The strong effect of temperature on the adsorbed S/Cl ratio is obviously related to the opposite temperature dependence for HCl and SO 2 adsorption. The decrease of adsorption with temperature for SO 2 [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF] is the normal behavior expected for adsorption. This is because in the expression for the Gibbs free energy G = H -TS for the adsorption reaction, the -TS term increases with temperature and should therefore shift the equilibrium to the side with higher entropy; the entropy of a molecular species is always higher in the gas phase than adsorbed on a surface. The increase in HCl adsorption with temperature is therefore very unusual, but this kind of behavior is sometimes observed for chemisorption (e.g. [START_REF] Batzill | Surface science studies of gas sensing materials: SnO 2[END_REF][START_REF] Duran-Munoz | Analysis of the CO 2 chemisorption reaction mechanism in lithium oxosilicate (Li 8 SiO 6 ): a new option for high-temperature CO 2 capture[END_REF], i.e. if the adsorption process involves the formation of strong chemical bonds. In such a situation, an activation energy may have to be overcome for the reaction, so that only a fraction of sites is involved in the adsorption process at low temperature, while this fraction increases at higher temperatures. Once all possible surface sites are activated, adsorption should decrease upon further increase with temperature, because of the thermodynamic argument mentioned above. Accordingly, the data presented here for HCl adsorption and equation 7 should not be extrapolated to temperatures far above those for which they were calibrated (i.e. beyond 150 ˚C).

The strong temperature effect on adsorption as shown in Figure 7 needs to be taken into account whenever volcanic gas compositions are to be derived from analyses of volatile species adsorbed on ash surfaces. This means that estimates of volcanic fluxes of HCl that were obtained from SO 2 flux measurements scaled by the S/Cl ratio in adsorbates should be treated with considerable caution; the same may also apply to other gas species. The frequently made assumption of equal adsorption of different species is only valid under very specific conditions (see also [START_REF] Bagnato | Leachate analyses of volcanic ashes from Stromboli volcano: A proxy for the volcanic gas plume composition?[END_REF]. For example, Figure 7 shows that for a gas with an S/Cl ratio of 1, equal adsorption, resulting in an S/Cl ratio of 1 also in the adsorbate, only occurs at a temperature near -20 ˚C. However, for an S/Cl ratio in the gas of 10, the temperature where no fractionation occurs decreases to near -50 ˚C.

The conclusions reached here on the strong effect of temperature on the fractionation of SO 2 and HCl during adsorption are actually quite consistent with a number of field observations. S/Cl ratios of ashes vary widely, < 0.01 to > 100, and often by orders of magnitude even within the same eruption (see Supplementary Table 2). This is likely not only due to variations in gas composition, but also to the fractionation of S and Cl between gas and ash. [START_REF] Edmonds | Rainwater and ash leachate analysis as proxies for plume chemistry at Soufrière Hills volcano, Montserrat[END_REF] measured the SO 2 /HCl ratio of gases from three eruption periods of the Soufrière Hills volcano on Montserrat between 1996 and 2002, using both open path infrared spectroscopy and the analysis of ash leachates. For the phase I and phase II major dome building periods, a SO 2 /HCl molar ratio near 0.1 was spectroscopically detected in the plume and ash analyses gave similar (although more scattered) numbers. This is consistent with Figure 7, which suggests that for an S/Cl ratio of 0.1, no fractionation occurs between ash and gas phase near + 30 ˚C -a very plausible temperature for a low-altitude tropospheric plume in a tropical climate. [START_REF] Bagnato | Scavenging of sulphur, halogens and trace metals by volcanic ash: The 2010 Eyjafjallajökull eruption[END_REF] studied ash leachates of the 2010

Eyafjallajökull eruption on Iceland and found major discrepancies between the S/Cl ratio adsorbed on ash surfaces and that measured in the volcanic gases. For the highly explosive phase III eruption, S/Cl ratios from ash leachates are mostly between 0.4 and 0.6, while direct gas measurements yielded significantly higher molar S/Cl ratios of 1.5 -3.3. Inspection of Figure 7 suggests that these data may be reconciled by assuming a temperature of adsorption near 0 ˚C, quite plausible for a tropospheric plume during spring in Iceland. Note that these ash samples were taken at horizontal distances from the vent up to more than 50 km, where the plume will have cooled down to ambient atmospheric temperature; closer to the vent, larger variations in adsorbed S/Cl ratio are observed, which may be related to variations in plume temperature and also plume height (4 -9 km; [START_REF] Petersen | The impact of the atmosphere on the Eyjafjallajökull 2010 eruption plume[END_REF]. Even larger fractionations of S and Cl between ash and gas were observed for the basaltic fire fountain phase; the S/Cl ratio in the gas was measured to be between 5 and 25 while a molar ratio of S/Cl of 0.14 was measured on the ash. According to Figure 7, this could imply equilibration between gas and ash at temperatures around + 50 ˚C, which is plausible for a fire fountain eruption, as there may be very significant radiative heating of particles after they left the vent and the plume likely entrained less ambient air than for the highly explosive phase III.

In the previous discussion, we have referred to Figure 7, which is based on equation 7 and strictly valid only for a dacitic composition. Glass composition will have some effect on the S/Cl fractionation, which needs to be considered. However, this effect is much smaller than the variation due to temperature. For a given gas composition, Figure 7 suggests variations in the ratio of adsorbed S and Cl by four orders of magnitude over just 250 ˚C. Compared to this, the compositional effect on the amount of adsorbed HCl (Fig. 2) is only about a factor of 2 and the effect of glass composition on SO 2 adsorption is even smaller (Schmauss and Kepper 2014).

Therefore, we believe that the conclusions derived from comparing the curves in Figure 7 with ash leachate analyses are at least approximately correct, although additional calibrations of the effect of ash composition on the S/Cl fractionation between plume gas and ash surface are certainly desirable. In general, our discussion shows that a large part of the apparent discrepancies between gas composition and the composition of gas leachates can be accounted for by the effect of temperature on adsorption. This does not rule out that other factors, in particular alteration processes may also be important under some circumstances. However, for fresh, unaltered ash it may indeed be possible to derive gas compositions from leachate analyses, if reasonable estimates of plume temperature can be made. Moreover, on a local scale close to a vent, the S/Cl ratio may actually provide a sensitive thermometer for the temperature in the plume.

Stratospheric HCl yield and ozone depletion

With the experimental data on HCl adsorption obtained in this study, the fraction of volcanic HCl reaching the stratosphere during Plinian eruptions can be better constrained. As noted above, HCl adsorption increases with temperature, at least up to 150 ˚C. Accordingly, one may suspect that most of the adsorption may occur in the relatively hot parts of the volcanic plume. However, ascent velocities in a typical Plinian eruption column are > 100 m s -1 [START_REF] Textor | Volcanic particle aggregation in explosive eruption columns. Part II: Numerical experiments[END_REF], implying that ash particles reach the stratosphere within 1 -2 minutes. This very much limits the time available for high-temperature, near-vent interaction between volcanic gases and ash.

The highest rate constant for HCl adsorption measured in this study (Table 4) was 1.8

. 10 -4 s -1 . For a residence time of 120 s, this would allow only 2 % of the equilibrium surface concentration to be adsorbed on the ash. Accordingly, it is likely that most of the adsorption occurs in the dilute parts of the volcanic plumes after it has cooled down to the ambient temperature at the respective altitude.

A rough estimate of the magnitude of HCl adsorption can be obtained by considering the HCl surface concentration that would result from the (hypothetical) total adsorption of all the HCl in the plume. This concentration is simply the ratio of the total mass of HCl in the plume to the total available surface area of the ash. According to [START_REF] Woods | Particle fallout, thermal disequilibrium and volcanic plumes[END_REF] fine ash with a grain size < 100 m accounts for > 50 wt.

% of total deposit for Plinian eruptions. Measurements of fine ash samples from various volcanoes yielded specific surfaces of 1.1 -2.1 m 2 / g [START_REF] Delmelle | Surface area, porosity and water adsorption properties of fine volcanic ash particles[END_REF].

The ash is produced by the exsolution of gas bubbles, which ultimately disrupt and fragment the melt. Subduction zone magmas that are responsible for the most prominent explosive eruptions typically have pre-eruptive water contents near 5 wt. % (e.g. [START_REF] Métrich | Volatile abundances in basaltic magmas and their degassing paths tracked by melt inclusions[END_REF]. Water is also the main component of the gas plume, with HCl concentrations ranging from < 0.05 to > 5 mole %, the majority of data probably being in the 0.1 to 1 mole % range [START_REF] Symonds | Volcanic-gas studies: Methods, results, and applications[END_REF][START_REF] Fischer | Fluxes of volatiles (H 2 O, CO 2 , N 2 , Cl, F) from arc volcanoes[END_REF]).

These data from volcanic gases are in rough agreement with the Cl/ H 2 O ratio in melt inclusions from primitive arc basalts [START_REF] Métrich | Volatile abundances in basaltic magmas and their degassing paths tracked by melt inclusions[END_REF]. If one therefore assumed a mass ratio of volcanic gas to (glassy) ash of 0.05 (equivalent to 5 % H 2 O in the pre-eruptive magma), 1 mole % HCl in the gas and a surface of 1 m 2 /g for the total ash (or 50 % of fine ash with 2 m 2 /g), adsorbing all the HCl on the ash would yield a surface concentration of 1.01 mg/m 2 . Comparing this number to the experimental data in Figure 2 and 3 suggests that such surface concentrations may be easily reached at room temperature or above; however, they may not be achievable at the much lower temperatures prevailing in the stratosphere. The situation would be very different, however, for an initial HCl concentration in the gas of 0.1 mole %; the corresponding surface concentration of 0.10 mg/m 2 is easily reached even at low temperatures and at low HCl partial pressure. This rough calculation already shows that the initial HCl concentration in the plume, which likely reflects the pre-eruptive

Cl/H 2 O ratio in the magma, largely controls the fraction of HCl sequestered by ash and therefore, together with scavenging by hydrometeors, the effective stratospheric HCl loading.

For a fully quantitative treatment of HCl adsorption, the equilibrium between the gas phase and ash surface as described by equation ( 5) has to be considered together with mass balance constraints under the conditions prevailing in the umbrella part of a volcanic plume. For our calculations, we assume a plume model similar to that described by [START_REF] Textor | Volcanic particle aggregation in explosive eruption columns. Part II: Numerical experiments[END_REF]. The umbrella part of the plume is located at an altitude near 20 km with a temperature around 220 K, containing about 100 g of ash per kg of plume mixture. Assuming an initial gas mass fraction in the erupting material of 5 wt. % (see above), this implies a dilution of volcanic gases by entrained air by a factor of 200. The calculations are, however, rather insensitive to this dilution factor, since according to equation ( 5), adsorption only increases with p HCl 0.27 , i.e. a ten-fold increase in the dilution factor would only decrease adsorption by a factor of 1.9. For a volcanic gas with 1 mole % HCl, the partial pressure of HCl decreases from 10 mbar at the vent to 0.0022 mbar in the umbrella cloud by the combined effects of the increasing altitude and dilution by entrained air. This number does not include possible scavenging of HCl by hydrometeors (see [START_REF] Textor | Injection of gases into the stratosphere by explosive volcanic eruptions[END_REF]. Under the given conditions, equation ( 5) predicts a concentration of HCl adsorbed on the surface of ash of 0.026 mg/m 2 in equilibrium with this HCl partial pressure, less than 3 % of the 1.01 mg/m 2 required for total adsorption (see above). Since during the adsorption process, the partial pressure of HCl in the gas decreases until equilibrium is reached, the real number for the equilibrium surface concentration is slightly smaller.

Following the method described by [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF], one obtains virtually the same value of 0.026 mg/m 2 . This number appears small, but the resulting bulk Cl concentration (26-52 mg/kg for a specific surface of 1-2 m 2 /g) overlaps well

with the low end of concentrations reported for natural ashes (Fig. 6). Stratospheric ash samples are likely under-represented in Figure 6, because many eruptions did not reach the stratosphere and even for large eruptions, the ashes likely contain particles that did not reach the stratosphere. For less explosive eruptions, particle velocities are smaller and the plume may be less diluted by entrained air. Ash might therefore be exposed to higher HCl partial pressures for longer times and at higher temperatures, leading to stronger adsorption. This is a possible explanation for the fact that the surface concentrations predicted for adsorption in the stratosphere are at the lower end of the data for natural ash samples. However, the physical conditions for adsorption likely vary considerably between individual eruptions and are influenced by parameters such as the actual wind field that are hard to include in general models.

The calculation above clearly shows that for high initial HCl concentrations (> 1 mole %) in the gas phase of a Plinian eruption, adsorption by ash does not significantly reduce the stratospheric HCl loading. However, the adsorption effect becomes significant for low initial HCl concentrations. For example, for an initial HCl content in the volcanic gas of 0.1 mole %, an equilibrium surface concentration of 0.013 mg/m 2 is obtained, 13 % of the value required for total absorption (see above). The relationship between HCl content in the initial volcanic gas and the adsorption on ash is illustrated in Figure 8. Physically, this relationship is due to the fact that the total amount of HCl in the plume increases proportionally to the initial HCl molar fraction and therefore the HCl partial pressure; however, the amount of HCl adsorbed on ash only increases with p HCl 0.27 so that at higher p HCl , the fraction of HCl that is not removed by adsorption increases. In general, the efficiency of adsorption on ash surfaces in dilute eruption plumes is much smaller for HCl than for SO 2 [START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF].

The pre-eruptive Cl contents and Cl/H 2 O ratios in subduction zone volcanics tend to be high [START_REF] Métrich | Volatile abundances in basaltic magmas and their degassing paths tracked by melt inclusions[END_REF]Wallace 2008, Kutterolf et al. 2013). Accordingly, HCl contents in the fluid phase near 1 mole % and higher will be common for Plinian eruptions in subduction zone settings. In such a situation, HCl adsorption on ash will not be able to significantly reduce the amount of HCl reaching the stratosphere. Although there is still much work to be done to understand these processes, previous studies [START_REF] Textor | Injection of gases into the stratosphere by explosive volcanic eruptions[END_REF] have suggested limited efficiency of HCl scavenging by liquid water and by ice in the eruption column, with more than 25 % of the total HCl reaching the stratosphere. Our results therefore support the hypothesis that significant and perhaps even catastrophic ozone depletion due to the HCl yield of giant eruptions is possible [START_REF] Kutterolf | Combined bromine and chlorine release from large explosive volcanic eruptions: A threat to stratospheric ozone?[END_REF][START_REF] Black | Acid rain and ozone depletion from pulsed Siberian Traps magmatism[END_REF].

Conclusions

The interaction of volcanic gases with ash particles could potentially limit the environmental impact of explosive eruptions. We provide new experimental data on the kinetics and thermodynamics of HCl adsorption on volcanic ash. Unlike for SO 2 , HCl adsorption increases with temperature, so that large temperature-dependent fractionations between HCl and SO 2 occur during the adsorption process. Extracting volcanic gas compositions from analyses of ash leachates therefore requires correcting for this effect. Although HCl adsorption increases with temperature, the slow rate of the process implies that most of the adsorption occurs in the cold, dilute parts of a volcanic plume. For low initial HCl concentrations in the volcanic gas (0.1 mole % and below), the depletion of HCl by adsorption can be significant. However, for HCl concentrations of 1 mole % and above, adsorption on ashes is unlikely to reduce the stratospheric HCl yield of Plinian eruptions. Therefore, it appears quite plausible that the HCl yield of large explosive eruptions may cause significant ozone depletion in the stratosphere. S2 for data and sources). The data are compared with the maximum surface concentrations according to the experimental data for HCl adsorption on dacite glass (vertical lines), assuming two different specific surface areas (1 m 2 /g for fine ash, 0.1 m 2 /g for coarser ash) and adsorption at three different temperatures (150 ˚C, 21 ˚C, -76 ˚C). Note that some samples may contain a contribution of Cl-bearing alteration products from other sources, e.g., evaporites, marine aerosols or pre-eruptive interaction with saline groundwaters, altered dome rocks or recycled volcanic ejecta.

(

  Figure 6 are completely consistent with earlier experimental results for fluorine.

Fig. 1 .

 1 Fig. 1. Kinetics of HCl adsorption on dacitic glass at room temperature and for an

Fig. 2 .

 2 Fig. 2. HCl adsorption and desorption isotherms for (a) andesitic, (b) dacitic, and (c)

Fig. 3 .

 3 Fig. 3. Effect of temperature on the adsorption of HCl on dacite glass. Error bars are

Fig. 4 .

 4 Fig. 4. Multiple regression model for the adsorption of HCl on dacite glass. Surface concentrations c are in mg/m 2 .

Fig. 5 .

 5 Fig. 5. HCl adsorption on a natural obsidian from the 1739 A.D. eruption of Vulcano

Fig. 6 .

 6 Fig. 6. Concentrations of chlorine adsorbed on volcanic ashes from recent eruptions

Fig. 7 .

 7 Fig. 7. Calculated fractionation of SO 2 and HCl between volcanic gas and the surface

Fig. 8 .

 8 Fig. 8. Fraction of HCl adsorbed on volcanic ash in the umbrella part of a

  allow testing this assumption. The adsorption of HCl and SO 2 can likely be treated independently, without considering direct interactions, because the concentration of these gases, particularly in the dilute part of the plume, is very low and therefore, none of them will fully cover the glass surface. Moreover, the experimental data of this study and of[START_REF] Schmauss | Adsorption of sulfur dioxide on volcanic ashes[END_REF] suggest that HCl and SO 2 actually react with different surface sites, since both the dependence of adsorption on glass composition and on temperature are very different. For SO 2 , glass composition has little influence on adsorption, while the total alkali content seems to control the reactivity with HCl. HCl adsorption increases with temperature, while the adsorption of SO 2 decreases with temperature.

	ln (c SO2 /c HCl ) adsorbed = 2855 T -1 + 0.28 ln (p SO2 /p HCl ) -10.58	(6)
	At the low pressures involved, the ratio of the partial pressures p SO2 /p HCl is virtually
	identical with the SO 2 /HCl molar ratio in the gas. To convert the ratio of surface
	concentrations c SO2 /c HCl , which are given in mg/m 2 , into a molar ratio of S/Cl, the
	numbers have to be divided by the ratio of the molar masses of SO 2 and HCl. This
	adds a linear term of -ln (64.06/36.46) = -0.56 to the equation, which then becomes:
	ln (S/Cl) adsorbed = 2855 T -1 + 0.28 ln (S/Cl) gas -11.14	(7)
	Schmauss and Keppler (2014) showed that SO 2 adsorption on dacite glass can be
	described by the equation
	ln c SO2 = 2140 T -1 + 0.29 ln p SO2 -9.32	(4)
	while in the present study, we observed that HCl adsorption follows the equation
	ln c HCl = -715.3 T -1 + 0.27 ln p HCl + 1.26	(5)
	In both equations, the coefficient in front of the ln p term is indistinguishable within
	experimental error. If one assumes an average coefficient of 0.28, both equations can
	be combined to

Table 1

 1 Chemical composition (wt. %) and specific surface A (m 2 /g) of synthetic glass samples and of a natural obsidian from Vulcano (Aeolian Islands, Italy) used for Numbers in parentheses are one standard deviation in the last digits. Fe 2 O 3 (t) is the total iron content given as Fe 2 O 3 . For dacite, three batches of sample were prepared.Batch 1 was used for experiments at room temperature, batch 2 at 150 ˚C, and batch 3 at -76 ˚C. Note that the synthetic compositions do not necessarily correspond to global averages of the respective rock type.

	adsorption experiments
		Rhyolite	Dacite	Dacite	Dacite	Andesite	Vulcano
			Batch 1	Batch 2	Batch 3		Obsidian
	SiO 2	76.45 (83)	67.08 (30)	67.02 (49)	66.68 (71)	56.77 (18)	73.47 (35)
	Al 2 O 3	12.37 (53)	16.49 (21)	16.81 (23)	16.96 (32)	21.63 (13)	13.49 (22)
	Fe 2 O 3 (t)						1.86 (13)
	MgO	3.14 (24)	4.96 (15)	4.97 (23)	5.14 (37)	7.31 (16)	0.11 (04)
	CaO	0.80 (07)	2.80 (10)	2.93 (11)	2.86 (16)	7.46 (10)	0.83 (10)
	Na 2 O	2.77 (08)	4.31 (09)	4.20 (05)	4.46 (09)	3.88 (09)	4.42 (08)
	K 2 O	3.33 (05)	3.15 (06)	3.00 (06)	2.92 (07)	1.77 (06)	5.10 (05)
	Total	98.86	98.79	98.93	99.02	98.82	99.28
	A	3.82	4.90	1.50	2.74	3.01	2.97

Table 2 758

 2 Rate constants  for the adsorption of HCl on glass surfaces 759 is the initial pressure of HCl vapor to which the sample was exposed in each step. The pressures are listed in the sequence of the steps of the 760 adsorption experiment. Numbers in brackets are estimated uncertainties.

	Andesite

p

Acknowledgements

We would like to thank Gerti Gollner and Julia Huber for technical assistance in the adsorption experiments and Lena Geiling for the determination of specific surfaces.

Constructive reviews by Tamsin Mather, Steffen Kutterolf and an anonymous referee helped to improve the manuscript.