Isaline Demangel

Agnes Zsofia Kovacs

Sylvain Richoz

Silvia Gardin

Leopold Krystyn

Andre Baldermann

Werner E Piller

Zsofia Kovacs

Development of early calcareous nannoplankton in the Northern Calcareous Alps (Austria) in the Upper Triassic

Keywords: Coccolithophorids, Calcareous nannofossils, Prinsiosphaera triassica, Quantification, Evolution. 1

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Calcareous nannoplankton were described for the first time in 1836 by the German biologist Ehrenberg, as small "flat", elliptical discs of "agaric-mineral" [START_REF] Tyrrell | Coccolithophores[END_REF]. After this https://doi.org/10.1016/j.gloplacha.2020.103254 first discovery, several researches in the Western Tethys (South Germany, Calcareous Alps of Italy and Austria) report the description and the first occurrence datum of the calcareous nannofossils during the Upper Triassic. Concerning the coccolithophorids, [START_REF] Prins | Evolution and stratigraphy of coccolothinids from the lower and middle lias[END_REF] described the first Crucirhabdus primulus from lower and middle Liassic sediments in England, France and Western Germany. Later [START_REF] Jafar | Significance of Late Triassic calcareous Nannoplankton from Austria and Southern Germany[END_REF] described the first Crucirhabdus minutus and record specimens of C. primulus in section of southern Bavaria (Germany) from the base of the Rhaetian. Two years later, [START_REF] Bown | Archaeozygodiscus gen. nov. and other Triassic coccoliths[END_REF] reports the first Archaeozygodiscus koessenensis from the marshi Zone (Late Rhaetian) in an Austrian section. [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] From the Upper Triassic onward, dinocysts, nannoliths and coccoliths composed a large part of the phytoplankton's total productivity, accumulating until today a huge amount of calcium carbonate on the ocean floor. This initiation of the pelagic carbonate production is a major change in the ocean chemistry representing the shift from the "Neritan Ocean" with infinite saturation dominated by biogenic shallow-water CaCO 3 to the "Cretan Ocean" with low saturation dominated by pelagic CaCO 3 and very stable deep ocean [CO 3 2-] ( [START_REF] Zeebe | A simple model for the CaCO3 saturation state of the ocean: the "Strangelove", the "Neritan", and the "Cretan" Ocean[END_REF]. To understand how the calcifying organisms will evolve with the current ocean acidification, the geological records can complement modern data by providing long time scale records. This critical nannoplankton appearance and evolution was quantified in two tethysian basins in South Italy (Preto et al., 2013a) with the main Rhaetian nannoliths, Prinsiosphaera triassica, building 0 to 10% of the rock volume during the Norian, 20 to 40 % in the middle Rhaetian and up to 60% in the upper Rhaetian, while coccolithophores remained subordinate. At Steinbergkogel (Austria), [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] published a semi-quantitative estimation of the calcareous nannofossils abundance in the Upper Triassic showing only few nannoliths and calcareous cysts in the mid -to late Norian and a significant increase in Prinsiosphaera triassica around the Norian-Rhaetian boundary. [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] and Preto et al. (2013a) improved the understanding of the early evolution of the calcareous nannofossils. To verify if the observations from the Italian sections are regional or global and to investigate the importance of the bathymetrical differences point out by Preto et al. (2013a) further investigation are however needed. Among other Steinbergkogel needed a detail quantification rather than semi-quantitatively estimation and a control on diagenesis. In this context, our study at Steinbergkogel first focuses on the eventual impact of diagenesis on the preservation and abundance of the calcareous nannofossils. The second aims is to proceed to detail investigations on the First Occurrence Datum (FOD) of the coccolithophorids by looking at middle to lower Norian sediment.

The final aim is to perform a quantitative estimation of the volume and palaeo-fluxes of the calcareous nannofossils during the Upper Triassic in Austrian hemipelagic highs.

Geological settings

During the Upper Ma; International Chronostratigraphic Chart v2017/02), the Northern Calcareous Alps (NCA) were located on the Western Tethys margin around 30°N (Fig 1)

and registered tropical climate with the maximum activity of the mega-monsoons system [START_REF] Robinson | Paleoclimatology and continental drift[END_REF][START_REF] Preto | Triassic climates -State of the art and perspectives -Palaeogeography[END_REF] modulated by Milankovitch cycles [START_REF] Ogg | Triassic[END_REF]. The Austrian Northern Calcareous Alps were during Middle and Upper Triassic a region of very large carbonate platforms [START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF]. During the late Carnian, the widespread Dachstein platform developed above the siliciclastic deposit link to the Carnian Humid Event (e.g. Ruffels et al., 2015). This platform is composed of intra-platform basins, large lagoons extending toward the south-east and fringing reefs open to the ocean, where Hallstatt facies pelagic limestones developed [START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF]. This deep shelf environment shows lateral and successive differences between massive light limestone and red condensed limestone [START_REF] Krystyn | Triassic conodont localities of the Salzkammergut region (Northern Calcareous Alps) -Geologischen Bundesanstalt[END_REF]. Indeed, the region was subject to diapirism of Permian evaporites creating hemipelagic high environments, where the red Hallstatt limestones were deposited at the top (Fig 1). This formation ends due to increasing terrigenous input during the early Rhaetian corresponding to the marly Zlambach Formation. The most recent study on the basin, conducted by [START_REF] Kenter | Slope angle and basin depth of the Triassic Platform-Basin Transition at the Gosaukamm, Austria[END_REF], proposed a depth of at least 300 meters, but probably 500m, based the clinoform geometry, sediment composition and geopetal fabrics in the Dachstein platform margin. indicating a low sedimentation rate and compaction after deposition [START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. The section ends with the transition to the grey marls of the Zlambach Formation linked with an increased sedimentation rate due to a higher level of siliciclastic input (Krystyn et al., 2007a). b) Overview of the Austroalpine units in the central NCA [START_REF] Leitner | Structure and evolution of a rocksalt-mudrock-tectonite: the haselgebirge in the Northern Calcareous Alps[END_REF]. c) Location of the studied sections near Hallstatt in Austria (modified after [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF]. d) Photograph of the STK 1A subsection of Steinbergkogel (from [START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. e) Photograph of the lower part of the STK 1B/C subsections of Steinbergkogel (from [START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. Numbers in the photographs correspond to the numbers indicated in figure 4 from Krystyn et al. (2007a).

Hallstatt limestones record an abundant and very diverse invertebrate fauna such as the 500 species of cephalopods mostly orthoceratids, nautiloids and ammonoids described in details by [START_REF] Mojsisovics | Das Gebirge um Hallstatt. Die Mollusken-Faunen der Zlambach-und Hallstätter Schichten[END_REF][START_REF] Mojsisovics | Das Gebirge um Hallstatt. Die Mollusken-Faunen der Zlambach-und Hallstätter Schichten[END_REF]. The bivalves, in particular halobiids, have been described by [START_REF] Kittl | Materialien zu einer Monographie der Halobiidae und Monotidae der Trias[END_REF].

Gastropods and brachiopods were respectively described by [START_REF] Koken | Die Gastropoden der Trias um Hallstatt[END_REF] and [START_REF] Bittner | Die Brachiopoden der alpinen Trias[END_REF]. Finally, few occurrences of reefal organisms are registered [START_REF] Krystyn | Triassic conodont localities of the Salzkammergut region (Northern Calcareous Alps) -Geologischen Bundesanstalt[END_REF]Krystyn et al., 2007b;[START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF]. The Hallstatt Formation is also composed of diverse microfauna including benthics foraminiferas, sponges spicules dominating the Rhaetian grey limestones [START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF], radiolarians, echinoderms (crinoids and echinoids), holothurian sclerites and conodonts [START_REF] Krystyn | Triassic conodont localities of the Salzkammergut region (Northern Calcareous Alps) -Geologischen Bundesanstalt[END_REF][START_REF] Mosher | Triassic conodonts from western North America and Europe and their correlation[END_REF]and Krystyn et al., 2007a, b). The conodonts and ammonoids have been used in parallel of abiotic stratigraphic tools (stable isotopes, magnetostratigraphy) for time control and high-resolution correlation (Fig 3) (Krystyn et al., 2007b[START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. Normal marine salinity occurs throughout all sections as indicated by the continuous presence of stenohaline sessile organisms (echinoderms). The diversity of benthic species link with the high density of traces fossil indicates oxic conditions at the sea bottom [START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF].

Description of the Upper Triassic calcareous nannofossils:

The GSSP candidate at Steinbergkogel is described as an excellent section for the Norian/Rhaetian boundary by Krystyn et al. (2007 a,b) but also as the greatest place for calcareous nannofossils birth investigation by [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] the latest Rhaetian and outlining three size groups, large (10-8 µm), medium (7-6 µm) and small (<5 µm) regarding the assemblages presents. P. triassica is the main carbonate producer species among the nannofossils during the Upper Triassic in the western Tethys [START_REF] Preto | Onset of significant pelagic carbonate accumulation after the Carnian Pluvial Event (CPE) in the western Tethys -Facies[END_REF].

The second nannoliths, ( Eo -) conusphaera zlambachensis (family Euconusphaeraceae) was first described by Moshkovitz (1982) as an elongated cone, truncated at both ends, composed in the inner part of 35-40 radial, inclined calcitic lamellae sinistrally turning and in the outer part of long smooth plates. Later, [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] suggest an evolution from a stubby shape with an average length of 3µm to an elongated form with an average length of 5µm.

Regarding the calcareous cysts, the three species have been all originally described by Jafar (1983). Thoracosphaera spp. corresponds to a bowl-or hat-shape with a size between 5 to 13 µm.

Living specimens are known to be a dinophyte which secretes a calcareous cell wall in the vegetative life phase. Orthopithonella spp. is a spherical solid with element perpendicular to the wall and a diameter around 9 µm. Obliquipithonella spp. is nearly similar to the previous species but possesses element oblique to the wall and the size is smaller with diameter around 6 µm.

Concerning the Upper Triassic coccoliths, Crucirhabdus primulus have been the first described [START_REF] Prins | Evolution and stratigraphy of coccolothinids from the lower and middle lias[END_REF]). This species with a size between 3 to 5 µm present an elliptical shape. [START_REF] Rood | Electron microscope studies of lower qnd middle Jurassic coccoliths[END_REF] and [START_REF] Janofske | Kalkige Nannofossillien aus der ober-Trias (Rhät)der Nördlichen kalkalpen. Berliner geowiss[END_REF] describe a loxolith rim with imbricated elements. [START_REF] Bown | The structural Development of Early Mesozoic Coccoliths and its Evolutionary and Taxonomic Significance[END_REF][START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] define the species with vertical to subvertical elements, corresponding to a protolith rim. The central area of the rim is bearing a cross. Later, Crucirhabdus minutus have been described

by [START_REF] Jafar | Significance of Late Triassic calcareous Nannoplankton from Austria and Southern Germany[END_REF] as a tiny elliptical coccolith with a size range between 1.6 to 2.4 µm. The inner rim present a cross supporting a central spine. Concerning the rim elements orientation, [START_REF] Bown | Archaeozygodiscus gen. nov. and other Triassic coccoliths[END_REF] defined a protolith rim, while [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] outlined both protolith and loxolith possibility. Finally, Archaeozygodiscus koessenensis, have been later described by [START_REF] Bown | Archaeozygodiscus gen. nov. and other Triassic coccoliths[END_REF] as an elliptical coccolith with loxolith structure rim imbricating in an anticlockwise direction and a size ranging from 1.9 to 3.2 µm. The inner part holds a thick bar with a circular hole in the middle. About their evolution, Bralower supported that C. primulus is the ancestor of a lineage which gave rise to C. minutus and A.

koessenensis . He justified this lineage with the row of apparition in the sediments and by the development of the rim structure, which is for him, protolithic in C. primulus , protolithic or loxolithic in C. minutus and loxolithic in A. koessenensis . In older studies, [START_REF] Thierstein | Mesozoic calcareous nannoplankton biostratigraphy of marine sediments[END_REF], [START_REF] Jafar | Significance of Late Triassic calcareous Nannoplankton from Austria and Southern Germany[END_REF], [START_REF] Bown | The structural Development of Early Mesozoic Coccoliths and its Evolutionary and Taxonomic Significance[END_REF] and recently [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] proposed an other lineage with C. minutus giving the rise to C. primulus , given an apparition order in contrary to [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF]. About their First Occurrence Datum, the most recent and biostratigraphically controlled report until now is given by [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] (see also [START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF] from an investigation at Steinbergkogel section.

Materials and methods

a. SEM sample preparation and quantification:

Through all the sections (Sommeraukogel, Steinbergkogel sections) (Figure 4) described above, a total of 26 samples have been carefully investigated under the SEM. In details, 3 samples of Sommeraukogel, 12 samples of ST4, 5 samples of STK 1A and 6 samples of STK 1B/C were observed. To select the studied samples an interval of 2 m for Sommeraukogel and ST 4, and of 1m for STK 1A, 1B/C have been chosen. In parallel, to check if lateral variations exist, beds 113 B and 12E respectively from section STK 1A and STK 1B/C, have been analysed regarding the abundance of nannofossils in differents lateral samples. For the bed 113 B (STK 1A), 6 samples have been investigated corresponding to the following lateral positions: 0m, 0.6m, 1m, 1.5m, 1.9m, 2.5m. For the bed 12E (STK 1B/C), 7 samples have been studied respectively at 1m, 2m, 3m, 4m, 5m, 6m, and 8m.

The use of SEM and of solid rocks was chosen instead of the more widely use smear/settling slide, because after several observations of smear slides under both light microscope and SEM no calcareous nannofossils were found. Most probably due to the extremely compact limestone enclosing the nannofossils and prevents their single "extraction" during the sample preparation. Thus, the sample preparation methods described by Preto et al. (2013a, b) have been followed with slight modifications regarding our facies and locality. Indeed, after a qualitative investigation of some solid samples with the Hitachi Tabletop Microscope TM3000 (Sorbonne University, UMP CNRS 7207 CR2P, Paris, France), the samples were cut in blocks of 1cm 2 . Then, they were polished with 600 and 1200 diamonds discs using deionized water. After, the blocks were etched for 15 seconds in 0.1% HCl, and cleaned for 7 seconds in ultrasonic bath with distillate water. The samples were dried for the night in the air oven at 50°C and finally coated with 1nm of platinum/palladium using Cressington Sputter Coater 208HR.

The quantitative estimation was done with a TESCAN MIRA 3 at Lund University. To reduce the effects of samples heterogeneity, surfaces perpendicular to the bedding were cut (Chayes, 1964).

Also, for two samples two transects, 1cm straight line along the block surface, were done to confirm the homogeneity of the quantification results. Each transects were observed to count each clear specimens, not including the potential small break part of P. triassica . The area of the frame (21.9 x 21.9 µm) was selected to be larger than the biggest calcareous nannofossils, in our case 11µm P. triassica specimens. The absolute number of nannofossils per cm² was calculated using the following equation of [START_REF] Bordiga | Absolute nannofossil abundance estimates: Quantifying the pros and cons of different techniques[END_REF] slightly modified: The total P. triassica carbonate flux could not be calculated in the standard way as described in several coccoliths carbonate fluxes studies (e.g., [START_REF] Young | Calculation of coccolith volume and its use in calibration of carbonate flux estimates[END_REF][START_REF] Bornemann | The impact of calcareous nannofossils on the pelagic carbonate accumulation across the Jurassic -Cretaceous boundary[END_REF][START_REF] Suchéras-Marx | Middle Jurassic coccolith fluxes: A novel approach by automated quantification[END_REF] due to the specific sample preparation method used in this study. Indeed, normally a certain amount of rock powder is used while we used solid piece of rock, thus the nannoliths carbonate flux was calculated by multiplying the total number of P. triassica per cm 3 ( X ) by the sedimentation rate of 3m/My as estimated by Schlager (1961) for the Hallstatt Limestones. To obtain, X, the total number of the P. triassica in 1 cm 3 , the following equation was use:

x = (N × A) ÷ (f × n) ( equation
X = x V rock × 1
(equation 2); with V rock (cm 3 ) = 1 cm 3 x mean diameter of P. triassica (cm) and x = absolute number of calcareous nannofossils per cm² of sediment, calculated previously. The diameter of specimens with distinct edges and clear half sphere visible were measured during the quantification and average for each samples.

In addition, to have an estimation of the percentage of P. triassica in the rock sample to compare to the study of Preto et al. (2013a), the total volume of P. triassica was calculated following the basic volume equation: V total , (equation 3) with r = mean radius and x = absolute = 4 3πr3 × x number of P. triassica per cm 2 . Then the percentage was compute following:

V rock V P . triassica × 100 (equation 4).

b. Inductively coupled plasma atomic emission spectroscopy (ICP-OES):

In order to investigated the impact of the diagenesis on the preservation of the sediments, inductively coupled plasma atomic emission spectrometry analyses were conducted on 18 samples at the University of Technology in Graz (Austria), respectively two from Sommeraukogel section, five from ST4, seven from STK 1A and four from STK 1B. For the digestion 0.050g of powder sample were put in 50mL flask, then 5 drops of HNO 3 69% and 50 mL of 2% HNO 3 bidestilled were added.

After shaking, all the flasks were put in air oven at 80°C during 12h to digest the dolomite. Finally, the analyses were carried out using a Perkin Elmer OPTIMA 8300 DV at the Technical University of Graz, to obtain the total concentration of traces elements (Al, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Si, Sr, Zn) in each sample, after calibration of the data in ug/L or ppm. To estimate the amount of diagenesis the ratio traces elements were calculated as followed for e.g Mg/Ca:

Atomic mass of Ca F raction of Ca (ppm) ×1000 Atomic mass of M g F raction of M g (ppm)
4. Results

a. Diagenesis:

The micrite of ten samples from the different sections were observed under the SEM to investigate dissolution or overgrowth patterns (Fig 5 Elemental concentration results are presented as ratios of the element of interest with the calcium content of the same sample. The Al/Ca vs. Fe/Ca cross-plot shows strong correlation for all sections with R 2 = 0.8424 (Fig 6). This correlation between the aluminium and the iron values indicated that the Fe belongs to the silicates. Thus, in this case, the iron values cannot be integrated in the cross plot Na+Sr/Mn. This cross-plot reveals an increase of both values from the base of ST4 to the top. The middle Norian values at Sommeraukogel are exceptions, they do not follow this trend, the values are higher than at ST4. The Mn/Ca does not show any correlation with Al/Ca (R 2 = 0.2148).

The Mn/Ca vs. Al/Ca cross-plot presents increasing values for both elements along all the sections, with however STK 1B presenting an anticorrelation (but based on only two points).

c. P. triassica abundance and carbonate flux:

The incertae sedis Prinsiosphaera triassica is the most abundant calcareous nannofossils in our sections and in general in Upper Triassic sediments [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF]Preto et al., 2013a). With a relative abundance above 87% in all samples except for ST4/2 (50%), those species clearly dominate the assemblage. The carbonate flux controlled by the absolute abundance of nannofossils show a global increase during the lowermost Rhaetian and a slight decreased toward the Zlambach Formation (Fig 12). Indeed, until the top of STK 1A section, the values vary between 0,02 and 2 N 10 -10 cm -2 My -1 . This stability is followed by an increased at the base of STK 1B reaching 6 N 10 -10 cm -2 My -1 . Finally, a slight decrease toward the Zlambach Formation with values between 2 and 4 N 10 -10 cm -2 My -1 characterized the top of the section. The absolute abundance values, from Sommeraukogel until the top of STK 1A varies between 397 and 52719 ( P. triassica /cm 2 ). They are marked by two major diminutions of abundance, one between samples ST4/6 and 10 with a 42 times decreased and a second between samples ST4/45 and STK 1A/103 with a reduction of factor 31. For the youngest section, STK 1B/C, the abundance varies between 50684 and 148094 ( P. triassica /cm 2 ).

The maximum value occur at the base of the subsection (STK 1B/16). This base of STK 1B presents a major increase with values 14 times more important than the 10944 ( P. triassica /cm 2 ) at the top of STK 1A. The top of the section shows a gradual decrease of the abundance characterized by a factor 2.7 between the lowest and the top samples. The average diameter of P. triassica does not vary significantly through the section (Fig 12).

Impact of diagenesis:

During the Upper Triassic, Steinbergkogel registered deep-shelf environment, with paleo-depth of at least 300 meters, but probably 500m [START_REF] Schlager | Das Zusammenwirken von Sedimentation und Bruchtektonik in den triadischen Hallstätterkalken der Ostalpen[END_REF], thus largely above the lysocline. Some dissolution already occurred in the water column during the sinking of the nannofossils. Also, in such environment the fabric-selective dissolution and overgrowth as observed in our samples might occur during the early stage of diagenesis [START_REF] Flügel | Microfacies of carbonate rocks. Analysis, Interpretation and Application[END_REF] immediately after deposition or burial [START_REF] Berner | Early diagenesis as a theoretical approach[END_REF]. Dissolution features are visible from the base until sample ST4/12. After a transition presenting both patterns, the recrystallization is visible from the sample ST4/38 to the top. As already described by [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF], some P. triassica specimens are affected by dissolution features. If we compare the specimens observed in our section with the classification scale of preservation stages created by [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF], the best-preserved specimens (stage one) with entire outer layer are absent. The P. triassica observed here were all etched from stage two (partly etched outer layer) to stage four (etched inner layer). This nannolith is also impacted by overgrowth features during the early diagenesis, with, crystallization of neocalcite on the whole surface affecting for example a P. triassica specimen in section STK 1A (Fig 5,A). This process occurred also for the calcareous cysts Orthopithonella and Obliquipithonella which are empty sphere during the life stage and have always been observed filled by calcite crystals. Finally, this recrystallization affected also coccoliths by the overgrowth of the rims and central elements preventing sometime the complete identification of the specimens.

It is assumed that changes in the trace elements composition can occur during the diagenesis [START_REF] Land | Late Pleistocene diagenesis and dolomitization, North Jamaica[END_REF][START_REF] Budd | Geochemical imprint of meteoric diagenesis in Holocene ooid sands, Schooner Cays, Bahamas: correlation of calcite cement geochemistry with extant groundwaters[END_REF][START_REF] Saller | Geochemistry of meteoric calcite cements in some Pleistocene limestones[END_REF]. Both recrystallization and dissolution can alterate the original signal by adding/removing preferentially trace elements to reach a new equilibrium with the pore water [START_REF] Brand | Chemical diagenesis of a multicomponent carbonate-system -1: trace elements[END_REF]. As the sediments studied here were deposited in deeper marine environment, none of them could have suffer meteoritic diagenesis. We wanted thus to evaluate rather if early and burial diagenesis could have an effect on the abundance of the nannofossils. To trace potential diagenesis Sr, Na, Mn, Fe can be particularly useful. Differences in partitional coefficients and in composition in marine versus pore water lead to a contrasting evolution of certain elements. Burial diagenesis impact can lead to depleted Sr and Na content and in contrary, to increased Mn and eventually Fe, Zn concentration (Turekian, 1972, Brand and[START_REF] Brand | Chemical diagenesis of a multicomponent carbonate-system -1: trace elements[END_REF] . At Steinbergkogel, most of these elements have low concentration. Manganese concentration varies between 22 and 237 ppm, Na + /Sr between 220 and 495 ppm with Sr values ranging from 137 to 215 ppm. Iron reaches high concentration; the values are between 385 and 2590 ppm. As noted before Fe and Al shows a strong correlation indicating detrital source for Fe and so this element was excluded when considering diagenesis effects. On the Sr+Na vs. Mn cross-plot no anticorrelation was observed, which would have been expected in case of increased diagenetic impact. Only the 4 samples of STK1B section showed a negligible anticorrelation. Furthermore, the generally low Sr values (average: 162 ppm) are consistent with the depositional environment characterized by low sedimentation rate in deeper settings [START_REF] Coimbra | New insights into geochemical behavior in ancient marine carbonates (Upper Jurassic Ammonitico Rosso): Nivel poxies for interpreting sea-level dynamics and palaeoceanography[END_REF] and are not the result of loss due to diagenesis. In several middle and upper Jurassic successions with Ammonitico Rosso type sediment similarly low values in the range from 50 to 250 ppm were reported (Price and Sellwood, 1994[START_REF] Préat | Petrography and biosedimentology of the Rosso Ammonitico Veronese (middle-upper Jurassic, north-eastern Italy)[END_REF][START_REF] Coimbra | New insights into geochemical behavior in ancient marine carbonates (Upper Jurassic Ammonitico Rosso): Nivel poxies for interpreting sea-level dynamics and palaeoceanography[END_REF]. Overall, these values were considered as close to the original signal, supporting a very early diagenesis with a very light influence on the preservation [START_REF] Price | Palaeotemperatures indicated by Upper Jurassic (Kimmeridgian -Tithonian) fossils from Mallorca determined by oxygen isotope composition[END_REF][START_REF] Derry | A burial diagenesis origin for the Ediacaran Shuram-Wonoka carbon isotope anomaly[END_REF][START_REF] Coimbra | Geochemical evidence for sediment provenance in mudstones and fossil-poor wackestones (Upper Jurassic, Majorca Island)[END_REF][START_REF] Coimbra | New insights into geochemical behavior in ancient marine carbonates (Upper Jurassic Ammonitico Rosso): Nivel poxies for interpreting sea-level dynamics and palaeoceanography[END_REF]. This observation is thus also valid for Steinbergkogel with no influence of the diagenesis on the abundance of the nannofossils.

b. Upper Triassic coccolithophorids: i. Lowest occurences of coccolithophorids

The oldest occurrences of the coccolithophorids have been reported just below the Norian/Rhaetian boundary by [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] in sediment from Steinbergkogel. Bown et al., (1991) report that the main location of evolutionary activity in both the Late Triassic and Early Jurassic seems to have been the subtropical/tropical Western Tethys region. Also [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF], suggested the Western Tethys, especially the Austrian Alps as the best place to document the earliest evolution of calcareous nannofossils with probably even older occurrence. In addition, the molecular clocks date the earliest origin of potentially calcifying haptophytes, so called Calcihaptophycidae, at 270-240 Ma for SSU rDNA and at 200 Ma for LSU rDNA [START_REF] Vargas | Origin and evolution of coccolithophores: from coastal hunters to oceanic farmers[END_REF]. Our results are going in the direction of the dates proposed in those genetic studies. Indeed, we report the oldest occurrence of coccoliths sp. in the Alaunian 2 (mid-Norian) in SO/15 (around 220 Ma). This specimen is badly preserved and only third of the rim shape is visible without clear rim elements. With caution, we propose a lowest occurrence of the coccolithophorids sp. at least in the middle Norian. In addition, other rims shapes more doubtful were observed at Sommeraukogel. Above, the specimen observed at ST4/2 (Upper Triassic) have a clear entire protolith rim visible, with a really small size (length of 2.13 μm and width of 1.57 μm) leaving no doubt for its identification as C. minutus. Belonging to upper Norian sediments, this specimen of C. minutus is the oldest report so far. Until now, the lowest occurrence was reported by [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] just above the Rhaetian boundary. Still at the base of this upper Triassic section at ST4/6, a well preserved specimen of A. koessenensis was observed with a clear loxolith rim and double bar crossing the central area, corresponding to the oldest A.

koessenensis report. During our quantification C. primulus could not be observed but in the same section [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] report the lowest occurrence of this species at STK B/28 corresponding to lower Rhaetian.

ii. Evolution of early coccolithophorids

The investigation of sediment from mid-Norian until Rhaetian allowed us to follow the early evolution of the coccolithophorids and the nannoliths appearing in the upper Triassic. The evolution trend observed in this study is in agreement with the report of [START_REF] Bown | The structural Development of Early Mesozoic Coccoliths and its Evolutionary and Taxonomic Significance[END_REF] and [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF], but in contradiction to [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] proposing C. primulus as the ancestor. With our proposed order of apparition and the lastest reports (see [START_REF] Bown | The structural Development of Early Mesozoic Coccoliths and its Evolutionary and Taxonomic Significance[END_REF][START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF][START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF], we suggest C. minutus as the ancestor of C. primulus , giving rise to the other genus, A.

koessenensis. Besides the evolution from protolith-subvertical rim toward a more loxolith rim, this proposed lineage is also logical regarding the size going from a small specimen with C. minutus (1,6 to 2,4 µm) to a bigger specimen with C. primulus (3 to 5 µm) through the middle size A. koessenensis

(1,9 to 3,2 µm). The first occurrence of C. zlambachensis is slightly after A. koessenensis and before C. primulus , in agreement with [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF]. During the upper Norian/lowermost Rhaetian the diversification rate of coccolithophorids is very low with only three species described. The diversification seems to be slow with millions years in between each new species apparition considering a sedimentation rate of 3m/1Ma as proposed by [START_REF] Schlager | Das Zusammenwirken von Sedimentation und Bruchtektonik in den triadischen Hallstätterkalken der Ostalpen[END_REF] for Hallstatt-type sediments. The diversification rate will accelerate only during the early Jurassic, with 1.7 species per million years. It is only during the late Cretaceous, 120 Ma after their apparition that coccolithophorids reach their maximum diversity (Bown et al., 1991).

iii. Doubtful incertae sedis :

One interrogation remains on the second type of calcisphere possessing axial rays around a central structure (Fig. 11). The mineral composition of those structures indicate CaCO 3 composition with pics for Ca, C, O and Mg. The spectrums do not show traces of Si which could be a proof of silica replacement during the diagenetic process. Thus, the hypothesis of siliceous microfossils such as radiolarians, or even diatoms or silicoflagellates could be excluded. The amount of Sr was very low as well, conclusive of a primary calcite structure rather than aragonitic. It was thus probably a undefined calcareous nannofossils species. Such specimen have been reported first by Di [START_REF] Nocera | Triassic nannoplankton limestones of deep basin origin in the central Mediterranean region[END_REF], in different Middle to Upper Triassic deep environment sediments from several localities of the Western Tethys (Italy, Austria, Montenegro, and Greece). They were described as Nannoconus sp.

with a circular shape and an axial canal. This one was irregularly filled with calcite and surrounded by radial structure probably formed by recrystallization. Later, [START_REF] Bellanca | Ritrovamento di ''Calcisfere'' e nannofossili calcarei in terreni carnici della Sicilia[END_REF] reports similar structures in Sicilian sections of Carnian age and arguing for a strong link to Nannoconus sp . Those Nannoconus sp. have conical or spherical shape composed of plates spirally arranged around a canal, which is close to the incertae sedis shape presented here. However, it is important to highlight that the Nannoconus sp. are known only from the Upper Jurassic, which is more than 50 Ma later [START_REF] Bown | Calcareous nannofossil biostratigraphy[END_REF]. Recently, in Upper Carnian sediment from Italy, [START_REF] Preto | Triassic and Jurassic calcareous nannofossils oft he Pizzo Mondello section: A SEM study[END_REF]2013a;[START_REF] Preto | Onset of significant pelagic carbonate accumulation after the Carnian Pluvial Event (CPE) in the western Tethys -Facies[END_REF] described those kind of specimens first as Thoracosphaera sp . then as calcispheres interpreted as calcareous dinocysts, from the " Pithonella " group, with epitaxial overgrowth as a consequence of the diagenesis. They explain these forms as a recrystallization artefact with crystals rays growing in open space, with rhombohedral terminations. However, we did not observed any of these rhombohedral terminations. It is thus not possible from our data to decipher if these specimens were recrystallised calcareous dynocysts [START_REF] Preto | Triassic and Jurassic calcareous nannofossils oft he Pizzo Mondello section: A SEM study[END_REF]2013a;[START_REF] Preto | Onset of significant pelagic carbonate accumulation after the Carnian Pluvial Event (CPE) in the western Tethys -Facies[END_REF] or very early appearance of Nannoconus sp . [START_REF] Nocera | Triassic nannoplankton limestones of deep basin origin in the central Mediterranean region[END_REF][START_REF] Bellanca | Ritrovamento di ''Calcisfere'' e nannofossili calcarei in terreni carnici della Sicilia[END_REF].

c. Upper Triassic Nannofossils quantification:

During quantification of nannofossils, one of the potential caveat is the effect of dissolution/overgrowth on the absolute abundance. The impact of the dissolution on the nannofossils can be strong but is very specific dependent for dissolution occurring above the lysocline (e.g., [START_REF] Berger | Deep-sea carbonates: Evidence for a coccolith lysocline[END_REF][START_REF] Roth | Distribution and dissolution of coccoliths in the South and central Pacific[END_REF][START_REF] Thierstein | Selective dissolution of Late Cretaceous and earliest Tertiary calcareous nannofossils: Experimental evidence[END_REF][START_REF] Gibbs | Identification of dissolution patterns in nannofossil assemblages: A high-resolution comparison of synchrnous records from Ceara Rise, ODP Leg 154[END_REF]. In Steinbergkogel, [START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF] hypothesized that variation of the preservation could impact the primary biological signal. Our careful control on diagenesis (see above) demonstrate however that if dissolution and overgrowth do have an influence on the preservation of the specimens, there are not affecting their abundance. This is confirmed by the fact that three samples with different abundance present similar dissolution features.

i. Lateral variation

The lateral variation into two beds, show clear change in abundance inside the same bed. The lateral variation is not often investigated during nannofossils quantification. In the Triassic time, [START_REF] Maurer | Quantification of input compositional variations of calciturbidites in Middle Triassic basinal succession (Seceda, Dolomites, Southern Alps)[END_REF] studied compositional variations of calciturbidites regarding the lithology and fossils assemblages and abundance in a basinal succession of the Dolomites (Southern Alps). They found a lateral variation of the fossils assemblages' link to a difference in lithology and they explain those results by a sorting effect in turbidity currents. The two beds investigated in Steinbergkogel correspond to a Hallstatt facies presenting no turbiditic events. Also, the influence of seafloor current (i.e. contourite) is nowadays often called to explain variation in assemblages and in abundances in deep-sea sedimentation [START_REF] Rebesco | Contourites and associated sediments controlled by deep-water circulation processes: State-of-the-art and future considerations[END_REF]. However, as report by different authors (e.g., [START_REF] Johnson | Sediment redistribution by bottom currents in the central Pacific[END_REF][START_REF] Ivanova | Late Pliocene-Pleistocene stratigraphy and history of formation of the loffe calcareous contourite drift, Western South Atlantic[END_REF], the bottom currents are sorting the microfossils and the grains by size showing a link between the two. In Steinbergkogel, investigation of thin-sections [START_REF] Richoz | End-Triassic crisis events recorded in platform and basin of the Austrian Alps. The Triassic/Jurassic and Norian/Rhaetian GSSPs[END_REF][START_REF] Richoz | The Upper Triassic events recorded in platform and basin of the Austrian Alps[END_REF], did not suggest any sorting of the grains, thus potential bottom current does not seem to be the right explanation. The most consistent hypothesis is the temporal variation. Indeed, even if the sample belongs to the same beds, they could have been collected at a few millimeters or centimeters of differences higher or lower along the same transect.

As report by [START_REF] Schlager | Das Zusammenwirken von Sedimentation und Bruchtektonik in den triadischen Hallstätterkalken der Ostalpen[END_REF], the sedimentation rate at Steinbergkogel is very slow with 3 meters deposed in 1Ma. Thus, 3 mm of difference in the sampling position, would create a variation of 1000 years between the ages of the two samples. This could explain such variation in the abundance results of those two beds and by consequence the second-order increases and decreases in the quantification of P. triassica .

ii. P. triassica abundance and carbonate flux:

The nannofossils assemblage observed in Steinbergkogel is typical for Upper Triassic sediments with all the Upper Triassic species described present. Moreover, as observed in all the previous study of nannofossils during the Upper Triassic in the Western Tethys [START_REF] Fisher | Electron Micrographs of Limestones and their Nannofossils[END_REF][START_REF] Wiedmann | Über Umfang und Stellung des Rhaetian[END_REF][START_REF] Jafar | Significance of Late Triassic calcareous Nannoplankton from Austria and Southern Germany[END_REF][START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF]Preto et al., 2013a), the nannoliths P.

triassica is fully dominating the assemblage with a relative abundance above 85% in most of samples.

The paleoflux from the main nannoliths of the Upper Triassic is important to estimate the contribution of those pelagic calcifiers to the carbonate production. The progressive increase of P. triassica along the Upper Norian to reach 6 (N 10 -10 cm -2 My -1 ) across the N/R boundary highlight the transition from P. triassica -poor to P. triassica -rich period predicting the success of those nannoliths in the Late Rhaetian. Indeed, even if the abundance of P. triassica only reach 4.2% per volume of rock in the early Rhaetian, the carbonate contribution of P. triassica increase by a factor of 15 just after the N/R boundary, corresponding to a first step before to reach the rock-forming abundance described by Preto However, the success time of P. triassica will be later in the upper Rhaetian. . The analysis of a sample from Eiberg section (NCA), corresponding to an intraplatform basin of the Upper Rhaetian confirm the observation of [START_REF] Preto | The calcareous nannofossil Prinsiosphaera achieved rock-forming abundances in the latest Triassic of western Tethys: consequences for the δ 13 C of bulk carbonate[END_REF]. The quantification of this sample follows the preparation and calculation method developed in this study, shows that in the Upper Rhaetian in Eiberg P. triassica represent 30% of the rock in cm 3 and 45 % of the rock-surface in cm 2 . The increase to rock-forming describe by Preto et al. (2013a), occurred only from the late Rhaetian but is preceded by at least one 15 fold increase during the lower Rhaetian as described above.

iii. P. triassica size evolution:

Concerning the size of P. triassica , first data published by [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] point out a size increase from 6 μm during the Mid-Norian to 9 μm in the upper Rhaetian. Then, [START_REF] Clémence | Bentho-planktonic evidence from the Austrian Alps for a decline in sea-surface carbonate production at the end of the Triassic[END_REF] described a size reduction during the latest Rhaetian with size ranging from < 5 μm up to 10 μm. In our samples, the size range is broad and vary from 4.93 μm to 12.92 μm (Fig 9). This last maximum size is 3 μm and 4 μm bigger than the biggest specimens seen respectively by [START_REF] Clémence | Bentho-planktonic evidence from the Austrian Alps for a decline in sea-surface carbonate production at the end of the Triassic[END_REF] and [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF]. In our dataset such size is not an exception because several specimens bigger than 11 μm have been measured. The size increased seen by [START_REF] Bralower | Significance of Upper Triassic nannofossils from the Southern Hemisphere (ODP Leg 122, Wombat Plateau[END_REF] and the size decreased during the Late Rhaetian seen by [START_REF] Clémence | Bentho-planktonic evidence from the Austrian Alps for a decline in sea-surface carbonate production at the end of the Triassic[END_REF] have not been observed in this study during the Norian to

Early Rhaetian, but our data come from a time-frame between these two studies.. The size is in general stable along all the sections with only few minor quick variations between one sample to another.

Conclusions

This detail quantitative investigation of the 3 sub-sections at Steinbergkogel, a candidate GSSP for the Norian-Rhaetian boundary, with an approach including careful diagenesis evaluation point out the relevance of this locality and in general of the Western Tethys to document the early evolution of the coccolithophorids. The results of this study highlight:

1. ICP-OES analysis shows only early marine diagenesis with a weak impact on the quantification results.

2. The lowest occurrence of coccoliths sp. leads in the middle Norian (SO/15). primulus, giving the rise to A.koessenensis in between . The size increase throughout this evolution and the rim structure evolve from protolith-subvertical rim to loxolith rim.

6. The incertae sedis, Prinsiosphaera triassica is dominating the calcareous nannofossils abundance during Upper Triassic.

7. The increase in abundance of P. triassica from the Upper Norian to the lower Rhaetian is interpreted as the beginning of their success in the late Rhaetian, but this nannoliths is still not rock-forming at this time.

Fig 1

 1 Fig 1 -a) Palaeogeographic reconstruction of the global world and the Western Tethyan margin during the Upper Triassic (from Hornung et al., 2007). With NCA = Northern Calcareous Alps, eCA = East Carpathians, HaB = Hallstatt Basin, Do = Dolomite, PiO = Pinde Ocean, LaB = Lagonegro Basin. b) Triassic stratigraphy of the NCA (middle sector) (from Mandl, 2000).

Fig 2

 2 Fig 2 -a) Distribution of the NCA in the frame of Austria (modified after[START_REF] Schorn | The structure of the Hallstatt evaporite body (Northern Calcareous Alps, Austria): A compressive diapir superposed by strike-slip shear?[END_REF]. b) Overview of the Austroalpine units in the central NCA[START_REF] Leitner | Structure and evolution of a rocksalt-mudrock-tectonite: the haselgebirge in the Northern Calcareous Alps[END_REF]. c) Location of the studied sections near Hallstatt in Austria (modified after[START_REF] Gardin | Where and when the earliest coccolithophores?[END_REF]. d) Photograph of the STK 1A subsection of Steinbergkogel (from[START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. e) Photograph of the lower part of the STK 1B/C subsections of Steinbergkogel (from[START_REF] Hüsing | Magnetostratigraphic dating of the proposed Rhaetian GSSP at Steinbergkogel (Upper Triassic, Austria): Implications for the Late Triassic time scale[END_REF]. Numbers in the photographs correspond to the numbers indicated in figure4fromKrystyn et al. (2007a).

Fig 3 -

 3 Fig 3 -Correlation of bioevents around the Norian/Rhaetian boundary at Steinbergkogel.

  1) With x = absolute number of calcareous nannofossils per cm² of sediment; N = total number of individuals counted; A = area of the block (1 cm²); f = area of one frame in cm²; and n = number of frame analysed.

Fig 4 -

 4 Fig 4 -Schematic lithology, biomarkers zones, magnetostratigraphy from Krystyn et al., 2007b) and sample location of Sommeraukogel, ST 4, and STK 1A, 1B/C sections.

Fig 5 -

 5 Fig 5 -SEM observation of the dissolution and overgrowth patterns. A-Recrystallized P. triassica from STK 1A/119A; B-Orthopithonella sp. refilled by secondary calcite crystal from STK 1B/29A; C-Overgrowth features in ST4/38; D-Dissolution patterns in ST4/2.

Fig 6 -

 6 Fig 6 -Cross plot between A: Aluminium and Iron; B: Manganese for all the sections.

Fig 9 -

 9 Fig 9 -SEM pictures representing ; A-Biggest P. triassica with half of the inner sphere visible composed of thin tabular calcite rhombohedral arranged in parallel group randomly positioned (SO/11). B-P. triassica with half of the inner sphere visible composed of block of calcite (STK 1B/29 A). C-Entire P. triassica with thin tabular calcite in parallel group visible (ST4/25).

Fig 12 -

 12 Fig 12 -P. triassica CaCO 3 paleofluxes (specimens 10 -10 cm -2 My -1 ) and absolute number (specimens/cm 2 ) plotted against the lithology, δ 13 C carb values, the percentage of P. triassica in the rock sample, size measurement of P. triassica with the black line corresponding to the mean diameters and the grey area of the range between minimum and maximum sizes. The lowest occurrences are represented by the black dots and the line above indicate the presence of the species.

Fig 13 -

 13 Fig 13 -Box plot representing the total number of nannofossils per transect of 1cm in STK 1A/113 and in STK 1C/12E.

  et al. (2013a) in the late Rhaetian. The factors boosting this croissance are still not yet understood. This first increase is quickly followed by the slight decrease in abundance occurring at the end of the section. This diminution co-occurs with the first occurrence of the second Upper Triassic nannoliths C. zlambachensis and a change in lithology with the transition to the Zlambach Formation. Those two observations raise the question of a possible competition between those two nannoliths and/or a possible negative feedback of the eutrophication occurring with the marly input of the Zlambach Formation. Indeed, nannoplanktons are sensitive to changes in the trophic levelThe change to a more marly lithology, possibly parallel to an increase in the nutrient concentration could have been unfavourable for P. triassica . Our quantification results are in accordance with the semi-quantitative values of Gardin et al. (2012) during the same time interval and at the same place. However, our percentages of P. triassica per volume of rock during the Norian in deep-shelf paleo-settings are slightly lower than the results published by Preto et al. (2013a) in shallow sea (Pizzo Mondelo) and open marine settings (Pignola-Abriola). For those two studies the time equivalence is only possible between ST4 and M.bidentata , M. hernsteini , P. andrusovi Zones (Norian). For ST4, our percentages are really low between 0.01 and 2.3 % (Fig 12). Preto et al. (2013a) report at Pizzo Mondelo (Sicanian Basin, Sicily -shallow sea) stable values below 3% during M. bidentata Zone (base of ST4), follow by an increase to 20% during M. hernsteini , P. andrusovi Zones (top of ST4). At Pignola-Abriola (Lagonegro Basin -Open Marine), Preto et al. (2013a) present values trending in opposition than the previous locality. Indeed at the end of M. bidentata Zone the volume percentages of P. triassica is around 10% and decrease below 5% during M. hersteini , P. andrusovi Zones (top of ST4). Note, that the analysis methods were different. Preto et al. (2013a) by using point-counting method obtained results for a surface resulting in a overestimation of the calculated amount per cm 3 , while we calculated a percentage for a volume of rock, with an equation underestimating the real abundance per cm 3 . Moreover, in our study only the complete specimens were counted, while Preto et al. (2013a) counted even the small single elements interpreted as broken P. triassica . During the late Rhaetian, at Pizzo Mondelo (Sicanian Basin, Sicily) they characterized P. triassica as rock-forming nannofossils with an abundance reaching up to 60% of the rock volume. At Pignola-Abriola, the maximum abundance occurring only in the M. ultima Zone is lower, around 20%. In consequence, Preto et al. (2013a) suggested that Prinsiosphaera was more common in shallower environment (Pizzo Mondello, Sicanian Basin) than in open marine setting (Pignola-Abriola, Lagonegro Basin). If P. triassica is effectively present during the whole time studied, it become a relevant composant only in the lowermost Rhaetian (around 4-5%), with some changes just after the N/R boundary interval.

3.

  The oldest C. minutus and A. koessenensis were observed in the Upper Norian (Base of ST4). 4. C. primulus was first observed from the Lower Rhaetian (Top of STK 1B). 5. The evolution of the first coccolithophorids is slow, from the ancestor C. minutus then C.

  reviewed all the different Upper Triassic reports of coccolithophorids to conclude for all of them to a Rhaetian age. In the same study, they reported the oldest occurrences of C. minutus and of Coccoliths spp. until now. Investigation at Steinbergkogel (Austria), a GSSP candidate for the Norian/Rhaetian boundary, permitted to find C. minutus specimens just above the Norian/Rhaetian boundary and tiny coccoliths spp. in the uppermost Norian.

Acknowledgements This research was granted by the Austrian Science Foundation (Project P 29497-P29 to Sylvain Richoz). Dorothee Hippler is acknowledge for her help for ICP-OES measurements and their analyses. Franz Tscherne for his work during the preparation of the SEM samples.


Clément Levard
email: levard@cerege.fr

Karima Hamdi-Alaoui

Isabelle Baudin

Amélie Guillon

Daniel Borschneck

Andrea Campos

Mohamed Bizi

Florence Benoit

Corinne Chanéac

Jérôme Labille

Corinne Chaneac

Silica-clay nanocomposites for the removal of antibiotics in the water usage cycle

Keywords: micropollutant, sustainable treatment, clay nanohybrid, antibiotics, water usage cycle Pharmaceutical class Antiepileptic Compounds Carbamazepine Ciprofloxacine

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

Introduction

Antibiotics are massively used in human and veterinary therapeutic applications. Their production and use lead to increasing detection of antibiotics in the environment, especially in effluents from wastewater treatment plants, but also in surface water and even groundwater.

The presence of these compounds in the water cycle can affect the quality of drinking water.

The resistance of some of these compounds to the conventional processes applied in drinking water supply chains has been evidenced in several national surveys. For example, in the United States, a federal survey identified 118 pharmaceutical micropollutants at the outlet of 25 drinking water production plants [START_REF] Furlong | Nationwide reconnaissance of contaminants of emerging concern in source and treated drinking waters of the United States: Pharmaceuticals[END_REF].

In this context, it is indispensable to strengthen the monitoring and treatment of these pharmaceutical pollutants at different stages of the water cycle. Two main treatment routes for pharmaceutical compounds are during the production of drinking water or during tertiary treatment of wastewater refining: the adsorption route and the chemical or biological oxidation route that were also widely explored for the elimination of other organic contaminants [START_REF] Pavithra | Removal of colorants from wastewater: A review on sources and treatment strategies[END_REF]. Activated Carbon (AC) is the most commonly used adsorbent medium for water treatment in industrial scale applications. The performance of AC in retaining a large number of pollutants is undeniable but is nevertheless sometimes insufficient. A wide range of other media are currently available for direct or polishing water treatment, with different characteristics (origin, activity, porosity, size, density, etc.). The best media for water treatment can be selected based on these properties in order to optimize the removal of the targeted pollutant and to be integrated in the existing treatment plant. On one hand, powdered activated carbon (PAC) in suspension can be directly added to the water to be treated, which has the advantage of being flexible as the dose can be adapted to the concentration of the pollutant. However, the main disadvantage of PAC is its high operating cost when used continuously, and the difficulty involved in separating the medium from the sludge or waste by settling or filtration at the disposal step. Granular activated carbon (GAC), on the other hand, uses a fixed-bed filter, usually located in one or two steps of the water treatment plant, for clarification and polishing. Compared with PAC, GAC is the most economical solution in larger systems or where organic contamination has to be controlled continuously. GAC performance and treatment reliability for the removal of organics are closely linked to the frequency of the carbon regeneration or replacement. In turn, the frequency is highly dependent on the adsorption affinity of the pollutants and on competition with the organic matter present in the water. This is particularly true with GAC used at the clarification step [START_REF] Knappe | Effects of activated carbon characteristics on organic contaminant removal[END_REF].

Considering the economic and technical limitations involved in the use of activated carbon in water treatment, developing an alternative process or material would be of great interest for water treatment suppliers. To be viable and applicable, such new processes should fulfill numerous conditions, including efficient pollutant elimination, easy integration into existing water treatment plants as an additional refining step, easy removal from the treated water after use, environmental sustainability, and low cost (PRASANNAMEDHA AND KUMAR, 2020).

Clay-based materials appear to be good alternative candidates in this context as they have a good adsorption capacity and ion exchange properties that can efficiently and selectively retain many micropollutants [START_REF] Besnault | Technical, economic and environmental evaluation of advanced tertiary treatments for micropollutants removal (oxidation and adsorption)[END_REF][START_REF] Zhu | Adsorbents based on montmorillonite for contaminant removal from water: A review[END_REF]. Clays can be modified, e.g. by bridging or grafting nanoparticles, to increase the surface exchange by avoiding the stacking of the clay layers, to adjust the interlayer distance to the size of the targeted micropollutants and in turn, improve the performance and adsorption selectivity (LEE AND [START_REF] Lee | Preparation and characterization of PMMA-Clay hybrid composite by emulsion polymerization[END_REF][START_REF] Jolivet | Iron Oxide Chemistry. From Molecular Clusters to Extended Solid Networks[END_REF][START_REF] Horner | Hydrothermal synthesis of large maghemite nanoparticles: Influence of the pH on the particle size[END_REF][START_REF] Banković | Synthesis and characterization of bentonites rich in beidellite with incorporated Al or Al-Fe oxide pillars[END_REF][START_REF] Brindley | Preparation and properties of some hydroxy-aluminium beidellites[END_REF][START_REF] Guégan | Tuning down the environmental interests of organoclays for emerging pollutants: Pharmaceuticals in presence of electrolytes[END_REF].

In this work, we developed and tested clay-silica nanoparticle hydrid nanostructures that can be an economic alternative to the activated carbon conventionally used in water treatment. The silica to clay ratio was adjusted to optimize the adsorption capacity of the nanocomposites (NCs). The efficiency of the resulting silica-clay NCs for the removal of ciprofloxacine was first tested on a model water. In the second step, real water contaminated by a selection of pharmaceutical micropollutants that are currently among the most recalcitrant to conventional treatments used in industrialised countries was used to assess the performance of the different NCs in realistic conditions.

Materials and Methods

Targeted pharmaceutical molecules

The pharmaceutical substances studied here were chosen based on consumption, occurrence in the water cycle, recalcitrance to conventional treatments, and potential risk for the environment. Based on these criteria, four antibiotics, doxycycline, ciprofloxacin, danofloxacin and sulfamethoxazole, and an anticonvulsant, carbamazepine, were selected (Table 1).

Table 1. Main use and characteristics of the pharmaceuticals studied here

The treatability of chemical compounds in water by adsorption on an hydrophobic medium, such as activated carbon, is often classified according to their respective log Kow (measurement of the differential solubility of chemical compounds in two different media), higher log Kow giving higher expected adsorption. Carbamazepine and sulfamethoxazole are thus expected to undergo strong adsorption, while ciprofloxacine is expected to adsorb more weakly, and danofloxacine and doxycycline should be less adsorbed. Performance tests have been carried out at laboratory, pilot and industrial scale for the elimination of carbamazepine [START_REF] Sudhakaran | Hybridization of natural systems with advanced treatment processes for organic micropollutant removals: New concepts in multi-barrier treatment[END_REF][START_REF] Akpinar | Rapid and Efficient Removal of Carbamazepine from Water by UiO-67[END_REF][START_REF] Bizi | Activated Carbon and the Principal Mineral Constituents of a Natural Soil in the Presence of Carbamazepine[END_REF][START_REF] He | Removal of the environmental pollutant carbamazepine using molecular imprinted adsorbents: Molecular simulation, adsorption properties, and mechanisms[END_REF] and sulfamethoxazole [START_REF] Stackelberg | Efficiency of Conventional Drinking-Water-Treatment Processes in Removal of Pharmaceuticals and Other Organic Compounds[END_REF]. Efficient removal of both compounds was obtained by adsorption on PAC [START_REF] Margot | Treatment of micropollutants in municipal wastewater: Ozone or powdered activated carbon?[END_REF][START_REF] Besnault | Technical, economic and environmental evaluation of advanced tertiary treatments for micropollutants removal (oxidation and adsorption)[END_REF].

AC is also well known as a non-specific but efficient treatment solution for removal of a wide range of hydrophobic compounds in various matrixes (resources, drinking water, wastewater, industrial effluents, etc.). However, the cost of AC is high and finding les costly alternative media is a real challenge for water treatment. That is why several works on different alternative adsorbent materials applied for micro-pollutants treatment have been undertaken. The materials include clay-based media [START_REF] Yu | Adsorption characteristics of selected pharmaceuticals and an endocrine disrupting compound-Naproxen, carbamazepine and nonylphenolon activated carbon[END_REF][START_REF] Zhang | Sorption and desorption of carbamazepine from water by smectite clays[END_REF] which have shown some adsorption capacity. The elimination of ciprofloxacine has been studied at laboratory scale on various media including carbon, clay, and zeolithes [START_REF] Wang | Preparation of a specific bamboo based activated carbon and its application for ciprofloxacin removal[END_REF][START_REF] Li | Zeolitic imidazolate framework-8 derived nanoporous carbon as an effective and recyclable adsorbent for removal of ciprofloxacin antibiotics from water[END_REF] ROSTAMIAN AND BEHNEJAD, 2017; BIZI AND EL BACHRA, 2020). However, these authors did not study the application or the treatment efficiency of clay-based nanocomposites in realistic conditions for the removal of several compounds with different solubility, size, hydrophobicity and charge, such as the ones targeted here. Finally, few laboratory-scale studies on danofloxacine and doxycycline treatments have focused on the difficulty of treating doxycycline by adsorption (ALI AND [START_REF] Ali | Adsorption behavior of doxycycline antibiotic on NaY zeolite from wheat ( Triticum aestivum ) straws ash[END_REF][START_REF] Liu | Facile synthesis of Cu(II) impregnated biochar with enhanced adsorption activity for the removal of doxycycline hydrochloride from water[END_REF].

Nanocomposite synthesis

The clay material used in this work was collected from Nador under the trade name Monador (North East Morocco, North Africa). It consists of a relatively pure Na,Ca montmorillonite. The cationic exchange capacity of the raw clay sieved to 20 µm was determined at 141 meq/100g. For this study, the finest fraction of the clay (< 2 µm) was recovered for the synthesis of the silica-clay NCs as follows: 20 g of the sieved clay were dispersed in 500 mL of ultrapure water by magnetic stirring at 500 rpm for 24 h. After 30 minutes, ultrasonic treatment was performed for 10 minutes. The clay suspension was then poured into a 25 cm glass column and left to settle for 20 h. According to Stokes law and considering a clay density of 2.6 g.cm -3 , 20 h correspond to a sedimentation cutoff close to 2 µm at 25 cm in height. The supernatant containing the size fraction below 2 µm was recovered and freeze dried, for further preparation.

The silica nanoparticles used in this work were obtained from "Clariant Corporation" under the trade name Klebosol® 30R50. They were characterised by a primary particle size of 80 nm [START_REF] Bizi | Stability and flocculation of nanosilica by conventional polymer[END_REF], also confirmed in hydrodynamic size in pure water measured by dynamic light scattering (82.4 ± 0.6 nm).

Silica-clay nanocomposites (NCs) were synthesized as follows. Clay was dispersed in Milli-Q water at a concentration of 20 g/L. The suspension was stirred for 24 h at 500 rpm to favor layer exfoliation. Then, a volume of SiO 2 nanoparticles was added to the suspension to reach 5 or 50 wt% of the clay substrate. At this neutral pH, both clay and silica are negatively charged and are stable in suspension. The mixture was homogenized by magnetic stirring for one minute, and then immediately frozen in liquid nitrogen to maintain the homogenous distribution of silica and clay components. Water was then removed by freeze drying, and the recovered solid material was heated at 500°C for 4 hours to consolidate the architecture of the aggregates. In order to obtain material reference, separate pure silica and clay materials were also heated at 500°C for comparison with NCs referred as SiO 2 -NP-500°C and Clay-500°C.

Characterization of the materials

Scanning electron microscopy (SEM) analysis was carried out using a Zeiss Gemini 500 high resolution field emission scanning electron microscope equipped with an in-lens secondary electron detector. Images of uncoated samples were acquired at low voltages for surface sensitivity and also to avoid beam damage. X-ray diffraction (XRD) analysis was performed using a Panalytical X'Pert Pro diffractometer running at 40kV and 40mA equipped with Co K radiation ( = 1.79 Å). Samples were deposited on low background silicon plates and scanned in a 2 range of 4-75° with a step size of 0.033° and a time step of 5.1s.

Removal efficiency of ciprofloxacin in a model effluent

Elimination of ciprofloxacin from a model effluent was studied as a preliminary approach to optimize the synthesis of the sorbent material. Evian water (pH= 7.5-7.8) was used to mimic the ionic composition of a typical surface water. Ciprofloxacin in the aqueous medium was quantified by measuring the specific absorbance peak at 272 nm wavelength. A Jasco V-650 UV-visible spectrophotometer (Jasco, Japan) was used for this purpose, with quartz cells of 1 cm optical pathway. A calibration curve was first measured showing good linearity from 0.5 to 20 mg/L (R 2 = 0.9999). Every absorbance measurement was processed in triplicate.

Ciprofloxacin was first spiked into water at 5 mg/L. The sorbent material was then added at a concentration of 50 mg/L and the reactor was kept under agitation for three hours by magnetic stirring at 650 rpm. Aliquots of the mixture were sampled at different times and filtered at 0.45 µm to remove the sorbent material and the retained ciprofloxacin. The supernatant was then analysed by UV absorbance to measure the concentration of ciprofloxacin remaining free in solution. X-ray Photoelectron Spectroscopy (XPS) was performed using a Kratos -Axis NOVA XPS equiped with a monochromatic Al K source to assess adsorption mechanism (150 Watts).

Nanocomposite dispersibility and potential for recovery after treatment

After use as a sorbent material during a tertiary or polishing treatment step, the NCs should be easily recovered from the liquid medium in an additional quaternary step before the treated water is released. This can be achieved by the rapid sedimentation of the material in the liquid batch when agitation is stopped, which depends on the size and density of the dispersed units.

Dispersion or sedimentation of the synthesized materials in aqueous medium was measured using laser diffraction and turbidity profiles. Firstly, 200 mg/L of NC was introduced in Evian water, that mimicked the ionic composition of a typical surface water, and maintained under constant agitation by magnetic stirring for four hours. A Malvern Mastersizer 3000 (Malvern Instruments, Worcester, UK) connected to the agitated system via a peristaltic pump in recirculation mode was used to measure the size distribution of the different NCs over time. For comparison with reference clay materials, the same procedure was used for pure montmorillonite clay before and after calcination at 500°C. Secondly, at t = 4h, a 20 mL aliquot of the medium was sampled under agitation and poured into the sedimentation tube. A Turbiscan apparatus (Formulaction, Toulouse, Fance) was used to measure the turbidity profile along the tube height according to time. Measurements were recorded every 25 sec during 30 minutes. The suspension clarification was analyzed via the intensity of the transmitted light increasing with time, while the formation of sediments was measured via the backscattered light intensity in the bottom of the tube (lower 1 mm). A 2% relative increase was reasonably used to distinguish the top of the solid deposit from the initial background backscattered by the liquid suspension.

Removal efficiency in real groundwater

After optimization of the synthesis and tests of NCs efficiency in model water, clay-silica NCs were tested in real groundwater spiked with the five selected micropollutants at realistic concentrations. Activated carbon, currently used in conventional water treatment, was also studied for the purpose of comparison.

Groundwater from the Paris region (France) was used for this experiment (pH = 7.5, dissolved organic carbon: 1.5 mg/L, UV absorbance at 254 nm = 4 m -1 , expressed as optical density). The main chemical elements constituting the geochemical composition were analyzed. Results are given in mg/L: Al (< 0.05), Ca (72.4), K (3.3), Mg (8.59), Na (15.3), P (< 0.05) and Zn (0.04). In order to control the presence of residual pollutants in this groundwater before exposition, the concentration of the five studied micropollutants was determined. Only carbamazepine and sulfamethoxazole were detected in the groundwater, at respectively 10 and 5 ng/L, giving the chemical background.

The five micropollutants ciprofloxacine, danofloxacine, doxycycline, carbamazepine and sulfamethoxazole were spiked into water at individual concentrations of 2 µg/L. The sorbent material was then added to the medium at a concentration of 50 mg/L, as processed in preliminary tests on the model effluent, and the mixture was agitated continuously for 48 h. Filtration at 0.45 µm on a glass fiber filter was then processed to remove the sorbent material and other particulate matter from the liquid medium.

An analytical method was developed to quantify the targeted micropollutants in this matrix and also in other complex waters (surface water, wastewater, etc.). Solid phase extraction on HLB cartridge (Waters) at pH 3 was performed. Then, after washing and drying, an elution was realised with 5 mL methanol. Analyses were liquid chromatography coupled with mass spectrometry. A triple quadrupole mass spectrometer, TSQ Quantum (Thermo Fisher Scientific) equipped with a HSS T3 column (Waters ® ) was used. The mobile phase followed a water/methanol gradient + 0.1% formic acid, circulating at a solvent flow rate of 200 µL/min, with an injected volume of 10 µL and a temperature of 30°C. Detection was achieved by two MS/MS transitions in positive ionization mode with electrospray source. Quantification was performed by internal calibration using compounds labelled with D or 13 C. The limits of detection obtained ranged between 1 and 5 ng/L. Since silica is amorphous, the X-ray diffraction pattern of the clay-based materials is consistent with a montmorillonite (Figure 1, left). The first peak corresponds to the (001) reflection (basal spacing) and is characteristic of layer stacking. For raw clay, the interlayer distance is 12.5Å, typical of hydrated Na-montmorillonite. When heated to 500°C, the lamellar structure is preserved and the (001) reflection is shifted to 9.6Å, indicating interlayer dehydration. Regarding NCs, although NC-95/5 was very similar to Clay-500°C, the first NC-50/50 peak became less intense and broader with a slight shift at lower angles, suggesting increased disorder in layer stacking and increased interlayer distance. However, no change in the order inside the layer was observed. The increase in structural desorder is in good agreement with the measured apparent density (Figure 2, right) that is more than one-order lower for NC-50/50 than for Clay-500°C (decrease in the apparent density by a fator of 14). In addition, -although not observed by X-ray diffraction, 5% of SiO 2 Nps in NC-95/5 is enough to strongly affect apparent density by one order of magnitude compared to Clay-500°C. NPs observed on the clay substrate for NC-95/5 confirms that the method of preparation used here satisfactorily delivered an apparently homogenous hybrid material. Moreover, irregularities, microcavities and the non-compact and very porous nature of the observed structures in addition to the low apparent density (probable high macroporosity) will favor the rapid diffusion of water and micropollutants. Beyond these structural considerations, the reactivity of the NCs was assessed in both model effluents and in realistic conditions.

Results

Characterization of materials

Removal efficiency of ciprofloxacin in model effluents

Ciprofloxacin was used for this first proof of concept thanks to the ease of UV-visible spectroscopy. In addition to the initial kinetics, the removal efficiency measured after 3 hours provides insight into sorbent capacity (Figure 4, orange dots), which ranged from 1% for SiO 2 -NPs-500°C to 53.5% for clay and NC-95/5. The best results were obtained for raw clay and NC-95/5 with a removal efficiency > 50%. Both products were well-hydrated after 3 hours and probably had similar surface areas and therefore similar adsorption capacity. This was not the case of NC-50/50, which showed reasonable but lower removal efficiency than for NC-95/5 and clay that can be attributed to the high fraction of SiO 2 -NPs in the nanocomposite that has no affinity for ciprofloxacin, as shown in Figure 3.

The reason why NCs are more efficient sorbent material than clay-500°C is certainly linked to the disordered and porous structure obtained in the NCs compared to clay-500°C, which is more favorable for fast diffusion of water and pollutants inside the material. In absence of NP inside the clay, the heating step at 500°C leads to sheet stacking that results in a dense structure that does not have the desired properties.

Although not responsive to ciprofloxacin, SiO 2 -NPs play a significant positive role in the kinetics of ciprofloxacin adsorption. Based on kinetic and removal efficiencies, NC-95/5 appears to be the most appropriate candidate for efficient removal of ciprofloxacin. Further analysis of the NC-95/5 sample was performed to check its crystallinity after adsorption of ciprofloxacin using XRD and to assess adsorption mechanism using XPS.

The mineralogy of NC-95/5 is preserved after adsorption as shown in Figure S1. The (001) reflection distance is shifted from 9.6Å (before adsorption) to 14.4Å (after adsorption). This basal reflection observed after adsorption of ciprofloxacin is typical of hydrated Camontmorillonite which is consistent with the fact that Evian water contains 80 mg/L of Ca 2+ . In addition to NC-95/5, calcium carbonate phases were detected which is, once again, consistent with the ionic composiiton of Evian water that contains bicarbonates (360 mg/L) which has favored the precipitation of calcite and aragonite (Figure S1).

XPS analysis was performed on NC-95/5 before and after sorption of ciprofloxacine to investigate sorption mechanism. However, although XPS confirms the presence of ciprofloxacin on the NC-95/5 substrate (specific signature of Fluor), it does not allow to identify the sorption mechanisms mainly because of the presence of a significant fraction of adventious carbon classicaly observed in XPS measurements (see Figures S2,S3, S4 and S5 in SI).

In addition to surface reactivity, the recoverability of the particles after treatment is an important aspect to be taken into consideration and was tested on the different sorbents NC-95/5, NC50/50, clay and clay-500. Laser diffraction was used to measure the dispersion of the hybrid material in water compared to raw clay and to clay-500°C. It proved that heating at 500°C causes the formation of irreversible aggregates that can withstand the dispersion step in aqueous medium. Indeed, after 4 h of agitation in water, neither clay-500°C nor the NCs displayed a particle size below 1 µm, which is roughly the upper limit of the colloidal size range. These three materials remained in an aggregated form with sizes ranging from 5 to 200 µm, while after 4 h of agitation, the clay was rapidly dispersed in sub-micrometer units. Colloidal dispersion of the montmorillonite clay usually implies continuous swelling, due to hydration of the inter-lamellar cations toward multiple layers of water molecules. Here the calcination step at 500°C led to dehydration of those cations and shrinkage of the interlamellar space, which likely reduced access to the water molecules from the outside when the material was redispersed in water. This resulted in the irreversible aggregation of the clay particles with no noticeable difference between the NCs and the clay-500°C.

Nanocomposite and clay recovery from liquid media

The larger aggregate size obtained here certainly favors more rapid sedimentation of the NCs, which should facilitate recovery from the water treatment plant after use. This was confirmed by the sedimentation trends shown in figure 6. After 4 h of agitation of the materials in synthetic surface water, the sedimentation rate was measured in terms of supernatant clarification and of formation of a sediment layer. The NCs clearly showed the highest sedimentation rates, reaching 30-35% of average supernatant clarification in 30 minutes (Figure 6A). In comparison, the pure clay, which still contained sub-micrometer dispersed units, sedimented at the lowest rate, reaching only 15-20% in the same time period. Clay-500°C displayed different sedimentation behavior characterized by a higher rate in the first 5 minutes, followed by a slower rate. This suggests coexistence of high-density aggregates that settled out rapidly, and of lower density aggregates that remained in suspension longer. These respective behaviors are corroborated by the formation of the sediment layer shown in Figure 6B. The change in sediment height over time points to higher kinetics of the nanocomposites than of pure clay, reaching approximately 0.7 mm for NCs compared with 0.35 mm in 30 min for pure clay. Again, clay-500°C underwent more rapid sediment formation in the first 5 minutes, after which the process slowed down. It is important to note that the change in sediment height over time results not only from the sedimentation rate but also from apparent sediment density. As the NCs are characterized by lower apparent density than pure clay, the sediments formed are expected to result in a thicker layer at a given mass of material. Such a contribution of the material density cannot be distinguished from that of the sedimentation rate in Figure 6B.

Of note, in the water treatment, the sedimentation efficiency of particulate matter is usually adjusted by the adding chemicals such as coagulants and/or flocculants. So any increase in sedimentation caused by the sorbent material, as obtained in our case, could also lead to lower chemical dosage and new sludge characteristics and behavior (more compact, easier to treat and to dispose of). [START_REF] Labille | Flocculation of colloidal clay by bacterial polysaccharides: Effect of macromolecule charge and structure[END_REF]. Na-Montmorillonite has also been shown to have good affinity for phenolic compounds [START_REF] Djebbar | Adsorption of phenol on natural clay[END_REF], which may explain the relatively good removal efficiency of doxycycline. Finally, the last two contaminants that

were not removed at all have neither carboxylic nor phenolic groups. They both have amino groups that were neutrally charged at the pH of our experiment. Adsorption of aniline (the phenyl group attached to an amino group) on Montmorillonite has been studied in the past and showed maximum adsorption when the pH of the solution was approximately equal to the pKa of the anilinium ion deprotonation reaction (pH 4.5-5) [START_REF] Essington | Adsorption of aniline and toluidines on montmorillonite[END_REF]. Minimum adsorption was observed at pH above 7, which could partially explain the low affinity of clay for sulfamethoxazole and carbamazepine.

Conclusion

Silica-clay nanocomposites were designed specifically for the removal of antibiotics in potable water treatment plants as an alternative to powder activated carbon. Among the five pharmaceuticals tested, the silica-clay nanocomposites enabled efficient removal of three molecules, ciprofloxacin, danofloxacin and doxycyclin, making them promising sorbent materials. In the hybrid structure of the nanocomposites, although non-reactive, the SiO 2 -NPs are homogenously distributed and strongly affect clay stacking by creating disorder which appears to favor the diffusion of water and micropollutants in the material. NC-95/5 showed the highest reactivity among the synthesized products. In addition, the design of the nanocomposites includes a calcination step, which resulted in the formation of non-reversible aggregates > 1 µm. After being used use as a sorbent in suspension, the material can thus easily be recovered from the water using normal filtration or sedimentation techniques. Finally, the use of natural clay is beneficial from an environmental point of view and the low loading of SiO 2 -NPs is advantageous in terms of the cost of synthesis.

As a possible next step in this work, NCs could be tested with other specific pollutants that are not easily treated by activated carbon due to their unique properties (hydrophilic, polar, mobile, small molecules), such as persistent and mobile organic compounds (PMOCs) [START_REF] Arp | Ranking REACH registered neutral, ionizable and ionic organic chemicals based on their aquatic persistency and mobility[END_REF]. PMOCs, such as pesticide metabolites, per-and poly fluoro alkyl substances etc., are sufficiently mobile in an aquatic environment to enter drinking water resources, and sufficiently persistent to survive current water treatment processes. Treatment of such compounds is a real challenge for environmental and sanitary risk management. Another possible perspective is to increase the efficiency of NCs by replacing the poorly reactive SiO 2 -NPs by Al-or Fe-based NPs that have a stronger affinity for these micropollutants. Finally, as an alternative to micropollutant removal through adsorption, recent publications pointed to the interest of oxydoreductases (such as peroxidases, polyphenol oxidases), hydrolases (such as proteases, esterases, lipases and cellulases) and lyases for wastewater treatment and especially for the degradation of phenol, aromatic compounds and hormones (DE CAZES et al., 2014a). The interest of using laccases and esterases for the degradation of antibiotics has recently been demonstrated and more generally, the biodegradation treatment by enzymes fixed on an adsorbent support like clay or membrane has been proposed as promising treatment solution [START_REF] Magnan | Immobilization of lipase on a ceramic membrane: activity and stability[END_REF]DE CAZES et al., 2014a;DE CAZES et al., 2014b;[START_REF] De Cazes | Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation[END_REF]. In this perspective, NCs could be used as a support for enzyme grafting.

Figure 1 .

 1 Figure 1. X-ray diffraction patterns (left) and apparent density measured in a volume of 230 mg of material (right) of the initial clay, nanocomposites and references heated at 500°C

Figure 2 .

 2 Figure 2. SEM images of NC-50/50 (left) and NC-95/5 (right)

Figure 3 .

 3 Figure 3. Removal kinetics of ciprofloxacin from a model of surface water by adsorption to the sorbent materials: clay, SiO 2 -NPs-500°C and NCs

Figure 4 .

 4 Figure 4. Removal kinetics (Figure 3) calculated on the first 4 experimental points (after 10 minutes of reaction) are represented on the primary y-axis (blue bars). Removal efficiencies after 3 hours are represented on the secondary y-axis (orange dots).

Figure 5 :

 5 Figure 5: Size distribution of the clay and NCs in synthetic surface water, at t 0 (left, just after dispersion) and after 4h of agitation (right).

Figure 6 :

 6 Figure 6: Kinetics of supernatant clarification (A) and sediment formation (B) measured respectively by light transmission and backscattering in the upper 39 mm and bottom 1 mm of the sedimentation tube for clay-based materials

Table 2 .

 2 Adsorption performances of the 5 micropollutants on activated carbon, NCs and the needed to explain this trend. However, based on the chemical composition of the different compounds, one can hypothesize that some properties may control the affinity between NCs and organics. The two antibiotics that are efficiently removed by NCs are the only ones that have a carboxylic functional group (Table1), which is known to be a good complexing group, with pKa values of 5.86/8.24 and 6.07/8.56 for Ciprofloxacin and Danofloxacin, respectively. At neutral pH, the carboxylic groups are deprotonated and most probably react with the positive Al groups of the clay (

	reference materials
	Sorbent	Cipro.	Dano	Sulfa	Doxy	Carba
	(50 mg/L)
	Activated	100%	100%	100%	100%	100%
	carbon
	MMT/SiO 2	99%	99%	0%	34%	3%
	(50/50)
	MMT/SiO 2	100%	100%	0%	60%	0%
	(95/5)
	clay-500	94%	95%	0%	41%	0%

Funding

This study was done within the framework of the POLPHARMA Project ANR-15-CE04-0007.

The authors gratefully acknowledge the French National Research Agency for financial support.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for pubication

Not applicable

Competing interests

The authors declare that they have no competing interests Authors' contributions CL, IB, AG and JL have designed the experiment, CL, KH-A, DB, AC, IB, AG and JL have performed the experiments, All authors have actively participated to the interpretation of the data, writting of the manuscript and approved the final manuscript.