Fatima-Zahra Ait-Itto

Mathieu Martinez

Gregory D Price

Ait Abdellah

Addi

Synchronization of the astronomical time scales in the Early Toarcian: a link between anoxia, carbon-cycle perturbation, mass extinction and volcanism

Keywords: Astrochronology, Toarcian, carbon cycle, Middle Atlas, oceanic anoxic events

The Late Pliensbachian-Early Toarcian is a pivotal time in the Mesozoic era, marked by pronounced carbon-isotope excursions, biotic crises and major climatic and oceanographic changes. Here we present new high-resolution carbon-isotope and magnetic-susceptibility measurements from an expanded hemipelagic Late Pliensbachian-Early Toarcian section from the Middle Atlas Basin (Morocco). Our new astronomical calibration allows the construction of an orbital time scale based on the 100-kyr eccentricity cycle. The Early Toarcian Polymorphum Zone contains 10 to 10.5 repetitions of the 100-kyr eccentricity both in the carbon-isotope and the magnetic-susceptibility data, leading to an average duration of 1.00 ± 0.08 myr. We also show that the Late Pliensbachian-Early Toarcian global carbon-cycle perturbation has an average duration of 0.24 ± 0.02 myr. These durations are comparable to previous astrochronological time scales provided for this time interval in the most complete sections of the Tethyan area, and longer than what has been provided in condensed sections.

Anchoring this framework on published radiometric ages and astrochronological time scales, we estimate that the carbon-cycle perturbation of the Late Pliensbachian-Early Toarcian corresponds with the early phase of the Karoo and Chonke Aike large igneous provinces.

Likewise, our new age constraints confirm that the Toarcian oceanic anoxic event is synchronous to the main phase of the Ferrar volcanic activity. Thus, these successive and short phases of the volcanic activity may have been at the origin of the successive phases of the mass extinctions observed in marine biotas in the Pliensbachian and Toarcian times.

Introduction

The Early Toarcian (184.15 -174.1 Ma) was a time of global warming events [START_REF] Dera | Climatic ups and downs in a disturbed Jurassic world[END_REF], mass extinctions [START_REF] Little | Early Jurassic mass extinction: a global long-term event[END_REF], and pronounced negative carbon-isotope excursions recorded in marine carbonates and organic matter, brachiopods, biomarkers and fossil wood [START_REF] Hesselbo | Carbon-isotope record of the Early Jurassic (Toarcian) Oceanic Anoxic Event from fossil wood and marine carbonate (Lusitanian Basin, Portugal)[END_REF]Suan et al., 2008a;[START_REF] Ait-Itto | Bulk-carbonate and belemnite carbon-isotope records across the Pliensbachian-Toarcian boundary on the northern margin of Gondwana (Issouka, Middle Atlas, Morocco)[END_REF]. These events coincide with carbonate production demises [START_REF] Bassoullet | Le Toarcien inférieur : une période de crise dans les bassins et sur les plate-formes carbonatées de l'Europe du Nord-Ouest et de la Téthys[END_REF][START_REF] Wilmsen | Biosedimentology of the Early Jurassic post-extinction carbonate depositional system, central High Atlas rift basin, Morocco[END_REF] and widespread oceanic anoxia [START_REF] Jenkyns | The Early Toarcian anoxic event: stratigraphic, sedimentary and geochemical evidence[END_REF][START_REF] Hesselbo | Carbon-isotope record of the Early Jurassic (Toarcian) Oceanic Anoxic Event from fossil wood and marine carbonate (Lusitanian Basin, Portugal)[END_REF].

Although large amounts of data have been produced, the timing and rhythms of the environmental perturbations are still debated, leading to controversies on the mechanisms at the onset of the climatic changes in the Early Toarcian (e.g. [START_REF] Kemp | Astronomical pacing of methane release in the Early Jurassic period[END_REF]Suan et al., 2008b).

For instance, [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF] assess the duration of Pliensbachian-Toarcian event (P-To event) at 0.18 to 0.27 myr, while in the Peniche section (Lusitanian Basin, Portugal) the duration of this event is evaluated as 0.05 myr (Suan et al., 2008b). Similarly, the estimates of duration of the Polymorphum Zone vary from 90 kyr to 1.15 myr (Matiolli and Pittet, 2004;Suan et al., 2008b[START_REF] Boulila | Astronomical calibration of the Toarcian Stage: Implications for sequence stratigraphy and duration of the early Toarcian OAE[END_REF][START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF][START_REF] Ruebsam | Chronology of the Early Toarcian environmental crisis in the Lorraine Sub-Basin (NE Paris Basin)[END_REF][START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. The main reason of these differences is the occurrence of four discontinuity events identified by correlations ofδ 13 C and sedimentological features throughout the Tethyan margins, which are due to changes in the sea level observed in the earliest Toarcian [START_REF] Pittet | Carbon isotope evidence for sedimentary discontinuities in the lower Toarcian of the Lusitanian Basin (Portugal): Sea level change at the onset of the Oceanic Anoxic Event[END_REF]. The section studied here in Issouka, Morocco, is expanded compared to the sedimentary series from the northern Tethyan margin. It provides the opportunity to fill the gap in the time scale of the Toarcian Stage and to establish a detailed chronology of the succession of the environmental disturbances occurring in the Early Toarcian.

Geological setting

The geological history of Morocco was influenced by two important events, starting in the early Mesozoic with the opening of the north Atlantic and western Tethys and the collision of Africa and Europe during the middle Cenozoic [START_REF] Michard | Eléments de géologie Marocaine[END_REF]. These events led to the formation of fault-bounded basins, which are made up of several smaller depocenters, separated by synsedimentary highs [START_REF] Studer | Déformations synsédimentaires en compression pendant le Lias supérieur et le Dogger, au Tizi n'lrhil (Haut Atlas central de Midelt, Maroc)[END_REF]. The Middle Atlas is one of these small fault-bounded basins (Fig. 1). It constitutes a part of a Meso-Cenozoic intracontinental chain, namely the Moroccan Atlas [START_REF] Michard | Eléments de géologie Marocaine[END_REF]. The Middle Atlas of Morocco is structurally dominated by four NE-SW trending anticlines and is mainly constituted of Lower and Middle Jurassic formations [START_REF] Du Dresnay | Extension et développement des phénomènes récifaux Jurassique dans le domaine Atlasique Marocain, particulièrement au Lias moyen[END_REF]). The basin is bounded by the Saïs Plain and the front of the Rifian Nappes in the North, by the Guercif Basin in the northeast, by the Moulouya Plain to the southeast, and by the Hercynian Central Massif in the West.

The Middle Atlas Basin is deep in the center and shallows towards the northern and southern basin margins [START_REF] Du Dresnay | Extension et développement des phénomènes récifaux Jurassique dans le domaine Atlasique Marocain, particulièrement au Lias moyen[END_REF]). The study area during the Early Toarcian was located at a palaeolatitude of ~20°N [START_REF] Bassoulet | Mid Toarcian[END_REF] (Fig. 1A). The sedimentary evolution and palaeogeographic differentiation is controlled by tectonic activity, combined with the rate of sedimentation and global eustatic variations [START_REF] Wilmsen | Biosedimentology of the Early Jurassic post-extinction carbonate depositional system, central High Atlas rift basin, Morocco[END_REF]. The rapid transition from shallow marine carbonates to hemipelagic marls has been taken to reflect a major deepening phase across the entire Middle and High Atlas area [START_REF] Wilmsen | Biosedimentology of the Early Jurassic post-extinction carbonate depositional system, central High Atlas rift basin, Morocco[END_REF]. The Pliensbachian-Toarcian transition coincides with a dislocation of the Lower Jurassic carbonate platform [START_REF] Lachkar | Early Jurassic normal faulting in a carbonate extensional basin: characterization of tectonically driven platform drowning (High Atlas rift, Morocco)[END_REF][START_REF] Ait Addi | Sedimentary evolution and palaeogeography of mid Jurassic deposits of the Central High Atlas, Morocco[END_REF] with Toarcian deposits dominated by marls lying upon Upper Pliensbachian shallow marine limestones and calcareous marls. This drowning episode is linked to the eustatic sea-level rise of the Early Toarcian described in Europe and Africa (e.g. [START_REF] Hallam | Estimates of the amount and rate of the of sea level change across the Rhaetian-Hettangian and Pliensbachian-Toarcian boundary (latest Triassic to Early Jurassic)[END_REF].

The biostratigraphy in the Middle Atlas has been established with ammonites [START_REF] El Hammichi | Les faunes d'Ammonites du Toarcien-Aalénien du Moyen Atlas sud-occidental (Maroc)[END_REF] with further biostratigraphic data provided by benthic foraminifera [START_REF] Bejjaji | Foraminiferal biostratigraphy of the Toarcian deposits (Lower Jurassic) from the Middle Atlas (Morocco). Comparison with western Tethyan areas[END_REF]. The ammonite zonation in the Issouka section is based on the Mediterranean zonation. Notably, the ammonites Emaciaticeras emaciatum of the Emaciatum Zone, Dactylioceras polymorphum of the Toarcian Polymorphum Zone and Hildaites in the Semicelatum Zone have been identified (e.g. El Hammichi et al., 2008). Furthermore, the occurrence of the benthic foraminifera Lenticulina sublaevis in the Issouka section is correlated by [START_REF] Bejjaji | Foraminiferal biostratigraphy of the Toarcian deposits (Lower Jurassic) from the Middle Atlas (Morocco). Comparison with western Tethyan areas[END_REF] to the Emaciatum ammonite zone of the Pliensbachian, whilst Lenticulina bochardi and Lenticulina toarcense are correlated with the Toarcian Polymorphum Zone and Lenticulina obonensis with the Serpentinus Zone.

The Issouka section is situated near the village of Issouka, ~25 km southwest of Immouzer Marmoucha, in the Middle Atlas (N33° 26' 55.56";W4° 20' 33.83";Fig. 1). The section begins with centimeter thick limestone-marl alternations (Fig. 2). The limestone beds contain a rich ammonite fauna, with also belemnites, echinoids and brachiopods. Foraminifera suggested a Late Pliensbachian age [START_REF] Bejjaji | Foraminiferal biostratigraphy of the Toarcian deposits (Lower Jurassic) from the Middle Atlas (Morocco). Comparison with western Tethyan areas[END_REF]Fig. 2A). The early Toarcian succession starts with green marls and marl-limestone alternations, rich in foraminifera, belemnites, echinoids and gastropods. The Toarcian deposits are generally hemipelagic and correspond to deep marine environments [START_REF] Bejjaji | Foraminiferal biostratigraphy of the Toarcian deposits (Lower Jurassic) from the Middle Atlas (Morocco). Comparison with western Tethyan areas[END_REF]Fig. 2B).

Material and methods

A total of 430 bulk-rock samples were collected with an even sample distance of 10 cm in the Issouka section. The samples were recovered from up to 15 cm below the surface, to minimize the effects of surface weathering. The sampled interval encompasses the end of the Pliensbachian Stage to the lowermost part of the Levisoni ammonite Zone. Such a range allows the Polymorphum Zone to be entirely sampled. The bulk-rock samples were then powdered using a metal ring grinder and analyzed for stables isotopes, magnetic susceptibility and calcium carbonate contents.

Carbon isotopes

A total of 430 samples were analyzed for stable isotopes at the University of Plymouth. Using 200 to 300 micrograms of carbonate, stable isotope data were generated on a VG Optima mass spectrometer with a Gilson autosampler. Isotope ratios were calibrated using NBS19 standards and are given in δ notation relative to the Vienna Pee Dee Belemnite (VPDB). Reproducibility was generally better than 0.1 ‰ for samples and standard material.

Magnetic susceptibility

The magnetic susceptibility (MS) of the 430 powdered samples was measured with a Kappabridge KLY-3. The samples are placed in small plastic cubes of 10 cm 3 and then introduced inside the instrument using the "pick-up" unit. We measured the cube empty and then with samples for blank correction. The blank-corrected MS values are normalized to their mass and volume. The resulting values are reported as mass-specific MS (m 3 .kg -1 ).

Calcium carbonate content

The samples were analyzed for their calcium carbonate content using a Bernard calcimeter at the Geosciences and Environment Laboratory, Cadi Ayyad University. The values are given with a precision between 1 and 5% [START_REF] Lamas | Selection of the most appropriate method to determine the carbonate content for engineering purposes with particular regard to marls[END_REF].

Spectral analysis

Prior to spectral analysis, the long-term trend of both series was measured and subtracted from the series to ensure stationarity. The detrend procedures removed the high powers in the frequencies close to 0, while not creating new spectral peaks in the low frequencies (see [START_REF] Vaughan | Cyclostratigraphy: data filtering as a source of spurious spectral peaks[END_REF]. Best-fit linear trend and LOWESS smoothing curves [START_REF] Cleveland | Robust Locally Weighted Regression and Smoothing Scatterplots[END_REF] were applied. The exact coefficients depend on each of the series and are detailed in the results. Following the removal of this long-term trend, the multi-taper spectral analyses, using three 2π prolate tapers (2π-MTM) with robust red-noise modelling [START_REF] Mann | Robust estimation of background noise and signal detection in climatic time series[END_REF] modified in [START_REF] Meyers | Astrochron: An R Package for Astrochronology[END_REF] were applied to evaluate lithological cycles as a possible record of astronomically forced sedimentation following the LOWSPEC approach [START_REF] Meyers | Seeing red in cyclic stratigraphy: Spectral noise estimation for astrochronology[END_REF]. The significance levels were then calculated assuming a chi-square distribution of the spectral background. We used the time-frequency weighted fast Fourier transforms (T-F WFFT; [START_REF] Martinez | Astrochronology of the Valanginian-Hauterivian stages (Early Cretaceous): chronological relationships between the Paraná-Etendeka large igneous province, the Weissert and the Faraoni events[END_REF] to characterize the evolution of the periods through the sedimentary series. Taner low-pass and band-pass filters [START_REF] Taner | Attributes Revisited[END_REF] were then applied to isolate each signal interpreted as an orbital forcing and calculate the duration of the Polymorphum Zone by cycle counting. Orbital tuning procedures are then used to anchor the sedimentary cycles to their corresponding orbital periods, allowing depth-time conversions to be done. Durations are then estimated by calculating the difference of relative ages. Error margins of each of the time intervals include the difference between the average and the calculated durations and the uncertainty in the orbital frequencies.

Results

Carbonate carbon isotopes

The carbon-isotope profile of the Issouka section shows relatively high values in the Pliensbachian part of the section, ranging from +1.7‰ to +2.7‰ (Fig. 3A). From 5 m to 5.6 m, the  13 C decreases from 2.5‰ to -0.6‰. This abrupt decrease of 3‰ in the  13 C observed at the base of the Toarcian is related to the Pliensbachian-Toarcian event (P-To event; Suan et al., 2008a;[START_REF] Ait-Itto | Bulk-carbonate and belemnite carbon-isotope records across the Pliensbachian-Toarcian boundary on the northern margin of Gondwana (Issouka, Middle Atlas, Morocco)[END_REF]. The  13 C values then remain low from 5 m to 23 m. In this interval, the values show gentle fluctuations from 5 to 17 m. At 17 m, the  13 C abruptly increase by 1‰ before they gently decrease until 23 m to -2‰. The values then progressively increase from 23 m to 30 m, changing from -2‰ to 1‰. Above 30 m to the top of the section, the values slightly increase from 1 to 2‰.

Magnetic susceptibility

The magnetic susceptibility curve (Fig. 3E) firstly shows from the base of the series to level 5.1 m low values, ranging from 9.65x10 -9 to 3.15x10 -8 m 3 .kg -1 . This interval corresponds to the limestone-dominated part of the formation of Pliensbachian age. From 5.2 m to the top of the series, MS values increase with an average value 1.04x10 -7 m 3 .kg -1 . The interval from 27 m to 35 m shows lower values of the MS in a more carbonated interval. Calcium carbonate content and MS values display a strong inverse correlation (r = -0.91, Fig. 4), indicating that the lithology mainly controls the MS variations.

Calcium carbonate content

The carbonate content series (Fig. 3D) firstly show high values (>80% on average) from the base of the series to 5.1 m, in the interval of the limestone beds of the Pliensbachian. From 5.2 m to 8.4 m, the values rapidly decrease from 91% to 8.6%. From 5.2 m to 18 m, the CaCO3 values remain low with average values around 22%. The transition between the first to the second interval is more progressive than observed in the δ 13 C series (Fig. 3A). From 18 m to 42.9 m CaCO3 series show high-amplitude cycles and high-frequency fluctuations from 60% and 20% linked to the limestone-marl alternations (Fig. 3D).

Spectral analyses

Carbon-isotope signal

The  13 C shows a rapid decrease at 5.2 m, and a rapid increase at 16.6 m (Fig. 3A). The series was stationarized using a best-fit linear trend from 0 to 5.1 m, a LOWESS regression with a coefficient of 0.6 from 5.2 to 16.5 m and a LOWESS regression with a coefficient of 0.3 from 16.6 to 42.9 m. The LOWESS coefficients have been chosen in such a way that they approximatively cover the same length of the section (i.e. ~7 m on average), which represents ca. 20% of the Toarcian part. This choice decreased the high powers at frequencies near 0, while not creating spurious spectral peaks in the low frequencies [START_REF] Vaughan | Cyclostratigraphy: data filtering as a source of spurious spectral peaks[END_REF]. The three subseries were then standardized (average = 0, standard deviation = 1) and stacked. Two points at 11.7 m and 12 m showing outstandingly low values when detrending may bias the spectrum in the high frequencies and have been removed from the series before detrending. The evolutive spectrum of the  13 C series shows a high-power spectral band in the low frequencies evolving having a period of 4.1 to 4.2 m from the base of the series to level 24 m (Fig. 3C). The period of this band then decreases to 3 m, from level 24 m to level 31 m, and stabilizes at 3 m from level 31 m to the top of the series. These changes in the period of the ~4-m peak likely reflects variations in the sedimentation rate. These distort the sedimentary record of the orbital cycles by decreasing the p-value of the low frequency and flattening the spectrum at high frequencies [START_REF] Weedon | Time-Series Analysis and Cyclostratigraphy: Examining stratigraphic records of environmental cycles[END_REF][START_REF] Kemp | Optimizing significance testing of astronomical forcing in cyclostratigraphy[END_REF][START_REF] Martinez | Testing the impact of stratigraphic uncertainty on spectral analyses of sedimentary series[END_REF]. We thus decided to analyze the frequency content of two sub-intervals of the series in which variations in the sedimentation rate are limited. The range of each of the sub-intervals is given in Fig. 3C. We compared the frequencies of the carbon isotopes in each interval with the orbital frequencies calculated for the Toarcian [START_REF] Waltham | Milankovitch Period Uncertainties and Their Impact on Cyclostratigraphy[END_REF], using the average spectral misfit (ASM; [START_REF] Meyers | Quantification of deep-time orbital forcing by average spectral misfit[END_REF]. The ASM method compares the sedimentary frequencies converted into time to the orbital frequencies and provides a quantitative assessment on the most likely sedimentation rate in each sub-interval (see supplementary materials). Spectra of Interval 1, from levels 0 to 31 m, show significant periods above the 99% confidence level (CL) at 4.2 m (frequency: 0.2404 cycles.m -1 ), and above the 95% CL at 1.2 m (frequency: 0.8013 cycles.m -1 ), 0.83 m (frequency: 1.202 cycles.m -1 ), and 0.31 m (Fig. 5A,C). Spectra of Interval 2, from levels 24 m to the top of the series show significant peaks above the 95% CL at 3.2 m (frequency: 0.3158 cycles.m -1 ), 0.63 m (1.579 cycles.m -1 ) and 0.33 m (Fig.

5B, D).

As noted above, various astrochronological frameworks estimate the duration of the Polymorphum Zone from 0.09 myr to 1.15 myr [START_REF] Mattioli | Spatial and temporal distribution of calcareous nannofossils along a proximal-distal transect in the Lower Jurassic of the Umbria-Marche Basin (central Italy)[END_REF]Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF][START_REF] Boulila | Astronomical calibration of the Toarcian Stage: Implications for sequence stratigraphy and duration of the early Toarcian OAE[END_REF][START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF][START_REF] Boulila | A review of tempo and scale of the early Jurassic Toarcian OAE: implications for carbon cycle and sea level variations[END_REF], implying a sedimentation rate varying from 400 m/myr to 31 m/myr. When converting m-cycles to time-cycles, the peaks at ~0.3 m have periods below the Milankovitch band whatever the sedimentation rate considered, so were excluded from the ASM analysis. Testing ASM over 500 sedimentation rates ranging from 0.4 cm/kyr to 40 cm/kyr leads to a most likely sedimentation rate of 3.9 cm/kyr (39 m/myr) in Interval 1, and to a most likely sedimentation rate of 3.2 cm/kyr (32 m/myr) in Interval 2 (Fig. 5E,F). The H0-significance level at the most likely sedimentation rate is below the critical significance level below which we can consider without ambiguity these spectra reflect an orbital forcing. In Interval 1, peaks at 4.2 m, 1.2 m and 0.83 m are respectively due to the 100-kyr eccentricity, the obliquity and the precession cycles. Notice than the peak of 1.2 m leads to an obliquity period which is shorter than expected in the theory (here 29.4 kyr vs. 37.5 kyr in [START_REF] Waltham | Milankovitch Period Uncertainties and Their Impact on Cyclostratigraphy[END_REF]. In Interval 2, peaks at 3.2 m and 0.63 m are respectively due to the 100-kyr eccentricity and the precession cycles. In addition, the peak at 0.33 m has a mean period of 10 kyr, which corresponds to the semi-precession, the first harmonic of the precession [START_REF] Berger | Equatorial insolation: from precession harmonics to eccentricity frequencies[END_REF].

The evolutive spectral analysis indicates the 100-kyr cycle is the most continuous throughout the studied series (Fig. 3C). We filtered the 100-kyr eccentricity recorded in the  13 C series and used it as target cycle for calibrating the series in time. We filtered the 100-kyr cycle using a Taner low-pass filter with a frequency cut of 0.3953 cycles.m -1 and a roll-off rate of 10 36 . The output filter indicates that the Polymorphum Zone contains 10 repetitions of the 100-kyr eccentricity cycle (Fig. 3B).

Magnetic susceptibility

The MS series is marked by a rapid increase of values from 5.0 m to 6.8 m. The trend of the series is then gentler. The series was stationarized using a best-fit linear regression from 0 to 5.2 m and a LOWESS smoothing with a coefficient of 0.2 in the remainder of the series, which represents 7.5 m of the section. The two subseries were then standardized (average = 0, standard deviation = 1) and stacked. The evolutive spectrum of the MS series shows a band in the low frequencies evolving from 3.1 m to 3.7 m in the lower part of the series, from the base of the series to level 20 m (Fig. 3G). Then, this period decreases to 2.9 m from level 26 m to the top of the series. 2π-MTM spectra have been generated from Interval 1 (from the base of the series to level 26 m), and from Interval 2 (from 21 m to the top of the series) (Fig. 6). Spectra of Interval 1 show spectral peaks over the 99% CL at 3.3 m and 0.38 m, and peaks over the 95% CL at 0.81 m and 0.23 m (Fig. 6A,C). Spectra of Interval 2 show peaks over the 99% CL at 2.9 m, 2.0 m, 0.27 m and 0.22 m, and a peak exceeding the 95% CL at 1.0 m (Fig. 6B,D).

Assuming from ASM applied on the  13 C spectra that Interval 1 has a mean sedimentation rate of 39 m/myr, the peaks of 3.3 m, 0.81 m and 0.38 m are attributed to the 100-kyr eccentricity, the precession and the semi-precession, respectively. Assuming from ASM that Interval 2 has a mean sedimentation rate of 32 m/myr, the peak of 2.9 m, 1.0 m and 0.27 m are respectively attributed to the 100-kyr eccentricity, the obliquity and the semiprecession (Fig. 6).

In Interval 1, the period of the 100-kyr cycle is lower in the MS series than in the  13 C.

On the evolutive spectral analyses, this is particularly obvious in the first 10 m of the series, when the lithology evolves from the limestones of the Pliensbachian to the marls of the Toarcian (Fig. 3). The 100-kyr eccentricity recorded in the MS series was isolated using a Taner lowpass filter with a frequency cut of 0.4070 cycles.m -1 and a roll-off rate of 10 36 (Fig. 3). The output filter indicates that the Polymorphum Zone clearly contains ~10.5 repetitions of the 100kyr eccentricity cycle compared to the 10 repetitions observed in the  13 C (Fig. 3).

Discussion

Astrochronology of the the Polymorphum Zone

The  13 C and the MS signals both display 12 complete short eccentricity cycles along the studied section. In the astronomical models computed for the last 20 myr [START_REF] Laskar | A longterm numerical solution for the insolation quantities of the Earth[END_REF], the average duration of 12 consecutive short eccentricity cycles is 1149 ± 63 kyr (2), so that the average duration of a short eccentricity cycle 95.8 ± 5.2 kyr (2). Assuming a constant sedimentation rate between two successive anchor points of the 100-kyr cycle obtained from the filters of the  13 C and the MS signals, the durations calculated of the Polymorphum Zone range from 0.97 myr with the  13 C signal, to 1.02 myr with the MS signal. In the spectra of the untuned series, the period of the 100-kyr cycle is longer in the  13 C than in the MS (Figs. 3,5,6). The difference is particularly noticeable in the first 10 m (Fig. 3). Below level 5 m, the filter of 100-kyr cycle in the  13 C is inversely correlated to the filter of the 100-kyr in the MS signal.

After 5 m, i.e. after the start of the P-To event, the two filters are in phase. The limestone beds in the Pliensbachian of the atlasic basins originate from exports from the neritic environments, in which the  13 C was higher than in pelagic environments [START_REF] Wilmsen | Biosedimentology of the Early Jurassic post-extinction carbonate depositional system, central High Atlas rift basin, Morocco[END_REF]. In the Pliensbachian, the  13 C is higher in limestone beds. The P-To Event corresponds to a demise in the neritic carbonate production. Fluctuations in the  13 C thus correspond to fluctuations in the pelagic production, which is usually higher during the deposit of marlier intervals, when detrital and nutrient increased the primary productivity [START_REF] Mutterlose | Milankovtich-scale palaeoclimate changes in pale-dark bedding rhythms from the Early Cretaceous (Hauterivian and Barremian) of eastern England and northern Germany[END_REF][START_REF] Mattioli | Spatial and temporal distribution of calcareous nannofossils along a proximal-distal transect in the Lower Jurassic of the Umbria-Marche Basin (central Italy)[END_REF]. The demise of the carbonate platforms thus changed the phasing between the MS and the  13 C, which is a source of uncertainty in the calculation of the durations. Another change of phasing occurs at 28 m, where the  13 C and the CaCO3 content increase. As the phasing between the orbital configuration and the proxies is still unclear, we choose to retain the average duration of 1.00 ± 0.08 myr for the Polymorphum Zone (the uncertainty includes the age model and the duration of a short eccentricity period, see supplementary materials).

The spectrum of the calibrated  13 C shows a high power at 96 kyr (expected when calibrated to the short eccentricity), 32.4 kyr (main obliquity), 24.1 and 19.7 kyr (precession), and 13.2 to 11.0 kyr (half-precession) (Fig. 7A). In addition, a peak exceeding the 95% confidence level appears at 400 kyr, which corresponds to the long-eccentricity cycle. The spectrum of the calibrated MS shows a high power at 95.7 kyr (expected), 65.5 kyr, 31.9 kyr (main obliquity), from 24.4 to 19.1 kyr (precession), and from 12.1 to 10.6 kyr (half precession) (Fig. 7B). In the  13 C and MS signals, the precession cycle has slightly longer periods than expected in the theory [START_REF] Waltham | Milankovitch Period Uncertainties and Their Impact on Cyclostratigraphy[END_REF]. This may be the consequence of a precession cycle missing per short eccentricity. In the astronomical solutions, the amplitude of the precession band is modulated by the 405-kyr and the 100-kyr eccentricity cycles, while the amplitude of the 100-kyr cycle is modulated by the 400-kyr cycle [START_REF] Laskar | A longterm numerical solution for the insolation quantities of the Earth[END_REF]. The Taner band-pass filter of the precession cycle in the  13 C series was performed with frequency cuts of 3.583 x 10 -2 cycles/kyr and 5.417 x 10 -2 cycles/kyr, and a roll-off rate of 10 36 (Fig. 8B,E). We calculated the amplitude modulation (AM) of the precession cycles using a Hilbert transform. The spectrum of the AM of the precession filter shows cycles at 340 kyr (near the 405-kyr eccentricity), and at 123 and 82.8 kyr (near the periods of 124 and 95 kyr of the short eccentricity) (Fig. 8F). We filtered the 100-kyr band of the AM of the precession filter using a Taner band-pass filter with frequency cuts of 6.250 x 10 -3 cycles/kyr and 1.558 x 10 -2 cycles/kyr and a roll-off rate of 10 36 (Fig. 8F). The AM of this filter shows a cycle at 340 kyr (Fig. 8G).

Similarly, the spectrum of the AM calculated on the direct filter of the 100-kyr band shows a cycle at 436 kyr (Fig. 8H). Thus, the AM of the precession and the 100-kyr eccentricity show similar patterns as expected in the astronomical models. The filters of the two bands shows 11 cycles of short eccentricity within the Polymorphum Zone (Fig. 8C), while the various filters of the 405-kyr cycles in the AM of the precession and the 100-kyr cycles show from 3 to 4 repetitions of the 405-kyr cycle (Fig. 8A,C,D). The difference between the direct filter and the filter of the AM is explained by the residence time of carbon in the ocean, which creates a delay in the response of the  13 C to the orbital forcing [START_REF] Laurin | Orbital Signals in Carbon Isotopes: Phase Distortion as a Signature of the Carbon Cycle[END_REF]. In this context of hemipelagic sediments, with the limestone beds originating from exports from neritic environments, the difference may also be due to the change of the carbonate source.

The duration of the Polymorphum Zone from this study is longer than the duration of ~0.8 myr assessed in the Peniche section (Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF] and the Foum Tillicht section [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. The correlation of these sections led to a duration of a composite Polymorphum Zone of 0.9-1.0 myr, which agrees with the duration calculated here. In addition, the duration calculated here is in close agreement with the duration estimated from Central Italy, in which 11 bundles of marl-limestone alternations have been related to the 100-kyr eccentricity cycle [START_REF] Mattioli | Spatial and temporal distribution of calcareous nannofossils along a proximal-distal transect in the Lower Jurassic of the Umbria-Marche Basin (central Italy)[END_REF].

The series of condensation affecting the Pliensbachian-Toarcian transition strongly affects the duration assessments in the Northern Tethyan margin. For instance, in the Paris Basin, the duration of the Tenuicostatum Zone (equivalent to Polymorphum) varies from 0.09 myr to 0.5 myr [START_REF] Boulila | Astronomical calibration of the Toarcian Stage: Implications for sequence stratigraphy and duration of the early Toarcian OAE[END_REF][START_REF] Ruebsam | Chronology of the Early Toarcian environmental crisis in the Lorraine Sub-Basin (NE Paris Basin)[END_REF]. Correlations of  13 C curves indicate that these sections are affected by a series of condensation events, leading to an underestimation of the duration of the Tenuicostatum Zone [START_REF] Pittet | Carbon isotope evidence for sedimentary discontinuities in the lower Toarcian of the Lusitanian Basin (Portugal): Sea level change at the onset of the Oceanic Anoxic Event[END_REF]. Recently, [START_REF] Boulila | A review of tempo and scale of the early Jurassic Toarcian OAE: implications for carbon cycle and sea level variations[END_REF] suggested that the 100-kyr eccentricity cycles identified in Suan et al. (2008b) and [START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF] are the record of the obliquity cycle. However, this would imply a hiatus of ~800 kyr in the middle of the Tenuicostatum Zone, which is not indicated in the  13 C data [START_REF] Pittet | Carbon isotope evidence for sedimentary discontinuities in the lower Toarcian of the Lusitanian Basin (Portugal): Sea level change at the onset of the Oceanic Anoxic Event[END_REF]. In addition, detailed observations of the sedimentological pattern of Peniche indicate than these cycles correspond to bundles of 4-6 marl-limestone alternations, which is related to the hierarchy in between the short eccentricity and precession (Suan et al., 2008b). We thus retain here for the Peniche section the astrochronological models from Suan et al. (2008b) and [START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF].

Duration of the P-To Event

The duration of the P-To event varies from ~50 kyr to 0.27 myr from various astrochronological models (Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF][START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. Uncertainties are due to the condensation event often observed at the Pliensbachian-Toarcian boundary [START_REF] Pittet | Carbon isotope evidence for sedimentary discontinuities in the lower Toarcian of the Lusitanian Basin (Portugal): Sea level change at the onset of the Oceanic Anoxic Event[END_REF] and to the difficulty in defining the upper boundary of the P-To event [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. The P-To event indeed starts with an abrupt decrease of 2 to 3‰ the  13 C and change in lithology from carbonated to detrital sediment (Suan et al., 2008a;[START_REF] Bodin | Toarcian carbon isotope shifts and nutrient changes from the Northern margin of Gondwana (High Atlas, Morocco, Jurassic): Palaeoenvironmental implications[END_REF]. Then, the  13 C values gently increase, making the definition of an upper limit of the P-To event problematic. [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF] defined two options to define the upper boundary of the P-To event: (i) the end of the decrease of the  13 C and CaCO3 values; (ii) the local increase of the  13 C values in the lower part of the Polymorphum Zone, corresponding to a maximum of the filter of the 405-kyr band in the  13 C. In the Issouka section, the filter of the 405-kyr band was calculated on the calibrated series (high frequency cut: 0.005833 cycles/kyr; roll-off rate: 10 36 ) and converted into depths shows that this maximum is at 13.1 m (Fig. 9). We suggest using this marker as the end of the P-To event, as it can be observed both in the Central High Atlas and the Middle Atlas. From the  13 C and the MS age models, the duration of the P-To event as defined here varies from 0.23 to 0.25 myr, with an average duration of 0.24 ± 0.02 myr (this uncertainty takes into account the uncertainty of the age model and the uncertainty of the period of the eccentricity, see supplementary materials). These durations are in agreement with Option 1 from the Foum Tillicht section, which vary from 0.25 to 0.29 myr, with an average duration of 0.27 myr [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. This duration of the P-To event appears stable in Moroccan sections, ranging from 0.23 to 0.29 myr.

A synthetic astrochronology for the Tethyan area in the Early Toarcian

The Polymorphum Zone at Issouka shows 10 to 10.5 repetitions of the 100-kyr eccentricity cycles. In comparison, the sedimentary records of Foum Tillicht and Peniche show a total of eight 100-kyr eccentricity cycles for the Polymorphum Zone. Lithological patterns in the Umbria-Marche Basin tend to indicate that the Polymorphum Zone contains ten to eleven repetitions of 100-kyr cycle [START_REF] Mattioli | Spatial and temporal distribution of calcareous nannofossils along a proximal-distal transect in the Lower Jurassic of the Umbria-Marche Basin (central Italy)[END_REF]. Four discontinuities identified in the Pliensbachian-Toarcian transition have affected the completeness of the sedimentary record in the Foum Tillicht and the Peniche sections. In particular, at Peniche, the Pliensbachian-Toarcian transition appears condensed, while in Foum Tillicht the top of Polymorphum is missing [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF]. In Issouka, Foum Tillicht and Peniche,  13 C has been measured at a high resolution and astrochronologies have been produced, making it possible to correlate the sections at a Milankovitch scale.

Based on the astrochronological frameworks produced in this study and in [START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF] and [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF], we can correlate these three sections at the eccentricity scale (Fig. 9). Radiometric ages have been anchored on stratigraphic ages at the Triassic-Jurassic boundary, in the Hettangian and the Sinemurian [START_REF] Ruhl | Astronomical constraints on the duration of the Early Jurassic Pliensbachian Stage and global climatic fluctuations[END_REF]. A U-Pb age has been proposed for the uppermost part of the Tenuicostatum Zone at 183.22 ± 0.26 Ma in Southern Peru [START_REF] Sell | Evaluating the temporal link between the Karoo LIP and climatic-biologic events of the Toarcian Stage with high-precision U-Pb geochronology[END_REF]. The lack of intercalibration with astrochronology makes it difficult to anchor it on our time scale. Conversely, the Triassic-Jurassic boundary have been bracketed at 201.36 ± 0.17 Ma with U-Pb ages from the Pucara Basin (Peru) and the New York Canyon (Nevada, USA), recalibrated applying version 3.0 of EARTHTIME tracer [START_REF] Wotzlaw | Towards accurate numerical calibration of the Late Triassic: high-precision U-Pb geochronology constraints on the duration of the Rhaetian[END_REF]. The duration of the interval from the base of the Jurassic to the base of the Toarcian have been assessed at 17.21 myr [START_REF] Ikeda | A 70 million year astronomical time scale for the deep-sea bedded chert sequence (Inuyama, Japan): Implications for Triassic-Jurassic geochronology[END_REF] [START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF]Fig. 9), i.e. 183.12 ± 0.66 Ma, -The T-OAE encompasses 11 short-eccentricity cycles (Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF][START_REF] Thibault | The wider context of the Lower Jurassic Toarcian oceanic anoxic event in Yorkshire coastal outcrops, UK[END_REF], corresponding to a duration of 1.05 ± 0.06 myr (Fig. 9).

The top of the T-OAE is thus dated here at 182.07 ± 0.72 Ma,

Origins of P-To and T-OAE disturbances

The large negative CIEs observed in the Early Toarcian, the P-To event and the T-OAE, imply the injection of large amount of light carbon in the oceanic system [START_REF] Suan | Calibrating the magnitude of the Toarcian carbon cycle perturbation[END_REF]. This has been interpreted as dissociation of methane hydrate, maybe orbitally paced [START_REF] Kemp | Astronomical pacing of methane release in the Early Jurassic period[END_REF], or production of thermogenic methane due to volcanic intrusion during the Karoo-Ferrar activity [START_REF] Svensen | Hydrothermal venting of greenhouse gases triggering Early Jurassic global warming[END_REF]. The long duration of the T-OAE (Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF] implies that the amount of methane needed to maintain the negative excursion for such a long time would exceed the volume of gas hydrate reservoir (Suan et al., 2008b). In addition, the interval of the T-OAE display a positive excursion in Hg/TOC, associated with a volcanic activity [START_REF] Percival | Globally enhanced mercury deposition during the end-Pliensbachian extinction and Toarcian OAE: a link to the Karoo-Ferrar Large Igneous Province[END_REF]. CA-ID-TIMS U-Pb ages indicate that the Ferrar province activity occurred from 182.779 ± 0.033 Ma and lasted 349 ± 49 kyr [START_REF] Burgess | High-precision geochronology links the Ferrar large igneous province with early-Jurassic ocean anoxia and biotic crisis[END_REF]. These ages fall in the range of the ages assessed here for the T-OAE (183.12 ± 0.66 Ma to 182.07 ± 0.72 Ma; Fig. 9). All these observations tend to favour the crucial role of the Karoo-Ferrar activity on the inception of the T-OAE. Hg/TOC data from Yorkshire and the Peniche sections show a positive excursion within the P-To event, which tend to indicate the concomitance between the P-To event and volcanic activity. The P-To event started 184.15 ± 0.58 Ma and ended at 183.91 ± 0.60 Ma (Fig. 9), so that the P-To event appears significantly older than the activity of the Ferrar province. Dating of the early phase of the Karoo province tends to indicate that the Karoo province started earlier than the Ferrar [START_REF] Burgess | High-precision geochronology links the Ferrar large igneous province with early-Jurassic ocean anoxia and biotic crisis[END_REF][START_REF] Moulin | Eruptive history of the Karoo lava flows and their impact on early Jurassic environmental change[END_REF]. Notably the oldest 40 Ar/ 39 Ar ages of the Karoo activity have been found in the southern part of the Barkly East Formation (South Africa), within the Omega and the Moshesh's Ford units (Moulin et al., 2017 and references therein). Other ages older than 183 Ma have been reported in the Northern part of the Chonke Aike LIP (South America) in the Rio Negro Province and in the eastern part of the Chubut Province [START_REF] Féraud | 40Ar/39Ar dating of the Jurassic volcanic province of Patagonia: migrating magmatism related to Gondwana break-up and subduction[END_REF]. The location and the thicknesses of the formations in which these older 40 Ar/ 39 Ar and and U/Pb ages have been provided are, to our knowledge, much more limited than that of the main phase of the Karoo-Ferrar activity (~183 Ma). While this main phase is likely to be responsible for the inception of the T-OAE, limited volcanic activities in the early phase of the Karoo and Chonke Aike LIP may have affected the environmental and ecological perturbations recorded at the Pliensbachian-Toarcian boundary.

Conclusions

The carbon isotope and MS variations in the Late Pliensbachian-Early Toarcian, performed in the expanded hemipelagic in Issouka section from the Middle Atlas Basin, Morocco, reveal superposed frequencies consistent with Milankovitch forcing (eccentricity, obliquity, and precession). The Polymorphum Zone contains 10 to 10.5 repetitions of the 100-kyr eccentricity cycle, so that its duration is assessed here at 1.00 ± 0.08 myr. The duration of the P-To event is in addition assessed at 0.24 ± 0.02 myr. The recognition of the 100-kyr and the 405-kyr eccentricity enabled us to establish a stratigraphic framework between the Northern and the Southern Tethyan margins. Anchoring this framework on published radiometric ages and astrochronological time scales, we could estimate the age of the base of the Toarcian as 184.15 ± 0.58 Ma, the top of the P-To event as 183.91 ± 0.60 Ma, the base of the Toarcian OAE as 183.12 ± 0.66 Ma, and the top of the Toarcian OAE as 182.07 ± 0.72 Ma. The age of the Toarcian OAE fits with the ages published for the Ferrar volcanic activity. Conversely, the age of the P-To event seems to correspond with early phase of the Karoo and Chonke Aike activity. Thus, the successive and short phases of the volcanic activity of the Chonke Aike, Karoo and Ferrar provinces may have been the smoking gun of the successive phases of the mass extinction observed in marine biotas in the Pliensbachian and Toarcian times. and 2 with confidence levels calculated with the Mann and Lees (1996) method (ML96) with a Tukey's endpoint rule [START_REF] Meyers | Astrochron: An R Package for Astrochronology[END_REF]. C. and D. Spectra of intervals 1 and 2 with confidence levels calculated with the LOWSPEC method [START_REF] Meyers | Seeing red in cyclic stratigraphy: Spectral noise estimation for astrochronology[END_REF]. E. and F. H0 significance levels from the ASM applied on intervals 1 and 2. The red dashed line indicates the sedimentation rate for which the sedimentary frequencies fit the best with the orbital frequencies. with confidence levels calculated with the Mann and Lees (1996) method (ML96) with a Tukey's endpoint rule [START_REF] Meyers | Astrochron: An R Package for Astrochronology[END_REF]. C. and D. Spectra of intervals 1 and 2 with confidence levels calculated with the LOWSPEC method [START_REF] Meyers | Seeing red in cyclic stratigraphy: Spectral noise estimation for astrochronology[END_REF]. [START_REF] Martinez | Orbital chronology of the Pliensbachian-Toarcian transition from the Central High Atlas Basin (Morocco)[END_REF], the Middle Atlas Basin (present study), the Peniche section (Suan et al., 2008b;[START_REF] Huang | Pacing of the Toarcian Oceanic Anoxic Event (Early Jurassic) from astronomical correlation of marine sections[END_REF] and the Yorkshire area [START_REF] Thibault | The wider context of the Lower Jurassic Toarcian oceanic anoxic event in Yorkshire coastal outcrops, UK[END_REF].

  , which matches with the time scale suggested in Ruhl et al. (2016) with an uncertainty of ± one 405-kyr cycle. It positions the base of the Toarcian at 184.15 ± 0.58 Ma. The uncertainty retained here corresponds to the sum of uncertainties of astrochronology and radiochronology. Other ages are as follows (see also supplementary materials for details of the calculations): -The average duration of the P-To event is 0.24 ± 0.02 myr, positioning the top of the P-To event at 183.91 ± 0.60 myr, -The average duration of the Tenuicostatum/Polymorphum Zone is 1.00 ± 0.08 myr, positioning the top of Tenuicostatum/Polymorphum at 183.15 ± 0.66 Ma. Notice that this age fits with the age of Sell et al. (2014) of 183.22 ± 0.26 Ma for the uppermost part of the Tenuicostatum Zone, -The base of the T-OAE occurs 0.03 myr after the end of the Polymorphum Zone

Fig. 1 .

 1 Fig. 1. Geological setting of the Issouka section. A. Palaeogeographic map of the western Tethys during

Fig. 2 .

 2 Fig. 2. The different facies in the Issouka section. A. Limestone of the late Pliensbachian with

Fig. 3 .

 3 Fig. 3. Raw data, spectrograms and filters of the 100-kyr eccentricity band of the carbon isotope ( 13 C)

Fig. 4 .

 4 Fig. 4. Cross-plot of the magnetic susceptibility (MS) versus calcium carbonate content

Fig. 5 .

 5 Fig. 5. 2-MTM spectra and ASM results of the  13 C series. A. and B. Spectra of intervals 1

Fig. 6 .

 6 Fig. 6. 2-MTM spectra of the magnetic susceptibility. A. and B. Spectra of intervals 1 and 2

Fig. 7 .

 7 Fig. 7. 2π-MTM spectra of the (A.) calibrated  13 C series, and (B.) calibrated MS series. The main periods are labeled in kyr. The theoretical period of the corresponding astronomical cycle is displayed in brackets. Abbreviations: E: 400-kyr eccentricity; e: 100-kyr eccentricity; O: obliquity.

Fig. 8 .

 8 Fig. 8. Comparison between the direct filters of the eccentricity on the calibrated  13 C signal

Fig. 9 .

 9 Fig. 9. Correlations of astronomical time scales in between the Central High Atlas Basin

Acknowledgements

This paper benefitted considerably from 3 constructive reviews. We would like to thank also the IAS Postgraduate Grant Scheme for the financial support that Fatima-Zahra Ait-Itto received in 2016 for making the isotopic analyses at Plymouth University. Mathieu Martinez was supported by ERC Consolidator Grant Earthsequencing.


L J Nonat

Charbonnière

Cyrille Charpentier

Jérémy Salaam

Alexandre Lecointre

Dr Olivier Jeannin

Dr Aline Nonat

Loïc J Charbonnière

Mr C Charpentier

Mr J Salaam

Dr Mr A Lecointre

Phosphonated podand type ligand for the complexation of lanthanide cations

Introduction

Lanthanide complexes are displaying exceptional magnetic and spectroscopic properties that make this family of complexes of particular interest in the design of probes for biomedical applications in the various fields of MRI imaging, 1 luminescence microscopy 2 or bio-analysis. 3 However, despite the plethora of complexes that one can find in the literature, there is still room for the design of new ligands and structures displaying improved properties and phosphonated ligands, while still scarce in the literature, are among such. 4 The coordination chemistry of phosphonate functions with Ln cations can be quite complicated by the various binding modes which can be adopted. 5 It is not rare to see that the three oxygen atoms of the function can take part into coordination of numerous Ln cations in the solid state, such as the pentadentate coordination observed in some structures of aminophosphonate complexes of Gd, 6 or the bridging mono-or bidentate modes of Ln helical structures. 7 However, phosphonate functions are also bringing numerous advantages, replacing favourably their carboxylate counterparts on a thermodynamic point of view 8 with a larger steric hindrance that may allow to exclude water molecules from the first coordination sphere such as in the DOTP complexes, 9 having a double negative charge around neutral pH, and bringing very important second sphere interactions, which can be advantageously used for MRI contrast agents for example. 10 The second sphere interactions of phosphonated complexes of lanthanide cations are also at the origin of intriguing behaviours in solution. It was soon recognized by Horrocks and co-workers that Eu 11 or Ce 12 with polyaminophosphonated ligands like ethylenediaminetetrakis(methylphosphonic) acid or diethylenetriaminepentakis(methylphosphonic) acid formed M:L complexes of 1:1 and 2:1 stoechiometric coefficients, the composition of which was strongly dependent on the pH of the solutions. The presence of numerous phosphonate functions resulted in potentially large negative charges on the complexes, which may interact with additional Ln 3+ cations in the solution to form polynuclear species. In view of recent results on polyphosphonated Ln complexes integrating other analyticals tools such as mass spectrometry, 13,14 it appears that the composition of such adducts can be even larger with the potential formation of tri-to pentanuclear species, the composition of which can be adjusted to obtained pure 10.1002/ejic.201900069

Accepted Manuscript

heteropolynuclear species with exceptional spectroscopic properties such as molecular upconversion in heavy water 15 and more recently in pure water. 16 In an effort to understand the structure/activity relationship in these edifices, we anticipated that ligand LH6 (Scheme 1), with its C3 symmetry, might be of particular interest to form polyphosphonated complexes with Ln cations and potentially more elaborated structures.

In this contribution, the coordination behaviour of LH6 with different Ln 3+ cations is questioned with different spectroscopic and analytical means, revealing once again a quite complex coordination ability.

Scheme 1

Results and discussion

Synthesis of the ligand. The tripodal ligand L can be readily obtained at the gram scale according to the synthetic procedure presented in Scheme 2.

Scheme 2. Synthetic procedure for the preparation of ligand L ((C2H5N)3 is acetaldehyde ammonia trimer) 17 Starting from the commercially available 2-chloromethyl-pyridine 1, N-oxydation using m-CPBA in chloroform afforded the corresponding pyridine-N-oxyde, which is functionalized 10.1002/ejic.201900069

Accepted Manuscript

into the o-phosphonic ethyl ester 2 by reaction with ethyl chloroformate and triethyl phosphite according to literature procedures. 13 The formation of the tripod was achieved in 42% yield by adaptation of a procedure developed by Mikata and co-workers using acetaldehyde ammonia trimer. 17 Interestingly, other attempts conducted with NH4Cl as amine source seemed to show that the alkylation steps are successively favoured, the third and second alkylations being more rapid than the first one, potentially as a result of an increase of the nucleophilicity of the formed amines upon introduction of the methyl-pyridyl substituents. Although there is a supplementary pyridyl unit in our case, this point may be related to the increase of the pKa values of the amines observed in aminomethyl phosphonic acid upon introduction of the phosphonic acid moieties. 8a, 18 The ligand was finally obtained by acid hydrolysis of the ethyl phosphonic esters followed. Dissolution of the ligand in hot water followed by slow cooling of the solution afforded nice cubic crystals which could be used for the solid state structure determination by X-ray diffraction. Crystal data and structure refinement are summarized in Table S1 (Supp.

Inf.). The structure is composed of a molecule of the ligand in its fully protonated state LH6

and two water molecules. A striking feature of the protonation is that the pyridyl phosphonic acid functions are found in a zwitterionic state, each pyridyl ring being protonated while the phosphonic acids are mono-deprotonated. The different labile proton atoms are implicated in an intricate network of H bonding interactions, also implying the two water molecules. Another 10.1002/ejic.201900069

Accepted Manuscript

striking feature of the ligand is the formation of an intramolecular π-π stacking interaction between two pyridyl rings with their mean centroid distance at 3.608 Å and an angle of 23.7°

between the average planes formed by the aromatic rings. Noteworthy, the orientation of the two rings are opposite, with the pyridinium bonds pointing in opposite direction, better than being aligned. This may result from a lower energy of the interaction between the dipoles, together with a lower steric interaction of the phosphonic acid functions which would have overlap otherwise. Also of interest, ligands are related by pairs in which, one of the phosphonate function is hydrogen bonded to two pyridinium H atoms of the second ligand. Finally, and intermolecular H bonding interactions is present in a ligand between the H and O atoms of two phosphonate functions (green dotted line in Figure 1).

Spectroscopic titrations of L by Ln 3+ cations.

In order to get insights into the coordination behaviour of the ligand with lanthanide cations, we first performed a series of spectrophotometric titrations monitored by UV-Vis electronic absorption spectroscopy and luminescence spectroscopy. In a typical experiment, 19 the spectra of the ligand solution were recorded after addition of increasing aliquots of the metal in a buffered aqueous solution (TRIS/HCl 0.01 M, pH = 7.1). Figure 2 As can be seen in Figure 2, the UV-Vis absorption spectrum of the free ligand is dominated by the intense π→π* transitions of the pyridine units at 267 nm ( = 14 700 M -1 .cm -1 ). During the different titration experiments, this maximum is almost not displaced and only the relative intensity of this transition and its shape, a shoulder appearing at ca 275 nm upon Eu addition, could be used in the fitting of the data. In contrast the luminescence titrations were particularly informative. In the case of Eu, addition of the metal immediately led to the observation of the typical emission spectra of Eu complexes with the 5 D0 → 7 FJ (J = 0 to 4) transitions of Eu. 20 Further additions of EuCl3 in the solution gave rise to an increase of the emission intensity of all transitions (Figure 2). Above one equivalent of lanthanide salt, strong changes in the shape of the transitions were observed, indicating the formation of additional species in solution. The most significant changes were observed on the 5 D0 → 7 F2 and 5 D0 → 7 F4 transitions, with a concomitant decrease of the emission at 616 nm and 690 nm, respectively, between 1.0 and 2.0 equivalent of added Eu 3+ followed by a plateau for higher Eu 3+ concentrations. Moreover, strong variations were also noticed on the 5 D0 → 7 F4 transition at 700 nm, for which the emission intensity kept increasing until the addition of 3 equivalents of EuCl3 (Figure 4). The global analysis of the UV-Vis absorption and spectrofluorometric titration data with europium was performed by using the Specfit software. 21 Data could be nicely reproduced by implementing a model including two coordination complexes of 1:1 and 2:1 Eu:L stoichiometric ratios, respectively, and a third species corresponding to the formation of larger supramolecular adducts, which was attributed to a 3:1 stoichiometry. Similar trends were also observed for the absorption and emission titrations with TbCl3 (Figure S1 andS2). The evolution of the luminescence intensities recorded for Eu 3+ at 615 nm and Tb 3+ at 545 nm as well as their corresponding fits are displayed in Figure S3 and Figure S4. Results were indicative of the formation of a highly stable 1:1 complex, which conditional stability constant is too high to be determined accurately by the given method. However, the conditional stepwise formation constants of the Eu2L and Eu3L species were estimated to log β(Eu2L) = 5.

Isolation of the [LnL] complexes and X-ray crystal structure of the Eu complex

The [LnL] complexes (Ln = Eu, Tb and Yb) could be isolated from equimolar mixtures of the ligand and LnCl3 salts in water after basicification with NaOH. The complexes were isolated by precipitation and the elemental analysis confirmed the presence of salts. However, the isolated compounds could be characterized by mass spectrometry, evidencing the presence of the peaks for a [LnL] composition with the expected isotopic distributions (Figure S7-12). In the case of europium, slow crystallization of a solution of the complex in water afforded microcrystals suitable for analysis by X-ray diffraction. Crystal data and structure refinement are summarized in Table S1.

Accepted Manuscript

Eu(OPO)2Eu metallacycle is formed, as often observed in dimeric Ln phosphonate complexes. 22,23 The Eu cations are nine coodinated by the central N atom, the three N atoms of the pyridyl rings, three O atoms of the three phosphonate functions, an O atom of a phosphonate function from the neighbouring EuL complex, and a water molecule. It is worth noting that the analogue ligand bearing three carboxylic acid functions in place of the phosphonic ones in our ligand has been extensively studied, 24 and the X-ray crystal structure of the Eu complex also displayed a coordination number of nine. Interestingly, both complexes crystallize as dimers, in which the two monomeric [EuL] units are linked together by the bridging of carboxylate or phosphonate functions. However, the supplementary stacking interaction is not observed in the case of carboxylate functions, as was observed for the free ligands (vide supra).

Accepted Manuscript

resulted in a flattening of the angles formed by the two opposite pyridyl strands. Considering the three atoms composing a tridentate unit (the tertiary N atom, the N atom of the pyridyl and the O atom of the carboxylate or phosphonate units), the average angles between the planes of the coordinating arms in opposite directions are 21.4° for the phosphonated complex (44.1° for the carboxylated one), with an almost planar pentadentate unit formed by these opposite strands as observed in disubstituted 6,6"-terpyridyl pentadentate ligands. 25

Spectroscopic properties of the ligand and of the [LnL] complexes (Ln = Eu, Tb and Gd)

Table 2 summarizes the main spectroscopic properties of the ligand and complexes in water (TRIS buffer, pH 7.0 or pD 7.4). As can be seen in Figure 8 and Table 2, the UV-Vis absorption spectrum of the ligand and complexes are very similar, with a maximum around 267-270 nm, corresponding to π→π* transitions centred on the ligand. To get insights into the ligand centred energy levels in the complexes, the spectroscopically silent Gd complex was prepared by mixing stoichiometric amounts of the ligand and Gd in water. The fluorescence spectrum was recorded at room temperature, while the phosphorescence spectrum was recorded in the time-resolved mode (time delay = 5 ms) at 77 K in the presence of glycerol (Figure 8). The 1 ππ* and 3 ππ* level were determined to be respectively at 33600 and 24600 cm -1 and the phosphorescence lifetime was determined to be 73 ms at 77 K. A comparison of the energy levels of the singlet absorption band and of the 3 ππ* state for the Gd complex of L with that of the La, Lu or Gd complexes of some pyridyl carboxylated analogues 27 reveals that the carboxylate function systematically displaces the energy levels towards lower energy. This shift corresponds to ca 800 cm -1 for the singlet absorption band (from 267-270 nm for L and its Ln complexes to 274 nm for pyridyl carboxylate ligands) and ca 600 cm -1 for the 3 ππ* (at 24600 cm -1 for GdL and ca 24 000 cm -1

for the carboxylated ligands 27 ). This energy lowering is to be associated to the higher electronic delocalization in the carboxylated version as a result of the sp 2 character of the carbon atom, in contrast to the sp 3 character of the phosphorous atom. [EuL] and [YbL] in water upon ligand excitation (normalized to their maximum of emission, exc = 269 nm, H2O, TRIS/HCl 0.01 M, pH 7.0).

Upon excitation into the ligand absorption bands, all complexes (Ln = Eu, Tb and Yb) display the characteristic emission pattern of the f-f Ln emission bands in the green ( 5 D4 → 7 FJ, transitions of Tb with J = 6 to 3), red ( 5 D0 → 7 FJ, transitions of Eu with J = 0 to 4) and NIR ( 2 F5/2 → 2 F7/2, transition of Yb) regions (Figure 9). The observed overall luminescence quantum yields are modest for all complexes and almost half of those generally observed for luminescent complexes based on carboxylated pyridine. 26,28 All luminescence decay profile could be nicely fitted with mono-exponential components, pointing to single species in solutions or to different species being in rapid kinetic equilibrium. In the case of Eu and Tb, the hydration numbers were determined to be one according to the methodology developed by Beeby and co-workers. 26 The presence of a water molecule in the first coordination sphere is probably an important factor explaining the modest luminescence quantum yield. In addition, the energy gap between the 5 D4 or 5 D0 excited states of Tb or Eu, and the triplet state of the ligand amount to 4400 and 7200 cm -1 , respectively. These gaps are quite important and are not favourable to good 3 ππ* → Ln* energy transfer according to the criteria proposed by Latva and co-workers. 29 Unfortunately, the excited state lifetime of the Yb complex was too short to be accurately determined in water probably because of the presence of a water molecule in the first coordination sphere. Unless they are perfectly shielded from first sphere water molecules, 28 Yb S7). The corresponding [(YbLH2)2H] -dimer displays a broad peak around 1400 m/z unit (Figure S11) and small peaks corresponding to the doubly charged dimer [(TbLHNa)2] 2-(at 707.44 m/z for example) appear between the peaks of the singly charged monomer (Figure S10). However, it is very difficult to interpret such mass spectrometry data as the main peak of the monomer observed may result from the dissociation of the dimers in the electrospray process, while the appearance of peaks for the dimer may also result from association of monomers during the evaporation of the solvent from the micro droplets. Finally, NMR spectra of the Eu and Yb complexes point to a clear C3 symmetry in D2O solutions at room temperature (Supp. Inf. Figure S13 andS14). When coordinated by bridging phosphonate functions as observed in the solid state structure of Eu, the dimer would be expected to have a C2 symmetry at best, with three sets of non-equivalent pyridyl arms. Observation of a single set thus points to the presence of a monomer in solution, or to a very rapid exchange between the isomers of a dimer, with a dissociative mechanism.

0,00E+00 1,00E+05 2,00E+05 3,00E+05 4,00E+05

Conclusion

Ligand LH6 could readily be synthesized at the gram scale using a four step protocol.

The C3 symmetrical ligand appeared to be well suited for the coordination with lanthanide cations and the various spectroscopic titrations revealed the formation of species having 1:1;

1:2 and 1:3 stoichiometric ratii, the thermodynamic stability of the species with the 1:1 ratio appearing very large at neutral pH. At equimolar concentrations of ligand and lanthanide cations, the 1:1 species could be isolated and characterized, as evidenced by the X-ray crystal structure of the Eu complex. The structure showed the cation to be embedded into the cavity Phosphorescence lifetimes were measured on the same instrument working in the Multi-Channel Spectroscopy (MCS) mode, using a Xenon flash lamp as the excitation source. For short μs lifetimes, the intensity decay was corrected for the lamp intensity decay profile using a scattering solution of Ludox in water. Luminescence quantum yields were measured according to conventional procedures, [START_REF]Molecular Fluorescence: Principles and Applications[END_REF] with optically diluted solutions (optical density < 0.05), using [Ru(bipy)3Cl3] in water (Φ = 0.04) [START_REF] Ishida | [END_REF] as reference for Eu, a bipyridine Tb complex, [TbL(H2O)] in water (Φ = 0.31) 35 as reference for Tb, and cardiogreen (IR125) in MeOH (Φ = 0.078) for Yb. 36 The errors are estimated to 10% on the lifetimes and 15% on the luminescence quantum yields.

Crystallography. Data were collected on a APEX II Brucker AXS diffractometer with Mo-Kα radiation (λ = 0.71073 Å). Structures were solved by direct methods (SIR92) 37 and refined (SHELXL-2014/7) 38 by full-matrix least-squares methods as implemented in the WinGX software package. 39 An empirical absorption (multi-scan) correction was applied. Hydrogen atoms were introduced at calculated positions (riding model) included in structure factor calculation but not refined except for H atoms of the two water molecules in the structure of the ligand which have been found in the Fourier difference map and refined with DFIX and DANG restraint. H atoms on water molecule in the structure of the Eu complex have been omitted in the refinement. Refinement parameters are summarized in Table 1. Crystallographic data have been deposited to the Cambridge Crystallographic Data Center (CCDC number 1876208 for the ligand and 1876207 for the Eu complex).

10.1002/ejic.201900069

Accepted Manuscript

Figure 1 .

 1 Figure 1. X-ray crystal structure of a dimer of the ligand LH6 in the solid state. H atoms have been omitted for the sake of clarity except for those of pyridinium and phosphonate functions. Blue double arrows represent intramolecular stacking interactions and black and green dotted lines represent intermolecular and intramolecular hydrogen bonds, respectively.

Figure 2 .

 2 Figure 2. Evolution of the UV-Vis absorption spectra of a solution of L upon titration by EuCl3 (0.01 M TRIS/HCl, pH = 7.1).

Figure 3 .

 3 Figure 3. Evolution of the luminescence spectra of a solution of L upon titration by EuCl3 (0.01 M TRIS/HCl, pH = 7.1, exc = 267 nm). Inset: Evolution of the emission intensity at 616 nm as a function of the [Eu 3+ ]/[L] ratio. [L] = 5.0×10 -5 M.

Figure 4 .

 4 Figure 4. a) Enlargement of the variations on the 5 D0 → 7 F4 transition of a solution of L upon titration by EuCl3 (0.01 M TRIS/HCl, pH = 7.1, exc = 267 nm). b) Evolution of the emission intensity at 689.5 nm and 700 nm as a function of the [Eu 3+ ]/[L] ratio.

  4(1) and log β(Eu3L) = 4.4(3), respectively ([L] = 5.0 × 10 -5 M, TRIS/HCl 0.01M, pH = 7.1, λexc = 267 nm). The corresponding species distribution diagram is depicted in Figure 5. The calculated emission (Ln = Eu and Tb) and absorption spectra (Ln = Eu) of the LnL, Ln2L and Ln3L species are displayed in Figure S5-S6. It is worth mentioning that at that pH, the protonation states of the different species could not be determined accurately, and pH dependence of the emission spectra of a equimolar mixture of Eu and L clearly shows large variations in the pH range from 4.52 to 10.95 (Figure S7).

Figure 5 .

 5 Figure 5. Theoretical representation of the species formed upon addition of LnCl3 (Ln = Eu, Tb) in an aqueous solution of ligand L ([L] = 5.00 × 10 -5 M, TRIS/HCl 0.01M, pH = 7.1, calculated with Log βLnL = 15.7, Log β(Ln)2L = 21.1 and Log β(Ln)3L = 25.5).

Figure 6 .

 6 Figure 6. X-ray crystal structure of the [EuL] complex showing the assembly of dimers, interconnected through the network of Na + cations, along the b axis (top left) or perpendicular to the b axis (top right), the different coordination modes of the phosphonate functions (bottom left) and the numerous interactions resulting in the stabilisation of the dimeric units (bottom right, the double arrow highlights the ππ stacking interaction). Eu = green, C = grey, N = blue, P = orange, O = red, Na = violet.

Figure 7 .

 7 Figure 7. Views along the Ntert-Eu bonds of the coordination spheres of Eu for the carboxylated analogue (left) 24 and the phosphonated one (right, CO and PO respectively refer to the oxygen atoms of the bridging carboxylate and phosphonate functions (see text) and H2O to the coordinated water molecule).

Figure 8 .

 8 Figure 8. UV-Vis absorption spectra of [TbL] (H2O, blue), and fluorescence (green, r.t.) and phosphorescence (red, 77 K, TRIS-HCl, pH 7.0/Glycerol 9/1 mixture) spectra of the [GdL] complex.

Table 2 .

 2 Main spectroscopic properties of the [LnL] complexes in aqueous solutions (TRIS/H(D)Cl, pH = 7.0 or pD = 7.4).

		Absorption		Luminescence
		max / nm	H2O / µs	q a	H2O / %
		( / M -1 .cm -1 )	D2O / µs)		D2O / %)
	L	268 (14 500)		-	-
	EuL	267 (12 300)	580 (1 910)	1.1	4.4 (19)
	TbL	267 (12 300)	1570 (2 490)	0.9	11 (18)
	YbL	270 (14 200)	-b (17.0)	-	0.1 (1.2)

a calculated according to reference 26. estimated errors are ± 1 nm for , 5 % on , 10 % on  and 15 % on . b too short to be determined.

  rarely reach the µs timescale. However, in deuterated water the complex displays very decent quantum yield (1.2 %) and lifetime (15 µs).When comparing the solid state structure and the data in solution, a puzzling question arises. If the dimer is disrupted in solution, the bridging phosphonate functions should live the

	10.1002/ejic.201900069
	7,00E+05
	6,00E+05
	5,00E+05
	(a.u.)
	place to an additional water molecule, resulting in a hydration number of two, while one is obtained for Eu and Tb. When considering mass spectrometric data (Figure S7-S12), molecular Intensity peaks corresponding to the monomers are clearly observed. However, one can notice the
	450 presence of small additional peaks in the spectrum of Eu corresponding to dimers, such as the 550 650 750 850 950 1050 Wavelength (nm) [(EuLH2)2Na] -observed at 1378.87 m/z units (calculated for C36H34N8P6O18Eu2H4Na; 1378.87, Figure	Accepted Manuscript

complex

  by the three pyridyl arms of the ligand with the phosphonate functions being implicated in a large network of H + , Na + or Eu 3+ bonding interactions. The structure also revealed the tendency of two complexes to associate in the solid state thanks to the concomitant action of ππ stacking interactions, intercomplex phosphonate bridges and intermediate Na + bridging. In solution, the spectroscopic properties of the Eu and Tb complexes are in agreement with the presence of a water molecule in the first coordination sphere of the complexes, in agreement with the solid state structure, but electrospray mass spectrometry and NMR data both point to a monomeric structure in solution. Whether these results can be interpreted by rapid exchange within the monomeric units in a dimeric species or by a dissociation with a decrease of the coordination number is unfortunately not fully elucidated. Solvents and starting materials were purchased from Aldrich, Acros and Alfa Aesar and used without further purification. IR spectra were recorded on a Perkin Elmer Spectrum One Spectrophotometer as solid samples and only the most significant absorption bands are given in cm -1 . Elemental analysis and mass spectrometry analysis were carried out by the Service Commun d'Analyses of the University of Strasbourg. Electrospray ionization mass measurements were performed on a microTOF II spectrometer in the negative mode using water as solvent.1 H, 31 P and13 C NMR spectra and 2D experiments were recorded on Avance 300 and Avance 400 spectrometers operating at 300 and 400 MHz for 1 H, respectively. Chemical shifts are reported in ppm, with residual protonated solvent as internal reference.31 When unspecified, : 699.96; found: 699.96 (100 %). Anal. Calcd for C18H18N4O9P3Yb . 9NaCl . 3H2O: C, 16.89; H, 1.89; N, 4.38. Found: C, 17.24; H, 2.06; N, 4.37. IR (cm -1 , ATR): ν 3203, 1653, 1593, 1558, 1457, 1129, 1062, 970. Spectroscopic measurements. UV/vis absorption spectra were recorded on a Perkin Elmer lambda 950 or on a Specord spectrometer from Jena Analytics. Steady state emission spectra were recorded on an Edinburgh Instrument FLP920 spectrometer working with a continuous 450W Xe Lamp and a red sensitive R928 photomultiplier from Hamamatsu in Pelletier housing for visible detection (230 to 900 nm) or a Hamamatsu R5 509-72 photomultiplier for the Vis-NIR part. All spectra were corrected for the instrumental functions. For emission spectra upon UV excitation, a 399 nm cutoff filter was used to eliminate second order artifacts.

	10.1002/ejic.201900069 10.1002/ejic.201900069
	[C18H19N4O9P3Yb]
	Experimental section General Methods. coupling constants refer to H-H coupling. The given pH values are corrected for the deuterium isotopic effects (pD = apparent pH + 0.4). 32 Compound 2 was obtained according to literature procedures. 13	Accepted Manuscript Accepted Manuscript
	Synthesis of compound 3. A mixture of compound 2 (2.0 g, 7.6 mmol, 4.5 eq.), acetaldehyde
	ammonia trimer trihydrate (310 mg, 1.7 mmol, 1.0 eq.), potassium carbonate (1.05 g, 7.6 mmol,
	4.5 eq.), and potassium iodine (1.27 g, 7.6 mmol, 4.5 eq.) in acetonitrile (30 mL) was refluxed

formed for 2 hours. The reaction mixture was cooled to r.t and the solvent was evaporated. The residue was extracted with CH2Cl2/water, dried over Na2SO4, filtered and evaporated under vacuum.

The crude product was purified by alumina gel column chromatography (eluent: CH2Cl2/MeOH +

Acknowledgements. The French National Research Agency (ANR) is gratefully acknowledge for financial support (Neutrinos project n°16-CE09-0015-02).


MBBS, MSc Toby M Maher
email: t.maher@imperial.ac.uk

MD Lisa H Lancaster

MD Stéphane Jouneau

MD Lake Morrison

MD David J Lederer

MD Maria Molina-Molina

MD Elisabeth Bendstrup

MD Klaus-Uwe Kirchgaessler

PhD Frank Gilberg

PhD Judit Axmann

MS Ute Petzinger

MD Paul W Noble

Pirfenidone Treatment in Individuals with Idiopathic Pulmonary Fibrosis: Impact of Timing of Treatment Initiation

A c c e p t e d m a n u s c r i p t

Gilead and Savara/Serendex; and fees for conferences from Actelion, AstraZeneca, BMS, Chiesi, GlaxoSmithKline, Mundipharma, and Pfizer. L. Morrison reports funding for advisory board participation and disease-state education from Boehringer Ingelheim and Roche-Genentech. L. Morrison's institution, Duke University, has received funding for clinical trials from Boehringer Ingelheim, Celgene, Nitto Denko, Novartis, and Roche-Genentech, received funding for a patient registry from Boehringer Ingelheim and the Pulmonary Fibrosis Foundation, and is negotiating funding from Galapagos for a potential clinical trial. D.J. Lederer has received consulting fees from, and served on advisory boards for, Roche-Genentech and Veracyte, served on advisory boards for Boehringer Ingelheim, and received consulting fees from FibroGen, Global Blood Therapeutics, ImmuneWorks, Philips Respironics, and Sanofi Genzyme. D.J. Lederer's institution, Columbia University, has received funding for clinical trials in IPF from Boehringer Ingelheim, FibroGen, and Global Blood Therapeutics, and received consulting fees from the Pulmonary Fibrosis Foundation. M. Molina-Molina, or her institution, has received grants from AstraZeneca, Boehringer Ingelheim, BRN, Chiesi, GlaxoSmithKline, InterMune, and Roche. E. Bendstrup has received unrestricted grants, funding for clinical trials in IPF, or speaker fees, and has served on advisory boards for, Boehringer Ingelheim and Roche, received speaker fees from AstraZeneca, and received funding for clinical trials in IPF from Sanofi outside of the submitted work. K-U. Kirchgaessler and J. Axmann are employees and shareholders of F. Hoffmann-La Roche, Ltd. F. Gilberg is an employee of F. Hoffmann-La Roche, Ltd. U. Petzinger was an employee of Clinipace-Accovion GmbH -a company contracted by F. Hoffmann-La Roche, Ltd. to perform analyses of study data -at the time of this study. P.W. Noble was a member of the ASCEND and CAPACITY study steering committees and was a consultant for Boehringer Ingelheim, Genentech, and InterMune/Roche. Intervention with an antifibrotic as early as possible in the disease course might be the most appropriate strategy to preserve lung capacity [START_REF] Albera | Efficacy of pirfenidone in patients with idiopathic pulmonary fibrosis with more preserved lung function[END_REF]. However, many physicians are reluctant to initiate antifibrotics at diagnosis, and delay treatment until disease progression is observed [START_REF] Maher | Identifying Barriers to Idiopathic Pulmonary Fibrosis Treatment: A Survey of Patient and Physician Views[END_REF].

Running

Furthermore, certain countries do not reimburse antifibrotic treatment for individuals with preserved lung function (percent predicted forced vital capacity [FVC] >80%) [START_REF] Maher | Identifying Barriers to Idiopathic Pulmonary Fibrosis Treatment: A Survey of Patient and Physician Views[END_REF]. These post hoc analyses aimed to assess: (1) FVC decline during long-term pirfenidone treatment in RECAP in individuals with IPF categorized by baseline percent predicted FVC; and (2) the impact of deferring pirfenidone treatment on annual FVC decline in individuals with IPF during CAPACITY (3) and RECAP [START_REF] Costabel | An Open-Label Study of the Long-Term Safety of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis (RECAP)[END_REF].

Methods

RECAP (NCT00662038) was an open-label extension study including individuals who had

completed the double-blind, placebo-controlled trials of pirfenidone in individuals with IPF (ASCEND [NCT01366209]; CAPACITY [NCT00287716/NCT00287729]); the methods and primary outcomes of RECAP have been described previously [START_REF] Costabel | An Open-Label Study of the Long-Term Safety of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis (RECAP)[END_REF]. Individuals who previously received pirfenidone or placebo treatment for 72-120 weeks in CAPACITY and received pirfenidone 2403 mg/day during RECAP were included in the analyses. Individuals from ASCEND were not included due to lack of FVC follow-up data.

A c c e p t e d m a n u s c r i p t

Association of baseline FVC (at entry into RECAP) with rate of FVC decline during RECAP (first aim) was assessed over 180 weeks using change from baseline in percent predicted FVC, categorized by baseline percent predicted FVC (<50%, ≥50% to <60%, ≥60% to <70%, ≥70% to <80%, ≥80% to <90%, and ≥90%).

Association of timing of pirfenidone initiation with annual FVC decline (mL/year) during CAPACITY and RECAP (second aim) was assessed over 220 weeks by categorizing individuals who completed CAPACITY and enrolled in RECAP by CAPACITY treatment group (pirfenidone 2403 mg/day or placebo; the pirfenidone 1197 mg/day group was not included). Annual FVC decline was calculated for Weeks 0-120 (CAPACITY), Weeks 72-120 (the transition period), and Week 120 onwards (RECAP), as described in Figure 1. This analysis was also stratified based on CAPACITY study of origin (004 or 006).

Results

FVC decline by baseline lung function. Overall, 584 individuals who entered RECAP with

baseline FVC values were included in this analysis: median age, 69.0 years; male, 71.9%; white, 97.8%; median body mass index (BMI), 28.9 kg/m 2 . At baseline, 28.6%, 52.2%, and 19.2% of individuals had a Gender, Age, Physiology Index of I, II, and III, respectively. Mean percent predicted FVC and hemoglobin-corrected carbon monoxide diffusing capacity (DLco) at baseline in individuals with available data were 70.9% and 41.1%, respectively (baseline FVC: <50%, n=54; ≥50% to <60%, n=113; ≥60% to <70%, n=136; ≥70% to <80%, n=123; ≥80% to <90%, n=84; ≥90%, n=74).

A c c e p t e d m a n u s c r i p t

For all baseline FVC subgroups, mean declines in percent predicted FVC over 180 weeks (2.5-4.3%) and annual rates of FVC decline (101.1-181.0 mL) during RECAP were similar (Figure 2).

FVC decline and timing of pirfenidone initiation.

Overall, 485 CAPACITY participants (n=236, pirfenidone 2403 mg/day; n=249, placebo) were enrolled in RECAP and had FVC value(s) recorded in the transition period. Demographics were similar between treatment groups (CAPACITY) and previous treatment groups (RECAP): median age, 67.0-69.0 years; male, 69.9-73.5%; white, 97.5-97.7%; median BMI, 29.0-30.0 kg/m 2 . Median percent predicted FVC and DLco at baseline were similar between pirfenidone and placebo groups in CAPACITY (FVC: 73.7% and 72.1%; DLco: 45.6% and 45.4%, respectively), and previous pirfenidone and placebo groups in RECAP (FVC: 69.8% and 69.4%; DLco: 40.4% and 40.1%, respectively).

During CAPACITY, annual rate of FVC decline was 142.0 mL and 182.3 mL (-40.3 mL difference) in pirfenidone and placebo groups, respectively (Figure 3). During RECAP, annual rate of FVC decline for previous pirfenidone and placebo groups, respectively, was 155.2 and 151.9 mL (3.3 mL difference) in the transition period and 145.3 and 140.9 mL (4.4 mL difference) after Week 120.

  Head: Impact of timing of pirfenidone initiation [42/50 characters] Key Words: Idiopathic pulmonary fibrosis, antifibrotic, pirfenidone, early intervention, preserved lung function Word Count: 1000 A c c e p t e d m a n u s c r i p t To the Editor: Idiopathic pulmonary fibrosis (IPF) is a debilitating, progressive, fatal, fibrosing lung disease (1, 2). Pirfenidone and nintedanib are oral antifibrotics with demonstrated efficacy in reducing lung function decline in individuals with IPF, independent of baseline lung function (3-7).

FVC

  decline in the placebo group in CAPACITY Study 006 was attenuated (3); therefore, this analysis was stratified based on CAPACITY study of origin. The difference between annual rate of FVC decline in the pirfenidone and placebo groups was larger during Study 004 (155.8 vs 212.1 mL) than Study 006 (128.6 vs 151.8 mL). Corresponding rates after CAPACITY were 123.2 vs 123.6 mL (previous Study 004) and 187.1 vs 184.7 mL (previous Study 006) during the

A c c e

  p t e d m a n u s c r i p t transition period and 138.5 vs 137.7 mL (previous Study 004) and 152.3 vs 144.4 mL (previous Study 006) after Week 120. Discussion These post hoc analyses of CAPACITY and RECAP found that long-term pirfenidone treatment had similar efficacy regardless of baseline FVC, and there was no effect of prior treatment on FVC change during RECAP. Importantly, loss of lung function that occurred before pirfenidone initiation was not recovered after initiation in RECAP, confirming that delaying antifibrotic treatment results in increased irreversible FVC loss. The efficacy of pirfenidone in reducing FVC decline was maintained for >4 years, with little change in annual rate of FVC decline after >1 year of treatment in individuals who received pirfenidone during CAPACITY. These results are in line with previous analyses indicating that pirfenidone treatment is beneficial in individuals with IPF, regardless of stage of lung function or time since diagnosis at initiation (5, 10, 11). These findings are limited by the fact that they represent post hoc exploratory analyses, and that RECAP was an open-label extension study with no placebo group. Long-term follow-up might have introduced selection bias towards individuals with more preserved lung function over time, since they were less likely to discontinue treatment during CAPACITY or RECAP.Additionally, individuals enrolled in CAPACITY had fewer comorbidities than are observed in unselected populations (3); thus, the benefits of initiating pirfenidone in individuals with more comorbidities could not be determined.Overall, these results add weight to evidence supporting pirfenidone initiation at diagnosis in individuals with IPF to prevent irreversible loss of lung function.

Figure 1 .

 1 Figure 1. Treatment periods for calculating annual FVC decline during CAPACITY and RECAP to

Figure 2 .Figure 3 .

 23 Figure 2. Rate of lung function decline over 180 weeks by baseline percent predicted FVC

Acknowledgments Medical writing support was provided by Rebekah Waters, PhD, of CMC AFFINITY, a division of McCann Health Medical Communications Ltd., Manchester, UK, funded by F. Hoffmann-La Roche, Ltd. Data sharing statement Qualified researchers may request access to individual patient level data through the clinical study data request platform (www.clinicalstudydatarequest.com). Further details on Roche's criteria for eligible studies are available here (https://clinicalstudydatarequest.com/Study-Sponsors/Study-Sponsors-Roche.aspx). For further details on Roche's Global Policy on the Sharing of Clinical Information and how to request access to related clinical study documents, see here (https://www.roche.com/research_and_development/who_we_are_how_we_work/clinical_tri als/our_commitment_to_data_sharing.htm).

, contributed to the letter from the outset, and read and approved the final draft. All authors vouch for the accuracy of the content included in the final letter. This study was sponsored by F. Hoffmann-La Roche, Ltd., which was involved in study design, collection, analysis, and interpretation of the data. T.M. Maher is supported by an NIHR Clinician Scientist Fellowship (NIHR Ref: CS-2013-13-017) and a British Lung Foundation Chair in Respiratory Research (C17-3) and has received industryacademic research funding from GlaxoSmithKline R&D and UCB, served on a clinical trial advisory board for GlaxoSmithKline R&D, received stock options from Apellis, and received consultancy or speaker fees from AstraZeneca, Bayer, Biogen Idec, Boehringer Ingelheim, Cipla, GlaxoSmithKline R&D, ProMetic, Roche, Samumed, and UCB. L.H. Lancaster reports research grants, prior advisory board, and disease-state education from Genentech, research grants and disease-state education from Boehringer Ingelheim, research grants from Celgene and Novartis, research grants and consulting fees from Galapagos and Bellerophon, and consulting fees from Theravance and Magnolia Therapeutics outside of the submitted work. S. Jouneau has received funding for clinical research trials, fees for advisory boards or consultancy and conferences, and financial support for clinical and basic research from AIRB, Boehringer Ingelheim, and Roche; funding for clinical research trials, fees for conferences, and financial support for clinical and basic research from LVL; fees for advisory boards or consultancy and conferences, and financial support for clinical and basic research from Novartis; funding for clinical research trials from