Accepted Manuscript

Quantification of paleo-aquifer changes using clumped isotopes in

subaqueous carbonate speleothems

Fernando Gázquez, Andrea Columbu, Jo De Waele, Sebastian

F.M. Breitenbach, Ci-Rong Huang, Chuan-Chou Shen, Yanbin
Lu, José-María Calaforra, Maryline J. Mleneck Vautravers, David
A. Hodell

PII: S0009-2541(18)30302-4
DOI: doi:10.1016/j.chemgeo.2018.05.046
Reference: CHEMGE 18799
To appear in: Chemical Geology
Received date: 6 April 2018
Revised date: 26 May 2018
Accepted date: 31 May 2018

Please cite this article as: Fernando Gázquez, Andrea Columbu, Jo De Waele, Sebastian
F.M. Breitenbach, Ci-Rong Huang, Chuan-Chou Shen, Yanbin Lu, José-María Calaforra,
Maryline J. Mleneck Vautravers, David A. Hodell , Quantification of paleo-aquifer
changes using clumped isotopes in subaqueous carbonate speleothems. Chemge (2017),
doi:10.1016/j.chemgeo.2018.05.046

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 ACCEPTED MANUSCRIPT

Quantification of paleo-aquifer changes using clumped

isotopes in subaqueous carbonate speleothems
Fernando Gázquez1*, Andrea Columbu2, Jo De Waele2, Sebastian F.M.
Breitenbach3, Ci-Rong Huang4, Chuan-Chou Shen4, Yanbin Lu5, José-María
Calaforra6, Maryline J. Mleneck Vautravers7 and David A. Hodell7

(1) School of Earth and Environmental Sciences. University of St. Andrews. St Andrews,
KY16 9AL, Scotland, United Kingdom. [email protected]

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(2) Italian Institute of Speleology, Department of Biological, Geological and Environmental
Sciences, University of Bologna, Via Zamboni, 67, 40126 Bologna, Italy.
(3) Sediment & Isotope Geology, Institute of Geology, Mineralogy & Geophysics. Ruhr,

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Universität Bochum, Universitätsstr, Bochum, Germany

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(4) High-Precision Mass Spectrometry and Environmental Change Laboratory (HISPEC),
Department of Geosciences, National Taiwan University, Taipei City 106, Taiwan ROC.
(5) Department of Earth Sciences, University of Minnesota, 310 Pillsbury Drive SE,
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Minneapolis, MN 55455-0231, USA.
(6) Water Resources and Environmental Geology Research Group. University of Almería.
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Ctra. Sacramento s/n, 04120, Almeria, Spain

(7) Department of Earth Sciences, University of Cambridge, Downing Street, Cambridge,
CB2 3EQ, United Kingdom.
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*corresponding author
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Abstract
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Here we track the water-table position and temperature of the Mount San Giovanni
aquifer (Iglesiente-Sulcis mining district, SW Sardinia, Italy) during the past 600 ka by
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determining the ages (U-Th dating) and stable isotope compositions (18O, 13C and
47) of a variety of subaqueous carbonate speleothems (e.g. calcite spars, dogtooth
calcite crystals and calcite coatings). Clumped isotopes (47) provide quantitative
estimates of carbonate formation temperatures (and thus water temperatures) that are
independent of the oxygen isotope composition of water (18Ow). Then, the 18Ow of
the paleo-water has been reconstructed from the clumped isotope temperature (T47)
and the 18O of the carbonate (18Oc). We find that high-temperature calcite spars
formed already before 600 ka at temperatures above ~120 oC. Lower-temperature
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spars (~70 oC) precipitated at ~400 ka, and cold-water subaqueous speleothems (~10-
20 oC) formed in perched ponds at different levels of the karst systems between 410
ka and 110 ka, while coeval precipitation of subaerial flowstones occurred in the upper
levels of the shallower caves until 82 ka. We infer that the groundwater level dropped
by ~120 m from ~400 to ~250 ka, with a relatively rapid rate of ~0.8 mm/y. Considering
the tectonic stability of Sardinia during the Quaternary, this high rate derives from
climate driven geomorphological processes at the surface rather than tectonic
uplifting. The 18O values of the paleo-aquifer water range from -6.0±0.7 ‰ during MIS

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5c, and similar to modern cave water values (-5.1 ‰), to -7.7±0.4 ‰ during the colder

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MIS 8. These values indicate that the groundwater reflected the 18O signal of meteoric
water, with no significant contributions from metasomatism and metamorphic waters.

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The observed 18Ow variability can be explained by glacial/interglacial paleoclimate
changes affecting rainfall 18O (and thus groundwater 18O). We conclude that
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clumped isotope thermometry on subaqueous carbonate speleothems is a powerful
tool for tracking paleo-aquifer temperatures and 18Ow reconstructions.
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Keywords: calcite-aragonite, clumped isotopes, hydrothermal aquifer; hypogene

karst, water temperature reconstruction, U/Th dating.

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1. Introduction

Hypogene speleogenesis caused by rising of CO2/H2S-rich fluids is, contrarily to what

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was thought only a decade years ago, a common karstification process that generated
dozens of caves worldwide (see Klimchouk et al., 2017, for a recent review on
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hypogenic caves). Signs of hydrothermal fluid-rock interaction in caves include

geomorphological features (Audra et al., 2002, 2007, 2009; De Waele and Forti, 2006;
Klimchouk, 2007, 2009; Stafford et al., 2008; Palmer, 2011; Calaforra and De Waele,
2011; Klimchouk et al., 2012; De Waele et al., 2013, 2016), evidence for bedrock
alteration (Dublyansky and Spötl, 2009; Palmer and Palmer, 2012; Plan et al., 2012;
Gázquez and Calaforra, 2013) and characteristic cave minerals and speleothems
(Polyak and Provencio, 2001; Davis, 2002, 2012; Audra et al., 2009, 2015; Forti, 2010;
Pagliara et al., 2010; Leél-Őssy et al., 2011; De Waele et al., 2013, 2016; Gázquez et
al., 2013a, 2017), often with distinctive geochemical compositions (LaRock and
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Cunningham, 1995; Dublyansky and Spötl, 2009; Palmer and Palmer, 2012; Decker
et al., 2015).

Unlike epigenic caves, corrosion/precipitation of carbonates in hypogene systems is

not controlled by CO2 inputs from the soil and vegetation coverage or exchange with
external atmospheric air, but has its origin in deeper processes (metasomatism and
metamorphic mechanisms releasing CO2; Klimchouk et al., 2017) or in the dissolution
of the carbonate host rock as thermal fluids ascend. Carbonate corrosion/precipitation

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is given by the reaction:

CaCO3 (s) + CO2 (g)↑ + H2O ↔ 2HCO3- + Ca2+

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Ca2+-HCO3--rich waters can precipitate carbonate minerals (i.e. calcite or aragonite)

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when the equilibrium reaction displaces to the left. This is generally accompanied by
CO2-degassing, evaporation and/or water temperature lowering (Audra et al., 2009).
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Such conditions are prone to occur when the fluids rise to the upper level of
hydrothermal aquifers and the hydrostatic pressure decreases, as water approaches
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the top of the aquifer, moving away from the deeper thermal source. Indeed, the
shallower sectors of many hypogene karst systems generally show greater abundance
of secondary carbonate formations, compared to deeper areas that often display
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mainly dissolution features (Audra et al., 2009; Gázquez and Calaforra, 2013).
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Carbonate speleothems can be classified into subaqueous and subaerial, depending

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on their position with respect to the water table at the time of mineral precipitation
Subaqueous speleothems can furthermore be subdivided into phreatic (usually formed
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tens of meters below the water table), epiphreatic (formed in shallower aquifer levels
that can be emerged because of occasional water table lowering) (Jennings, 1985)
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and vadose (formed in pools/lakes in caves well above the water table). Note that in
other studies, the term “phreatic” has been used to refer to overgrowth speleothems
(POS) deposited close to the water table in anchialine caves (e.g. Fornós et al., 2002),
which would be classified as epiphreatic speleothems following the classification used
in the present study.

Typical subaqueous carbonate speleothems in hypogene caves include calcite spars

and dogtooth crystals (LaRock and Cunningham, 1995; De Waele et al., 2013;
Gázquez et al., 2013b; Decker et al., 2015; Vardanjani et al., 2017), and mammillary
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coatings (Polyak et al., 2013; De Waele et al., 2017; Gázquez et al., 2017), whereas
folia (e.g. Audra et al., 2009; Leél-Őssy et al., 2011; Davis, 2012; Gázquez and
Calaforra, 2013; Polyak et al., 2013), calcite rafts and calcite raft cones (e.g. Jones,
1989; Hill and Forti, 1997; Davis, 2000; Piccini, 2000; Audra et al., 2002; Leél-Őssy et
al., 2011; Polyak et al., 2013; Gázquez and Calaforra, 2013; Piccini et al., 2015), are
typical of shallower (epi)phreatic levels or the oscillation zone. The mechanisms
involved in the formation of each type of speleothem are generally associated with
specific environmental conditions. For example, calcite raft cones form exclusively in

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relatively shallow (less than few meters deep) subterranean water pools, and
therefore, can be used as indicators of paleo-aquifer levels (Gázquez and Calaforra,

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2013; De Waele et al., 2017; Kovacs et al., 2017). However, it is worth mentioning that

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some types of subaqueous carbonate speleothems are not exclusive of hypogene
caves but are also found in low-temperature epigene caves (e.g. cave cones and folia;
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Wildberger, 1987; Plan and De Waele, 2011; Davis, 2012; Fornós et al., 2002;
D’Angeli et al., 2015a; De Waele et al., 2017; Kovacs et al., 2017).
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Assuming that subaqueous carbonate speleothems precipitate in isotopic equilibrium

with water and no significant kinetic effects occur during carbonate crystallization, the
oxygen isotope composition of calcite can be used to reconstruct the water
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temperature at the time of mineral precipitation. This is because the oxygen isotope
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fractionation factor () between HCO3- (previously equilibrated with H2O) and CaCO3
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is sensitive to temperature (Urey, 1948). This temperature relationship for calcite

precipitation in cave environments was postulated by Coplen (2007) to be represented
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by the expression:

1000 ln 𝛼 = 17.4 (103 𝑇 −1 ) − 28.6 (Equation 1)

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where,  depends on the oxygen isotope compositions (standardized with respect to

the international reference material Vienna Standard Mean Ocean Water, V-SMOW)
of the carbonate (18Oc) and the water (18Ow) at the time of mineral precipitation, and
T is the water temperature in K. 18Oc can be easily determined by isotope ratio mass
spectrometry (Spötl and Vennemann, 2003), while 18Ow is generally obtained from
the analysis of fluid inclusions trapped in calcite (Arienzo et al., 2013) or 18Ow in
groundwaters of known age (Talma and Vogel, 1992).
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An alternative to the 18Oc-18Ow approach is the determination of the actual formation

temperature of carbonates via clumped isotope thermometry. This paleothermometer
is based on the temperature dependence of the 13C-18O clumping into bonds within
the carbonate mineral lattice, expressed as 47 (Ghosh et al., 2006; Eiler, 2007) and
is independent of the 18Ow for temperature calculations. Instead, 18Ow can be back-
calculated from 18Oc and the clumped temperature (T47). Unfortunately, the
application of clumped isotopes to subaerial speleothems (i.e. stalagmites) has been

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limited in many cases because of the lack of understanding of
equilibrium/disequilibrium processes during carbonate precipitation from thin water

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films in vadose cave environments (Affek et al., 2008; Daëron et al., 2011; Kluge et
al., 2014a). However, recent clumped isotope analyses of mammillary coatings in

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Devil’s Hole (Nevada, US) suggest that subaqueous carbonate formed under isotope
equilibrium conditions between 180 ka and 27 ka, and therefore, water temperatures
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could be calculated (Kluge et al., 2014b). Also, T47 obtained from recent subaqueous
cave pearls formed in different climatic settings, matches the water temperature of the
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pool waters from which it formed (Breitenbach et al., 2018). This suggests that, under
certain conditions, subaqueous cave carbonates can precipitate in thermodynamic
and isotopic equilibrium with water and clumped isotope thermometry can be used for
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paleo-temperature reconstructions. In addition to low-temperature carbonates,

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clumped isotope thermometry has been recently applied to infer the temperature of
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paleo-waters by using calcite spars (Budd et al., 2013) and hydrothermal dolomite
(Ferry et al., 2011) formed in deep environments.
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Here we combine clumped isotope thermometry and classic 18O and 13C
measurements in calcite to reconstruct paleo-water temperature and 18Ow in Mount
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San Giovanni (SW Sardinia, Italy) during the Late Quaternary (<600 ka to 82 ka). The
various crystalline habits and arrangement of speleothems in the caves of Mount San
Giovanni suggest that carbonate formed under different environmental conditions,
including changing distances from the water surface (subaqueous/subaerial
conditions), water geochemistry and temperatures. However, the exact environmental
settings in which these speleothems crystallized have not been constrained yet. In
addition to temperature, clumped isotopes on subaqueous speleothems have been
used to calculate 18Ow values of the paleo-aquifer. The significance of the different
types of speleothems and potential paleoclimatic implications are discussed,
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regarding oscillations in the water table, water geochemistry and temperature changes
with time.

2. Geological setting

Mount San Giovanni Mine is located in the Iglesiente region (SW Sardinia, Italy), a Pb-
Zn mining district active since pre-Roman times (Cidu et al., 2009) (Fig. 1). Most mines
exploited Mississippi Valley Type ore bodies hosted in Lower Cambrian limestones

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and dolostones (Bechstadt and Boni, 1996). The carbonate succession starts with
grey dolostones (Santa Barbara Fm.), followed by darker massive dolostones and a

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thick succession of whitish-grey intensely karstified limestones (San Giovanni Fm.).

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The San Giovanni Mine exploited a very rich E-W aligned, 2 km long and 700 m wide
ore body (Brusca and Dessau, 1968) hosted in the southern limb of the Iglesias
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syncline (Civita et al., 1983). This ore was excavated from the top of the mountain
(400 m a.s.l.) to around 250 m below sea level, and around 100 km of mine galleries
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have been excavated through Mount San Giovanni. Ore exploitations lasted until
1998, when pumping operations in a nearby (connected) mine was discontinued in
1997 and the water table rapidly rose back to its original level at around 60 m a.s.l.
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(Cidu et al., 2011).

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During tunnel construction many karst voids were intercepted. A total of over 50 caves
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have been mapped in this mine, most of which lack a natural entrance (so called “mine
caves”; Messina et al., 2005; Pagliara et al., 2010; De Waele et al., 2013; 2017;
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Gázquez et al., 2013b). Despite the fact that some caves are known since a very long
time (the most interesting from a scientific point of view being Santa Barbara 1 cave,
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discovered in April 1952; Rossetti and Zucchini, 1956), multi-disciplinary scientific

researches began only recently (Pagliara et al. 2010; Caddeo et al., 2011; De Waele
et al. 2013; Gázquez et al., 2013b).

Mine levels hosting significant numbers of caves are located between 170 and 226 m
a.s.l. (Figs. 2 and 3). In the Peloggio tunnel (218 m a.s.l.) a sequence of small caves
was intercepted, including Crovassa Azzurra cave (Caddeo et al., 2011). This cave is
composed of two superposed chambers, connected by a shaft, and hosts a wide
variety of speleothems and mineralizations, like blue aragonite flowstones, and calcite
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dogtooth spars. The Idina gallery (150 m a.s.l.) intercepts a large mineralized karst
void extending from this level up to the surface (370 m a.s.l.). Between 160 and 180
m a.s.l. a section of a speleothem-rich natural cave has been saved from mining
activities and is known as Crovassa Ricchi in Argento cave. The richest mine tunnel
is known as Albert (~226 m a.s.l.), which intercepted at least eight important caves.
Massa Riccardo cave is located in its upper portion and descends into a large room,
the floor of which is located at around 205 m a.s.l. (Figs. 2 and 3).

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3. Materials and methods

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3.1. Samples

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In 2005 a core 8 cm in diameter and 0.5 m long (sample GA-core) was retrieved from
an aragonite/calcite flowstone located at ~2 m below the cave entrance of Crovassa
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Azzurra (216 m a.s.l., Figs. 3 and 4E). The core was sampled using a hand-held water-
cooled HILTI® drilling machine with a 30 cm long drill bit and extensions. The core was
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split lengthwise and 11 subsamples for U-Th dating were taken (Fig. 4D). In the 2012
sampling campaign, the void left by the 2005 core extraction was found filled with
dripwater, and a sample of this water was collected for isotopic analysis.
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In addition to the cores samples, carbonate samples from three caves of the Mount
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San Giovanni mine were collected in 2012 (Table 1, Fig. 3). Six samples were
collected in Crovassa Azzurra cave. A fragment of calcite coatings detached from the
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cave ceiling was collected at ~5 m below the cave entrance (208 m a.s.l., Fig. 3). The
inner part of this sample consists of a pristine scalenohedral calcite spar (~10 cm in
size) that is coated with a layered calcite crust (~15 cm thick) composed of whitish and
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brownish laminae (Fig. 4A). Two additional scalenohedral calcite spars (~15 cm in size
each) were collected from the cave floor at 205 m a.s.l.. Also, three dogtooth calcite
speleothems were taken from the cave walls at ~12 m below the cave entrance (at
201 m a.s.l. in Fig. 3 and Fig. 4B). A fragment of blue aragonite flowstone was collected
from the cave floor (208 m a.s.l.) as well as a sample of the carbonate host rock.

In Crovassa Ricchi in Argento cave, four scalenohedral calcite spars were collected in
2012 from the floor in different parts of the cave, at around 180 m a.s.l. (Figs. 3). In
Massa Riccardo cave (Fig. 3), fragments of a whitish calcite flowstone (~20 cm thick)
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that deposited on a scalenohedral calcite spar (Fig. 4D), were collected at 206 m a.s.l..
Two samples of euhedral calcite, composed of aggregates of ~3 cm in size planar
calcite crystals resembling “roses” were collected from the cave wall (Fig. 4C) at 208
m a.s.l.. A fragment of the carbonate host rock was taken from the wall of Massa
Riccardo cave.

Homogeneous carbonate powders from the cleanest part of all the specimens
described above were obtained using a Dremel® drill tool. In addition, we consider for

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interpretations the U-Th age of a mammillary calcite coating (cave clouds) taken in
2005 in Santa Barbara 2 cave at 65 m a.s.l. and reported by De Waele and Forti (2006)

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(SB2-CC in Fig. 3).

3.2. U-Th dating

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Seven carbonate subsamples, 100-200 mg each, were selected from calcite spars,
dogtooth calcite crystals, aragonite flowstones and calcite crusts, and eleven
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subsamples of drilled flowstone cores from Crovassa Azzurra cave were analyzed for
U-Th dating at the High-precision mass spectrometry and environment change lab
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(HISPEC) at the National Taiwan University. We followed the chemical procedure

described in Edwards et al., (1987) and Shen et al. (2012) to separate uranium and
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thorium. A gravimetrically calibrated (Cheng et al., 2013) triple-spike, 229Th-233U-236U,

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isotope dilution method was employed to correct mass bias and determine U-Th
contents and isotopic compositions (Cheng et al., 2000; Shen et al., 2012). U-Th
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isotopic compositions and 230Th dates were determined by a multi-collector inductively

coupled plasma mass spectrometer (MC-ICPMS), Thermo Fisher Neptune (Shen et
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al., 2012; Cheng et al., 2013). Half-lives of U-Th nuclides used for 230Th age calculation
are given in Cheng et al. (2013). Ages were corrected by assuming an initial
230Th/232Th atomic ratio of 4.4±2.2 x10-6 (see Table S1 for details). Uncertainties in the
U-Th isotopic data and uncorrected-corrected ages are given at the two sigma (2)
level or two standard deviations of the mean (2m) unless otherwise stated (Table S1).

3.3. 18O and 13C in carbonates

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Powdered carbonate samples (n=32, ~200 g each) were flushed with CP grade
helium in Exetainer tubes (Labco®), then acidified with 104% H3PO4 and left to react
for 1h at 70 oC. Stable carbon and oxygen isotopes of CO2 were measured using a
ThermoScientific GasBench II, equipped with a CTC autosampler coupled to a
MAT253 mass spectrometer (Spötl and Vennemann, 2003) in the Godwin Laboratory
at the University of Cambridge, UK. Analytical precision was estimated at ±0.03‰ for
13C and ±0.08 ‰ for 18O by repeated analysis of an inhouse Carrara Marble

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standard. Results are reported in ‰ relative to the Vienna PeeDee Belemnite (V-PDB)
reference.

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3.4. Clumped isotopes

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Seven samples of subaqueous carbonate speleothems (i.e. calcite spars, dogtooth
calcite crystals and calcite coatings), were investigated for their clumped isotope
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composition (47, Table 1 and supplementary Table S2). Note that no samples from
the GA-core were analyzed for 47, since it is interpreted as having formed in subaerial
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conditions and may have been affected by isotopic disequilibrium during precipitation
(Affek et al., 2008; Daëron et al., 2011; Kluge et al., 2014a). Powder aliquots of 120 to
140 g each were loaded in borosilicate vials and analyzed on a KIEL-IV device,
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coupled to a ThermoFinnigan MAT253 mass spectrometer in the Godwin Laboratory

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at the University of Cambridge, UK (for details see Breitenbach et al., 2018). Between
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16 and 37 aliquots per sample were analyzed over a period of several months. Acid
digestion using 104 % orthophosphoric acid was performed at 70 oC. The four
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carbonate standards ETH1, ETH2, ETH3 and ETH4 were used for normalization
(Meckler et al., 2014) and errors are reported as 2-standard error of the mean of the
aliquots measured. The relationship between 47 and temperature for low temperature
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carbonate speleothems (i.e. 47 >0.5‰) was determined using our inhouse
calibration, which is based on natural subaqueous cave carbonates formed at
temperatures between 3 and 47 oC (Breitenbach et al. 2018, 47 =
(0.0447±0.006)*106/T2+(0.149±0.07); where T in K and 47 in ‰; Eq. 2). For higher
temperatures (i.e. 47 <0.5‰), we used the 47-temperature calibration of Kele et al.
(2015) (47 = (0.044±0.005)*106/T2+(0.205±0.0504); where T in K and 47 in ‰; Eq.
3). Both calibration equations were obtained using the same parameters and
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standards, but the Breitenbach et al. (2018) calibration is used here for low-
temperature samples, so it is based on the same type of subaqueous cave carbonates.

4. Results

4.1. U-Th dating

238U content in the dated samples varies between aragonite and calcite, although in
both polymorphs concentrations are relatively high (Table S1). In aragonite, 238U is

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always higher than 1000 ppb, while in calcite the average is 476 ppb (min: 8 ppb, max:
1642 ppb). The content of non-authigenic 232Th is higher for calcite and lower for

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aragonite (Table S1); however, the 230Th/232Th activity ratio (which together with the
error associated with the calculation of 238U/234U/230Th/232Th rules the uncertainties

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associated with the final ages) is generally high (>50). Consequently, all ages reported
have relatively low 2errors except sample GA-CORE-2, which is close to the U-Th
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dating limit).

U-Th results attest that speleothems in the Mount San Giovanni karst system formed
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between >600 ka and 82.3±0.3 ka BP (Table 1). The “>600 ka” notation refers to
samples out of the range of the dating method, which is currently between 600 and
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800 ka BP depending on the U-Th characteristics of the sample under analysis

(Hellstrom, 2003; Cheng et al., 2013). The flowstone core sampled in Crovassa
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Azzurra (GA-core) started to grow beyond this limit, with basal ages of 684±244 and
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>500 ka BP (GA-core-2 and 3, Table 1). Its middle section, mostly characterized by
cm-long fiber calcite, grew from 570±53 to 506±19 ka BP (GA-core 4 and 5), although
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an evident hiatus occurs between 587±44 and 581±51 ka BP (GA-core 11 and 12),
indicated by the presence of muddy-like brownish materials. A similar hiatus appears
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above samples GA-core 5, and the stratigraphically subsequent (younger) age is

228±2 ka BP (GA-core 6). From here, a peculiar blue aragonitic flowstone deposited
without apparent major growth interruptions until 82.3±0.3 ka BP (see table 1 for
intermediate ages). In the same cave, a similar blue aragonitic flowstone yielded an
age of 383±0.5 ka BP (GA-BA-2). This indicates that environmental conditions for the
formation of subaerial blue aragonite flowstone were suitable at least since this time.
Scalenohedral calcite spar and the overlying calcite crusts, formed slightly earlier,
between 394±13 and 410±14 ka BP (GA-05-1 and 2). Conversely, dogtooth calcite
crystals, sampled in the lower part of Crovassa Azzurra cave, were forming
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contemporaneously with the blue aragonite flowstone, considering their ages of

216±1.3 and 109±0.5 ka BP (GA-01-1 and 2). In Massa Riccardo cave, calcite roses
deposited since at least 264±0.6 ka BP (sample MS-01-2). The scalenohedral calcite
spar from Crovassa Ricchi in Argento returned out-of-range ages (>600 ka BP, GRA-
01-1). Finally, the mammillary calcite from Santa Barbara 2 cave (SB2-CC) is 247±17
kyr old, as reported by De Waele and Forti (2006).

4.2. 18O and 13C analyses in carbonates and clumped isotopes (47)

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thermometry

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Carbonate oxygen and carbon isotope values show two distinct groups of samples
(Fig. 5). A first cluster is characterized by relatively low 18Oc values (-18.8 ‰ to -13.6

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‰), i.e. distinctly lower than the Cambrian carbonate host rock (-11.7 ‰ to -10.4 ‰),
and relatively high and homogeneous 13C values (-0.3 ‰ to +0.2 ‰), similar to the
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host rock (-0.8 ‰ to -0.2 ‰). This cluster comprises all calcite spars, except one (MS-
06-03) from Massa Riccardo cave that shows considerably higher 18O (-7.0 ‰) and
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more depleted 13C (-8.4 ‰) values.

The second cluster comprises the remaining samples, which show rather
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homogeneous 18O values, ranging -5.2 ‰ to -4.1 ‰, but a wider range of 13C, from
-11.1 ‰ to -4.3 ‰. In general, aragonite flowstone shows more positive 13C values
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(>-10 ‰), while most samples (i.e. calcite flowstones and calcite “roses”) are grouped
around -10.5 ‰. The dogtooth calcite crystals from Crovassa Azzurra cave show
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intermediate 13C values (-8.5 ‰ to -6.2 ‰).

The seven samples analyzed for clumped isotopes show 47 values ranging from
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0.476 ‰ to 0.708 ‰ (Table 1 and supplementary Table S2). The uncertainties of the
clumped isotope analyses are between 0.004 ‰ and 0.009 ‰ (1SE) or 0.008 to 0.018
‰ as margin of error at the 95% confidence level. All the clumped isotope uncertainties
and propagated errors for T47 and 18Ow are given as 2-standard errors of the mean
(2SE). The spar samples GA-03-1, GRA-01-1 and GRA-01-3 showed the lowest 47
value (0.476±0.018 ‰, 0.485±0.009 ‰ and 0.491±0.012 ‰, respectively), whereas
the calcite crust GA-05-03 (0.696±0.012) and the dogtooth crystals (GA-01-01 and
GA-01-02; 0.680±0.013 ‰ and 0.694±0.013 ‰, respectively) of Crovassa Azzurra
 ACCEPTED MANUSCRIPT

cave and the calcite “roses” MS-01 of Massa Riccardo cave (0.708±0.008 ‰) display
higher 47. After calibration using eq. 2 for 47 values >0.5 and eq. 3 for 47 values
<0.5, the higher formation temperature corresponds to the calcite spars GA-03-01
(130±14 oC), GRA-01-1 (123±6 oC) and GRA-01-3 (119±9 oC), whereas the rest of the
samples formed at lower temperatures, ranging from 10±2 oC to 17±3 oC (Table 1).

5. Discussion

5.1. Clumped isotope thermometry and paleo-aquifer18Ow reconstruction

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The clumped isotope results suggest that the paleo-aquifer of Mount St. Giovanni

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experienced pronounced cooling from the earlier (>600 ka) stages, when calcite spar
precipitated under deep-phreatic conditions, to the later (<410 ka) phases of

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epiphreatic and subaerial speleothem formation. According to the 47 data from calcite
spars (samples GA-03-1, GRA-01-1 and GRA-01-3, Table 1), the water temperature
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in the caves of the Mount San Giovanni was very high (probably >120 oC) before >600
ka, but lowered until 410±14 ka and ranged from 10±2 oC to 17±3 oC till at least ca.
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109±0.5 ka. Given the limited number of samples analyzed and the analytical
uncertainty of the measurements, small T 47 variations (e.g. 13±3 oC to 17±3 oC) are
not interpreted here. Monitoring in Santa Barbara 1 cave carried out between 2001
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and 2003 has shown cave temperature to range between 15 and 16°C, and these
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temperatures are comparable to those measured from time to time in the other caves
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located roughly at the same altitude (Chiesi, 2005). This suggests that water
temperature at the ~210 m level has been cold (i.e. 10 to 20 oC) from at least 410±14
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ka until present.

Combining T47 and 18Oc from the same speleothem, the 18Ow of the paleo-water
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can be calculated. The 18Ow value of the speleothem-forming water at ~410 ka was -
6.4±0.7 ‰ (sample GA-05-3) during the warmer-than-today marine isotope stage 11
(MIS 11). During the similarly warmer MIS 7 (~216 ka) and MIS 5c (~109 ka) the 18Ow
were -6.0±0.7 ‰ and -6.0±0.7 ‰, respectively. These values roughly agree with the
modern water 18Ow in the cave (-5.1 ‰) and the annual weighted mean 18Ow of
rainfall in Sardinia (-4.8±1.8 ‰, n=16, at the IAEA stations at Cagliari and Capo
Caccia, between 2001 and 2002). Previous investigations of the microthermometry of
fluid inclusions coupled with 18Oc measurements on epiphreatic calcite speleothems
 ACCEPTED MANUSCRIPT

(i.e. carbonates formed in pools) of unknown ages in caves of Masua mine (~15 km
North from Mount San Giovanni), produced also low temperatures (15-20 oC) and
18Ow values of -4 to -5 ‰ (De Vivo et al., 1987), in good agreement with our
observations. Interestingly, the 18Ow of the speleothem-forming water at ~264 ka,
during the colder MIS 8 (-7.7±0.4 ‰,) was ~2 ‰ lower than the modern 18Ow in the
cave. These results suggest that the 18Ow of the cave water (and thus that of the
rainfall over Sardinia) may have varied at least ~2 ‰ during the past 400 ka, including

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warmer (MIS 11, MIS 7 and MIS 5c) and colder (MIS 8) periods. Such differences
might be explained by the temperature effect on the 18O of rainfall that tends to be

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lower during colder periods (Clark and Fritz, 1997), or other paleoclimatically driven
processes affecting rainfall at glacial/interglacial timescale (Fairchild et al., 2006).

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The undated – although likely >600 ka, according to other U-Th dated spars – high-
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temperature calcite spar from Crovassa Azzurra and Crovassa Ricchi in Argento
caves (GA-03-1, GRA-01-1 and GRA-01-3) show 18Ow values between -3.1±1.5 ‰
and -4.3±1.0 ‰. These values are slightly higher than the modern 18O observations
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of the rainfall and cave water and also higher than the reconstructed <600 ka-old
paleo-water 18O. This may have been related to seawater ingression in the aquifer
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and seawater/freshwater mixing during the early stages of hydrothermal calcite

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precipitation (probably during the Neogene/early Quaternary), paleo-aquifer

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evaporation because of the “boiling” that enriched the water in 18O or some
contributions of water from deep geological processes (e.g. metasomatism and
metamorphic water), which generally shows higher 18O (Clark and Fritz, 1997).
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It can be assumed that the 18Ow of the recharge to the aquifer is equal to the mean
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18Ow of the rainfall in this region and varied between -5 ‰ and -6.5 ‰ during the past
600 kyrs. Then, using mean 18Ow of 5.5±1 ‰, we apply the 18Oc-18Ow approach to
estimate the formation temperatures of all our carbonate speleothems, including those
that were not analyzed for clumped isotopes (using eq. 1 for calcite samples, and the
temperature equation for inorganic aragonite by Kim et al., 2007). Most speleothems
formed at temperatures between 11 oC and 20 oC, except for calcite spars which return
precipitation temperatures ranging from 33 oC to 110 oC (T, 18Oc-18Ow in Table 1). The
discrepancies between the 47-temperatures and the 18O 18
c- Ow-approach
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temperatures (up to ~20 oC in the case of some high-temperature spars) are assumed
to be caused by temporal variations in 18Ow of the paleo-water, as discussed above.

5.2 Paleohydrological changes at Mount San Giovanni

Clumped isotope thermometry yields solid quantitative estimates of paleo-water
temperatures from subaqueous carbonate speleothems. Along with U-Th ages, and
stratigraphic/speleological observations (e.g. speleothem characterization), we

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reconstruct the evolution of the Mount San Giovanni paleo-aquifer during the Late
Quaternary. In this karst system, subaqueous speleothem precipitation occurred in

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three different environments: i) phreatic settings, i.e. tens to hundreds of meters below

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the water table; ii) epiphreatic environments, i.e. less than tens of meters below the
water table and; iii) long-standing or ephemeral cave pools, located above the local
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water table. However, much earlier, and before the groundwater reached calcium
carbonate saturation and speleothems precipitated, karstification
(corrosion/dissolution of the carbonate host rock) was the prevailing mechanism.
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The earliest stages of speleogenesis (formation of karstic voids) date back to the
Cambrian-Ordovician (De Waele et al., 2013), when the upwelling of thermal water
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and oxidation of sulfide ore bodies during repeated cycles of marine to continental
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environmental changes (Bechstadt and Boni, 1996) led to karstification via sulfuric
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acid speleogenesis (Fig. 6A). Active oxidation of polymetallic sulfide ore bodies
occurred because of enhanced circulation of meteoric water inside the system (Fig.
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6B); this oxygen-rich water forced intense alteration of sulfides and partial re-
mobilization of ore mineralizations (De Vivo et al. 1987; Cortecci et al. 1989). Typical
hypogene morphologies related to rising thermal fluids, including bubble trails, cupolas
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and megascallops (De Waele et al., 2013), probably developed during the more recent
karstification interval since the Oligo-Miocene, up to present.

From the Oligocene to the Pliocene subaerial and subaqueous conditions alternated
in the entire Iglesiente region, with periods of seawater ingression into the karst (De
Vivo et al., 1987). Subsequent stages were marked by water temperature lowering, as
the solution reached the supersaturation in quartz (SiO 2) and calcite. First, quartz
precipitated from highly saline solutions (13.3–23.3 % NaCl eq.) at high temperature
(70–112 °C) (De Vivo et al., 1987). Subsequently, the water temperature decreased
 ACCEPTED MANUSCRIPT

and rhombohedral and scalenohedral spars of calcite (sometimes tens of centimetres

in size) precipitated in voids of the bedrock from low-saline solutions, as suggested by
less saline fluid inclusions in calcite spar crystals in Masua Mine (0.5–2.7 % NaCl eq.)
(De Vivo et al., 1987). Our U-Th data indicate that the earlier stages of hydrothermal
calcite precipitation occurred before 600 ka BP, when the temperature of the aquifer
was probably higher than 120 oC. The high-temperature calcite spar GRA-01-1 from
Crovassa Ricchi in Argento cave is older than 600 ka BP, most likely
contemporaneous to GRA-01-2, GRA-01-3 and GRA-01-4 (Table 1), as well as the

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spars GA-03-1 and GA-03-2 found in Crovassa Azzurra cave. Most high-temperature
18O, with 18Oc values ranging from -18.8 ‰ to -13.6

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spars are relatively depleted in
‰, typical of high temperature carbonates (e.g. LaRock and Cunningham, 1995;

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Dublyansky and Spötl, 2009; Palmer and Palmer, 2012; Decker et al., 2015), whereas
their 13C values are within a narrow range from -0.5 ‰ to +0.2 ‰. These carbon
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isotope values are similar to the Cambrian carbonate host rock (-0.8 ‰ to -0.2 ‰) and
suggest that the main source of dissolved inorganic carbon during this early stage was
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marine bedrock, with insignificant contributions from other sources (e.g. CO2 from the
overlying soil and vegetation) (Fig. 5).

Although the exact age of the early phases of calcite spar precipitation has not been
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determined, thermal conditions might have occurred much earlier than 600 ka BP (Fig.
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6C). In fact, a phreatic stage of barite precipitation at around 50 °C has been

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suggested for the nearby Corona ‘e Sa Craba Cave, ca. 10 km south of Mt. San
Giovanni and at around 270 m a.s.l.. Also there, the age of this episode remains
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unknown, although it has been estimated to have occurred during the Lower Miocene
(ca. 20 Ma), when volcanic activity characterized this part of Sardinia (Sauro et al.,
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2014).

Thermal activity persisted until at least ~400 ka BP (Fig. 6D), as suggested from
relatively low 18Oc at ~208 m a.s.l. in Crovassa Azzurra cave (~70 oC at 394±13 ka
BP; GA-05-1). However, even before that time (i.e. 684±244 and 570±53 ka BP)
subaerial carbonate flowstones (the base of the GA-core) formed at low temperature
(~12-16 oC) in the upper chamber of this cave, at 216 m a.s.l., where no sign of
hydrothermal calcite precipitation has been found from ca. 600 ka to 83 ka BP. This
testifies that, at ~500-400 ka, carbonate deposition in two sectors of the same cave
occurred in radically different hydrogeochemical environments (Fig. 6D). This can be
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explained by the upper cave levels were affected by meteoric cold-water infiltration
and were relatively isolated from the lower levels by more than 10 m of carbonate rock.

The age of the cold-water calcite coatings (~13±3 oC at 410±14 ka; sample GA-05-3)
deposited over the GA-05-1 spar, and thus stratigraphically younger than the euhedral
calcite crystals, overlaps with the final stage of high-temperature hydrothermal activity
recorded by this spar. Apparently, a shift from hydrothermal to low-temperature
conditions occurred in the caves at ~400 ka at the ~208 m level. A plausible

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explanation for this sudden change is a rapid drop of the local water table and
replacement of thermal water at the ~208 m level (e.g. the bottoms of Crovassa

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Azzurra, Massa Riccardo and Santa Barbara 1 caves) by cold water that occupied
perched ponds fed by meteoric seepage (Fig. 6E). The local groundwater level may

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be tens of meters below the perched ponds, and although the aquifer water
temperature may still be warm, this did not significantly affect the temperature of the
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perched waters that likely adjusted to the average external environmental temperature
during some phases of MIS 11. The coexistence of a thermal aquifer and colder
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perched pools at higher levels in the same karst system and disconnected from the
main groundwater level, has been reported from hydrothermal systems elsewhere
(Gázquez et al., 2012; 2013a). The relatively low 13C values found in samples
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younger than 400 ka (-11.1 ‰ to -4.3 ‰), compared to the older calcite spars support
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the greater contribution of edaphic CO2, and minor input of CO2 of deep/hypogene
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origin, including Cambrian dolostone dissolution. The 13C values in low-temperature

carbonate are consistent with CO2 (and derived HCO3-) from plants (e.g. Bar-Matthews
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et al., 2010) and carbonate precipitation in perched pools disconnected from a main
hydrothermal reservoir.
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After ~400 ka, no evidence is found that the local aquifer reoccupied the ~208 m level.
Interestingly, mammillary calcite coatings (cave clouds) deposited under ephiphreatic
conditions in the lower level of Santa Barbara 2 cave at 247±17 ka (sample SB2-CC;
De Waele and Forti, 2006), probably in a relatively warm aquifer. Cave clouds
generally form in epiphreatic, warm environments, by a slow mechanism of CO2
degassing close to the water table (some tens of meters below). Thus, at ~247 ka BP
the water table probably was around 90 m a.s.l., at the same time that the calcite
“roses” formed in a cold water perched pound in Massa Riccardo cave at 208 m a.s.l..
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In synthesis, our data suggest that the phreatic level may have dropped from 208 m
a.s.l. at ~400 ka BP, to around 90 m a.s.l. at 247±17 ka BP. The latter altitude is a
conservative estimate, considering that the dated cave cloud in Santa Barbara 2 was
not the highest one in this location (mammillary calcite cave clouds occur up to 10 m
higher) (De Waele and Forti, 2006), and these peculiar speleothems can also form
very close to the water surface (Hill and Polyak, 2010). Thus, the groundwater level
dropped at least 120 m in 147±30 kyrs, which is a rate of 0.82 +0.2/-0.14 mm/year. Similar
values are found in rapidly uplifting areas (Picotti and Pazzaglia, 2008; Columbu et al.,

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2015), but Sardinia is thought to be a relatively stable terrain at least for the last million
years. A minimal (<0.1 mm/year) uplift rate locally affected Sardinia (D’Angeli et al.,

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2015b) during the Late Quaternary, constituting ~14±3 m absolute groundwater falling

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over this 147±30 ka timeframe. Nevertheless, future detailed studies will investigate
other processes triggering the remaining ~100 m drop. At this point of the research,
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we exclude higher uplift rates affecting the SW area of Sardinia and we point toward
a prolonged downwearing of the phyllites (Fig. 1) surrounding the karst terrains at the
surface. This low permeability rock, subjected to higher surface dismantling with
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respect to carbonates, constitutes an optimal impermeable boundary of the karst

reservoir. Climate-driven processes (i.e. glacioeustatic sea-level variations, rainfall
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perturbations, etc.) might have intensified their weathering and incision, accelerating
the drainage of the aquifer and thus the groundwater drop.
E
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6. Conclusions
CE

Clumped isotope thermometry, along with U-Th series dating of different types of
carbonate speleothems from caves of the Mount San Giovanni mine have been used
to reconstruct the temperature, isotope composition (18Ow) and the position of the
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groundwater level during the Late Quaternary. Subaqueous calcite spars were already
forming before 600 ka at temperatures above ~120 oC, from a solution with 18Ow
values slightly higher than the modern infiltration water in this region and the paleo-
water 18Ow during the Late Quaternary reconstructed here.

Lower-temperature spars (~70 oC) and subaqueous flowstones continued precipitating

at 393±14 ka (MIS 11) at 208 m a.s.l., indicative of a cooling aquifer, while subaerial
flowstones were forming at ~500 ka in upper level of the cave. Subsequent,
stratigraphically younger, cold-water subaqueous carbonate coatings covering these
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spars formed in perched cave pools at approximately the same time (410±14 ka),
within analytical errors. At 265±6 ka, the aquifer temperature remained cold, whereas
subaqueous dogtooth calcite crystals formed between 216±1 ka and 109±0.5 ka in a
subterranean pool at lower temperatures. Subaerial flowstones continued forming
above 210 m a.s.l., for at least 300 ka (from 383 to 82 ka), stopping their growth only
when climate became dry during MIS 5b.

Our data suggest that the water level fell by ~120 m from ~400 to ~250 ka; which is a

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rapid rate of ~0.8 +0.2/-0.14 mm/year, never observed for the tectonically stable Sardinian
landmass. Therefore, we suggest that such a high rate has been the result of climate-

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driven surface weathering of less permeable bounding geological formations (i.e.

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phyllites) that, prior to ~400 ka, confined the karst reservoir.

The reconstructed 18Ow values of the paleo-aquifer during MIS 11, MIS 8, MIS 7 and
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MIS 5c differ by less than 2 ‰ from the modern cave water and meteoric water in the
region. This 18Ow results can be of interest for future quantitative paleo-temperature
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reconstructions from subaerial speleothems in Sardinia and the Mediterranean region.

We conclude that subaqueous speleothems, formed in either phreatic or epiphreatic
environments are a powerful tool for reconstructions of environmental changes in
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paleo-aquifers, with potential implications for paleoclimate and paleohydrology

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researches.
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Acknowledgements
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We thank James Rolfe and Ian Mather for technical assistance in the Godwin
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Laboratory. This study was supported by the NERC project, ID E/M003752/1 to D.A.H.
The sampling surveys were supported by the Water Resources and Environmental
Geology Research Group (University of Almería). S.F.M.B., F.G. and D.A.H.
acknowledge support from the European Union’s Horizon 2020 Research and
Innovation programme for project “QUEST” (Marie Sklodowska-Curie grant
agreement No. 691037). U-Th dating was supported by grants from Taiwan ROC
MOST (104-2119-M-002-003, 105-2119-M-002-001 to C.-C.S.) and the Research
Center for Future Earth, National Taiwan University (107L901001 to C.-C.S.). The
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authors are grateful to Professor Philippe Audra and two anonymous reviews, who
contributed to improve the original manuscript.

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FIGURES AND TABLE CAPTIONS

Figure 1. Geological map of the Iglesiente-Sulcis mining district (SW Sardinia, Italy)
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and location of the Mount San Giovanni mine (after Caddeo et al., 2011).

Figure 2. Cross-section of the Mount S. Giovanni mine (after De Waele et al., 2013)
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with the locations of the studied caves, the main ore bodies and the mine galleries
and shafts indicated in grey. Sampled caves in this study are shown in red.
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Figure 3. Topographic profile of caves Crovassa Azzurra, Crovassa Ricchi in Argento,

Massa Riccardo and Santa Barbara (after Messina et al., 2005), showing the sampling
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sites: GA-05 (calcite spar and calcite coatings); GA-01 (calcite dogtooth crystals); GA-
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core (flowstone core); GA-03 (calcite spars); GA-BA (blue aragonite flowstone); GRA-
01 (calcite spars); MS-01 (calcite roses); MS-06 (calcite spar and flowstone); GA-host
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and MS-host (host rock); SB2-CC. Mammillary calcite coatings (cave clouds) analyzed
by De Waele and Forti (2006). Note that the relative positions of the caves in the X
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axis have been modified for graphical representation.

Fig. 4. Overview on the speleothem samples analyzed in this study; A. Scalenohedral

calcite spar (subsample GA-05-1) covered with white and brownish calcite coatings
(subsamples GA-05-3 and GA-05-2, respectively) collected in Crovassa Azzurra cave;
B. Dogtooth calcite crystals from the lower levels of Crovassa Azzurra cave
(subsamples GA-01-1, GA-01-2 and GA-01-3); C. Calcite “roses” from Massa
Riccardo cave (subsamples MS-01-1 and MS-01-2); D. Scalenohedral calcite spar
(subsample MS-06-3) covered with white calcite flowstone (subsamples MS-06-1 from
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the top and MS-06-2 from the base) collected in Massa Riccardo cave. E and F.
Sampling of the flowstone core in Crovassa Azzurra cave (sample GA-core) and
position of eleven subsamples for U-Th dating.

Fig. 5. Oxygen and carbon isotopes in carbonate samples from the caves of the Mount
San Giovanni mine.

Figure 6. Genetic evolution of the Mount San Giovanni caves. Estimated water table
levels and temperatures are given in connection with precipitation of different types of

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speleothems: A. First stages of hypogene speleogenesis at high temperature during
Cambrian; B. Intense karstification during the Carboniferous, probably related to

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H2SO4 produced due to oxidation of polymetallic sulfide ore bodies after increased O 2-

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rich meteoric water input; C. The first stages of hydrothermal calcite precipitation took
place before 600 ka BP at temperature ~120 oC; D. Precipitation of hydrothermal (~70
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oC) calcite spars and subaerial speleothem in the upper level of the karst coexisted
between ~500 and ~400 ka BP; E. At ~400 ka BP, the water temperature decreased
(~14 oC) and subaqueous calcite coatings formed in a perched pound covering
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hydrothermal spars formed during previous stages in Crovassa Azzurra cave. At the
same time, subaerial aragonite flowstones formed in the upper level of this cave; F.
D

Different types of epiphreatic speleothems (calcite dogtooth crystals and calcite roses)
precipitated at low temperature (~10 oC to 20 oC) in perched pounds between 265 and
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109 ka BP at the ~208 m level, while mammillary calcite coatings formed in deeper
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levels (~65 m a.s.l.) from the still warm, aquifer water. Meanwhile, aragonite flowstone
kept growing until 82 ka BP. Note that the depth scale is illustrative and may have
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varied with time because of the exhumation of the karts.

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Table 1. Sample locations and descriptions, isotopic analyses, U-Th ages (±2SD) (see
supplementary Table S1 for details) and temperature estimates using clumped
isotopes thermometry (T47 ± 2SE). The number of aliquots analyzed (n) is given. The
actual 18Ow (± 2SE) of the paleo-aquifer has been calculated from the T47 and 18Oc
using equation 1 in the main text (see supplementary Table S2 for calculations and
propagation of analytical uncertainties). Water temperatures are also calculated using
the 18Oc-18Ow approach (T18Oc-18Ow), assuming that the paleo-18Ow was -5.5±1 ‰
(2SE) for all samples, a value similar to the modern cave water (see discussion for
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details), using eq. 1 for calcite samples and the paleotemperature equation of Kim et
al. (2007) for aragonite samples. The 18Oc-18Ow approach gives temperature
uncertainties of ±5oC (2SE). *sample analyzed by De Waele and Forti (2006).

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18Oc 13C 47 18Ow

Depth Age T(18Oc-18Ow)
Sample Description Formation environment (‰ (‰ (‰) T 47 (0C) (‰
(m a.s.l) (ka) (0C)
VPDB) VPDB) VSMOW)
CROVASSA AZZURRA
GA-05-1 208 Scalenohedral calcite spar Subaqueous (high temperature) -13.6 -0.5 394±13 69 - - -
GA-05-2 208 Calcite crust (brown layer) Subaqueous (low temperature) -4.6 -9.7 - 17 - - -
GA-05-3 208 Calcite crust (white layer) Subaqueous (low temperature) -4.6 -7.9 410±14 17 0.696±0.012 (n=19) 13±3 -6.4±0.7
GA-01-1
GA-01-2
201
201
Dogtooth calcite crystals
Dogtooth calcite crystals
Subaqueous (low temperature)
Subaqueous (low temperature)
-5.1
-4.3
-8.1
-6.3
109±0.5
216±1.3

P T 20
16
0.680±0.013 (n=16)
0.694±0.013 (n=20)
17±3
14±3
-6.0±0.7
-6.0±0.7

I
GA-01-3 201 Dogtooth calcite crystals Subaqueous (low temperature) -4.2 -6.2 - 16 - - -
GA-BA-1 208 Aragonite flowstone (white layer) Subaerial (low temperature) -4.3 -5.7 - 12 - - -
GA-BA-2
GA-03-1
GA-03-2
208
205
205
Aragonite flowstone (blue layer)
Scalenohedral calcite spar
Scalenohedral calcite spar
Subaerial (low temperature)
Subaqueous (high temperature)
Subaqueous (high temperature)
-4.1
-18.8
-15.6
-4.3
-0.3

C
0.1 R
383±10
-
-
11
109
83
-
0.476±0.018 (n=20)
-
-
130±14
-
-
-3.1±1.5
-

S
GA-core-2 216 Flowstone core (36 cm from the top) Subaerial (low temperature) -5.1 -8.9 684±244 16 - - -
GA-core-3 216 Flowstone core (31.5 cm from the top) Subaerial (low temperature) -4.8 -11.1 >600 14 - - -
GA-core-4
GA-core-11
GA-core-12
216
216
216
Flowstone core (27 cm from the top)
Flowstone core (22.5 cm from the top)
Flowstone core (21.5 cm from the top)
Subaerial (low temperature)
Subaerial (low temperature)
Subaerial (low temperature)
-4.3
-4.5

N
-4.6 U -9.1
-8.2
-6.2
570±53
587±44
581±51
12
14
15
-
-
-
-
-
-
-
-
-

A
GA-core-5 216 Flowstone core (16.6 cm from the top) Subaerial (low temperature) -4.1 -8.4 506±19 11 - - -
GA-core-6 216 Flowstone core (12 cm from the top)-aragonite Subaerial (low temperature) -4.8 -7.6 228±2 14 - - -

M
GA-core-7 216 Flowstone core (8.5 cm from the top)-aragonite Subaerial (low temperature) -4.4 -7.4 208±1 12 - - -
GA-core-8 216 Flowstone core (6 cm from the top)-aragonite Subaerial (low temperature) -5.2 -7.2 198±1 16 - - -
GA-core-9 216 Flowstone core (3.6 cm from the top)-aragonite Subaerial (low temperature) -4.9 -6.8 193.6±0.8 14 - - -
GA-core-10
GA-host
MASSA RICCARDO
216
210
Flowstone core (0.5 cm from the top)
Host rock

E D
Subaerial (low temperature)
-
-4.5
-10.4
-6.5
-0.8
82.3±0.3
-
13
-
-
-
-
-
-
-

MS-06-1
MS-06-2
MS-06-3
206
206
206
Calcite flowstone (top)
Calcite flowstone (bottom)
Scalenohedral calcite spar
P T Subaerial (low temperature)
Subaerial (low temperature)
Subaqueous (medium temperature)
-4.4
-5.1
-7.0
-10.1
-10.2
-8.4
-
-
-
16
20
29
-
-
-
-
-
-
-
-
-
MS-01-1
MS-01-2
MS-host
208
208
207
Calcite “roses”
Calcite “roses”
Host rock
C E Subaqueous (low temperature)
Subaqueous (low temperature)
-
-4.4
-5.2
-11.7
-10.4
-10.4
-0.2
-
265±6
-
16
23
-
-
0.708±0.008 (n=37)
-
-
10±2
-
-
-7.7±0.4
-
CROVASSA RICCHI IN ARGENTO
GRA-01-1
GRA-01-2
GRA-01-3
GRA-01-4
180
180
180
180
A C
Scalenohedral calcite spar
Scalenohedral calcite spar
Scalenohedral calcite spar
Scalenohedral calcite spar
Subaqueous (high temperature)
Subaqueous (high temperature)
Subaqueous (high temperature)
Subaqueous (high temperature)
-18.8
-18.2
-18.8
-18.6
-0.3
0.2
0.1
0.2
>600
-
-
-
109
105
109
109
0.485±0.009 (n=20)
-
0.491±0.012 (n=18)
-
123±6
-
119±9
-
-3.8±0.6
-
-4.3±1.0
-
SANTA BARBARA 2 CAVE
SB2-CC* 65 Mammillary calcite coatings Subaqueous (medium temperature?) - - 247±17 - - - -

Table 1.
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Highlights:
We apply clumped isotopes on subaqueous speleothems to reconstruct changes in paleo-
aquifer water temperature and 18Ow.
The 18Ow of the paleo-water is then reconstructed from the clumped isotope temperature and
the 18O of the carbonate.
Hydrothermal spars (~120°C) formed before 400 ka, while low-temperature (10 to 17°C)
speleothems did from 400 to 82 ka.
The 18O values of the paleo-aquifer ranged from -6.0±0.7 ‰ to -6.4±0.7 ‰ during MIS 5c, MIS7

PT
and MIS11, similar to modern values.
The 18Ow of the paleo-aquifer was ~2‰ lower during the colder MIS 8.

RI
SC
NU
MA
E D
PT
CE
AC
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6