diff --git "a/B9AzT4oBgHgl3EQfh_2U/content/tmp_files/2301.01493v1.pdf.txt" "b/B9AzT4oBgHgl3EQfh_2U/content/tmp_files/2301.01493v1.pdf.txt" new file mode 100644--- /dev/null +++ "b/B9AzT4oBgHgl3EQfh_2U/content/tmp_files/2301.01493v1.pdf.txt" @@ -0,0 +1,2628 @@ + +This is the ‘accepted manuscript’ version of our paper: + + + + +Therese Weissbach, Tobias Kluge, Stéphane Affolter, Markus +C. Leuenberger, Hubert Vonhof, Dana F.C. Riechelmann, Jens +Fohlmeister, Marie-Christin Juhl, Benedikt Hemmer, Yao Wu, +Sophie F. Warken, Martina Schmidt, Norbert Frank, Werner +Aeschbach, 2023, Constraints for precise and accurate fluid +inclusion stable isotope analysis using water-vapour saturated +CRDS techniques, Chemical Geology 617, 121268. +https://doi.org/10.1016/j.chemgeo.2022.121268 + + +Please contact the corresponding author, if you want to discuss +the content. + + + + + + + +© 2023. This manuscript version is made available under the +CC-BY-NC-ND 4.0 license +http://creativecommons.org/licenses/by-nc-nd/4.0/ + +Constraints for precise and accurate fluid inclusion stable isotope +analysis using water-vapour saturated CRDS techniques +Therese Weissbach1,2, Tobias Kluge1,2,3,4,*, Stéphane Affolter5, Markus C. Leuenberger6, Hubert +Vonhof7, Dana F.C. Riechelmann8, Jens Fohlmeister9, 10, Marie-Christin Juhl2, Benedikt Hemmer2, Yao +Wu2, Sophie F. Warken2,11, Martina Schmidt2, Norbert Frank2, Werner Aeschbach2, 3 +1Heidelberg Graduate School of Fundamental Physics, Heidelberg University, Im Neuenheimer Feld +226, 69120 Heidelberg, Germany +2Institute of Environmental Physics, Heidelberg University, Im Neuenheimer Feld 229, 69120 +Heidelberg, Germany +3Heidelberg Center for the Environment, Heidelberg University, Im Neuenheimer Feld 229, 69120 +Heidelberg, Germany +4now at: Institute of Applied Geosciences, Karlsruhe Institute of Technology, Adenauerring 20b, +76131 Karlsruhe, Germany +5Department of Environmental Sciences, University of Basel, Bernoullistrassse 30/32, 4056 Basel, +Switzerland +6Climate and Environmental Physics Division, Physics Institute and Oeschger Centre for Climate +Change Research, University of Bern, Sidlerstrasse 5, 3012 Bern, Switzerland +7Climate Geochemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 +Mainz, Germany +8Institute for Geosciences, Johannes Gutenberg University Mainz, Johann-Joachim-Becher-Weg 21, +55128 Mainz, Germany +9Federal Office for Radiation Protection, Köpenicker Allee 120-130, 10318 Berlin, Germany +10GFZ German Research Centre for Geosciences, Section ‘Climate Dynamics and Landscape +Development’, Telegrafenberg, 14473 Potsdam, Germany +11Institute of Earth Sciences, Heidelberg University, Im Neuenheimer Feld 234, 69120 Heidelberg, +Germany +*corresponding author: tobias.kluge@kit.edu + +Journal: Chemical Geology + +Highlights: + +Laser-based fluid inclusion analysis with water-vapour purged extraction enables high +precision (δ2H≤±1.5‰, δ18O ≤±0.5‰) + +Biasing effects (memory, adsorption, amount) in fluid inclusion isotope analysis are negligible +for ≥1 µl water/g calcite + +Isotopic interference is negligible for sample isotope ratios within 10‰(δ18O) and 50‰(δ2H) +of the water vapour background + +Reconstructed temperatures of a 20th century stalagmite trace the recent warming of 1 °C in +Central Europe + +Abstract + +Hydrogen (δ2H) and oxygen (δ18O) isotopes of water extracted from speleothem fluid +inclusions are important proxies used for paleoclimate reconstruction. In our study we use a cavity +ring-down laser spectroscopy system for analysis and modified the approach of Affolter et al. (2014) +for sample extraction. The method is based on crushing of small sub-gram speleothem samples in a +heated and continuously water-vapour purged extraction line. The following points were identified: + +Injection of reference water shows a precision (1σ) of 0.4-0.5 ‰ for δ18O values and 1.1-1.9 ‰ +for δ2H values for water amounts of 0.1-0.5 µl, which improves with increasing water amount to 0.1- +0.3 ‰ and 0.2-0.7 ‰, respectively, above 1 µl. The accuracy of measurements of water injections and +water-filled glass capillaries crushed in the system is better than 0.08 ‰ for δ18O and 0.3 ‰ for δ2H +values. The reproducibility (1σ) based on replicate analysis of speleothem fluid inclusion samples with +water amounts > 0.2 µl is 0.5 ‰ for δ18O and 1.2 ‰ for δ2H values, respectively. Isotopic differences +between the water vapour background of the extraction system and the fluid inclusions have no +significant impact on the measured fluid inclusion isotope values if they are within 10 ‰ for δ18O and +50 ‰ for δ2H values of the background. Tests of potential adsorption effects with inclusion free spar +calcite confirm that the isotope values are unaffected by adsorption for water contents of about 1 µl +(fluid inclusion) water per g of carbonate or above. +Fluid inclusion analysis on three different modern to late Holocene speleothems from caves in +northwest Germany resulted in δ18O and δ2H values that follow the relationship as defined by the +meteoric water line and that correspond to the local drip water. Yet, due to potential isotope exchange +reactions for oxygen atoms, hydrogen isotope measurements are preferentially to be used for +temperature reconstructions. We demonstrate this in a case study with a Romanian stalagmite, for +which we reconstruct the 20th century warming with an amplitude of approximately 1 °C, with a +precision for each data point of better than ±0.5 °C. + +Keywords: laser spectroscopy, water isotopes, cavity-ring-down measurement, speleothems, +paleoclimate, small samples + +1. Introduction +Speleothem fluid inclusions can provide direct insight into past climatic conditions as they are +a unique archive for the original drip water and the corresponding meteoric water (e.g., Griffiths et al., +2010; Affolter et al., 2014; Labuhn et al., 2015; Warken et al., 2022). Fluid inclusion water isotope ratios +(δ18O and δ2H values) are increasingly usedas proxies in hydrology and paleoclimate studies (e.g., +McGarry et al., 2004; Demény et al., 2017; Millo et al., 2017; Affolter et al., 2019; Wilcox et al., 2020; +Matthews et al., 2021). Two physically different measurement principles, laser spectroscopy (mainly +cavity ring-down spectroscopy - CRDS) and isotope ratio mass spectrometry (IRMS), allow determining +the isotopic composition of speleothem fluid inclusion water (CRDS: Arienzo et al., 2013; Affolter et +al., 2014; Uemura et al., 2016; Dassié et al., 2018; IRMS: Dennis et al., 2001; Vonhof et al., 2006; +Dublyansky and Spötl, 2009). Although CRDS and IRMS systems yield comparable results (de Graaf et +al., 2020) challenges remain for both methods regarding precise and reproducible analysis of small +water amounts. Often only a single measurement attempt is possible due to low growth rates of the +speleothems (often 10-100 µm/a) or intended high resolution. Water contents in natural speleothems +range from ~ 0 up to several 10 µl per g (McDermott et al., 2006). The necessary water sample amount +(depending on the setup 0.05-0.2 µl, e.g., Dublyanksy and Spötl, 2009; Uemura et al., 2016) limits the +temporal resolution and restricts analytical repetition. +Fluid inclusion water for isotope analysis is released either by crushing (e.g., Schwarcz et al., +1976; Dennis et al., 2001; Vonhof et al., 2006; Dublyansky and Spötl, 2009; Demény et al., 2013) or +thermal decrepitation (e.g., Yonge, 1982; McGarry et al., 2004; Verheyden et al., 2008). Thermal +decrepitation has the disadvantage that structurally-bound water with a very low δ2H value may be +released during extraction, resulting in large isotopic shifts of up to 30 ‰ in comparison to parent cave +drip water (Yonge, 1982; Matthews et al., 2000; McGarry et al., 2004; Verheyden et al., 2008). This +analytical artefact can be largely avoided by crushing the sample mechanically. For fluid inclusion +analysis using IRMS (Schwarcz et al., 1976; Harmon et al., 1978; 1979), water was extracted by crushing +the sample under vacuum conditions and then subsequently converted to water vapour followed by +conversion into directly measurable gases such as H2 for H isotopic analysis. Fluid inclusion δ18O values +were initially not measured but calculated from measured δ2H values via the relationship between +δ18O and δ2H values of the meteoric water line (e.g., Schwarcz et al., 1976). In recent extraction systems +the oxygen in fluid inclusion water is converted to CO gas during a high temperature reaction with +glassy carbon which is then used for analysis of δ18O values in an IRMS (e.g., Dublyansky and Spötl, +2009). The first combined method for oxygen and hydrogen measurements with an off-line crushing +method and dual-inlet IRMS was developed by Dennis et al. (2001). It achieved good precision of ± 0.4 +‰ for δ18O and ± 3 ‰ for δ2H values, but required a comparatively large sample with water amounts +of 1-3 μl (see also Matthews et al., 2000). A reduction in sample amount down to 0.1 μl, which + +corresponds to 0.1 g of calcite for samples that contain 1 µl of water per gram, was achieved by Vonhof +et al. (2006) by combining off-line preparation and continuous-flow mass spectrometry. This technique +enables a faster analysis of 0.1 - 0.2 μl sized samples with a precision of ± 0.5 ‰ for δ18O and ± 1.5 ‰ +for δ2H values (Vonhof et al., 2006; Dublyansky and Spötl, 2009; de Graaf et al., 2020). +Laser spectroscopy is less expensive and represents a reliable, precise and easy technique to +directly measure stable water isotopes (Brand et al., 2009; Gupta et al., 2009). The first application +using CRDS to measure fluid inclusions in speleothems was developed by Arienzo et al. (2013). They +used a CRDS analyser with a stainless-steel line heated to 115 °C that was constantly flushed with dry +nitrogen as a carrier gas. It achieved comparable precisions as the traditional IRMS technique, with ± +0.5 ‰ for δ18O and ± 2.0 ‰ for δ2H values. The development of another analysis system using off-axis +integrated cavity output spectroscopy (OA-ICOS) achieved similar precision (Czuppon et al., 2014). The +latest analytical systems are able to measure released water volumes in the nano-litre range (50 to +260 nl) with a precision of ± 0.3 ‰ for δ18O and ± 1.6 ‰ for δ2H values using the CRDS technique +(Uemura et al., 2016). +The above discussed fluid inclusion extraction lines of Arienzo et al. (2013), Czuppon et al. +(2014), and Uemura et al. (2016) are working with dry carrier gas and low water vapour concentrations +in the analyser cavity, which may influence the stable isotope measurements by adsorption and cause +memory effects. The measured isotopic signal needs to be corrected for, e.g., the isotopic dependence +on the water vapour concentration (Uemura et al., 2016) or the memory effect (e.g., van Geldern and +Barth, 2012). The memory effect in the analyser cavity is due to limitations during the removal of all +gas between two measurements preventing the full desorption of water molecules from the cavity +walls. A standard technique to deal with memory effects in liquid water analysis is the repeated +injection of the same water sample. The measured signal converges exponentially towards the actual +sample signal (e.g., van Geldern and Barth, 2012). However, multiple crushing steps on the same +sample are typically not feasible for fluid inclusion measurements of speleothems since the amount of +water of these samples is often too low to split it in several aliquots (sub-μl range). The adsorption +issue was addressed by Affolter et al. (2014) with an extraction line that is continuously purged with a +moist gas providing a water vapour background with constant and known δ18O and δ2H values. The +extraction line with a “wet” N2 gas allows reproducible and precise measurement of released fluid +inclusion water. The continuous heating of the system enables the instantaneous evaporation of the +water released from inclusions followed by spectroscopic analysis of the resulting mixture of +background and sample water vapour. The main advantage of the water-vapour flushing is that this +procedure avoids additional corrections of the measured water stable isotopes related to memory +effects. The achievable standard deviations of the measurements with this analytical system are +smaller than 0.4 ‰ for δ18O and 1.5 ‰ δ2H values, which is comparable with the traditional IRMS + +technique (Vonhof et al., 2006; Dublyansky and Spötl, 2009) and CRDS setups working with a dry carrier +gas (Arienzo et al., 2013; Czuppon et al., 2014). +One important application of isotope fluid inclusion studies is the reconstruction of +paleotemperatures using the oxygen isotope fractionation between water and calcite. The +temperature reconstruction was initially based on the measurement of fluid inclusion δ2H values from +which the fluid inclusion δ18O values were calculated using the δ2H-δ18O relationship of the global +meteoric water line (Craig, 1961). Combined with the δ18O value of the calcite, temperatures were +calculated from the oxygen isotope fractionation between the carbonate mineral and water. This +indirect approach achieved a reported precision of about ± 2 °C in the early studies of Schwarcz et al. +(1976). In the decades prior to 2010, direct temperature calculation from measured fluid inclusion δ18O +values has been rare due to severe challenges in its analysis with standard mass spectrometric +approaches. Therefore, mostly the indirect way of first converting measured δ2H values into fluid +inclusion water δ18O values was used (e.g., Matthews et al., 2000). More recent studies provided +comparable temperature precision based on inclusion water δ18O values: ± 1.3 °C (van Breukelen et +al., 2008), ± 0.9-2.1 °C (Meckler et al., 2015); ± 2.7 °C (Arienzo et al., 2015), and ± 0.6-3.1 °C (Uemura +et al., 2016). The uncertainty of the indirect paleotemperature reconstruction from δ2H variations in +speleothem fluid inclusions is similar: ± 1.5 °C (Zhang et al., 2008) and ± 0.9-2.5 °C (Meckler et al., +2015). More recently, a better precision has been achieved when using the rainfall δ2H/T relation in +mid-latitudes: ± 0.2-0.5 °C (Affolter et al., 2019). +In this study, we systematically assess the measurement method of stable water isotopes in +speleothem fluid inclusion analysis using CRDS, including in particular the effect of sample amount +(water amount per analysis), water adsorption on freshly crushed calcite surfaces, influence of the +isotope values of the water vapour background on the sample signal, as well as the external +reproducibility of speleothem fluid inclusion samples (using adjacent aliquots along growth layers). In +addition, we present a recent case study allowing to determine paleo-temperature trends in the 1°C +range. + +2. Methods and site description +2.1 Water extraction from fluid inclusions + +At the Institute of Environmental Physics (Heidelberg) water from speleothem fluid inclusions +is extracted within a system that is constantly purged by an artificially prepared moist gas, leading to +a water vapour background with known δ18O and δ2H values (Fig. 1). In the extraction line this stable +water vapour background is generated by mixing water of a known isotopic composition into a dry +nitrogen gas flow (300 ml/min). A peristaltic pump (Ismatec -REGLO Digital, Wertheim, Germany) +continuously supplies small amounts of water (1 μl/min) to the line through a T-injection port with a + +septum (Fig. 1 A). A constant temperature of 120 °C ensures a complete and immediate evaporation. +Instant water evaporation is induced in a fused silica capillary, which slightly touches the heated base +of the port. A two-litre mixing cavity placed after the T-injection port generates a stable water vapour +background and compensates fluctuations caused by the peristaltic pump cycles. The nitrogen flow is +controlled by a mass flow controller (Analyt MTC, model GFC-17, Müllheim, Germany) and creates a +constant overpressure of 0.5 bar. The flow rate is 40 ml/min into the CRDS analyser (L2130-i, Picarro, +Santa Clara, USA). The surplus gas stream is vented through a purge capillary before the crusher unit. +With this setup the water vapour concentration in the CRDS cavity ranges between 6000 and 8000 +ppmV, but the cavity can also be adjusted to higher or lower water vapour concentrations if needed. +We have chosen a range between 6000 and8000 ppmV to allow for the detection and analysis of small +fluid inclusion water amounts (sufficiently high ratio of water vapour from the sample relative to the +background) but also to provide a background water vapour concentration that prevents memory +effects. +The sample (speleothem fragment or glass capillary)is inserted in a copper tube (Fig. 1 B) which +is connected to the extraction and measurement system. Due to limitation of the copper tube with +respect to length and diameter, compact mineral pieces with rectangular dimensions of 6 mm x 6 mm +x 10-20 mm are preferred. The copper tube including the sample is purged for at least 30 min until +water vapour concentration and isotope values reach a constant signal. The stability of the water +vapour background is verified by monitoring the standard deviation of the water vapour concentration. +When the standard deviation of the water vapour concentration remained less than 20 ppmV for 30 +minutes, the speleothem sample is crushed by compressing the copper tube from the outside with a +hydraulic press at 200-300 bar. This compression in the heated system leads to the release and +immediate evaporation of inclusion water and a sudden pressure increase. This pressure increase +could cause gas flow not only towards the CRDS but also in direction of the purge capillary and may +provoke sample gas loss. Therefore, a reflux valve is installed between the 2l mixing cavity and the +crushing unit to prevent a backflow and loss of the sample. In general, a very good crushing efficiency +has been achieved with an average grain size of 37 μm after the crushing (Weißbach, 2020). A +reproducibility test related to the crushing procedure showed similar particle size distributions for five +different speleothem samples investigated with laser diffraction (Analysette-22 Micro Tec) after +crushing with the hydraulic system. +An injection port is situated next to the copper tube, allowing to mimic a water release from a +mineral sample and helps to evaluate and control accuracy and precision of the δ18O and δ2H values +from small (inclusion) water samples. An additional small mixing cavity (400 ml) directly after the +crushing unit prolongs the generated sample signal from an usually few seconds lasting peak to a well + +measurable signal with a duration of several minutes. After the mixing cavity the gas proceeds to the +L2130-i isotope and gas concentration analyser (Picarro). + +Figure 1: Fluid inclusion line for extraction and measurement of stable oxygen and hydrogen isotopes of fluid +inclusions in speleothems. Water with a known isotope composition is mixed into a nitrogen gas flow to create a +stable water vapour background (position A). The purge capillary reduces the background vapour flow from 280 +to 40 ml/min as required for the CRDS analyser (L2130-i, Picarro). The two mixing cavities provide a smoothing of +the background vapour signal and a dispersion of the measurement signal. The speleothem sample or the glass +capillary is placed in a copper tube and installed at position B inside the heated oven. In order to prevent the +backflow of the water vapour from the freshly crushed sample, a reflux valve is installed. The flow directions are +indicated as blue arrows. [black/white for figures in print, colour online] + +2.2 Analysis and data evaluation +The water vapour concentration and the δ18O and δ2H values are determined with the L2130-i +isotope and gas concentration analyser of Picarro. The L2130-i analyser is based on wavelength- +scanned CRDS in the spectral range from 7183.5 to 7184 cm-1 and uses a multi-pass cell that creates a + +N2 +tankgas +injection +glass +port +capillary +peristalticpump +CRDS +background +heatingtape +40 +water +ml/min +(120°C) +purge capillary +~280 +ml/min +refluxvalve +mixing cavity -21 +filter +injection +port +oven +mixingcavity-400ml +loadedcopper +(120C) +tubelong effective absorption path length of about 12 km (Aemisegger et al., 2012). A stable cavity +temperature of 80°C ± 0.002 °C is maintained). The cavity pressure is set to 66.66 hPa (50 Torr). The +water isotopologue lines pertaining to 18O and 2H are measured simultaneously over a 0.8 s interval. +In our setup the L2130i allows isotope measurements in the water vapour concentration range +between 1 000 and 50 000 ppmV. +In constant flow mode the actual measured signal is composed of a background signal and a peak +signal after crushing and must be integrated over a corresponding time interval (Fig. 2). The evaluation +routine follows the approach of Affolter et al. (2014) but was extended by the correction of a potential +level change in the water vapour background (Weißbach, 2020). The import of the data and the +evaluation was carried out with the assistance of the Python script IsoFluid (https: +//github.com/bhemmer/IsoFluid and http://doi.org/10.5281/zenodo.5911265). IsoFluid determines +the sample or calibration standard peak start and end based on a slope criterion that compares the +start and end slope with the background slope of the water vapour concentration in user-defined time +intervals. The sample isotope value (δ18Osample) is calculated by subtraction of the background signal +(index back) from the measured signal of the mixed gas signal (index mix) and follows the approach of +Affolter et al. (2014): +𝛿��𝑂������ = +∫ +𝛿��𝑂������(𝑡) ∗ 𝐻�𝑂������(𝑡) ∙ 𝑑𝑡 +�� +�� +∫ +𝐻�𝑂������(𝑡) +�� +�� +∙ 𝑑𝑡 += +∫ +𝛿��𝑂���(𝑡) ∗ 𝐻�𝑂���(𝑡) ∙ 𝑑𝑡 − ∫ +𝛿��𝑂����(𝑡) ∗ 𝐻�𝑂����(𝑡) ∙ 𝑑𝑡 +�� +�� +�� +�� +∫ +𝐻�𝑂���(𝑡) +�� +�� +∙ 𝑑𝑡 − ∫ +𝐻�𝑂����(𝑡) +�� +�� +∙ 𝑑𝑡 + + +(Eq.1) +H2O refers to the related water amount. The calculation for δ2H values is correspondent. +All uncertainties are reported at the 1σ level. + + +Figure 2: Water vapour signal in the CRDS before, during, and after speleothem sample crushing. Background +intervals (orange) are used to determine the value of the water background during a sample analysis (blue +shaded). [black/white for figures in print, colour online] + +2.3 Water vapour background calibration +For the calibration of the oxygen and hydrogen isotope signal of the water vapour background +five independently measured in-house reference waters were used (Table 1). Water from +Willersinnweiher water (WW) was not used for calibration but for precision and accuracy assessment. + +Table 1: In - house reference waters for isotopic calibration of the fluid inclusions CRDS system, Isotope +values were independently measured at the Institute of Environmental Physics (IUP) at Heidelberg +University. Uncertainties are given as 1σ errors. +Water type +Code +δ18O values +(‰ VSMOW) +δ2H values +(‰ VSMOW) +Artificially evaporated water +AE +3.8 ± 0.3 +-21.79 ± 1.8 +Ocean water +Kona +-0.05 ± 0.08 +0.5 ± 0.7 +Lake +Water +- +Willersinnweiher +(S. +Germany) +WW +-0.32 ± 0.11 +-18.8 ± 0.4 +De-ionized local tap water +VE +-8.57 ± 0.08 +-61.0 ± 0.7 +Alpine Water +VCL +-13.04 ± 0.08 +-98.3 ± 0.7 + +8500 +samplepeak +water vapour concentration [ppmV] +8000 +background interval +background interval +7500 +peak - +start +peak-end +linearregression +7000 +11:15 +11:30 +11:45 +12:00 +12:15 +12:30 +time [hour]Alpine Water - Colle Gniffeti ice core +CC +-15.13 ± 0.08 +-110.6 ± 0.7 +North Greenland water-surface snow +NG +-26.54 ± 0.08 +-212.1 ± 0.7 + + + + + +The isotope values of these five reference waters were determined independently with a Los +Gatos Research LGR1 analyser. These reference waters cover a range of −26.5 up to -0.05 ‰ in δ18O +values and −212.1 up to 0.5 ‰ in δ2H values (both VSMOW) (Table 1), which includes the relevant +range for speleothem samples. Five different isotope background values were realised by using the +corresponding reference water as supply, injected with the peristaltic pump into the system. Once a +sufficiently stable water vapour concentration was achieved in the preparation line (standard deviation +below 20 ppmV for 30 min) the isotope signal was averaged over 60 minutes, which results in a +standard deviation of 0.2 ‰ and 0.7 ‰ for the δ18O and δ2H background values, respectively. Figure 3 +shows the CRDS-measured isotope value against the reference value (Table 1). The value of the water +vapour background was constantly monitored via repeated measurements of the in-house reference +waters, and has remained constant over several years. + +Figure 3: The calibration of δ18O and δ2H values of the water vapour background results from a linear regression +(red lines). The calibration equation is y = (0.994 ± 0.007 · x + 2.30 ± 0.14) ‰ for δ18O values (R²=0.999) and y = +(0.980 ± 0.003 · x−7.77 ± 0.38) ‰ for δ2H values(R²=0.999). Both calibrations remained constant over several +years. Isotope data on the reference waters used for water vapour background calibration are given in Table 1. +The residuals from the linear regression indicate that calibrated values are within 0.08 ‰ (δ18O) and 0.35 ‰ +(δ2H) of the expected reference value. Furthermore, the residuals show a random distribution. Uncertainties on +the 1σ level in the calibration graphs are smaller than the symbol size. [black/white for figures in print, colour +online] + +residuals (%o) +0.08 +8180 +(0%) +0.30 +2H +0.04 +0.15 +S +8 +0.00 +dual +0.00 +-0.15 +-0.04 +0 +-0.08 +-0.30 +0 +-30 +-25 +-20 +-15 +-10 +-5 +0 +-250 +-200 +-150 +-100 +-50 +0 +50 +50 +0 +Kona +1:1 +(% VSMOW) +1:1 +(%。 VSMOW) +0 +-5 +Kona +VE +-10 +-50 +VCL +VE +-15 +CC +-100 +VCL +linearregression +pected +CC +-20 +-150 +exp +-25 +NG +linearregression +-200 +NG +-30 +-250 +-30 +-25 +-20 +-15 +-10 +-5 +0 +-250 +-200 +-150 +-100 +-50 +0 +50 +8180. +measured (%。 VSMOW)2.4 Water amount calibration +A precise water amount calibration is necessary for determining the exact amount of released +water from the crushed speleothem calcite. The released water amount is a major parameter for the +calculation of the fluid inclusion isotope value and is determined via water vapour signal integration +(see Eq.1). Isotope values could also be calculated with the time-integrated water vapour mixing ratio +alone, however, knowledge of the released water amounts is recommended for uncertainty +assessment (amount dependence) and for assessment of speleothem growth conditions (fluid +inclusion water yield). Typically, volume calibrations are carried out by injecting water in the μl range +with syringes (here: SGE 1BR-7RAX and 5BR-7RAX and Hamilton 70001KH and 75N), however, the +variability of the calculated water amount only using syringe injections is significant and can be as high +as 10 % (inset in Fig. 4, Weißbach, 2020). +Here we present a water amount calibration method with glass capillaries that follows the +approach of Kluge et al. (2008). The glass capillaries (borosilicate, Hirschmann) can be filled with 0.1- +5.0 μl water at varying isotopic composition. They can be closed airtight by melting both ends. The size +of the filled capillary can be adjusted to the size of the crushing cell down to a minimum length of +approximately 1 cm. The exact volume is determined by scanning the capillary with a high-resolution +office scanner and comparison with the pre-marked 1 μl labels on the capillary. The volume uncertainty +of the glass capillary water amount is ± 0.025 μl and was determined by five repetitions of the manual +evaluation of a scan. The accuracy is given by the uncertainty of the pre-marked 1 µl labels (± 0.003 +µl). Water-filled capillaries were analysed weekly to monitor the stability of the water amount +calibration (Fig. 4). The uncertainty of the water amount determination from the calibration is +approximately ± 0.02 µl at a water volume of 1 µl and ± 0.04 µl at 2.5 µl using the 1σ uncertainty of +the linear regression. In general, we rarely observed outliers in the water amount calibration when +using glass capillaries. + + +Figure 4: The time-integrated measured volume signal in ppmV*s as given by the Picarro analyser is plotted +against the water volume of the glass capillaries. The resulting linear regression y = (5.9 × 10−7 ·x−0.011) μl +(R²=0.999) is used to determine the released amount of water from speleothem samples. In total 45 capillaries +were measured for calibration spanning a water amount range from 0.2 up to 4.3 μl. The upper inset shows a +glass capillary filled with about 0.5 µl of water (in the middle of the capillary). The lower inset shows the +comparison of water injections with different syringes (blue and red symbols) and the glass capillaries (black +circles). The uncertainties are given on the 1σ level. [black/white for figures in print, colour online] + +2.5 Site and sample description +Hüttenbläserschacht Cave (Germany) +For direct comparison with rainwater δ18O values and measured drip water isotope values, we +selected a suite of modern and late Holocene samples from the Hüttenbläserschacht Cave, located +only a few 100 meters west of the well-monitored Bunker Cave in northwest Germany (e.g., +Riechelmann et al., 2011). Both caves are situated in the upper Middle Devonian limestone in Iserlohn +(Sauerland). Hüttenbläserschacht Cave hosts pool spar calcites that are expected to provide fluid +inclusion isotope values close to drip water as they grow under the water table of the pools. Pool spars +from this cave have already been investigated by Kluge et al. (2013) using clumped isotope ∆47 and +calcite δ18O values for calculation of the (drip) water δ18O value. Calcite was actively precipitating in +the pools (e.g., abundant calcite rafts) at the time of pool spar removal. 230Th-U disequilibrium dating + +8.0x106 +label +watercolumn +I area (ppmV*s) +6.0x106 +integrated signal +4.0x106 +3x106 +linearregression +oglass capillary +syringes +Hamilton +2x106 +SGE +2.0x106 +1x106 +0 +0.0 +0.5 +1.0 +1.5 +2.0 +0.0 +0 +1 +2 +3 +4 +5 +water volume capillary (μl)at Heidelberg Academy of Sciences provided radiometric ages of one pool spar and one raft sample of +0.05 ± 0.27 ka BP and 0.36 ± 0.12 ka BP, respectively (Supplemental material S1), corroborating the +assumption that the pool spars and rafts are modern age. +Cloşani Cave (Romania) +For the second case study we selected a 20th century stalagmite (Stam 4) from Cloşani Cave, +Romania (e.g., Constantin and Lauritzen, 1999). A monitoring program from 2010 to 2012 and 2015 +demonstrated a stable cave environment with an air temperature of 11.4 ± 0.5 °C and a relative +humidity close to 100 % (Warken et al., 2018). The isotopic composition of the drip water in direct +vicinity (1 m) of the former location of Stam 4 showed no seasonal cycle and was constant throughout +the monitoring. The mean dripwater δ18O value was −9.6 ± 0.2‰ and −66.3 ± 1.7‰ for δ2H values. +Stalagmite Stam 4 has a total length of 6 cm and an average growth rate of 510 μm per year, as +deduced from counting of layers related to annual cycles in the concentration of various elements +(Supplemental material S2). The speleothem grew actively until the removal in spring 2010 C.E. as drip +water was feeding the stalagmite. The recent growth of the stalagmite was further constrained by the +detection of the 20th century radiocarbon bomb spike, which was imprinted by the transport of the +atmospheric signal into the speleothem (see Supplemental material S3). Combined layer counting and +radiocarbon measurements suggest a growth period from 1910 to 2010 C.E. For the fluid inclusion +study, pieces were taken from the peripheral part of Stam 4 with a distance of approximately 1 to 1.5 +cm from the actual growth axis (see Supplementary Fig. S2). + +3. Results +3.1 Precision of isotope measurements +The precision of isotopic measurements (Fig. 5, Table 2) was quantified using the standard +deviation of repeated analyses of the reference waters injected via syringes (VE water; Table 1) and +independently cross-checked with water-filled glass capillaries using VE and WW reference waters. The +injected water amount using syringes) varied between 0.1 and 4.0 μl. +Using syringe injection method, a clear decrease of the standard deviation of these isotope +analyses with increasing water amount becomes apparent (Fig. 5, Table 2). The standard deviation +decreases strongest between 0.1 and 1 µL and reaches values between 0.1 and0.3 ‰ for δ18O and +between 0.2 and0.7 ‰ for δ2H values, for samples larger than 1 µl (Supplementary Table S4). For +smaller water amounts, i.e., of 0.5 µl and below, the isotope values of the injections show a +significantly larger scatter, leading to standard deviations between 0.4 and0.5 ‰ for δ18O and between + +1.1 and1.9 ‰for δ2H values. These uncertainties are based on an exponential fit of the standard +deviation against the water volume using repeated measurements at a given water volume (Fig. 5). +For the determination of the precision, reference water sealed in glass capillaries was crushed in +the fluid inclusion system. Consistent with the results from the water injections, the precision for +isotopic analyses of water released from crushing glass capillaries is between 0.07 and0.10 ‰ for δ18O +values and between 0.3 and0.4 ‰ for δ2H values, for water amounts above 0.5 µl. Smaller water +amounts resulted in a significant increase in the uncertainty and are expressed through a lower +precision (Table 2). + +Table 2: Measurement precision and accuracy in dependence of the water amount. The precision +(± 1σ) was determined from repeated injections of isotopically well-characterized water standards +using syringes. The accuracy (given at the 1σ level) was assessed by measurement of reference water +both from injections and by release from crushing of sealed glass capillaries and comparison with the +independently determined isotope values (Table 1). The error estimate for the accuracy assumes a +Gaussian distribution and includes the uncertainty of the expected value (VE water: ± 0.08 ‰ for δ18O, +± 0.7 ‰ for δ2H, WW water: ± 0.11 ‰ for δ18O, ± 0.4 ‰ for δ2H). n represents the number of analyses +in the investigated water volume range. The water isotope values are given relative to VSMOW. +Type +Reference +water +Water +volume (µl) +Precision (1σ) +δ18O value(‰) + +δ2H +value +(‰) +Accuracy (1σ) +δ18O +value +(‰) + + δ2H +value(‰) +n +Water +injection +VE + + + + + + + + +0.1 +0.54 +1.8 + + +6 + + +0.2 +0.34 +1.6 + + +6 + + +0.3 +0.49 +1.6 + + +6 + + +0.4 +0.53 +1.1 + + +6 + + +0.5 +0.58 +1.4 + + +6 + + +1 +0.17 +0.4 + + +16 + + +2 +0.21 +0.4 + + +11 + + +3 +0.16 +0.4 + + +11 + + +4 +0.10 +0.1 + + +9 + + +Mean all +0.35 +1.0 +0.09 ± 0.19 +0.3 ± 0.7 +77 + + +Mean < 1µl +0.50 +1.5 +0.12 ± 0.22 +0.3 ± 0.7 +30 + + +Mean ≥ 1µl +0.16 +0.3 +0.08 ± 0.12 +0.3 ± 0.7 +47 +Glass +capillary + + + + + + + + +WW +0.3-4.3 +0.42 +0.4 +0.10 ± 0.44 +0.1 ± 0.6 +16 + +WW +> 0.5 +0.07 +0.3 +-0.02 ± 0.13 +0.1 ± 0.5 +14 + +VE +0.3-4.1 +0.30 +1.3 +0.14 ± 0.31 +0.6 ± 1.5 +17 + +VE +> 0.5 +0.10 +0.4 +-0.03 ± 0.13 +0.1 ± 0.8 +9 + + + +Figure 5: Upper panels: precision (1σ) of the isotope measurements for varying amounts using the water injection +method. The red lines represent the least-square exponential fits to the data. The standard deviation decreases +with increasing water amount for both δ18O and δ2H values. . Lower panel: accuracy determination for varying +water amounts based on individual injections (open circles). The related mean values with their 1σ standard +deviation are shown as filled dots with error bars. The black horizontal lines represent the reference value for VE +- tap water (δ18O=- 8.57 ‰, δ2H =- 61.0 ‰) with its uncertainty band (grey shading, ± 0.08‰ for δ18O, ± 0.7 ‰ for +δ2H values). [black/white for figures in print, colour online] + + + + + +2.0 +0.6 +18 +1α standard deviation (%o) +0.5 +5 + standard deviation ( +0.4 +1.0 +0.3 +0.2 +0.5 +0.1 +0.0 +0.0 +0 +1 +2 +3 +4 +0 +1 +2 +3 +4 +water amount (μl) +water amount (μl) +-57 +singlevalue +8H [%。 VSMOW] +-58 +meanvaluewithstdv +8 +-60 +-61 +-62 +-63 +6180 [%VSMOW] +-8.0 +-8.5 +-9.0 +9.5 +singlevalue +meanvaluewithstdy +-10.0 +3 +1 +2 +4 +volume [u]3.2 Accuracy of isotope analysis of micro-litre water amounts +The accuracy of the water δ18O and δ2H values was assessed for reference waters (Table 1) by the +injection with syringes and by crushing glass capillaries. The injected water amounts covered the +typical range of water extracted from inclusions (Fig. 5). The glass capillaries were filled with reference +water with similar water amounts between 0.3 and 4.3 µl and were crushed in the copper tube with +the same hydraulic press as the stalagmite samples. +Considering the water isotope mean values of all measurements performed with the glass +capillaries, the δ18O value deviated from the expected reference values (Table 1) by 0.10 ± 0.44 ‰ for +WW water (n=16) and by 0.14 ± 0.31 ‰ for VE water (n=17) (Table 2). For δ2H values the deviation +from the reference value was 0.1 ± 0.6 ‰ for WW water (n=16) and 0.6 ± 1.50 ‰ for VE water (n=17). +Considering only those measurements with water amounts above 0.5 µl reduces the uncertainty. For +this selection, the δ18O value of both reference waters deviates on average from the expected +reference value by -0.02 ± 0.13 ‰ for WW water (n=14) and -0.03 ± 0.13 ‰ for VE water (n=9). For +δ2H values the deviation from the reference value was 0.1 ± 0.5 ‰ for WW water (n=14) and 0.1 ± 0.8 +‰ for VE water (n=9). +The accuracy as determined by crushing of water-filled glass capillaries is confirmed by the +injection-based data (Table 2). Overall, the δ18O value of the injected VE water deviated from the +expected value on average by 0.09 ± 0.19 ‰ (n=77), that of the δ2H value by 0.3 ± 0.7 ‰ (n=77). + +3.3 Adsorption and/or desorption on the calcite surface +Adsorption on a calcite surface and, in particular, on freshly crushed carbonate with a large surface +to volume ratio provides the possibility to alter the isotope values of the fluid inclusion water (Dennis +et al., 2001). Therefore, an artificial fluid inclusion system (speleothem analogue) as described by +Dennis et al. (2001) has been prepared to quantify the influence of adsorption on the measured +isotopic signal in our setup. We measured water vapour released from a water-filled glass capillary in +direct contact with inclusion-free Iceland spar carbonate. The compact Iceland spar pieces (0.45-0.81 +g) as well as the released water of the capillaries (1.4-3.7 µl water) represent a speleothem sample +with a water content of 2.2 up to 7.8 μl per g calcite. In total, we prepared and analysed five artificial +fluid inclusion - calcite systems in the range between 5.2 and7.8 µl/g and three at about 2.2 µl/g and +compared them to water-filled glass capillaries without additional calcite. The crushing of the compact +Iceland spar pieces provided fresh and fine-grained calcite for interaction and adsorption testing. The +measurements suggest that the adsorption of water molecules on the calcite surfaces does not affect +the measured isotopic signal in the investigated water/calcite ratio range (Fig. 6). Both measured +oxygen and hydrogen isotope values accurately match the expected value. With a standard deviation + +of ± 0.05 ‰ for δ18O and ± 0.22 ‰ for δ2H values (high water/calcite ratio, n=5) and ± 0.15 ‰ for δ18O +and ± 0.31 ‰ for δ2H values (low water/calcite ratio, n=3) in both adsorption tests, a good +reproducibility of the individual measurements was achieved. We observed that after crushing of +Iceland spar (0.25 g) 0.023 μl water was adsorbed on the crushed calcite from the moist carrier gas +(Supplementary Fig. S3), which corresponds to a ratio of approximately 0.1 μl water per g calcite. Thus, +for low water contents of < 0.1 µl per g calcite an influence of adsorption on the released water amount +and the isotopic values probably cannot be excluded. Therefore, we rejected all fluid inclusion samples +with water amounts below 0.1 µl based on this observation (see Weißbach, 2020). + +Figure 6: Isotopic values measured for the artificial inclusion calcite system, for which compact Iceland spar pieces +were crushed together with VE water - filled glass capillaries (triangles). Open circles indicate water - filled glass +capillaries (VE) without calcite addition, measured for comparison. An isotopic fractionation due to adsorption of +water molecules on the calcite surface is not detectable for the investigated water content range of 5.2-7.8 µl +water/g calcite (left side) and for 2.22±0.08 µl water/g calcite (right). Marginal differences in the isotope values +between left and right panel are within the expected variations in the reference water isotope values due to a +several year time lag between both experimental series. The uncertainties are displayed on the 1σ level. +[black/white for figures in print, colour online] + +3.4 Isotopic effect of the water vapour background + +5.2 - 7.8 μl/g +~2.2 μl/g +?H (% VSMOW) +-58 +144 +-60 +444 +D +-62 +(%。VSMOW) +8.0 +丰 +-8.4 +0-8.8 +-9.2 +water plus +only water +waterplus +only water +iceland spar +iceland sparThe potential influence of the isotope ratio of the water vapour background on that of the +measured sample could be relevant for speleothem samples whose isotopic composition strongly +differs from that of the water vapour background. For testing this potential effect, we injected our VE +water standard on four different water vapour backgrounds with different isotopic composition (Fig. +7). We used VE-water as injection fluid, because its isotopic composition is comparable to the majority +of fluid inclusion of speleothems from mid-latitudes. For each water vapour background 3.0 μl of VE +water were injected five times. For the background waters with the two most extreme isotope +compositions we additionally injected 1.0 µl of VE (n=6) to assess the robustness also for smaller water +amounts. . +If VE water is injected on VE background water vapour, the average isotope value corresponds to +the expected value within uncertainty. A deviation from the expected isotope value is notable for +injections on a different water vapour background. For example, VE injections on a negative water +vapour background (NG, δ18O = -26.54 ± 0.08 ‰, δ2H = -212.1 ± 0.7 ‰, Table 1) yield a deviation of ++0.40 ‰ for δ18O and +2.9 ‰ for δ2H values from the reference values. VE injections on a background, +which is based on lake water (WW) with higher isotope values compared to VE water, yield deviations +of -0.15 ‰ for δ18O and -0.3 ‰ for δ2H values. Tests with injected water amounts of 1 µl corroborate +the observed trend (Fig.7). The standard deviation of repeated water injections is independent from +the isotopic composition of the water vapour background. The effect of the isotopic difference +between the samples and the background water vapour exceeds the measurement uncertainty only +for differences larger than 10 ‰ (δ18O). ). This experiment highlights that it is not necessary to correct +samples when using background water with an isotope composition close to the paleoclimate samples. +For speleothem measurements we used VE water as background water. + +Figure 7: Deviation of the measured injection isotope value relative to the expected value. The deviation is related +to the difference between the isotope signal of the injection and that of the water vapour background. Single +injections are shown as open circles and the mean values as filled circles s. The green andblue lines indicate the +linear regression with all individual 3 µl measurements. An increasing deviation between the measured and +expected isotopic signal is observed for an increasing difference between injection isotope value and that of the + +0.8 +oxygen +hydrogen +D +3 μl +1 μl +0.6 +3 μl +1 μl +(0%) +singleinjections +O +singleinjections +-expected) ( +meanvaluewithuncertainty +expected)( +3 +mean value with uncertainty +0.4 +linearregression +Viation(measured +0.2 +1 +0.0 +-0.2 +devi +T: +VE +VE. +injections +injections +-0.4 +uo +on wWIAE +VE +cC +CC +NG +AE +ww +VE +NG +-10 +-5 +0 +5 +10 +15 +20 +-50 +50 +100 +150 +200 +deviation (injection-background signal)(%o) +deviation(injectionbackgroundsignal)(%o)water vapour background. The uncertainty of the expected value (black horizontal line) is shown as grey envelope. +Measurement uncertainties are given on the 1σ level. [black/white for figures in print, colour online] + +3.5 Case applications +Case example 1: Modern – late Holocene sinter samples +The two fluid inclusion replicates of each modern or late Holocene sample from +Hüttenbläserschacht Cave reproduce very well and are within uncertainty of each other (Table 3). +Related standard deviations of the mean (0.2 and 1.6 ‰ for δ18O and δ2H values, respectively) are +comparable (δ18O values) or slightly larger (δ2H values) than the measurement precision in this water +amount range (0.5-3.0 µl, Fig. 5). The mean fluid inclusion δ18O value of -7.6 ± 0.2 ‰ is identical to the +calculated drip-water value of Kluge et al. (2013) of -7.6 ± 0.3 ‰, independently confirming the former +finding. Drip water in Hüttenbläserschacht Cave was not monitored but should be close to the +neighbouring Bunker Cave and shares the same karst aquifer with comparable residence times of a +few years (e.g., Kluge et al., 2010). Fluid inclusion isotope values are close to the mean drip water +values from Bunker Cave of -7.9 ± 0.2 ‰ for δ18O and -53.3 ± 1.6 ‰ for δ2H values (Riechelmann et al., +2017). +Table 3: Measurement of fluid inclusions in three CaCO3 spar samples from Hüttenbläserschacht Cave +(Germany). Each sample was split in two to allow for a replication test. ‘Avg.’ refers to the average of the two +analyses. For comparison also the calculated pool water δ18O value of Kluge et al. (2013) is shown that uses an +independent temperature estimate and clumped isotope ∆47 for correction of kinetic isotope effects. The Bunker +Cave drip water is taken from Riechelmann et al. (2017). Uncertainties are given on the 1σ level. + +ID +Sample weight +(g) +Water +amount +(µl) +Water +content +(µl/g) +δ18O value + (‰ VSMOW) +δ2H value + (‰ VSMOW) +Pond A +A-1 +0.52 +0.24 +0.46 +-7.5 ± 0.5 +-53.2 ± 1.5 + +A-2 +0.52 +0.31 +0.60 +-7.9 ± 0.5 +-51.9 ± 1.5 + +Avg. +- +- +- +-7.7 +-52.5 +Pond B +B-1 +0.57 +0.43 +0.76 +-7.6 ± 0.5 +-49.7 ± 1.5 + +B-2 +0.65 +0.42 +0.64 +-7.8 ± 0.5 +-48.6 ± 1.5 + +Avg. +- +- +- +-7.7 +-49.1 +Pond C (little +pond) +C-1 +0.59 +1.78 +3.02 +-7.3 ± 0.3 +-51.1 ± 1.0 + +C-2 +0.45 +0.48 +1.08 +-7.7 ± 0.5 +-51.0 ± 1.5 + +Avg. + + + +-7.5 +-51.0 +Average all + + + + +-7.6 ± 0.2 +-50.9 ± 1.6 + +Reconstructed +after Kluge et al. +(2013) + + + + +-7.6 ± 0.3 + +Drip +water +Bunker Cave + + + + + + +range +2006- +2013 + + + + +-8.5 to -7.0 +-48 to -58 +mean +2006- +2013 + + + + +-7.9 ± 0.2 +-53.3 ± 1.6 + +Case example 2: Speleothem sample from the 20th century – Stam 4 from Cloşani Cave +Comparison with current drip water and reproducibility assessment +We sampled calcite pieces at the outer surface of the stalagmite for comparison with current drip +water. It can be assumed that recent calcite precipitated there and accordingly, recent drip water is +enclosed in the fluid inclusions. The water yields during crushing were between 0.49 and 1.38 µl/g with +a mean of 0.93 ± 0.28 µl/g (one sample was excluded due to a low water amount of 0.18 µl) +(Supplementary Table S3). The mean value of 13 fluid inclusion measurements of samples from the +outer stalagmite layer is δ18O = −9.5 ± 0.5 ‰ and δ2H = −64.6 ± 1.2 ‰ (Supplementary Table S3). These +values agree within uncertainty with the mean of the related drip site CL3 of δ18O = −9.6 ± 0.2 ‰ and +δ2H = −66.3 ± 1.7 ‰ (Fig. 8). The 13 individual measurements reproduce with a standard deviation of +0.5 ‰ and 1.2 ‰ for δ18O and δ2H values, respectively, which is slightly higher than the analytical +uncertainty based on the standard deviation of repeated syringe injection for water amounts between +0.5-and 1.7 µl (0.2-0.4 ‰ for δ18O values, 0.4-1.1 ‰ for δ2H values, Fig. 5). The standard deviation for +the 13 individual speleothem analyses is also comparable to that of other CRDS systems and similar +water amount ranges, such as of Arienzo et al. (2013) with ± 0.5/2.0 ‰ for δ18O/δ2H values and Affolter +et al. (2014) with ± 0.5/1.5 ‰ for δ18O/δ2H values . The precision of the Stam4 sample analysis also +compares well with traditional IRMS measurement techniques which achieve a precision of ± 0.5 ‰ +for δ18O and ± 2.0 ‰ for δ2H values for water amounts > 0.2 µL (Dublyansky and Spötl, 2009). + + + +Figure 8: Fluid inclusion water isotope ratios of samples from the outer stalagmite surface (light blue dots), with +corresponding mean value (dark blue). Drip water data from the same cave chamber where Stam 4 was removed +(light green triangles; drip site CL3) and its mean value (dark green) agree with the fluid inclusion results. Both drip +water and fluid inclusion data match the local meteoric water line (LMWL) of Cluj-Napoca of δ2H = 8.03 · δ18O + +11.29 ‰ (Cozma et al., 2017). The uncertainties are given on the 1σ level. [black/white for figures in print, colour +online] + +Fluid inclusion analysis of samples along the growth axis +We used the stalagmite pieces closest to the growth axis of Stam 4 for paleo-drip water and - +temperature reconstruction (Fig. S2). Where possible, the reproducibility of the individual +measurements was tested with a second set of fluid inclusion samples, extracted adjacent to the first +set of samples (Table 4). The second set had a larger distance from the growth axis than the first set. +The samples corresponding to the same growth period are grouped in levels, indicated by letters A-K +(Fig. 9). For sample level D, only the second sample is used because the first sample is close to the +applied water amount limit and contains only 0.18 μl. On average, the δ2H values of sample and +replicate are largely consistent (mean deviation: 0.1 ± 0.8 ‰). The same is observed for the inclusion +water δ18O value (mean deviation: 0.31 ± 0.51 ‰). In addition the water content of the different levels +appears characteristic. For level D and E with 5 replicates each, the water content varies only 0.1 µl/g +(excluding one sample each with low total water amount). For the other levels, a higher scatter has + +LMWL +FI-Stam4 +-60 +FImeanwithstdy +dripwater (CL3) +dripwatermeanwithstdv +-62- +8’H [% VSMOW] +-64 +-66 +-68 +-70- +-11.0 +-10.5 +-10.0 +-9.5 +-9.0 +-8.5 +8180 [%。 VSMOW]been observed, potentially due to a general heterogeneity of the speleothem inclusion distribution +(e.g., Muñoz-García et al., 2012). Generally, the water content was between 0.45 and 1.66 µl/g, +suggesting minimal or negligible influence of adsorption on the freshly crushed surface (Table 4). Fluid +inclusion δ18O values vary between -10.4 ‰ and -8.0 ‰ and, with one exception (level C), follow a +temporal trend towards higher values towards more recent times (Fig. 9, Table 4). + +Table 4: Measurement results of fluid inclusion samples of stalagmite Stam-4 from Cloşani +Cave (Romania). Several samples were cut from individual layers that reflect contemporaneously +grown carbonate and allow for replication tests. The fractionation factor 18α(CaCO3-H2O) between +water and CaCO3 was calculated based on the difference between the calcite δ18O values (averaged +over the edge length of the fluid inclusion sample of typically 5 mm) and the fluid inclusion water δ18O +values. The temperature T18O,cc was determined using the 18α(CaCO3-H2O) - T relationship proposed by +Kim and O’Neil (1997). TH is related to the relative temperature change calculated using the δ2H- +temperature relationship in rainfall (4.72‰/°C) and was referenced to top level K and the current cave +temperature. T18O, Fi refers to the temperature difference relative to sample level K with the modern +cave temperature as reference and was calculated using the δ18O-T relationship in rainfall (0.59‰/°C). +Samples in grey are not included in the interpretation and discussion as the water amount was 0.19 µl +or below. Samples closest to the growth axis (‘1’ closest, higher numbers are further away) were used +for temperature assessment based on the classical carbonate thermometer. Samples A1 and A2 were +the oldest samples and were excluded from the discussion as they belong to the stalagmite base with +unclear chronology. The age corresponds to the mean age of each sample level. Dft: distance from top. +ID +Dft +(mm) +Age +(year AD) +Sample +weight +(g) +Water +amount +(µl) +Water +content +(µl/g) +δ2H value + (‰ +VSMOW) +δ18O value +(‰ +VSMOW) +18α +(CaCO3- +H2O) (‰) +T18O,cc +(°C) +T18O,Fi (°C) +TH (°C) +A1 + +unknown +0.58 +0.30 +0.52 +-65.4 ± 1.5 +-9.5 ± 0.5 + + + + +A2 + + +0.49 +0.29 +0.59 +-59.7 ± 1.5 +-9.8 ± 0.5 + + + + +B1 +48.2 +1928 +0.42 +0.40 +0.95 +-64.1 ± 1.5 +-9.6 ± 0.5 +31.8 ± 0.5 +7.7 +± +2.2 +9.4 ± 0.2 +10.4 ± 0.5 +B2 + + +0.49 +0.37 +0.75 +-64.7 ± 1.5 +-9.6 ± 0.5 + + + + +B3 + + +0.53 +0.40 +0.76 +-64.8 ± 1.5 +-8.9 ± 0.5 + + + + +B4 + + +0.52 +0.29 +0.56 +-66.3 ± 1.5 +-9.1 ± 0.5 + + + + +B5 + + +0.42 +0.19 +0.45 +-68.5 ± 1.5 +-9.4 ± 0.5 + + + + +C1 +43.9 +1937 +0.49 +0.81 +1.66 +-57.6 ± 1.0 +-8.0 ± 0.3 +30.7 ± 0.5 +12.7 +± +2.3 +12.1 ± 0.1 +11.8 ± 0.4 +C2 + + +0.42 +0.51 +1.21 +-59.6 ± 1.0 +-8.5 ± 0.3 + + + + +C3 + + +0.54 +0.44 +0.81 +-60.4 ± 1.5 +-9.0 ± 0.5 + + + + +D1 + + +0.32 +0.18 +0.57 +-63.8 ± 1.5 +-8.5 ± 0.5 + + + + +D2 +39.0 +1944 +0.51 +0.42 +0.83 +-63.8 ± 1.5 +-10.0 ± 0.5 +32.6 ± 0.5 +4.3 +± +2.1 +8.7 ± 0.2 +10.5 ± 0.5 +D3 + + +0.56 +0.50 +0.89 +-63.7 ± 1.5 +-10.4 ± 0.5 + + + + + +D4 + + +0.55 +0.54 +0.98 +-64.0 ± 1.5 +-10.1 ± 0.5 + + + + +D5 + + +0.40 +0.35 +0.88 +-61.7 ± 1.5 +-9.0 ± 0.5 + + + + +E1 +34.5 +1952 +0.49 +0.38 +0.78 +-62.9 ± 1.5 +-10.3 ± 0.5 +32.7 ± 0.5 +3.5 +± +2.1 +8.2 ± 0.2 +10.7 ± 0.5 +E2 + + +0.58 +0.46 +0.78 +-62.5 ± 1.5 +-9.4 ± 0.5 + + + + +E3 + + +0.54 +0.44 +0.82 +-62.8 ± 1.5 +-10.4 ± 0.5 + + + + +E4 + + +0.53 +0.32 +0.60 +-63.1 ± 1.5 +-10.0 ± 0.5 + + + + +E5 + + +0.25 +0.14 +0.55 +-59.4 ± 1.5 +-9.0 ± 0.5 + + + + +F1 +30.1 +1960 +0.47 +0.35 +0.75 +-63.5 ± 1.5 +-9.6 ± 0.5 +31.9 ± 0.5 +7.0 +± +2.2 +9.4 ± 0.2 +10.5 ± 0.5 +F2 + + +0.58 +0.82 +1.4 +-63.2 ± 1.0 +-9.4 ± 0.3 + + + + +F3 + + +0.56 +0.87 +1.56 +-59.8 ± 1.0 +-8.3 ± 0.3 + + + + +G1 +26.2 +1968 +0.46 +0.40 +0.87 +-62.0 ± 1.5 +-9.0 ± 0.5 +31.3 ± 0.5 +9.7 +± +2.2 +10.4 ± 0.2 +10.9 ± 0.5 +G2 + + +0.50 +0.76 +1.53 +-61.8 ± 1.0 +-8.9 ± 0.3 + + + + +H1 +22.1 +1977 +0.40 +0.46 +1.16 +-61.6 ± 1.0 +-9.1 ± 0.3 +31.9 ± 0.5 +7.2 +± +2.2 +10.2 ± 0.1 +10.9 ± 0.4 +H2 + + +0.35 +0.46 +1.30 +-61.0 ± 1.0 +-8.8 ± 0.3 + + + + +I +17.8 +1990 +0.39 +0.28 +0.72 +-60.3 ± 1.5 +-9.3 ± 0.5 +32.0 ± 0.5 +6.8 +± +2.2 +9.9 ± 0.2 +11.2 ± 0.5 +J +10.9 +2004 +0.50 +0.43 +0.86 +-59.2 ± 1.5 +-8.7 ± 0.5 +31.3 ± 0.5 +9.9 +± +2.2 +10.9 ± 0.2 +11.4 ± 0.5 +K +3.9 +2008 +0.46 +0.43 +0.94 +-59.4 ± 1.5 +-8.4 ± 0.5 +30.7 ± 0.5 +12.3 +± +2.3 +11.4 ± 0.2 +11.4 ± 0.5 + +4. Discussion +4.1 Constraints for precise and accurate fluid inclusion isotope data +The presented setup allows for a good reproducibility with respect to isotope measurements +of pure water samples in the µl range, either injected via a syringe or by crushing of water-filled glass +capillaries in the copper tube (similar to speleothems samples; section 3.3). The achievable precision +is 0.4-0.5 ‰ for δ18O and 1.1-1.9 ‰ for δ2H analyses at extracted water amounts between 0.1 µl and +0.5 µl and decreases with increasing water amount to ± 0.1-0.3 ‰ for δ18O and ± 0.2-0.7 ‰ for δ2H +measurements at extracted water amounts >1 µl (Fig. 5). The improved precision with increasing water +amount is consistent with the observations of Dassié et al. (2018) who reported similar precision of +0.2-0.3 ‰ for δ18O and 0.6-2.6 ‰ for δ2H values for 0.2-1 µl as well as a strong increase of the +uncertainty at water amounts lower than 0.1 µL. Replicate analyses of calcite samples from the +outermost surface of a Romanian stalagmite corroborate the precision as determined by crushing of +water-filled glass capillaries and water injections. +Adsorption of water on freshly crushed surfaces appears negligible for water contents of about +1 µl water per g calcite or above Dennis et al. (2001) similarly observed a decreasing adsorption +influence at increasing H2O/CaCO3 ratios at room temperature. However, an adsorption effect could + +be relevant if the water content in the crushed samples approaches 0.1 µl/g or is below this value. We +therefore recommend to use the water content as one parameter to check the robustness of the +analysis and to carefully assess or conservatively reject samples with water contents below 0.1 µl/g. +We observed a small dependence of the measured isotope value on the water vapour +background (Fig. 7).. After injection of a certain water amount, the δ18O value of the (hypothetically) +well mixed water vapour consisting of background and injection water is an amount-weighted mixture +of both δ18O values. For background water with relatively depleted values such as North Greenland +Water (NG, -δ18O =-26.5 ‰ and δ2H = -212.1 ‰) this would mean that the δ18O value of the VE water +with δ18O = -8.57 ‰ and δ2H = -61.0 ‰ is higher than the mixed water. For example, if the background +to injection volume is 1.8:3.0, the isotopic composition of the mixture is expected to be δ18O = -15.3 +‰ and δ2H = -117.7 ‰.Given the short residence time of the water vapour in the mixing cavity before +the measurement in the CRDS, a full isotopic mixing is not reached. The kinetically slower molecules +containing an 18O atom remain preferentially in the gas stream compared to the faster molecules +containing only 16O atoms that preferentially take part in the mixing with the background water. Thus, +for this case example it is expected that the injection water isotopes are slightly higher relative to the +background and the mixed signal. Conversely, for a positive background as the WW water, the isotope +value of the VE injection is more negative relative to the isotope value of the hypothetical fully mixed +gas stream. Due to the kinetic behaviour of 18O, the injection stays more negative relative to the +expected value for this background. The adsorption effects and the influence of kinetic isotope +exchange are similar for the 1 µl and 3 µl injections (Fig. 7). + For water amounts in the µl range this dependence on the vapour background isotope value +is relevant if the isotopic composition of the fluid inclusions is significantly different from the +background (> 10 ‰ for δ18O and > 50 ‰ for δ2H). Otherwise, the potential effect of the isotopic +difference to the background water vapour is within the analytical uncertainty of water samples +between 0.1 and 1.0 µl. The maximum expected deviations are < 0.25 ‰ for δ18O and < 1.0 ‰ for δ2H +values, if the sample is within the 10 ‰ range of the water vapour background for δ18O and 50 ‰ for +δ2H values. For water amounts larger than 1 µl the acceptable deviation between sample and +background water isotope values reduces in relation to the higher measurement precision at higher +water amounts (Fig. 5). + +4.2 Paleotemperature calculation from Stam 4 using fluid inclusion isotopes +For calculation of the CaCO3-H2O isotope fractionation, we averaged the calcite δ18O values +which correspond to the growth period of the spatially larger fluid inclusion sample (Fig. S4). The +calculated fractionation factor 18α(CaCO3-H2O) between calcite and fluid inclusion water yields values +between 30.7 and 32.9 ‰. This range would correspond to temperatures between 3.5 ± 1.5 °C and + +12.5 ± 1.5 °C using the 18α(CaCO3-H2O)-T relationship of Kim and O’Neil (1997) (Table 4). The calculated +absolute temperatures deviate slightly from these values depending on the used 18α(CaCO3-H2O)-T +relationship (e.g., Démeny et al., 2010; Tremaine et al., 2011). However, relative differences between +the coldest and warmest periods and the trend in the data set is largely independent of the selected +fractionation-temperature relationship as most experimental and empirical studies yield similar +18α(CaCO3-H2O)-T slopes. Following an apparent change of 2.2 ‰ in 18α(CaCO3-H2O) a temperature +change of about 9°C would formally correspond to the growth period of the stalagmite. This +temperature difference is much larger compared to that observed at local meteorological stations with +maximum and minimum mean annual air temperature differing by approximately 3°C. This discrepancy +suggests that the temperature trend related to 18α(CaCO3-H2O) in the stalagmite has been enhanced, +e.g., by stronger isotopic disequilibrium. As the measured fluid inclusion water isotopes correspond to +the meteoric water line (Fig. S5), post-depositional and other significantly altering effects are unlikely +for the water-filled inclusions. However, mineral formation in speleothems often takes place in a non- +equilibrium regime (Deininger et al., 2021) and may also have influenced the calcite δ18O values of +Stam 4 due to a high growth rate and strong seasonal variations in prior calcite precipitation (PCP, +Warken et al., 2018).. We refrain from correcting the disequilibrium effect in calcite δ18O values due +to the related large and hardly quantifiable uncertainties and only focus on the fluid inclusion δ2H +values in the following. Note, that it may be possible in other cases to derive temperature variations +from the oxygen isotope fractionation between fluid and calcite if the degree of PCP is negligible or +constant and the length of drip interval has not changed significantly during growth. +Affolter et al. (2019) demonstrated that δ2H values and its temperature relationship in +rainwater of mid-latitudes can be used to deduce temperature changes throughout the Holocene. In +stalagmite Stam 4, a long-term trend towards higher δ2H values is observed from the oldest to +youngest fluid inclusion samples (Fig. 9). A significant increase for δ2H values of +4.8 ± 2.1 ‰ was +identified between sample level F and K and similarly between B and C (Fig. 9 C). This transfers into to +a temperature change of +1.0 ± 0.4 °C using the relationship between the isotopic composition of +precipitation and temperature for Central Europe of +0.59 ± 0.04 ‰/°C for oxygen and +4.72 ± +0.32‰/°C for hydrogen isotopes (Rozanski et al., 1992). Since stalagmite Stam 4 from Cloşani Cave +grew under continental climatic influence, the mean value for Central Europe seems to be the best +reference for the determination of the relative temperature change with the δ2H/T relationship. GNIP +(Global Network of Isotopes in Precipitation) stations and other weather stations in Hungary, Austria, +Slovakia and Poland with more than 10 years of isotope analysis show similar slopes of +3.9-5.4 ‰/°C +(Demény et al., 2021). Considering the observed range of the rainfall δ2H-temperature slopes in Central +and Eastern Europe by Gaussian error propagation, the uncertainty increases slightly to 0.5°C. + + With the confirmed recent growth of the stalagmite, the topmost stalagmite piece is assigned +to the year 2010 C.E.( year of stalagmite removal). Annual growth layers provide a possibility to assign +ages to all other sample depths (Supplementary Fig. S2). Temperature changes ΔT relative to the +reference level B is close to zero up to ca. 1960 C.E. (3 cm distance from top, level F), followed by an +increase of 1.0 ± 0.4 °C at the stalagmite top (Fig. 9F). The mean annual air temperature for the time +period from 1928 to 2008 C.E. at the meteorological station Drobeta/Turnu Severin, which is located +in the vicinity of the cave, shows a similar temperature increase of about 1 °C from 1980 until 2008 +C.E. (Fig. 9G). This is consistent with the general trend in Romania, which experienced a 0.8 °C increase +for the period of 1901-2012 C.E. (Ministry of Environment and Climate Change, 2013). The +temperature change determined from δ2H values in the fluid inclusions corresponds well to the trend +and magnitude measured in the mean annual air temperature of the region (Fig. 9). Directly +interpreting the fluid inclusion δ18O values using the rainfall δ18O-T relationship for Central Europe of ++0.59 ± 0.04 ‰/°C by Rozanski et al. (1992) also leads to a temperature increase, albeit with a higher +amplitude of 2.0 ± 1.1°C relative to level B, but within uncertainty consistent with the temperature +reconstruction using δ2H values (Table 4). + + + +growthaxis[cm] +5 +A +B +C +D +E +Base +level +A +B +c +D +E +F +G +H +K +a (% VSMOW) +T +-8 +B +II +-9 +-10 +-11°H (% VSMOW) +-60 +65 +(0%) +33 +D +32 +30 +(82H) (%) +E +reference +0 +TevelB +2 +reference13 +(C) +MAAT +11 +10 +1920 +1940 +1960 +1980 +2000 +yearFigure 9: A) Stam 4 with assignment of sample pieces based on visual correlation with the growth axis. Layers B to +K were used for temperature reconstruction. The small inset shows alternation of fluid inclusion-rich and inclusion- +poor layers. Winter layers yield very little inclusions, while summer layers include abundant air- and water-filled +inclusions. Width of the image is ca. 3 mm. B) Fluid inclusion δ18O values C) δ2H values D) Fractionation factor α +between calcite and inclusion water. E) Change in δ2H values relative to level B (lowest temperature). F) Inferred +temperature change relative to level B. A trend is visible for Δ(δ2H) as well as for ΔT from the stalagmite bottom +to the top. Using the δ2H/T relationship of 4.72 ± 0.32 ‰/T (Rozanski et al., 1992) a total increase of ΔT =1.0 ± 0.5 +°C is observed within the growth period of the investigated stalagmite. G) Mean annual air temperature (MAAT) +of the Drobeta/Turnu Severin station in the cave region (thin black line, Klein Tank et al., 2002) for the last 100 +years with a 10-year running mean (red line) . The 10 year-smoothing interval corresponds to the average age that +is covered by the fluid inclusion samples. The uncertainties are given on the 1σ level. [black/white for figures in +print, colour online] + +4.3 Paleotemperature reconstruction using fluid inclusions +Our study supports the conclusion of previous publications (e.g., Affolter et al., 2014; Uemura +et al., 2016; de Graaf et al., 2020) that an accurate and precise determination of the isotope +composition of micro-litre water amounts is possible. Our setup is able to produce small errors, which +are in the same range as the precision in the previous fluid inclusion isotope studies(Dublyansky and +Spötl,2009; Arienzo et al., 2013); Affolter et al., 2014),; Uemura et al. 2016; Dassié et al.,2018). In these +studies a precision of 0.3-0.5 ‰ for δ18O and 0.7-1.9 ‰ for δ2H values in the water amount range of +0.1-1.0 µl, and 0.1-0.3 ‰ for δ18O and 0.2-0.7 ‰ for δ2H values at water amounts > 1 µl was +demonstrated. The analytical precision determines the currently achievable temperature precision. +In principle, three possible ways of temperature calculation from fluid inclusion isotopes exist: +a) from the temperature-dependent oxygen isotope fractionation between calcite and fluid inclusion +water (e.g., Arienzo et al., 2015; Labuhn et al., 2015), b) indirectly via transfer of the fluid inclusion δ2H +value to the corresponding water δ18O value using the δ18O-δ2H relationship of the meteoric water line +and then using the oxygen isotope fractionation between carbonate and water for temperature +calculation (Zhang et al., 2008; Meckler et al., 2015), and c) from the hydrogen isotopes using a locally +valid δ2H-temperature relationship of the rainfall (e.g., Affolter et al., 2019). Of the three methods for +temperature reconstruction the first two (a and b) show the highest uncertainty of 0.6-3.1 °C (Van +Breukelen et al., 2008; Zhang et al., 2008; Arienzo et al., 2015; Meckler et al., 2015; Uemura et al., +2016). The highest achievable temperature precision in the case of the best analytical fluid inclusion +δ18O precision of 0.1-0.2 ‰ (and a calcite δ18O uncertainty <0.1 ‰) is 0.6-1.1 °C. Approaches a and b +are additionally affected by the potential influence of disequilibrium isotope fractionation during +carbonate mineral formation (e.g., Deininger et al., 2021), causing too high temperatures or an +unrealistically large temperature spread in case of significant changes of isotopic disequilibrium. + +Furthermore, diagenetic exchange between host calcite and fluid inclusion water could further alter +the water δ18O value (Demeny et al., 2016; Uemura et al., 2020). The precision of the temperature +reconstruction directly from fluid inclusion δ2H values depends critically on the value of the rainfall +δ2H/T relationship and the availability of well-defined rainfall δ2H/T functions at the study site. For the +Central European region a value of 4.72 ± 0.32 ‰/°C of Rozanski et al. (1992) can be used and can yield +a temperature precision of 0.2 °C for released water amounts of ~0.5 µl if the analytical precision is +~1.0 ‰ for δ2H measurements. The uncertainty of the rainfall δ2H/T function is negligible for our case +study but could be relevant in case of a reduced temperature dependence of the rainfall δ2H values. +At locations with a stronger temperature dependence of the rainfall δ2H value an even better precision +is possible, e.g., ± 0.13 °C for the average of Swiss stations, which show a slope of 7.44 ‰/°C (Rozanski +et al., 1992) and for the typical analytical uncertainty of our setup. + The temperature resolution of this method is slightly reduced at lower latitudes (e.g., ± 0.55 +°C at Hong Kong with a δ2H rainfall-temperature relationship of 2 ‰/°C; Rozanski et al., 1992). Note +that temperature estimates using fluid inclusion δ2H values and the rainfall δ2H/T relationship without +climatic reference points are relative, i.e., they record only temperature changes. With an anchor, e.g., +modern reference temperature and rainfall δ2H values, absolute temperatures can be also inferred +from fluid inclusion δ2H values. The application of the rainfall δ2H/T relationship for calculating +temperature changes from fluid inclusion δ2H values also requires the δ2H/T relationship to be +constrained for the past. Information on the rainfall isotope systematic and the δ2H/T relationship in +the past can be gained for example from groundwater studies (Darling, 2004) in combination with +noble gas temperatures (e.g., Kreuzer et al., 2009; Varsány et al., 2011, Túri et al., 2020). The +uncertainty of the δ2H/T relationship needs to be considered and likely decreases the achievable +precision for pre-Holocene speleothems as the uncertainty for the δ2H/T relationship increases when +applying the modern or Holocene relationship back in time. +Affolter et al. (2019) used the δ2H/T relationship for temperature reconstruction from fluid +inclusions throughout the Holocene and achieved a precision of 0.2-0.5 °C for a Swiss stalagmite. Our +analytical approach allows for the same temperature resolution and with measurements of stalagmite +Stam 4 from Romania confidently verified the recent 20th century warming. Both studies together +illustrate the potential of the inclusion-based methodology for tracing and reconstructing minor +temperature fluctuations of < 2 °C during the Holocene and, at sufficient temporal resolution (requiring +high stalagmite growth rates), also of sub-degree changes such as the recent anthropogenic warming +trend. + +5. Summary and conclusion + +Fluid inclusion isotope analysis using CRDS measurements after mechanical sample crushing +benefits from fluid extraction and measurement under a constant and controlled water vapour +background. The specific isotope and water volume calibration of the CRDS system remained valid for +several years. For assessing the fluid inclusion extraction and measurement performance we used +syringe injections and boro-silicate glass capillaries filled with reference water. We have shown that +out setup has no drift in the isotope values for smaller water amounts and that the memory effect for +this system is negligible when using an isotopically appropriate background water vapour. The water +vapour background should be chosen such that the isotope values of sample and background do not +deviate significantly (maximum 10 ‰ for δ18O and 50 ‰ for δ2H values). +Direct comparison of calcite powder-free and -filled extraction tubes proved that the adsorption of +water on the speleothem surface has no effect on the measured isotope signal if the water content is +larger than 1 µl water per g calcite. For samples with a water content below 0.1 µl/g calcite results +have to be checked as we observed a corresponding adsorption of the water vapour background on +freshly crushed calcite. Related to the above-mentioned constraints, the precision (1σ) of isotope +measurements for aliquots of water from speleothem fluid inclusions improves with increasing water +amount. It is 0.4-0.5 ‰ for δ18O and 1.1-1.9 ‰ for δ2H values for water samples between0.1 and0.5 +µl, which is comparable to other CRDS systems and IRMS techniques. This value was further confirmed +by replicated measurements of adjacent samples of the Romanian stalagmite Stam 4 (standard +deviation of 0.5 and 1.2 ‰ for δ18O andδ2H values). For water amounts larger than1 µl the precision +improves to 0.1-0.3 ‰ for δ18O and 0.2-0.7 ‰ for δ2H. +Analysis of fluid inclusions of recent pool spars from a German cave shows good agreement +between drip water and fluid inclusion isotope values. Similarly, the δ18O and δ2H values of a Romanian +stalagmite, grown during the 20th century, reflect the isotopic composition of the modern drip water +within uncertainty. In the same case study, we observed a T-trend from δ18O values, which is +inconsistent with local weather records, suggesting a major influence disequilibrium and kinetic effects +on the speleothem calcite δ18O signal of Stam 4. The isotopic disequilibrium causes a significant +overestimation of the temperature changes calculated from the oxygen isotope fractionation between +calcite and water (in our case 9 °C difference instead of ca. 1°C). In contrast, hydrogen isotopes are not +involved in calcite precipitation and therefore provide a relatively undisturbed link to the stable +isotopic composition of drip and rain water. Using the δ2H-temperature relationship in rainfall we +obtained a temperature increase for Cloşani Cave of +1.0 ± 0.5 °C between 1960 and 2010, which is in +excellent agreement with the local temperature record. Thus, applying the local rainfall δ2H-- +temperature relation on fluid inclusion δ2H variations appears to be a reliable method to determine +mean annual air temperatures for mid-latitude speleothems. The achieved precision furthermore +highlights the potential of fluid inclusion isotope studies in speleothems for high resolution + +paleoclimate reconstruction, given that the rainfall isotope relationship is significantly linked to +temperature and is available for the studied area and valid for past periods. + +Acknowledgements + +The project was funded by DFG Grant KL 2391/2-1 and supported by the Heidelberg +Graduate School for Physics in the context of grant GSC 129. We thank Sylvia Riechelmann and +Jasper Wassenburg for collection of Stam 4, Silviu Constantin and Mihai Terente for +monitoring, Christoph Spötl for drip water analysis at CL3, Regina Mertz-Kraus for LA-ICP-MS +element analysis and Sven Brömme for calcite δ18O and δ13C analysis on Stam 4. We thank the +editor Michael E. Böttcher and three anonymous reviewers for their very detailed comments +and suggestions that helped to improve the manuscript. +Data availability + +Data of this study are summarized in Tables 1-4 and Supplementary Table S1-S3. Raw +data related to Figs. 3-6 are given in the Appendix of Weißbach (2020), available at +https://doi.org/10.11588/heidok.00028559 +Competing interests statement + +The authors declare the absence of competing interests. + +References +Aemisegger, F., Sturm, P., Graf, P., Sodemann, H., Pfahl, S., Knohl, A., Wernli, H., 2012. 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Ac., 72, +1014-1026. + +Supplementary Material +for +Constraints for precise and accurate fluid inclusion stable isotope +analysis using water-vapour saturated CRDS techniques +by +Therese Weissbach, Tobias Kluge, Stéphane Affolter, Markus C. Leuenberger, Hubert Vonhof, Dana +F.C. Riechelmann, Jens Fohlmeister, Marie-Christin Juhl, Benedikt Hemmer, Yao Wu, Sophie Warken, +Martina Schmidt, Norbert Frank, Werner Aeschbach + +S1) Hüttenbläserschacht Cave – sample dating +Table S1: Pieces of speleothem samples (pool spars and rafts) collected at Hüttenbläserschacht Cave +were dated using the Th-U disequilibrium method at the Heidelberg Academy of Sciences. The +analytical procedure followed the methods described in Fohlmeister et al. (2012). + +Sample +232Th +[ppb] +238U +[ppb] +230Th +[fg/g] +± +(234U/238U) +± +(230Th/238U) +± +Age +Age + + + + + + + + + +Corr. +uncor. + + + + + + + + + + + +Hinterm +Ballsaal +13.0 382.1 +58.8 6.7 +1.1407 0.0030 +0.0005 0.0011 +0.04±0.26 +0.90±0.08 +Kristall- +häutchen +3.1 321.9 +34.3 2.5 +1.2159 0.0053 +0.0040 0.0005 0.3557±0.13 0.5862±0.04 + +S2) Closani Cave and Stam 4 annual layer counting +Cloşani Cave is located on the southern slope of the Carpathians at an altitude of 433 m above sea +level and developed in massive limestones of Upper Jurassic-Aptian age (Constantin and Lauritzen, +1999). The cave is overlain by about 30 m of rock overburden. A monitoring programme showed +microclimatic stability for the cave interior with a mean air temperature of 11.4 ± 0.5 °C and a relative +humidity close to 100% for 2010-2012 and 2015 (Warken et al., 2018). The cave air pCO2 pattern +follows a strong seasonal cycle with high values in late summer (up to 8000 ppmV) and lower values +during winter (2000 ppmV). Water infiltration occurs predominantly during winter time (October - +March) where 75 to 100% of the meteoric precipitation is available for infiltration. Warken et al. (2018) +showed that calcite precipitation is favoured during winter time and reduced in summer, as a result of +seasonally varying CO2 concentrations in the cave air and related equilibrium DIC concentrations. The +water isotopic composition of the drip water in direct vicinity (1 m) of the former location of Stam 4 +shows no seasonal cycle and is constant with a mean value of −9.6 ± 0.2‰ for δ18O and −66.3 ± 1.7‰ +for δ2H. + +The relatively small and fast-grown stalagmite Stam 4 was collected from the “laboratory passage” in +the cave in 2010. It has a total length of 6 cm and an average growth rate of 510 μm per year, as +deduced from counting of elemental layers. Both summer and winter layers are clearly detectable in +the thin sections, whereas winter layers show a compact structure with a lower number of inclusions +and the milky-white porous summer layers contain abundant air- and water-filled inclusions. This +layering in Stam 4 was induced by the strongly changing pCO2 in the cave air, resulting in a seasonal +change in growth rate and corresponding seasonal cycles in Sr and Ba in the stalagmite calcite. Similar +seasonal Sr and Ba pattern have also been observed e.g., by Treble et al. (2003), Mattey et al. (2010), +and Warken et al. (2018). The visible annual layers in stalagmite Stam 4 are not as pronounced as the +annual cycles in the measured high-resolution Ba concentration. Ba concentration was measured with +a LA-ICP-MS (Agilent 7500 ce with Laser UP-213, Institute of Geosciences Mainz) at 4.3 µm resolution. +The minima of this record were counted five times. These five counted layer series were cross-dated +to each other. Layers have to be counted at minimum three times, layers only counted once or twice +were deleted from the time series. For each layer a mean value of layer thickness was calculated from +the five layer thickness series to a master chronology. This layer thickness chronology results in a +growth of Stam 4 from 1910 to 2010, the year of sampling under an active drip site. +S3) Radiocarbon dating +Four samples were drilled with a hand-held dental burr (1 mm). Calcite powder was acidified in vacuum +with HCl. The emerging CO2 was combusted to C with H2 and an iron catalyst at 575°C (Fohlmeister et +al., 2011). Measurements were performed with a MICADAS AMS system (Synal et al., 2007) in the +Klaus-Tschira laboratory Mannheim. The results for the four samples show a typical speleothem +radiocarbon bomb spike (Tab. S1, Fig. S1), constraining recent growth of the speleothem. +Table S2: Radiocarbon measurement results. Radiocarbon results and errors are expressed in fraction +modern (fm). +MAMS lab nr. +depth [mm] +14C [fm] +14C error [fm] +14709 0.5 +1.0416 +0.0029 +14710 18.7 +1.0730 +0.0029 +14711 39 +0.9265 +0.0025 +14712 55 +0.9133 +0.0024 + + + +Fig. S1: Radiocarbon measurements (black) over depth (bottom-axis), plotted to fit the atmospheric +radiocarbon anomaly (blue, top x-axis) in the mid to late 20th century. + + +Year [A.D.] +1900 +1920 +1940 +1960 +1980 +2000 +180 +180 +160. +160 +140- +140 +120 +120 +100 +100 +80 +80 +1900 +1920 +1940 +1960 +1980 +2000 +distancefromtop[mm]Additional figures + +Fig. S2: Age assignment of the fluid inclusion samples +A) Fluid inclusion sample pieces (labelled B to K) are shown on the left half of the stalagmite slab. The +red lines illustrate the assignment of the individual sample blocks to the growth axis. The visible +lamination was used as guideline for correlation. Sample A is related to the base and due to a disturbed +growth structure does not allow to assign any age. Due to the intrinsic uncertainties of this procedure +(for details see Weißbach, 2020) we associated age ranges to the individual fluid inclusion samples B +to K. +B) Age depth model with distance from top (dft) in cm. The chronology was established by layer +counting and additional 14C measurements (see S1 and S2). + + +2025 +B +2010- +1995- +2 +1980- +H +[C.E.] +GFED +1965- +3 +year +1950 +C +1935- +E5 +B +5 +growth axis +1920 +Base +1905- +5 +4 +3 +2 +1 +0 +dft[cm] +Fig. S3: Water vapour adsorption by the artificial fluid inclusion system. Water vapour concentration +during crushing of 0.25 g Iceland spar. The decrease of the water vapour concentration indicates an +adsorption of water molecules on the freshly crushed calcite. Using the water amount calibration, it +corresponds to about 0.023 µl of water adsorption. The reference water vapour background is marked +in orange with interpolated linear fit as dashed line. The small inset shows examples of compact and +inclusion-free pieces of Iceland spar. + + +6920+ +6900 +6880- +1 cm +6860 +water +background +6840 +water +background +6820 +iceland spar +6800- +crush +:35 +:40 +:45 +:50 +:55 +11:00 +:05 +:10 +time [hour] +Fig. S4: from top to bottom: relative temperature change derived from α(CaCO3-H2O) relative to +sample level B (orange dots); fractionation factor α(CaCO3-H2O) (grey squares); calcite δ18O values +(green triangles) corresponding to intervals with an edge length of 0.5 cm of the fluid inclusion sample +pieces, with smoothed higher-resolution data (green line); fluid inclusion δ18O (blue triangles). For a +better overview the depth (dft) errors of α(CaCO3-H2O) and the calculated temperature change are not +shown, but are the same as for the fluid inclusions δ18O. + + +level +B +C +D +E +F +G +K +6 +△(T) [K] +3 +0 +reference levelB +-3 +-6 +33.6 +32.9 +32.2 +31.5 +30.8 +30.1 +-7.5 +-8.0 +8180. +-8.5 +-8 +-9 +-10 +180 +-11 +5 +4 +3 +2 +0 +dft [cm] +Fig. S5: Samples B-K of stalagmite Stam 4 with replicates from the same growth phases (Table 4) +displayed relative to the meteoric water line. The aliquots closest to the growth axis of the stalagmite +are shown as red circles. + + + + + + + + + +-45 +closest to axis +-50 +replicates +8H (% VSMOW) +55 +60 +65 +-70 +-75 +-11 +-10 +-9 +-8 +-7 +9- +-5 +s180 (% VSMOW)Additional Tables +Table S3: Fluid inclusion data from the outermost layer of stalagmite Stam 4. The distance to the +growth axis increases with higher Roman numbers. Arabic numbers indicate replicates with similar +distance from the growth axis. Samples in grey are not included in the interpretation and discussion as +the water amount was below 0.2 µl. + +ID +Sample +weight (g) +Water +(µl) +Water content +(µl/g) +δ2H (‰ +VSMOW) +δ18O +(‰ VSMOW) +I-1 +0.69 +0.30 +0.52 +-65.4 ± 1.5 +-9.5 ± 0.5 +II-1 +0.61 +0.40 +0.95 +-64.1 ± 1.5 +-9.6 ± 0.5 +II-2 +0.30 +0.37 +0.75 +-64.7 ± 1.5 +-9.6 ± 0.5 +II-3 +0.37 +0.40 +0.76 +-64.8 ± 1.5 +-8.9 ± 0.5 +II-4 +0.38 +0.29 +0.56 +-68.5 ± 1.5 +-9.1 ± 0.5 +III-1 +0.38 +0.81 +1.66 +-59.6 ± 1.5 +-8.0 ± 0.5 +III-2 +0.40 +0.51 +1.21 +-60.4 ± 1.5 +-8.5 ± 0.5 +III-3 +0.44 +0.44 +0.81 +-63.8 ± 1.5 +-9.0 ± 0.5 +IV-1 +0.41 +0.18 +0.57 +-63.8 ± 1.5 +-8.5 ± 0.5 +IV-2 +0.47 +0.42 +0.83 +-63.7 ± 1.5 +-10.0 ± 0.5 +V-1 +0.23 +0.38 +0.78 +-62.5 ± 1.5 +-10.3 ± 0.5 +V-2 +0.58 +0.46 +0.78 +-62.8 ± 1.5 +-9.4 ± 0.5 +VI +0.47 +0.35 +0.75 +-63.2 ± 1.5 +-9.6 ± 0.5 +VII +0.46 +0.43 +0.94 +-65.4 ± 1.5 +-8.4 ± 0.5 + + + + + + + +Table S4: Precision of fluid inclusion δ18O and δ2H measurements, interpolated from repeated water +injections and crushing of water-filled glass capillaries. The values refer to an exponential fit to the +standard deviation at various water amounts (Fig.5). The precision at 0.02-0.1 µl are extrapolated using +the exponential fit. + +Water +amount +(µl) +Precision (1σ) +δ18O +(‰) + + δ2H + (‰) +0.02 +0.55 +2.08 +0.05 +0.54 +2.00 +0.08 +0.53 +1.92 +0.1 +0.53 +1.87 +0.2 +0.50 +1.65 +0.3 +0.47 +1.45 +0.4 +0.44 +1.28 +0.5 +0.42 +1.14 +0.6 +0.40 +1.01 +0.7 +0.37 +0.90 +0.8 +0.35 +0.81 +0.9 +0.34 +0.73 +1.0 +0.32 +0.66 +2.0 +0.20 +0.33 +3.0 +0.14 +0.26 +4.0 +0.11 +0.24 + + + + + + + +Additional references: +Fohlmeister, J., Kromer, B., Mangini, A., 2011. The influence of soil organic matter age spectrum on +the reconstruction of atmospheric 14C levels via stalagmites. Radiocarbon 53(1), 99–115. +Fohlmeister, J., Schröder-Ritzrau, A., Scholz, D., Spötl, C., Riechelmann, D. F. C., Mudelsee, M., +Wackerbarth, A., Gerdes, A., Riechelmann, S., Immenhauser, A., Richter, D. K., and Mangini, +A., 2012. Bunker Cave stalagmites: an archive for central European Holocene climate +variability. Clim. Past, 8, 1751–1764, doi:10.5194/cp-8-1751-2012. +Mattey, D. P., Fairchild, I.J., Atkinson, T. C., Latin, J.-P., Ainsworth, M., Durell, R., 2010. Seasonal +microclimate control of calcite fabrics, stable isotopes and trace elements in modern +speleothem from St Michaels Cave, Gibraltar. Geological Society, London, Special +Publications, 336, 323-344. +Synal , H.-A., Stocker, M., Suter, M., 2007. MICADAS: a new compact radiocarbon AMS system. Nucl. +Instr. Meth. Phys. Res. B, 259, 7-13. +Treble, P., Shelley, J.M.G., Chappell, J., 2003. Comparison of high resolution sub-annual records of +trace elements in a modern (1911–1992) speleothem with instrumental climate data from +southwest Australia. Earth Planet. Sci. Lett.216, 141–153. + +