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Combined use of conventional and clumped carbonate stable isotopes to identify hydrothermal isotopic alteration in cave walls

Abstract

Alteration of conventional carbonate stable isotopes (δ18O, δ13C) in cave walls has been shown to be a useful tool to identify cave formation driven by deep-seated processes, i.e., hypogene karstification. If combined with a prior information on the paleowater stable isotope composition, further insights can be obtained on the temperature and the source of the paleowater. Clumped isotope composition (Δ47) of carbonates is an independent measurement of temperature, and if combined with the conventional stable isotopes, can provide information on the paleowater stable isotope composition. On the example of Provalata Cave (N. Macedonia), we apply for the first time, both conventional and clumped stable isotope analysis, and identify two different isotope alteration trends, reflecting two distinct hydrothermal events: an older, hotter one, where isotope alteration was likely related to isotope diffusion, lowering the δ18O values of the carbonate; and a younger one, related to the cave formation by low-temperature CO2-rich thermal waters, with dissolution-reprecipitation as the alteration mechanism, causing decrease in δ18O values, and unexpected increase in δ13C values. The findings are further corroborated by additional insight from optical petrography and cathodoluminescence microscopy, as well as fluid inclusion analysis of secondary calcite crystals related to the cave forming phase.

Introduction

Hydrothermal caves are one of the main genetic type of caves, a sub-type of hypogene caves, whose origin is related to deep-seated processes (source of acidity and recharge to the soluble rock formation is from depth), contrary to the most commonly found caves whose origin is connected to surface-related processes (i.e., epigene caves; source of acidity and recharge is from the surface)1,2,3. Hydrothermal caves are formed by thermal waters that have generally higher CO2 concentrations, and dissolve carbonate bedrock as they cool down along their rising flow path due to inverse relationship of calcite solubility and temperature4. At shallower levels of the aquifer, decrease in pressure facilitates CO2 degassing, leading to supersaturation and precipitation of calcite5. They are generally strongly structure controlled and develop a characteristic suite of cave morphologies and deposits2,6,7.

While some active thermal caves are accessible and offer possibility to study some of the related processes occurring in shallower conditions8,9, most of the known hydrothermal caves are fossil, and became accessible only after being intercepted by surface erosion10. Identification of characteristic morphologies has been the most common approach to identify the hydrothermal origin of caves11, however equifinality of some morphological features (e.g., convectional forms such as cupolas, pockets etc.) makes this approach not resolutive. Study of secondary minerals has been proven to be a more reliable approach, with microthermometry and stable isotope composition used to confirm hydrothermal origin and identify the sources of fluids as well as their formation temperature12,13,14,15,16.

Although secondary minerals might have been deposited from thermal waters, their hydrothermal origin is not a proof of the primary hydrothermal origin of the cave14, unless their relationship to the cave origin can be unambiguously demonstrated4. Likewise, the absence of hydrothermal minerals does not exclude a hydrothermal origin of a cave, as they might not precipitate at all, and later modification in shallower settings can modify and overprint the primary hydrothermal features. Isotope alteration of bedrock, an approach developed for and most commonly used in hydrothermal ore-related studies17,18,19, has also been successfully applied to cave wall bedrock. Recent studies, based on alteration of conventional carbonate stable isotope compositions (expressed as δ18O and δ13C values) in cave walls, have demonstrated a hypogene origin in the presence or absence of related cave minerals15,20,21. Furthermore, it has been found that alteration halos do not develop in epigene caves22 and this approach can be used to confirm or dismiss hypogene-related cave origin23. The most commonly identified alteration in cave walls is lowering of the bedrock δ18O values, as a result of the interaction with low-δ18O high-temperature meteoric waters, as well as lowering of bedrock δ13C values due to interaction with dissolved inorganic carbon (DIC) with lower δ13C values12,14,15,22.

Recent developments in carbonate clumped isotope thermometry24 allow an independent estimate of the mineral formation temperature, that, if combined with the conventional oxygen isotope composition, can be used to reconstruct the paleofluid oxygen composition25. Carbonate clumped isotope thermometry has found wide applications, such as in reconstructing metamorphic and exhumation histories of marble formations26,27, sediment diagenesis28,29, as well as speleothem studies30,31,32. The application of clumped isotope thermometry to subaerially formed speleothems has proven challenging, due to disequilibrium effects related to various controls (e.g., rapid CO2 degassing), leading to overestimation of formation temperatures33,34. However, subaqueous speleothems, especially slowly precipitating ones, were found most likely to have precipitated in equilibrium, thus providing more reliable results31,32,35,36,37.

Here we expand on the cave wall isotope alteration approach22 by applying both clumped and conventional stable isotope analyses on carbonates. On the example of Provalata Cave (N. Macedonia), we show for the first time, that the characteristics of two distinct hydrothermal events causing different cave wall isotope alterations can be identified, the second one responsible for cave formation. We support our findings by additional insight obtained by optical petrography and cathodoluminescence microscopy, as well as fluid inclusion analysis of secondary calcite minerals, and reconstruct the subsequent evolution of the cave.

Study area

Hypogene karst in Mariovo (N. Macedonia) is found at several localities, at the intersection of low topography and major faults, and is developed mainly in calcite and dolomite marble, that are part of NNW-SSE oriented nappes along the eastern part the Pelagonian massif38 (Supplementary Fig. S1). Melnica locality in Mariovo is an output zone of a hypogene karst system, where caves and cave remnants developed in calcite marble, dolomite marble and carbonate breccia, and a small lukewarm Melnica Spring, are found (Supplementary Fig. S2). Provalata Cave is a small fossil cave and the most remarkable example of this system. It was formed in calcite marble in two successive phases, first by CO2-rich thermal waters in a phreatic setting, and then by sulfuric acid in a dominantly vadose setting, both leaving characteristic suites of cave morphologies and associated minerals39. Sulfuric acid speleogenesis (SAS), that was active at ca. 1.6 Ma, left abundant gypsum deposits formed as a replacement of carbonate bedrock and calcite spar39,40. The calcite spar is found as coatings, up to 0.5 m thick, that cap the hydrothermal carbonic phase.

From Provalata Cave we collected two wall-drilled cores41 (C2 and C3) that cover cave wall bedrock and calcite coatings, and two additional samples cut from calcite coatings covering the cave wall (PROV03 and PR20). Furthermore, we collected samples along the NNW-SSE nappes to better characterize the primary isotopic composition of the calcite marble. Details of the sampled material are given in the Supplementary Information.

Results and discussion

Macroscopic observations

The two cores, C2 and C3, have length of ~ 30 and ~ 6 cm, with calcite marble representing ~ 15 and ~ 4 cm, respectively. In addition to primary grey-colored marble, two distinct types of color alteration are visible: a narrow rim (< 2 cm) with intense white discoloration at the contact of the cave wall and the overlying calcite coating, and a dispersed pale grey discoloration in the inner part (Fig. 1, Supplementary Fig. S5). The thickness of the calcite coatings is significantly different (~ 15 cm and ~ 2 cm), with the one at C3 likely largely dissolved in the subsequent SAS phase.

Figure 1
figure 1

Conventional and clumped stable isotope composition of cores C2 (a) and C3 (b) from Provalata Cave. Average values and ranges (± 1SD) for calcite marble in the wider area are given by brown lines and brown bands, respectively.

Conventional stable isotope composition

The grey calcite marble sections in both cores have similar conventional stable isotope composition, with calcite oxygen (δ18Occ) and carbon (δ13Ccc) isotope values that are within the range of values obtained for samples along the NNW-SSE nappes (Figs. 1, 2). This range of values is essentially the same as what was found from a quarry ~ 30 km to the north42, indicating that the grey sections in the cores are isotopically unaltered.

Figure 2
figure 2

Conventional and clumped stable isotope composition of the studied carbonate samples. (a) Conventional stable isotope δ18Occ − δ13Ccc plot. Black lines indicate the two trends of covariation of δ18Occ and δ13Ccc values from Provalata Cave calcite coatings. (b) Clumped isotope composition (Δ47) vs δ18Occ. Equilibrium curves based on the equation of Daëron et al.31 for a given water stable isotope composition (δ18Ow) are shown in purple. (c) Clumped isotope composition vs δ13Ccc. Apparent clumped isotope-based temperatures are also indicated in (b) and (c). Shaded brown and grey areas show range of values for the unaltered calcite marble and the narrow white rim, respectively.

The two discolored sections of the rock wall have altered δ18Occ and δ13Ccc values that show two distinctive trends. The pale grey parts show no change in δ13Ccc and decrease in δ18Occ, with maximum shift of up to − 5.4‰ Δδ18O and only + 0.1‰ Δδ13C (where ΔX = XalteredMAX − XunalteredMEAN, and X is δ18Occ or δ13Ccc). The white narrow rim shows change in both δ13Ccc and δ18Occ, with Δδ18O of − 6.7‰ to − 8.3‰ and Δδ13C of + 3.3‰ to + 3.2‰, for C2 and C3, respectively, with values trending towards the isotopic composition of the overlying calcite coatings (Figs. 1, 2). The observed shift to lower δ18Occ is commonly found in hypogene caves with reported Δδ18O ranging from − 15‰ to − 2‰, reflecting interaction with thermal water of predominantly meteoric origin14,20,22,23,43,44,45 (Fig. 3). However, the δ13Ccc values are commonly reported to be either unchanged or shifted to lower values, with Δδ13C from − 12 to + 1‰, due to interaction with fluid with low δ13C DIC values (δ13CDIC), reflecting predominantly organic-derived carbon14,20,22,23,43,44,45 (Fig. 3). The shift to higher δ13Ccc values observed in the narrow rims in cores C2 and C3, suggests that the paleowater at Provalata Cave had high δ13CDIC. This is in agreement with recent geochemical data from nearby lukewarm Melnica Spring, where it was found that up to 54% of the carbon in the DIC is from metamorphic CO2 with δ13C of + 4.5‰46.

Figure 3
figure 3

Extent of observed stable isotope alteration in carbonate bedrock walls of hypogene caves. (a) Alteration vectors in δ18Occ − δ13Ccc space. (b) Magnitude of alteration (Δδ18Occ − Δδ13Ccc). 1 and 2—Provalata Cave (this study); 3 to 8—Hypogene caves in Austria22 (3—Mitterschneidkar-Durchgangshohle, 4—Entrische Kirche, 5—Stegbachgraben, 6—Obir Caves, 7—Güntherhohle, 8—Marchenhohle); 9—F10 Cave, Dolomites, Italy23; 10—Cupp-Coutunn/Promeszutochnaya, Turkmenistan14; 11 and 12—Hypogene caves in Crimea, Ukraine20; 13 and 14—Movile Cave, Romania44, 15—Plavecká jaskyňa, Slovakia45; 16 and 17—Carlsbad Cavern, USA43.

The calcite coatings have low δ18Occ (mean − 11.8‰) and very high δ13Ccc (mean + 7.3‰), with a relatively small range of values (Table 1). They show covariation in δ18Occ–δ13Ccc space along two distinct trends, one with positive and one with negative slope (Fig. 2).

Table 1 Summary statistics of the stable isotope composition of wall bedrock and calcite coatings from Provalata Cave.

Clumped isotope composition

The clumped isotope compositions follow a similar pattern. The grey calcite marble has the lowest Δ47 values (mean 0.323 ± 0.016‰), reflecting highest apparent temperatures (mean 206 ± 22 °C) (Figs. 1, 2, Table 1). Similar values were also found for the calcite marble along the NNW-SSE nappes (Figs. 1, 2). They are in the range of equilibrium blocking temperatures for the carbonate clumped thermometer24,27,47, and considering their unaltered δ18Occ and δ13Ccc, likely reflect blocking temperatures from the last metamorphic event. Such temperatures are in the range of zircon fission track (FT) closure temperatures, and FT ages from the eastern part of the Pelagonian massif relate them to Late Cretaceous-Paleocene thrusting48,49. The altered sections have increased Δ47 values showing two distinct clumped isotope compositions, that reflect mean apparent temperatures of 147 ± 23 °C in the pale grey section and 54 ± 13 °C in the white rim (Table 1). The apparent temperatures of the two altered sections, combined with their distinct conventional stable isotope composition (e.g., δ13Ccc), imply that the two alterations were related to two distinct hydrothermal events.

The calcite coatings have the highest Δ47 values, reflecting apparent temperatures of 11–34 °C (mean 22 ± 6 °C; Table 1). The onset of calcite deposition in C2 is at 12 °C and in C3 at 33 °C (Table S5), and all calcite coating samples show variation in temperature along their growth (Fig. 1). This variation shows that the evolution of the hydrothermal cave after the onset of calcite deposition was not following a simple cooling and degassing due to shift to shallower conditions, as generally suggested for such systems4, but experienced pulsations in temperature, with at least one subsequent heating event of up to ~ 34 °C.

We calculated the water oxygen isotope composition (δ18Ow) of the fluid in equilibrium with calcite for all of the identified sections, using the calcite-water oxygen equilibrium equation31, the measured δ18Occ and the clumped isotope-based apparent temperature (see also fluid inclusion section). The unaltered calcite marble has δ18Ow values that are within the range for metamorphic fluids50. However, as the apparent temperatures of the unaltered marble represent equilibrium blocking temperatures, their calculated δ18Ow values are not considered as representative. The pale grey section has lower δ18Ow values, which are within the range for magmatic fluids50. The secondary calcite minerals have the lowest values, reflecting largely meteoric water composition, while the narrow rim has intermediate δ18Ow values. The apparent temperatures for the pale grey section are within the range of temperatures (100 to 200 °C) of the ore mineralization at the nearby Carlin-type-like Allchar deposit51 (Supplementary Fig. S1), that is related to circulation of magmatic fluids with variable contribution of meteoric water, and associated with a ca. 5 Ma volcano-plutonic center of the Kožuf-Voras volcanic system52,53,54.

Microscopic petrography and alteration mechanisms

The macroscopic and stable isotope observations of the alteration are also supported by the microscopic petrography in both plane-polarized transmitted and cathodoluminescent light (Fig. 4). The grey marble section contains large (up to few mm) calcite crystals, with dark blue luminescence. The pale grey section shows similar size calcite crystals having violet luminescence, found in different positions: throughout the crystal; diminishing inwards from crystal boundaries; or along crystal defects. The narrow white rim section shows remnants of large crystals with dark blue or violet luminescence that are surrounded by a large number of smaller size (up to 50 μm) crystals with orange luminescence, similar to the overlying calcite coating (Fig. 4, Supplementary Fig. S6).

Figure 4
figure 4

Photomicrographs of C2 core showing different sections of the calcite marble cave wall in plane-polarized transmitted and cathodoluminescent light. (a) Overview of the thin section from the outer part of the core with location of the closer examined sections. (b) View of the unaltered section of the marble having large (mm size) crystals with dark blue luminescence. (c) A cross-section from the unaltered (left) through pale grey (middle) to white rim section, showing dominantly dark blue, violet and orange luminescence, respectively. (d) Part of the white rim, with remnants of a large calcite crystal with dark blue luminescence that changes outwards to violet, surrounded by smaller crystals with orange luminescence. (e, f) Close-up view of the white altered rim section, showing very small calcite crystals filling up pore spaces. An extended version of this figure is found in Supplementary Fig. S6.

The blue luminescence in the grey marble reflects intrinsic luminescence55,56, and supports the primary (unaltered) character of this section as also indicated by the isotopic composition. The similar luminescence of the small calcite crystals in the narrow rim to the one of the overlying calcite coatings suggests deposition from the same (or similar) fluid. This is also supported by the white rim δ13Ccc and δ18Occ values, as they are shifted towards the composition of the overlying calcite coating. A continuous transition between blue, violet and orange cathodoluminescence colors can occur due to the relative and absolute intensity of the intrinsic and orange luminescence55. However, in the absence of additional elemental analyses, it is not possible to gain more insight from the cathodoluminescence on the conditions under which calcite precipitation or alteration occurred.

Carbon and oxygen isotopes in calcite can exchange with fluids by either dissolution-reprecipitation, when mineral and fluid are far from chemical equilibrium, or by diffusion, when mineral and fluid are at or close to chemical equilibrium57. The exchange might also first proceed by dissolution-reprecipitation until chemical equilibrium is achieved and then by diffusion58. Rate of diffusion of carbon and oxygen in minerals is strongly temperature-controlled57, but in the presence of water, oxygen diffusion is greatly increased, while carbon diffusion is largely unaffected59,60,61. The alteration of oxygen isotopes with no change in carbon isotopes in the pale grey section, as well as the variation in cathodoluminescence towards the crystal surface, suggest hydrothermal alteration by diffusion. Experimental studies on oxygen isotope diffusion in calcite61,62 suggest that the water effect to oxygen diffusion in calcite is primarily a surface process, facilitating oxygen exchange between calcite surface and fluids. However, while water enhances oxygen isotope diffusion at the calcite crystal surface, to alter the clumped isotope composition, reordering of C-O bonds requires bulk mineral increase in oxygen diffusivity62,63, which is achieved only at higher temperatures and longer times25,47. This implies that dissolution-reprecipitation is the more likely process to explain the alteration of the clumped isotope composition at lower temperatures. Additionally, a combination of increased diffusivity, dissolution-reprecipitation, as well as pressure-induced structural changes can increase the reordering rates62. Although our stable isotope data is not detailed enough to investigate variation along a single crystal, the luminescence halo seen along boundaries of some crystal grains, and no apparent sign of dissolution-reprecipitation along the crystals, support the oxygen diffusion interpretation. Thus, the variation of δ18Occ and Δ47 in the pale grey section most likely reflects the degree of isotopic exchange that occurred between the hydrothermal fluid and the calcite crystals, with alteration progressing further in smaller size crystals and crystals with defects, while creating a halo in larger crystals. The lowest apparent temperature in the pale grey section (~ 110 °C) is found in the sample with the lowest δ18Occ values, that likely reflects the temperature of the hydrothermal fluid.

For the narrow rim, the microscopic observations (both under transmitted and cathodoluminescent light) clearly point to dissolution-reprecipitation as the alteration mechanism, where previously formed porosity is filled with secondary calcite that continues further from the cave wall as calcite coating. Thus, the stable isotope composition along the white rim reflects the frequency of secondary calcite minerals, i.e., the linear trends seen in δ18Occ − δ13Ccc, Δ47 − δ13Ccc, and Δ47 − δ18Occ space (Fig. 2) reflect the mixing of the isotopic compositions of the secondary calcite and the calcite marble, the latter varying between the compositions of the grey and pale grey sections. Based on the lowest apparent temperature in the white rim, this alteration was caused by a low-temperature (< 35 °C) hydrothermal fluid.

Fluid inclusions in secondary calcite minerals

Provalata calcite samples are rich in fluid inclusions that are dominated by primary (intra-crystalline) inclusions, mostly as large size inclusions in palisade crystals, but also as clouds of small ‘thorn’ shaped inclusions (Supplementary Fig. S7). Their water stable isotope composition indicates meteoric origin, with δ18Ow values ranging between − 13.9‰ and − 11.6‰ and δ2Hw values between − 87.0‰ and − 76.0‰ (Supplementary Table S1). The data fall between the local and Mediterranean meteoric water lines, with lower values than what was found for the cold and lukewarm springs in the area46 (Fig. 5).

Figure 5
figure 5

Isotope composition of carbonate and fluid inclusions from Provalata Cave calcite. (a) Water stable isotope composition (δ18Ow, δ2Hw). Global (GMWL), Mediterranean (MMWL) and local (LMWL46) meteoric water lines are included. Samples for which both carbonate and water stable isotope data are available have black outlines. (b) Carbonate stable isotope composition (δ18Occ, Δ47) and clumped based apparent temperatures. (c) Calcite-water oxygen fractionation factors for Provalata Cave calcite compared to equilibrium curves of Kim and O’Neil67 and Daëron et al.31. (d) Helium isotope composition (R/RA, where R and RA are the 3He/4He ratio of the sample and air respectively) in a three-component helium mixing plot (atmospheric He: R/RA = 1, 4He/20Ne = 0.318, crustal He: R/RA = 0.02, 4He/20Ne = 1000, mantle He: R/RA = 8, 4He/20Ne = 100064,65,66). Numbers indicate stratigraphic position of the subsample within the calcite coating sample.

Noble gas data show predominance of crustal He in the fluid inclusions, with R/RA values (where R and RA are the 3He/4He ratio of the sample and air, respectively) of 0.37 to 0.50 and 4He/20Ne values of 1.5 to 2.4. The three-component (air, crust, mantle64,65,66) helium mixing model (Fig. 5, Supplementary Table S2) shows that the mantle component is low (2–4%) and the atmospheric component ranges from 13 to 22%.

For the samples with combined conventional and clumped carbonate stable isotope data, and fluid inclusion water stable isotope data (i.e., formation T, δ18Ow and δ18Occ; n = 4), the calcite-water oxygen isotope fractionation was calculated (Fig. 5). The data fall above the experimental equilibrium line of Kim and O’Neil67, and exactly on the empirical equilibrium curve based on slow growing mammillary calcites from Devils Hole and Corchia Cave31. The latter is almost identical to the one proposed by Coplen68 and to the theoretical prediction by Watkins et al.69, indicating that Provalata calcite formed under conditions at or close to thermodynamic equilibrium. Considering this, the paleowater δ18Ow was calculated for the samples for which no fluid inclusion data were available, using δ18Occ, Δ47-based apparent temperature and the calcite-water equilibrium equation31. The calculated δ18Ow values range from − 13.4‰ to − 9.6‰, with uncertainties of 0.5–1.5‰, due to propagation of larger uncertainties in apparent temperatures.

Timing of the calcite coating deposition

The calcite coating has been estimated to form during the end of Pliocene or Early Pleistocene39. The low U concentrations in C2 samples suggested that the samples are not readily suitable for U–Pb chronology. Three uranium-series analyses were used to suggest that the coating formed during the Early to Middle Pleistocene. If Pliocene in age, both the 230Th/238U activity and δ234U value would measure 1 and 0‰, respectively, which is secular equilibrium in well preserved, unaltered calcite. C2 core exhibits densely crystalline calcite that yielded 230Th/238U activities of 1.012, 1.028, and 1.027, and δ234U values of 10, 22, and 8‰, respectively for samples C2-1, C2-2a, and C2-2b (Supplementary Table S3). The U-series results indicate, given no alteration, that the calcite coating formed after 2.5 Ma. During the subsequent SAS phase, when the cave reached the water table, in addition to gypsum replacing calcite minerals, potassium sulfate minerals (e.g., alunite, jarosite) formed at the contact of sulfuric acid and clay deposits39. The 1.6 Ma Ar–Ar age of alunite formed during this phase39 thus provides the minimum age limit for the deposition of the calcite coating.

The evolution of the secondary calcite minerals

The calcite coatings in Provalata Cave show small variation in their δ18Occ and δ13Ccc values and a strong covariation that follows two lines: one with positive slope and another with negative slope (Fig. 6). For the samples with positive δ18Occ − δ13Ccc slope, the T-δ18Ow relationship also follows a positive slope (~ 0.2‰/°C), indicating that the δ18Ow values are a result of mixing of two fluids: one cold (< 10 °C) with low δ18Ow (< − 13‰) and another hot (> 35 °C) with higher δ18Ow (> − 9.5‰). The cold one clearly represents a relatively shallow, meteoric water, while the hot one indicates a deep-seated origin. Magmatic waters have δ18Ow in the range of + 6‰ to + 10‰ and metamorphic waters in the range of + 5‰ to + 25‰50, thus based on the δ18Ow/T slope seen in the samples, and considering magmatic or metamorphic origin, the estimated temperatures of the hot component are between 100 and 200 °C, respectively.

Figure 6
figure 6

Stable isotope composition of calcite coatings from Provalata Cave. (a) Covariation of δ18Occ and δ13Ccc values following generally two lines, one with positive, and one with negative slope. (b) Variation of δ18Ow (from fluid inclusions or calculated) with temperature. Dashed purple lines show equilibrium lines for a given δ18Occ. (c) Variation of δ13Ccc with temperature. Green and purple full lines show curves of modeled variation of δ13Ccc with temperature change, with δ13CDIC at + 4‰ and + 1‰, respectively, for a selected fH2CO3 (indicated for each curve) and 0.1 mol fraction of carbon in the degassed CO2 (Xc). Closed system calcite precipitation (no degassing) is shown with dashed lines. See Supplementary Information for explanation of calculation and additional modeled curves. (d) Variation of δ18Ow with δ13Ccc. For each sample the stratigraphic sequence of stable isotope data is indicated to observe the trends in stable isotope change. Encircled symbols show noble gas fluid inclusion samples and associated values indicate R/RA values. See text for details.

While the water composition is clearly a mixture of two components, the positive correlation in δ18Occ − δ13Ccc space cannot be explained by calcite precipitation due to mixing of two fluids. First, the two fluids will need to be highly saturated in calcite, as mixing of two fluids saturated in calcite at different equilibrium concentrations of Ca2+ produces a mixture with lower calcite saturation due to the curvature of the calcite equilibrium line, that will lead to calcite dissolution (i.e., mixing corrosion)70,71,72. Second, the very small change observed in the δ13Ccc values would require two fluids of such contrasting origin, such as meteoric and deep-seated, to have similar carbon isotopic composition, which is highly unlikely. Furthermore, while mixing of two fluids with similar isotopic composition and contrasting temperatures can produce correlation in δ18Occ − δ13Ccc space, it is unlikely that two fluids will have similar isotopic composition, indicating similar origin, but have contrasting temperature73. Thus, it is more reasonable to assume calcite precipitation from one fluid under a temperature effect, then due to mixing of two fluids73. This implies that the water mixing occurred prior to calcite precipitation, and likely contributed to the cave passage formation by mixing corrosion.

The calcite that marks the onset of deposition at C2 core has very high δ13Ccc for a DIC dominated by a meteoric (soil) sourced carbon. The δ13Ccc depends on the δ13CDIC, T and pH (distribution of carbon species, mainly H2CO3 and HCO3, having significantly different fractionation with calcite74). For such high δ13Ccc value (+ 7.9‰) at low temperature (12 °C), if the calcite precipitates in isotopic equilibrium from fluid with pH > 6, both δ13CDIC and δ13C of the CO213Cext) should be higher than 0‰, suggesting a deep-seated origin of the CO2. At pH above 7 the δ13CDIC should be >  + 5.4‰, and δ13Cext >  + 7.5‰, with more than 41% of the carbon in DIC coming from carbonate dissolution (see Supplementary Information). However, in such case the alkalinity will be low (Supplementary Fig. S10), and if some soil CO2 is present (as expected in shallow meteoric waters), the deep-seated CO2 must have even higher δ13C, suggesting that the combination of higher pH with such high δ13CDIC values is not likely. Thus, it is most likely that the onset of calcite deposition at C2 was from low temperature, slightly acidic fluid, where the water was of meteoric origin, but the CO2 was mostly of deep-seated origin. As the subsequent mixing with deep-seated hotter fluid occurred prior to calcite precipitation, this supports the interpretation46 that meteoric and deep-seated fluids converged at depth along a fault structure with dominantly metamorphic CO2 flux.

Since calcite precipitation is accompanied with temperature change in our samples (Figs. 1, 2, 6), the δ13Ccc values can be modeled in terms of change in temperature with or without CO2 degassing75. The modeled curves show characteristic slopes of T-δ13Ccc for a range of H2CO3 fractions (fH2CO3) in DIC (Fig. 6; Supplementary Information). At C2, the calcite precipitation follows two lines in T-δ13Ccc space with similar slightly negative slope, that based on our modeling (Fig. 6), indicates low degassing and lower pH of the fluid. The shift to lower intercept with same slope suggests either lowering of δ13CDIC or some variation in the degassing. Similar pattern is also seen in the first part of C3 core. The larger vertical shift in the rest of the samples can be due to δ13CDIC change, higher degassing, general shift to higher pH (e.g., lowering of pCO2 in the fluid), or a combination. These samples show also larger shift in δ18Ow (< 2‰). While increase in pH has no direct effect on oxygen isotopes, concurrent increase in pH and δ18Ow can occur by increased water–rock interaction. However, this is unlikely as the low temperature setting does not favor such large water–rock oxygen isotope exchange.

Another possibility is a change in the primary isotopic composition of both DIC and water. Lowering of δ13CDIC of the deep component is a possible explanation (e.g., increase in mantle-derived CO2), but increase in mantle-derived fluids cannot explain the observed increase in δ18Ow, and noble gas fluid inclusion data show minor and relatively constant mantle He component. Variation in the δ18Ow of the shallow component is very likely, and such increase in values can be due to climate variations (e.g., higher values in warmer and/or drier conditions and vice-versa76). Although the effect of δ18Ow change in the shallow component can be seen in the mixture δ18Ow, the δ13CDIC change in the shallow component (carrying a soil δ13C signal, e.g., − 25 to − 10‰76) will be overprinted by the large fraction of deep-derived CO2 (with endogenic δ13C signal, e.g., − 7‰ to − 4‰ for mantle, or > 0‰ for metamorphic CO250,77. If the deep-sourced CO2 flux decreases, pCO2 will decrease, leading to an increase in pH and lowering of the δ13Ccc, but also relative increase in the shallow component carbon fraction, and further lowering of δ13CDIC and δ13Ccc. This is supported by the noble gas fluid inclusion data, where δ13Ccc decrease is accompanied with R/Ra increase and 4He/20Ne decrease (Fig. 6), that reflects concurrent increase of the atmospheric He component (from 13 to 22%) and decrease of the crustal He component (from 84 to 76%), with constant minor mantle He component (2–4%).

In other words, climatically controlled variation in the isotopic composition of the cold end-member (δ18Ow, δ13CDIC) is seen in the mixture δ18Ow but generally masked in the mixture δ13CDIC by the high amount of deep-sourced CO2. Thus, the offset from the positive δ18Occ - δ13Ccc covariation in Provalata calcite reflects a phase of lowering of deep CO2 contribution.

The evolution of the hydrothermal cave system: a brief summary

The grey calcite marble of the cave walls reflects primary isotopic composition, that was first altered in color and isotopic composition by hydrothermal water (~ 110 °C) of magmatic or mixed magmatic-meteoric origin (Fig. 7). It was likely close to chemical equilibrium with the rock, and caused only lowering of δ18Occ, probably by oxygen diffusion between water and rock. The alteration was pervasive, and appearing not bound to a major fissure, although the hydrogeological setting under which it operated is unclear. This event probably corresponds to an earlier volcanic activity of the Kožuf-Voras system (6.5–1.8 Ma52,53,54). The second alteration (narrow rim along cave walls), is related to the cave-forming phase, when convectional features developed along high-angle fractures of a NW–SE oriented fault zone39, related to Late Miocene-Quaternary extension48,49. Cave passages formed by dissolution and enlargement of fractures during cooling of rising low-temperature (< 35 °C) thermal waters (of mixed meteoric and deep-seated origin), rich in metamorphic CO2, likely enhanced by mixing corrosion. As dissolution progressed perpendicular to cave walls, porosity was increased in the first few centimeters. Cooling continued, but the system shifted to shallower settings, forcing CO2 degassing and calcite precipitation, first filling up pore spaces, and progressing into thick calcite coatings. The thermal evolution and δ13Ccc values of the coatings suggest that after the initial cooling, another heat pulse followed, and variation in pCO2 controlled the variation in δ13Ccc. The calcite deposition took place between 2.5 Ma and 1.6 Ma and is likely connected to the youngest volcanic activity of the Kožuf-Voras system (3.0–1.8 Ma), focused in its southwestern part52,53,54, close to the study site (Supplementary Fig. S1). It is possible that both alteration phases are part of the long-term evolution of the same hydrothermal system, that first commenced at deeper settings with pervasive alteration, and then evolved along newly formed extension-related high-angle faults with high CO2 flux, creating cave passages.

Figure 7
figure 7

Conceptual model of the evolution of the studied hydrothermal phases in Provalata Cave with associated alteration mechanisms and calcite deposits presented schematically at three scales: hydrogeological setting, cave and cave wall. The thickness of the blue and red groundwater flowlines in each phase indicates the relative influence of the shallow epigene and deep hypogene component, respectively. (a) Pervasive alteration of color and δ18Occ in calcite marble by hydrothermal water (~ 110 °C) with oxygen diffusion along crystal boundaries as the most likely alteration mechanism. The hydrogeological setting is unclear. (b) Development of cave passages along high-angle fractures due to cooling of low-temperature (< 35 °C) thermal waters rich in metamorphic CO2, likely enhanced by mixing corrosion. Porosity increases perpendicularly to the cave wall. (c) Shift to shallower depth leads to CO2 degassing and calcite precipitation, filling up pore spaces. (d) Large decrease in temperature and δ18Ow, indicating increased influence of the shallow groundwater component. Calcite with high δ13Ccc values precipitates outward from the cave wall. (e) Increase in temperature and δ18Ow, likely related to nearby volcanic activity, leads to precipitation of calcite with lower δ13Ccc. (f) Temperature and δ18Ow decrease. Lowering of pCO2 leads to decrease in calcite δ13Ccc.

Conclusions

We show that by combined use of conventional and clumped stable isotope analysis of carbonates in the study of cave wall alteration by thermal waters we can better constrain the character of the isotopic alteration, as well as unambiguously identify different alteration events.

On the example of Provalata Cave we demonstrate the primary isotopic character of the unaltered calcite marble, and identify two distinct hydrothermal phases causing different alterations. The older one was pervasive with flow along smaller structures and crystal boundaries, causing lowering of δ18Occ values likely due to diffusion-controlled isotope exchange with fluid at or close to chemical equilibrium with the rock, at higher temperature (~ 110 °C). The second one was related to cave formation by fluid flow along a fracture, i.e., progressed perpendicularly inward from the fracture (cave wall), and proceeded with dissolution-reprecipitation-controlled alteration by lower-temperature fluids (< 35 °C) that transitioned from being calcite-undersaturated (dissolution) to calcite-saturated (precipitation).

While most of the examples of cave wall isotope alteration reported in the literature show lowering of δ18Occ and either unchanged or lowered δ13Ccc, our findings show a rare example of isotope alteration with lowered δ18Occ and significantly increased δ13Ccc. We expect that such case is not a unique one and can be expected at sites with high metamorphic CO2 flux. We also show a non-linear thermal evolution of the hydrothermal cave system, suggesting that simple cooling, as usually assumed for such systems, is likely simplifying the evolution, especially for karst systems related to volcanic areas.

Methods

Stable isotope analysis of carbonates

Conventional and clumped stable isotope analyses of carbonates were carried out at the Isotope Climatology and Environmental Research Centre (ICER), Institute for Nuclear Research (ATOMKI), Debrecen.

Conventional carbon and oxygen stable isotope analyses were carried out on hand-drilled carbonate powders with an automated GASBENCH II sample preparation device (phosphoric acid digestion at 72 °C) attached to a Thermo Finnigan DeltaPLUS XP isotope ratio mass spectrometer (IRMS)78. Conventional stable isotope results are expressed as δ18O and δ13C values relative to Vienna Pee-Dee Belemnite (VPDB). The precision of the measurements is ≤ 0.08‰ for δ13C and ≤ 0.1‰ for δ18O.

Clumped isotope analysis was done on a Thermo Scientific 253 Plus IRMS, after phosphoric acid digestion at 70 °C using a Thermo Scientific Kiel IV automatic carbonate device. Each carbonate sample measurement consisted of at least 10 replicate analyses of 100–120 μg aliquots that were measured alongside carbonate standard samples (ETH1, ETH2, ETH3, ETH4, and IAEA-C2) with assigned values79,80. Data evaluation was done on the Easotope application81, with the CO2 clumped ETH PBL replicate analyses method and IUPAC parameters82,83,84,85. Δ47 results are given in the I-CDES90 scale80, and apparent temperatures in °C were calculated based on the Δ47-temperature calibration from Anderson et al.86, with temperature uncertainties propagated from the 1σ standard error (SE) of the Δ47 value. Simultaneously during clumped isotope analysis, on the same samples, conventional carbonate stable isotope composition was also determined.

Petrography

Double-polished sections for petrographic observations were prepared employing low-speed precision sawing and polishing, which minimize thermal and mechanical stresses on the samples. Fluid inclusion observations of secondary calcite were carried out on a transmitted-light microscope (Nikon Eclipse E 400 POL) at the Institute of Geology, University of Innsbruck and petrographic observations of calcite marble from cave walls were carried out on a transmitted-light microscope (Olympus BX53 equipped with Olympus DP23 camera) at ICER, ATOMKI, Debrecen. Additionally, cathodoluminescence studies were carried out at the Karst Research Institute ZRC SAZU, Postojna, on a Technosyn® cold cathodoluminescence luminoscope (CITL CL8200 MK4) with a 14 kV electron beam and an electron current of 350–400 μA, mounted on a Nikon Eclipse E600 biological petrographic microscope. The thin sections, examined in transmitted light and cathodoluminescent light, were photographed using a Nikon Coolpix 990 digital camera.

Fluid inclusion

Fluid inclusion analyses were carried out at three laboratories. Water stable isotope composition was determined at the Institute of Geology, University of Innsbruck, and at the Institute for Geological and Geochemical Research (IGGR), Budapest. Noble gas isotope composition (3He, 4He, 20Ne, 22Ne) was determined at ICER, ATOMKI.

At Innsbruck sample PR20 was crushed in a heated crushing cell87, and after cryogenic focusing, the water was transported by He flow into the high-temperature reactor of the TC/EA unit (ThermoFisher) and pyrolyzed into H2 and CO at 1400 °C. The evolved gases were separated in a chromatographic column and analyzed using a Thermo Fisher Delta V Advantage IRMS. Detailed description of the method can be found in Dublyansky and Spötl88. The remaining samples were analyzed at the IGGR in Budapest. Sample chips of about 0.5–1 g were crushed under vacuum in stainless steel tubes, after which the released water was purified by vacuum distillation and introduced into a Los Gatos liquid water isotope analyzer (LWIA-24d). Details of the method are described in Demény et al.89. Results are expressed as δ2H and δ18O relative to Vienna Standard Mean Ocean Water (VSMOW). Analytical precision is 0.5‰ for δ18O and better than 1.5‰ (Innsbruck) or 2‰ (IGGR) for δ2H.

For noble gas isotope analyses, 1.3 to 2.3 g of calcite samples were loaded into stainless-steel holders with a magnetic ball and baked at ~ 60 °C for 10–12 h in vacuum before the measurements. Gas was extracted by single-step crushing (~ 100 strokes) at room temperature (22 °C). The relatively low number of strokes was applied for each measurement in order to avoid significant contribution of an in-situ component. Helium isotope abundances and ratios were determined by a HELIX- SFT mass spectrometer, whereas a VG-5400 mass spectrometer was used for neon. The analytical procedures are described in more detail in Papp et al.90 and Molnár et al.91.

Uranium-series

Three small pieces of densely crystalline calcite coating (core C2) were dissolved in 15 N HNO3, doped with a 229Th, 233U, and 236U mixed spike, and cleaned and separated U and Th in anion exchange resin column chemistry following the methods of Asmerom et al.92. Measurements were made on a Thermo-Neptune multicollector inductively coupled plasma mass spectrometer at the Department of Earth and Planetary Sciences, University of New Mexico. U and Th were measured separately as static runs where all isotopes are measured in faraday cups except for 230Th and 234U, which were measured in a secondary electron multiplier. Efficiency between the cups and multiplier were determined using an in-house Th standard and the U standard NBL-112. Decay constants and a detailed description of the uranium-series methods are in Cheng et al.93. The measured δ234U value for standard NBL-112 (CRM-112a) is -38.5‰, where δ234U = [(234UA/238UA) – 1] × 1000, and A refers to activity.

Modeling of carbon isotope composition

To model the δ13Ccc values in terms of calcite precipitation due to change in temperature with or without CO2 degassing, we used the equation of Zheng75 with a modification, that instead of considering two extreme cases of HCO3-dominant or H2CO3-dominant fluid, for which only fractionation of calcite-HCO3 or calcite-H2CO3 is used, respectively, we select a set of fractions of H2CO3 in the DIC of the fluid (where DIC = fH2CO3 + (1-f) × HCO3), that itself reflects fluid pH at a given T. For a selected δ13CDIC, range of temperatures and fH2CO3, first we calculate the δ13C of the CO2 in equilibrium with the fluid (δ13CCO2) and then we calculate δ13Ccc values using the Rayleigh model equation of Zheng75.

To model the fluid composition from which calcite can precipitate in isotopic equilibrium recording temperature and δ13Ccc as found for the onset of calcite deposition in C2 core, first we calculate the fractions of H2CO3 and HCO3 in PHREEQC Version 3 software94 for a range of pH values, with a fixed temperature and calcium and DIC concentrations determined by forcing charge balance to the solution at fixed calcite saturation index. Then we calculate the δ13CCO2 and δ13CDIC, and using the modeled chemical composition for dissolution of only calcite or calcite and dolomite, we determine the isotopic composition of the carbon from CO2 not accounted for by dissolution of carbonate rocks (external CO2; δ13Cext).

Detailed explanation of the calculations is given in the Supplementary Information.

Consent to publish

Consent was obtained from the identifiable person in Fig. S4b (Marjan Temovski) to publish image in an online open access publication.

Data availability

All data generated or analysed during this study are included in this published article and its Supplementary Information file.

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Acknowledgements

This research was supported by the European Union and the State of Hungary, co-financed by the European Regional Development Fund in the project of GINOP-2.3.2-15-2016-00009 ‘ICER’. Part of this research was also supported by the MTA Postdoctoral Fellowship Programme (No. PP-030/2015). Fieldwork was carried out under Research Permit No. 11-1654/6 issued in 2016 by the Ministry of Environment and Physical Planning of the Republic of Macedonia. We thank Zlatko Angeleski, Martin Dameski and Darko Nedanoski from SK Zlatovrv Prilep for assistance during fieldwork.

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Open access funding provided by ELKH Institute for Nuclear Research.

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M.Te. designed the research, wrote the manuscript, and prepared the figures. M.Te., L.P. and M.Tú. performed field work. I.F. and M.Te. performed conventional stable isotope analysis of carbonate. L.R. and M.Tú. performed clumped isotope analysis of carbonate. Y.D., K.M., and B.O. performed microscope analysis. Y.D. and A.D. performed fluid inclusion stable isotope analysis. K.M. and L.P. performed fluid inclusion noble gas analysis. V.P. and Y.A. performed U-series analysis. P.A. contributed to the final version of the manuscript. All authors reviewed the manuscript.

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Correspondence to Marjan Temovski.

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Temovski, M., Rinyu, L., Futó, I. et al. Combined use of conventional and clumped carbonate stable isotopes to identify hydrothermal isotopic alteration in cave walls. Sci Rep 12, 9202 (2022). https://doi.org/10.1038/s41598-022-12929-4

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