Carbonate biological hard tissues are valuable archives of environmental information. However, this information can be blurred or even completely
lost as hard tissues undergo diagenetic alteration. This is more likely to occur in aragonitic skeletons because bioaragonite often transforms into
calcite during diagenesis. For reliably using aragonitic skeletons as geochemical proxies, it is necessary to understand in depth the diagenetic
alteration processes that they undergo. Several works have recently investigated the hydrothermal alteration of aragonitic hard tissues during short-term experiments at high temperatures (
Calcium carbonate hard tissues are valuable geochemical proxies for deciphering the past climate dynamics and environmental changes. However, the greatest challenge that these biological archives face lies in their capacity to retain their pristine signature after the death of the calcifying organism, since from this moment the skeletons become highly prone to alteration. The original microstructure and chemical composition of the biocarbonate structural hard tissue can become partially, in the best case, to totally, in the worst case, obliterated, and so does the environmental-derived information recorded in it (Brand, 1989; Swart, 2015; Casella et al., 2018; Pederson et al., 2019a, b, 2020).
Biocarbonate structural materials are composites of biopolymers and calcium carbonate mineral phases (mostly calcite and/or aragonite, rarely and much less abundantly vaterite) (Weiner and Dove, 2003). The alteration of biocarbonate hard tissues is influenced by both external-environment-related and internal-archive-related factors. Main external factors are alteration time, the degree of geochemical disequilibrium with the environment, the physicochemical conditions in the depositional environment, the porosity and permeability of the sediments, the chemical composition of the pore fluids, and the fluid–rock ratio, among others (Pederson et al., 2019a, b, 2020). On the other hand, some internal factors that influence the alteration kinetics of the calcium carbonate skeletons are the concerned carbonate phase, the original microstructure and texture of the mineral component, the fabric, and amount and distribution of the organic matter within the composite hard tissue (Gaffey, 1988; Gaffey et al., 1991, Casella et al., 2018; Pederson et al., 2020). At the conditions that prevail in natural diagenetic environments, calcite is the stable calcium carbonate polymorph while aragonite is the more soluble, thermodynamically metastable phase (Plummer and Mackenzie, 1974; Plummer and Busenberg, 1982; Sass et al., 1983; Walter and Morse, 1984; Bischoff et al., 1987, 1993; Redfern et al., 1989; Navrotsky, 2004; Morse et al., 2007; Gebauer et al., 2008; Radha et al., 2010; Gebauer and Cölfen, 2011; Radha and Navrotsky, 2013). In the presence of an aqueous phase, aragonite crystals can transform into calcite through a coupled dissolution–crystallization reaction. The progress of the reaction is driven by the difference in solubility between the two carbonate phases and is facilitated by the generation of porosity (Berner, 1975; Bischoff, 1969, 1968; Fyfe and Bischoff, 1965; Cardew and Davey, 1985; Mucci et al., 1989; Putnis and Putnis, 2007; Putnis, 2009; Ruiz-Agudo et al., 2014; Sun et al., 2015). The result of this dissolution–crystallization reaction is the formation of calcite pseudomorphs after aragonite that consist of blocky calcite crystals (Perdikouri et al., 2008, 2011, 2013). It is noteworthy that biogenic calcite also is metastable with respect to abiogenic calcite and that calcite biominerals may also undergo dissolution–recrystallization reactions that result in the diagenetic overprint of their isotopic notations, as was recently demonstrated experimentally (Bernard et al., 2017; Cisneros-Lázaro et al., 2021, 2022).
The metastable nature of aragonite explains that aragonitic skeletons have a lower potential of becoming preserved in the geologic record, relative to
their calcitic counterparts (James et al., 2005; Cherns and Wright, 2011; Cherns et al., 2011, Janiszewska et al., 2018; Wright et al., 2003; Wright
and Cherns, 2004). Lowenstam (1954) and Hallam and O'Hara (1962) estimated that, during diagenesis, most aragonitic carbonates would be replaced by
calcite within a few to thousands of years. However, under low-temperature regimes, in shallow environments enriched in organic matter, the
transformation of aragonite into calcite is precluded and the progress of diagenetic alteration is restricted to its very first stages (Hall et al., 1967; Seuß et al. 2009; Janiszewska et al., 2018). The resulting structural and chemical
changes between the pristine and altered skeletons are then very subtle and difficult to trace. In this work, we aim to disclose the subtle
microstructural and chemical changes undergone by aragonitic hard tissues during low-temperature, long-time hydrothermal alteration. We conducted
long-term (4 and 6 months) hydrothermal alteration experiments at 80
Three aragonitic hard tissues from animals with very different microstructures were chosen for this study. Shells of the modern bivalve
For the alteration experiments, pieces of the hard tissues were cut into slices with a diamond 6
For all alteration experiments, three pieces of the pristine skeletons of modern
One of the three pieces of every altered sample was crushed in an agate mortar and measured with powder X-ray diffraction (XRD) for phase composition
evaluation. The analysis was performed using
The sample that was used for XRD measurements was recovered from the XRD holder and was further used for TGA measurements for the determination of
organic matter content within the pristine and altered sample. TGA measurements were conducted with a Q500 TGA. The samples were heated from room
temperature to 1000
Overview images for the visualization of the different microstructures within a shell or skeletal element were taken with a Keyence 3D laser scanning confocal microscope (VK-X1000 series). The second shell or skeleton segments were embedded in epoxy resin and polished down with eight sequential polishing steps for obtaining a highly even sample surface. Laser confocal microscopy imaging was conducted on uncoated samples.
The sub-micrometre and nanometre structure of the shells and skeletal elements was scanned with AFM. AFM imaging was done in non-coated, epoxy-embedded and highly polished sample surfaces. Samples were imaged in contact mode with a JPK NanoWizard II AFM using silicon nitride cantilevers. Scans of lateral and vertical deflection traces were analysed with the NanoWizard® IP image processing software by using the “gold” scale for colour. The lateral and vertical deflection traces are the result of the interaction between the cantilever tip and the sample surface. Height traces were used to generate 3D models of the nanoscale topography of some of the samples.
To visualize major microstructural elements in the pristine and the altered samples, the aragonitic hard tissues were imaged with FE-SEM and analysed
with EBSD. The remaining piece of the altered samples, together with pristine skeletons, was embedded into epoxy resin and polished with several
mechanical grinding and polishing steps down to a grain size of 1
FE-SEM imaging and EBSD measurements were carried out in a Hitachi SU5000 field emission SEM, equipped with an Nordlys Oxford EBSD detector. The SEM
was operated at 20
EBSD band contrast gives the signal strength of the EBSD–Kikuchi diffraction pattern and is displayed as a greyscale component in a map. The strength of the EBSD signal is high when a crystal is detected (bright in the map), whereas it is weak or absent when a polymer, such as organic matter or epoxy resin, is scanned (dark/black on the map). Crystal co-orientation statistics are derived from Kikuchi diffraction patterns measured at each image pixel of an EBSD map. Crystal co-orientation is given by the MUD (multiple of uniform (random) distribution) value. A high MUD indicates high crystal co-orientation, while low MUD values reflect low to random crystallite or/and mineral unit co-orientation. Pole figures are stereographic projections of the orientations of crystallographic axes or plane normals measured at all pixels of an EBSD map.
The term texture relates to the distribution of crystal orientations within a material and is illustrated by pole figures, showing either colour-coded
orientation data or contoured versions of density distributions of
EBSD measurements allow us to distinguish between individual grains and, hence, to obtain grain-related parameters such as grain area and grain
boundaries. A grain in an EBSD map is defined as a region that is completely surrounded by boundaries across which the misorientation angle relative
to the neighbouring grains is larger than a critical value. In this study we use a critical misorientation value of 2
The pristine skeletons of
EBSD colour-coded orientation maps with their corresponding pole figures depicting aragonite microstructure and texture in
Vertical deflection AFM images of
The shell of the bivalve
EBSD colour-coded orientation maps with their corresponding pole figures depicting the microstructure and texture of biogenic aragonite of the
Vertical deflection AFM images depicting
Three-dimensional surface reconstruction of height AFM measurements of aragonitic nacre tablets for
The pristine shell of the gastropod
Colour-coded orientation maps with their corresponding pole figures derived from EBSD scans depicting the microstructure and texture of
Vertical deflection AFM images of
The modern skeleton of the scleractinian coral
The interaction of the hard tissues of
TGA measurements show a reduction in the organic matter content of
Laser confocal microscopy imaging was used to obtain overview images of sample surfaces to visualize main differences between the pristine and the
altered samples (Figs. A4, A7 and A11). In the case of
The microstructural evolution of the altered samples was followed with FE-SEM and EBSD measurements, and the sub-microstructure to nanostructural evolution of
the altered samples was recorded with AFM imaging. FE-SEM images of
For
The microstructure of the stony coral
Relative frequency versus grain size/grain area for pristine and hydrothermally altered
Statistical evaluation of grain size gained from EBSD measurements allows us to quantitatively evaluate grain size distribution in the pristine and
altered samples. The area of grains was grouped into clusters of 0.15
Change in aragonite grain area/grain size with increasing hydrothermal alteration for inner and outer layers of
For the granular microstructure of
The two microstructures of
For
The effect of laboratory-based hydrothermal alteration of several aragonitic biominerals has recently been studied in great detail (Ritter et al.,
2017; Casella et al., 2017, 2018; Pederson et al.,
2019a, b, 2020). A multi-analytical approach was used to characterize the hard tissues, both pristine and after their alteration at temperatures
above 100
Structural materials secreted by organisms are composites of biopolymers and minerals arranged in hierarchical architectures (Weiner and Dove, 2003). In these composites, mineral and organic matrices are intricately interrelated at all scale levels. This study, as well as previous works, (Casella et al., 2018; Pederson et al., 2019a, b, 2020) deciphered the main intermediate steps undergone by aragonitic microstructures during hydrothermal alteration. The alteration usually starts with the degradation of the biopolymers which are incorporated into the biocarbonate material. The fabric of the organic matter within the hard tissue is either a network of organic fibrils and/or a sequence of organic membranes. The degradation of the organic matter constitutes the first step of the hydrothermal alteration process and leads to the second alteration step, which consists in the formation of a network of pores that permeate the biomineral and facilitate the circulation of the hydrothermal fluid (Casella et al., 2017, 2018). As a result of this rather extensive phenomenon, local dissolution of bioaragonite occurs concomitant to the precipitation of new, non-biogenic, aragonite (Casella et al., 2018). The precipitation of abiogenic aragonite is the third step of the hydrothermal alteration and results in an increase in aragonite grain size in the altered samples, relative to the size of the grains in their pristine counterparts (Casella et al., 2018). The fourth step of the hydrothermal alteration finally consists in the progressive replacement of both biogenic and newly formed non-biogenic aragonite by new, non-biogenic, calcite crystals (Casella et al., 2017, 2018). In this case, the extent of the replacement depends on a variety of factors, such as temperature, time, secondary porosity network features, and fluid / solid ratio, and might be stopped before a complete replacement takes place (Sandberg and Hudson, 1983). Ritter et al. (2017) have also shown that the composition of the hydrothermal fluids can induce changes in the sequence of these alteration steps.
Temperature and time are key parameters in defining the extent of the alteration of the aragonitic hard tissues when these are exposed to interaction
with hydrothermal fluids (Ritter et al., 2017; Pederson et al., 2019a). Regardless of the specific microstructure of the biocarbonate material, longer
interactions and higher temperatures lead to more extensive alterations and stronger overprints of the pristine features of the aragonitic
skeletons. However, we observed that some characteristics of the pristine hard tissue make aragonitic biomaterials particularly resistant to
hydrothermal alteration.
The primary porosity of the biomaterial defines the initial surface area of the microstructure that is exposed to the hydrothermal fluid and can
react with it. Primary porosity strongly influences the very early stages of the alteration process (Casella et al., 2018; Greiner et al., 2018). The amount, fabric, distribution and composition of the organic matter within the hard tissue define the characteristics of the secondary
porosity network that results from biopolymer degradation during the first step of the hydrothermal alteration process. This secondary porosity
network adds to the primary porosity and provides new pathways for the infiltration and circulation of the hydrothermal fluid within the biomaterial
(Jonas et al., 2017; Casella et al., 2018). The tortuosity and permeability of this network, which depend on the shape, size and interconnectivity
of its constituting pores (Forjanes et al., 2020a), define the extent of hydrothermal fluid infiltration through the hard tissue (Casella et al.,
2018). Microstructures in biological hard tissues result from an intimate interlinkage between minerals and organic matter, at all scale levels. This
interlinkage determines that the architecture of the mineral component can influence the kinetics of the degradation of the organics and, thereby,
modulate the formation of the secondary porosity network. Mineral microarchitecture and biopolymer characteristics are taxon- or even
species-specific (Carter and Clark, 1985). This explains that different biological aragonitic hard tissues show different susceptibilities to
hydrothermal alteration such that, while exposed to identical hydrothermal alteration conditions, some undergo a complete overprint of their
pristine features, while others remain virtually unaltered (this study and Casella et al., 2018). The solubility of the bioaragonite depends on factors such as crystal morphology, composition and amount of occluded biopolymers. Biogenic
aragonite can incorporate small amounts of The third step that marks the progress of the alteration of aragonitic hard tissues involves the dissolution of biogenic aragonite and the
precipitation of non-biogenic aragonite. Consequently, the fourth step is given by the dissolution of both aragonite types, biogenic and
non-biogenic, and the concomitant precipitation of abiogenic calcite. Small differences in aragonite solubility influence the development of the
dissolution–crystallization reactions and affect the kinetics of the entire alteration process and mechanism.
It should be noted that, in the presence of fluids, porosity networks have a transient nature (Putnis, 2015). The dissolution–crystallization reactions that take place during the third and fourth steps of the hydrothermal alteration process are connected to solubility and molar volume changes. When, as a result of these changes, a partial or total obliteration of the biological hard tissue porosity network takes place in the course of the third alteration step, the fourth step of the alteration process cannot progress; it is either hindered or even totally precluded (Putnis et al., 2005; Jonas et al., 2014; Putnis, 2015).
Main intermediate stages of alteration reached by the different aragonitic microstructures from this work when hydrothermally altered at 80
Figure 10 summarizes the different steps of the hydrothermal alteration process experienced by the outer and inner granular shell layers of the
bivalve
At 175
At temperatures above 160
The behaviour of
The Arrhenius equation predicts double reaction rate constants as temperature increases by 10
Biopolymer degradation, which defines the first step of the hydrothermal alteration process and generates the secondary porosity network, is, indeed,
a temperature-dependent process (Moussout et al., 2016). Biopolymers decompose through processes that involve depolymerization, bond scission, loss of
functional groups and formation of free radicals (Gaffey, 1988; Gaffey et al., 1991). Under dry conditions, these processes take place very slowly up
to temperatures that depend on the composition and the structure of the biopolymer. Biopolymer decomposition temperatures can be as high at
250
The organic component of biological hard tissues consists of complex mixtures of polysaccharides, proteins, glycoproteins, and glycosaminoglycans, and
these degrade at different temperatures and rates (Gaffey, 1988; Gaffey et al., 1991; Tiwari and Raj, 2015) and are species-specific (Marie et al.,
2011; Drake et al., 2013; Le Pabic et al., 2017). Accordingly, there will be slightly different degradation pathways for the organic matter of the
hard tissues of the different organisms (Keenan and Engel, 2017). In addition, water-soluble and water-insoluble macromolecules are found in the structural
materials of biocarbonates (Weiner and Traub, 1984; Goffredo et al., 2011; Sancho-Tomás et al., 2013). Therefore, it is likely that, prior to the
complete degradation of the biopolymers, the organic matrices will reorganize and some of their soluble components will be released into the
alteration fluid, especially at low temperatures. Since biopolymers contain a variety of functional groups, this release can influence the progress of
the hydrothermal alteration. It is well known that active moieties like peptide or carboxylic groups affect both the dissolution and the
crystallization of calcium carbonate polymorphs through a variety of mechanisms. This influence is especially important when, as it occurs in the
experiments conducted in this study, the fluid phase contains
The organic matrix of
In contrast, significant decomposition of organics is found in the altered shells of
Our study shows that the difference in dissolution/decomposition of organics present within the hard tissues is a key parameter in the generation of
different volumes of secondary porosity, which adds up to the primary porosity initially present in the studied biomaterials. Accordingly, the hard
tissue of
The altered shell of
When the hydrothermal overprint starts, the fluid is equilibrated with atmospheric
Two main factors might promote the formation of abiogenic aragonite instead of abiogenic calcite: (i) the presence of
The size distribution of aragonite crystals is different between pristine and altered
The strongest replacement of aragonite by abiogenic calcite during hydrothermal alteration at 80
The decay of biopolymers also generates a certain volume of secondary porosity in the prismatic shell layer. The less structured microstructure of
this layer might induce that this porosity has also a high interconnectivity, even if its volume is smaller, relative to that of the nacreous shell
layer. In both layers, secondary porosity guarantees the interaction with the hydrothermal fluid over an enlarged surface of the mineralized
tissue. During the first 4 months of hydrothermal alteration there is no significant replacement of aragonite by calcite (Fig. A2). Therefore, it
can be assumed that once the hydrothermal fluid equilibrates locally with the biogenic aragonite in the pores and becomes supersaturated with respect
to abiogenic aragonite, aragonite nucleation will take place. Aragonite nucleation triggers the dissolution of biogenic aragonite and the
crystallization of abiogenic aragonite through the formation of a dissolution–crystallization loop (Putnis, 2002, 2009; Ruiz-Agudo
et al., 2014). The evolution in crystal size distribution in the prismatic and the nacreous layers in
Studies of the hydrothermal alteration of carbonate biominerals under conditions mimicking those found in diagenetic environments (Jonas et al., 2017;
Casella et al., 2017, 2018; Ritter et al., 2017; Pederson et al., 2019a, b; 2020) provide valuable information on the ability of different biological
archives to retain their pristine signature and help to identify the parameters that underlie this ability. Thus, these studies may have far-reaching
implications for taphonomic, palaeoecological and biostratigraphic research as they can contribute to define objective criteria for evaluating the
reliability of microstructural, chemical and isotopic notations derived from these archives. It is well known that most aragonitic skeletons undergo
extensive replacement by calcite during burial diagenesis. However, some favourable taphonomic scenarios (rapid lithification, storm plasters, anoxic
bottoms, high sedimentation rates, etc.) can result in the preservation of aragonite (James et al., 2005; Cherns et al., 2008). It is often assumed
that aragonite preservation equals lack of diagenetic alteration. Our results show that this assumption can be baseless. In contact with a burial-like
fluid at 80
The susceptibility of biocarbonates to resist diagenetic overprint is strongly influenced by several primary features, such as the carbonate phase, the microstructure of the mineral component, or the distribution and fabric of the organic matter within the composite hard tissue, among others. Laboratory-based hydrothermal alteration experiments offer important insights into the fate of biocarbonate hard tissues when responding to diagenetic alteration. While previous studies investigated the effect of high-temperature and short-term hydrothermal alteration on the change in biocarbonate microstructures (Casella et al., 2017, 2018; Pederson et al., 2019a, b, 2020), the emphasis of this work is placed on the influence of low-temperature and long-term hydrothermal overprint processes of biologically secreted aragonite microstructures. The experimental conditions used in this work more closely approach those common in burial diagenetic environments
We deduce from our study the following conclusions.
We identify several intermediate stages during the overprint of aragonitic hard tissues: (I) decomposition of biopolymers, (II) gain of
secondary porosity, (III) dissolution of the biogenic aragonite and precipitation of abiogenic aragonite, and (IV) replacement of the biogenic and
abiogenic aragonite by abiogenic calcite. Depending on the composition, fabric and pattern of distribution of the organic matter within the biological hard tissue, hydrothermal
alteration induces the formation of secondary porosity. The latter porosity adds to the primary porosity and facilitates the penetration and
circulation of the hydrothermal fluid. The porosity network greatly affects the kinetics of the alteration process, irrespective of the alteration temperature. The tortuosity and
permeability of the porosity network defines the extent of infiltration and percolation of the alteration fluid into the hard tissue. This explains
that different biological aragonitic hard tissues show different susceptibilities to hydrothermal alteration. For similar alteration conditions,
some microstructures undergo significant to complete overprint of their pristine features, while others remain virtually unaffected. For most aragonitic microstructures, except for the microstructure of Precipitation of abiogenic aragonite occurs always prior to calcite precipitation. This is supported by the observation of several processes
affecting the aragonitic microstructures which take place without a phase change. These processes are the increase in the size of the aragonite
crystals, the amalgamation of adjacent crystals and the decrease in crystal co-orientation strength for amalgamated crystals. Our results have major implications for palaeoenvironmental reconstruction based on proxy data gained from fossil archives. Our findings suggest
that, due to diagenesis, most fossil carbonate hard tissues are overprinted to some extent, even if they do not show clear signs of carbonate phase
change and microstructure destruction. When diagenetic alteration does not involve mineralogical change, it can be easily overlooked by conventional
characterization approaches involving the use of standard analytical techniques. Findings in this work provide evidence that the unequivocal
detection of the subtle changes undergone by aragonitic hard tissues at early stages of diagenesis requires the application of multi-analytical
protocols that incorporate high-resolution tools.
Preparation of
X-ray diffractograms of the hydrothermally altered shells and skeletons of
Thermogravimetric analysis data of pristine and altered hard tissues of
Laser confocal microscopy images of
FE-SEM images of
EBSD band contrast and phase maps illustrating the differences in the microstructure and mineralogy between the pristine and the most altered shell of
Laser confocal microscopy images of
FE-SEM images depicting the internal structure of
FE-SEM images showing the
EBSD band contrast and phase maps illustrating differences in microstructure and mineralogy between the pristine and the most altered shells of
Laser confocal microscopy images of
SEM images showing the
EBSD band contrast and phase maps illustrating the differences in microstructure and mineralogy between the pristine and the most altered skeletons of
Exemplary Rietveld refinement plot for an altered
The software needed to process the SEM and EBSD data (AZTec and CHANNEL 5 HKL) and the AFM data (JPK NanoWizard II AFM) requires a subscription and cannot be shared publicly. The software required to perform the mineralogical quantification of the XRD results (FullProf Suite) can be found at
This paper is mainly based on EBSD data sets which usually contain over 500 000 single measurement points each. The data sets are too large in data volume and cannot be stored in volume-limited databases; thus, our data are unsuitable for public access. In case specific EBSD data are required, please contact the corresponding authors. Raw XRD and TGA data appear in Figs. A2 and A3, respectively.
PF, EG, LFD and WWS designed the study. NAL provided sample material. PF performed the experiments. PF, MG, MSR and SVV conducted the analyses and carried out the evaluation and merging of data. PF, EG, JMA and LFD drafted the manuscript. All authors contributed to discussions and the final manuscript.
The contact author has declared that none of the authors has any competing interests.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank Moritz Zenkert, Xiaofei Yin and Ana Vicente Montaña for their support with sample preparation. We are grateful for the helpful reviews by Theodore Present and two anonymous referees, which have considerably improved the quality of this work.
This study was supported by the Spanish Ministry of Economy and Competitiveness under project CGL2016-77138-C2-1-P and the Spanish Ministry of Science and Innovation under project PID2021-125467NB-I00. Pablo Forjanes acknowledges a FPU predoctoral contract (FPU17/01689) from the Spanish Ministry of Universities. We acknowledge the European Union's Horizon 2020 research and innovation programme (BASE-LiNE Earth, grant no. 643084) and the German Research Council programme (GR 9/1234).We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
This paper was edited by Aninda Mazumdar and reviewed by Theodore Present and two anonymous referees.