Description: Brachiopods present a key fossil group for Phanerozoic palaeo-environmental and palaeo-oceanographical reconstructions, owing to their good preservation and abundance in the geological record. Yet to date, hardly any geochemical proxies have been calibrated in cultured brachiopods and only little is known on the mechanisms that control the incorporation of various key elements into brachiopod calcite. To evaluate the feasibility and robustness of multiple Element/Ca ratios as proxies in brachiopods, specifically Li/Ca, B/Ca, Na/Ca, Mg/Ca, Sr/Ca, Ba/Ca, as well as Li/Mg, we cultured Magellania venosa, Terebratella dorsata and Pajaudina atlantica under controlled experimental settings over a period of more than two years with closely monitored ambient conditions, carbonate system parameters and elemental composition of the culture medium. The experimental setup comprised of two control aquariums (pH0 = 8.0 and 8.15, T = 10 °C) and treatments where pCO2−pH (pH1 = 7.6 and pH2 = 7.35), temperature (T = 16 °C) and chemical composition of the culture medium were manipulated. Our results indicate that the incorporation of Li and Mg is strongly influenced by temperature, growth effects as well as carbonate chemistry, complicating the use of Li/Ca, Mg/Ca and Li/Mg ratios as straightforward reliable proxies. Boron partitioning varied greatly between the treatments, however without a clear link to carbonate system parameters or other environmental factors. The partitioning of both Ba and Na varied between individuals, but was not systematically affected by changes in the ambient conditions. We highlight Sr as a potential proxy for DIC, based on a positive trend between Sr partitioning and carbonate chemistry in the culture medium. To explain the observed dependency and provide a quantitative framework for exploring elemental variations, we devise the first biomineralisation model for brachiopods, which results in a close agreement between modelled and measured Sr distribution coefficients. We propose that in order to sustain shell growth under increased DIC, a decreased influx of Ca2+ to the calcifying fluid is necessary, driving the preferential substitution of Sr2+ for Ca2+ in the crystal lattice. Finally, we conducted micro-computed tomography analyses of the shells grown in the different experimental treatments. We present pore space – punctae – content quantification that indicates that shells built under increased environmental stress, and in particular elevated temperature, contain relatively more pore space than calcite, suggesting this parameter as a potential novel proxy for physiological stress and even environmental conditions.

Description: Carbonate hardparts of marine organisms are frequently explored archives of their ambient seawater composition. Among the various materials used, the hardparts of molluscs are particularly relevant because their accretionary growth allows for the compilation of time-resolved proxy data sets. Here, we explore to which extent the calcium isotope values of aragonitic cephalopod hardparts represent proxies for seawater composition or record a trophic level signal. In a first step, we collect Ca isotope data from a diverse set of modern cephalopods, including Nautilus, Sepia and Spirula. These taxa differ in their external/internal hardparts, life spans, trophic levels, habitat depth, hinterland geology, and resulting runoff. In a second step, we have put these concepts to the test and explore the calcium isotope signatures of Lower Cretaceous (lower Albian) well-preserved cephalopods (nautiloids, ammonoids and belemnites) and bivalve shells from Madagascar. In the sense of a methodological comparison, two measurement approaches (double spike (δ44/40Ca, TIMS) and bracketing standard (δ44/42Ca, MC-ICP-MS) techniques are applied. Calcium from organic matter and intra-shell variations on bulk δ44/40Ca isotope values are explored. Four protocols (H2O2, NaOH, HClO4, incineration up to 250 °C including subsequent rinsing with deionised water and Ca removal) are applied to destroy organic matter in the shell material and to dissolve any non‑carbonate bound Ca, i.e., its metastable secondary Ca reservoir. These are equally successful, and the Ca isotope values of the purified carbonate phase plot on the equilibrium mass fractionation line. Samples not treated to destroy or remove their organic matter do not fall on the equilibrium mass fractionation line. Limitations of sample pre-treatment are reflected by a slight shift towards lower values in the case of incineration experiments at 650 °C. This feature is best explained in the context of a mineralogical phase transformation of aragonite to calcite. Calcium isotope values show no significant variability when sampling hardparts with different microstructures (callus, nacre, prismatic layer) and mineralised during different ontogenetic phases (outer shell, septa). Perhaps most relevant, mean δ44/40Ca (‰ SRM-915a) isotope values for Nautilus (0.72‰ ±0.19 2SD), Sepia (0.79‰ ±0.18 2SD), and Spirula (0.50‰ ±0.18 2SD) lack evidence for a significant control by trophic levels. Nitrogen isotope data and known diets for all three cephalopods serve as a litmus test for these data, which do not correlate with this dietary proxy information. These results are considered encouraging and suggest that Ca isotope values of cephalopod shells are proxies for their ambient seawater Ca isotope signatures. According to our data, however, it remains unclear to which extend cephalopod hardparts record the Ca isotopic composition of past seawater. Specifically, the data from three different modern species point to a mean Δ44/40Ca(ceph-seawater) fractionation of −1.21‰ (± 0.15 2SD). Based on our work, we present a best practice guide for cephalopod Ca isotope analysis and interpretation.

Description: To study the incorporation of magnesium (Mg) and strontium (Sr) in calcite precipitated over synthetic calcite seeds (overgrowths) as a function of the precipitation rate (R*, μmol/m2 h), we performed precipitation experiments wherein temperature and precipitation rates were decoupled at intervals of approximately 3.63–5.22 μmol/m2 h. In most sample reactions, high‑magnesium calcite (HMC) overgrowths co-precipitated with aragonite from the stirred solutions exposed to an atmosphere of NH3 and CO2 gases throughout the spontaneous decomposition of (NH4)2CO3. The percentage of aragonite in the solid CaCO3 increased with both temperature and dissolved inorganic carbon (DIC). The order of reaction with respect to the [DIC] is temperature dependent and is 1.9, 2.4, and 2.9 at temperatures of 12.5, 25.0, and 37.5 °C, respectively. The magnesium distribution coefficient (DMg) increases significantly with increasing R*, temperature, and Mg/Ca ratio in the fluid. The strontium distribution coefficient (DSr) increases with R* and with increasing MgCO3 concentrations in the calcite overgrowths. However, it is independent of temperature.

Description: Calcium (Ca) isotopes (δ44/42Ca) in serum and urine have been suggested as novel sensitive markers of bone calcification. The response of δ44/42Ca to acute changes in Ca homeostasis, has not yet been demonstrated. We measured serum Ca and δ44/42Ca in rats maintained on a standard and a 50% Ca reduced diet for 4 weeks, and after injection of 1 mg/kg of the calcimimetic AMG-416, 24 h prior to sacrifice. AMG-416 decreased serum Ca by a maximum of 0.38 ± 0.10 and 0.53 ± 0.35 mmol/l after 12 and 6 h, respectively, in the standard and low-Ca diet groups (p = 0.0006/0.02), while serum δ44/42Ca did not change over 24 h in both groups. Urinary Ca concentrations were higher 24 h after AMG-416 injection in both groups (p = 0.03/0.06), urine δ44/42Ca was not different compared to the untreated control groups. Our data does not show acute changes in δ44/42Ca in response to a single dose of AMG-416 within 24 h after injection, possibly due to a lack of bone calcification.

Description: Background Prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) have an increased fracture risk. Exploring biomarkers for early bone loss detection is of great interest. Methods Pre-planned substudy of the ARNEO-trial (NCT03080116): a double blind, randomised, placebo -controlled phase 2 trial performed in high-risk PCa patients without bone metastases between March 2019 and April 2021. Patients were 1:1 randomised to treatment with gonadotropin-releasing hormone antagonist (degarelix) + androgen receptor signalling inhibitor (ARSI; apalutamide) versus degarelix + matching placebo for 12 weeks prior to prostatectomy. Before and following ADT, serum and 24-h urinary samples were collected. Primary endpoints were changes in calcium-phosphate homeostasis and bone biomarkers. Findings Of the 89 randomised patients, 43 in the degarelix + apalutamide and 44 patients in the degarelix + placebo group were included in this substudy. Serum corrected calcium levels increased similarly in both treatment arms (mean difference +0.04 mmol/L, 95% confidence interval, 0.02; 0.06), and parathyroid hormone and 1,25-dihydroxyvitamin D3 levels decreased. Bone resorption markers increased, and stable calcium isotope ratios reflecting net bone mineral balance decreased in serum and urine similarly in both groups. Interpretation This exploratory substudy suggests that 12 weeks of ADT in non-metastatic PCa patients results in early bone loss. Additional treatment with ARSI does not seem to more negatively influence bone loss in the early phase. Future studies should address if these early biomarkers are able to predict fracture risk, and can be implemented in clinical practice for follow-up of bone health in PCa patients under ADT.

Description: Highlights • Sr isotope composition in different minerals is an excellent proxy, that can be used to reconstruct the environmental conditions of their precipitation. In order to study Strontium (Sr) isotope fractionation during the precipitation of strontianite (SrCO3) as a function of the specific precipitation rate (R*) and temperature (T), strontianite was precipitated at 12.5, 25.0 and 37.5 °C by diffusing NH3 and CO2 gases into aqueous solutions. The specific precipitation rate R* (mol/cm2.h) for every sample was determined by applying the initial rate method. The mean isotope difference between bulk solution and precipitate (∆88/86Srstrontianite-solution) was found to be −0.279 ± 0.005‰ (2σmean) independent of both rate and temperature. Hence, Sr isotope fractionation in strontianite is completely different from that in calcite and aragonite, where a strong dependency from both rate and temperature can be observed. The latter is interpreted to reflect the competition between Sr2+ and Ca2+ ions for incorporation into the calcium carbonate crystal lattice, which is absent during the precipitation of pure strontianite. The isotope difference between strontianite and bulk solution then simply reflects the intermolecular forces in the aqueous solutions as well as the kinetic effect. The difference in the (∆88/86Srstrontianite-solution between experiments then reflects the dehydration energy of Sr ions in the adsorption layer of SrCO3.

Description: Dysregulated calcium homeostasis is common in chronic kidney disease and causally associated with disorders of bone mineralization. However, radiological measures and biomarkers do not allow accurate evaluation of bone calcium balance. Non-radioactive calcium isotopes, 42Ca and 44Ca, are present in our diet and sequestered into body compartments following principles of kinetic isotope fractionation. Isotopically light 42Ca is preferentially incorporated into bone, while heavier 44Ca is excreted. The ratio (44/42Caserum) increases when bone formation exceeds resorption and vice versa, reflecting bone calcium balance. We measured these calcium isotopes by inductively coupled plasma mass-spectrometry in blood, urine and feces of 42 children with chronic kidney disease and 92 receiving dialysis therapy. We compared the isotope ratios with bone biomarkers and determined total bone mineral content by dual-energy x-ray absorptiometry and peripheral quantitative CT expressed as age-adjusted z-scores. The 44/42Caserum ratio positively correlated with serum calcium, 25-hydroxyvitamin D and alkaline phosphatases and inversely with serum parathyroid hormone and other bone resorption markers. The 44/42Caserum ratio positively correlated with age-adjusted z-scores of tibial trabecular bone mineral density and total bone mineral content measured by peripheral quantitative CT, and hip bone mineral density measured by dual-energy X-ray absorptiometry. Significant and independent predictors of total bone mineral content, measured by, were the 44/42Caserum ratio and parathyroid hormone. The 44/42Caserum ratio, repeated after four weeks, highly correlated with baseline values. When adjusted for calcium-containing medications and kidney impairment, the 44/42Caserum ratio in patients receiving dialysis was 157% lower than that of age-matched children and 29% lower than levels in elderly women with osteoporosis, implying significantly lower bone mineral content. Thus, calcium isotope ratios may provide a novel, sensitive and non-invasive method of assessing bone calcium balance in chronic kidney disease.

Description: The Ca isotope composition of calcite and aragonite can provide insights into their formation conditions. The accurate interpretation of the Ca isotope composition of natural samples, however, requires precise knowledge of the equilibrium Ca isotope fractionation between the solid and fluid phases. In this study, the three-isotope method with 42Ca, 43Ca and 44Ca has been used to estimate the equilibrium isotope fractionation of Ca between the CaCO3 minerals calcite and aragonite and the Ca2+ aquo ion. Reactive fluids were enriched with 43Ca and equilibrated with synthetic calcite and aragonite of natural Ca isotope distribution up to 3745 h at 25 °C. The isotopic composition of solids and fluids was measured using MC-ICP-MS and the estimated equilibrium fractionation for calcite-Ca2+(aq) and aragonite-Ca2+(aq) was Δ44/42Casolid-fluid=−0.02±0.13‰ and −0.80±0.10‰, respectively. Textural observations of the reacted solids suggest that isotope equilibration in aragonite experiments occurs via extensive Ostwald ripening, yielding large crystalline needles at the end of the experiments. In contrast, calcite did not exhibit an observable increase in size during the course of the experiments. Isotope exchange rates in the case of calcite are similar to those reported in previous studies and ∼4 orders of magnitude lower than the far-from-equilibrium calcite dissolution rate. Calcium isotope exchange rates for aragonite are more rapid than calcite driven by a greater extent of Ostwald ripening occurring via dissolution/precipitation reactions. The results of this study suggest that the Ca isotope compositions of calcite and aragonite crystals in chemical equilibrium, but isotope disequilibrium, with natural fluids could be significantly altered without overt evidence of diagenesis, especially in the case of calcite. The extent to which Ca isotope compositions are altered, however, would depend strongly on the environmental conditions, such as fluid:solid ratio and permeability of the solid facilitating fluid transport.

Description: Lithium has proven a powerful tracer of weathering processes and chemical seawater evolution. Skeletal components of marine calcifying organisms, and in particular brachiopods, present promising archives of Li signatures. However, Li incorporation mechanisms and potential influence from biological processes or environmental conditions require a careful assessment. In order to constrain Li systematics in brachiopod shells, we present Li concentrations and isotope compositions for 11 calcitic brachiopod species collected from six different geographic regions, paralleled with data from culturing experiments where brachiopods were grown under varying environmental conditions and seawater chemistry (pH–pCO2, temperature, Mg/Ca ratio). The recent brachiopod specimens collected across different temperate and polar environments showed broadly consistent δ7Li values ranging from 25.2 to 28.1‰ (with mean δ7Li of 26.9 ± 1.5‰), irrespective of taxonomic rank, indicating that incorporation of Li isotopes into brachiopod shells is not strongly affected by vital effects related to differences among species. This results in Δ7Licalcite–seawater values (per mil difference in 7Li/6Li between brachiopod calcite shell and seawater) from −2.9‰ to −5.8‰ (with mean Δ7Licalcite–seawater value of −3.6‰), which is larger than the Δ7Licalcite–seawater values calculated based on data from planktonic foraminifera (~0‰ to ~−4‰). This range of values is further supported by results from brachiopods cultured experimentally. Under controlled culturing conditions simulating the natural marine environment, the Δ7Licalcite–seawater for Magellania venosa was −2.5‰ and not affected by an increase in temperature from 10 to 16 °C. In contrast, a decrease in Mg/Ca (or Li/Ca) ratio of seawater by addition of CaCl2 as well as elevated pCO2, and hence low-pH conditions, resulted in an increased Δ7Licalcite-seawater up to −4.6‰. Collectively, our results indicate that brachiopods represent valuable archives and provide an envelope for robust Li-based reconstruction of seawater evolution over the Phanerozoic.

Description: Local carbonate cycling in lagoon-estuarine systems, involving processes such as inorganic and biogenic carbonate precipitation/dissolution, represents an important but poorly constrained component of the coastal carbon budget. This study investigates the sensitivity of stable Sr isotope tracer (δ88/86Sr) with respect to carbonate saturation and salinity of local waters in the Coorong, Lower Lakes and Murray Mouth (CLLMM) estuary in South Australia. The CLLMM has an extensive range of salinity from fresh to hypersaline (from ∼0 to over 100 PSU), with corresponding variations in water chemistry and major ion composition that in turn controls mineral saturation states, and thus CaCO3 precipitation/dissolution in local waters. Here we use the novel δ88/86Sr tracer in tandem with the more established radiogenic Sr isotope ratio (87Sr/86Sr), where the latter is a robust proxy for Sr sources and thus water provenance. We also produced a geochemical (PHREEQC) model of calcium carbonate (CaCO3) saturation changes across this unique lagoon-estuarine system. The results indicate a systematically increasing trend of δ88/86Sr (from ∼0.25‰ to ∼0.45‰) with increasing salinity and CaCO3 (aragonite, calcite) saturation indices of the coastal waters, which in turn suggest an overall control of carbonate dissolution/precipitation processes on the stable Sr isotope composition in the CLLMM system. This was further corroborated by Ca isotope data (δ44/40Ca) published previously on the same samples from the Coorong, as well as a quantitative simulation of local carbonate removal in the lagoon based on Rayleigh modelling and Sr isotope data. Overall, our results confirm that a coupled Sr isotope approach (combining 87Sr/86Sr and δ88/86Sr) can be used to constrain not only the main water sources (continental versus marine Sr) but also local CaCO3 dissolution/precipitation processes, and thus inorganic carbon and coastal carbonate cycling in the CLLMM system. Finally, this coupled δ88/86Sr and 87Sr/86Sr approach can be potentially applied to fossil carbonate archives to reconstruct paleo-hydrology and salinity changes in the CLLMM and/or other carbonate-producing coastal systems.