Rationale: Acid digestion of carbonates to release CO 2 is a crucial and sensitive step in sample preparation for clumped isotope analysis. In addition to data reduction and instrumental artefacts, many other uncertainties in the clumped isotope analysis of carbonates arise from the method used for the preparation of CO 2 .We describe here an in-house-designed reaction vessel that circumvents degassing and contamination problems commonly associated with the McCrea-type digestion protocols. Methods:We designed a leak-free break seal reaction vessel (made of Pyrex™) suitable for prolonged acid digestion at 25°C. Using this new vessel, several carbonate reference materials widely used in the clumped isotope community and other inhouse laboratory standards were acid-digested and analysed for their δ 13 C, δ 18 O and Δ 47 values with a dual inlet MAT 253 isotope ratio mass spectrometer following standard gas chromatography purification and data evaluation protocols.Results: Long-term reproducibility in Δ 47 determination was established using international references and in-house working standards as follows (mean and SE):Carrara-1 (0.395 ± 0.002‰, n = 43), Carrara-2 (0.441 ± 0.003‰, n = 22), OMC (0.587 ± 0.004‰, n = 16), NBS 19 (0.393 ± 0.005‰, n = 10), NBS 18 (0.473 ± 0.003‰, n = 5), ETH 1 (0.271 ± 0.005‰, n = 7), ETH 3 (0.698 ± 0.005‰, n = 3), MZ (0.715 ± 0.002‰, n = 3) and several others. Conclusions:A new method using a break seal tube was found to be efficient for the clumped isotope analysis of carbonates that require longer reaction time at 25°C. This method yields good precision in Δ 47 analysis and was found to be suitable for acid digestions at any desired temperature.

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The solar salterns in Tuticorin, India, are man-made, saline to hypersaline systems hosting some uniquely adapted populations of microorganisms and eukaryotic algae that have not been fully characterized. Two visually different microbial mats (termed ‘white’ and ‘green’) developing on the reservoir ponds (53 PSU) were isolated from the salterns. Firstly, archaeal and bacterial diversity in different vertical layers of the mats were analyzed. Culture-independent 16S rRNA gene analysis revealed that both bacteria and archaea were rich in their diversity. The top layers had a higher representation of halophilic archaea Halobacteriaceae, phylum Chloroflexi, and classes Anaerolineae, Delta- and Gamma- Proteobacteria than the deeper sections, indicating that a salinity gradient exists within the mats. Limited presence of Cyanobacteria and detection of algae-associated bacteria, such as Phycisphaerae, Phaeodactylibacter and Oceanicaulis likely implied that eukaryotic algae and other phototrophs could be the primary producers within the mat ecosystem. Secondly, predictive metabolic pathway analysis using the 16S rRNA gene data revealed that in addition to the regulatory microbial functions, methane and nitrogen metabolisms were prevalent. Finally, stable carbon and nitrogen isotopic compositions determined from both mat samples showed that the δ13Corg and δ15Norg values increased slightly with depth, ranging from − 16.42 to − 14.73‰, and 11.17 to 13.55‰, respectively. The isotopic signature along the microbial mat profile followed a pattern that is distinctive to the community composition and net metabolic activities, and comparable to saline mats in other salterns. The results and discussions presented here by merging culture-independent studies, predictive metabolic analyses and isotopic characterization, provide a collective strategy to understand the compositional and functional characteristics of microbial mats in saline environments.

The Cretaceous greenhouse climate was accompanied by major changes in Earth’s hydrological cycle, but seasonally resolved hydroclimatic reconstructions for this anomalously warm period are rare. We measured the δ18O and CO2 clumped isotope Δ47 of the seasonal growth bands in carbonate shells of the mollusc Villorita cyprinoides (Black Clam) growing in the Cochin estuary, in southern India. These tandem records accurately reconstruct seasonal changes in sea surface temperature (SST) and seawater δ18O, allowing us to document freshwater discharge into the estuary, and make inferences about rainfall amount. The same analytical approach was applied to well-preserved fossil remains of the Cretaceous (Early Maastrichtian) mollusc Phygraea (Phygraea) vesicularis from the nearby Kallankuruchchi Formation in the Cauvery Basin of southern India. The palaeoenvironmental record shows that, unlike present-day India, where summer rainfall predominates, most rainfall in Cretaceous India occurred in winter. During the Early Maastrichtian, the Indian plate was positioned at ~30°S latitude, where present-day rainfall and storm activity is also concentrated in winter. The good match of the Cretaceous climate and present-day climate at ~30°S suggests that the large-scale atmospheric circulation and seasonal hydroclimate patterns were similar to, although probably more intense than, those at present.

Seawater incursion and freshwater discharges into the tropical Mandovi Zuari (MZ) estuary is investigated here using stable oxygen isotope data on monthly water samples from locations spatially separated within the estuary. Surface water samples were analysed for d 18 O and salinity relationship, d 18 O ¼ 0:0843 Â S À 2:1; where freshwater end member is designated as À2.1%; similar to the composition recorded for the rain water. We have estimated percentage of monthly freshwater Cuxes into the estuary using mass balance equation adopting an isotopic value of seawater end member. Our estimates showed freshwater contribution to the estuary water during monsoon time was 63%, while in the postmonsoon season the fraction drops to 34.7%. The contribution of the freshwater registered a minimum value of 6.8% during the pre-monsoon season. Further, we analysed the seasonal growth band secreted by the mollusc from the same estuary to understand the potential of mollusc as a recorder of seasonal water composition. The d 18 O of mollusc shell growth layers varied over a range between À4.3% and À2.1%. We interpreted this as seasonal signal assuming the growth rate from the culture experiment. The d 18 O of estuary water and observed temperature are used to simulate the isotopic composition of seasonal growth bands. The lighter d 18 O value of À4.3% precipitated during the month of July 2010, which coincides with the time of low productivity (d 13 C = À3.5%). While the heavier d 18 O (À2.1%) is recorded in the growth layer generated during November 2010 deBning the period of post-monsoon growth.