Temperature Estimates of Lower Miocene (Burdigalian) Coastal Water of Southern India Using a Revised Otolith “Clumped” Isotope Paleothermometer

Carbonate clumped isotope thermometry is based on the ordering of 13C and 18O in the carbonate lattice and is based on the relative abundance of 13C18O16O in CO2 produced through acid digestion of carbonate minerals. The major advantage of this technique is its non‐dependency on the δ18O value of water from which the carbonate precipitated. Ghosh et al. (2007, https://doi.org/10.1016/j.gca.2007.03.015) previously published calibration data for fish otoliths referenced to heated gases and used the Gonfiantini 17O parameter set in their data evaluation. Herein, we present a new clumped isotope (Δ47) calibration for aragonitic fish otoliths in the absolute reference frame using the International Union of Pure and Applied Chemistry 17O correction. Our revised calibration equation for otolith is: Δ47CDES=0.0364±0.005×(106)T2+0.2619±0.0657(R2=0.9,p−value<0.001) . To test the accuracy of this calibration, we apply it to otoliths of modern Lutjanus lutjanus from the Bay of Bengal. The estimated average temperature (22.3°C ± 4.2°C) for the Bay of Bengal and δ18OV‐SMOW composition of waters of −1.7‰ (±0.5) are consistent with the onsite observations. We also apply the new calibration to well‐preserved otoliths of “genus Ambassidarum” sp. and “genus Gobiidarum” sp. from lower Miocene (Burdigalian) sediments of the Quilon Formation, India to quantify coastal water conditions. Estimated average environmental water temperatures in their habitats were 12.9°C ± 1.7°C, and the average δ18OV‐SMOW of ambient waters calculated yielded a value between −3.5‰ and −2.6‰ (V‐SMOW) (mean: −2.9‰ ± 0.4) and −4.4‰, respectively. These results indicate δ18O values reflect the kinetic effects impacting the δ18O of fish otoliths independently of Δ47, although we cannot fully preclude diagenesis.


Introduction
Otoliths are accretionary body parts deposited within the inner ear of fishes. They accrete new crystalline and protein material onto their exterior surface daily. Incorporated within these accreted layers are minor and trace elements along with the aragonitic mineralization. Because the majority of fishes are ectothermic, meaning they do not have physiological mechanisms to regulate their body temperature, these accretionary body parts deposited within the inner ear of fishes are used as recorders of the environment and are thus, an essential archive for paleo-climatic studies (Thorrold et al., 1997). Assuming fish otoliths form under equilibrium conditions, their stable isotope ratios of carbon (δ 13 C) and oxygen (δ 18 O) may therefore be used for the estimation of temperatures of their habitats (Fay, 1984;Morris & Kittleman, 1967). Being monomineralic (aragonitic), they can be screened for diagenetic effects based on the determination of the occurrence of secondary calcite using bulk mineralogical analyses (such as XRD or FTIR) and assessment of the degree of local structural ordering using micro Raman techniques (Toffolo, 2021). Previous studies employing the δ 18 O composition of otoliths demonstrated that the accretionary growth layers is in equilibrium with the water in which the host fish lives (Kalish, 1991b;Patterson et al., 2013;Thorrold et al., 1997). This equilibrium relationship has been applied to track environmental temperatures and migration routes for species of modern fish, constrained by the water δ 18 O value (Campana & Neilson, 1985).
Abstract Carbonate clumped isotope thermometry is based on the ordering of 13 C and 18 O in the carbonate lattice and is based on the relative abundance of 13 C 18 O 16 O in CO 2 produced through acid digestion of carbonate minerals. The major advantage of this technique is its non-dependency on the δ 18 O value of water from which the carbonate precipitated. Ghosh et al. (2007Ghosh et al. ( , https://doi.org/10.1016Ghosh et al. ( /j. gca.2007.015) previously published calibration data for fish otoliths referenced to heated gases and used the Gonfiantini 17 O parameter set in their data evaluation. Herein, we present a new clumped isotope (Δ 47 ) calibration for aragonitic fish otoliths in the absolute reference frame using the International Union of Pure and Applied Chemistry 17 O correction. Our revised calibration equation for otolith is:     6 2 47 CDES 2 10 Δ 0.0364 0.005 0.2619 0.0657 0.9, value 0.001 To test the accuracy of this calibration, we apply it to otoliths of modern Lutjanus lutjanus from the Bay of Bengal. The estimated average temperature (22.3°C ± 4.2°C) for the Bay of Bengal and δ 18 O V-SMOW composition of waters of −1.7‰ (±0.5) are consistent with the onsite observations. We also apply the new calibration to well-preserved otoliths of "genus Ambassidarum" sp. and "genus Gobiidarum" sp. from lower Miocene (Burdigalian) sediments of the Quilon Formation, India to quantify coastal water conditions. Estimated average environmental water temperatures in their habitats were 12.9°C ± 1.7°C, and the average δ 18 O V-SMOW of ambient waters calculated yielded a value between −3.5‰ and −2.6‰ (V-SMOW) (mean: −2.9‰ ± 0.4) and −4.4‰, respectively. These results indicate δ 18 O values reflect the kinetic effects impacting the δ 18 O of fish otoliths independently of Δ 47 , although we cannot fully preclude diagenesis.
PRASANNA ET AL.
A significant limitation of δ 18 O based paleo-environment reconstruction is partitioning of this combined signature of temperature and water isotopes into meaningful climatic signals. The application of carbonate δ 18 O in reconstructing paleo-environments suffers from limited constraints on the isotopic composition of paleo-environmental waters. This is because the equilibrium formation of aragonite in otoliths, with respect to oxygen isotopic composition, depends on the isotopic composition and temperature of the water. Thus, an assumption of either the isotopic composition or temperature of the water is required to determine the other when using conventional oxygen isotope thermometry. This limitation may be circumvented with the "carbonate clumped isotope thermometer" (Eiler, 2011;Ghosh et al., 2006;Schauble et al., 2006). A significant advantage of using this isotope geothermometer is that it relies on a homogeneous equilibrium (reaction among components of a single phase). Therefore, it can rigorously constrain the temperature of carbonate growth without any prior knowledge of the isotopic composition of its parent water.
The foundation of this thermometer was the empirical relationship established by analyzing calcite precipitated at controlled temperatures ranging between 5°C and 50°C, reported on a stochastic reference frame derived by analyzing CO 2 heated to 1,000°C . The early empirical calibration for the carbonate clumped isotope thermometer was applied to biogenic corals from deep and surface ocean locations with known growth temperatures . The calibrations was later extended to otolith specimens from modern fishes sampled across the latitudinal transect of the Atlantic Ocean (between 54°S and 65°N), and other species from the tropical western Pacific (Ghosh et al., 2007) with growth temperatures varying between 2°C and 25°C. These observations showed that the Δ 47 values of otoliths varied with growth temperature, making it a potential archive for paleo-climate reconstruction. Since these early studies, the clumped isotope proxy has been applied to various geological archives and there have been significant advances in calibrations, analytical developments, standardization, and data reduction efforts (Bernasconi et al., 2021;Daëron, 2021;Daëron et al., 2016;Dennis et al., 2011;Fernandez et al., 2017;Kelson et al., 2017;Meckler et al., 2014;Petersen et al., 2019;Saenger et al., 2021). These advances were necessary because of discrepancies in Δ 47 -temperature (T) calibrations and Δ 47 data acquisition regimes. Improvements have since been made in temperature (Δ 47 -T) calibrations and overall Δ 47 data evaluation but important discrepancies remain and are thought to relate to methodological differences among laboratories, for example, sample preparation and acid digestion techniques (Swart et al., 2019), choice of 17 O correction parameters (Daëron et al., 2016;Schauer et al., 2016), mineralogy and acid fractionation dependencies (Petersen et al., 2019), standardization of measurements (Bernasconi et al., 2021;Dennis et al., 2011), among others. For example, differences in calibration slopes among laboratories are partly due to the choice of 17 O correction parameters (International Union of Pure and Applied Chemistry [IUPAC]) parameters versus Gonfiantini parameters (Daëron et al., 2016;Gonfiantini et al., 1995;Schauer et al., 2016) adopted in the data processing. Thus, the use of IUPAC parameters is recommended since it allows convergence of most datasets to statistically similar calibration slopes within reasonable analytical uncertainties (Petersen et al., 2019). These parameter sets relate to the differences in the choice of the slope of the triple oxygen isotope line (λ) that describes the fractionation of 17 O relative to 18 O, and the absolute 13 C/ 12 C and 17 O/ 16 O, 18 O/ 16 O ratios of VPDB and VSMOW, respectively (see Brand et al., 2010;Daëron et al., 2016 for details).
Given the recent progress in the development of the clumped isotope proxy and community-wise consensus on acquiring Δ 47 data through carbonate based standardization (Bernasconi et al., 2021) and IUPAC parameters (Petersen et al., 2019), we revisit the Δ 47 -T relationship published for otoliths (Ghosh et al., 2007) and further assess its applicability to select samples from a Miocene habitat. It must be noted that, the Ghosh et al. (2007) otolith calibration was derived based on Δ 47 data acquired through the conventional heated gas anchors and Gonfiantini parameter sets. In addition, despite the potential for otolith clumped isotope composition as a useful paleo-climate archive, we are aware of a few studies assessing its reliability. For example, carbonate clumped-isotope thermometry was used to detect and reconstruct prehistoric processing methods in skeletal aragonitic molluscs (shells) and fish (otoliths) . In this paper, we reanalyzed some of the otoliths reported by Ghosh et al. (2007) Zachos et al., 2001). During MMCO, the global annual surface temperature was on average about ∼3°C-4°C higher compared to present-day. Interestingly, climate models predict MMCO-like temperature conditions for the 22nd century given the current CO 2 emission rates (You, 2010;You et al., 2009). MMCO has been widely accepted as a possible analog of climate change in the future (Lee et al., 2021;You, 2010 (Froese & Pauly, 2021) for the individual fish species. We established average growth temperatures with a precision of ±2°C based on the latitude, longitude, and depth of the fish habitat using data from https://www.nodc.noaa.gov. The formation temperature of the carbonate in the fish otoliths was inferred to be the mean temperature of the habitat, averaged over the specimens' estimated lifetimes.
Additionally, five specimens of L. lutjanus otoliths were collected at Chennai, India from a commercial fishing catch from Bay of Bengal water in October 2011 with a typical fish weight ∼50-70 g. L. lutjanus species are "cold-blooded" (i.e., they cannot regulate their body temperature), schooling species that live around coral reefs and coastal areas in tropical marine waters of the Indo-West Pacific. The general habitat of these fishes is bathydemersal marine with a depth range of 0-96 m.

Fossil Otoliths
Fossil otolith samples belonging to "genus Ambassidarum" sp. and "genus Gobiidarum" sp. were recovered from the carbonaceous clay unit of Quilon Formation exposed at the Pozhikkara cliff section along the Kerala coast, southwest India ( Figure 2). Based on previous studies, the depositional settings of the recovered otoliths were ascribed to be an open marine shelf with local coral reef occurrences in settings shallower than 20 m proximal to the coast (Narayanan et al., 2007;Raha et al., 1983). The age of the Quilon Formation is assigned as lower 4 of 14 Miocene (Burdigalian) based on the foraminiferal biostratigraphy (Jacob & Sastri, 1952;Raju, 1978;Rasheed & Ramachandran, 1978). This lower Miocene (Burdigalian) age also corresponds to the global warming event MMCO (∼17-15 Ma). Details on the stratigraphy and age of the Quilon Formation are provided as Text S1 in Supporting Information S1.  Table 1.

Figure 2.
Geographic locations of Pozikkara cliff section, Quilon Formation, southwest India and its lithostratigraphy of the location. Miocene otoliths "genus Ambassidarum" and "genus Gobiidarum" were sampled from the carbonaceous clay layer of the section.
Geographically, modern-day Ambassid fishes (family Ambassidae) are widely distributed throughout the shallow waters of the Indo-Pacific region (i.e., South Africa, India, New Guinea, and Australia) (Martin & Heemstra, 2015) while Gobiids (Family: Gobiidae) are essentially marine and brackish water, bottom dwellers sustaining tropical and sub-tropical shallow waters.
The lower Miocene (Burdigalian) fish otolith specimens for paleo-temperature reconstruction were recovered following the procedure as described in Kapur et al. (2019), but, without the utilization of chemicals. Preservation of otoliths was assessed using SEM and cathodoluminescence imaging of thin sections and XRD analysis of powder (Text S2 and S3 in Supporting Information S1). The results suggest that the analyzed otoliths have a composition of ∼ >85% aragonite, and the trace element geochemistry is consistent with minimum diagenetic alteration. However, the fact that otoliths are not 100% aragonite suggests some alteration.

Carbonate Clumped Isotope Thermometry
Carbonate clumped isotope paleo-thermometry is based upon the principle that the formation temperature of carbonates is proportional to the relative abundance of 13 C 18 O 16 O 2 in carbonate ion groups determined through the measurement of 13 C 18 O 16 O in CO 2 generated through acid digestion. The abundance of these multiply substituted isotopologues in carbonate minerals is governed by thermodynamic parameters (Hill et al., 2014(Hill et al., , 2020Pramanik et al., 2020;Schauble et al., 2006) Sample R 47 ratios are normalised to a reference gas whose composition has achieved a stochastic distribution.

Sample Treatment for Carbonate Clumped Isotope Analysis
The modern fish otoliths of L. lutjanus from the Bay of Bengal and the lower Miocene (Burdigalian) fossil otoliths specimens belonging to "genus Ambassidarum" sp. and "genus Gobiidarum" sp. used for proxy validation and paleo-environment reconstruction were analyzed in the OASIS Stable Isotope Lab, Centre for Earth Sciences, Indian Institute of Science (IISc), Bangalore, following the "breakseal" method described in Fosu et al. (2019). All samples were cleaned with 30% H 2 O 2 for 12 hr to remove any organic material. The samples were subsequently weighed, finely crushed, and stored in glass vials. An aliquot weighing ∼5-8 mg was digested individually by reacting with anhydrous 105% phosphoric acid (H 3 PO 4 ) at 25°C; for several hours (>16 hr). The produced CO 2 was extracted cryogenically in a vacuum line apparatus using liquid nitrogen (at ∼−196°C) and an ethanol + liquid nitrogen "slush" (held at ∼−90°C). In this way, the CO 2 was transferred through a series of U-traps avoiding potential re-equilibration with H 2 O. The CO 2 gas (devoid of H 2 O and other trace gases) was further purified to remove organic and other potential contaminants (hydrocarbons and halocarbons) by use of a gas chromatograph (custom-made Porapaq-Q column, 2 m length, 1/8″ diameter, 80-100 mesh) held at −10°C. Samples gas was transferred through the Porapaq trap using ultra-high purity He streams at 10 mL/min. CO 2 yields were monitored with a capacitance manometer before and after cleaning to ensure complete transfer; samples with low yield were discarded. The purified CO 2 was analyzed in dual-inlet mode on a Thermo Fisher Scientific MAT 253 mass spectrometer within a few hours after cleaning.
The MAT 253 Isotope Ratio Mass Spectrometer (IRMS) was configured to simultaneously analyze masses 44-49 of CO 2 . Masses 47-49 were measured with 10 12 Ω resistors. Masses 48 and 49 were monitored to ensure that there were no isobaric interferences due to the presence of contaminants (Huntington et al., 2009), given that these interferences can lead to spurious Δ 47 measurements. Each analysis involved 60 measurement cycles of the sample CO 2 and reference CO 2 (six acquisition lines with 10 cycles each, with a signal integration time of 8 s). The compression in the dual inlet bellows was sufficient to maintain the CO 2 mass-44 ion beam intensity at a voltage of 10-12 V for the start of each acquisition. Sample analyses were covered in two different sessions. The analysis for modern fish otoliths of L. lutjanus from the Bay of Bengal was carried out in June-August 2013. The working gas (a high purity 99.999% CO 2 from Linde AG, Munich, Germany) used in this session has a composition of δ 13 C: −4‰ VPDB and δ 18 O: 25.19‰ VSMOW. Otoliths specimens belonging to "genus Ambassidarum" sp. and "genus Gobiidarum" sp., from the Quilon Formation, were analyzed in February 2019. The composition of the working gas during this period was −3.92‰ and 25.58‰ for δ 13 C and δ 18 O, respectively.
Modern otoliths from LACM used for the revised calibration experiment were analyzed in the Tripati Stable Isotope Lab at the University of California Los Angeles (UCLA), using methods described in Defliese and Tripati (2020). All otolith samples were finely crushed and stored in glass vials. The NuCarb automated carbonate device, interfaced with the Nu Instruments Perspective IRMS, allows small amounts of carbonate samples (15-150 μg) to be reacted in separate reaction vials maintained at 70°C. The pre-weighed samples along with carbonate standards, including ETH 1-4 (Bernasconi et al., 2018), as well as several in-house standards, were loaded into individual vials and reacted sequentially by controlled injection of ∼1 ml of phosphoric acid. The evolved CO 2 was purified using a Porapak trap and transferred cryogenically through a series of cold finger apparatus whose temperature is regulated by varying the amount of liquid nitrogen. The cleaned gas was ultimately transferred via micro inlet followed by isotopic measurement on the high resolution, high sensitivity Nu Perspective IRMS. Crucial steps such as vial positioning, the vial pump out, delivery of acid, CO 2 collection, sample clean-up, and transfer via the micro-volume, matching of reference, and sample intensities before data acquisition were all automated.
The Nu Instruments Perspective mass spectrometer at UCLA is a relatively new design. The Perspective has a Nier-type ion source and magnetic sector; however, it features a pair of quadratic lenses after the magnetic sector, and cups 47, 48, and 49 are shielded by small electrostatic sector analyzer that can be tuned to screen out secondary background ions. Analyses were performed in micro-volume mode, with balanced sample and reference gas volumes that allow the gas to deplete at precisely matched rates, thereby increasing the efficacy of gas usage. Each cycle lasts around 60 s, and measurements range from 70 to 30 nA for the mass 44 signal. The working gas used was Oztech CO 2 (Safford, AZ, USA) that has a δ 13 C value of −3.6‰ (VPDB) and δ 18 O value of 25.0‰ (VSMOW).

Standardization
The evaluation of Δ 47 data in both IISc and UCLA laboratories follow a near-identical approach. Differences arise from the full carbonate-based standardization scheme used in UCLA whereas a combination of equilibrated gases and carbonate standards were used in the IISc lab. Δ 47 values of carbonate standards common to both labs are however comparable and are within the analytical uncertainties of measurements in the respective laboratories. For example, Carrara marble from UCLA (CM Tile) yielded a mean value of 0.380 ± 0.028 (1σ, n = 20), whereas, Carrara marble standard from IISc (MARJ1) yielded a mean value of 0.393 ± 0.025 (1σ, n = 14).
Briefly, Δ 47 results at IISc were standardized using a set of equilibrated CO 2 gases of varying bulk isotopic composition in combination with several carbonate standards (Carrara-1, OMC, IAEA-603, NBS-19, and NBS-18). The CO 2 gases were equilibrated at 25°C by exchange with water for a minimum of 72 hr and at 1,000°C by heating in an oven (in flame-sealed quartz tubes) for 2 hr. All equilibrated gases were subject to the same cleaning and handling procedure as carbonate samples/standards, as discussed above, except that they were not reacted with phosphoric acid. The analytical data were evaluated using the Easotope software (John & Bowen, 2016).
At UCLA, a carbonate based standardization method (Bernasconi et al., 2018(Bernasconi et al., , 2021Defliese & Tripati, 2020;Upadhyay et al., 2021) involving carbonate standards (ETH 1-4) of varying bulk isotopic and Δ 47 composition was used. The performance of the mass spectrometer and the consistency of the carbonate-based standardization were monitored using additional standards such as IAEA-C1, IAEA-C2 and Merck and in-house carbonate standards such as Carrara marble, Veinstrom, CM Tile, and Caramel Chalk. The carbonate based normalization has the benefit of avoiding the time intensive preparation of equilibrated gases, and ensuring identical treatment of both standards and samples. However, this requires analyzing multiple standards during the run (preferably a 50:50 sample to standard ratio), reducing sample throughput. All the data were processed using the Easotope software (John & Bowen, 2016) The external precision (calculated from replicate analyses of an in-house laboratory standard) was 0.05‰ for δ 13 C and 0.1‰ for δ 18 O. The external precision of Δ 47 measurements was typically 0.03‰, which is consistent with the shot-noise limits for ion-beam intensities and the analytical duration (≥1 hr) consisting of several repeat analysis of sample CO 2 . Furthermore, R 48 and R 49 values were routinely monitored for all sample gases as a quality check.

Reconstruction δ 18 O and δ 13 C of the Environmental Water
The environmental water composition of present day Bay of Bengal was determined using the relationship between temperature and the 18 O/ 16 O fractionation factor in marine habitat (1,000 ln α = 18.56 (10 3 TK −1 ) − 32.54) by Thorrold et al. (1997). Since previous studies have indicated the depositional settings to be shallow marine proximal to the coast (Narayanan et al., 2007;Raha et al., 1983), the paleo-environmental water composition was determined assuming a brackish habitat (1,000 ln α = 18.39 (10 3 TK −1 ) -34.56) by Willmes et al. (2019). The effect of the choice of fractionation factor on the final reconstructed δ 18 O-water is discussed in Text S4 in Supporting Information S1. Carbon isotope ratios of the samples were also used to estimate the δ 13 C composition of paleo-water using a mass balance approach (Kalish, 1991a). First, the percentage of metabolically derived carbon in the modern otoliths was calculated using the δ 13 C values, δ 13 C of seawater, and carbon reservoirs in the form of metabolic by-products (Text S5 in Supporting Information S1). Following Kalish (1991b), the proportional contribution of metabolic carbon in the body fluid was estimated, from which the paleo-seawater δ 13 C was subsequently derived.

Revised Otolith Calibration
Normalization schemes for Δ 47 values can affect data interpretation, thus, we herein consider two prominent scales, the traditional Carbon Dioxide Equilibrium Scale (CDES) and the recently introduced Intercarb-Carbon Dioxide Equilibrium Scale (I-CDES).
First, we compare the mean Δ 47 value determined for each otolith sample against independent constraints on growth temperature as shown in Figure 3. The equation obtained for otoliths on the CDES, anchored to the ETH 1-4 standards (Bernasconi et al., 2018), is as follows:     where ∆ 47 is in ‰, T (temperature) is in K, and the two-tailed p-values are calculated using a t-test. Even if the carbonate standards anchored to the otolith data set are limited to ETH 1-4 (as in Equation 2), the Δ 47 -T calibration equation in I-CDES is indistinguishable from Equation 3.
A comparison of these results to other published calibrations that were derived using the same IUPAC parameter set and data normalization (Bernasconi et al., 2018;Defliese & Tripati, 2020;Jautzy et al., 2020;Petersen et al., 2019) is illustrated in Figure 3. These studies employed either a fully carbonate-based standardization as adopted in this study (Bernasconi et al., 2018;Jautzy et al., 2020) or equilibrated gases or a combination of equilibrated gases and carbonate standards (Defliese & Tripati, 2020;Petersen et al., 2019).

Modern Otoliths
The results of five modern Bigeye snapper (L. lutjanus) otoliths from the Bay of Bengal are plotted in Figure 4. The average δ 18 O and δ 13 C values of five individual samples analyzed in this study are −1.94 ± 0.3‰ and −4.1 ± 0.29‰, respectively. The reconstructed body temperature based on clumped isotope analysis suggests a range of 18.4°C-28.4°C and a mean value of 22.3°C ± 4.2°C in comparison to 25°C-28.5°C in the mixed layer of Bay of Bengal observed in the modern day (Anilkumar et al., 2006).

The Effect of 17 O Correction and Data Standardization on Δ 47 -T Calibration
The revised Δ 47 -T calibration equation for otolith using the IUPAC parameters yields a slope and intercept of 0.0364 ± 0.005 and 0.2642 ± 0.0657, respectively. In contrast, using the Gonfiantini parameters the calibration yield a calibration slope and intercept of 0.0335 ± 0.005 and 0.2956 ± 0.0664, respectively (Text S6 in Supporting Information S1). These slopes and intercepts obtained using the different isotopic parameter sets are statistically indistinguishable and the maximum difference in reconstructed temperatures at a given calibration point is 0.7°C which is within the analytical uncertainty of our measurements. This suggests that the use of carbonate-based standardization minimizes potential differences between the slopes and intercepts in comparison to the exclusive use of equilibrated gases. In effect, the dependence of the calibration on the choice of 17 O correction parameters is minimized following consistent normalization of Δ 47 data with carbonate standards.
Despite the agreement in the different slopes and intercepts (using IUPAC vs. Gonfiantini parameters) for the otolith calibration, minor differences remain when compared to other Δ 47 -T calibrations that also adopt a full carbonate-based standardization or intersperse carbonates with equilibrated gases such as those from Bernasconi et al. (2018), Jautzy et al. (2020), Defliese and Tripati (2020), and Petersen et al. (2019). Besides the differences in mineralogy (calcite, aragonite, dolomite, or a composite) and sample types (inorganic, biogenic or a mix), it is clear from Figure 3 that calibration slopes are probably sensitive to methodological differences amongst laboratories. For the Δ 47 -T otoliths calibration, the differences in slopes for both CDES and I-CDES are statistically significant (P = 0.046 see Text S7 in Supporting Information S1 for details). The slope of the Defliese and Tripati (2020) calibration is identical to the slope of the otolith data set (this study; both data sets generated in the same laboratory) which is not the case for the other published calibrations (Figure 3). The recent Intercarb standardization exercise (Bernasconi et al., 2021) and uncertainty propagation methods (Daëron, 2021) aim to resolve these inter-laboratory differences.
In addition to the substantial errors in reconstructed temperature introduced by analytical uncertainties and other uncertainties related to the slopes and intercepts of Δ 47 -T calibrations, recent studies suggest that the direct measurement of triple oxygen isotope compositions (Δ 17 O) of reference materials may improve the accuracy and precision of Δ 47 determinations (Saenger et al., 2021). Traditionally, laboratories assume the Δ 17 O value of the carbonates (or CO 2 analyte) to be zero (i.e., Δ 17 O = 0). Such an assumption has the potential to yield small inaccuracies in Δ 47 reconstructed values, depending on the composition of the mass spectrometer working gas and data normalization procedures, that can potentially be up to 0.6°C-5.8°C (Saenger et al., 2021). While the choice of 17 O correction parameters yields negligible differences in the slopes and intercepts of the otolith Δ 47 -T calibrations (anchored to ETH standards), the use of the accurate Δ 17 O values of these and other commercial standards Saenger et al., 2021;Wostbrock et al., 2020) may improve the agreement between inter-laboratory calibrations and the overall accuracy of Δ 47 measurements.

Bigeye Snapper (L. lutjanus)
The reconstructed temperature of 22.3°C (this study) is within ±2°C of the previously measured sea surface temperature (SST) over the coastal Bay of Bengal (Anilkumar et al., 2006). The δ 18 O of water reconstructed from otoliths belonging to L. lutjanus is −1.7 (±0.5‰) ( Table 2) which is identical to previously reported depleted oxygen isotope signals (i.e., δ 18 O is <−1‰) for the Bay of Bengal surface waters during the winter months of November to February (Achyuthan et al., 2013).

Miocene Reconstruction
The lower Miocene (Burdigalian) strata that were sampled fall within a unique geologic time referred to as the "Middle Miocene Climatic Optimum (MMCO)" when the global annual surface temperatures were on average about ∼3°C-6°C higher compared to present-day and pCO 2 levels were similar to modern levels (Böhme, 2003;Tripati et al., 2009). In the present study, the estimated range of Δ 47 based temperatures in the "genus Ambassidarum" sp. is 12.1°C-14.3°C (mean: 13.5°C ± 1°C), while Δ 47 temperature obtained for "genus Gobiidarum" sp. is 10.2°C (Table 3). These reconstructed temperatures are significantly lower than those inferred from pollen, Mg/Ca and fossil records. Reuter et al. (2013) in their report on the Early/Middle Miocene (∼17-15 Ma) pollen assemblages from the siliciclastic Ambalapuzha Formation at the coastal cliffs of Varkala in southwest Kerala, India, suggested a mean annual air temperature of 22.2°C-26.6°C based on the coexistence approach. However, Mg/Ca-SST based temperature estimates from mixed layers in the Eastern Arabian Sea (EAS) off the coast of western India suggest a temperature of ∼27.7°C between ∼16 and 15 Ma (Yang et al., 2020).
Because other proxies point to the higher atmospheric and mixed layer temperatures, we interpret the cooler temperatures inferred from our analysis of otoliths to reflect formation in an upwelling environment. Fewer studies (Faul et al., 2000;Peeters et al., 2002) on modern and fossil foraminifera have shown that organisms that calcify in an upwelling environment yield higher δ 18 O values and lower δ 13 C values. Faul et al. (2000) used the relationship between modern surface hydrography and core-top planktonic foraminiferal abundances and their isotopic composition to interpret upwelling and productivity changes in the eastern equatorial Pacific over the last 20,000 years. Peeters et al. (2002) based on the oxygen isotope composition of the foraminifera shells and vertical shell concentration profiles suggest that the depth habitat for planktonic foraminifera species (Globigerina bulloides and Globigerinoides ruber) is shallower during upwelling. Further, the carbon isotope composition recorded in the above-mentioned foraminifera species decreases as a result of lower dissolved inorganic carbon (δ 13 C DIC ) values in upwelled waters. Our reconstructed δ 13 C values of ambient seawater are between −1.5‰ and −0.2‰ compared to previously reported δ 13 C DIC 0.5‰-0.9‰ for Arabian Sea surface waters (Prasanna et al., 2016). There exists a relationship between otoliths δ 13 C and reconstructed temperature (Text S8 in  Thorrold et al. (1997). Oxygen isotope signals of Bay of Bengal surface waters during the winter months of November to February is <−1‰ (Achyuthan et al., 2013).  Supporting Information S1). This value could indicate that in this region, the fish were at or near the coast with carbon input from either surface runoff or through the resurgence of cold bottom water with isotopically lighter carbon to the surface. The recorded changes in the isotopic values and temperatures (this study) may be attributed to localized upwelling considering that previous literature generally argues that upwelling causes higher δ 18 O and lower δ 13 C values, in response to mixing with upwelled deeper, colder waters. Recently, Bertucci et al. (2018) utilized stable isotopes on modern Whitemouth croaker otoliths to test for upwelling conditions. Their results suggest that otolith samples from the eastern coast of Brazil, grown under the direct influence of the Cabo Frio upwelling were characterized by their low water temperatures and higher δ 18 O-lower δ 13 C values compared to Whitemouth croaker otoliths from the western coast of Brazil which grew under less upwelling influence. It is interesting to note that, several previous studies have also argued in favor of the prevalence of monsoonal upwelling in the Southern Arabian Sea during the Miocene (Gupta et al., 2015;Singh et al., 2011;Zhuang et al., 2017).
In the present study, the environmental water δ 18 O composition calculated from the "genus Ambassidarum" sp. yielded a value between −3.5‰ and −2.6‰ (V-SMOW) (mean: −2.9‰ ± 0.4) while of "genus Gobiidarum" sp. is −4.4‰. However, reported local δ 18 O values for seawater varies between ∼−0.82‰ and 1.99‰ during the middle Miocene (∼16 to 11 Ma) interval (Yang et al., 2020). We consider here two possibilities: (a) cryptic diagenesis in the altered part of the otoliths that show ultrastructure and mineralogy preserved (see Wang et al., 2020), and (b) that the changes in the δ 18 O values are due to kinetic effects impacting the δ 18 O of fish otoliths independently of Δ 47 (see Ghosh et al., 2006;Kimball et al., 2016;Thiagarajan et al., 2011).
An assessment of diagenetic effects on the samples (using cathodoluminescence imaging and XRD) suggest that the otoliths are composed of ∼>85% aragonite. The presence of secondary calcites may be due to the dissolution and reprecipitation of aragonite caused by the rapid influence of the hydrologic regime on the unstable otoliths aragonite under certain conditions (Weiner, 2010). The secondary calcite preferentially precipitated at ambient conditions because it is more stable than aragonite. They likely precipitated from an extraneous solution that enabled aragonite dissolution. Through this mechanism, the original composition of the otolith and isotopic signatures can be compromised. The dissolution and reprecipitation occurs on a nanoscale and affects the surface of single aragonite crystals. Therefore, a very small quantity of water is sufficient to trigger the reaction (Pingitore, 1976).
The latter scenario (kinetic effects) is also likely in the analyzed fish otoliths, causing changes in their δ 18 O values. Resolving biases due to kinetic effects in (bio) mineralized carbonates would be enhanced by paired Δ 47 and Δ 48 in future investigations, enabling further constraints on the processes responsible for isotopic disequilibrium (Bajnai et al., 2020).

Conclusions
We present a new Δ 47 -T calibration based on modern fish otolith.  (Anilkumar et al., 2006). The δ 18 O of water reconstructed from these otoliths is −1.7‰ (±0.5) which closely matches with the depleted oxygen isotope signals of Bay of Bengal surface waters during the winter months of November to February (Achyuthan et al., 2013), hence demonstrating the validity of the proposed Δ 47 -T calibration.
In addition, we attempt to quantify lower Miocene (Burdigalian) coastal conditions in southwest India utilizing fossil otoliths. The revised Δ 47 -T calibration, which was successfully tested in the case of modern L. lutjanus otoliths from the Bay of Bengal was applied to lower Miocene (Burdigalian) otoliths herein identified as belonging to "genus Ambassidarum" sp. and "genus Gobiidarum" sp. The clumped isotope-based temperature estimates (this study) of 12.1°C-14.3°C (mean: 13.5°C ± 1°C) for "genus Ambassidarum" sp., and 10.2°C for "genus Gobiidarum" sp. certainly provides constraints on coastal conditions, possibly in a region of upwelling during the MMCO, although we can not preclude diagenesis. The present investigation also opens future research avenues for assessing and predicting the coastal ecosystem in a warm climate. However, the readers should be cautious about the presence of kinetic effects influencing δ 18 O or cryptic diagenesis with aragonite-aragonite