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J Paleolimnol (2015) 53:35–45 DOI 10.1007/s10933-014-9805-3 ORIGINAL PAPER Oxygen isotope analysis of multiple, single ostracod valves as a proxy for combined variability in seasonal temperature and lake water oxygen isotopes Yama Dixit • David A. Hodell • Rajiv Sinha Cameron A. Petrie • Received: 18 May 2014 / Accepted: 1 October 2014 / Published online: 11 October 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Paleoclimate studies in lakes typically use oxygen isotopic ratios in samples that consist of multiple ostracod specimens, to obtain an average d18O value that reflects the mean temperature and d18O of lake water over the life spans of the combined individuals measured. This approach overlooks potential information on seasonal climate variability that is recorded in the single valves of short-lived ostracods. Here we estimate seasonal variability in ostracod d18O by measuring 10–30 individual carapaces of Cyprideis torosa in selected stratigraphic levels of a sediment core from paleolake Riwasa in Haryana, India. The mean d18O values of ostracod populations show a general decrease from 9.6 to 8.3 kyr BP, which was interpreted previously as resulting from strengthening of the Indian summer monsoon during the early Holocene. The d18O measurements of single ostracods within samples show a large range (up to *15 %) and standard deviation (up to ±3.3), suggesting high seasonal variability in the hydrology of this playa lake. The great variability is ascribed to changes in both seasonal temperature (16 °C) and d18O of lake water in a drying water body. The latter is attributable to the Rayleigh distillation process, described using a Craig– Gordon model for isotopic fractionation during evaporation from an open water body. Our results suggest that the range of d18O values measured in single ostracod carapaces is useful to evaluate seasonal changes in lake temperature and hydrology. Even with great intra-sample d18O variability, however, the mean d18O of multiple (more than 10) ostracods can be used to infer long-term climate trends. Keywords Single ostracod Paleolake Oxygen isotopes Indian summer monsoon Introduction Y. Dixit (&) D. A. Hodell Godwin Laboratory for Palaeoclimate Research, Department of Earth Sciences, University of Cambridge, Cambridge CB2 3EQ, UK e-mail: [email protected] R. Sinha Department of Civil Engineering, Indian Institute of Technology, Kanpur, India C. A. Petrie Department of Archaeology and Anthropology, University of Cambridge, Cambridge CB2 3DZ, UK Isotopic measurements of ostracods from lake sediment cores are used extensively for paleoclimate reconstruction in continental settings. It is common to measure samples containing more than ten ostracod valves, depending on size, to obtain an average d18O value for each stratigraphic level (Decrouy et al. 2011; Pérez et al. 2013). Ostracod assemblages within a single stratigraphic layer are composed of multiple generations of shells that formed at different times, and the d18O of multiple valves reflects the average 123 36 conditions over which the individuals lived. Ostracods molt and secrete new calcareous carapaces within a short time, on the order of days to months (Horne et al. 2002). The range of isotopic values measured on individual ostracod valves from a single stratigraphic level can be used to estimate the seasonal range of d18O variability (Schwalb et al. 2002; Holmes 2008; Escobar et al. 2010). Previous studies focused on the number of ostracod valves per stratigraphic level needed to provide reliable estimates of mean conditions (DeDeckker 1983; Chivas et al. 1986; Holmes 2008). A few studies used intra-sample d18O and d13C variability for seasonal paleolimnological reconstruction (Heaton et al. 1995; Xia et al. 1997; Jones et al. 2002; Escobar et al. 2010). Schwalb et al. (2002) collected valves of dead ostracodes from ephemeral ponds and lakes in Patagonia, Argentina and stable isotope data showed a wide scatter, suggesting that each sample provides a snapshot of the seasonal history of the host water. Xia et al. (1997) studied single ostracod valve geochemistry (d18O, Mg/Ca, and Sr/Ca) in Candona rawsoni from lakes of the American Midwest and suggested that 10–15 valves from each stratigraphic level are required to remove high-frequency variability. Jones et al. (2002) studied the optimal sample size required to obtain reliable estimates of the mean climate signal and reported up to an 8 % range in d18O within samples from lake sediments in Turkey. Escobar et al. (2010) used ostracods in lacustrine sediments from the Yucatan Peninsula to calculate optimal sample size and assess the potential for using intra-sample d18O variability in ostracod and gastropod shells to infer short-term climate change. Here we present oxygen isotope measurements on more than ten individual shells of the ostracod Cyprideis torosa in 19 stratigraphic levels from the early Holocene of paleolake Riwasa, located on the semi-arid plains of Northwest (NW) India (Fig. 1). The objective of this study was to measure d18O of multiple, single ostracods in each stratigraphic level and determine whether the data provide information on seasonal climate variability. The d18O values of individual ostracod valves within a stratigraphic horizon showed very high variability, ranging from 4.3 to *15 %. This high within-sample variability can be attributed to both changes in temperature and hydrology. We used the Craig–Gordon model for fractionation during evaporation, which is based on 123 J Paleolimnol (2015) 53:35–45 the Rayleigh process of distillation, to explain the seasonal changes in temperature and lake water d18O in this playa. The study complements Dixit et al. (2014b), who measured 10–20 specimens of C. torosa in samples from a nearby well section at Riwasa to reconstruct long-term changes in the intensity of the Indian summer monsoon. Study area We collected sediment cores from paleolake Riwasa, at the northeast edge of the Thar Desert in NW India (Fig. 1). Paleolake Riwasa is represented by a lensoidal body of silty clay spread over more than 2,000 km2 of the Bhiwani District of Haryana. The Riwasa area is principally composed of four geomorphic units: granite-rhyolite inselbergs, sand sheets, sand dunes and shallow lacustrine depressions (Saini et al. 2005) (Fig. 1). The thickness of the lacustrine sediments varies from 0.4 to 2.5 m (Saini et al. 2005). The lake sediments contain abundant, well-preserved fossil ostracod and gastropod shells. Today, the region receives 300–500 mm of rainfall annually, of which about 80 % falls during the summer season (June to September) from the Bay of Bengal arm of the southwest summer monsoon. The remaining 20 % falls during winter months (Saini et al. 2005). Climate data from the nearest meteorological station at Hissar, located 70 km northwest of Riwasa, indicate mean annual precipitation is 446 mm. Mean of the minimum monthly air temperatures over the last 50 years ranged from 17.4 °C in January to 32.8 °C in May (Indian Meteorological Department 2000). Riwasa was a closed-basin lake during the Holocene that received water by direct rainfall on the lake and runoff from the catchment. Greatest hydrologic loss from the basin occurred by evaporation. Materials and methods In December 2008, two holes (25.1 and 34.4 m deep) were drilled into the dry lakebed near Riwasa village (N28°470 20.900 , E075°570 29.700 ) using a Calyx drilling system. Complete recovery of the sequence was not achieved because of the occurrence of a 12-cm-thick hardground that was encountered at 1.67 m. This hardground occurs at a level of 108–120 cm in a nearby exposed well section located *60 m J Paleolimnol (2015) 53:35–45 37 Fig. 1 a Map of northwest India showing Riwasa (black triangle), b Riwasa region showing core sampling location (star) and well-section (circle). Also shown in the map are the villages of Riwasa and Dhani Mahu (black shapes), lacustrine deposits (blue) and inselbergs (black with white outlines). (Color figure online) northwest of the coring site (Dixit et al. 2014b). The hardground was dated at 8.2 kyr BP, and formed when Lake Riwasa dried completely (Dixit et al. 2014b). For this study, we used sediment from the cored section below the hardground at 167 cm. The sequence between 237 and 167 cm was deposited in the early Holocene and contains abundant, well-preserved ostracods that are suitable for isotopic analyses. We did not measure individual ostracods below 237 cm because of low ostracod abundance below this level. 123 38 Between 10 and 30 well-preserved specimens of the adult ostracod C. torosa were picked from each stratigraphic horizon for oxygen isotopic measurement. C. torosa inhabits a wide range of aquatic habitats with low Alk/Ca (Mezquita et al. 2005). This species is mainly found in the meiobenthos on soft sediments in shallow brackish environments where large amounts of organic detritus are present. C. torosa is euryhaline and tolerates salinities up to 60 % (Heip 1976). It requires permanent water to reproduce because its eggs cannot withstand desiccation (Anadón et al. 1986). Temperature plays an important role in the life cycle of this species and the minimum temperature required for the moulting stage is more than 15 °C for complete development of adult C. torosa (Heip 1976). In Belgian lakes, C. torosa has one generation annually and moulting occurs only during the summer season when temperatures are above 15 °C (Herman et al. 1983; Marco-Barba et al. 2012). Because the minimum mean monthly air temperature ranges from 17.4 to 32.8 °C, and for shallow lakes like Riwasa the mean lake water temperature follows the ambient air temperature closely (Haslett 2001), we assume C. torosa moults throughout the year in this region. C. torosa secretes its shell at near oxygen isotopic equilibrium with lake water (Durazzi 1977; MarcoBarba et al. 2012). Males and females show no difference in isotopic values (Marco-Barba et al. 2012). The drill core was sampled at *4-cm intervals between 237 and 167 cm, corresponding to *9.6–8.3 kyr BP. Individual valves of adult specimens of the ostracod C. torosa were picked from the sieved 425–500-lm fraction of each sample. Prior to isotope analysis, ostracod valves were cleaned ultrasonically in methanol to remove organic material, and rinsed in water before drying. Valves were subsequently checked for cleanliness using a binocular microscope. Individual ostracod valves were weighed and loaded into glass vials for oxygen isotope analysis. The average weight of a C. torosa valve was *63 lg and permitted isotope analysis of single valves using a VG PRISM Mass Spectrometer and Multicarb preparation system in the Godwin Laboratory at the University of Cambridge. Analytical precision for d18O was estimated at ±0.1 % by repeated analysis of Carrara Marble. Results are reported relative to Vienna Pee Dee Belemnite (VPDB). 123 J Paleolimnol (2015) 53:35–45 Table 1 Isotopic composition of oxygen and deuterium in a few groundwater samples taken from wells near Riwasa and adjacent areas Location d18O dD N28°45.8850 : E075°57.0240 -3.03 -28.73 N28°47.1360 : E075°57.3830 -7.76 -53.55 N28°48.0600 : E075°57.4480 -4.68 -37.23 0 -5.57 -39.9 N28°47.7840 : E075°57.8460 -4.58 -34.1 N28°48.3840 : E075°58.2920 -5.41 -48.93 0 N28°48.613 : E075°57.251 Carbonate content was measured on bulk sediments that were dried at 60 °C and ground with a mortar and pestle. Weight percent CaCO3 was determined by coulometric titration (Engleman et al. 1985). Analytical precision was estimated by measurement of 94 reagent grade CaCO3 (100 %) standards that yielded a mean and standard deviation (1r) of 99.97 ± 0.82 %. Samples of groundwater from six wells around Riwasa village were collected in April 2010 (Table 1). Oxygen and hydrogen isotopes in groundwater samples were measured by cavity ringdown laser spectroscopy (CRDS) using an L1102-i Picarro water isotope analyzer and A0211 high-precision vaporizer. Each sample was injected nine times into the vaporizer. Memory effects from previous samples were avoided by rejecting the first three analyses. Values for the final six injections were averaged with in-run precision of less than ±0.1 % for d18O and ±1 % for dD (1 standard deviation). Calibration of results to V-SMOW was achieved by analyzing internal standards before and after the six water samples. Internal samples were calibrated against V-SMOW, GISP and SLAP. All results are reported in parts per thousand (%) relative to V-SMOW. Results Lithostratigraphy The sediment core is characterized by yellow–brown, well-sorted aeolian sand from the base up to 320 cm (Unit I), which grades to grayish-brown shelly sand (Unit II) from 320 to 229 cm. For this study, we used sediments from 237 to 229 cm from Unit II, and from 229 to 167 cm in Unit III, the latter consisting of J Paleolimnol (2015) 53:35–45 39 Fig. 2 Lithostratigraphy and correlation of calcium carbonate content between the Riwasa drill core and well section of Dixit et al. (2014a, b). The highest carbonate content at 167 cm in the core correlates with the base of the hardground in the well section at 120 cm. The hardground was poorly recovered by the Calyx drill. The mining of kankar and backfilling of sediments led to reworking of the top 1.5 m of sediments in the drill cores, introducing an offset of 47 cm between the drill cores and well section. The core depths were corrected by 47 cm and the age model of the well section was applied massive calcareous grayish-white lacustrine mud with abundant gastropod and ostracod shells. Unit III in the drill cores corresponds to the interval between 180 and 120 cm in the well section described by Dixit et al. (2014b) (Fig. 2). The top of Unit III in the well section is capped by a 12-cm hardground (Dixit et al. 2014b). In the drill cores, a few calcretic remains of the hardground were recovered at 167 cm, but the Calyx drill did not recover this layer completely. The mining of kankar and backfilling of sediments led to reworking of the top 1.5 m of sediments in the drill cores, introducing an offset of 47 cm between the drill cores and well section. Core depths were adjusted by 47 cm by correlating the calcium carbonate content from the well section to the drill cores (Fig. 2). The chronology of the drill cores was established by correlation to the well section, adopting the radiocarbon dates of Dixit et al. (2014b) (Fig. 2). Oxygen isotopes of ostracods The data consist of 320 individual oxygen isotope measurements from 19 stratigraphic horizons deposited during the early Holocene. The number of individual ostracod (C. torosa) shell measurements (n) from each horizon ranged from 10 to 30. The mean d18O shows a general decrease from *?2.7 to *1.9 % up-section, between 9.6 and 8.3 kyr BP. The d18O record of individual specimens of C. torosa within each stratigraphic horizon shows very high variability. At Riwasa, the average intra-sample d18O range is 8.7 %, and varies from a minimum of 4.3 to a maximum 14.9 % during this period, with the maximum range (-7.9 to ?7.1 %) at 9.2 kyr BP. The intra-horizon single-shell oxygen isotope variability is unrelated to sample size (Fig. 3). The standard deviation of d18O measurements within stratigraphic levels is ±2.6 %, ranging from ±1.4 to 3.6 % (Table 2). Oxygen isotopes of groundwater Oxygen isotopic analyses of groundwater yielded a mean value of -5.2 %. The best-fit line of d18O versus dD for groundwater from Riwasa is dD = 5.5 9 d18O - 12.148 (Fig. 4). The slope of this line is significantly less than the local meteoric water line (LMWL) calculated from the nearest International Atomic Energy Agency (IAEA) station at New Delhi, which is dD = 7.15 9 d18O ? 2.60 (Pang et al. 2004). The slope of Riwasa groundwater 123 40 J Paleolimnol (2015) 53:35–45 Fig. 3 a Single ostracod shell variability for each stratigraphic horizon from *9.6 to 8.2 kyr BP. Blue diamonds represent d18O measurements of single ostracod valves of C. torosa, red diamonds represent the mean value for each sample, and the errors bars are one standard deviation. The black line is a regression line through the mean d18O values. b The standard deviation about the mean of d18O measurements in each sample. (Color figure online) samples is 5.5, which is indicative of evaporation during rainfall and/or evapotranspiration from soils during recharge (Bhattacharya et al. 1985; Darling et al. 2006) Lake water isotope modeling Paleolake Riwasa was a closed basin during the Holocene and the only significant water loss from the lake was via evaporation. The change in oxygen isotope ratios from a drying water body can be described by the Craig–Gordon equation (1965), which takes into account evaporation and the changing isotopic composition between lake water and atmospheric water. Calculations were performed for a temperature range of 17–33 °C. The assumption made in the calculations is that water loss results from evaporation and that the conditions of evaporation (e.g. temperature, relative humidity) remained unchanged. The evolution of oxygen isotope ratios as a function of the fraction of residual lake water undergoing progressive evaporation is calculated from the following relationship (Gonfiantini 1986): 123 d18 Olake ¼ ðd0 A=BÞ f B þ A=B where d0 is the initial isotopic composition of water, f is the fraction of residual water in the lake, A/B is the isotopic composition that water attains in its final evaporation stages when f approaches zero and A and B are: ðhda þ De þ e=aÞ ð1 h þ DeÞ ðh De e=aÞ B¼ ð1 h þ DeÞ A¼ da is the d18O of the atmosphere, which can be calculated from the ambient temperature in Riwasa as, d18O = 0.39T (°C)–22.8; given the average annual temperature range at Riwasa, the calculations were performed from 17 to 33 °C, with a step of 2 °C; aw is the thermodynamic activity of water: aw = -0.000543/ f2 - 0.018521/f ? 0.99931; De, the kinetic enrichment factor: De = 0.0142 (1-h/aw); a, equilibrium fractionation factor: a = exp (1137/T2 - 0.4156/T - 0.00207) (Majoube 1971); T is the temperature in Kelvin; e is total isotopic enrichment factor: e = a - 1 J Paleolimnol (2015) 53:35–45 41 Table 2 Summary statistics for single-valve ostracod d18O from 20 stratigraphic horizons of paleolake Riwasa Depth (cm) Age (kyr BP) n Cyprideis torosa d18O Mean SD 167 8.30 18 -1.9 1.9 172 8.37 18 -2.2 2.5 173 8.40 10 -1.4 1.4 177 8.44 20 -2.2 1.9 181 8.51 15 0.6 3.1 186 8.58 14 -0.6 2.7 189 8.63 10 -1.6 3.2 191 8.65 15 0.8 3.1 196 8.72 15 0.1 3.3 201 8.80 30 0.3 2.8 205 210 8.88 8.99 30 20 -0.2 1.3 2.0 2.4 215 9.09 24 1.2 2.7 220 9.20 25 1.5 3.3 221 9.24 10 0.7 2.5 225 9.31 15 1.3 2.1 229 9.42 10 0.6 2.7 234 9.52 10 0.0 2.4 237 9.59 10 2.7 1.9 The Riwasa best-fit line intersects the LMWL at d18O = -8.9 % and dD = -60.7 %, and this value is taken as the starting point for evaporation (Fig. 4). Because Riwasa was predominantly a NaCl lake, indicated by the abundance of C. torosa, which thrives in NaCl-rich waters, and the thermodynamic activity of NaCl solution (aw) is only valid up to 10 % fractional residual water (Yadav 1997), calculations for the evolution of lake-water d18O were carried out until f = 0.2 and results are shown in Table 3. Discussion Single ostracod d18O as a proxy for seasonal climate variability Assuming the ostracod calcite is precipitated in isotopic equilibrium with the lake water (Durazzi 1977; Marco-Barba et al. 2012), the d18O of C. torosa reflects the temperature and d18O of lake water at the time of moulting. C. torosa moults throughout the year Fig. 4 dD–d18O plot of groundwater samples (black) collected in the vicinity of paleolake Riwasa. Delhi MWL is the local meteoric water line calculated using precipitation data from New Delhi (Pang et al. 2004). The best-fit line (BFL) for Riwasa samples falls below the Delhi MWL with r2 = 0.84 and is described by dD = 5.5 9 d18O - 12.148; the slope of the line (5.5) is typical of evaporating water bodies. The Riwasa BFL intersects the Delhi MWL at -8.9 and -60.7 % for d18O and dD, respectively, which equals the isotopic composition of precipitation at Riwasa and the initial composition taken for modeling of the lake water in this region and therefore records the seasonal range of both temperature and lake water d18O. The annual temperature range is about 15 °C, which could only account for up to 3.8 % variation in d18O, assuming C. torosa undergoes continuous moulting. The d18O of lake water is controlled by the amount and d18O of rainfall and evaporation. Riwasa receives 80 % of its total rainfall during the summer monsoon (June to September), with a weighted mean of -10.7 %, and the rest during winter, with a weighted mean of ?1 %. Changes in monsoon timing and intensity affect the lake water d18O by both altering the d18O of rainfall and changing the hydrologic balance (evaporation/ precipitation) of the lake (Dixit et al. 2014a). Results suggest that the d18O of lake water varies greatly with changing lake volume and temperature. Previously, Yadav (1997) measured the stable isotopic composition of Sambhar Lake and adjacent ground waters to study the evolution of the oxygen isotopic composition of Sambhar Lake during the annual precipitation and evaporation cycle. The study suggested that the d18O of atmospheric water vapor is the 123 42 Table 3 Parameters used to calculate the evolution of d18O in drying lake Riwasa at a range of temperature and corresponding atmospheric oxygen isotopic compositions following the Craig–Gordon model for initial oxygen isotopic composition (d0) of -8.9 %, the intersection of the evaporative and MWL J Paleolimnol (2015) 53:35–45 Temperature (°C) d18Oa Lake water d18O at fraction residual lake water 1 0.8 0.6 0.4 0.2 17 -16.2 -8.9 -6.1 -3.8 2.5 21.2 19 -15.4 -8.9 -7.1 -4.3 1.4 18.1 21 23 -14.6 -13.8 -8.9 -8.9 -7.3 -7.5 -4.8 -5.3 0.2 -0.9 14.9 11.9 25 -13.1 -8.9 -7.7 -5.9 -2.2 8.7 27 -12.3 -8.9 -7.9 -6.4 -3.4 5.6 29 -11.5 -8.9 -8.1 -6.9 -4.5 2.5 31 -10.7 -8.9 -8.3 -7.5 -5.7 -0.6 33 -9.9 -8.9 -8.5 -7.9 -6.9 -3.8 Fig. 5 a Modeled changes in the d18O of lake water with evaporation and temperature using the Craig–Gordon (1965) equation and assuming an initial lake water d18O = -8.9 %. b Modeled d18O of ostracod calcite as a function of both temperature and changing lake water d18O with evaporation most sensitive variable affecting the evolution of lake water d18O and the back-condensation of isotopically light atmospheric water vapor dominantly controls the evolution of lake water d18O during the annual evaporation cycle (Yadav 1997). For the temperature range of 17–33 °C in the Riwasa region, the evolution of d18O of lake water is shown in Fig. 5a. As the d18O of ostracod calcite depends on both the temperature and the isotopic composition of water in which the shell was formed, the following relationship can be used to calculate the d18O of ostracod calcite (Shackleton 1974): dw, obtained from the Craig–Gordon equation and various temperatures within the seasonal temperature range 17–33 °C at a step of 2 °C (Fig. 5b). The calculated d18O values of calcite encompass the range of observed d18O variability of up to *15 % in a single stratigraphic level. We conclude that the high intra-sample d18O variability observed for paleolake Riwasa is explained by its high surface to volume ratio and the consequent large seasonal changes in temperature and evaporation/precipitation under a monsoon climate. T ¼ 16:9 4:38 ðdC dW Þ þ 0:1 ðdC dW Þ2 where dc is the oxygen isotopic value of shell calcite relative to PDB and dw is the oxygen isotopic value of the lake water relative to SMOW. We solved the equation for dc, using the lake water isotopic values, 123 Long-term changes in mean d18O of ostracods Despite the great inter-shell d18O variability within a sample, the mean d18O of all ostracod valves in a sample shows a general decreasing trend from 9.6 to 8.3 kyr BP (Fig. 3). The regression line through the J Paleolimnol (2015) 53:35–45 Fig. 6 The ostracod d18O variability and regression lines of drill cores (red diamonds) and Riwasa well section (purple circles) between *9.6 and 8.3 kyr BP. The slopes of both the lines show a decrease in mean d18O during the early Holocene. (Color figure online) mean d18O has a slope of *?2.3 % per kyr. A similar trend occurs in the well section, with a slope of *?2.7 % per kyr, which was interpreted as resulting from increased summer monsoon rainfall and decreased E/P in the region (Fig. 6) (Dixit et al. 2014b). Intensification of the summer monsoon during the early Holocene is also recorded in marine records from the Arabian Sea (Overpeck et al. 1996; Schulz et al. 1998; Anand et al. 2008), speleothems from Qunf cave in Oman (Fleitmann et al. 2003), and speleothems and lake sediments from China (Hodell et al. 1999; Wang et al. 2005; Zhang et al. 2011; Cai et al. 2012). Our results suggest that the mean d18O of [10 single ostracod valves from each stratigraphic level records the mean climate state and provides a reliable record of long-term climate variability. We used the student t test to estimate the optimum sample size, which is the minimum number of individual valves that achieves acceptable error for each stratigraphic level: n ¼ ðt s=EÞ2 where t is the student-t value for a given level of significance, s is the standard deviation, and E is the acceptable error (Holmes 2008). We assume 10 % acceptable error based on the observed d18O change over the entire stratigraphic sequence, which is 4.6 %. We estimate 10 valves as a minimum sample size to achieve a 10 % error, which is consistent with previous studies (Heaton et al. 1995; Xia et al. 1997; Holmes 2008; Escobar et al. 2010). 43 To test whether the long-term decrease in d18O in the Riwasa well section could occur merely by chance, we tested if sample means are statistically different from one another, given the observed d18O variability of single valve measurements. We used the student-t test to construct a 19 by 19 matrix of p-values of significance for each set of pairs in the sample population. The mean of the oldest sample at 9.6 kyr BP is significantly different from sample means from 8.8 to 8.3 kyr BP. Similarly, the sample mean of the youngest sample at 8.3 kyr BP is significantly different from the sample means at 9.6 to 8.65 kyr BP. We further tested if the slope of the regression line through the mean d18O is significantly different from zero. The small p value (1.593 9 10-5) indicates a high probability that the slope is significantly different from zero. Holmes (2008) suggested that the ultimate test of whether low-frequency trends are significant is to replicate results in a parallel stratigraphic section. We replicated the results of this study, in the well section and drill cores. Despite the great intra-sample d18O variability, the mean d18O of more than ten ostracod valves reflects long-term trends in the mean hydrologic conditions of the lake (Fig. 6). Summary and conclusions We measured d18O of single ostracod valves from selected stratigraphic levels in the early Holocene sequence of paleolake Riwasa. Our results indicate high d18O variability of single ostracods in each sample, with a range of values from 4.3 to 14.9 %. This high variability is superimposed on a longer-term trend of decreasing mean d18O values between 9.6 and 8.3 kyr BP. The great within-sample d18O range can be explained by seasonal changes in temperature and lake water d18O in this playa lake. Despite this high intra-sample variability, a long-term decrease in mean d18O was discerned by measuring more than ten ostracod valves per sample. This trend is related to intensification of the Indian monsoon in the early Holocene (Dixit et al. 2014b). Acknowledgments This work was supported by the Natural Environment Research Council (NE/H011463/1). Yama Dixit was funded by the Gates Cambridge Trust and Learning and Research Funds from St. John’s College, Cambridge. We thank Mike Hall, James Rolfe and Jeannie Booth for analytical 123 44 assistance. Many thanks to Prof. R. N. Singh, (BHU), Vikas Pawar and Sandeep Mallik for logistical field support. Ajit Singh helped with sediment core sampling. Thanks also to Thomas Guilderson for AMS radiocarbon dating at the Center for Accelerator Mass Spectrometry (CAMS), Lawrence Livermore National Laboratory (California, USA) and Ayan Bhowmik for helpful discussions. References Anadón P, Deckker P, Julià R (1986) The Pleistocene lake deposits of the NE Baza Basin (Spain): salinity variations and ostracod succession. 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