Calibration of δ 18 O of laboratory-cultured deep-sea benthic foraminiferal shells in function of temperature

Calibration of δO of laboratory-cultured deep-sea benthic foraminiferal shells in function of temperature C. Barras, J.-C. Duplessy, E. Geslin, E. Michel, and F. J. Jorissen Laboratory of Recent and Fossil Bio-Indicators (BIAF), UPRES EA 2644, CNRS-INSU UMS 3281 OSUNA, Angers University, 49045 Angers Cedex, France Laboratory of Marine Bio-Indicators (LEBIM), Ile d’Yeu, France Laboratoire des Sciences du Climat et de l’Environnement (LSCE), CEA-CNRS-UVSQ, UMR1572, Gif-sur-Yvette, France Received: 16 December 2009 – Accepted: 17 December 2009 – Published: 18 January 2010 Correspondence to: C. Barras (christine.barras@univ-angers.fr) Published by Copernicus Publications on behalf of the European Geosciences Union.


Introduction
Stable oxygen isotopes of carbonate microfossils are one of the most widely used tools in paleoceanography.The temperature dependency of oxygen isotope fractionation has previously been quantified on the basis of inorganically precipitated calcite (Urey, 1947;McCrea, 1950;O'Neil et al., 1969;Kim and O'Neil, 1997), and has been verified for living organisms in field and/or laboratory cultures of corals (Reynaud-Vaganay et al., 1999), molluscs (Epstein et al., 1953) and planktonic foraminifera (Erez and Figures Back Close

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Printer-friendly Version Interactive Discussion Luz, 1983;Bouvier-Soumagnac and Duplessy, 1985;Bouvier-Soumagnac et al., 1986;Bemis et al., 1998).For benthic foraminifera, until now, all existing temperature calibrations are based on core top material.On the sea floor, not only temperature and the isotopic composition of the seawater influence the 18 O/ 16 O composition of foraminiferal calcite, but also other factors, such as the carbonate ion effect (Spero et al., 1997;Zeebe, 1999;Rathmann and Kunhert, 2008), microhabitat effect (McCorkle et al., 1997), vital effects (Duplessy et al., 1970) and diagenetic processes may strongly influence the δ 18 O of carbonate microfossils.Since all these factors are interfering in the natural environment, only culture experiments can precisely reveal the influence of a single parameter, such as temperature.
Several laboratory studies have been performed to study the oxygen isotopic fractionation in planktonic and shallow water benthic foraminifera (e.g., Erez and Luz, 1983;Bouvier-Soumagnac and Duplessy, 1986;Chandler et al., 1996;Spero and Lea, 1996;Spero et al., 1997;Bemis et al., 1998).On the contrary, experiments with deep-sea benthic foraminifera are very scarce (Wilson-Finelli et al., 1998;McCorkle et al., 2008;Filipsson et al., 2010).Actually, the growth of deep-sea benthic foraminifera takes much longer than for planktonic foraminifera so that the experiments in stable conditions have to last for periods extending to several months.However, benthic foraminifera present the indisputable advantage that they can reproduce in the laboratory (Hintz et al., 2004;McCorkle et al., 2008;Barras et al., 2009;Filipsson et al., 2010).It is therefore possible to measure the isotopic composition of shells entirely calcified in controlled conditions.
In order to obtain the results presented in this paper, we developed new laboratory protocols to produce large quantities of Bulimina marginata shells in controlled and stable conditions and over a large range of temperatures (4-19 • C), making it possible to investigate the influence of temperature on the δ 18 O of deep-sea benthic foraminiferal calcite.The large amount of foraminiferal shells produced allowed us also to investigate the effect of test size on isotopic fractionation. 2 Material and methods

Experimental protocols
For this study, adult specimens of B. marginata, sampled in the Bay of Biscay at 450 and 650 m depth, were used in different experiments to obtain reproduction and subsequent growth of the juveniles (detailed protocol in Barras et al., 2009).Before their introduction in the experiments, adults specimens were labelled using calcein-tagging method (Bernhard et al., 2004) in order to distinguish specimens that totally calcified their shells in our controlled experiments (not fluorescent) from the adults initially added (partly fluorescent).Two different laboratory setups were used to obtain reproduction and growth of B. marginata under stable physico-chemical conditions: 1) a closed system (CS I and CS II ), with 25 l microfiltrated (0.45 µm) natural seawater circulating through a reservoir and different experiment bottles, and 2) a Petri dish system (PD) where half of the seawater was renewed twice per week.Between 30 and 190 adult specimens of B. marginata were introduced in each experiment, which were regularly fed with fresh Phaeodactylum tricornutum diatoms.In all experiments, which lasted from 43 to 108 days, we obtained production and growth of juveniles of Bulimina marginata.Therefore, the isotopic composition of foraminiferal calcite was measured on tests of Bulimina marginata entirely calcified under controlled laboratory conditions (not fluorescent specimens).
Temperature was recorded inside the incubators (standard deviations range from 0.1 to 1.1 decrease of pH by 0.3 units between the start and the end (average of 7.79±0.09,NBS-scale) occurred in the six experiments, whereas alkalinity remained stable, and similar to the other systems (2487±14 µmol l −1 ).In the hypothetical case of linear growth of the shells during the experimental period, this gradual decrease of pH by 0.3 units could theoretically result in a positive δ 18 O shift of about 0.15‰ of the newly formed foraminifera, due to the carbonate ion effect (Zeebe, 1999).However, benthic foraminifera do not have a uniform growth, chamber addition being faster during early ontogenetic stages (Bradshaw, 1957;Stouff et al., 1999;Barras et al., 2009).

Analytical procedures
Oxygen isotopic analyses were performed on 10 to 150 entire specimens of B.
marginata.In order to study the ontogenetic effect on the 18 O/ 16 O ratios of the shells of deep-sea benthic foraminifera, specimens were separated into different size fractions (length measurements with microscale).Observation of the shells under the stereomicroscope showed that they were very clean (i.e.transparent, free of mineral adherences), therefore specimens were only rinsed with deionised water before analyses.
All tests were then roasted at 380 were converted from VSMOW to VPDB by subtracting 0.27‰ (Hut, 1987).We applied linear regression to our data sets since this provided equally good fits as quadratic regression.The choice of linear or quadratic equations was discussed by Bemis et al. (1998).The coefficient of determination (R 2 ) and the standard errors on the slope and intercept are indicated for each equation.

Influence of temperature on the δ 18 O of cultured foraminifera
Knowing that shell size may have an effect on isotope ratio in foraminifera (Spero and Lea, 1996;Bemis et al., 1998;Elderfield et al., 2002;Schmiedl et al., 2004) and given size fraction (Fig. 1).For the ≤150 and 150-200 µm size fractions, where sufficient data are available, we used Lin's test (Lin, 1989) to estimate the concordance of the regression lines for the three systems.For all cases, we obtained concordance correlation coefficients above 0.990, confirming the high degree of similarity of the data obtained with the three systems.Therefore, the eventual effect of the pH decrease in CS II was not high enough to significantly shift the δ 18 O of foraminifera calcified in these experiments.Since the δ 18 O of B. marginata appears to be independent of the applied protocol, in the following text we will no longer distinguish the three experimental setups.
The linear equations which best describe the relationship between temperature and Equations ( 1)-( 3) exhibit similar slopes considering the standard errors on the slope estimates.For these three size fractions, the relative influence of temperature on the oxygen isotopic composition of B. marginata is −0.22‰ • C −1 .For the >250 µm size fraction, the linear regression between δ 18 O f −δ 18 O w and temperature presents a steeper slope (Eq.4).However, the linear regression for this size fraction is less well defined than that obtained for the smaller size fractions, since data are available only for three different temperatures and only few individuals attained a size larger than 250 µm.Further experimental work is needed to refine this Eq.( 4), which we will not consider in the remaining part of this paper.

Influence of shell size on the δ 18 O of cultured foraminifera
Interestingly, there is an increase in the intercept values with increasing size fraction (15.25, 15.73 and 16.00, respectively for the size fractions ≤150, 150-200 and 200-250 µm; Fig. 1e), indicating a shift towards higher δ 18 O values with increasing size.In Fig. 2, individual δ 18 O measurements are presented as a function of test size for the three temperatures for which we had a sufficient amount of different size fractions to obtain a reliable regression equation (p<0.01).Figure 2 shows that at 10.2, 12.7 and 14.7 • C, the δ 18 O of the foraminiferal tests increases by 0.0012-0.0022‰µm −1 , with determination coefficients (R 2 ) between 0.4 and 0.7 (p<0.01).The influence of size on oxygen isotopic composition is well established for planktonic foraminifera (Spero and Lea, 1996;Bemis et al., 1998;Elderfield et al., 2002), whereas previous field-based studies of size-dependent trends in benthic foraminiferal isotopic values have been in-Introduction

Conclusions References
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Full conclusive (Vincent et al., 1981;Dunbar and Wefer, 1984;Grossman, 1987;Corliss et al., 2002).Generally, in these studies, benthic foraminifera do not show a significant change in δ 18 O with size.Schmiedl et al. (2004), however, who studied the ontogenetic effect on the isotopic fractionation of Uvigerina mediterranea, found a 0.3-0.4‰enrichment in δ 18 O over a size range of 175 to 1250 µm.This enrichment was particularly strong in the early growth stages (100-300 µm) and became weaker for adult forms.
If we compare the slope of their logarithmic correlation equation for these younger stages (the size fraction we studied) with our data, their δ 18 O versus test size curve has an average slope of about 0.001‰ µm the averaged size effect on δ 18 O composition of B. marginata is 0.0014‰ µm −1 .It appears therefore that an ontogenetic effect on oxygen isotope fractionation exists also for benthic foraminifera and cannot be neglected in paleoceanographic studies.Since the regression lines of δ 18 O f −δ 18 O w versus test size are more or less parallel for the tested temperatures, we conclude that the mechanism responsible for this small ontogenetic effect is independent of calcification temperature.We recommend to perform measurements in a size range not larger than 50 µm to fully exploit the 0.07‰ accuracy of mass-spectrometric analyses.precipitated calcite for a temperature range between 10 and 40 • C (Eq. 5).

Comparison with equilibrium calcite as defined by
We compared our experimental calibration equations with the Kim and O'Neil (1997) equation because this equation was established in controlled laboratory conditions as in our study, and measurements were performed on inorganic calcite, free of vital effects.The three experimental regression curves we determined for size fractions smaller than 250 µm exhibit similar slopes as the least square regression line applied to the quadratic relationship of Kim and O'Neil (1997) over the studied temperature range (Fig. 3).Therefore, the influence of temperature on the δ 18 O of calcite is similar, and independent of test size.Furthermore, the offsets of the foraminiferal curves with respect to the inorganic carbonate curve are very small.Regression lines ( 2) and (3) fit well with the Kim and O'Neil (1997)  conspicuous ontogenetic effect on δ 18 O values of about 0.0014‰ µm −1 that should be taken into account in order to produce accurate paleoclimatic reconstructions.Bulimina marginata specimens with a test length between 150 and 250 µm calcify very close to the equilibrium calcite as defined by Kim and O'Neil (1997).Finally, these experiments, leading to reliable data, proved that the foraminiferal treatment protocols developed for this study could be applied in future studies to investigate the impact of other physicochemical parameters (salinity, carbonate chemistry, etc.) on benthic foraminiferal shell composition (isotopes, trace metals, etc.).

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• C during 45 min to remove all organic matter.The 18 O/ 16 O ratio of foraminiferal calcite was measured with Isoprim and VG-Optima massspectrometers.Results are expressed as δ=((R sample −R standard )/R standard ) * 1000, where R is the 18 O/ 16 O isotopic ratio.The analytical precision of the δ 18 O analyses is ±0.05‰ relative to the VPDB (Vienna Pee Dee Belemnite) standard.Seawater δ 18 O (δ 18 O w ) were measured by equilibrating water samples with pure CO 2 which was subsequently analysed with a Finnigan Mass spectrometer.The analytical precision of the δ 18 O analyses is ±0.05‰ relative to the VSMOW (Vienna Standard Mean Ocean Water) standard.In order to determine the relationship between temperature and δ 18 O of B. marginata shells, we calculated least square regressions of the isotopic difference between foraminiferal shell and seawater (δ 18 O f −δ 18 O w ) versus temperature.The δ 18 O w data Figures Back Close , our data were treated according to four different size fractions to consider this possible effect on B. marginata: ≤150 µm, 150-200 µm, 200-250 µm and >250 µm.For each of the four size fractions, we plotted the oxygen isotopic composition of the foraminiferal shell of B. marginata (δ 18 O f −δ 18 O w ) as a function of the different temperatures tested in the experiments (Fig. 1a-d).The 18 O/ 16 O composition of B. marginata appears similar for the 3 experimental protocols (CS I , CS II and PD) for a given temperature

δ
18 O of foraminiferal tests entirely calcified in controlled laboratory conditions are, for = 15.73(±0.14)− 4.49(±0.13)* (δ 18 O f −δ 18 O w ) for 150 − 200µm (2) T ( • C) = 16.00(±0.33)− 4.61(±0.37)* (δ 18 O f −δ 18 O w ) for 200 − 250µm (3) T ( • C) = 16.93(±0.23)− 5.31(±0.23)* (δ 18 O f −δ 18 O w ) for > 250µm (4) Kim and O'Neil (1997) Among the numerous paleotemperature equations published since the 1950's, Kim and O'Neil (1997) reinvestigated the relationship of O'Neil (1969) based on inorganically equation (taking into account the standard errors), suggesting that for the 150-200 and 200-250 µm size classes, the biological effect is negligible.Over the investigated temperature range of 4 to 19 • C, the difference on the temperature estimates between Eqs. (2), (3) and the Kim and O'Neil equation is at most 0.7 • C.However, the calibration equation of cultured B. marginata for the 200-250 µm fraction is closer to Kim and O'Neil relationship than the equations derived for smaller size fractions.Additional measurements are necessary to accurately study the δ 18 O of size fractions larger than 250 µm.4ConclusionsThe new protocols developed for this study allowed us to obtain reproduction and calcification of the deep-sea benthic foraminifer Bulimina marginata in controlled conditions at 12 different temperatures between 4 and 19 • C. In general, a 1 • C decrease in calcification temperature increases the δ 18 O of Bulimina marginata by +0.22‰, irrespective of the size fraction and culture setup considered.This effect is similar to the thermodynamical effect observed for inorganic calcite.However, our data show a small but Introduction
• C depending on the incubator).Culture water samples were collected every 3 to Introduction −1 which is similar to the size effect found in our experiments.Also McCorkle et al. (2008) and Filipsson et al. (2010) observed an ontogenetic effect on the δ 18 O of Bulimina aculeata/marginata shells obtained in laboratory experiments.On the basis of all our 83 δ 18O measurements performed on specimens of B. marginata which totally calcified in controlled conditions, we applied a multiple regression that takes into account δ 18 O of the shells, calcification temperature (4-19 • C) as well as test size (50-300 µm).According to this multiple regression, Lesourd, M., and Debenay, J.-P.: Laboratory observations on asexual reproduction (schizogony) and ontogeny of Ammonia tepida with comments on the life cycle, J. Foramin.Res., 29, 75-84, 1999.Urey, H. C.: The thermodynamic properties of isotopic substances, J.Chem.Soc., 562-581,  1947.Figures