ect of carbonate ion concentration and irradiance on calcification in foraminifera

Effect of carbonate ion concentration and irradiance on calcification in foraminifera F. Lombard, R. E. da Rocha, J. Bijma, and J.-P. Gattuso LSCE/IPSL, laboratoire CEA/CNRS/UVSQ, LSCE-Vallée, Bât. 12, avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX, France Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany INSU-CNRS, Laboratoire d’Océanographie de Villefranche-sur-mer, B.P. 28, 06234 Villefranche-sur-mer Cedex, France UPMC University of Paris 06, Observatoire Océanologique de Villefranche-sur-mer, 06230 Villefranche-sur-mer, France currently at: DTU Aqua, Technical University of Denmark, Kavalergården 6, 2920 Charlottenlund, Denmark Received: 27 July 2009 – Accepted: 10 August 2009 – Published: 1 September 2009 Correspondence to: F. Lombard (fla@aqua.dtu.dk) Published by Copernicus Publications on behalf of the European Geosciences Union.


Introduction
Due mostly to human activities, the atmospheric carbon dioxide (CO 2 ) partial pressure is currently increasing and, depending on the socio-economic scenarios, will reach 490 to 1250 ppmv by 2100 (Prentice et al., 2001).About 25% of the total anthropogenic CO 2 emissions have been absorbed by the ocean (Sabine et al., 2004).However, absorption of large quantities of atmospheric carbon implies changes in the carbonate system equilibrium, notably a decrease in pH and carbonate ion concentration ). pH has already decreased by 0.1 units compared to pre-industrial values and will further decrease by 0.3 to 0.4 unit by 2100 (Feely et al., 2004;Orr et al., 2005).Such changes may significantly influence the calcification rates of various organisms.Negative impact of ocean acidification on calcification have been reported in coccolithophores, pteropods, corals and commercial shellfish (e.g., Riebesell et al., 2000;Introduction Conclusions References Tables Figures

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Full strains may be unaffected to elevated pCO 2 (e.g., Iglesias-Rodriguez et al., 2008).Reducing the calcification rate of planktonic organisms can have opposite effects on the carbon cycle.Firstly, it decreases the positive feedback of calcification on atmospheric CO 2 (Gattuso et al., 1999;Wolf-Gladrow et al., 1999).Secondly, ocean acidification will decrease the role of ballast that calcium carbonate has by facilitating the export of organic matter to the deep ocean (Armstrong et al., 2002;Klaas and Archer, 2002).Understanding the possible effect of ocean acidification therefore requires investigating the response of the major calcifying organisms.Planktonic foraminifera are widespread calcifying protozoa, responsible for 32-80% of the global deep-ocean calcite fluxes (Schiebel, 2002).Moy et al. (2009) reported that the modern shell weight of G. bulloides is 30 to 35% lower than that measured from the sediments.They attributed the difference to reduced calcification in response to ocean acidification.Few experimental results also indicated that ocean acidification can impact planktonic foraminifera, notably by reducing their shell thickness and weight (Bijma et al., 1999;Russell et al., 2004).However, these results were obtained as a by-product of geochemical study focusing on shell composition and did not provide any quantitative estimates of calcification rates.
This article presents a re-analysis of results from different geochemical experiments, designed to provide quantitative estimates of the effect of ocean acidification on foraminifera' calcification. 3 ] was manipulated by adding NaOH or HCl to filtered sea water.Foraminifera were kept in this modified seawater in closed borosilicate glass culture vessels of 125 ml, with no headspace to prevent exchange with atmospheric CO 2 .
The carbonate chemistry of the solutions was analysed by measuring alkalinity via Gran titration using a Metrohm open-cell autotitrator with a mean precision of 10 µEq kg −1 , calibrated against certified reference material provided by A. Dickson.
Seawater pH and culture media pH were determined potentiometrically and calibrated with standard NIST buffers and are reported on the NBS scale.Alkalinity and pH were measured at the start and termination of the experiments and used to calculate initial and final carbonate chemistry using CO2SYS (Lewis and Wallace, 1998) and the dissociation constants of Mehrbach et al. (1973) refitted by Dickson and Millero (1987).
Globigerinoides sacculifer was grown at 26(±1) • C, 36.2(±0.2) salinity.Data include measurements of the initial and final size (µm), the survival time (∆t; days from collection to gametogenesis), and final weight of the shell (W f ; µg) of each specimen measured prior to isotopic analysis.Only individuals that underwent gametogenesis and grew at least one chamber were used for later analysis.The shell length vs. weight regression obtained under "ambient" [CO 2− 3 ] (233 µmol kg −1 , Fig. 1, Table 1) was used to estimate the initial shell weight (W i ; µg) from the measured initial shell size.Initial and final organic carbon weight of each foraminifera were calculated using a conversion factor (0.089 pg C µm −3 ; Michaels et al., 1995) assuming spherical shells.
The geometric average weight (W org ; µg C) was then calculated.In order to estimate calcification rates independently from the individual size, it was normalized per unit of cytoplasmic carbon (C; µg µg C −1 d −1 ): Data for O. universa were directly taken from the results obtained at 22 • C were used.Results from their experiment I and II, even though similar, were kept separate because the numbers of specimens per sample were different.The average shell length (µm) and weight (µg) of mature specimens were used to estimate the length-weight relationship within each condition.Unfortunately, critical measurements, such as initial size or survival time were not reported.
The survival time in the laboratory (∆t) was assumed to be 7.4 days as it was the mean survival time at 22 • C observed in experiments carried out at the Catalina Island laboratory (Lombard et al., 2009).All specimens grew a spherical chamber that represented 95% of the final shell weight (Lea et al., 1995;Russell et al., 2004).The initial (pre-spherical) weight of the shell (W i ) was therefore estimated to represent 5% of the final weight.The organic carbon weight (W org ) was calculated from the final size of adult O. universa (spherical form) and the specific conversion factor of 0.018 pg C µm −3 reported by Michaels et al. (1995).The calcification rate was then calculated as described in Eq. ( 1).

Results
In the G. sacculifer experiments, the average initial size was 396(±92) µm with minimum size of 190 µm and maximum size 716 µm (Table 1).Irradiance had a strong effect on both ∆t and final size.In LL condition the individuals reproduced on average two days sooner and at a smaller size (about 100 µm less) than under HL.The different [CO2− 3 ] conditions had no or only little effect on ∆t and the final size of the animals (Table 1).Only the final shell weight seemed to be influenced by [CO 2− 3 ], and individuals had generally heavier shells when grown under high [CO 2− 3 ] conditions (t-test, P <0.001 in all cases).This indicates that the shell thickness is influenced by [CO 2− 3 ] but not the general growth pattern.However, ∆t and the initial and final shell sizes influenced the final shell weight and a better indicator of calcification, insensitive to these parameters must be used.Introduction

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Interactive Discussion
The relationships between shell size and weight (Fig. 1, Table 2) better represented the influence of [CO 3 ] conditions are larger under HL conditions (Fig. 1a) than under LL conditions (Fig. 1b).For G. sacculifer, for all conditions, the exponents b of the regressions was not significantly different at various [CO whereas a is significantly different (covariance analysis on log-transformed data; Table 2).Under HL, for a given size, G. sacculifer grown at low [CO 2− 3 ] (72, 124 and 139 µmol kg −1 ), was lighter than at "ambient" (233 µmol kg −1 ) and 504 µmol kg −1 conditions and heaviest at high [CO 3 ] was larger for the largest specimens.Similar observations were made for O. universa: the exponents of all relationships were not significantly different and can be approximated by a mean exponent b of 3.42, but the parameter a is significantly different for the different relationships (Table 2).O. universa shell weights increased with increasing [CO 2− 3 ] (Fig. 1c).
Figure 2 shows the shell length and weight as a function of [CO 3 ] (µmol kg −1 ) as well as the standard deviation of these relationships was: .07 for G. sacculifer (HL)

Discussion and conclusions
The observation that [CO 2− 3 ] has an effect on the shell weight of foraminifera is consistent with previous studies (Bijma et al., 1999(Bijma et al., , 2002;;Russell et al., 2004).However, there is, up to now, no quantitative estimate of the response of foraminifera calcification to changes in the seawater carbonate chemistry.The final shell weight was impacted by both the initial shell weight and the time needed till gametogenesis (∆t).For O. universa, the weight of the initial shell, calcified in the field, was negligible (∼5% of final weight), whereas for G. sacculifer, it was about half of the final weight.∆t also varies under the two different light conditions used for G. sacculifer, with gametogenesis occurring two days sooner under high irradiance than under low irradiance.In contrast to previous estimates, the rate of calcification normalized per unit biomass was either not influenced or only slightly influenced by the initial shell size and ∆t.Hence, the [CO 2− 3 ] impact on the final weight was certainly biased in G. sacculifer and normalizing the mass increase by the time required to precipitate it, effectively calculating the rate calcification, should lead to a better approximation of the [CO 2− 3 ] effect on the calcite production.To our knowledge, this is the first report providing a first order estimate of the [CO 2− 3 ] effect on calcification rates of planktonic foraminifera.Our estimate of the calcification rate was, however, not free of biases, particularly in the case of O. universa.The initial shell size and survival time of this species in culture were not available and were estimated from independent observations.Therefore, the organic weight could not be calculated as the average weight during the experiment, but only as a function of the final shell weight.This uncertainty influences the calcification estimates of O. universa, but does not affect the conclusion that calcification decreases as a function of decreasing [CO 2− 3 ] and the final weight observations.Such bias does not occur with G. sacculifer because all the required data were available.Foraminifera calcify intermittently.They calcify new chambers every few days within only a few hours (e.g., Spero, 1988;Hemleben et al., 1989).They add an additional layer of so-called gametogenic calcite just before undergoing gametogenesis, which Introduction

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Full can account for 4 to 20% of the final weight of the shell of O. universa (Hamilton et al., 2008).Hence, foraminiferal calcification is not a constant process and our estimates are averages over the culture period involving primary, secondary and gametogenic calcite.O. universa produces a thin juvenile trochospiral test and, at the end of its life cycle, a large thick spherical chamber.This massive calcification is responsible for the high calcification rate calculated for this species (Fig. 3), which is not representative of the calcification rate when growing its trochospiral shell.It should be noted that O. universa produces this final spherical chamber over a period of several days of continued calcification.
Irradiance had a significant effect both on growth and calcification of G. sacculifer.
At low irradiance, the time between collection and reproduction (∆t) was shorter and the final shell weight as well as the rates of calcification were lower compared to high irradiance (Figs.1-3, Table 1).Calcification was 30% lower in LL than in HL.This is consistent with measurements made on G. sacculifer (Erez, 1983) and O. universa (Lea et al., 1995) which indicated rates of calcification 3 to 4 times higher in the light than in the dark, corresponding to a 66-75% decrease in dark conditions.Similar observations have also been made on other photosynthetic calcifying organisms such as zooxanthellate corals (Gattuso et al., 1999;Moya et al., 2006;Schutter et al., 2008), stressing the strong interactions between irradiance and calcification rate.The final shell weight (Figs. 1 and 2) as well as the calcification rate (Fig. 3) clearly depended on [CO 2− 3 ].Over the full range of [CO 2− 3 ] tested, calcification rates increased between 34 and 44% for G. sacculifer and 34 to 41% for O. universa, resulting in a shell weight increase between 24 to 34% for G. sacculifer and 64 to 87% for O. universa.Based on these results, the potential impact of ocean acidification on foraminifera calcite production can be estimated.For this, we assume that, in the surface ocean, year 2100 (Orr et al., 2005).Under these conditions, the present rate of calcification of G. sacculifer and O. universa would be 1.5 to 3.5% lower than preindustrial values and 5 to 10% lower than during the LGM.The present calcification would correspond to a decrease of the final shell weight of 1.1-1.6%(G.sacculifer LL and HL) to 5-7% (O.universa) compared to preindustrial conditions and 3.4-4.8%(G.sacculifer LL and HL) to 15-20% (O.universa) compared to LGM conditions.These estimated differences between present, preindustrial and LGM foraminifera weights are in the same range of values (but slightly lower) than observed in sediment cores.Globigerinoides ruber is presently 11% lighter than preindustrial specimens and 20% than LGM specimens (de Moel et al., 2009).Individuals of Globigerina bulloides sampled in sediment cores exhibit a 30 to 35% decrease in weight since the LGM (Barker and Elderfield, 2002;Moy et al., 2009).Our results suggested that in 2100, the rate of calcification of G. sacculifer and O. universa could be reduced by 6 to 13% compared to present rates, leading to shell weights reduction of 20 to 27% for O. universa and of 4 to 6% for G. sacculifer.The magnitude of this potential decrease is consistent with that projected for some zooxanthellate corals (Langdon and Atkinson, 2005) and oysters (Gazeau et al., 2007), and lower than other observations on corals (Langdon and Atkinson, 2005), mussels (Gazeau et al., 2007) or pteropods (Comeau et al., 2009).Other planktonic foraminifera may have a higher sensitivity to [CO 2− 3 ].Indeed, O. universa and G. sacculifer have numerous symbiotic algae that facilitate their calcification, whereas numerous other species, notably temperate to cold water species that are naturally exposed to waters more depleted in CO 2− 3 , are not symbiotic.Hence, it is possible that the decrease of [CO 2− 3 ] may have a larger effect on non-symbiotic species than on symbiotic species.This may explain the larger decrease of shell weight between LGM to modern conditions observed for the non-symbiotic species G. bulloides (Barker and Elderfield, 2002;Moy et al., 2009) compared to the symbiotic G. ruber (de Moel et al., 2009).Consequently, there is a need to assess the effect of [CO estimate the effect of reduced pH on shell dissolution during sedimentation, but also to estimate the combined effect of decreased pH and elevated temperature.Indeed, at higher temperatures, foraminifera are usually more abundant (B é and Tolderlund, 1971), have higher growth rates (Lombard et al., 2009) and larger shell sizes (Schmidt et al., 2006).Hence, the predicted increase in temperature could increase the production of calcite by foraminifera, counteracting the negative impact of ocean acidification.
The combined effect of temperature and [CO 2− 3 ] thus need to be investigated in order to estimate the impact of global environmental changes on foraminifera.

BGD Introduction
Full Schutter, M., Van Velthoven, B., Janse, M., Osinga, R., Janssen, M., Wijffels, R., and Verreth, J.: The effect of irradiance on long-term skeletal growth and net photosynthesis in Galaxea fascicularis under four light conditions, J. Exp.Mar. Biol. Ecol., 367, 75-80, 2008.8597 Spero, H. J.: Ultrastructural examination of chamber morphogenesis and biomineralization in the planktonic foraminifer Orbulina universa, Mar. Biol., 99, 9-20, 1988. 8596 were still influenced by ∆t and the initial size of the individuals.Only under ambient [CO 2− 3 ] (233 µmol kg −1 ) was the shell-length-weight relationship not significantly different between HL and LL.The HL and LL data at ambient [CO 2− 3 ] were therefore combined and used to estimate the initial shell weight of individuals based on the initial shell size.On average, the initial shell weight represented 35% of the final weight under HL and 61% under LL.Consequently, the shell size-weight differences observed between the various [CO 2− (455 and 566 µmol kg −1 ).Only two groups can be identified at LL with heavier shells at [CO 2− 3 ] of 233, 455, and 566 µmol kg −1 and lighter shells at concentrations of 72, 124, 139 and, surprisingly, for 504 µmol kg −1 .In both conditions, the difference in weight as a function of increasing [CO 2− of shell size.Since the initial weight of G. sacculifer accounts for a large part of the final weight, only a final size of 700 µm was considered in order to minimize the pre-culture (field-grown) contribution to shell mass.For a similar size, the final shell weight for both G. sacculifer and O. universa increased significantly with increasing [was greater for O. universa compared to G. sacculifer, greater for large individuals of O. universa and also greater under HL than under LL for G. sacculifer.The final shell weight of G. sacculifer obtained in LL was 20 to 26% lower than under HL.From previous weights and survival time measurements, calcification rates normalized per unit biomass were calculated.The biomass-normalized rate of calcification significantly decreased with decreasing

Table A1 of
Russell et al. (2004)and only Introduction 2−

Table 1 .
G. sacculifer initial and final mean size (Is and F s), final weight (F w) and estimated initial weight (I e w), duration of the experiment from collection till gametogenesis (∆t) and mean weight increase (∆w) under the different [CO 2− 3 ], total alkalinity (T A), pH and irradiance levels.See TableA1ofRussel et al. (2004)for similar information on O. universa.

Table 2 .
Parameters of relationships between length (L) and shell weight (W ) obtained for the different species, under different [CO 2− 3 ] and light conditions for G. sacculifer.All the relationships are expressed as W =aL b .Covariance analyses on log-transformed data were used to test the effect of [CO 2− 3 ] and irradiance on the final shell weight.* : P <0.01; * * : P <0.005; * * * : P <0.0001.