Controls on ostracod valve geochemistry: Part 2. Carbon and oxygen isotope compositions

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Geochimica et Cosmochimica Acta 75 (2011) 7380–7399

Controls on ostracod valve geochemistry: Part 2. Carbonand oxygen isotope compositions

Laurent Decrouy a,⇑, Torsten Walter Vennemann a, Daniel Ariztegui b

a Institut de Mineralogie et Geochimie, Faculte des Geosciences et de l’Environnement, Universite de Lausanne,

L’Anthropole, 1015 Lausanne, Switzerlandb Section des Sciences de la Terre et de l’Environnement, Faculte des Sciences, Universite de Geneve, Rue des Maraıchers 13,

1205 Geneve, Switzerland

Available online 9 September 2011

Abstract

The stable carbon and oxygen isotope compositions of fossil ostracods are powerful tools to estimate past environmentaland climatic conditions. The basis for such interpretations is that the calcite of the valves reflects the isotopic composition ofwater and its temperature of formation. However, calcite of ostracods is known not to form in isotopic equilibrium with waterand different species may have different offsets from inorganic precipitates of calcite formed under the same conditions. Toestimate the fractionation during ostracod valve calcification, the oxygen and carbon isotope compositions of 15 species livingin Lake Geneva were related to their autoecology and the environmental parameters measured during their growth. Theresults indicate that: (1) Oxygen isotope fractionation is similar for all species of Candoninae with an enrichment in 18O ofmore than 3& relative to equilibrium values for inorganic calcite. Oxygen isotope fractionation for Cytheroidea is less dis-criminative relative to the heavy oxygen, with enrichments in 18O for these species of 1.7 to 2.3&. Oxygen isotope fractiona-tions for Cyprididae are in-between those of Candoninae and Cytheroidea. The difference in oxygen isotope fractionationbetween ostracods and inorganic calcite has been interpreted as resulting from a vital effect. (2) Comparison with previouswork suggests that oxygen isotope fractionation may depend on the total and relative ion content of water. (3) Carbon isotopecompositions of ostracod valves are generally in equilibrium with DIC. The specimens’ d13C values are mainly controlled byseasonal variations in d13CDIC of bottom water or variation thereof in sediment pore water. (4) Incomplete valve calcificationhas an effect on carbon and oxygen isotope compositions of ostracod valves. Preferential incorporation of CO3

2� at the begin-ning of valve calcification may explain this effect. (5) Results presented here as well as results from synthetic carbonate growthindicate that different growth rates or low pH within the calcification site cannot be the cause of oxygen isotope ‘vital effects’in ostracods. Two mechanisms that might enrich the 18O of ostracod valves are deprotonation of HCO3

� that may also con-tribute to valve calcification, and effects comparable to salt effects with high concentrations of Ca and/or Mg within the cal-cification site that may also cause a higher temperature dependency of oxygen isotope fractionation.� 2011 Elsevier Ltd. All rights reserved.

1. INTRODUCTION

Ostracods are micro-crustaceans enclosed in a low-Mgcalcite shell. Like other crustaceans, ostracods grow bysuccessive moulting. Their shells, which are surrounded by

0016-7037/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.gca.2011.09.008

⇑ Corresponding author. Tel.: +41 21 692 44 49; fax: +41 21 69243 05.

E-mail address: [email protected] (L. Decrouy).

chitinous membranes, fossilize easily and are often well-preserved and hence abundant in sedimentary records(Oertli, 1975). Ostracod valves have, in addition, an advan-tage over bulk sediments for geochemical studies. Byseparating the ostracod valves from the sediment, onlyauthigenic material can be analysed and any detrital influ-ence avoided (e.g., Lister, 1988). Furthermore, ostracodsare benthic animals that can populate the deepest zones oflakes where water temperature is constant. Under such con-ditions, variations of ostracod oxygen isotope compositions

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Control on oxygen and carbon isotope fractionations in ostracods 7381

directly reflect changes of the isotopic composition of lakewater and thus of climate (e.g., von Grafenstein, 2002).

Many studies have used the stable isotope compositionof ostracod fossils to reconstruct palaeoenvironmental con-ditions (e.g., von Grafenstein et al., 1999a; Ricketts et al.,2001; Schwalb, 2003; Belis and Ariztegui, 2004; Janz andVennemann, 2005; Anadon et al., 2006; Tutken et al.,2006). The use of ostracod valves as a geochemical archiveis based on the assumption that the organism crystallisedits carapace in equilibrium with water. If a vital effect is pres-ent, it should be constant over a large range of environmen-tal conditions. The presence of vital effects on isotopiccompositions of ostracods was postulated in vonGrafenstein et al. (1992). They suggested that it is largelyphylogenetic. Growing ostracods in the laboratory, Xiaand co-authors (1997) proved that ostracods do not crystal-lise at equilibrium but have d18O values higher than pre-dicted for equilibrium growth of inorganic calcite.Studying living ostracods in German lakes, von Grafensteinand co-authors (1999b) were able to show that the vital ef-fect of different species was neither dependant on tempera-ture nor on the isotopic composition of water. Laterstudies on living ostracods inhabiting an environment withconstant geochemical and physical conditions confirmedthese results (Keatings et al., 2002). However, Keatingset al. (2002) suggested different mechanisms to be responsi-ble for the non-equilibrium fractionation of ostracods rela-tive to previous results for inorganic calcium carbonateprecipitation (Kim and O’Neil, 1997; Zeebe, 1999). Keatingsand co-authors (2002) also obtained vital offsets for oxygenthat were about 0.8& higher than those published in vonGrafenstein et al. (1999b). This discrepancy correspondsto a non-negligible change in temperature of 3 �C and is be-yond analytical uncertainties. First results on the isotopecomposition of ostracod valves from sediments of LakeGeneva showed the same discrepancy (Decrouy, 2004).These preliminary results indicate the need to investigate indetail the stable isotope fractionation during ostracod valvecalcification in Lake Geneva, if the ostracod fossils of thislake are to be used for palaeoenvironmental interpretations.In addition, new knowledge on ostracod biomineralisationprocesses (e.g., Keyser and Walter, 2004) and on the inor-ganic calcium carbonate systems (Kim et al., 2006, 2007) per-mits a re-evaluation of the different hypotheses proposed toexplain the isotopic non-equilibrium in ostracods, as well asto suggest several mechanisms that may be responsible forisotopic non-equilibrium calcite formation in ostracods.

In the present study, the stable isotope compositions ofliving ostracods belonging to 15 species collected at one-month intervals during a one-year cycle at five sites from2 to 70 m water depths in Lake Geneva are examined. De-tailed knowledge on ostracod autoecology (Decrouy, 2009;Decrouy et al., in press) and environmental parameters(Decrouy et al., 2011) form the basis of the interpretation.The relation between the oxygen isotope composition ofostracods and that of water as well as temperature of waterwas examined for juveniles and adult males and females atdifferent depths. Precise knowledge on the species-specificlife-cycles permits an evaluation of the oxygen isotopefractionations. Carbon isotope compositions of ostracod

fossil valves are more difficult to interpret (e.g., Filippiet al., 1999; Schwalb, 2003) and are often not discussed atall. The complex interaction between variations in the isoto-pic composition of dissolved inorganic carbon (DIC) inmicroenvironments and the species microhabitat prefer-ences may help explain the apparent non-coherence ob-served in ostracod carbon isotope compositions (e.g., vonGrafenstein et al., 1999b; Decrouy et al., 2011). Here, spe-cial attention was paid to the relation between ostracodautoecology (life-cycle and microhabitat preferences) andcarbon isotope composition, which allows the different bio-logical and environmental controls on the carbon isotopecomposition of ostracod valves to be evaluated.

2. GEOGRAPHICAL SETTINGS AND METHODS

2.1. Study sites

Samples of ostracods were periodically taken in the natu-ral environment of Lake Geneva. This large freshwater lakelies between Switzerland and France and is made up of alarge western basin (“Grand-Lac”) and a smaller eastern ba-sin (“Petit-Lac”; Fig. 1A). Five sampling sites situated in the“Petit-Lac” and representing different water depths (2, 5, 13,33, and 70 m; Fig. 1B) have been sampled for living ostracodson a monthly basis during a one year-cycle. More informa-tion about Lake Geneva is given in Decrouy et al. (2011).

2.2. Material

2.2.1. Monitoring of physical and chemical parameters

Water temperature and pH were measured every monthat the five sites. In addition, water temperature was mea-sured continuously every 3 h from January 2006 to July2007 with data-loggers (Vemco Minilog) installed at thefour shallower sites (2, 5, 13, and 33 m water depths). Bot-tom as well as interstitial pore water was sampled periodi-cally at the five sites. Measurement techniques, samplingmethods, analytical procedures and results on water tem-perature and major ion composition are given in a compan-ion paper (Decrouy et al., 2011).

2.2.2. Living ostracods

At 13, 33, and 70 m water depths, undisturbed sedimentsrecovered with the help of a short gravity-corer were usedto collect living ostracods. When meteorological conditionswere calm, 4 to 5 cores were taken, otherwise only 2 to 3cores. At 2 and 5 m water depths, a sediment grab was usedto recover pebbles, sand, and algae. Sediments of all siteswere placed in flasks closed with a pierced lid, stored duringtransport to the laboratory of the University of Lausannein a cold box and kept refrigerated at approximately 6 �Cuntil further processing.

To facilitate ostracod separation, samples were washedin a 200 lm mesh sieve. Residues were transferred withtap water into Petri dishes. Living ostracods were takenup using Pasteur pipettes under a stereomicroscope andkilled in 30% alcohol. The animals were then stored in pureethanol. All samples were processed during 24–48 h, rarely72 h, following sampling.

Fig. 1. (A) Geographical setting of Lake Geneva and (B) location of the five sampling sites at 2, 5, 13, 33, and 70 m water depths.

δ13C

(‰ V

PDB)

δ18O

(‰ V

PDB)

std δ13Cstd δ18O

0.190.16

0.130.11

0.110.09

0.080.08

0.060.07

samplesize

range(mV)

<2 2-3 3-5 5-7 >7

5 10 15

1.75

2

2.25

2.5

0

-2

-1.75

-1.5

(‰)

Total intensities read on IRMS cup m/z=44/45/46 for 1st peak of measurement (Vs)

Fig. 2. Stable isotope compositions of carbon (black dots) andoxygen (grey triangle) of the internal standard (Carrara Marble)corrected for sample size and calibrated relative to VPDB used torecalculate delta values of ostracod samples. Dashed lines representvalue of the internal standard of 2.05& for carbon and �1.70& foroxygen, solid lines represent the 1-sigma standard deviation fordifferent sample size ranges. Note the decrease of the precision withdecreasing sample size, whereas accuracy remains stable over wholesample size range.

7382 L. Decrouy et al. / Geochimica et Cosmochimica Acta 75 (2011) 7380–7399

Dead ostracods were kept in a 4% NaOH solution for4 h to remove organic tissue. Empty and disarticulatedvalves were then thoroughly rinsed with tap water, distilledwater, and ethanol (Merck absolute R.G. for analyses), andpermitted to dry at room temperature (for ostracod pre-treatment methods, see Danielopol et al., 2002; Keatingset al., 2006; Mischke et al., 2008). All valves were then sep-arated according to species, gender, and instars followingDanielopol (1969) and Meisch (2000) and weighed sepa-rately (in general adults and A-1 instars) or by batches(small valves and A-2 instars). For analyses, valves wererecombined to reach acceptable sample size (P50 lg whenpossible). Valves of different development stages were notmixed and valves of different gender were in general ana-lysed separately. For dates with a very low number of spec-imens, valves of two different sampling sessions, but issuedfrom the same sampling site, were combined but only if theenvironmental conditions experienced by the animals wereconsidered analogous. Depending on weight and numberof available valves, one sample can consist of one to morethan forty valves. All samples were then washed under astereomicroscope with a 000 paint brush with Milli-Q water(18 Xm) to remove any impurities and rinsed with pure eth-anol before being placed in borosilicate vials for isotopicanalyses. These last manipulations were carried out in theclean lab to avoid dust contamination, especially for furthertrace element analyses.

2.3. Analytical procedures

Carbon and oxygen isotope composition of ostracodshells and standards were determined with a Gasbench IIcoupled to a ThermoFinniganplusXL isotope ratio massspectrometer (IRMS) at the Stable Isotope Laboratory ofthe University of Lausanne following the method of aciddigestion at 70 �C (Spotl and Vennemann, 2003). Becausesubsequent trace element analyses were planned, acidificat-ion was done using Merck Suprapur orthophosphoric acidhaving originally 65% weight of acid. The acid was distilledto about 96% H3PO4 by extracting water on a vacuum line.

The orthophosphoric acid was placed in a silicate glass(SiO2–pure glass), which was gently warmed up at the sametime that air and water were pumped out under vacuum. Toassess the distillation of the acid, the acid weight percentwas periodically determined using a gravimetrical method(Burman et al., 2005). The final acid was approximately96 wt.% H3PO4. For isotopic analyses, an internal labora-tory standard (Carrara Marble) calibrated to VPDB wasused to normalised the final d-values. For small samples(in general < 70 lg), values had to be corrected as a func-tion of the sample weight (estimated with 1st peak areafor m/z = 44, 45, and 46), following the method suggestedby Spotl and Vennemann (2003). Fig. 2 shows the d18O


and d13C values of all standards, corrected when necessaryfor sample size, used during the analysis period. The preci-sion decreases with decreasing sample size but accuracy ispreserved over the whole sample size range. For large sam-ples, the standard deviation for all analyses is 0.06& forcarbon and 0.07& for oxygen. Uncertainty increases to0.19& for carbon and 0.16& for oxygen for the smallestsamples (approximately 20–30 lg).

Oxygen and carbon isotope composition of carbonateare reported in the d-notation relative to VPDB (ViennaPeedee Belemnites) following the expression:

dX ¼ ðRsample=Rstandard � 1Þ � 1000 ð1Þ

where dX is the delta value of the sample for element X (Oor C) in parts per thousand (“per mil,” &) and R is the mo-lar ratio of the heavy to light isotope in the sample and inan international standard, respectively.

3. DETERMINATION OF OXYGEN ISOTOPE

FRACTIONATION FACTORS AND CALCULATIONS

FOR THE ISOTOPIC COMPOSITION OF AN

EQUILIBRIUM CALCITE

Differences between the d values of two chemical phases,X and Y, may be expressed using the isotopic fractionationfactor (aX–Y) where

aX–Y ¼ ð1000þ dXÞ=ð1000þ dYÞ ð2aÞ

or the isotope enrichment factor (eX–Y) where

eX–Y ¼ ðaX–Y � 1Þ � 1000 ð2bÞ

The isotope enrichment factor can be further approximatedby

eX–Y � DXY ¼ dX � dY ð2cÞ

3.1. Determination of oxygen isotope fractionation factors

and oxygen isotope composition of calcite grown under

equilibrium

Knowing the oxygen isotope composition of water andcalcite, the oxygen isotope fractionation factor (acalcite–water)can be calculated using Eq. (2a):

acalcite–water ¼ ð1000þ d18OcalciteÞ=ð1000þ d18OwaterÞ ð2dÞ

where d18Ocalcite and d18Owater are the respective oxygen iso-tope compositions of calcite and water expressed using del-ta notation relative to VSMOW. To convert d18O valuesgiven relative to VPDB to those relative to VSMOW, theexpression suggested by Coplen et al. (1983) was used:

d18OVSMOW ¼ 1:03091 � d18OVPDB þ 30:91 ð3Þ

acalcite–water is temperature dependent and here the expres-sion of acalcite–water and temperature as suggested by Kimand O’Neil (1997) was used:

1000 � ln acalcite–water ¼ a � ð1000=T Þ � b ð4aÞ

where T is the water temperature in Kelvin and a and b areunknown coefficients that must be determined using exper-imental data. The study of Kim and O’Neil (1997) suggests

the following expression for inorganic calcite precipitated atequilibrium:

1000 � ln acalcite–water ¼ 18:03 � ð1000=T Þ � 32:42 ð4bÞ

If oxygen isotope composition of water and temperatureare known, the expected d18O value of calcite formed inequilibrium can be assessed using expression (2d) and (4b).

3.2. Determination of carbon isotope composition of calcite

grown under equilibrium

The model proposed by Keatings et al. (2002) was usedto calculate the expected carbon isotope composition of cal-cite grown in equilibrium with water.

“The water’s total dissolved inorganic carbon (DIC)may be regarded as a mixture of dissolved CO2, HCO3

�,and CO3

�. For simplicity we will denote these three speciesas a, b, and c, and their d13C values as da, db, and dc,respectively.

If f is the concentration of a species as a fraction of theDIC, and noting that fa + fb + fc = 1, then for isotope massbalance:

dDIC ¼ fa � da þ fc � dc þ ð1� fa � fcÞ � db ð5aÞ

Using enrichment factors to express the d13C value of a car-bon species in terms of its difference from that of HCO3

�,and noting that the approximation eX–Y � dX – dY is accu-rate enough for the carbon isotope enrichment factors usedhere, the following relationship can be derived:

dcalcite ¼ dDIC þ ecalcite–b � ðfa � ea–b þ fc � ec–bÞ ð5bÞ

This relationship will be used to calculate the expected equi-librium d13C values for calcite from measurements of thed13C value of the DIC, determination of carbon speciation(f) based on the water chemistry, and published determina-tions of the equilibrium enrichment factors (Romaneket al., 1992; Zhang et al., 1995).”

4. RESULTS

Carbon and oxygen isotope compositions of ostracodvalves (d13Costra and d18Oostra) were determined for 15 spe-cies belonging to the family Candonidae (Candona candida,Candona neglecta, Fabaeformiscandona caudata, Pseudocan-

dona compressa, and Cypria ophtalmica forma lacustris), thefamily Cyprididae (Prionocypris zenkeri, Herpetocypris rep-

tans, Isocypris beauchampi, Cypridopsis vidua, Plesiocyprid-

opsis newtoni, Potamocypris similis, and Potamocypris

smaragdina), and the superfamily Cytheroidea (Limnocy-

there inopinata, Limnocytherina sanctipatricii, and Cytheris-

sa lacustris). Given the restrictions of space, onlysummarised results are given in this manuscript. Completelists of raw results can be found in Decrouy (2009).Candona candida was chosen as a representative species(see Table 1 and Figs. 3, 4 and 7).

4.1. Oxygen isotope results

To examine the relation between the oxygen isotopecompositions of ostracods (d18Oostra), water (d18OH2O),

Table 1Geochemical data of adults and juveniles of Candona candida (see text for explanation).

Depth (m) Age Sampling No. ofvalves

d13Costra

(& VPDB)Std. d18Oostra

(& VPDB)Std. Calc. time Tc Date d18OH2O d18OH2O

(& VSMOW)acalcite–water Vital effect

(&)d18O calc. Tc

(�C)

33 Ad O 10.25.06 5 �3.25 0.18 �7.33 0.15 2 weeks 7.3 1 month �12.31 1.0361 3.75 5.5Q 11.28.06 6 �6.64 0.06 �8.18 0.08 1 month 8.4 1 month �12.38 1.0353 3.12 8.9S 01.09.07 8 �6.54 0.06 �8.37 0.08 O.T. – November 07 �12.38 1.0351 – 9.7U 02.15.07 6 �6.56 0.06 �8.15 0.08 O.T. – November 07 �12.38 1.0353 – 8.7W 03.12.07 2 �6.35 0.18 �8.06 0.15 O.T. – November 07 �12.38 1.0354 – 8.3Y 04.10.07 2 �6.47 0.18 �8.18 0.15 O.T. – November 07 �12.38 1.0353 – 8.9aa 05.01.07 2 �7.08 0.18 �7.96 0.15 O.T. – November 07 �12.38 1.0355 – 7.9

A-1 M 10.04.06 4 �5.89 0.21 �7.91 0.14 2 months 7.2 1 month �12.49 1.0357 3.24 7.2O 10.25.06 6 �6.00 0.21 �7.99 0.14 2 months 7.4 1 month �12.31 1.0354 3.03 8.4

A-2 I 07.24.06 6 �7.18 0.16 �7.69 0.14 3 weeks 7.1 1 month �12.43 1.0359 3.28 6.5K 08.31.06 6 �6.33 0.16 �7.78 0.14 3 weeks 7.0 1 month �12.33 1.0357 3.33 7.3M 10.04.06 8 �6.99 0.16 �7.77 0.14 3 weeks 7.3 1 month �12.49 1.0358 3.21 6.6

13 Ad F 06.12.06 2 �6.31 0.18 �8.25 0.15 O.T. – 1 month �12.45 1.0353 – 8.9P 11.15.06 4 �3.76 0.18 �9.18 0.15 1 month 14.0 1 month �12.38 1.0343 3.38 13.4R 12.12.06 2 �4.33 0.18 �8.20 0.15 1 month 10.3 1 month �12.26 1.0351 3.41 9.5T 01.16.07 2 �6.09 0.18 �8.11 0.15 3 months 7.8 1 month �12.21 1.0352 2.89 9.4X 03.27.07 2 �6.85 0.18 �8.43 0.15 O.T. – January 07 �12.21 1.0349 – 10.8

A-1 P 11.15.06 10 �5.39 0.10 �9.76 0.06 2 months 14.4 1 month �12.38 1.0337 2.87 16.1R 12.12.06 4 �5.40 0.21 �9.17 0.14 2 months 12.4 1 month �12.26 1.0341 2.90 14.0

A-2 F 06.12.06 9 �5.39 0.05 �8.59 0.03 3 weeks 10.3 1 month �12.45 1.0349 3.17 10.4H 07.11.06 14 �5.75 0.08 �9.01 0.06 3 weeks 13.1 1 month �12.40 1.0344 3.36 12.6J 08.10.06 13 �5.17 0.08 �9.30 0.06 3 weeks 14.4 1 month �12.39 1.0341 3.25 13.9L 09.12.06 11 �6.17 0.05 �9.22 0.03 3 weeks 14.9 1 month �12.30 1.0341 3.43 14.0N 10.10.06 18 �5.68 0.08 �9.28 0.06 3 weeks 14.6 1 month �12.28 1.0340 3.40 14.3

7384L

.D

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etC

osm

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Acta

75(2011)

7380–7399

AdA-1A-2A-3A-4

AdA-1A-2A-3A-4

δ18O (‰ VPDB) 1000 / T (°K)

T (°

C)

T (°

C)

1000

ln α

calc

ite-w

ater

13 m 33 m

3.45 3.5 3.55 3.630

31

32

33

34

35

36 y = 18.01 x - 29.151r2 = 0.92

0

4

8

12

16

20

24

AB D F H J L N P R T V X Z02468

10121416

A C E G I K M O Q S U W Y aa

-8

-7

-6

-5

-4

-3

-10-9.5-8.5-8-7.5-7

A B

C D

13 m Adults A-1 A-233 m Adults A-1 A-2

δ13 C

(‰ V

PDB)

-9

Dev

elop

men

tst

ages

Dev

elop

men

tst

ages

Apr May Jun Ju

lAug Sep NovOct Dec Ja

nFe

bMar Apr

20062007Apr May Ju

n Jul

Aug Sep NovOct Dec Jan

Feb

Mar Apr

20062007

Fig. 3. Oxygen isotope composition of adult and juvenile valves of Candona candida: (A) carbon versus oxygen isotopic compositions forspecimens collected at 13 and 33 m; (B) oxygen isotope fractionation factors (acalcite–water) versus valve crystallisation temperatures calculatedon the basis of the species life cycle and linear regression best fits for specimens collected at 13 and 33 m (‘estimated’ valve crystallisationtemperatures in the text); (C) water temperature at 13 m water depth and crystallisation temperatures recalculated on the basis of ostracodsample d18O values, water sample d18O values and linear coefficient assessed in aforementioned sub-figure (3B; ‘d18O recalculatedcrystallisation temperatures’ in the text) with subjacent illustration of the ostracod life cycle; (D) same as (C) but for 33 m water depth. Smallersymbols represent samples that were not considered for linear regression calculations; grey scaled box within illustrations of ostracod life cycleillustrate the monthly relative abundance of the specific instar to the entire population, the higher the proportion is, the darker the box, date ofsampling sessions are illustrated by black triangles and their respective code (A to aa); horizontal bars illustrate “calcification periods” used tocalculate mean water temperature during valve calcification (see text for more explanations).


and water temperature, it is necessary to estimate the oxy-gen isotope composition of the water in which the ostracodmoulted and the temperature at the time of valve calcifica-tion (Tc). d18OH2O values measured throughout the year areconstant, ranging from �12.2& to �12.5& VSMOW. Incontrast, water temperature varies, especially in the littoralzone where temperatures range from 2.1 to 28 �C. The exacttime at which ostracods moulted (i.e., time of valve calcifi-cation) is unknown and has to be estimated. A first estimatecan be made on the basis of the species autoecology. Thelife-cycles were studied for all species sampled in the“Petit-Lac” (Decrouy, 2009). The life-cycle of C. candida

is illustrated in Fig. 3C and D. On the basis of these dia-grams, it is possible to postulate the approximate time ofmoulting. For example, moulting periods of the last juve-nile stage A-1 to adult at 13 m water depths begins inNovember. As no specimen of the last juvenile stage A-1was found after December, all adults found after Januarymust have reached maturity between November and Janu-ary and survived until being sampled.

The water temperature during valve calcification can beestimated using a ‘best fit’ correlation method. While theexact time during which the valve was calcified is unknown,

it is safe to assume that the ostracods moulted at a precisetime during a certain period preceding the sampling. More-over, for a high number of specimens analysed or for rela-tively constant temperatures, mean temperature precedingsampling must reflect the temperature prevailing duringvalve calcification. Average temperatures preceding thesampling were thus calculated on the basis of the continu-ous water temperature record for increasing time precedingsampling, with the value for one day corresponding tomean water temperature over the 24 h preceding sampling,the value for 1 week corresponding to mean water temper-ature over the 7 days preceding sampling, and so on. Aver-age temperatures were calculated for 1, 2, 3, 4, 5, and 6 days– 1, 2, and 3 weeks – 1, 2, 3, 4, 5, and 6 months. The lapse oftime used for the calculation (for example 3 weeks) is re-ferred to as ‘calcification time’ and the respective tempera-ture as ‘calcification temperature’ (Tc).

For carbonates crystallised at low temperature in equilib-rium, 103 lnacalcite–water is inversely proportional to 103/T(Kim and O’Neil, 1997). Hence, if ostracod calcite grows in“equilibrium” or with a constant ‘vital offset’ as suggestedby several authors (e.g., von Grafenstein et al., 1999b;Keatings et al., 2002), then 103 lnacalcite–water must plot

Keatings et al. (2002)C. candida adultC. candida juvenilePs. rostrata adultPs. rostrata juvenile

von Grafenstein et al. (1999b)C. candida adult

Xia et al. (1997)C. rawsoni

Wetterich et al. (2008)F. pedata

1000 / T (°K)y = 18.01 x - 29.15r2 = 0.92 n = 22

y = 23.76 x - 49.60r2 = 0.90 n = 117

y = 22.12 x - 43.83r2 = 0.89 n = 18

y = 20.83 x - 39.08r2 = 0.98 n = 31

y = 21.10 x - 41.40r2 = 0.97 n = 23

y = 21.10 x - 40.08r2 = 0.97 n = 188

1000

ln α

calc

ite-w

ater

C. candida

C. neglecta

F. caudata

Candoninae

y = 18.03 x - 32.42

y = 21.70 x - 43.75

y = 21.56 x - 42.80

3.35 3.4 3.45 3.5 3.55 3.629

30

31

32

33

34

35

36

37

( )

1°C

1‰

Ps. compressa

C. ophtalmica f. lacustris

Fig. 4. Oxygen isotope fractionation factors (acalcite–water) versus ‘estimated’ valve crystallisation temperatures of adult and juvenileCandonidae. Ostracod data are from Xia et al. (1997), von Grafenstein et al. (1999b), Keatings et al. (2002), Wetterich et al. (2008) and thepresent study. Fractionation factors determined by Kim and O’Neil (1997) of synthetic calcites crystallised from calcium-bearing solutions of5, 15 and 25 mM (respectively bold, dashed, and dotted line in the lower part of the graph) are also represented in each graph to facilitatecomparison.


linearly against 103/T. Given that this is valid, the higher thecorrelation coefficient (r2) between 103 lnacalcite–water and103/T values is, the better is the estimate of Tc.

As such, different ‘calcification times’ and their respec-tive ‘calcification temperatures’ are used in order to getthe highest r-coefficient possible. Note that the ‘calcificationtime’ is at first equivalent for all samples, that is the valuesof 103 lnacalcite–water for each sample are compared with ‘cal-cification temperature’ calculated for the average of, forexample, one month preceding sampling (= ‘calcificationtime’). The correlation factor is then calculated and, in asecond step, another ‘calcification time’ can be used. Thevalue of 103 lnacalcite–water for each sample is then comparedwith the ‘calcification temperature’ calculated for the aver-age of two, three weeks, and 2 months preceding samples.The ‘calcification time’ corresponding to the relation103 lnacalcite–water versus ‘calcification temperature’ withthe highest correlation factor is then considered as beingthe best estimate of the period during which ostracodsmoult before being sampled. Once this has been done,inspection of all values may reveal that some points fall sys-tematically off a linear distribution of the values. It is con-sidered that these outliers do not reflect the conditionsprevailing before sampling. This is the case, for example,for the valves collected at the end of March at 13 m (sam-pling X), which have a 103 lnacalcite–water value reflecting ahigh water temperature. These valves were sampled aftera period of low water temperature (small black squares inFig. 3B with 1000/T = 3.573 and 1000 lna(calcite–water) =34.26). It is obvious on the basis of the species life-cycle that

this sample contains valves that must have calcified betweenNovember and January while the temperature was higher.This sample is, therefore, discarded and labelled as out oftime (O.T.) in the database (see Table 1). With all O.T. sam-ples discarded, the correlation coefficient between 103 lnacal-

cite–water and 103/T will obviously increase. For Candona

candida, for example, correlation factor r2 is 0.75 with theO.T. values included, but 0.90 when these values arediscarded.

The ‘calcification time’ was generally found to rangefrom one week to several months. However, for certain spe-cies, specific environmental conditions, mainly water tem-perature, have to be fulfilled before the animal can moultto adult stage. Once this limiting temperature requirementis fulfilled, the specimen can moult to develop the subse-quent stage (i.e., A-1 to Adult or A-2 to A-1), and this oftenhappens within a short period of time. The carapace ofthese first adults (or A-1) must then have formed in onemonth or less, because for the other preceding months thetemperature was still too low to moult. For such individu-als, the ‘calcification times’ are thus not arbitrarily variedbut are fixed on the basis of the species life-cycle. SampleO at 33 m depth (see Fig. 3D and Table 1) is one suchexample. Such adjustment of the ‘calcification time’ for aspecific sample can also apply for the last occurrence ofspecimens of a certain development stage. Adults sampledat 13 m on the 16th January 2007 (sampling session “T”)must have formed their shell between November andJanuary (Fig. 3C). For these individuals, ‘calcification time’must be longer than that for the other adults. Hence, a


‘calcification time’ of three months was chosen for this sam-ple on the basis of the species life-cycle (Table 1).

A last optimisation can be done by using slightly differ-ent ‘calcification times’ for all data except the two casesmentioned above. Once again, the ‘calcification time’ hav-ing the best fit is retained for the further calculation. Differ-ent values for the isotopic composition of water, i.e., usingthe value measured at the time of sampling or values of pre-vious sampling sessions, in accord with estimated ‘calcifica-tion time’, can be used. These last modifications change thecoefficients for the best-fit regression lines only slightly. ForCandona candida, for example, the coefficients are a = 18.23and b = �30.07 and r2 = 0.90 when the d18OH2O valuesused to calculate the fractionation factors correspond tothe sampling date. When the d18OH2O values of the priormonth are used, the coefficient are a = 18.01 andb = �29.15 and r2 = 0.92. Thus, using different d18OH2O

values in this case leads to a change of a 1 to 3& of theregression coefficients. All these manipulations were at firstdone independently for each site and for each developmentstage. If the regression lines obtained for the different sitesand the different development stages and/or gender weresimilar, values were taken together and only one uniqueregression line was calculated for the species. No differencesbetween the regression lines were noted for the same speciesat the different sites. Fractionation factors are equivalentfor females and males as well as for the different develop-ment stages, except in the case of Herpetocypris reptans.The homogeneity of the fractionation factors obtained forthe different sites and the different development stages canbe assessed for C. candida in Fig. 3. These results are inagreement with those of a previous study that also didnot report any differences between the different instars(von Grafenstein et al., 1999b).

The reason why oxygen isotope fractionation is differentamong juveniles and adults of Herpetocypris reptans is asyet unclear, but may be linked to the large size of this spe-cies compared to other common freshwater species andhence a distinct vital or kinetic effect during growth and/or a local control of the direct growth environment, wheresuch effects are not evened out as less shells were used forany one analyses.

Using Eqs. (2d) and (4a), measured d18OH2O values, andmeasured d18Oostra values, it is possible to recalculate watertemperature during valve calcification for each sample(‘d18O calc. Tc’ in Table 1). Results for C. candida are com-pared to water temperature on Fig. 3C and D. The horizon-tal bars in the graphs represent ‘calcification time’. Thiskind of representation, together with the subjacent life-cy-cles, permits the pertinence of determined ‘calcificationtime’ to be tested. Note that the same graph can be con-structed using only the raw data, d18Oostra being placedon a second inverse Y-axis and adjusted to the water tem-perature record. For both Y-axis scales, the relation1& � 0.25 �C has to be used. This is possible becaused18OH2O values are homogeneous.

Isotopic fractionation factors of inorganic calcite crys-tallised in solutions of 5, 15, and 25 mM of calcium deter-mined by Kim and O’Neil (1997) are also represented inFigs. 3B and 4 for comparison with the oxygen isotope frac-

tionation among different ostracod species and betweenostracods and synthetic calcite. These authors consider,for several reasons, that calcite grown in the most dilutesolution, i.e., 5 mM of Ca2+, crystallised in equilibriumwith water. The fractionation factor determined for thissolution is thus taken to represent equilibrium for thewater–calcite system.

To conclude, information given in Fig. 3 permits theoxygen isotope data to be evaluated and summarised. Iden-tical figures can be given for each species (see Decrouy,2009).

4.2. Carbon isotope results

A comparison of the d13C values of the ostracods withmonthly d13CDIC values does not indicate any obvious cor-relation. This does not necessarily mean that ostracodvalves do not form in isotopic equilibrium with the DIC,but instead may be explained by: (1) that the d13CDIC valuemeasured once every month does not reflect the precised13CDIC value prevailing during valve calcification or (2)the d13CDIC values measured in the water overlying the sed-iment are not equal to the d13CDIC value in the microenvi-ronment where calcification occurred. The first hypothesisis supported by a linear positive correlation betweend13CDIC values measured at 2, 5, and 13 m water depthsevery month and the water temperature at the time of sam-pling. In contrast, the correlation is absent when d13CDIC

values are compared to the mean temperature calculatedfor different periods of time preceding sampling (Decrouyet al., 2011). This suggests that d13CDIC values reflect thecondition at the time of sampling only and not the meanconditions prevailing during the preceding periods. Thisalso implies that the values may vary from one day to thenext (for a detailed discussion, see Decrouy et al., 2011).As ostracod d13C measurements represent one to tens ofvalves, the d13Costra value reflects the condition prevailingduring the exact time of valve calcification in the case wherea single ostracod is analysed, or a mean condition over anextended period when a number of ostracods have beenanalysed. In both cases d13CDIC values simply cannot beused to estimate the d13CDIC values prevailing during valvecalcification. Positive correlations between d13Costra valuesand ‘calcification temperature’ are obtained for certain spe-cies though (Decrouy, 2009). As d13CDIC and water temper-ature taken at the time of sampling are positively correlated(Decrouy et al., 2011), this observation supports thehypothesis that d13Costra values reflect d13CDIC values dur-ing valve calcification. Consequently, d13Costra values werenot examined as a function of their sampling dates in orderto assess the carbon isotope fractionation but examined asannual means. Box plots were used in Fig. 5 to represent thecarbon isotope composition of females, males, adults, andA-1 and A-2 juvenile stages of Candonidae, Cyprididae,and Cytheroidea.

Concerning possibility (2) above, it is evident thatd13Costra values should be compared to d13CDIC values ofthe ambient water within which the ostracod calcified itscarapace. Hence, assessing the spatial variability of d13CDIC

values is crucial for this point. Sampled water was limited

1000 / T (°K)P. zenkeri

I. beauchampi

Cyprididae

y = 18.03 x - 32.42y = 21.70 x - 43.75y = 21.56 x - 42.80

y = 20.06 x - 37.20r2 = 0.95 n = 34

y = 18.24 x - 30.37r2 = 0.88 n = 12

y = 19.56 x - 35.35r2 = 0.99 n = 10

y = 18.71 x - 32.09r2 = 0.91 n = 17

y = 23.89 x - 50.621r2 = 0.98 n = 6

y = 22.72 x - 46.17r2 = 0.99 n = 7

y = 18.66 x - 32.45r2 = 0.96 n = 11

1000

ln α

calc

ite-w

ater

H. reptans adult

H. reptans juvenile

C. vidua

Plesio. & Potamo.

3.35 3.4 3.45 3.5 3.55 3.628

29

30

31

32

33

34

35

1°C

1‰

Keatings et al. (2002) H. reptans adult H. reptans juvenileChivas et al. (2002) A. robusta

Li and Liu (2010) E. mareotica

Fig. 5. Same as for Fig. 4 but for Cyprididae. Ostracod data are from Chivas et al. (2002), Keatings et al. (2002), Li and Liu (2010) and thepresent study.


to the layers occurring a few centimetres above the sedi-ment–water interface (‘bottom water’). As such, thed13CDIC values were measured on lake water, which, be-cause of the action of waves and bottom currents, is likelyto be quite homogeneous but may be different from thatwithin the sediment pore space. Ostracods are benthic ani-mals and it can be assumed that the location where calcifi-cation occurs is fixed by the species-specific microhabitatpreferences. d13CDIC values in these microenvironmentscan be different compared to those in ‘open’ water column.For example, phytophylous species generally feed on algae.Due to the photosynthetic growth of the algae the DIC ofthe ambient fluid in this microenvironment can be relativelydepleted in 12C compared to the water column above thismicroenvironment. In contrast, infaunal species that liveburrowed within the sediment, where water DIC can bestrongly affected by release of CO2 enriched in 12C due toremineralisation of organic matter may also have lowerd13C values. An epifaunal habitat (i.e., living on the surfaceof the sediment) presents the same problems because theDIC pool at the sediment–water can contain DIC addedby diffusion from the underlying sediment. Mean d13CDIC

values of interstitial pore water at the three deepest sites(13, 33, and 70 m) can be approximated though, as is out-lined in a parallel study on the sediment interstitial porewater geochemistry (Decrouy et al., 2011).

To examine whether ostracod valves crystallised in equi-librium with DIC or not, the d13Costra values are comparedto the d13C values of a calcite (d13CCaCO3) that precipitatesin equilibrium with DIC according to Eq. (5b) in Figs. 7and 8. d13CCaCO3 values were calculated for all monthlywater samples and for interstitial water samples using themeasured d13CDIC values, pH, and temperature.

5. DISCUSSION

5.1. Biomineralisation in ostracods: state of the art

In order to discuss the different mechanisms that mayaccount for isotopic fractionation during valve calcifica-tion, it is necessary to briefly describe what is known aboutthe structure and biomineralisation processes of ostracodvalves. Biomineralisation in ostracods has not been as in-tensely studied as for other organisms such as foraminifera,corals, or microbial mats (e.g., Dove et al., 2003). However,several studies have investigated ostracod biomineralisation(e.g., Turpen and Angell, 1971; Keyser and Walter, 2004).

The carapace of ostracods consists of two dorsally artic-ulated valves, which in most groups, including all freshwaterspecies, are mineralised with low magnesium calcite(Kesling, 1951; Sohn, 1958). Like other crustaceans, ostrac-ods develop by successive moulting (ecdysis). Only the laststage (the adult) is fully formed and sexually mature, butall development stages possess a more or less calcified cuticle.At each moult, the cuticle and the carapace is shed and dis-carded. A new carapace is calcified in a period of a few hours(Turpen and Angell, 1971) to a few days (Roca andWansard, 1997). Turpen and Angell (1971) showed that, inHerpetocypris, the calcium in the valve is derived from theambient water and neither recycled during moulting norstored in the animal prior to moulting. However, Fassbinder(1912) was able to culture fully calcified Cypridopsis vidua incalcium free water, suggesting that metabolic sources of cal-cium and metabolic fluids must also play a role in the forma-tion of valve calcite. The calcified shell consists of smallcrystallites embedded in a chitinous and protein matrix (Bateand East, 1972, 1975; Langer, 1973; Rosenfeld, 1979;


Keyser, 1982). The shell can be completely made up ofcalcite crystals as in Cytheroidea, or composed of parallelchitinous lamellae together with a layer of crystallites as inCypridoidea (Keyser and Walter, 2004). On the basis ofSEM images and XRF-analyses of animals selected at differ-ent times during the moulting process, the latter authors pro-posed a general succession of processes for valvecalcification: “Prior to moulting, the ostracods begin pro-ducing shells by storing a huge amount of calcium phosphategranules together with chitin precursors in the outer epider-mal cells. These granules release their contents into the extra-cellular space directly outside the epidermal cells. Thismaterial is transformed into small platelets, each about thesize of one granule. The platelets are no longer made up ofcalcium phosphate but of calcium carbonate and they disin-tegrate into small granular structures, which appear to beamorphous calcite. This granular substance then forms thecrystals, which, in connection with chitin and proteins,builds the shell of the ostracods. This final step is notachieved in some species, for instance in the genus Cypria,and the shells consist mainly of amorphous material. How-ever, in shells of adult specimens of other genera all theamorphous material has crystallised and no amorphousmaterial is left. In the larval stages, in contrast, crystallisa-tion is not complete and the animals have weaker shells”.

Besides biomineralisation processes, some morphologi-cal and geochemical particularities are also of interest tothe present discussion. Growth rate and calcification areboth influenced by the temperature of water. In general,development is faster at high temperature, i.e., the time be-tween successive moults is shorter and life span is shorter(Geiger, 1990). Ostracods bred in the laboratory at rela-tively low temperatures with respect to the species autoecol-ogy, have low survival rates associated with slowdevelopment and weak as well as slow calcification thatmay account for the low survival rate (Roca and Wansard,1997; Xia et al., 1997; Mezquita et al., 1999). In contrast,valve size of natural specimens is inversely proportionalto water temperature (Kamiya, 1988; Cronin et al., 2005).Moreover, shell weight of fully calcified specimens collectedin natural environments is also inversely correlated to thetemperature of formation (data from Palacios-Fest andDettman, 2001).

5.2. Oxygen isotope fractionation

A synthesis of the results for Candonidae, Cyprididae,and Cytheroidea from Decrouy (2009) is presented inFigs. 4–6. Results from previous studies (Xia et al., 1997;von Grafenstein et al., 1999b; Chivas et al., 2002; Keatingset al., 2002; Wetterich et al., 2008; Li and Liu, 2010) arealso represented on the respective plots for comparison.Some points to note in Figs. 4–6 include:

(1) A comparison between oxygen isotope fractionationfactors of ostracods and synthetic calcite formed inaqueous solutions clearly supports previous observa-tions that ostracod valves do not crystallise in equi-librium, but are enriched in 18O compared to theinorganic carbonate. In addition, the regression lines

of the fractionation factors versus temperatureobtained for certain species are not parallel to the linefor equilibrium calcite. Using a slightly differentapproach and the expression proposed for inorganiccalcite by Friedman and O’Neil (1977), vonGrafenstein and co-authors (1999b) observed that,for a given species, the difference between ostracodand inorganic calcite d18O values was not tempera-ture dependent, that is the oxygen isotope fractiona-tions between ostracods and water can be describedusing a constant species-specific ‘vital offset’ relativeto inorganic calcite. Yet, the present dataset indicatesthat for Candoninae, and to a lesser extent for Cyp-rididae, the ‘vital offset’ may even increase with lowertemperatures of crystallisation (Figs. 4 and 5). Thisobservation needs to be confirmed as slopes deter-mined for the fractionation of oxygen isotopes foreach species may be affected by uncertainties in theevaluation of the exact calcification time and, there-fore, water temperature during valve calcification.In addition, this finding, if revealed correct, wouldhave important consequences on the use of ostracodstable isotope composition as water palaeo-thermom-eter, further emphasising the need of additional stud-ies on oxygen isotope fractionation during ostracodvalve calcification.
It is important to mention that the equation of Fried-man and O’Neil uses a second order (106/T2) rela-tionship. The extrapolation of their expression tolow temperatures results in a steepening of the slopeclose to 0 �C. Thus, the difference between an inor-ganic calcite precipitate calculated according toKim and O’Neil (1997) and Friedman and O’Neil(1977) is 0.29& at 20 �C but increases to 0.89& at4 �C. Hence, when results from von Grafensteinand co-authors (1999b) and from the present studyare compared to inorganic calcite calculated accord-ing to Friedman and O’Neil (1977), temperaturedependence for the ‘vital offsets’ as described aboveis difficult to distinguish. For the following discus-sion, the relation given by Kim and O’Neil (1997) isused because: (1) at low temperatures a linear rela-tionship between 103/T and 103 lnacalcite–water isexpected from theoretical considerations (Kim andO’Neil, 1997; Chacko et al., 2001), and (2) the resultsfrom Kim and O’Neil (1997) are based on experi-ments done at low temperatures and hence are takento approximate oxygen isotope fractionations at lowtemperature better than high temperature experi-ments extrapolated to lower temperature such as inFriedman and O’Neil (1977). This difference betweenthe equations given in Friedman and O’Neil (1977)and Kim and O’Neil (1997) illustrates the need of fur-ther experimental verifications of oxygen isotopefractionations at low temperatures, and may alsolead to confusion when, for example, the “vital off-sets” estimated based on inorganic fractionations ofFriedman and O’Neil (1977), such as that of vonGrafenstein et al. (1999a,b) are compared to thoseof studies using Kim and O’Neil (1997).

von Grafenstein et al. (1999b) L. inopinata C. lacustris

1000 / T (°K)L. inopinata

L. sanctipatricii

Limnocytherinae

y = 18.03 x - 32.42

y = 21.70 x - 43.75

y = 21.56 x - 42.80

3.35 3.4 3.45 3.5 3.55 3.629

30

31

32

33

34

35

y = 19.27 x - 35.08r2 = 0.98 n = 23

y = 18.51 x - 32.47r2 = 0.95 n = 14

y = 18.01 x - 30.55r2 = 0.96 n = 9

y = 19.97 x - 37.12r2 = 0.94 n = 26

1°C

1‰

1000

ln α

calc

ite-w

ater

C. lacustris (juvenile)

Fig. 6. Same as for Fig. 4 but for Cytheroidea. Ostracod data are from von Grafenstein et al. (1999b) and the present study.


(2) Oxygen isotope fractionation factors are similarwithin each sub-family, but vary between differentfamilies. In addition, different oxygen isotope frac-tionation factors are obtained between sub-familieswithin the family Candonidae (i.e., Candoninaesub-family versus Cypria ophtalmica forma lacustris

that belongs to the Cyclocypridinae sub-family;Fig. 4). This might also be the case for fractionationfactors between sub-families belonging to other fam-ilies such as the families Cyprididae, Limnocytheri-nae or Cytherideidae. However, this cannot betested as the number of ostracod species in LakeGeneva is limited. However, fractionation factorsobtained for the different species within the familyCyprididae are similar (Fig. 5). Hence, to simplifythe following discussion, the fractionation factorsare regrouped into three groups according to theirtaxonomic affiliation: species belonging to the sub-family Candoninae will be collectively referred to asCandoninae, with the same applying to the speciesof the family of Cyprididae and those of the super-family Cytheroidea. Calcification processes areassumed to be equivalent within the same genus,sub-family, or even family, as is suggested by similaroffsets in the oxygen isotope fractionations within afamily, but it is different between taxonomically dis-tant species, perhaps due to different biomineralisa-tion processes.

(3) The lines determined for Candoninae plus the onedetermined for Cypria ophtalmica forma lacustris

are parallel to the line of synthetic calcites grown inconcentrated Ca2+ solutions (15 and 25 mM). How-ever, the lines determined for Candoninae have anoffset of approximately +1& in comparison to syn-thetic calcite that grows in the most concentrated

Ca2+ solution (Fig. 4). In contrast, the lines obtainedfor Cytheroidea are parallel to that of synthetic cal-cite grown at equilibrium (5 mM), but have a higheroffset in comparison to the latter (Fig. 6). Slopesdetermined for Cyprididae are different from thoseof the other families. This may be an intermediatestate between both extremes (Fig. 5). Given these dif-ferences between Candoninae and Cyprididae rela-tive to inorganic calcite growth, the vital effects forthese two species may be related to two distinctmechanisms. One could be a temperature indepen-dent enrichment in 18O (i.e., a parallel shift of ostra-cod 103 lnacalcite–water towards higher values). Theother could be responsible for the slope steepeningand additional enrichment in 18O. The first one isreferred to here as the ‘primary isotope vital effect’,whereas the second is referred to as the ‘secondaryisotope vital effect’. The ‘primary effect’ may be dom-inant for Cytheroidea, whereas a combination ofboth effects is needed to explain the values obtainedfor Cyprididae and Candoninae. This interpretationis supported by differences observed between valvestructures of the Cyprididae and Cytheroidea(Keyser and Walter, 2004).

(4) Oxygen isotope fractionation factors determined byKeatings et al. (2002) for Candona candida, Pseudo-

candona rostrata, and Herpetocypris reptans in twosmall spring-fed ponds in southern England are inagreement with the results of this study (Figs. 4 and5). 103 lnacalcite–water estimated for Candona candida,Limnocythere inopinata and Cytherissa lacustris col-lected in the Ammersee and the Starnberger See insouthern Germany by von Grafenstein et al.(1999b) are, in contrast, depleted by approximately0.4& relative to the results presented here and those


of Keatings and co-authors (2002). This discrepancyis even up to one per mil if published ‘vital offsets’from von Grafenstein and co-authors (1999b) areused. The difference is also approximately 1.0& forresults from Candona rawsoni cultivated at 25 �Cin vitro by Xia et al. (1997). Wetterich andco-authors (2008) determined a vital offset forFabaeformiscandona pedata that is even 1.5& lowerthan the results presented here (Fig. 4). In addition,results for Cyprididae from two studies in more sal-ine environments showed that the vital effect can varyeven more when extreme environmental conditionsare given. Chivas et al. (2002) showed that at 15–25 �C, the oxygen isotope composition of Australocy-

pris robusta is approximately 0.7& higher thaninorganically precipitated calcite at the same temper-ature. Li and Liu (2010) obtained oxygen isotopecompositions for Eucypris mareotica that were evenlower than the value expected for calcite in equilib-rium with water, clearly indicating that the vital effectwithin a family may vary by several per mil over largechanges in environmental conditions (Fig. 5).

pH, or more precisely the relative abundance of bicar-bonate ions, can affect oxygen isotope fractionation inforaminifera (Spero et al., 1997; Zeebe, 1999) and, by infer-ence, this may also be true for ostracods. Cultures by Xiaet al. (1997) were performed with a pH of 8.6, the pH ofthe lakes studied by von Grafenstein et al. (1999b) was be-tween 7.9 and 8.5, values for Lake Geneva were between 7.5and 9, and the pH in the ponds studied by Keatings et al.(2002) was 6.9. The lack of a relationship between pH ofthe different studies and vital effect offsets supports theproposition by Keatings et al. (2002) that oxygen isotopefractionation is not affected by the pH of the water. Liand Liu (2010) suggest, on the basis of Li et al. (1997), thathigher salinity (and/or alkalinity) leads to lower 18O–16Ofractionation. However, the experiments conducted by Chi-vas et al. (2002) had higher salinity than the experimentalcultures of Li and Liu (2010), but the vital offset was higherthan that determined by Li and Liu (2010), whereas theopposite would be expected (Fig. 5). Hence, additionalparameters may control magnitude of the ‘vital effect’.The chemical composition of water, for example, may varyfrom one site to the other. Further studies such as labora-tory cultures of ostracods under different but controlledconditions (mainly different pH and chemical compositionsof water), are required to resolve the reasons for the offsetsrelative to inorganic calcite–water fractionations. A con-stant offset is crucial for studies using the ostracod valveisotopic composition as a palaeoenvironmental proxy.

Last but not least, results presented here do not dis-credit the validity of previous studies on ostracods as pal-aeoenvironmental proxies. Oxygen isotopic compositionsof ostracods have been reliably used to determine oxygenisotope composition of water in continental as well asmarine environments (e.g., Holmes and Chivas, 2002;von Grafenstein, 2002; Janz and Vennemann, 2005)and, given the knowledge of a vital offset, this use isjustified.

5.3. Carbon isotope compositions

As mentioned in Section 4.2, it is difficult to demonstratewhether or not the carbon isotope composition of ostracodsis in equilibrium with that of the DIC of water, and hencethe assessment of a vital effect for carbon isotope fraction-ation is also complicated. von Grafenstein et al. (1999b) ob-served an enrichment in 13C of approximately 1& betweenCandoninae d13 C values and d13CDIC values. This enrich-ment corresponds to the carbon isotope enrichment be-tween calcite and HCO3

�. This suggests that the carbonisotopic composition of Candoninae is in equilibrium withwater DIC. However, other species, such as Cytherissa

lacustris, a typical infaunal species, or Limnocythere inopi-

nata, which is believed to be mostly epifaunal, have d13Cvalues that are much lower compared to Candoninae. vonGrafenstein and co-workers attributed these relative deple-tions in 13C to variation of d13CDIC values in interstitialwater for the former species or vital effects for the latter.Better quantification of pore-water d13CDIC variationswithin species-specific microhabitats would be necessaryto estimate true vital effects (von Grafenstein et al.,1999b). Keatings and co-authors (2002), using a slightly dif-ferent method to calculate the expected carbon isotopecomposition of an “equilibrium” calcite, confirmed thatostracod valves are generally in equilibrium with DIC. Re-sults obtained in lakes (von Grafenstein et al., 1999b, pres-ent study) are difficult to interpret because d13CDIC valuesin littoral zones experience large seasonal variations. More-over, ostracods living in the profundal zones of lakes aremostly infaunal and variations of interstitial pore waterd13CDIC values would need to be known. To remedy thesecomplications, microhabitat preferences and life-cycleshave also been examined but are reported elsewhere(Decrouy, 2009; Decrouy et al., in press).

Given the limited space and an improved readability, theresults are not discussed for each species individually here.Equivalent autoecological characteristics are grouped. Twomain groups emerge from Figs. 7 and 8. The first one con-sists of epifaunal forms found in the littoral zones, while thesecond of infaunal forms from the profundal zones. Envi-ronmental factors controlling the d13CDIC values in ostra-cod microhabitats are discussed in Decrouy et al. (2011).The present discussion is limited to the carbon isotope frac-tionation, influence of species-specific autoecology, andeventual isotopic vital effects.

Group 1: Pseudocandona compressa, Prionocypris zenk-

eri, Herpetocypris reptans (summer generation), Isocypris

beauchampi, Cypridopsis vidua, Plesiocypridopsis newtoni,Potamocypris similis, Potamocypris smaragdina, Limnocy-

there inopinata, and Limnocytherina sanctipatricii are con-sidered as epifaunal as they live above or on the sediment(Meisch, 2000; Decrouy, 2009; Decrouy et al., in press,).Thus, the d13C values of their valves should correspondto those of open water DIC at the time of valve calcifica-tion. For all these taxa, a more or less well-expressed nega-tive correlation between d13Costra and d18Oostra values wasfound. For Pseudocandona compressa, r2 is 0.36** withn = 55; for Prionocypris zenkeri, r2 is 0.5 with n = 11; forepifaunal Herpetocypris reptans, r2 is 0.62*** with n = 60;

-10-9-8-7-6-5-4-3

n=6 n=2 n=3 n=11 n=19 n=14 n=9

-10-9-8-7-6-5-4-3

n=5 n=2 n=5 n=12 n=19 n=6 n=8 n=9 n=5 n=12 n=11

-10-9-8-7-6-5-4-3

n=26 n=32 n=16 n=16 n=17

-10-9-8-7-6-5-4-3

n=6 n=10 n=6 n=16

-10-9-8-7-6-5-4-3

n=7 n=3 n=7

δ13 C

calc

ite (‰

VPD

B)

Cyp

ria o

phta

lmic

afo

rma

lacu

stris

Ad A-1 A-2 Am A-1 A-2Af Ad A-1 A-2 Am Ad A-1 A-2 Ad

Botto

mw

ater

Porewater

0-1c

m

1-2c

m

Pseudocandona compressa

Fabaeformiscandona caudata

Candonaneglecta

Candonacandida

Fig. 7. Carbon isotope composition of Candonidae at 2, 5, 13, 33, and 70 m water depths represented with box plots (minimal value, 1stquartile, median (bold line), average (fine line), 3rd quartile, and maximal value). Crosses are used when sample abundance is low or foroutliers. n represents the number of analyses, Ad for adult valve, Af for female adult valves, Am for male adult valves, A-1 for valve belongingto the last juvenile stage A-1, A-2 for valves belonging to penultimate juvenile stage A-2. Carbon isotope composition of a calcite inequilibrium with DIC is represented on the right side of each graph to allow for a comparison. Open circles represent the values of a calcitethat crystallised in bottom water, marker size being proportional to water temperature at the time of sampling. Open diamonds representvalues of a calcite that crystallised in pore water of the top centimetres of sediment. Grey lines illustrate the average along sediment depthprofiles (see text for explanation).


for Isocypris beauchampi, r2 is 0.72 with n = 5; for Cyprid-opsinae (Cypridopsis vidua, Plesiocypridopsis newtoni, Pot-

amocypris similis, and Potamocypris smaragdina), r2 is0.50** with n = 30; and for Limnocytherinae (Limnocythere

inopinata and Limnocytherina sanctipatricii), r2 is 0.61***

with n = 19 (� for p < 0.05, �� for p < 0.01, and �� forp < 0.001; Decrouy, 2009). This indicates that d13Costra val-ues are higher when the water temperature is higher, in con-cert with the seasonality of d13C values of supernatantwater DIC. All stenochronic species (Prionocypris zenkeri,Cypridopsis vidua, Plesiocypridopsis newtoni, Potamocypris

similis, and Potamocypris smaragdina) except L. inopinata

and L. sanctipatricii, have d13Costra values that correspondto values expected for an equilibrium calcite (d13CCaCO3)that precipitated during the respective development period,suggesting that these species crystallised their valves in equi-librium with DIC (Fig. 8). For two species, d13Costra valuesare approximately 2–3& lower than expected, hence adepletion in 13C suggests a vital effect for shells of Limno-cytherinae. The same results were obtained for Limnocy-

there inopinata in the Ammersee and the Starnberger See(von Grafenstein et al., 1999b). For eurychronic forms(Pseudocandona compressa, and Isocypris beauchampi),d13Costra values of the different development stages corre-

Prio

nocy

pris

zenk

eri

Cyp

ridop

sis

vidu

a

Isoc

ypris

beau

cham

piHerpetocyprisreptans

Ples

iocy

prid

opsi

s&

Pota

moc

ypris

Ad Aw As A-1 A-2 Ad Ad Ad

n=6 n=16 n=3 n=1 n=7

n=4 n=10 n=1 n=2 n=12 n=6

n=13 n=16 n=19 n=7 n=3

n=2

n=5

Botto

mw

ater

Porewater

0-1c

m

2-3c

m

-10-9-8-7-6-5-4-3

-10-9-8-7-6-5-4-3

-10-9-8-7-6-5-4-3

-10-9-8-7-6-5-4-3

δ13 C

calc

ite (‰

VPD

B)

Lim

nocy

ther

ein

opin

ata Cytherissa lacustrisLimnocytherina

sanctipatricii

Ad A-1 Ad A-1 A-2

n=2 n=6 n=2 n=55 n=24 n=13

n=5

n=4

n=27 n=19 n=13

n=14 n=20 n=13

Ad

-10-9-8-7-6-5-4-3

1-2c

m

Fig. 8. Same as for Fig. 7 but for Cyprididae and Cytheroidea. Aw and As represent adults belonging to Herpetocypris reptans thatcrystallised in winter and summer, respectively.


spond to the d13CCaCO3 values expected for the respectivemoulting periods, suggesting that these species crystallisedtheir vales in equilibrium with DIC (Figs. 7 and 8). Previousresults on Cypridopsis vidua, and Candoninae support theseresults (von Grafenstein et al., 1999b; Keatings et al., 2002).

Group 2: Based on their autoecology, the species in thisgroup have an infaunal behaviour: Candona candida,Candona neglecta, Fabaeformiscandona caudata, summergeneration of Herpetocypris reptans at 13 m, and Cytherissa

lacustris. For these species, d13Costra values are not expectedto vary seasonally but must, in contrast, reflect the intersti-tial water d13CDIC values with respect to the specific sedi-ment penetration depth of each specimen. The carbonisotope composition of dissolved inorganic carbon of inter-stitial water decreases in d13CDIC values with increasingwater depth at the same site. d13CDIC values decrease withincreasing sediment depth (Decrouy et al., 2011). The studyof ostracod sediment penetration depth indicates that indi-

viduals of Candona candida, Candona neglecta, and Cyther-

issa lacustris are found deeper within the sediment inprofundal sites. In addition, the specific sediment penetra-tion depth of each development stage of Candona neglecta

and summer generation of Herpetocypris reptans increaseswith increasing development stage (Decrouy et al., 2011).Thus, d13Costra values must decrease with increasing waterdepth for Candona candida, Candona candida and Cytheris-

sa lacustris and d13Costra values of each development stageof Candona neglecta and Herpetocypris reptans are expectedto decrease with increasing development stage. In Figs. 7and 8, patterns observed for the three species indicate thatd13Costra values reflect variations of d13CDIC of pore wateraccording to the specimens’ specific microenvironment pref-erence. Given that carbon isotopes are incorporated inequilibrium, the comparison of d13Costra values of Cytheris-

sa lacustris and Herpetocypris reptans with d13CCaCO3 ofpore water indicates that these individuals calcified their


valves at sediment depths of approximately 0.5 cm (Fig. 8),which corresponds to the microenvironment preferencefound for these species. This indicates that these two speciescrystallise their valves at or near isotopic equilibrium withDIC. Results for Herpetocypris reptans at 2 and 5 m furthersupport this interpretation. Yet, comparison of d13Costra

values of Candona candida, Candona neglecta, as well asFabaeformiscandona caudata and d13CDIC of pore waterindicates a shallower sediment penetration depth comparedto the actual depth at which specimens of this species werefound in the sediment (Fig. 7). This suggests either that thesediment penetration depth was overestimated in this case,or that the valves of this species are enriched in 13C com-pared to equilibrium. Sediment penetration depths ob-tained from studies of specimen distributions are thoughtto be overestimated because of active dwelling of the ostrac-ods during sediment subsampling (Decrouy et al., in press).In addition, results of previous studies indicate that speciesbelonging to Candoninae sub-family crystallise their valvesin equilibrium with DIC (von Grafenstein et al., 1999b;Keatings et al., 2002). Hence, the hypothesis of crystallisa-tion at equilibrium with DIC together with overestimationof sediment penetration depth for Candona neglecta is pre-ferred as an explanation for the patterns observed in Fig. 7for this species.

Keatings and co-authors (2002) observed 0.8& lowerd13C values of Herpetocypris reptans compared to equilib-rium values of a synthetic calcite. The higher variability ind13CDIC found in Lake Geneva cannot validate nor inval-idate such a small carbon isotopic disequilibrium. Micro-environment effects might also differ from the two studysites and explain the differences observed for these twostudies. von Grafenstein et al. (1999b) measured d13Costra

values for Cytherissa lacustris lower than those ofd13CDIC. In addition, these authors observed an increaseof d13Costra values with development stage. These authorsattributed their higher d13Costra values for adults to deeperhabitats. In deep sediments, d13CDIC values are higherbecause of the release of CO2 enriched in 13C producedduring methanogenesis. Such an explanation is not appli-cable for Lake Geneva for different reasons. Althoughmethanogenesis was detected between 3 and 5 cm in thesediment at 70 m water depth (Decrouy et al., 2011), al-most no ostracods were found at this depth within the sed-iment (Decrouy et al., in press). Secondly, an increase ind13Costra values of Cytherissa lacustris with developmentstage is measured at the site of 13 m water depth(Fig. 8), even though no methanogenesis was detected inthe top five centimetres of sediment. Thirdly, autoecolog-ical data suggest a similar habitat for juveniles and foradults.

One species, Cypria ophtalmica forma lacustris, canneither be included in Group 1 nor Group 2. This speciesis believed to be an epifaunal and/or of phytophylousform (Hiller, 1972). Yet, autoecological and carbon isoto-pic data suggest that in lakes, this species inhabits theinterstices of the sediment (Fig. 7). The rather scarceautoecological data obtained so far may hint at a moresuperficial habitat. If so, this species would have a largenegative vital effect for carbon isotopes.

5.4. Effect of incomplete valve calcification

Xia and co-authors (1997) observed that the ostracodsthey bred at 15 �C were less calcified (lower shell weight)and had lower d18O values compared to calcite in equilib-rium with water (symbol in brackets in Fig. 4). In addition,development and survival rates were much lower at 15 �Cthan at 25 �C. Optimum life temperature for Candona raw-

soni is 25 �C (Xia et al., 1997). The authors thus interpretedthe relatively 18O-poor oxygen isotopic compositions andthe lower shell calcification at 15 �C as being due to growthunder stress conditions and a slower calcification process.In contrast, rapid precipitation of carbonate in organismssuch as corals is expected to discriminate less between 16Oand 18O, leading to lower d18O values compared to equilib-rium values (McConnaughey, 1989). A decrease of d18Ovalues with increasing precipitation rate has also been ob-served in synthetic carbonate minerals (Kim et al., 2006).

During the one-year sampling in Lake Geneva, someostracods were found to be only partially calcified. In ourcase, incomplete calcification of the valve is attributed tothe fact that these specimens were sampled just after ecdysisand were killed before complete valve calcification. Asincompletely calcified valves are easily recognised on thebasis of their fragility during sample preparation suchvalves may be excluded from analyses or analysed sepa-rately. d18O values for these samples were similar to thoseof other samples. However, these samples can have d13Cvalues of 1 to 3& higher than normally calcified valves.For example, the carapace with the highest d13Costra valuesmeasured for an adult Candona candida at 33 m (outlier va-lue symbolised with the cross in Fig. 7) had only 1/4 of thespecies typical weight. Thus, incomplete valve calcificationhas an effect on the stable isotope composition: d13C valuesare higher in Lake Geneva, whereas Xia and co-workers(1997) found lower d18O values compared to the bulkpopulations.

The crystallisation site in ostracods can been envisagedas occurring from a solution separated from the externalenvironment by organic membranes (see Decrouy, 2009).Chemical exchange between both internal solution and lakewater must occur through this membrane or via body fluidsof the ostracod. In addition, this exchange and the mineralprecipitation within the membrane may be ‘biologically’controlled. Thus, the internal solution can be seen as a finitereservoir of DIC that has to be continuously refilled via per-meability of the organic membranes and/or substances se-creted by epidermal cells. Knowledge on mineralisationprocesses in ostracods is quite limited but it is generallyagreed that ostracods have a strong control on the timingof moulting and thus on valve calcification.

Precipitating instantaneously different fractions ofwitherite (BaCO3) from a finite DIC reservoir, Kim andco-authors (2006) demonstrated that CO3

2� was preferen-tially incorporated into the growing witherite crystal. AsO-isotopic fractionation between water and CO3

2� is lowerin comparison to the other DIC species, the isotopic com-position of carbonate that equilibrated only with a smallamount of the original DIC reservoir has lower d18O values.This phenomenon may explain the oxygen isotope effect


observed for incomplete ostracod valve calcification. Beforevalve calcification, a certain amount of DIC is present inthe internal solution. At the beginning of valve calcification,the organism has to induce precipitation of the calcite. Thisforced precipitation may be comparable to the instanta-neous precipitation of synthetic carbonate in a finite reser-voir. Hence, the first calcite that mineralises mayincorporate a larger proportion of CO3

2�. As calcite isrecrystallised at later stages of valve calcification, the isoto-pic composition of CO3

2� is slowly erased as the DIC poolis being consumed and/or as the carbonate re-equilibrateswith the other DIC species dissolved in water.

Carbon isotope enrichment factor between CO32� and

HCO3� ranges between 2.3& and 3.3& from 20 to 4 �C

(Zhang et al., 1995). Hence, if more CO32� is incorporated,

the d13C value is expected to be higher compared to anequilibrium with all DIC phases. Unfortunately, Xia andco-authors (1997) did not report the carbon isotopic com-position of their cultured ostracods. However, an enrich-ment of 1 to 3& is observed for non-completely calcifiedvalves in the present dataset. This could provide further evi-dence that CO3

2� is preferentially incorporated during theinitial phase of valve calcification.

Homogeneity of the isotope compositions of well-calcified samples implies that the initial calcium carbonateformed at the beginning of valve calcification must re-equilibrate to reach its final constant values. Once the firstcalcium carbonate has crystallised, re-equilibration is onlypossible via re-crystallisation of the minerals. Using SEMmicrophotographs and EDX-analyses, Keyser and Walter(2004) demonstrated that the ostracod valve calcificationprocess consist of successive precipitation and re-crystalli-sation of different mineralogical phases. Thus, it is possiblethat the isotopic composition of preferential CO3

2� uptakeis erased during final re-crystallisation.

The fact that the effect of incomplete valve calcificationwas observed only for carbon isotope compositions and notfor oxygen isotopes in the present study may be due to re-equilibration of oxygen isotopes between the DIC speciesand/or carbonate and water, while this exchange may bemore sluggish for carbon. This may be the case becausethe pool of oxygen is much larger than the pool of carbonin the solution present during calcification. This effect ofincomplete valve calcification may also be more importantunder stressed laboratory growth and hence affect both car-bon and oxygen isotope compositions.

5.5. Biomineralisation processes and the understanding of

isotopic vital effects in ostracods

5.5.1. Effect of pH at the crystallisation site

Keatings et al. (2002) suggested that the non-equilibriumfractionation of oxygen and carbon isotopes for Herpetocy-

pris reptans might be explained by a fixed pH at the site ofcalcite crystallisation and controlled by the organism. Thisinterpretation is based on the proposition of Zeebe (1999)that oxygen isotope fractionation between the carbonateions and water is dependant on the pH of the solution.According to Zeebe (1999): “The different hydrate carbon-ate species can be expressed with S = [H2CO3] +

[HCO3�] + [CO3

2�] and the relative proportion of thesecarbonate species is a function of the pH. Provided that cal-cium carbonate is formed from a mixture of the carbonatespecies in proportion to their relative contribution to S, theoxygen isotope fractionation factor between calciumcarbonate will reflect the balance of the different oxygenisotope fractionation factors between the different carbon-ate species and water according to their relative proportionto S. As oxygen isotope fractionation factors between thedifferent carbonate species and water decrease accordingto the sequence aH2CO3–water > aHCO3–water > aCO3–water,and as increasing pH leads to the deprotonation sequence[H2CO3]! [HCO3

�]! [CO32�], the d18O value of calcium

carbonate decreases with increasing pH”. Zeebe (1999)hence attributed the “non-equilibrium” fractionation factorobtained by Kim and O’Neil (1997) in 15 and 25 mM Ca2+

solution to precipitation at lower pH, at a pH of 6.9 and 6.6for 15 and 25 mM Ca2+ solutions, respectively (Zeebe,1999). The similarity between the oxygen isotope fractiona-tions of ostracods and those of synthetic calcite precipitatedin 25 mM Ca2+ solution lead Keatings et al. (2002) to sug-gest that pH at the site of calcite precipitation in ostracod isrelatively low (67).

Kim et al. (2006) demonstrated with a sequence ofexperiments that oxygen isotope fractionation for calciumcarbonate precipitated under equilibrium is not pH depen-dent. This was also postulated theoretically by Deines(2005), and suggests that pH at the calcification site cannotexplain the enrichment in 18O observed in ostracod valves.Studies of the pH during moulting of ostracods would beneeded to resolve this controversy.

5.5.2. Effect of calcification rate

von Grafenstein et al. (1999b) noted that species with theshortest instar development have higher d18O values com-pared to species developing slowly. On the basis of thisobservation, it was suggested that the differences in oxygenisotope fractionations observed among the different speciesmight be correlated to the speed of valve calcification. Thedataset for Lake Geneva indicates that this is not valid forthe ostracods measured in this study. For example, Candona

neglecta develops rather slowly but continuously at 70 mwater depths, and discontinuously at 13 m, with a long sum-mer without moulting but fast development when environ-mental conditions are favourable once more. In spite ofthese different developments, the oxygen isotope fraction-ation of C. neglecta is constant at all sites. Another observa-tion supporting that development rate has no influence onisotopic fractionation is that both Limnocythere inopinata

and Limnocytherina sanctipatricii have lower 103 lnacalcite–water even though both species develop particularlyquick (for species development, see Decrouy, 2009).

However, rate of calcite growth may not necessarily be re-lated to the rate of instar development. As long as the truecalcification rate is unknown, it is not possible to verify its ef-fect on the isotopic composition of the carbonate. As men-tioned above, kinetic effects linked to forced calcificationduring initial valve calcification may affect the isotopic com-positions of incompletely calcified valves, but this effect maybe subsequently erased during the later valve calcification.


5.5.3. Deprotonation of HCO3� as source of CO3

2�

Depending on the pH, carbonate ions available for car-bonate mineralisation in the internal crystallisation site areH2CO3, HCO3

�, and CO32�. CO3

2� is the first carbonateion to precipitate, whereas HCO3

� and H2CO3 must bedeprotonated before being added to the mineral. Theamount of DIC present in the internal solution before crys-tallisation of calcite is presumably not sufficient to provideall the carbonate for calcification of the whole shell. Trans-fer of DIC from water or body fluids towards the crystall-ising solution is thus required for complete valvecalcification. If mass transport is rapid, i.e., faster thanDIC is consumed for calcite precipitation, and if carbonatespecies are not fractionated during incorporation and trans-fer to the calcification site, and if calcite precipitation is suf-ficiently slow so that no kinetic fractionation occurs, thenthe calcite must be formed in equilibrium with the water.In most natural waters, the dominant dissolved carbonatespecies is HCO3

�. Thus, it is plausible that the main speciespresent in the calcification site is HCO3

�. Under instanta-neous precipitation of synthetic calcium carbonate in aclosed system, the ions incorporated in the minerals pre-serve their original isotopic composition. At a high pH,the first carbonate that crystallises has a d18O value corre-sponding to the isotopic fractionation between CO3

2� andwater. In contrast, calcium carbonate formed from theinstantaneous precipitation of all the DIC at neutral pHhas a d18O value corresponding to HCO3

� – water fraction-ation (Kim et al., 2006). As aHCO3–water is higher thanacalcite–water, the d18O value of calcite precipitated rapidlyfrom a large DIC pool in a closed system is higher than thatfor calcite grown in equilibrium.

In the case of the ostracods, incorporation of HCO3� or

of CO32� with or without equilibration with the ambient

water may explain the observed vital offset. Non-equilib-rium fractionation may be related either to diffusion via apostulated membrane or be the result of the rate of incorpo-ration of the ions into the calcifications sites as the calciticcuticle is growing.

5.5.4. Salt effect

Kim et al. (2007) observed that a high concentration ofMg2+ has an effect on the oxygen isotope fractionation,similarly to the so-called “salt-effects”.

Many studies demonstrated that the O-isotope compo-sition of CO2 in equilibrium with highly concentrated sal-ine solutions is lower in 18O compared to that of CO2 inthe absence of the salts (O’Neil and Truesdell, 1991;Horita et al., 1993a,b). O’Neil and Truesdell (1991) pro-posed two mechanisms to explain this phenomenon, mea-sured using the CO2–H2O equilibration technique: (1)fractionation among the three oxygen-bearing species inthe system (bound or solvation water, free water, andCO2), and (2) modification of the structure of water inthe presence of ions. It can be inferred from the firstmechanism that Mg2+ ions, being structure-makers, willpreferentially attract 18O-rich water molecules to theirhydration spheres, leaving the free water proportionallyenriched in 16O. Therefore, a CaCO3 precipitating fromsuch a solution is expected to be in equilibrium with the

free water and, when fractionation factors are calculatedon the basis of the isotope activity-ratio (this ratio refersto the oxygen isotope composition of CO2 in equilibriumwith the saline solution), results would be identical tothose in dilute systems. Kim et al. (2007) observed a posi-tive shift of 0.4& for aragonite precipitated in highlyMg2+ concentrated solutions when fractionation factorsare calculated on the basis of the isotope activity-ratio.To explain this discrepancy, they suggested that either:(1) the isotopic activity composition of concentrated solu-tions determined by the CO2–H2O equilibration techniquedoes not reflect the true isotope activity ratios of the con-centrated solution or (2) the presence of high concentra-tions of Mg2+ ions modifies the mechanism(s) ofaragonite precipitation and results in its relative enrich-ment in 18O. The mechanism(s) of oxygen isotope salt ef-fects in mineral–water systems hence also remains uncleareven though the work by O’Neil and Truesdell (1991) doesimply that a high salt content has an effect on the oxygenisotope fractionation in the carbonate–water system.O’Neil and Truesdell (1991) noted that the salt effect ishigher for strong structure-making ions relative to weakerstructure-making ions. The importance of the salt effectfollows the sequence Al+3 > Mg2+ > Ca2+. In addition,the authors showed that the salt effect increases withdecreasing water temperature. The slopes determined byKim and O’Neil (1997) and illustrated in Figs. 3 and 4for solutions of different concentrations are parallel butat a critical concentration the slope of the lines becomesteeper. These observations are similar to those of thepresent study. For the three systems (CO2-‘salt enrichedsolution’, calcite-‘Ca2+ enriched solution’, and ostracod-water), the lines have a positive offset from the equilibriumline (i.e., lower salt content for CO2 and calcite). The frac-tionation lines for the ostracods have steeper slopes com-pared to the inorganic calcite line. Within a single system,the lines are parallel, however, at a certain critical Ca2+

concentration, or between Cytheroidea and Cypridoidea,the slopes change to be steeper (see differences betweenCandoninae and Cytheroidea in Figs. 4 and 6). Thismay suggest that disequilibrium observed for synthetic cal-cite precipitated in Ca2+ concentrated solution as well asthat precipitated for the ostracods may be due to thechange of the water structure related to the addition ofsalts. As Ca2+ ions are structure-making, the d18O valueof the hydration spheres is believed to be higher than thatof the non-bound water molecule. If this is correct, oxygenisotopes of the ions incorporated and/or adsorbed to thesurface of the growing mineral may actually approachequilibrium with water of the hydration spheres. Whiledifficult to prove, this interpretation is supported by thechanges in Mg2+ concentration in carbonate formed dur-ing valve calcification of ostracods (e.g., Chivas et al.,1983, 1986; Palacios-Fest and Dettman, 2001). In addi-tion, a large amount of Ca2+ is released from epidermalcells into calcification sites during carbonate formation(Keyser and Walter, 2004). Thus, salt concentrationsmay be very high in calcification sites at the beginningof valve calcification. Small-scale structural studies of thecarapace in parallel with studies of isotopic and trace ele-


ment compositions for different moulting stages of the spe-cies may help to resolve the relation of other ions andhence “salt effects” on the vital effects.

6. CONCLUSIONS

Studies of stable isotope compositions of calcite valvesof living ostracods collected at monthly intervals over thecourse of one year in parallel with studies on their autoecol-ogy and environmental parameters allow new constraints tobe placed on variations in the oxygen and carbon isotopefractionations between ostracods and water or DIC inwater:

(1) Oxygen isotope fractionation is equivalent for all spe-cies belonging to the sub-family Candoninae andleads to an enrichment in 18O of more than 3& rela-tive to equilibrium values estimated for inorganic cal-cite. Slopes of the oxygen isotope fractionation lineswhen plotted against temperature for these ostracodsare the same as those for synthetic calcite precipitatedfrom solutions with high Ca2+ content (Kim andO’Neil, 1997). Oxygen isotope fractionation for spe-cies belonging to the superfamily Cytheroidea haslower enrichments in 18O of 1.7–2.3& relative to syn-thetic calcite. Oxygen isotope fractionation for thesetaxa has the same temperature dependency and henceslope compared to that for synthetic calcite grown indilute Ca2+ solution. Oxygen isotope fractionationsdetermined for species belonging to the familyCyprididae plot in-between the results of the twopreviously cited families. The difference in oxygenisotope fractionation between ostracods andinorganic calcite has been interpreted to result from‘vital effects’.

(2) Results of this study and from carbonate precipita-tion experiments indicate that two mechanismsmight be responsible for an enrichment in 18O ofostracod valves relative to synthetic calcite formedin equilibrium with water. The first is deprotona-tion of HCO3

� that may be important in the calci-fication of valves from all ostracod taxa and resultin a temperature independent enrichment in 18O.The second mechanism, linked to a “salt-effect”may be only important for Cypridoidea and resultsin an additional enrichment in 18O as well as ahigher temperature dependency of oxygen isotopefractionation.

(3) Regrouping results from previous studies with thoseof the present study indicates that the vital offsetsare different within the same sub-family (e.g., Can-donidae) or same family (e.g., Cyprididae). This sug-gests that the extent of the “vital offset” within afamily or a sub-family may change from one locationto the other. These variations may be caused by dif-ferences in the chemical composition of water.

(4) Given the large natural variability, carbon isotopecompositions of ostracod valves were generally esti-mated to approach equilibrium with DIC. The spec-imens’ d13Costra values are controlled by seasonal

variations of d13CDIC and variations of d13CDIC insediment pore waters of the habitat of the ostracods.Species having thin valves (Cypria ophtalmica formalacustris, Limnocythere inopinata, and Limnocytheri-

na sanctipatricii) have d13Costra values that are lowercompared to inorganic calcite in equilibrium withDIC. This relative depletion in 13C has been inter-preted as a ‘vital effect’ related to the biomineralisa-tion processes.

(5) Incomplete valve calcification has an effect on thecarbon and oxygen isotope composition of ostracodvalves. Preferential incorporation of CO3

2� at thebeginning of valve calcification but a change towardsother carbonate species in solution may explain thiseffect. The isotopic composition of this initial carbon-ate phase may be erased as the valves recrystalliseduring later stages of calcification.

ACKNOWLEDGEMENTS

The research presented in this manuscript was generouslyfunded by Swiss National Science Foundation (SNF) Projects(SNF – 200021-107958 and 200020-119935). The authors wouldalso like to express their gratitude towards the Institute F.-A. Forelfor providing the boat and the limnological material necessary forsediment sampling, many colleagues and friends for their help withthe field work and Dan Danielopol for advice on ostracod identifi-cation. Thanks are also due to Steffen Mischke, Antje Schwalb andan anonymous reviewer for their constructive comments andsuggestions.

In addition, the authors would like to express their gratitude tothe late Stephanie Chemin, and to Gregory Grosjean, the “Brigadedu Lac de Lausanne”, and the “Police de la navigation de Geneve”

for the installation of the data-loggers during several divingcampaigns.

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Controls on ostracod valve geochemistry: Part 2. Carbon and oxygen isotope compositions

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