IR Laser Extraction Technique Applied to Oxygen Isotope Analysis of

An IR-laser fluorination technique is reported here for analyzing the oxygen isotope composition (δ18O) of microscopic biogenic silica grains (phytol...
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Anal. Chem. 2008, 80, 2372-2378

IR Laser Extraction Technique Applied to Oxygen Isotope Analysis of Small Biogenic Silica Samples Julien Crespin, Anne Alexandre,* Florence Sylvestre, Corinne Sonzogni, Christine Paille`s, and Vincent Garreta

CEREGE, CNRS, Universite´ Paul Ce´ zanne Aix-Marseille III and IRD, Europoˆ le de l’Arbois, BP 80, 13545, Aix en Provence Cedex 04, France

An IR-laser fluorination technique is reported here for analyzing the oxygen isotope composition (δ18O) of microscopic biogenic silica grains (phytoliths and diatoms). Performed after a controlled isotopic exchanged (CIE) procedure, the laser fluorination technique that allows one to visually check the success of the fluorination reaction is faster than the conventional fluorination technique and allows analyzing δ18O of small to minute samples (1.60.3 mg) as required for high-resolution paleoenvironmental reconstructions. The long-term reproducibility achieved with the IR laser-heating fluorination/O2 δ18O analysis is lower than or equal to (0.26‰ (1 SD; n ) 99) for phytoliths and (0.17‰ (1 SD; n ) 47) for diatoms. When several CIE are taken into account in the SD calculation, the resulting reproducibility is lower than or equal to (0.51‰ for phytoliths (1 SD; n ) 99; CIE > 5) and (0.54‰ (1 SD; n ) 47; CIE ) 13) for diatoms. A minimum reproducibility of (0.5‰ leads to an estimated uncertainty on δ18Osilica close to (0.5‰. Resulting uncertainties on reconstructed temperature and δ18Oforming water are, respectively, (2°C and (0.5‰ and fit in the precisions required for intertropical paleoenvironmental reconstructions. Several methodological points such as optimal extraction protocols and the necessity or not of performing two CIE prior to oxygen extraction are assessed. Investigating the oxygen isotope composition of biogenic silica (δ18Osilica) such as phytoliths and diatoms is of particular interest for continental paleoclimatic reconstructions as the oxygen isotopic composition of a mineral reflects both temperature and isotopic composition of the solution from which it precipitates (δ18 Oforming water). Diatoms are micrometric unicellular algae (5400 µm) that precipitate in isotopic equilibrium.1-5 δ18Osilica values of diatoms from lacustrine sediments are commonly investigated * Corresponding author. E-mail: [email protected]. Fax: +33 4 42 97 15 40. (1) Labeyrie, L. Nature 1974, 248, 40-42. (2) Juillet-Leclerc, A.; Labeyrie, L. Earth Planet. Sci. Lett. 1987, 84, 69-74. (3) Matheney, R. K.; Knauth, L. P. Geochim. Cosmochim. Acta 1989, 53, 32073214. (4) Brandriss, M. E.; O’Neil, J. R.; Edlund, M. B.; Stoermer, E. F. Geochim. Cosmochim. Acta 1998, 62, 1119-1125. (5) Moschen, R.; Lu ¨cke, A.; Schleser, G.H. Geophys. Res. Lett. 2005, 32, L07708; doi:10.1029/2004GL022167.

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for reconstructing past temperature and trends in lake moisture balance (e.g., refs 6-10). Phytoliths are micrometric particles (< 60-100 µm) that form in plants tissues, inside or between the cells.11 Shahack-Gross et al.12 and Webb and Longstaffe13-16 demonstrated that when phytoliths precipitate in nontranspiring tissues, their δ18Osilica value is related to the δ18Otissue water values and to the growing season temperature. Thus, δ18Osilica value of phytoliths formed in nontranspiring tissue, such as wood,17 should reflect atmospheric temperature and δ18O values of soil water, in turn controlled by (1) δ18O values of meteoric water, (2) potential input of groundwater, and (3) isotopic enrichment due to evaporation.18 Although the potential of the δ18O value of continental biogenic silica as a proxy of atmospheric temperature and part of the water cycle is very promising, it faces analytical problems. Up to now, the conventional fluorination technique19 is commonly used for extracting oxygen from the biogenic silica grains.6,20,4,21,13 This method is time-consuming and the success of the fluorination (6) Shemesh, A.; Charles, C. D.; Fairbanks, R. G. Science 1992, 256, 14341436. (7) Rietti-Shati, M.; Shemesh, A.; Karlen, W. Science 1998, 281, 980-982. (8) Barker, P. A.; Street-Perrot, F. A.; Leng, M. J.; Greenwood, P. B.; Swain, D. L.; Perrott, R. A.; Telford, R. J.; Ficken, K. J. Science 2001, 292, 23072310. (9) Polissar, P. J.; Abbott, M. B.; Shemesh, A.; Wolfe, A. P.; Bradley, R. S. Earth Planet. Sci. Lett. 2006, 242, 375-389. (10) Barker, P. A.; Leng, M. J.; Gasse, F.; Huang, Y. Earth Planet. Sci. Lett., in press. (11) Piperno, D. P. Phytolith Analysis-An Archaeological and Geological Perspective; Academic Press: New York, 1988. (12) Shahack-Gross, R.; Shemesh, A.; Yakir, D.; Weiner, S. Geochim. Cosmochim. Acta 1996, 60, 3949-3953. (13) Webb, E. A.; Longstaffe, F. J. Geochim. Cosmochim. Acta 2000, 64, 767780. (14) Webb, E. A.; Longstaffe, F. J. Geochim. Cosmochim. Acta 2002, 66, 18911904. (15) Webb, E. A.; Longstaffe, F. J. Geochim. Cosmochim. Acta 2003, 67, 14371449. (16) Webb, E. A.; Longstaffe, F. J. Geochim. Cosmochim. Acta 2006, 64, 767780. (17) Crespin, J.; Alexandre, A.; Sylvestre, F.; Sonzogni, C.; Hilbert, D. Abstracts of the 6th International Meeting on Phytolith Research; Barcelona, Spain, September 12-15, 2006. (18) Hsieh, J. C. C.; Chadwick, O. A.; Kelly, E. F.; Savin, S. M. Geoderma 1998, 82, 269-293. (19) Clayton, R. N.; Mayeda, T. K. Geochim. Cosmochim. Acta 1963, 27, 43-52. (20) Schmidt, M.; Botz, R.; Stoffers, P.; Anders, T.; Bohrmann, G. Geochim. Cosmochim. Acta 1997, 61, 2275-2280. (21) Leng, M.; Barker, P.; Greenwood, P.; Roberts, N.; Reed, J. J. Paleolim. 2001, 25, 343-349. 10.1021/ac071475c CCC: $40.75

© 2008 American Chemical Society Published on Web 02/28/2008

reaction cannot be checked prior to measuring the liberated oxygen yield. Moreover, it requires large amounts of samples (four to 10 mg). Considering the low natural abundances of phytoliths in soils22 and sediments23 and despite diatoms being more abundant in lake sediments,24 it is difficult to reach the required amount of purified material needed for high-resolution (decadal to centenal) paleoclimatic reconstructions. Additionally, the hydrous amorphous silica (SiO2, nH2O) that comprises phytoliths and diatoms contains hydroxyl and unstable silanol groups that are likely to exchange with oxygen atoms from the atmosphere or the solutions within which they are in contact.25 Two procedures are commonly followed for overcoming this problem: stepwise fluorination,26 which is rapid but requires from 5 mg21 to 30-70 mg of biogenic silica,20 and controlled isotopic exchange (CIE),25 which is longer but very efficient for small samples. Lu¨cke et al.27 already addressed these analytical problems and recently presented a new technique called iHTR (inductive hightemperature carbon reduction). This technique is very promising but requires that the laboratories already using the conventional or laser fluorination techniques completely change their equipment. In this paper, we present a new infrared (IR) laser-heating fluorination procedure for extracting oxygen from phytoliths and diatoms, after performing the CIE. It requires a smaller amount of sample than the other techniques and is faster than the conventional fluorination technique. Although laser fluorination is now commonly used for extracting oxygen from oxides and silica minerals, analyzing fine grains fractions has for a long time faced problems, including the loss of ejecta and inconsistent δ18O values.28,29 Alexandre et al.30 recently reported a laser protocol for fine quartz grains (1-50 µm) which prevents ejecta and gives a reproductibility better than 0.15‰. The laser protocols presented here for extracting oxygen from phytoliths and diatoms are adapted from Alexandre et al.30 Particular care is taken to estimate the reproducibility of the analyses through a long term measurement period (2 years) of small samples (1.6-0.3 mg). In order to discuss the efficiency of a given δ18Osilica value to be a proxy of temperature and δ18Owater, errors associated with the calculation of δ18Osilica values are estimated. Effects of several chemical treatment temperatures on the δ18Osilica values are also investigated. EXPERIMENTAL SECTION Materials. This paper focuses on two biogenic silica samples: a phytolith sample (MSG) made of grass phytoliths from Mascareignite soil (La Re´union Island31) and a diatom sample (KYO) composed of lacustrine diatoms from an Australian Mi(22) Alexandre, A.; Meunier, J.-D.; Colin, F.; Koud, J-M. Geochim. Cosmochim. Acta 1997, 61, 677-682. (23) Abrantes, F. Earth Planet. Sci. Lett. 2003, 209, 165-179. (24) Fro ¨hlich, F.; Servant-Vildary, S. Diat. Res. 1989, 4, 241-248. (25) Labeyrie, L.; Juillet, A. Geochim. Cosmochim. Acta 1982, 46, 967-975. (26) Thorleifson, J. T.; Knauth, L. P. Geol. Soc. Am. Abstr. Prog. 1984, 16, 675. (27) Lu ¨ cke, A.; Moschen, R.; Schleser, H. Geochim. Cosmochim. Acta 2005, 69, 1423-1433. (28) Fouillac, A-M.; Girard, J.-P. Chem. Geol. 1996, 130, 31-54. (29) Rumble, D.; Miller, M.F.; Franchi, I.A.; Greenwood, R.C. Geochim. Cosmochim. Acta, in press. (30) Alexandre, A.; Basile-Doelsch, I.; Sonzogni, C.; Sylvestre, F.; Parron, C.; Meunier, J.-D.; Colin, F. Geochim. Cosmochim. Acta 2006, 70, 2827-2835. (31) Meunier, J.-D.; Colin, F.; Alarcon, C. Geology 1999, 27, 835-838.

ocene diatomite of quasi-monospecific composition (Aulacoseira granulata). These samples were selected because of their natural abundance and purity. A quartz laboratory standard30 (Boulange´ 50-100 µm) was analyzed on each run. Routine analyses used 1.5 mg of quartz and 1.6 mg of phytoliths and diatoms. Additionally, a few aliquots of 0.3 mg of quartz and phytolith samples were also tested. Chemical Treatment of Phytoliths and Diatoms. The phytolith sample (MSG) was extracted from 10 g of dry soil slightly crushed and sieved at 2 mm, after the following steps:32 (1) dissolution of carbonates using HCl (1 N); (2) iron oxides reduction performed with trisodium citrate (C6H5Na3O7) at 88.4 g L-1 and 1 g of sodium dithionite (Na2O4S2, H2O); (3) oxidation of organic matter using H2O2 (30%) until the reaction subsides; (4) defloculation in a sodium hexametaphosphate Na(PO3)6 (5%) solution buffered at pH ) 7; (5) sieving of the samples at 60 µm; (6) clay removal by sedimentation or centrifugation; (7) densimetric separation of phytoliths carried out using a zinc bromide heavy liquid (ZnBr2) with a density of 2.3; (8) drying at least 24 h. Steps 1-4 were performed in centrifuging bottles. The supernatant was discarded after centrifugation without significant dilution. Because of the study from Juillet-Leclerc,33 a consensual chemical treatment temperature of 50-60 °C is commonly used for cleaning diatom and phytolith samples, although no study evaluated the oxygen isotopic shifts associated with higher chemical treatment temperatures. Within this aim, steps 2, 3, and 8 were processed at 40, 50, 60, 70, and 90 °C. The related phytolith subsamples were, respectively, named MSG 40, MSG 50, MSG 60, MSG 70, and MSG 90. The diatom sample (KYO) was recovered from the diatomite after the following steps (modified after ref 34): (1) dissolution of carbonates using HCl (1 N) for 3 h; (2) oxidation of organic matter with H2O2 (33%) for 3 h; (3) oxidation with HNO3 (65%) for 2 h. Steps 1-3 were repeated seven times and followed by rinsing with distilled water implying a more diluted final sample than for phytoliths; (4) rinsing with distilled water three times repeatedly; (5) filtration at 5 µm; (6) densimetric separation in heavy liquid (ZnBr2) with a density of 2.3; (7) drying for 24 h. The KYO samples were treated at 40, 50, 60, 70, and 90 °C. The related subsamples are, respectively, named KYO 40, KYO 50, KYO 60, KYO 70, and KYO 90. Additionally, an alternative faster and more efficient treatment is used for diatom cleaning:34 steps 1-3 are replaced by a unique dissolution/oxidation step using a mixture of HClO4 (70%) and HNO3 (65%), in a water bath at 50 °C, for 15 min. The sample was rinsed by dilution and oxidized four more times. This diatom subsample was named KYOHClO4. Controlled Isotopic Exchange (CIE). In order to fix the exchangeable oxygen at a known δ18O value, the CIE set up by Labeyrie and Juillet25 and now commonly used by others (e.g., refs 6, 13, and 20) was applied in this study. Twenty samples of (32) Kelly, E.F. Methods for Extracting Opal Phytoliths from Soils and Plant Material, internal report; Colorado State University, Fort Collins, 1990. (33) Juillet-Leclerc, A. Proceedings of the 8th International Diatom Symposium; Ricard, M., Ed.; Koeltz: Koenigstein, Germany, 1984; pp 733-736. (34) Battarbee, R. W.; Jones, V. J.; Flower, R. J.; Cameron, N. G.; Bennion, H. In Tracking Environmental Change Using Lake Sediments, Vol. 3: Terrestrial, Algal, and Siliceous Indicators; Smol, J. P.; Birks, H. J. B.; Last, W. M., Eds; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001; pp 155202.

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phytoliths and diatoms and nine quartz lab standards were loaded in a nickel holder. Samples were equilibrated in a quartz vacuum line for 6 h at 200 °C with a vapor made at 21 °C from a water of known δ18O value. The exchanged samples were then sintered at 1020 °C under high vacuum, for 6 h, to crystallize the silica. This isotopic exchange was processed twice, with two waters of known δ18O (δ18OH2O liquid 1 ) -8.14 ( 0.05‰ and δ18OH2O liquid 2 ) 40.37 ( 0.05‰). Samples were kept under vacuum, or in a desiccator, until they were loaded in the sample chamber of the oxygen extraction line. Oxygen Extraction Using the IR Laser-Heating Fluorination Technique and δ18O Measurement. Molecular O2 was extracted from silica in a laser extraction line close to the one described by Sharp.35 A Merchanteck 30 W CO2 IR laser was used. The nickel sample holder was loaded in the sample chamber, prefluorinated with 50 mbar of BrF5 for 1 h and pumped for several hours. In an atmosphere of 100 mbar of BrF5, samples were preheated for 20 s with a 2000 µm diameter laser beam, increasing the power of the laser beam until the particles start moving: from 0% to 3.6% of the laser full power for fine quartz, 2.2% for diatoms, and 2.6% for phytoliths. The laser emission was stopped after 30 s. Quartz grains and phytoliths were then heated with a 2000 µm diameter laser beam at 30-35% of its full power (11-12 W), starting at the center and slowly moving the laser beam following concentric circles until a bowl of liquid silica formed. Diatom samples were heated with a 2000 µm diameter laser beam, starting at the edge and progressively increasing the laser power from 0% up to 30-35% of its full power (11-12 W), slowly moving the laser beam following concentric circles. For all samples, when a bowl of liquid silica formed, the laser beam was then focused at 1000 µm of diameter until the liquid disappeared. The remaining particles were heated with a focused 200 µm of diameter laser beam. Laser emission was stopped when no more reaction to the laser beam occurred. Some residues remained for the diatom subsamples. They decreased from KYO 40 to KYO 90. These protocols prevented ejecta. The liberated oxygen was then purified and trapped by adsorption in a microvolume filled with 13X molecular sieve and cooled in liquid nitrogen. The oxygen gas was then heated at 100 °C and directly sent to the sample bellow of the dual-inlet mass spectrometer (ThermoQuest Finnigan Delta Plus). In order to get a sufficient 34/32 signal (2-3 V), the oxygen from 0.3 mg aliquots was concentrated in the mass spectrometer in an autocooled 800 µL microvolume filled with silica gel and directly connected to the dual-inlet system. The oxygen isotope results are expressed in standard δ-notation, relative to Vienna standard mean ocean water (V-SMOW).

δ18O )

(18O/16O)sample (18O/16O)standard - 1

× 1000 (‰)

Measured δ18O values of the samples were corrected on a daily basis using a quartz lab standard (δ18O“Boulange´ 50-100 µm”, 16.36 ( 0.09‰; n ) 16). Replicate analyses of the international standard NBS 28 (120-250 µm) during the calibration period gave an average of 9.6 ( 0.17‰ (n ) 13). (35) Sharp, Z. D. Geochim. Cosmochim. Acta 1990, 66, 2865-2873.

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Calculation of δ18Osilica Value. The δ18O value of exchanged oxygen (δ18Oexchanged 1 and 2) was calculated following eq 1:2

δ18Oexchanged ) δ18OH2O liquid + 1000 ln RH2O liquid-H2O vapor + 1000 ln RH2O vapor-Oexchanged (1) where δ18OH2O liquid 1 ) -8.14 ( 0.05 ‰ and δ18OH2O liquid 2 ) 40.37 ( 0.05 ‰; 1000 ln RH2O liquid-H2O vapor at 21 °C ) -9.66‰ according to the fractionation factor equation calculated by Majoube;36 RH2O vapor - Oexchanged is the fractionation factor of the silica-vapor system at 200 °C with 1000 ln RH2O vapor - O exchanged that approximates δ18Oexchanged - δ18OH2O vapor ) 13.5‰ at 200 °C, as estimated by Labeyrie and Juillet25 by plotting the percentage of exchanged oxygen vs measured δ18O diatom values and extrapolating to nonexchanged and totally exchanged diatom samples. We assume a standard deviation (SD) of 0.1‰ for both 1000 ln R H2O liquid-H2O vapor and δ18Oexchanged - δ18OH2O vapor. This leads to δ18Oexchanged 1 and δ18Oexchanged 2 values of -6.21 ( 0.15 and 42.32 ( 0.15, respectively. The percentage of exchangeable oxygen (X in %) is calculated as follows:2

δ18Osilica ) δ18Osilica )

100δ18Omeasured 1 - X(δ18Oexchanged 1) (100 - X) 100δ18Omeasured 2 - X(δ18Oexchanged 2) (100 - X)

(2)

(3)

or

X)

100(δ18Omeasured 1 - δ18Omeasured 2) (δ18Oexchanged 1 - δ18Oexchanged 2)

(4)

The δ18O value of the nonexchangeable silica (δ18Osilica in ‰ vs V-SMOW) is calculated using eq 2. Error Associated with the Calculation of δ18Osilica Value. In order to get the best estimate of error associated with the calculation of δ18Osilica, we present the results of a Monte Carlo simulation that determines the median and limits of the 90% confidence interval (q0.5, q0.05, and q0.95) of X and δ18Osilica, using R.37 X and δ18Osilica are computed 10 000 times, using 10 000 simulated values of the 6 variables taken into account in eqs 1-4: (1) 1000 ln RH2O liquid-H2O vapor, 1000 ln RH2O vapor-Oexchanged, δ18OH2O liquid 1 and δ18OH2O liquid 2 are randomly sampled from their normal distributions characterized by their mean and standard deviation (SD) given by the literature or measured at the laboratory. (2) δ18Omeasured 1 and δ18Omeasured 2 are randomly sampled from their empirical distributions (measured values in Table S-1), as the assumption of a normal distribution is not justified for a small number of samples (n in Table 2). Note that in the case of a high number of samples showing a normal distribution, limits of the 90% confidence interval should equal the mean value (1.6 SD. RESULTS AND DISCUSSION Reproducibility of δ18Omeasured Data. In order to measure with accuracy the reproducibility that one may expect from the (36) Majoube, M. J. Chim. Phys. Biol. 1971, 10, 1423-1436. (37) R Development Core Team. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0 (http://www.R-project.org.), 2006.

Table 1. Long Term δ18O Measurement (‰ vs V-SMOW) of 1.5 mg of Phytolith and Diatom Samples Performed after a Controlled Isotopic Exchange (CIE) with Water 1 (δ18O Water ) -8.14 ‰ vs V-SMOW) per controlled isotopic exchange (CIE)

whole set of analyses

δ18Omeasured 1 samples MSG 40

MSG 60

MSG 90

KYO 50

a

date CIE

na

mean

SD

mean SD

01/19/2006 02/07/2006 04/12/2006 04/26/2006 05/10/2006 05/31/2006 12/06/2006 03/07/2005 03/22/2005 06/02/2005 06/13/2005 07/04/2005 04/12/2006 04/26/2006 05/31/2006 03/22/2005 05/30/2005 06/13/2005 07/04/2005 07/12/2005 05/31/2006

7 8 7 5 6 3 1 3 6 6 6 7 4 3 2 6 5 4 4 4 2

36.73 36.72 37.04 36.76 36.18 36.34 36.64 36.63 37.37 37.07 37.75 37.54 36.31 36.68 37.19 33.53 33.88 34.30 34.07 34.11 34.34

0.31 0.11 0.19 0.26 0.20 0.47 0.06 0.12 0.29 0.17 0.29 0.16 0.40 0.12 0.32 0.20 0.10 0.35 0.29 0.11

07/04/2005 07/12/2005 01/19/2006 02/07/2006 04/12/2006 04/26/2006 05/31/2006 06/23/2006 07/03/2006 07/19/2006 10/16/2006 10/24/2006 01/03/2007

2 2 3 4 7 6 4 3 5 3 2 3 3

32.69 32.56 32.48 32.91 31.90 31.15 31.96 31.64 31.67 31.22 31.91 31.80 32.12

Lacustrine Diatoms 0.04 0.17 0.09 0.08 0.18 0.15 0.14 0.16 0.11 0.21 0.14 0.39 0.30 0.18

δ18Omeasured 1 mean, SD of the mean

nb

mean

mean SD

Phytoliths 0.26

36.63 ( 0.31

37

36.67

0.36

0.20

37.07 ( 0.45

37

37.17

0.51

0.23

34.04 ( 0.30

25

33.97

0.37

32.01 ( 0.54

47

31.92

0.54

Number of analyses per CIE. b Number of analyses per sample.

method presented in this study, SD on δ18O measurements of the 1.6 mg of phytolith and diatom samples were calculated over a long-term period (2 years, 151 and 123 analyses of phytoliths and diatoms, respectively). Mean and SD values are presented for δ18Omeasured 1 of MSG 40 (37 analyses, 7 CIE), MSG 60 (37 analyses, 8 CIE), MSG 90 (25 analyses, 6 CIE), and KYO 50 (47 analyses, 13 CIE) in Table 1. δ18Omeasured 1 and 2 values for individual analyses are presented in Table S-1. Mean δ18Omeasured 1 values are similar when they are calculated taking into account either the whole set of analyses or the average per isotopic exchange. When the whole procedure is taken into account (CIE/IR laser-heating fluorination/O2 δ18O analysis), i.e., when SD is calculated from the whole set of measured δ18O values per sample, it is lower than or equal to (0.51‰ for phytoliths and (0.54‰ for diatoms. When the exchange step is not taken into account, i.e., when SD is calculated from the mean measured δ18O values per CIE, average SD is lower than or equal to (0.26‰ for phytoliths (MSG 40) and (0.17‰ for diatoms (KYO 50). Comparison with techniques already available is difficult as measurement conditions (e.g., number of replicates, number of CIE) from which reproducibility is calculated are often incom-

pletely reported. Using the CIE/conventional fluorination/CO2 δ18O analysis method, Shahack-Gross et al.,12 following Shemesh et al.,6,38 present an average SD of 0.2‰ calculated from duplicate analyses of 11 diatom samples; Webb and Longstaffe13 present an average SD of 0.2‰ calculated from duplicate analysis of 8 phytolith samples produced from 2 CIE. Diatoms δ18O data given by Schmidt et al.20 show a SD ranging from 0.2‰ (5 replicates) to 0.9‰ (12 replicates). Using the stepwise fluorination/CO2 δ18O analysis method on a diatomite standard sample, Leng et al.21 gave a mean variance of 0.28‰, which is equivalent to a standard deviation of 0.53‰. Using the iHTR/CO δ18O analysis method, Lu¨cke et al.27 gave an average SD of 0.19‰ calculated from average δ18O values obtained from four analyses of a diatom standard per day, run over 6 days within several weeks. These values are close to or higher than the mean SD obtained from the long-term laser-heating fluorination technique/O2 δ18O analysis calibration (mean SD < 0.26‰ for phytoliths and < 0.17‰ for diatoms, Table 1). They are often lower than the SD obtained when including several exchange procedures (SD of 0.51‰ for (38) Shemesh, A.; Burckle, L. H.; Hays, J. D. Paleoceanography 1995, 10, 179196.

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Table 2. Estimation of Percentage of Exchangeable Oxygen (X) and δ18O Silica (‰ vs V-SMOW), for Both Phytolith and Diatom Samples Exchanged with Two Waters, Using Monte Carlo Simuationa δ18Omeasured 1b

δ18Omeasured 2b

90% confidence interval X q0.05f

q0.95f

δ18Osilica

δ18Osilica q0.5f

2.98 2.97 3.43 1.17 5.34 2.84

6.54 6.50 4.88 4.88 6.97 6.35

38.76 38.45 39.38 38.40 38.71 35.81

Lacustrine Diatoms 11 6.64 3.50 15 7.29 4.57 5 4.08 1.88 11 5.14 2.15 5 7.27 7.18 5 7.69 5.76

8.28 9.73 5.63 6.96 7.54 9.59

36.23 34.76 35.59 36.60 36.42 35.28

Xd q0.5f

samples

mean

SD

nc

mean

SD

nc

MSG 40 MSG 40-0.3 mg MSG 50 MSG 60 MSG 70 MSG 90

36.67 36.60 37.51 37.17 36.13 33.97

0.36 0.52 0.52 0.51 0.21 0.37

37 3 11 37 4 25

39.00 38.68 39.57 38.56 39.01 36.17

0.36 0.31 0.17 0.26 0.16 0.40

Phytoliths 12 4.80 4 4.51 3 4.22 5 2.72 4 6.08 6 4.56

KYO 40 KYO 50 KYO 60 KYO 70 KYO 90 KYOHClO4

33.78 31.92 33.95 34.55 33.48 32.21

0.19 0.54 0.28 0.30 0.10 0.36

6 47 6 5 2 5

36.68 35.41 35.93 36.96 36.95 35.94

0.85 0.54 0.52 0.64 0.16 0.52

90% confidence interval δ18Osilicae q0.05f

q0.95f

38.80 38.45 39.33 38.35 38.78 35.80

37.99 38.01 39.21 38.08 38.55 35.30

39.18 38.81 39.55 38.81 38.90 36.29

36.64 34.77 35.66 36.69 36.49 35.31

35.30 34.07 34.93 35.45 36.45 34.52

37.15 35.65 36.15 37.26 36.51 35.67

or empirical distributions: 1000 ln R H2O liquid-H2O vapor36 ) -9.66 ( 0.15‰; 1000 ln R ( 0.05‰; δ 18OH2O liquid 2 ) 40.37 ( 0.05‰; δ18Omeasured 1 and δ18Omeasured 2 (Table S-1). Silica calculated from eq 3 without Monte Carlo simulation is also given for comparison. b Mean and SD of δ18Omeasured 1 (CIE with water1, -8.14‰) δ18Omeasured 2 (CIE with water2, 40.37‰) in ‰ vs V-SMOW calculated over the whole set of analyses. c Number of analyses per sample. d Percentage of exchangable oxygen calculated following eq 4 using a Monte Carlo simulation. e Calculated following eq 3 using a Monte Carlo simulation. f q0.5, q0.05, and q0.95 are, respectively, the median, 5%, and 95% quantiles calculated using a Monte Carlo simulation .

a Six variables are randomly sampled from their normal 25 18 H2O vapor-Oexchanged ) 13.5 ( 0.15‰; δ OH2O liquid 1) -8.14

MSG 40 and 0.54‰ for KYO 50), which is the poorer but more reliable reproducibility we may expect from the CIE/ IR laserheating fluorination technique. Uncertainty Associated with δ18Osilica Values. The kind of simulation presented here for accurately estimating uncertainty associated with δ18Osilica values may be of particular interest for discussing, in future studies, the efficiency of δ18Osilica data as a proxy of continental temperature and δ18Osoil water (phytoliths) or δ18Olake water (diatoms). Given the CIE conditions presented in this study (i.e., two exchanged waters with δ18Owater close to δ18Osilica for the first one and lighter by about 50‰ for the second one), error bars (90% confidence interval) associated to the percentage of exchangeable oxygen (X) are close to or lower than 2% for the phytolith subsamples and close to or lower than 3% for the diatom subsamples. Error bars associated to the δ18Osilica values are close to (0.5‰ for the phytolith subsamples, close to or lower than (1‰ for the diatom subsamples (Table 2). The 90% confidence intervals of δ18Osilica vary proportionally to the reproducibility of the measurements (δ18Omeasured SD), with no relationship to the amount of exchangeable oxygen being evident (Table 2). In future studies, similar errors on δ18Osilica values for both fossil (weakly hydrous) and fresh (highly hydrous) materials are reasonably expected. Given the range of δ18Omeasured values of 35-39‰ and according to the opal fractionation factor obtained from phytoliths,12 an uncertainty of (0.5‰ on δ18Osilica leads to a reconstructed temperature uncertainty lower than (2°C. This is close to temperature uncertainties obtained using δ18O signature of phosphate and carbonate land and freshwater organisms39 and lower than uncertainties obtained by climatic reconstructions from pollen data.40 For a fixed temperature, uncertainty of (0.5‰ on δ18Osilica leads to an uncertainty of (0.5‰ on δ18Oforming water which is lower than the seasonal variations of δ18Oforming water of tropical soil waters (2-5‰,18) and of tropical lake waters.41,9 For compari2376 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

son, a 1-2‰ error in the estimation of soil water is expected from δ18O measurements of plant cellulose and its derivative.42 Accuracy and Reproducibility of δ18O Measurements of Samples as Small as 0.3 mg. The δ18O measurements of small samples (0.3 mg) of the quartz laboratory standard give an average δ18O value and SD of 16.36 ( 0.13‰ (n ) 6) similar to the average δ18O value and SD obtained for large samples (1.5 mg) (16.36 ( 0.09‰; n ) 16). The δ18O measurements of minute aliquots (0.3 mg) of the phytolith sample MSG 40 (MSG 40-0.3 mg) give average δ18Omeasured 1 and δ18Omeasured 2 values and SD close to the values obtained for larger samples (Table 2). The calculated X and δ18Osilica values are also similar, in the 90% confidence interval, to the values obtained for the larger sample MSG 40. These results demonstrate that quartz and biogenic silica samples as small as 0.3 mg can be accurately analyzed using the IR laser-heating fluorination technique, provided that it is associated with a mass spectrometer equipped with an autocooled microvolume that concentrates small quantities of O2 gas prior to their expansion into the dual inlet system. Effect of Temperature and Chemical Treatment on δ18Osilica Values. Phytolith and diatom samples are expected to respond differently to cleaning procedures, to isotopic exchange, and to laser-fluorination. Indeed, their primary surface area is different: 5.18 m2/g for the phytolith MSG sample;43 23.87 m2/g for a Miocene diatomite44 close to the KYO sample. Moreover, (39) Grimes, S. T.; Mattey, D. P.; Hooker, J. J.; Collinson, M. E. Geochim. Cosmochim. Acta 2003, 67, 4033-4047. (40) Peyron, O.; Jolly, D.; Bonnefille, R.; Guiot, J. Quat. Res. 2000, 54, 90-101. (41) Vallet-Coulomb, C.; Gasse, F.; Robison, L.; Ferry, L. J. Geochem. Explor. 2006, 88, 153-156. (42) Sternberg, L. S. L.; Anderson, W. T.; Morrison, K. Geochim.Cosmochim. Acta 2003, 67, 2561-2566. (43) Fraysse, F.; Pokrovsky, O. L.; Schott, J.; Meunier, J. D. Geochim. Cosmochim. Acta 2006, 70, 1939-1951. (44) Bustillo, M. A.; Fort, R.; Bustillo, M. Eur. J. Mineral. 1993, 5, 1195-1204.

Figure 1. δ18O silica values of phytolith (a) and diatom (b) samples versus chemical treatment temperature. Error bars represent the 5 and 95% quantiles (q0.05 and q0.95)

while phytoliths can contain less than 1% by weight of organic carbon, coming from the wall and from the cytoplasm of the plant cell in which they precipitate,45 the surface cell wall composition of fresh diatoms can contain more than 60% of organic carbon.46 Although this outermost part must be almost removed during the chemical treatment, some residues of organic matter located in the inner part of the cell wall may remain.47 To our knowledge, this inner organic matter has never been quantified. These differences must be kept in mind when interpreting Figure 1, which plots the δ18Osilica values of both phytolith and diatom samples versus chemical treatment temperature. The δ18Osilica values of phytoliths are very close when the chemical treatment temperature is lower than or equal to 70 °C; the mean δ18Osilica value being 38.8 ( 0.4‰ (1 SD). This demonstrates that there is no significant isotopic fractionation involved at these temperatures. The δ18Osilica value is significantly lower when the chemical treatment temperature reaches 90 °C (δ18Osilica value of 35.8‰). Dissolution/precipitation processes may explain this trend, in agreement with the thermodependent relationships between δ18O value of the forming water and δ18O value of the precipited silica phase. As phytolith chemical treatment is processed at almost constant volume, when the solution temperature increases from room temperature to 90 °C (e.g., at the beginning of the organic matter oxidation step), the solubility of amorphous silica increases41 and phytoliths start dissolving. Conversely, with decreasing temperature and for constant dissolved silica concentrations, the solution is more saturated and precipitation of amorphous silica takes place.48 In order to avoid precipitation processes, we recommend an optimal chemical treatment temperature of 70 °C for phytoliths. However higher (45) Smith, F. A.; Anderson, K. B. In Phytoliths: Applications in Earth Science and Human History; Meunier, J. D., Colin, F., Eds; A. A. Balkema Publishers: Rotterdam, The Netherlands, 2001; pp 317-327. (46) Gelabert, A.; Pokrovsky, O. S.; Schott, J.; Boudou, A.; Feurtet-Mazel, A.; Mielczarski, J.; Mielczarski, E.; Mesmer-Dudons, N.; Spalla, O. Geochim. Cosmochim. Acta 2004, 68, 4039-4058. (47) Sumper, M.; Kro ¨ger, N. J. Mater. Chem. 2004, 14, 2059-2065. (48) Gunnarsson, I.; Arnorsson, S. Geochim. Cosmochim. Acta 2000, 64, 22952307.

chemical treatment temperature may be suitable provided that dilution is performed after each heating step and before discarding the solution, as processed during the diatom treatment. δ18Osilica values of diatoms are close when the chemical treatment temperature ranges from 50 to 60 °C, whatever the extraction protocol used (HClO4 vs standard protocols), the mean δ18Osilica value being 35.2 ( 0.4‰ (1 SD). When chemical treatment temperature is lower (40 °C) or higher (70 and 90 °C), δ18Osilica values increase by about 1 ‰. Presence of postfluorination residues that decrease from KYO 40 to KYO 90 supports the presence of the remains of organic compounds, due to an incomplete cleaning. These organic remains would prevent total controlled isotopic exchange and reaction to the laser-fluorination and contribute to the high δ18Osilica value measured for the KYO sample cleaned at 40 °C. The high δ18Osilica values measured for the KYO sample cleaned at 70 or 90 °C may be explained differently, by the commencement of dissolution of the porous silica structure; the lighter isotope, forming weaker bonds and having a higher diffusion velocity than the heavier isotope, goes preferentially to the liquid phase. Scanning electron microscopy (SEM) supports this hypothesis: while surface ornamentations can be easily distinguished on diatom frustules from KYO 40 and KYO 50, frustules from KYO 70 are devoid of ornamentation and pitted. Although Tyler et al.49 investigated the effects of several preparation methods on δ18Osilica values of diatoms, to our knowledge, the impact of increasing temperature when using chemical treatment was never tested. Finally, the close δ18Osilica values of KYO 50, KYO 60, and KYOHClO4 suggest that for a chemical treatment temperature of 50-60 °C, whatever the cleaning protocol is, organic matter oxidation reaches a maximum while silica dissolution is minimal, leading to stable δ18Osilica values. This 50-60 °C chemical treatment temperature range is commonly used for diatom cleaning.34,38,4,20,50 Our results support that a chemical treatment temperature higher than 60 °C51 could induce potential isotopic fractionation due to dissolution of the frustule. One or Two Controlled Isotopic Exchanges? As suggested by Shemesh et al.,38 by choosing an exchange water leading to a δ18Oexchanged close to the δ18Osilica value, the δ18Omeasured value is no longer sensitive to the amount of exchangeable oxygen, and a second exchange with a lighter water can be avoided. However, as the difference between δ18Osilica and δ18Oexch is linked to the amount of exchangeable oxygen and to the real δ18Osilica value, it cannot be considered as constant from one sample to another. Thus, choosing a unique controlled exchange step adds an uncontrolled bias to the estimation of δ18Osilica that should be taken into account when discussing the data. For instance, Table 2 shows that for the range of exchangeable oxygen measured in our study (X ) 2-8%) and when δ18Osilica is, respectively, 3-7 ‰ lower than δ18Oexchanged 2, i.e., 1-5 ‰ lower than δ18OH2O liquid 2, the difference between δ18Omeasured 2 and δ18Osilica varies from 0.2 to 0.7‰. Thus, in order to make a significant use of δ18Osilica as an (49) Tyler, J. J.; Leng, M. J.; Sloane, H. J. J. Paleolimnol. 2007, 37, 491-497. (50) Schmidt, M.; Botz, R.; Rickert, D.; Bohrmann, G.; Hall, S. R.; Mann, S. Geochim. Cosmochim. Acta 2001, 65, 201-211. (51) Morley, D. W.; Leng, M. J.; Mackay, A. W.; Sloane, H. J.; Rioual, P.; Battarbee, R. W. J. Paleolim. 2004, 31, 391-401.

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accurate proxy of paleoclimate, we recommend performing two controlled exchange steps. CONCLUSION The IR-laser fluorination technique reported here for analyzing the oxygen isotope composition (δ18O) of phytoliths and diatoms gives long-term reproducibility close to the one obtained with techniques already available. Related to this reproducibility, uncertainty associated with the calculation of δ18Osilica, and assessed here for the first time, is good enough for using δ18Ophytoliths and diatoms as proxies of continental temperatures and δ18Osoil water (phytoliths) or δ18Olake water (diatoms). Performed after a controlled isotopic exchanged (CIE) procedure, the IR-laser protocol prevents ejecta and the success of the fluorination reaction can be checked prior to measuring the liberated oxygen yield. Particularly, if postfluorination residues are observed, this indicates that organic compounds were still present in the sample. In the case of diatoms, organic remains are trapped in significant amounts inside the outermost layer of the silica structure and insufficient cleaning may prevent complete controlled isotopic exchange and reaction to the laser-fluorination. Visual control on the efficiency of the laser fluorination allows detecting such a case. Because of their extremely low organic content, phytoliths do not face this problem. The laser-heating fluorination technique is faster than the conventional fluorination method and allows analyzing the oxygen

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isotopic composition (δ18Omeasured) of samples as small as 0.3 mg, as required for high-resolution paleoenvironmental reconstructions. ACKNOWLEDGMENT This research was conducted in the framework of J. Crespin’s Ph.D. thesis and was supported by CEREGE. A.A. is grateful to P. Girard, C. France-Lanord, and C. Guillemette for their advising when setting up the oxygen extraction line of the stable isotope laboratory. Thanks also to E. Webb, A. Shemesh, and M. Schmidt for their precious advice when we built up the equilibration line, to J.-P. Ambrosi for fruitful discussion on silica dissolution/ precipitation processes, and to C. Vallet-Coulomb for oxygen isotope analysis of waters. SUPPORTING INFORMATION AVAILABLE δ18O values for individual analyses (expressed in ‰ vs V-SMOW) of phytolith (MSG 40, MSG 40-0.3 mg, MSG 50, MSG 60, MSG 70, MSG 90) and diatom (KYO 40, KYO 50, KYO 60, KYO 70, KYO 90, KYO HClO4) samples, performed after controlled isotopic exchanges (CIE) with water 1 (δ18Owater 1 ) -8.14 ( 0.05‰) and water 2 (δ18Owater 2 ) 40.37 ( 0.05‰). This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 12, 2007. Accepted January 4, 2008. AC071475C