Solubilities of bone mineral from archaeological sites: the recrystallization window

Journal of

Archaeological SCIENCE Journal of Archaeological Science 31 (2004) 867–882 http://www.elsevier.com/locate/jas

Solubilities of bone mineral from archaeological sites: the recrystallization window Francesco Berna a*, Alan Matthews b, Stephen Weiner a a

b

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel Institute of Earth Sciences, Givat Ram Campus, Hebrew University, Jerusalem 91904, Israel

Received 14 October 2003; received in revised form 30 November 2003; accepted 4 December 2003

Abstract Bone mineral solubility is an important parameter for understanding the preservation of bones in the archaeological and palaeontological records. In this study we have measured the solubility of the carbonated hydroxyl apatite of sub-recent and fossil bones, as well as synthetic hydroxyl apatite in deionized water and in pH-buffered solutions. The solutions were open to the atmosphere and the pH values were around neutral; measurement conditions that are relevant to bone mineral preservation in nature, but that were absent from most previous studies. We obtained internally consistent results from both the water and the buffered experiments supporting the notion that we are measuring an inherent property of the mineral phase. We found that bone mineral is much more soluble than synthetic hydroxyl apatite. We measured the ionic activity products at “steady state” conditions and we identify a recrystallization window between pH 7.6 and 8.1, which defines the conditions under which bone crystals dissolve and reprecipitate as a more insoluble form of carbonated hydroxyl apatite. As these conditions are common in nature, most fossil bones will not maintain their original crystals with time. We also found that bones that contained small amounts of calcite did not dissolve at all during our experiments. These results provide a basis for better understanding the conditions in sediments under which bones are preserved and the relative states of preservation of bone. They also have important implications for the selection of the most appropriate bone samples for paleoenvironmental and paleodiet analyses and dating.  2004 Elsevier Ltd. All rights reserved. Keywords: Carbonated apatite; Hydroxyl apatite; Bone; Diagenesis; Solubility; Recrystallization

1. Introduction Bone is a major component of the archaeological and palaeontological records. Its mineral and macromolecular components may contain much information about the animal itself and the environment in which it lived. Extracting this information, however, depends upon an understanding of the stability of the components of bone with time. Here we focus on the stability of the mineral phase of bone, which is carbonated hydroxyl apatite [(Ca, Na, Mg)5(HPO4, PO4, CO3)3(OH, CO3)]. Bone mineral is also referred to as carbonated apatite or dahllite [25]. Field observations in archaeological sites show that bones are generally preserved when they co-exist with * Corresponding author. Fax: +972-8-9344136 E-mail address: [email protected] (F. Berna). 0305-4403/04/$ - see front matter  2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2003.12.003

calcite and authigenic carbonated apatite [(Ca, Na, Mg)5(PO4, CO3)3(OH, F)] in the sediments, but are absent—presumably due to dissolution—when more insoluble (hence, more stable) authigenic calcium aluminum phosphate minerals are present in the sediments [16,17,40,48,49]. Bone dissolution reflects changes in the chemical environment within the sediments. Even when bones do not dissolve, their mineral phase changes [36,46]. In order to better understand the changes that occur to bone crystals over time, as well as the precise conditions under which bone mineral dissolution and/or recrystallization take place, information is needed on the solubilities of fossil bones at different stages of diagenesis. Solubility of a fined-grained solid in essence reflects the nature of its crystal structure and, in particular, its surface characteristics. Solubility can thus provide insights into the driving mechanisms behind mineral diagenesis.

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A significant fraction of the carbonate ions in fresh bone substitutes for the phosphate groups of the hydroxyl apatite [Ca5(PO4)3OH] structure, resulting in a non-stoichiometric composition for bone mineral with respect to pure hydroxyl apatite [21]. Bone’s carbonated apatite crystals in vivo are plate-shaped and extremely small, with average dimensions of 50252–4 nm and a very large specific surface area of about 240 m2/g [46]. Both these factors contribute to its solubility. Bone crystals are not very different from those of synthetic carbonated hydroxyl apatite formed around neutral pH. TEM analyses show that such synthetic crystals are also plate-shaped and tend to be somewhat larger and less elongated than those found in bone [28]. Thus, it is not only the fact that the bone crystals form in the collagen framework that makes them plate-shaped and so small. One proposal is that the initially formed phase is not hydroxyl apatite, but octacalcium phosphate, a mineral similar to hydroxyl apatite and one that naturally has a plate-shape [4]. When bone is on the soil surface or buried in the ground, the crystals undergo changes. They become larger and acquire a needle-like morphology more similar to that of pure hydroxyl apatite [37,44]. These changes will decrease the solubility of altered bone as compared to fresh bone. Aspects of these changes can be monitored by the so-called crystallinity index of bone, which is measured by line width broadening using X-ray diffraction [1,41] and/or by the sharpening of two absorption peaks in the infrared spectrum; the so-called splitting factor [42,45]. The uptake of fluoride from the soil solution will lead to the formation of fluorinated carbonated hydroxyl apatite, francolite [Ca5(PO4, CO3)3F] and eventually to fluoroapatite [Ca5(PO4)3F]. The presence of a significant amount of fluoride substitutions and the formation of francolite is detectable by FTIR spectroscopy in the splitting factor region [9]. The progressive incorporation of fluoride in the lattice increases the stability of the apatite as compared to carbonated hydroxyl apatite resulting in fluoroapatite being more insoluble than pure hydroxyl apatite [22]. This explains why francolite is the usual constituents of the most ancient fossil bones such as the ones of dinosaurs [14]. The fluoridation of apatite depends on the availability of fluoride in the sediment solution which is very variable throughout different environments [22]. It is interesting to note that in the karstic caves of Israel most prehistoric bones have not transformed into francolite [47,48]. Solubility of relatively insoluble mineral phases is very difficult to measure directly, especially for minerals with as complex a structure and stoichiometry as carbonated hydroxyl apatite [3]. Furthermore in this study, we are interested in the solubilities of prehistoric bone mineral, which is often intimately associated with other minerals and has adsorbed or altered surface phases. It

is therefore not surprising that for the carbonated apatite of fossil bone mineral there are no measurements of the thermodynamic solubility, referred to as the solubility product (Ksp). The Ksp of fresh bone mineral [18] and synthetic hydroxyl apatite have been measured [26]. The conditions used to measure the solubility product require a closed system and a controlled atmosphere. Even then it is doubtful that the true Ksp is measured for bone apatite in that bone also contains about 20% by weight organic material. Moreover, if this organic material is initially removed from the bone, then it is questionable whether the mineral surface’s atomic structure is preserved in its in vivo state; any surface changes will certainly affect solubility [43]. As the primary aim of our study is to understand the driving mechanisms behind bone mineral diagenesis, we have opted for making the measurements under conditions open to the atmosphere and at pH conditions around neutral. We have also not pre-treated the bone samples. We therefore cannot demonstrate that equilibrium (in strictly thermodynamic terms) is obtained between the solid and the dissolved ions, and in reality we measure ion activity products (IAP) obtained after prolonged “steady state” conditions. We do show that under these conditions the results are internally consistent and appear to be relevant to natural conditions. The experimental strategy used was to first treat a series of sub-recent and fossil bones from archaeological sites by allowing them to reach steady state conditions in deionized water, and determine their relative solubilities. We then performed experiments with some of the subrecent bones using pH-buffered solutions in order to induce more mineral dissolution. For comparison, we used synthetic hydroxyl apatite in both experiments. We also regard this mineral phase as reasonable analog of a highly insoluble end-member of the bone mineral early diagenetic process.

2. Materials and methods Synthetic hydroxyl apatite (SHA) (Baker Chemical Co. 1-1436) and different mammalian bones were analysed. The latter includes three sub-recent bones (SR0, SR1 and SR2) and 3 fossil bones from the early Bronze Age (EB), the Chalcolithic (CL) and the Middle Paleolithic (MP). Sub-recent bone was used instead of fresh bone in order to avoid having to chemically remove the tissues and other organic components of the fresh bone, which could affect the surface properties of the bone crystals, especially if oxidants such as sodium hypochlorite are used [43]. For the same reason the bone samples were not pretreated with acetic acid [31]. Details of the bones are given in Table 1. Whole portions of long bones were selected, adhering sediments were mechanically removed and the samples

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Table 1 Description of the bones used in this study Sample no.

Archaeological period Mammal group

Age of deposition (years)

Location in Israel

Depositional environment

Collagen (wt%)

SR0 SR1 SR2

Sub-recent Sub-recent Sub-recent

Bovine Human Human

w1–2 w50 w50

Early Bronze Age Chalcolithic

Human Ovi-caprine

w5500 w7000

MP

Middle Palaeolithic

Cervids

w55 000

On soil surface Sandy soil Carbonate and clay rich cave sediment Sandy soil Anthropogenic sediment beneath clay layer Partially brecciated ashy deposit

26.5 21.7 28.0

EB CL

Nahal Galil (Haifa) Lahav Tzadik (Negev) Ein Tamar (Judean Desert) Azur (Coastal Plain) Ein Hashofet (Mt. Carmel) Kebara (Mt. Carmel)

were washed in deionized water (DIW). All the bone samples were then dried under a heat lamp, and homogenized by grinding them in an agate mortar and sieved to obtain the 63–125 µm sized fraction. The weight percent of collagen was determined by dissolving the samples in 1 N HCl and weighing the washed insoluble fraction. The 1 N HCl insoluble fraction as well as the mineral phase of the samples was investigated by Fourier transform infrared (FTIR) spectroscopy using KBr pellets and obtaining spectra at 4 cm
1 resolution with a Midac spectrometer. The IR splitting factor (IRSF) of the bone mineral phase was calculated according to the convention proposed by Weiner and BarYosef [45]. The shapes and dimensions of the crystals were analyzed using transmission electron microscopy (TEM) (Philips CM12) after disaggregating the crystals in sodium hypochlorite (Method 2 in [46]). The elemental compositions (Ca, P, Na, Mg) of the samples were analyzed by dissolving the samples in 3 N HCl and analyzing the solution by inductively coupled plasma optical emission spectrometry (ICP-OES-Spectro). Carbonate (CO3) contents were analyzed by dissolving the samples with 1 N HCl in a vacuum line and measuring the volume of produced CO2.

0.0 2.1 0.0

and Al (w10
7 M) in chloride forms were also initially added to all the buffered solutions to mimic reasonable soil solution concentrations [32]. The pH values and exact compositions of the different starting buffered solutions are given in Table 2. For all the experiments, the flasks were placed in a thermostated shaker (Innova 4430) maintained at 25.00.2 (C and kept in suspension at 250 rpm. The atmospheric CO2 partial pressure (pCO2=310
4 atm) was maintained constant by inserting a needle (diameter=2 mm) in the rubber stopper of the flasks allowing air exchange, but keeping evaporation negligible. Biological activity was inhibited by dissolving 10 mg l
1 of sodium azide (NaN3) into each flask. Dissolution of the mineral phase was monitored by measuring total Ca concentrations and pH at different time intervals. Five ml of the suspension were sampled in each measurement, filtered (Millipore 0.22 µm) and analyzed for pH and Ca by flame atomic absorption spectrometry (Perkin Elmer 5100 PC). 0.1% SrCl2 was added to the analyzed solutions to suppress phosphate interferences. The estimated analytical uncertainty was 5%. 2.2. Analyses of the “steady state” solutions and the composition of the residues

2.1. Experimental setup Two sets of dissolution experiment were performed: (1) in aqueous solutions with synthetic hydroxyl apatite, sub-recent and fossil bone samples; (2) in buffered solutions with synthetic hydroxyl apatite and only subrecent bone. An aliquot of 80 to 100 mg of each sample was pre-washed in Millipore water and placed in a 250 ml glass Erlenmeyer flask with 200–250 ml of solution. The water experiments were performed by using deionized water (MilliQ 18.2 M
cm) in each flask. For the buffered experiments the samples were placed in a series of 0.05 M Tris-Base (Bio-Lab ultrapure) solutions, buffered with 0.2 N HCl to different pH values, ranging from 6.2 to 7.8. The pH-buffered solutions had two main initial total P regimes, high (w10
3 M) and low (w10
6 M), prepared with crystalline H3PO4. Ca (w10
4 M)

When the released Ca had reached a prolonged “steady state” as determined by a plateau on the calcium release plot, the experiment was stopped and the suspensions were centrifuged for 30 min at 4000 rpm at 25 (C in a thermostated centrifuge (Eppendorf 5810R) and the supernatant was filtered (Millipore 0.22 µm). The residual solid phase was freeze-dried and stored under vacuum for analysis. The solutions were then analyzed for pH, total dissolved inorganic carbon (IC), and concentrations of total Ca, P, Na, Mg and Al. pH was determined with a Hamilton Skylite electrolyte single pore electrode with an error of 0.01 pH units as defined by the standard buffers. The total concentrations of Ca, P, Na, Mg and Al were analyzed by ICP-OES (Spectro). To ensure identical solution conditions, multiple Ca, P, Na, Mg and Al standards were prepared

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Table 2 Initial chemical composition of the 0.05 M Tris-Base solutions and resulting ionic strength (µ) at atmospheric CO2 partial pressure (pCO2=3.010
4 atm). These solution were used to dissolve SHA, SR0, SR1 and SR2 starting material Solution name

pH

Cl

Ca

P

Na

Mg

Al

µ

2.84E-07 5.76E-08 8.64E-08 9.05E-08 1.07E-07 6.99E-08 1.60E-07 1.28E-07 1.77E-07 w 1.48E-07 2.88E-07

2.59E-08 7.04E-08 w 1.11E-07 2.96E-08 2.04E-07 7.41E-08 1.33E-07 1.48E-07 2.48E-07 2.00E-07 3.37E-07

0.076 0.076 0.079 0.079 0.073 0.071 0.077 0.075 0.067 0.066 0.071 0.070

M 65/6 65/6A 65/3 65/3A 70/6 70/6A 70/3 70/3A 75/6 76/6A 75/3 76/3A

6.53 6.51 6.24 6.50 7.11 7.01 7.16 7.01 7.64 7.42 7.55 7.35

5.02E-02 5.02E-02 5.02E-02 5.02E-02 4.74E-02 4.55E-02 4.74E-02 4.55E-02 4.02E-02 4.02E-02 4.02E-02 4.02E-02

1.07E-04 1.10E-04 1.12E-04 1.06E-04 1.07E-04 1.06E-04 1.03E-04 1.03E-04 1.10E-04 1.06E-04 1.06E-04 1.04E-04

6.20E-07 6.30E-07 1.09E-03 1.10E-03 5.20E-07 6.49E-07 1.11E-03 1.08E-03 8.07E-07 5.07E-07 1.08E-03 1.07E-03

7.09E-05 6.77E-05 6.73E-05 6.52E-05 6.80E-05 6.59E-05 6.65E-05 6.42E-05 6.67E-05 6.65E-05 6.70E-05 7.02E-05

using Tris–HCl buffers. The analytical uncertainties are estimated as follows: P and Al 3%, Ca 4%, Mg 6%, Na 8%. IC total concentration was measured using a TOC-IC analyzer (Skalar FormacsHT) with an estimated reproducibility of 10% in the unbuffered water solution and of 23% in the pH-buffered solutions. The aliquots of freeze-dried residues from the dissolution experiments on the synthetic hydroxyl apatite and the bone samples were analyzed by FTIR to detect possible phase transformations and/or the precipitation of other Al phosphates.

is carbonated hydroxyl apatite (CA), defined as Ca(5
x)Na(2x/3)(PO4)(3
x)(CO3)xOH(1
x/3) by [2]. The CO3 stoichiometric coefficient (x) was determined by

3. Calculations

3 K IAPHA⫽(Ca2⫹)5 (PO3K 4 ) (OH )

(3)

The following set of calculations was performed with the analytical data obtained from the “steady state” solutions.

(3Kx) x IAPCA⫽(Ca2⫹)(5Kx) (Na⫹)2x/3 (PO3K (CO2K 4 ) 3 ) K (1Kx/3) (OH )

(4)

IAPCC⫽(Ca2⫹) (CO2K 3 )

(5)

3.1. Dissolved mineral phase The weight % of the dissolved phosphate phase (D) in the buffered solution experiments was calculated as follows: D⫽{[(PT⫺P0)/P%]/W}#100

(1)

where PT is the final weight (mg) of P measured in solution, P0 is the initial weight (mg) of P in the solutions, P% is the weight% P measured in the starting mineral phase and W is the initial weight (mg) of the starting mineral phase. The corresponding solubility (Sol) of the mineral phase is reported as mg of mineral phase per l of solution. 3.2. Stoichiometry models and ionic activity products The stoichiometry model adopted for hydroxyl apatite (HA) is Ca5(PO4)3OH, while for bone, it

x⫽[502.3 C]/[65.38 C⫹60.01·100]

(2)

where C is the CO3 weight % measured in the starting material, and 502.3 and 60.01 are the formula weights of hydroxyl apatite (Ca5(PO4)3OH) and CO3 respectively. The corresponding ionic activity products (IAP) for HA, CA and calcite CaCO3 (CC) were expressed as

where the round parentheses indicate the activities of the different ions. The concentration of the different ionic species and the ionic strength (µ) of the solutions were calculated using the reactions and the relative thermodynamic constants listed in Table 3. The activity coefficients (
i) for the various ions were calculated using the extended Debye–Hu¨ckel law: log
i⫽⫺AZ2i

公 1⫹Bi公

(6)

where µ is the ionic strength of the solution, A=
0.509 and B=0.328 at 25 (C, and the value of the hydrated ˚ ) are as follows: 9 for Al3+ and H+, 4.5 ionic radii ai (A 2
2+ 2+ for CO3 and HCO
3 , 6 for Ca , 8 for Mg , 4 for +
3
2
Na , 3.5 for OH , and 4 for PO4 , HPO4 and H2PO
4 [19].

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

diagrams by plotting pH as a function of log(H2PO
4 ), using the IAPCA values and the Ca2+ and Na+ activities as experimentally determined.

Table 3 Reaction and corresponding dissociation constants used in the calculation Reaction CO2(gas)+H2O=H2CO03 H2CO03=H++HCO
3 + 2
HCO
3 =H +CO3
2+ Ca +HCO3 =CaHCO+ 3 0 Ca2++CO2
3 =CaCO3 + 0
H3PO4=H +H2PO4 + 2
H2PO
4 =H +HPO4 3
+ =H +PO HPO2
4 4 2
2H2PO
4 =(H2PO4)2 2+ 2
Ca +HPO4 =CaHPO04 + Ca2++H2PO
4 =CaH2PO4 Al3++4H2O=Al(OH)4
+4H+

Constant at 25 (C
1.47

KC0=10 KC1=10
6.35 KC2=10
10.33 KCaHCO3=101.11 KCaCO30=103.22 KP1=10
2.148 KP2=10
7.198 KP3=10
12.375 KP4=10
0.35 KCaHPO40=102.407 KCaH2PO4+=100.707 KAl=10
23.33

Reference

4. Results

[33] [33] [33] [33] [33] [30] [30] [30] [22] [10] [10] [22]

3.3. Solubility diagrams Assuming congruent dissolution, the dissolution reaction of carbonated apatite in aqueous solution is Ca(5Kx)Na(2x/3)(PO4)(3Kx)(CO3)xOH(1Kx/3)⫽(5Kx)Ca2⫹ 2K ⫹(2x/3)Na⫹⫹(3Kx)(PO3K 4 )⫹x(CO3 ) ⫹(1Kx/3)OHK (7) The corresponding ionic activity product of carbonated apatite (IAPCA) is (3Kx) IAPCA⫽(Ca2⫹)(5Kx) (Na⫹)2x/3(PO3K 4 ) 2K x ⫺ (1Kx/3) (CO3 ) (OH )

(8)

Equation (8) can be rewritten as KlogIAPCA⫽K(5Kx)log(Ca2⫹)K2x/3 log(Na⫹)K 2K (3Kx)log(PO3K 4 )Kxlog(CO3 )K(1Kx/3) log(OHK) (9) Substituting the following terms in Equation (9), Klog(OHK)⫽KlogKwKpH

(10)

K Klog(PO3K 4 )⫽Klog(H2PO4 )KlogKP2KlogKP3K2pH (11)

Klog(CO2K 3 )⫽18.15K2pHKlog(CO2)(g)

(12)

at 25 (C and pCO2=310
4 atm and solving for pH, gives pH⫽[logIAPCAK(3Kx)log(H2POK 4 )K 2⫹ (5Kx)log(Ca )K(2x/3)log(Na⫹)K2.52x ⫹72.56]/(7Kx/3)

871

(13)

Assuming congruent dissolution, from Equation (13) it is therefore possible to draw “steady state” solubility

Transmission electron microscopy was used to characterize the shapes and sizes of the bone crystals analyzed, as well as the synthetic hydroxyl apatite crystals (Fig. 1). The synthetic hydroxyl apatite crystals are needle-shaped and have an average size of 163285 nm (N=108) and a surface area of about 150 m2/g (Fig. 1a). The crystals of the sub-recent bone samples (Fig. 1b) have average dimensions of 50252–4 nm (N=120) and a specific surface area of about 250 m2/g, which are comparable with measurements on crystals from fresh bones reported by Weiner and Price [46]. The crystals of the MP bone (Fig. 1c) have an average size of 58264–5 nm (N=50) and a specific surface area of around 200 m2/g. They are therefore slightly larger than the sub-recent bones. Other mineralogical and chemical properties of the samples are listed in Table 4. 4.1. Dissolution in water The major objective of our research was to determine the relative solubilities of the sub-recent and fossil carbonated apatite samples, under conditions that are relevant to natural aqueous environments. We therefore used deionized water as a solvent in the presence of atmospheric concentration of CO2 (410
4 atm). The three sub-recent bones (SR0, SR1 and SR2) together with three fossil bones from the early Bronze Age (EB), the Chalcolithic (CL) and the Middle Paleolithic (MP), were placed in water at 25 (C, shaken and left open to the atmosphere for up to 170 days. We also analyzed synthetic hydroxyl apatite (SHA), as a control. Monitoring the release of total Ca showed that a “steady state” was reached in all cases by 100 days (Fig. 2). A similar conclusion was drawn from a plot of the ratio of Ca/OH (Fig. 3). The compositions of the “steady state” water solutions are presented in Table 5. This table shows that all samples released calcium into solution, but two of the fossil bones (samples EB and CL) did not release any significant amounts of phosphate. These two bones contained small amounts of calcite (Table 4). The pH of their solutions approached pH 8.2 and their calculated ionic activity products for calcite (IAPCC) (data not shown) approached the solubility product (Ksp) of calcite, namely logKsp=
8.480 [33]. This indicates that these solutions almost reached saturation with respect to calcite, and we conclude that the presence of calcite prevents dissolution of the carbonated apatite. Table 5 also shows the calculated ionic activity products for carbonated hydroxyl apatite (IAPCA) of all the

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F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

Fig. 1. TEM images of some of the samples used. (a) Needle-shaped synthetic hydroxyl apatite crystals from sample SHA; (b) Plate-shaped carbonated apatite crystals from sub-recent bone SR1. (c) Carbonated apatite crystals of Middle Paleolithic (MP) fossil bone. Note the larger size of some of the MP carbonated apatite crystals (c) as compared to the SR1 crystals (b).

Table 4 Mineralogical and elemental characteristics of the mineral phases used Sample no. SHA SR0 SR1 SR2 EB CL MP a b

Mineral phasesa

HA CA CA CA CA+CC CA+CC CA

IRSFb

5.4 2.9 2.8 3.2 4.1 3.0 4.5

Ca

P

CO3

Na

Mg

wt%



wt%



wt%



wt%



wt%



Ca/P molar ratio

39.9 36.0 35.2 36.3 38.7 36.7 38.2

1.6 1.4 1.4 1.5 1.5 1.5 1.5

18.5 16.6 16.2 16.7 15.2 13.5 16.4

0.6 0.5 0.5 0.5 0.5 0.4 0.5

– 5.10 6.12 3.78 9.30 13.00 5.80

– 0.2 0.2 0.2 0.4 0.5 0.2

– 0.9 0.6 1.0 0.2 0.4 0.3

– 0.1 0.1 0.1 0.0 0.1 0.0

0.04 0.58 0.35 0.51 0.08 0.13 0.30

0.00 0.02 0.01 0.02 0.00 0.01 0.01

1.668 1.670 1.682 1.679 1.970 2.106 1.798

HA: hydroxyl apatite; CA: carbonated apatite; CC: calcite. Determined by FTIR spectroscopy. Infrared splitting factor [45].

other solution samples. The relative solubilities of the samples can thus be compared. In fact, the higher the IAPCA value of the solution the higher is the corresponding solubility of the dissolving sample. The three sub-recent bones have similar IAPCA values and they are higher than the Middle Paleolithic bone. The synthetic hydroxyl apatite sample (SHA) is the least soluble. These results are consistent with what is expected during diagenesis. The key question, however, is whether these values reflect the inherent solubility properties of the different mineral phases, especially as very small amounts of mineral actually dissolved in these experiments (D and Sol values in Table 5). We therefore performed another series of experiments in which a buffer was used not only to stabilize the pH, but also to cause more mineral to be dissolved. 4.2. Dissolution in pH-buffered solutions The sub-recent bones and synthetic hydroxyl apatite were treated for a period of 100 days or more in

solutions with low and high phosphate concentrations and buffered in three different pH ranges as shown in Fig. 4. Almost none of the high-phosphate solution experiments resulted in the release of additional phosphate, and hence net mineral dissolution was suppressed. These solutions will therefore not be discussed further. The release of calcium with respect to time for the low-phosphate solution experiments shows that a “steady state” was reached after 100 days, except in two experiments where 170 days were necessary (Fig. 4a and b). The analyses of the buffered solutions at “steady state” are shown in Table 6. All three sub-recent bone carbonated apatite samples released more Ca than the synthetic hydroxyl apatite under all experimental conditions, showing that in general bone carbonated apatite is more soluble than pure hydroxyl apatite. Table 6 also shows, as expected, that the lower the pH the more mineral dissolved (see column Sol). In all four samples the solutions buffered to the lowest pH also produced the lowest IAPCA values. The experiments in which the most mineral dissolved also have Ca/P molar

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

873

7.5E-04

6.5E-04

[Total Ca], M

5.5E-04

4.5E-04

3.5E-04

2.5E-04

1.5E-04

5.0E-05 0

25

50

75

Days

100

125

150

175

Fig. 2. Total calcium concentration (M) released in aqueous solutions at 25 (C and atmospheric pCO2 (3.0 10
4 atm) by synthetic hydroxyl apatite SHA (B), by carbonated apatite of sub-recent bones SR0 (,), SR1 (%) and SR2 ($), and of fossil bones EB (:), CL (6) and MP (:).

ratios closest to the ideal stoichiometric value for hydroxyl apatite (1.67). These experiments probably represent the closest approach to equilibrium and their IAPCA values approached the solubility products. An important issue in these experiments is if the unbuffered water experiments provide reliable IAPCA values. Comparison of the IAPCA values of the buffered solutions with the IAPCA values for the equilibrated water solution at the same pH (Tables 5 and 6) shows that they are almost indistinguishable. This agreement implies that the insolubility of these minerals in water did not affect the relative ionic concentrations of the results. We do not expect that the measured IAP values reflect true Ksp values of these mineral phases because of the conditions under which the experiments were performed. Based on the observations that we do reach “steady state” with respect to calcium released over 100 days (Figs. 2–4), the relative solubilities observed between the samples, the pH range in which dissolution occurred and the consistency between the water and buffered experiments, we conclude that the results should be relevant to conditions in natural sediments. They do not simply reflect the idiosyncrasies of a particular experimental design. The interpretation of these experiments is complicated, and some of the details of the results require

further comment. The pCO2 values calculated from measured total inorganic carbon in all of the water experiments were close to the values for atmospheric concentrations, indicating that in our experimental set-up equilibrium with the atmosphere was obtained. In the buffered experiments, almost an order of magnitude higher pCO2 values were measured in some experiments. We do not understand the reason for this, and we can only assume it is somehow related to the interference of the Tris buffer during the analyses, as possibly indicated by the poorer reproducibility in the pH-buffered solution as compared to the non-buffered experiments. We therefore assumed atmospheric pCO2 concentration (310
4 atm) for the IAPCA calculations of the buffered experiments. The Ca/P molar ratios of the solutions equilibrated with bone mineral in both the water and the buffered experiments all have ratios typically are higher than the stoichiometric values of the starting materials (Table 4). Thus a stoichiometric excess of calcium with respect to phosphate was released. Calculations of the concentration of Ca2+ that is in ion pairs with the different species of carbonic and phosphoric acid in solution show that these alone do not account for the observed differences excess of calcium in solution. The excess of Ca2+ is probably due to preferential dissolution of the

874

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882 2.5E+03

[Ca]/[OH], M/M

2.0E+03

1.5E+03

1.0E+03

5.0E+02

0.0E+00 0

20

40

60

80

Days

100

120

140

160

180

Fig. 3. Ca/OH ratio at 25 (C and atmospheric pCO2 (3.010
4 atm) reached in unbuffered water solutions by synthetic hydroxyl apatite SHA (B), by carbonated apatite of sub-recent bones SR0 (,), SR1 (%) and SR2 ($) and of fossil bones EB (:), CL (6) and MP (:).

Table 5 “Steady state” compositions of the unbuffered water solutions incubated at 25 (C and atmospheric pCO2 (w310
4 atm) with synthetic hydroxyl apatite (sample SHA) and sub-recent (SR0, SR1 and SR2) and fossil bones (EB, CL, MP) Sample name

Experiment no.

µ

pH

pCO2 atm

[Ca]

[P]

[Na] M

[Mg]

[Al]

SHA

4 5 6 88 89 90 1 2 3 85 86 87 91 92 93 94 95 96 44 45 46

0.001 0.002 0.001 0.002 0.001 0.003 0.004 0.004 0.003 0.003 0.002 0.003 0.002 0.002 0.002 0.003 0.003 0.003 0.001 0.002 0.002

7.16 7.17 7.19 7.99 8.02 7.80 7.67 7.56 7.65 7.82 7.88 7.85 8.06 8.13 8.04 8.16 8.21 8.25 7.65 7.80 7.67

7.2E-05 1.3E-04 1.1E-04 1.9E-04 9.1E-05 2.0E-04 6.1E-04 5.8E-04 4.0E-04 2.9E-04 2.0E-04 3.0E-04 1.7E-04 1.3E-04 1.7E-04 2.1E-04 2.0E-04 1.9E-04 2.7E-04 3.8E-04 3.9E-04

1.8E-04 1.9E-04 1.9E-04 2.6E-04 1.7E-04 3.5E-04 6.2E-04 6.6E-04 3.7E-04 3.4E-04 1.9E-04 3.3E-04 4.4E-04 3.6E-04 4.2E-04 6.4E-04 6.8E-04 6.5E-04 2.3E-04 4.3E-04 2.7E-04

1.3E-04 1.4E-04 1.4E-04 1.1E-04 6.4E-05 1.6E-04 2.5E-04 2.7E-04 1.3E-04 1.7E-04 9.2E-05 1.7E-04 1.2E-06 5.2E-07 1.1E-06 3.6E-06 3.6E-06 6.0E-06 1.8E-05 4.1E-05 1.1E-04

2.1E-04 2.1E-04 2.2E-04 1.6E-04 1.5E-04 1.6E-04 2.8E-04 2.6E-04 2.8E-04 1.8E-04 1.5E-04 1.5E-04 1.2E-04 1.1E-04 1.2E-04 1.3E-04 1.2E-04 1.2E-04 1.4E-04 9.6E-05 1.6E-04

3.2E-06 3.2E-06 3.3E-06 4.5E-05 3.8E-05 4.8E-05 3.0E-05 2.9E-05 2.5E-05 3.9E-05 3.2E-05 3.9E-05 6.5E-06 5.7E-06 6.6E-06 9.0E-06 9.6E-06 8.9E-06 3.0E-05 3.7E-05 3.2E-05

8.5E-08 5.1 5.2E-08 5.6 1.1E-07 5.3 3.7E-09 6.1 1.5E-08 3.7 0.0E+00 9.5 1.7E-07 13.7 8.2E-08 15.5 2.2E-07 7.5 2.2E-08 10.3 2.6E-08 5.3 1.5E-08 9.7 3.3E-08 0.1 3.3E-07 0.0 4.8E-08 0.1 8.9E-08 0.2 5.6E-08 0.2 7.4E-09 0.3 1.8E-07 0.8 8.2E-08 1.8 5.4E-07 4.7

SR0

SR1

SR2

EB

CL

MP

D Wt%

Sol mg/l

[Ca]/[P] molar ratio

IAPCA

22.0 23.8 23.1 20.0 11.8 30.3 47.0 52.5 25.0 32.3 17.0 31.1 0.3 0.1 0.2 0.8 0.8 1.4 3.3 7.6 20.3

1.36 1.37 1.39 2.38 2.66 2.15 2.54 2.39 2.83 1.97 2.06 1.99 356.2 685.3 375.3 178.0 186.4 107.0 13.27 10.53 2.47

3.9E-55 8.3E-55 9.4E-55 2.3E-48 1.3E-49 3.2E-48 8.5E-47 3.8E-47 1.8E-48 1.4E-48 4.0E-50 1.4E-48

1.1E-51 5.2E-49 2.4E-49

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

875

---------- [Initial P], M --------Low = 10-6 M

High = 10-3 M

2.5E-03

a

b

c

d

e

f

1.5E-03

5.0E-04 0.0E+00 2.5E-03 2.0E-03 1.5E-03 1.0E-03

pH 7.0-7.2

---------- [Total Ca], M ----------

1.0E-03

pH 7.4-7.6

2.0E-03

5.0E-04 0.0E+00 2.5E-03

1.5E-03 1.0E-03

pH 6.2-6.5

2.0E-03

5.0E-04 0.0E+00 0

25

50

75

100

125

150

175

0

25

50

75

100

125

150

175

--------- Days--------Fig. 4. Total calcium concentrations (M) released into the different buffered solutions (see Table 2) during the incubation at 25 (C and atmospheric pCO2 (w310
4 atm) of synthetic hydroxyl apatite SHA (B) and of sub-recent bones SR0 (,), SR1 (%) and SR2 ($).

carbonates substituting the phosphates, as was described for experiments with enamel and synthetic carbonated hydroxyl apatite [24]. Another possible contribution to this apparently non-stoichiometric dissolution is the formation of metastable Ca-phosphate phases when apatite dissolves in the presence of CO2 [5,13,20]. Note that the extremely high Ca/P ratios for the EB and CL bones (Table 5) are due to the differential solubility of calcite and apatite. It is possible that the high Ca/P values for the MP bone are also due to some effect of diagenesis. We do not understand why this should be reflected in only two of the three analyses. FTIR analyses of the residues of the bone samples do show a general broadening of the 1036 cm
1 absorption (v3 PO4 domain) and a shift towards lower absorptions. An additional absorption band at 1028 cm
1 is in fact characteristic of octacalcium phosphate (OCP) Ca4H(PO4)3·2.5H2O. On the other hand, the calculation of the ionic activity product of OCP shows that none of the solutions equilibrated with the bone or SHA samples is saturated with respect to OCP (Ksp for OCP=1.0610
47, [29]). Although the total concentration of Al increased in these experiments during the equilibration, none of the solutions equilibrated “inside”

the stability fields of the Al-phosphates [17,32]. Therefore no Al-phosphates were expected to form and this was confirmed by FTIR analyses. We are aware that small amounts (µM) of Al may prevent apatite from reaching equilibrium in solution [6]. 5. Discussion The major purpose of this study was to measure the solubilities and hence the stabilities of fossil bone mineral under semi-natural conditions. We have shown that in an open system at around neutral to alkaline pH the solubility of sub-recent bone mineral is significantly higher than that of a bone from the Middle Paleolithic, and that the fossil bones are more soluble than synthetic hydroxyl apatite. These observations were made in both unbuffered water and in pH-buffered experiments, suggesting that the observed relative solubilities reflect the inherent properties of the mineral phases under conditions open to the atmosphere. The two fossil bones that contained small amounts of calcite did not dissolve at all. One indication of the higher solubility of bone mineral compared to synthetic hydroxyl apatite, is seen in a

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F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

Table 6 “Steady state” compositions of the pH-buffered solutions incubated at 25 (C and atmospheric pCO2 (w310
4 atm) with synthetic hydroxyl apatite (sample SHA) and sub-recent bone samples SR0, SR1 and SR2 Sample name

Experiment Starting no. solution

µ

pH

[Ca]

[P]

[Na] M

[Mg]

[Al]

D Sol Wt% mg/l

关Ca兴⫺关Ca兴0 关P兴⫺关P兴0 molar ratio

IAPCA

SHA

28 29 16 17 12 13 83 59 60 65 66 81 82 57 58 63 64 79 80 55 56 61 62

0.078 0.078 0.074 0.074 0.067 0.067 0.085 0.077 0.080 0.072 0.072 0.086 0.086 0.080 0.080 0.073 0.073 0.083 0.083 0.078 0.078 0.071 0.071

6.68 6.68 7.24 7.23 7.83 7.82 6.77 7.15 7.05 7.49 7.48 6.76 6.84 7.01 7.04 7.49 7.45 6.73 6.63 7.00 7.02 7.36 7.36

4.5E-04 4.3E-04 3.0E-04 2.9E-04 1.9E-04 1.8E-04 1.7E-03 1.2E-03 1.6E-03 1.2E-03 1.3E-03 2.0E-03 1.9E-03 1.8E-03 1.8E-03 1.3E-03 1.3E-03 1.4E-03 1.5E-03 1.4E-03 1.4E-03 9.9E-04 8.9E-04

2.3E-04 2.2E-04 1.2E-04 1.3E-04 5.7E-05 6.0E-05 8.2E-04 5.0E-04 7.1E-04 3.4E-04 3.1E-04 8.6E-04 8.2E-04 7.2E-04 7.2E-04 5.1E-04 5.1E-04 6.9E-04 6.8E-04 6.2E-04 6.0E-04 4.0E-04 3.6E-04

1.3E-04 1.3E-04 1.3E-04 1.2E-04 1.6E-04 1.4E-04 1.6E-04 1.4E-04 1.6E-04 1.5E-04 1.7E-04 1.5E-04 1.6E-04 1.5E-04 1.5E-04 1.4E-04 1.4E-04 1.6E-04 1.6E-04 1.5E-04 1.5E-04 1.4E-04 1.5E-04

0.00 0.00 0.00 0.00 0.00 0.00 5.3E-05 5.1E-05 4.7E-05 5.0E-05 4.7E-05 2.2E-05 2.1E-05 1.3E-05 1.9E-05 2.0E-05 1.5E-05 4.0E-05 3.9E-05 3.8E-05 3.7E-05 3.2E-05 3.7E-05

6.4E-07 4.8E-07 2.7E-06 1.9E-06 6.6E-06 4.3E-06 3.6E-07 3.1E-07 4.9E-07 1.8E-06 2.1E-06 3.2E-07 1.6E-06 1.5E-06 1.4E-06 2.8E-06 2.8E-06 5.2E-07 3.9E-07 1.1E-06 1.0E-06 5.8E-07 9.7E-07

8.54 8.20 4.50 4.82 2.13 2.24 48.1 28.5 42.6 19.1 17.3 48.1 46.0 40.5 40.5 28.2 28.5 40.6 39.5 36.3 34.4 24.4 21.1

1.51 1.47 1.62 1.41 1.46 1.10 1.92 2.10 2.12 3.37 3.81 2.15 2.24 2.42 2.30 2.35 2.44 1.89 1.98 2.12 2.19 2.20 2.20

1.5E-57 1.1E-57 6.0E-56 6.0E-56 7.8E-55 5.5E-55 7.2E-51 5 E-50 1.5E-49 1.3E-48 1.1E-48 4.5E-50 4.7E-50 6.0E-49 7.2E-49 1.0E-47 8.2E-48 6.1E-52 1.3E-52 1.3E-50 1.5E-50 6.3E-50 3.3E-50

SR0

SR1

SR2

65/6 65/6 70/6 70/6 76/6 76/6 65/6A 70/6A 70/6A 76/6A 76/6A 65/6A 65/6A 70/6A 70/6A 76/6A 76/6A 65/6A 65/6A 70/6A 70/6A 76/6A 76/6A

plot of the amount of phosphate released into solution at steady state as a function of pH (Fig. 5). For any given pH value we tested, about 4 times more bone mineral dissolves than hydroxyl apatite. It is also of interest to estimate how long the dissolution process could take based on the rates of dissolution of the various bones in the unbuffered water experiments. If we assume that the hydrological regime in the sediments is such that the bone is completely flushed every 120 days, then it will take between 25 and 50 years for 1 g of bone to dissolve in pure water. In the context of archaeological and geological environments, this is very rapid. Fig. 6 is a plot of the boundaries between the stable solid phase (right hand side of the boundary) and the solution phase for the sub-recent and fossil bones we measured as a function of phosphate concentration and pH. These boundaries, represented by the lines on the chart, are referred to as isotherms. From the experimental measurements we made (also shown in Fig. 5) we conclude that, depending upon the concentration of phosphate in solution, bone will be stable in aqueous solution above pH 7.6–8.1, and dissolves below pH 7.6–7.2 at 25 (C. The point of interest is that there is a zone between the stability and the dissolution fields in which sub-recent bone mineral is not stable, but synthetic hydroxyl apatite is. What therefore happens to bone mineral when it is exposed to the chemical conditions in this field?

37.76 36.27 19.92 21.61 9.45 9.94 151.9 92.9 132.8 63.0 57.1 163.9 156.7 138.0 138.6 97.4 96.7 127.5 125.8 113.9 111.3 74.6 66.0

Fresh and sub-recent bone crystals will dissolve when they enter into this field. If the hydrological regime is aggressive, then the dissolved ions are rapidly removed from the bone and the mineral is lost. The more likely scenario is that within the pores of the bone, the hydrologic regime is fairly static. The concentration of dissolved ions will exceed the saturation values for a more insoluble form of carbonated apatite, and a new phase will precipitate that is more insoluble than the one that partially or totally dissolved. The most likely location for the new precipitate is on the surface of an existing crystal. As conditions change, this process will repeat itself. This process can be referred to as recrystallization. The end result is that even though the bone itself has not changed macroscopically, the crystal population becomes more and more insoluble, hence more stable. The shadowed area in Fig. 6 delineates the approximate conditions of pH and phosphate concentrations under which this replacement will occur, based on our experimental data. We call this region the “recrystallization window”. The boundaries of the recrystallization window are arbitrarily defined by the region in which the experimental points fall. Obviously, if we would have performed more experiments, the boundaries may have changed a little. Another question is whether it is valid to have the 25 (C isotherm for synthetic hydroxyl apatite as the boundary between the recrystallization window and

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

877

1E-03 9E-04 8E-04

[Total P], M

7E-04 6E-04 5E-04 4E-04 3E-04 2E-04 1E-04 0E+00 6.50

6.75

7.00

7.25

pH

7.50

7.75

8.00

8.25

Fig. 5. Total P concentrations (M) at steady state as a function of pH in the various types of carbonated apatite in pure water and in low-phosphate buffered solutions incubated at 25 (C and atmospheric pCO2 (3.0 10
4 atm). The symbols refer to hydroxyl apatite SHA (B) and carbonated apatite of the sub-recent bones SR0 (,), SR1 (%) and SR2 ($). The solid and dashed lines represent respectively the linear regressions for synthetic hydroxyl apatite (SHA) and cumulatively for the carbonated apatite of all three sub-recent bone samples. The linear relations found are: [P]SHA=
1.4110
4 pH+0.00115 (R2=0.972) for hydroxyl apatite and [P]CA=
5.7810
4 pH+0.00469 (R2=0.903) for the bone carbonated apatite (CA).

the dissolution field, especially bearing in mind that hydroxyl apatite does not normally form at ambient temperatures and pressures? It is only found in igneous rocks formed at high temperatures. The low temperature forms of hydroxyl apatite are always carbonated, and are hence more soluble than pure hydroxyl apatite. Studies of bone mineral diagenesis show that the more altered the bone, the more needle-shaped the crystallites tend to be [37,44]. We therefore think that the synthetic hydroxyl apatite crystals we analyzed, which are needleshaped, can be regarded as a good analog for endmembers of the lower boundary of the recrystallization window. The incorporation of fluoride in the lattice carbonated hydroxyl apatite will increase the stability of the bone mineral phase and shift the isotherms towards more acidic conditions. In order to compare these results with others reported in the literature, we also show in Fig. 6 the isotherm for synthetic hydroxyl apatite measured by McDowell et al. [26] under closed conditions in a CO2-free system. It is shifted significantly from the isotherm we obtained for our synthetic hydroxyl apatite in an open system. This could be due to a difference in the two synthetic minerals but is more likely due to the fact that in an open system CO2 is available as a source of acid for inducing dissolution. The latter of course, reflects the situation in nature. We therefore suggest that the isotherm deter-

mined in the open system is more relevant to natural conditions. Fig. 6 also shows the isotherm measured for fresh bone under a very high pCO2 (1.810
2 atm) atmosphere in a closed system [18]. The location of this isotherm does not seem reasonable based on our knowledge of bone under physiological conditions, where the pH is generally buffered at pH 7.5. Kanri et al.’s [18] data imply that bone mineral is essentially stable in vivo, but it is known that this is not the case and that even in vivo, bone mineral undergoes recrystallization [34]. We are therefore confident that our results reflect the varying stabilities of carbonated apatites in fossil bone in nature. Why are sub-recent to fresh bone crystals so much more soluble and hence unstable than fossil bone mineral and synthetic hydroxyl apatite? We note contributing factors, such as the huge surface area to volume ratio of the fresh bone crystals, the extensive substitution of carbonate for phosphate in the lattice, which causes disorder, and the fact that the crystals are just 2–4 nm thick. The fact that they are so thin means that many of the atoms are disordered simply due to their proximity to the surface. This must be an important destabilizing factor. In fact, the more relevant question may be why bone crystals are not much more soluble and do not spontaneously dissolve? As collagen is present in the sub-recent bones we analyzed and of course in fresh bone, it may conceivably

878

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882 3.0

ER AT NW EI ON TB EN EC R E B-R AT SU W N EM R EI ST FE ON UF SY LB NB ED SSI EI LL FO ON RO TB NT EN -CO EC 2 CO B- R R SU IN TE E WA ON IN HB I TE ES AT FR AP YL ER OX FF U DR NB EM HY I ST I TE SY AT EE AP -FR YL 2 CO OX IN DR ITE HY AT AP YL OX DR HY

3.5

-log(H2PO4-)

4.0

4.5

SOLID PHASE

SOLUTION

5.0

5.5

6.0 5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

pH

Fig. 6. “Steady state” stability relations (isotherms) calculated using Equation (13) for sub-recent and fossil bone carbonated apatite as well as for synthetic hydroxyl apatite in unbuffered water and pH-buffered solutions at 25 (C and at atmospheric pCO2 (3.010
4 atm). Activities of Ca2+ and Na+ are fixed at 2.010
4 and 1.610
4 respectively. The isotherms were plotted using averages of experimental IAPCA values. These IAPCA values are: 7.9410
55 in water and 1.2610
57 in pH-buffered solutions for synthetic hydroxyl apatite; 4.3010
48 in water and 5.4110
51 in pH-buffered solutions for the sub-recent bones; 2.5110
49 for fossil bone in water. Dashed line is the isotherm for synthetic hydroxyl apatite in CO2-free system with Ksp=4.710
59 as determined by McDowell et al. [26]. Dotted line is the equilibrium isotherm for fresh bone carbonated apatite with an empirical formula Ca4.27Mg0.08Na0.13(HPO4)0.11(CO3)0.58(PO4)2.48(OH)0.01 and Ksp=2.0210
43 measured in CO2 controlled aqueous system at pCO2=0.018 atm according to Kanri et al. [18]. The symbols refer to the “steady state” composition of the water solutions incubated at 25 (C and atmospheric pCO2 (3.010
4 atm) with synthetic hydroxyl apatite SHA (B), sub-recent bones SR0 (,), SR1 (%) and SR2 ($) and fossil bone MP (:). The shadowed area represents the “recrystallization window”. The arrows indicate the pH interval (7.55–8.05) in which bone carbonated apatite dissolved in the water experiments while hydroxyl apatite is stable. Note that the solubility of fossil bone carbonated apatite is intermediate between the solubility of synthetic hydroxyl apatite and sub-recent bone carbonated apatite.

influence the solubility and the recrystallization process of the crystals. This would occur if there is a strong interaction between the collagen and the mineral phase. Little is known in fresh bone about this interface, but the fact that recrystallization occurs in vivo in fresh bone, points to a rather weak interaction that allows the crystals to recrystallize even when inside the collagen fibril. In this study no correlation was found between the amount of collagen and relative solubility of the bone samples analyzed. There are, however, many other proteins in bone, some of which are acidic and readily adsorb on to the crystal surface. The most abundant is osteocalcin, which has 4
-carboxyglutamic acid residues that bind strongly to the apatite crystal surface [35]. This protein is preserved in fossils [7] and could influence the solubility and recrystallization of the bone mineral. A comparison of the Middle Paleolithic bone (MP) to the sub-recent bone (Fig. 2c and b) shows that recrystallization is an important process in fossilization. There

is a linear correlation between crystal size and the IR-derived splitting factor [44], even though the latter is also influenced by other factors such as atomic order. Splitting factor measurements of bones from archaeological sites vary between 2.8, the value for fresh bone, to around 7. A study of the splitting factors of bones in the Kebara cave (Israel) showed that, the splitting factors increased in bones closest to a front where bones dissolved completely [49]. These field observations are consistent with the notions that the recrystallization process continues while the conditions are such that the bones are in the “recrystallization window” and the process is faster in bones that are closer to a dissolution front where presumably the conditions in the sediment approach the boundary between the recrystallization window and the dissolution field (Fig. 6). When conditions in the sediments are outside the recrystallization window (pH below 7) and bone mineral dissolves, Al-phosphates that are more insoluble than

F. Berna et al. / Journal of Archaeological Science 31 (2004) 867–882

HYDROXYL APATITE

879

CARBONATED APATITE AUTHIGENIC

BONE

IRSF 2.6

In vivo 3.0 On soil surface slightly altered

3.4

Fossil altered

4.1

5.4

highly altered Synthetic

7.0

DEGREE OF RECRYSTALLIZATION

Fossil

Fig. 7. Infrared splitting factors (IRSF) and degree of recrystallization of bone and authigenic carbonated apatite as compared to synthetic hydroxyl apatite. We found that most of IRSF values for authigenic carbonated apatite exceed 3.4.

bone mineral form. Their stability fields relative to that of hydroxyl apatite have been reported by Karkanas et al. [17]. The presence of these more insoluble phases in the sediments implies that the conditions are such that bones could not have been preserved if they were once present. Note that the stability field designated by Karkanas et al. [17] used a solubility value (Gibbs free energy of formation (Gf)=
6322.9 kJ mol
1) for hydroxyl apatite reported by Nriagu [32]. The manner in which Nriagu determined this value was not given, but the value is almost exactly the same as the value we determined in our open system dissolution experiments for synthetic hydroxyl apatite (Gf=
6326.56.0 kJ mol
1). Thus, the boundary of the stability field defined by Karakanas et al. [17] between hydroxyl apatite and the other phosphate phases, is the same as we obtained experimentally. Carbonated apatite can also be formed de novo in the sediments, unrelated to the presence of bone [17,23]. This authigenic carbonated apatite commonly forms due to the reaction of phosphate released by the breakdown of organic matter with calcium carbonate present in the sediments. Not much is known about the stoichiometry, the shapes and the dimensions of these authigenic crystals. Schiegl et al. [38] reported that authigenic carbonated apatite is usually microcrystalline, but also forms as elongated needles inside soil voids. We noted that the

splitting factors of authigenic carbonated apatites from the sediments of different prehistoric caves (Kebara, Hayonim and Qesem caves) are mostly (76%) above 3.4 with average values of 4.11.2 (N=20). They are therefore significantly more ordered and presumably more insoluble than fresh bone crystals. Fig. 7 schematically illustrates the recrystallization process as tracked by splitting factor changes in bone and authigenic carbonated apatites. An open question is whether the authigenic crystals subsequently undergo recrystallization? We noted that essentially no phosphate dissolved in the experiments performed in water with the two fossil bones that contained calcite (samples EB and CL). We ascribe this phenomenon to the fact that calcite, being much more soluble than the bone apatite, “quickly” released Ca and carbonate, producing a high-calcium solution buffered to pH above 8.0. These conditions suppressed the release of phosphate ions. This hypothesis is supported by the data trends in Fig. 5, from which it can be extrapolated that neither bone mineral nor synthetic hydroxyl apatite will dissolve at or above pH 8.2. Thus, it can be predicted that, if bones are deposited together with calcite, as often occurs in archaeological sites, because, for example, wood ash is composed mainly of calcite [15], then the bones should be stable as long as calcite remains. In fact, if conditions are such that calcite is abundant and cements the sediment grains

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to form a breccia, then the bones will be stabilized for very long periods of time. The converse of this is that, if bones are present in a breccia, then the breccia may well have formed very early after bone deposition and before any bone dissolution could take place. The diagenesis of bone is generally attributed to combinations of five different processes: microbial degradation, tissue and collagen preservation, changes in porosity, variations in crystallinity, and uptake of extraneous elements [11]. Porosity, crystallinity and elemental uptake all depend on the solubility conditions prevailing within the bone. These, in turn, are a function of exactly where the mineral phase is within the recrystallization window and the nature of the hydrological regime of the sediments in which the bone is buried. Furthermore, the degradation of the tissue and the collagen can also drive the recrystallization process due to the release of acid and hence lowering of the pH as the organic material degrades [44]. Thus four of the five parameters affecting bone preservation are intimately related to dissolution and hence to each other. The data provided here on dissolution of bone mineral thus constitute a common basis for better understanding bone diagenesis. 5.1. Broader archaeological implications We can infer from splitting factor measurements of bone and authigenic carbonated apatites that bone crystals are more soluble than authigenic crystals. Authigenic carbonated apatite crystals can therefore be forming while bone crystals are dissolving. Thus, in principle, the presence of authigenic carbonated apatite in the sediments does not imply that bones in the same locality have not dissolved. In practice, however, it has been observed that authigenic carbonated apatite and bones co-exist in the sediments [17,40,47,48]. The reason is probably related to the fact that while the authigenic phase is forming, the bone mineral is recrystallizing and becoming more insoluble. It thus resembles more closely the authigenic mineral. Bone, dentin and enamel are all widely used by geochemists and archaeologists to reconstruct aspects of the paleoenvironment and for dating. There is much interest in being able to a priori identify the specimens with the most suitable mineral phases for these purposes. From the concepts presented here, it can be expected that bones and teeth buried in association with primary calcite, such as calcite containing loess and ash-derived calcite, will be relatively well preserved. We have, however, noted that the small size of bone crystals means that they are inherently unstable in almost any environment, including the in vivo environment where they also undergo recrystallization. Thus it is unlikely to find fossil bones that are still composed mainly of crystals formed in their in vivo environment. Bones that have

splitting factors the same as or similar to those in vivo (namely around 2.8), are clearly the best candidates. There are, however, uses of fossil bone which depend upon the rapid uptake of certain elements that are not initially in bone, such as uranium for dating purposes [39] or rare earth elements [12,43] for reconstructing paleoenvironmental conditions. Ideally these elements should be sealed into the crystal fabric as early as possible. It would seem that for this to occur optimally, the bone crystals should undergo rapid recrystallization and, in so doing, incorporate the appropriate elements into a more stable phase. For uranium series dating, enamel has proved [27]. Enamel crystals are much larger than those of bone and dentin [8]. A possible advantage of enamel crystals in this regard, is that once the uranium is taken up by the crystal, it is subsequently less easily removed. 6. Conclusions The chemical conditions under which bone carbonated apatite can recrystallize into authigenic apatite, the so-called recrystallization window, are restricted and lie in a narrow alkaline pH range. Thus, a small pH shift of the environment dictates the rules of bone preservation. Bone is likely to be best preserved only in sediments in which the pH of the pore solution is above 8.1, such as those saturated with respect to calcite. In alkaline to neutral conditions bone mineral will be preserved, but its mineral component will undergo recrystallization. In this process, the original crystals are replaced by more stable forms of apatite. The recrystallization process together with the precipitation of additional apatite will result in a general increase in crystallinity of the bone mineral phase. This recrystallization process will be more intense as the sediment pH approaches neutrality from the high-pH side. We predict that, below pH 7.5, the original bone mineral will be totally replaced by authigenc apatite. The degree of recrystallization of bone mineral can be monitored by the infrared splitting factor and can provide insight into the depositional chemical environment. When the pH of the sediment solutions drops below 7, fresh or recrystallized bone mineral will rapidly dissolve. These findings are important for dating and paleoenvironmental or paleodiet studies. In fact the diagenetic history of the bone and the extent of recrystallization are fundamental parameters when the most appropriate samples are selected for isotope or elemental uptake analyses.

Acknowledgements The authors express their gratitude to L. Addadi, P. Karkanas, J. Pasteris and G. Falini for their helpful

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comments during the preparation of this paper. This study was supported by a grant (No. 302/00) from the Israel Science Foundation to S.W. S.W. is the incumbent of the Dr Walter and Dr Trude Borchardt Professorial Chair in Structural Biology. F.B. is an E.U. Marie Curie Fellow.

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