Descripción de la tercera herramienta de extracción de información: cuestionario

A series of leaching tests were conducted to determine the most effective treatment for removal of diagenetic material while avoiding altering the primary composition of the tooth enamel (Section 4.1; Koch 1997, Nielsen-Marsh & Hedges 2000). Six samples were chosen for this investigation, four wombat teeth (two modern and two fossil) and two mineral standards (Florida Rock Phosphate NIST 120c and Durango mineral apatite).

Tooth enamel was separated and cleaned with a diamond saw. Enamel and Durango mineral apatite were ground with an agate mortar and pestle, while NIST120c was available in powder

117 form. Each sample was split into 11 sub-samples of approximately 20 mg (40 mg for NIST120c) and labelled a-k. A list of sample numbers and weights is presented in Table 4.1

Table 4.1. Leaching test sub-sample masses Modern WB503 Mass (mg) Modern WB69 Mass (mg) Fossil WT23A Mass (mg) Fossil WT01G Mass (mg) Mineral NIST120c Mass (mg) Mineral Durango Mass (mg)

M1-a 19.99 M2-a 19.91 F1-a 20.05 F2-a 20.56 S1-a 40.48 S2-a 21.88

M1-b 20.12 M2-b 19.3 F1-b 19.92 F2-b 20.19 S1-b 41.81 S2-b 21.63

M1-c 20.06 M2-c 19.99 F1-c 20.08 F2-c 20.44 S1-c 38.67 S2-c 20.13

M1-d 20.09 M2-d 19.1 F1-d 20.08 F2-d 20.23 S1-d 39.64 S2-d 20.97

M1-e 20.03 M2-e 20.29 F1-e 20.01 F2-e 20.28 S1-e 40.42 S2-e 20.85

M1-f 20.06 M2-f 19.36 F1-f 19.91 F2-f 20.35 S1-f 41.07 S2-f 21.07

M1-g 20.14 M2-g 19.79 F1-g 20.02 F2-g 19.99 S1-g 39.97 S2-g 20.84

M1-h 20.08 M2-h 19.7 F1-h 19.96 F2-h 20.15 S1-h 40.81 S2-h 21.21

M1-i 19.97 M2-i 20.95 F1-i 19.89 F2-i 20.63 S1-i 39.57 S2-i 20.83

M1-j 19.94 M2-j 16.07 F1-j 19.98 F2-j 20.36 S1-j 40.07 S2-j 20.44

M1-k 20.08 M2-k 20.17 F1-k 10.72 F2-k 20.26 S1-k 41.02 S2-k 20.8

To destroy organic matter, 30% H2O2 was added to sub-samples a-j at a ratio of 0.04 ml per 1 mg. Subsample k was not treated for organic matter removal. After 24 hours, each of the treated samples was centrifuged for five minutes and the supernatant decanted and discarded. Samples were then rinsed in ultra-high purity water (>18 MΩ), centrifuged and decanted twice, then oven-dried at 60ºC.

Sub-samples a-i were each leached in one of three treatment solutions (0.1 M sodium acetate buffer solution, pH 3.8; 0.1 M acetic acid, pH 2.7; and 1.0 M acetic acid, pH 2.2) for 4, 8 or 24 hours. Subsample j was untreated apart from organic matter removal while sub-sample k remained untreated. Table 4.2 shows each of the treatment strengths and treatment times applied to sub-samples a-i. After treatment, these sub-samples were centrifuged, decanted and rinsed twice, then oven-dried in the same manner following H2O2 treatment described above.

Table 4.2. Treatment strengths and times for sub-samples a-i.

4h 12h 24h

buffer a b c

0.1M d e f

1.0M g h i

All sub-samples were digested in 2 ml 1 M HNO3 for 24 hours then transferred to 10 ml tubes. A silver nitrate solution of 0.1 g AgNO3 in 1 ml 1 M HNO3 was added to each

118 sub-sample followed by drop-wise addition of 1 M NaOH, until a persistent yellow precipitant (silver phosphate) formed. They were left to precipitate overnight, before being centrifuged, decanted, rinsed twice and dried as above.

One or two 2.5-3.5 mg aliquots of each sub-sample were weighed into silver foil cups along with 0.5 mg graphite (see Appendix D for the final weights of each sample). These were dried in a vacuum oven at 82ºC for at least one hour. They were subsequently loaded into a sample tray for pyrolysis and δ18_{O measurement by Continuous Flow Isotope Ratio Mass} Spectrometry (CF-IRMS). The combustion chamber was set to 1200ºC to produce CO which was introduced via a helium carrier gas to the CF-IRMS fitted with three Faraday cups arranged to measure masses 28 (12C16O), 29 (13C16O) and 30 (12C18O).

At the start of each of two analytical runs, eight beet sugars of known δ18_{O composition were} measured as consistency standards in addition to two silver phosphate standards (NH4_2 and 2ndFS), which were run in pairs after every 15 sample measurements.

Figure 4.12 shows the results for a modern wombat tooth (M1), fossil wombat tooth (F1) and standard (S2). M1 shows a general trend of increasing δ18_{O with treatment time. The 0.1 M} acetic acid gives higher ratios than the buffer solution, while the 1.0 m acetic acid gives consistently high ratios. F1 conversely shows a general trend of decreasing ratios with treatment time and treatment strength. The Durango mineral apatite displays a decrease in δ18_{O with treatment time even with the weakest treatment (buffer solution). Unfortunately,} due to sample loss during preparation, the full results for the untreated, 0.1 M and 1.0 M acetic acid treatments are not available for this sample.

119 Figure 4.12. Results of leaching treatments for three samples. A: a modern wombat tooth (M1, WB503). B:

a fossil wombat tooth (F1, WT23A). C: Durango mineral apatite standard (S2). Note the different scale for S2. Note also that uncertainties are not shown, as they are generally smaller than data point markers.

120 The fossil sample shows a change in δ18_{O with H}

2O2 treatment, whereas the modern sample does not. This is unexpected as modern enamel would be expected to have higher organic matter content. This suggests the fossil tooth may have been contaminated by organic material with a significantly lower δ18_{O than the enamel apatite. The decrease in δ}18_{O in the mineral} apatite with treatment time is concerning, as mineral apatite should not contain any diagenetic material. These results could indicate that recrystallisation is occurring, altering hydroxyapatite to brushite (Lee-Thorp & Van Der Merwe 1991). On the other hand, these sub-samples were measured toward the end of the run, where the NH4_2 standard which was run in pairs after every 15 sample measurements shows a significant decrease in measured values (Figure 4.13). It is possible a similar decrease may have affected the measured values of the Durango mineral apatite. This would also explain why the sub-sample treated only with H2O2 (black circle in Figure 4.12C) gives a lower value than any of the treatments, as this was the last sample measured in the run. On the other hand, the second standard (2ndFS) continues to provide consistent results during this stage of measurement.

Figure 4.13. Results for silver phosphate standards, NH4_2 and 2ndFS. A pair of each standard was measured after every 15 samples.

Given the change in measured δ18_{O becomes stronger with increased treatment time and with} treatment strength for both the fossil and modern enamel sub-samples, and since the results for the mineral apatite potentially suggests recrystallisation with stronger treatments, the four-hour buffer solution treatment was selected for the main runs of wombat teeth. This is in

121 accordance with the findings of Koch et al. (1997) and Nielsen-Marsh & Hedges (2000), who also suggest short treatment times and reduced reagent strengths be applied to biological apatite, to avoid recrystallisation and chemical exchange.