Biochemical Procedures

Strontium isotope analysis was accomplished at the Isotope Geochemistry Laboratory at the University of North Carolina at Chapel Hill by the author with the assistance of Dr. Drew Coleman. Each tooth was cleaned by surface abrasion. Between 5 to 10 mg of dental enamel was extracted using a Brasseler hand-held dental drill fitted with a 0.3 mm round tungsten carbide bit, weighed on a Sartorius microbalance, and stored in 5 mL Savillex vials with deionized water prior to processing. Strontium was extracted by dissolving the powdered enamel in 500 μL of 7M HNO₃, then evaporating and redissolving it in 500 μL of 3.5M HNO₃. Sr-Spec^™ columns were cleaned and loaded with 50 to 100 μL of EiChrom SR-B100-S resin, and the enamel sample was centrifuged. The sample was loaded by pipette from the centrifuge vial and subjected to dropwise and bulk sample rinses with HNO₃. Strontium was eluted into a clean Savillex vial with deionized water, 25 μL of H₃PO₄ was added, and the water was allowed to evaporate on a hotplate. The sample was redissolved with 2 μL of TaCl₅. Half of the strontium was loaded onto a rhenium filament, and the ⁸⁷Sr/⁸⁶Sr isotope ratio was measured on a fully automated VG Micromass Sector 54 TIMS spectrometer in reference to standard NBS-987, which has a ratio of 0.710270 ± 0.000014 (absolute, 2σ), based on replicate analyses of the standard run over the same period as the samples. The internal precision for individual strontium runs was ± 0.000008 to 0.000013 (absolute, 2σ) standard error based on 100 triple-dynamic cycles of data collection.

Analysis of the light isotopes of carbon and oxygen was performed at the Stable Isotope Facility at the University of Bradford. The outer layer of each tooth was cleaned using a diamond dental burr. A single enamel sample of around 15 mg was extracted from the thickest part of the tooth wall, rather than the earlier-forming occlusal region, following the procedure of [48]. Between individuals, the dental burr was cleaned with 4M HNO₃, rinsed with deionized water, placed in an ultrasonic bath for five minutes, and swabbed with acetone.

The procedure for pre-treatment of enamel apatite is based on [99]. Each sample received 1.8 mL of NaOCl. Samples were rinsed with deionized water and centrifuged three times. 1.8 mL of 0.1M acetic acid was added, and the samples were again rinsed with deionized water and centrifuged three times. Samples were heat-dried overnight and freeze-dried before being weighed and loaded onto the IRMS. Rinsed and freeze-dried samples were weighed in duplicate and measured using a Finnigan Gasbench II connected to a Thermo Delta V Advantage continuous flow isotope ratio mass spectrometer. Enamel carbonate was reacted with anhydrous phosphoric acid at 70deg C to release CO₂ gas from which δ¹⁸O_VSMOW and δ¹³C_VPDB were determined using a CO₂ reference supply.

Data were normalized by means of a linear calibration equation derived from a plot of accepted versus measured values for two internal standards, Merck Spurapur CaCO₃ and OES (ostrich egg shell), and the NBS19 international standard. Ratios of δ¹⁸O and δ¹³C are reported, respectively, per international standards Vienna Standard Mean Ocean Water (VSMOW) and Vienna Pee Dee Belemnite (VPDB). Analytical precision was determined using an internal enamel laboratory standard to be ± 0.1‰ (1 stdev) for δ¹³C and ± 0.2‰ (1 stdev) for δ¹⁸O.