Collection of Stable Isotope Data

In order to examine the relationship between the skeletal indicators of poor health (detailed above), age, burial status, and evidence for age-at-weaning and dietary intake, stable isotope analysis of carbon and nitrogen was undertaken.

(i) Criteria for Inclusion in the sample

(a) Human Bone Samples

From the overall sample population of 967, 120 individuals were selected for isotopic sampling (see Appendix 4 for full sample list). Permission to sample the collections held by the Museum of London and MoLA only permitted the sampling of bone, and not the dentition. As different bones vary in their expression of isotopic signatures (Katzenberg 2008; Jørkov et al. 2008), it was decided that only rib bones would be selected for sampling; this also permitted samples from non-MoL collections to be used, and allowed for comparison with other published studies

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(Prowse et al. 2008). The sampling of ribs is considered less destructive due to their greater abundance, post-mortem fragmentation, and less diagnostic morphology. Therefore, only individuals with ribs preserved were considered for isotopic sampling.

Due to the focus of this work on childhood health and care, 100 of the samples were allocated to sub-adult individuals. Preference was given to the preterm, full term and infancy age categories, with approximately 30 samples taken from these groups. This was to ensure that the full weaning signal could be detected and an age-at-weaning could be identified (see Chapter 3). The remaining 70 samples were distributed equally between the other subadult age categories where possible. The last 20 samples were allocated between the adult age categories 7, 8, 9 and 10, to provide an adult comparison for childhood diet. Where insufficient individuals from an age category were available, the remaining quota was redistributed among the other groups. Individuals that could not be assigned an age or sex, in the case of adults, were not considered for isotopic sampling (see Appendix 4).

Within each age category, samples were evenly distributed, where possible, amongst four groups designed to allow the relationship between burial treatment, stress indicators, and dietary intake to be examined. These were:

1) Individuals with burial container present and no stress markers 2) Individuals with burial container present and stress markers 3) Individuals without burial container present and no stress markers 4) Individuals without burial container present and stress markers

In addition, care was taken to select samples equally across all four cemeteries and from both time periods.

Alongside the above samples, results from a further 4 individuals (3 adults and 1 subadult) were obtained from on-going research undertaken by Millard et al. (in prep) (see Appendix 4).

(b) Animal Bone Samples

In additional to the human samples taken, in order to interpret dietary intake from the isotopic ratios obtained, it is necessary to determine the isotopic ratios of the different food elements consumed. In order to obtain the isotopic end points of the animal portion of the diet, samples were taken from 50 animals (5 chicken, 5 cow, 5 pig, 5 sheep, and 5 marine fish) from

domestic contexts at two sites from Roman London (MFI 89 and ONE 94) (see Appendix 5). The faunal bones were identified by Redfern and Pipe, and only diagnostic bones were selected. Due to other ongoing research projects between LAARC and English Heritage, it was not

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possible to obtain samples for freshwater fish. Therefore, stable isotope values from other Romano-British sites and London excavations are used as proxies.

(ii) Sampling Protocol – Collagen Preparation

All bone samples were prepared in the Archaeology Department at Durham University. The collagen extraction method followed was a modified Longin (1971) method, with an additional ultrafiltration step (Brown et al. 1988).

From each individual, 2-3g of bone was taken by hand or, where necessary, using a hacksaw. Samples were cleaned either by hand or by shot blasting to remove markings or larger dirt deposits. Following cleaning, approximately 200mg of bone was weighed out and placed into 15ml test tubes. Dilute refrigerated 0.5M Hydrochloric acid (HCL) was then added to each test tube in order to demineralise the bone fragment. A regime of shaking the sample, and

replacing the HCL acid on alternate days for a period of approximately two weeks was adopted to maintain optimum demineralisation. Once the bone sample was demineralised, the extant material was rinsed three times in purified water to achieve a near neutral solution.

In order to gelatinise the protein, a weak acid solution (pH3) was added to the remaining bone tissue and the sample was heated in a heater block at 75°C for 24-48hrs, with each test tube sealed with a marble to prevent evaporation. The supernatant liquid was then filtered using an Ezee* filter (Evergreen Scientific, 5-8 microns) and centrifuged in an ultrafilter to remove degraded polypeptides and contaminants with a molecular weight of less than 30kDa. The supernatant was then placed in sealed, pre-weighed plastic test-tubes and freeze-dried (lyophilised) for 24-48hrs to remove any organic solvents and remaining water. Following lyophilisation, the remaining solid collagen tissue was then weighed, with the final weight subtracted from the starting weight to determine collagen yield.

If a sufficient yield has been obtained (see Chapter 4 for acceptable collagen criteria), 0.3mg of the extracted collagen was then weighed out and placed into a tin capsule to allow combustion in the IRMS. Each sample was weighed out in duplicate in order to allow measurement in separate runs, ensuring the results obtained were repeatable.

(iii) Stable Isotope Analysis

The carbon and nitrogen ratios of all the samples prepared for this study were analysed using a Costech elemental analyser online with a Thermo-Finnegan Delta V Advantage Isotope Ratio

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Mass Spectrometer. Internal standards and laboratory controls (nylon and gelatine of known isotopic ratio) were run alongside samples to ensure measurement quality.