Estructura poblacional

Sample 32 was a relatively large heavy pebble.

Figure 3.11 Author’s own images of samples from Site 4, during collection.

3.3.3 Sample Drying method development

Sampling and storage procedures were developed on site taking into consideration the limited facilities available. Material was collected from potential sites. Samples were then taken where possible from an initial depth between 2-5cm and then a deeper depth 5-10cm from the surface. This avoids material which would have been exposed to rain and so may not be truly representative of the underlying material. A rigid ‘plastic’

rod, marked at cm intervals, was hammered into the surface to the required depth then sample material excavated using a rigid long handled plastic palette knife. Samples were collected in clean screw topped glass jars and labelled, and taken back to the onsite laboratory.

Samples to be used in method development were divided into two, placed in polypropylene weighing boats and weighed. Samples were then either placed on the work bench and covered with aluminium foil or placed in a snap-lock polypropylene storage box together with a jar containing Self-indicating silica gel granules (GeeJay Chemicals Ltd). This silica gel contains a moisture sensitive indicator that changes colour orange to green as moisture is adsorbed giving a visual indication of the activity level of granules. The samples were examined daily and reweighed after 2-3 days. During this period the relative humidity was > 80%. The foil covered samples failed to dry suitably in these conditions and became contaminated by insects. The samples dried in the silica gel/box conditions showed a small initial mass loss (<2%) with the silica gel taking on a slight green colouration and no insect contamination. After 7 days, there was no further change in mass. Figure 3.12 illustrates the colour change on drying.

Sample 19 Sample 27

Sample 30, 2

fragments Sample

32

50

(Left)- Example Site 8 after 7 days drying (Right) Example Site 8 undried sample

Figure 3.12 Illustrating the effect of drying with silica gel, after 5-7 days

The dried samples were transferred to Ziploc bags, double bagged. The bags were placed in snap-lock polypropylene boxes, containing silica gel, for long-term storage. The silica gel drying and box storage method was adopted. Situations of sampled sites are presented in Appendix 1.4, Table 1.3.

The analytical techniques used in this investigation and their relationship to the potential functional hypotheses are presented in Table 3.3.

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Table 3.3 Summary of the analytical techniques used, rational for analysis and relationship to potential functional hypotheses.

Rational for analysis and relationship to potential functional hypothesis (Chapter 1 Section 1.9)

Technique- characteristic determined Sample

Characterisation

Comparison with published data

Anti- diarrhoeal

Antacid/acid neutralisation/gastro protection

Nutrition/

physiological

imbalance Detoxification

Anti-bacterial /anti-infective properties

Munsell colour characteristic  

Measurement of water content 

Loss on Ignition/ presence of carbonates/organic

material  

XRD –presence of clay or other minerals      

IR – presence of clay minerals/organic matter/water      

XRF– detectable elements present    

ICP– of elements released into gastric extract

Effects of geophagy material on potentially available Fe    

Laser diffraction particle size in variable pH-media

conditions/presence of clay sized particles    

Sample pH in water and KCl solution  

UV– analysis of gastric extract following exposure to

example PSM in gastric conditions  

Microbiological screening of gastric digest 

51

52

3.4 General Sample Preparation – Analytical methods

Several of the analytical techniques required dry sample material of less than 2mm size. Approx. one third of a sample from each site was gently processed in an agate mortar using a rubber pestle, to prevent cross contamination. The resulting material was sieved using a 2mm mesh stainless steel sieve. The sieved material was then Colour characterised and used to determine the Water content and Loss on Ignition (LOI).

The remaining material was stored in sealed containers, containing silica gel, for the remaining analyses.

Samples 33-36 from Site 5 (a potential Cebus monkey site) were discarded following examination in the laboratory at LJMU, as there were obvious signs of white thread-like material (probably fungal hyphae) contaminating the samples and condensation on the inner surface of the bags. Section 3.4.12, Table 3.16 provides a summary of samples collected and analyses undertaken.

3.4.1 Munsell© Colour Characterisation

Method

Using the standard method from Rowell (1994), previously sieved, (<2mm) air dried material was placed between clean glass slides to produce a smooth uniform surface. The top slide was removed and the sample colours determined using the Munsell® Soil Colour Charts, 1998 Revised Edition. Colours were determined between 11.00-14.00 hours in bright daylight conditions.

The value obtained from the charts e.g. 7.5YR 5/3 has three components, hue – colour specific (e.g.

7.5YR), value – light or darkness (e.g. 5) and chroma - colour intensity (e.g.3). Chart for Hue 7.5YR can be found (Appendix 1.4, Figure 1). A typical colour descriptor would take the form 7.5Y 5/3, colour ‘brown’. The results are presented in Section 3.5.1.

3.4.2 Determination of Water Content at 105

C

Soil materials contain non-mineral related water, the amount depending upon the presence of organic material and the preceding weather conditions. Even after air-drying the material will have residual water, the amount dependent upon the characteristics of the material and the humidity of the room used for air-drying.

Moisture content of collected air-dried soil samples may change during storage (fluctuations in air moisture, temperature, oxidation of organic matter, loss of volatile constituents). The storage conditions adopted were attempts to mitigate these changes.

Water content is determined after drying in an oven at 105⁰C. The methods used were those routinely employed by soil scientists (Rowell 1994, Pansu et al. 2007). The temperature maintained for a defined period of time, is sufficiently high to eliminate free forms of water and sufficiently low not to cause a significant loss of organic matter and unstable salts by volatilization (Pansu et al. 2007).

This analysis was conducted approx. 10 weeks after collection, following arrival of samples at the laboratory in Liverpool following the methods in Pansu et al. (2007 pages 1-13). All samples were analysed as a single batch.

53 Method

10.0±0.2g previously stored, dried, sieved < 2mm fraction of each sample was weighed into a pre-weighed porcelain crucible. The samples were then placed in a preheated oven at 105⁰C overnight (16 hours).

After removal samples were allowed to cool in a desiccator for 2 hours before weighing. All the determinations were made in a single session, to standardise conditions. A single determination was made on each sample, with multiple samples tested from each site, with the exception of Site 10 where only a single sample was obtained by the climbing team. Results are presented in Section 3.5.2.

3.4.3 Determination of Loss on Ignition (LOI)

The loss on ignition value is used as an estimate of the content of non-volatile organic matter in soil samples e.g. microorganisms, roots/organic waste products. Organic matter begins ignition at ~200⁰C and is completely depleted at about 550⁰C.

LOI may also contain a contribution from water linked to the mineral crystal lattice plus a little residual non-structural adsorbed water (Pansu et al. 2007). Additionally sulphide minerals, carbonates and metallic oxy-hydroxides can modify LOI values as well, via oxidation or dehydration and this may be significant where there is a high clay mineral content (Dean 1974, Santisteban et al. 2004).

Method

Organic matter content was determined by measuring the Loss on Ignition, (LOI), at 500⁰C ±25⁰C, for 2 hours in accordance with the European Standard Method EN 15934. Following reweighing of desiccator-stored samples used for the determination of water the oven dried samples in the porcelain crucibles were placed in a, previously heated muffle furnace and kept at 550⁰C ± 10⁰C for 2 hours. After 2 hours at 550⁰C the furnace door was released and whilst still hot the crucibles were placed in a desiccator containing fresh silica gel and allowed to cool before weighing. Results are presented in Section 3.5.3.

3.4.4 X-Ray Diffraction (XRD)

Each crystalline solid has its unique characteristic X-ray powder diffraction pattern that may be used as a fingerprint for its identification, seen both when analysed as a pure material and as part of a mixture. X-ray powder diffraction is used for the fingerprint characterization of crystalline materials and the determination of their crystal structure. Approx. 95% of solid materials are described as crystalline. When X-rays interact with crystalline material, an X-Ray Diffraction pattern is generated. XRD is commonly used for the qualitative and semi-quantitative determination of clay minerals (Kodama et al. 1989).

The X-ray radiation most commonly used is that emitted by a copper source, whose characteristic wavelength of the radiation is =1.5418Å. When the incident beam strikes a powder sample, diffraction occurs (Figure 3.13) in every possible orientation of 2theta (2θ).

Using the angles and intensities of the diffracted beams a three dimensional structure may be constructed and this subsequently used to identify materials. In order for there to be interference between the reflected waves, the path difference must be an integral number of wavelengths: nλ= 2x. The path difference between two incident waves: 2λ= 2d sinθ. This leads to the derivation of the Bragg equation: nλ = 2d sinθ;

54

Figure 3.13 Reflection of x-rays from two planes of atoms in a solid, from (Wilson 1987).

An example calculation, substituting the following: n=1, λ = 1.315Å, θ = 22.25 enables a value for d to be calculated:

2x1.315 = 2d sin(22.25); d = 1.541Å

The diffracted beam is detected by using a moveable detector such as a Geiger counter, which is connected to a chart recorder. In normal use, the counter is set to scan over a range of 2θ values at a constant angular velocity. Routinely, a 2θ range of 3 to 70 degrees is sufficient to cover the most useful part of the powder diffraction pattern for characterisations.