THE OUTER SHELL PRISMATIC LAYER.
4.3.4 SEM analysis
The number of the bands and position of the bands was determined by acetate peels (see section 1.2.2). However, to understand better how the organics changes in the shell, and to compare the position of each laser spot to the shell architecture at that particular point, Secondary Electron (SE) images were taken. The samples were
etched for 60 s using 0.1 N HCl. These samples were then thoroughly rinsed. Without etching, it was impossible to determine the growth checks from the growth bands.
The samples were carbon coated (as this provides a finer film than gold and thus a better resolution). It should be noted that by etching the samples, it caused the supporting calcium carbonate structure to dissolve, with the surface tension causing a crushing effect due to drying in air (Clark, 1980). Figure 4.6 shows images from
Mytilus californianuspolished, etched, and air-dried compared to polished and etched section prepared by critical-point etching taken from Clark (1980). It can be clearly seen that Critical Point Drying helps to maintain the dimensional stability of the sample. Unfortunately, it could not be used with the LA-ICPMS as the size of the sample prohibited it. Future work using smaller samples would be extremely interesting to further study the structure ofA. islandica.
SE images allow the growth check to be clearly distinguished (see Figure 4.7). The concentration or nature of the organics changed both laterally as well as between different growth checks. This is important when examining whether trace elements are affected by the presence of organics, either directly (e.g. they are hosted by it) or indirectly (e.g. it effects trace element partitioning through modification of the crystal nucleation and propagation).
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1m 1m 1m
Figure 4.6: Secondary Electron images from Mytilus californianus of the pallial myostracum (thick layer in middle of images) a) fractured section: organic matrix present but cannot be readily distinguished from the aragonite crystals b) polished and etched and air-dried c) polished and etched section prepared by critical-point drying. Images and captions taken from Clark (1980).
Figure 4.7: Secondary electron images of the growth check (1999) from the outer shell prismatic layer of shell 248. The main image highlights the distinct changes between the growth band (left) and the increase in organics indicating the growth check (right). The insert highlights position of spots compared to the growth checks. This in situcomparison is important particularly in this example, as it highlights that the spot missed the narrow growth check of 1999. Direction of growth is from left to right.
4.3.5 Data processing
The start position of each transect has been standardised relative to Transect 1 (T1) 250m from the periostracum. This is in order that the start position of each transect is comparable, as the width of the growth band decreases laterally away from the periostracum. Note that the distance axis was not otherwise altered; with the position of the annual growth, checks marked on the trace element fluctuations of the plots.
Data where the Li/Ca ratio exceeded 0.05 were removed. Li is a relatively constant measure of background and large changes in Li/Ca indicate changes in the raw Ca counts. Apparent decreases in Ca concentration result from cracks or imperfections within the shell. 27Al was used to provide a check on contamination since Al is present in the polishing media. Mass-27 counts also include a component from54Fe2+, which is also a surface contaminant. No high Al/Ca ratios were observed in PL248. Calibration was carried out using GLITTER© software under license by New Wave™, developed by van Achterberghet al.(2001). The software uses a linear fit of the count ratio of the internal normalising isotope, (in this case 43Ca) to the element of a standard to calculate the concentrations of the unknowns based on an estimate of the weight percent CaO (Table 4.1b) i.e. the trace element concentration in GLITTER©is calculated by:
Concni= (cpsni/abundancej)/yieldni
Where:
Concni= Concentration of element i in analysis n.
133 yieldn= cps per ppm of element i in analysis n.
Yield of element i in analysis n is determined by: yieldni= yieldnsx Int(yieldni/ yieldns)std
Where:
yieldns= cps per ppm of internal standard s in analysis n.
Int(yieldni/ yieldns)std= ratio of the yield of the internal standard s in analysis n,
interpolated over standard analyses.
The software selects a background and signal window for these calculations, which can then be reviewed by the user, to confirm that the most stable part of the signal has been selected. The review window (see Figure 4.8) indicates to the user where the signal is most intense for each element (by the colour scheme in the upper panel), the signal window chosen will be applied to all elements measured.
The review of the stability of the signal is important. If the signal is unstable, the results will be strongly influenced by the positioning of the sample window, with the calculated concentrations changing significantly. During one analysis session (data not shown), the signal decayed significantly due to the decay of detector sensitivity. This resulted in a change in concentration of the element concentration depending on the position of the signal window e.g. Sr/Ca changed by ~30%, whereas typical variation was ~1%.
The concentrations of the standards (plus typical concentrations in the samples) are shown on Table 4.1b.
The precision and accuracy are both estimated at 95% confidence (2. The Mean Detection Limit (MDL) is calculated at 99% confidence (i.e. 3) determined by Poisson counting statistics:
MDL= 2.3√2B
Where B= total counts in background interval
Figure 4.8: Typical signal selection window within GLITTER© showing signal from analysis of outer shell prismatic layer of A. islandica (shell 248 spot 7). The background signal selection is delimited by the first (green) box, with the sample signal denoted by the second (green) box. The colour scheme for the LA-ICPMS represents the count rates.
135 Isotope Mass Window Settling time (s) Sample time (s) Samples per peak Segment duration (s) Integration window (%) Detection mode Typical Limits of detection (ppm) 7Li 10 0.3 0.01 10 0.01 10 Both 24Mg 10 0.046 0.01 10 0.01 10 Both 2-5 43 Ca 40 0.029 0.01 10 0.04 10 Both 40-80 44Ca 10 0.001 0.01 10 0.01 10 Both 50-75 55 Mn 40 0.014 0.01 10 0.04 10 Both 0.2-0.6 88Sr 40 0.03 0.01 10 0.04 10 Both 0.03-0.05 137 Ba 40 0.033 0.01 10 0.04 10 Both 0.1-0.3
Table 4.1a: Experimental set-up for LA-ICPMS measurements (used for both standards and unknowns). 250 runs, with one pass were used each analysis. When both analog and counting are used to record signals at the same time, this is called “both” mode. Samples per peak refer to the measurement of slightly different masses for the same mass window.
Standard NIST610 NIST612 BCR MACS1 NIES OKA Samples CaO (%) 11.45 11.9298 7.12 56.0 54.3 56.0 56.0
Table 4.1b: CaO weight programmed into GLITTER© to calculate absolute concentration.
NBS610 NBS612 BCR MACS1 Element ppm mmol/ mol ppm mmol/ mol ppm mmol /mol ppm mmol/ mol Sr 497.40 0.57 76.15 0.09 337.00 0.38 200-240 0.23-0.27 Mg* 482.4 1.99 60.31 0.248 20986 86.38 Ba 424.1 0.31 37.74 0.027 684.00 0.50 100-150 0.073-0.11
Table 4.2: Certified concentrations within the standards. Where values are not available, cell is left blank. *Where values were quoted as oxides, they have been converted to ppm.
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Table 4.3: Details of analyses sessions with a log of transects, distances and years covered. Analysis of T3 was continued (cont) for PL248 on 3rd May 2006. Nomenclature for PL248 is same as that used for PL228, i.e. T1 is 250 m from the periostracum, T2 500m and T3 1000m.