LABORATORY METHODS

3.6.1. Foraminiferal Analyses (Fossil and Contemporary)

Sample preparation followed that outlined by Scott and Medioli (1980) and de Rijk (1995), wet sieving 10 cm3 of sediment through 500 µm and 63 µm sieves before transferring the >63 µm fraction into a wet splitter (Scott and Hermelin, 1993) and allowing the sample to settle out of suspension. Using a pipette, known volumes (usually 1/8th) of the >63 µm fraction (live and dead) were placed onto a spiral counting tray and counted wet under a Leica S8APO Stereo microscope at a magnification of x63 until a minimum of 100 (dead) counts was achieved (Patterson and Fishbein, 1989; Fatela and Taborda, 2002). The >500 µm fraction and supernatant was also checked for foraminifera before being discarded. Samples for foraminiferal analysis were stored in buffered ethanol with Rose Bengal added to aid in the identification of live specimens at the time of collection. As the protoplasm is stained bright red, it was assumed that any tests containing protoplasm were either alive or only recently dead at the time of collection (Murray, 1991). A continuing debate exists in modern foraminifera studies as to which assemblage best reflects a reliable model for the sampled environment. Several authors (Horton and Edwards, 2003; Leorri et al., 2010; Rossi et al., 2011) advise using dead assemblages for analogues of palaeoenvironmental change as they more accurately depict the modern environment in comparison with total or living assemblages which are more susceptible to seasonal (Murray, 1991) and/or post- depositional changes (Horton and Edwards, 2006). The number of live specimens counted in each sample was significantly lower than the dead fraction, and the majority of surface samples analysed were void of any living foraminifera. Where present, numbers of live foraminifera were below statistically confident limits even when considering the low species diversity of the studied environments. This observation confirms previous findings by Cosovic et al. (2006) who analysed recent foraminifera along the Croatian Adriatic seacoast concluding that dead tests are substantially more common compared to living assemblages, regardless of season when sampling took place. Similarly in a study of foraminiferal populations from the Gulf of Venice, dead foraminiferal assemblages were not only more diverse, but much more abundant in comparison to the living component (Serandrei-Barbero et al., 2003). As a result, only the ‘dead’ datasets were employed in the results and interpretation of the contemporary study as discussed in chapter 4. Raw counts of both surface (live and dead) and core foraminifera are provided Appendix B.

Foraminiferal taxonomy was confirmed through comparison with primary resources (e.g. Murray, 1973; 1979; 1991) and the vast array of scanning-electron microscope (SEM)

Page | 38 images of intertidal foraminifera in the published literature (e.g. Horton and Edwards, 2006). The lack of previous research on salt-marsh foraminifera from the eastern Adriatic coastline limits comparisons, however comprehensive studies of benthic foraminifera in the Adriatic Sea were frequently referenced (e.g. Jorissen, 1987; 1988) to aid the identification of calcareous taxa. The various calcareous species of Ammonia, Elphidium and

Quinqueloculina are grouped together at genus level as Ammonia spp., Elphidium spp. and

Quinqueloculina spp., respectively, following Hayward et al. (2004) and Horton and Edwards (2006). Using a Hitachi TM3000 Tabletop microscope, high-resolution ‘SEM’ style images of the main foraminiferal taxa encountered were captured and are presented in Appendix E.

3.6.2. Environmental Variable Analyses

To explore the potential mechanisms influencing surface foraminifera distributions, sediment samples were sub-sampled and analysed for environmental variables so that their significance on the foraminiferal dataset could be determined through multivariate statistical analyses, as presented in chapter 4. Where applicable, the same procedures were repeated for both contemporary and core material (e.g. loss-on-ignition, laser granulometry). Results from surface environmental variables are presented in chapter 4 whilst down-core trends are presented in chapter 5.

3.6.2.1. Salinity and pH

Conductivity (as an indicator of salinity) and pH were analysed simultaneously after creating a 1:2 soil to water mixture comprising 35 ml of sediment and 70 ml double distilled water. The mixture was thoroughly stirred using a glass rod and allowed to settle for approximately one hour before measurements commenced. The solution was then measured and repeated three times before an average was taken for each sample station. Measurements of pH were performed using a HANNA HI-98115 pH meter while conductivity measurements were recorded using a HANNA HI-9033 multi-range conductivity probe. Calibration solutions were first used, and periodically thereafter, to ensure the correct measurement of pH and conductivity, using pH solutions 4.01 and 7.01 and conductivity solution 1413 µs/cm (H170031). As the probe measures conductivity, which is used as a direct indication of salinity levels at each sample station, the equation given below was used to express conductivity as a function of salinity, presented as parts per thousand (‰) following Gehrels and Newman (2004). To ensure consistent results, the temperature of the analysed solution was also readily recorded.

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3.6.2.2. Organic Matter

The organic matter of each sample was determined by loss-on-ignition (LOI) (Ball, 1964) whereby the organic content in each sample is combusted to ash and carbon dioxide. To remove hydroscopic moisture, approximately 5 g of sediment from each sample was left overnight (typically 12 hours) in a Sanyo Convection Oven at 105°C. The samples were then re-weighed before ignition at 450°C for approximately four hours in a pre-heated muffle furnace. Samples were allowed to cool in desiccators before weighing using a Mettler Toledo high-precision analytical balance allowing down-core and surface trends of LOI to be calculated using the following equation:

where;

LOI450 = Loss-on-ignition at 450°C expressed as a percentage.

DW105 = Dry-weight (g) of the sample before ignition (after drying at 105°C). DW450 = Dry-weight (g) of the sample after ignition.

3.6.2.3. Particle Size Analysis

Substrate particle size distribution of both contemporary and core material was analysed using a Coulter laser diffraction granulometer (LS200). The variable organic content of salt- marsh sediments, as identified from LOI results, resulted in samples being pre-treated to remove unwanted organic particulates that may contaminate the minerogenic particle size results (Allen and Thornley, 2004; Gray et al., 2010). This involved sub-samples of sediment (between 0.5g and 5g depending on organic content) first being sieved through 2 mm to remove larger rootlets and stems, before being heated gently on a hot plate in a solution of 20% hydrogen peroxide (H2O2) and double distilled water. The reaction was continued until all organic material had been digested. Prior to inputting to the granulometer, clay-rich samples were disaggregated on a watch glass using Calgon. During the analysis, which measures particle size distributions between 4 and 2000 µm (0.004 and 2 mm), samples were sonicated to further aid even dispersion of the sediment. Finally, output data from the analyses were processed using the computer program GRADISTAT version 8 (Blott and Pye, 2001). For statistical analyses presented in chapter 4, the data were separated into the individual proportions of percentage sand, silt and clay using the size classes provided by GRADISTAT.

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3.6.2.4. Dry Bulk Density

Dry bulk density (DBD) analyses were performed to complement the analysis of short-lived radionuclides, providing information regarding changes in mass accumulation rate that may also be used to identify potential sediment compaction issues, often associated with minerogenic low-energy intertidal sediments (Brain et al., 2011). At a resolution of 1 cm throughout the core material, approximately 1 cm3 of sediment was carefully cut and its dimensions recorded before being weighed wet. Each sample was frozen and then placed in a freeze-drier to remove all water content. Finally the samples were re-weighed using a high- precision analytical balance and the dry bulk density calculated using the following equation:

Where;

DBD = Dry-bulk density (g/cm3) DW = Dry-weight of the sample (g) V = Volume of the sample (cm3)

Where the volume of core samples was unable to be achieved through the above procedure, due to compression (either during transportation or storage), a volume by displacement method was used following the principal that 1 mL of water has a volume of 1 cm3. An initial water level was subtracted from the final water level allowing volume to be calculated.