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2.4.1 Preparatory Methods

All the diatom samples were taken from either a known area of substratum or, in the case of epiphytic and rope samples, from a known dry weight of material. This allowed for quantitative preparatory techniques to be used which, although not comparable in all cases, did allow for estimates of cell numbers to be made relative to a substratum type. All the

preparatory techniques followed those of Battarbee (1986) with some adaptations where necessary. Glassware was thoroughly washed with detergent and a soft abrasive cloth prior to use, followed by 30 minutes soaking in hot 10% sodium hydroxide. The latter treatment ensured that any diatom valves from previous preparations would dissolve. The beakers were thoroughly rinsed with distilled water before the samples were added.

Epilithon and Tile Samples

The natural epilithic and tile samples were all prepared in the same way. The whole sample was transferred to a clean 100 ml glass beaker and 5 ml of hydrogen peroxide (H2O2) added. This was allowed to stand until any violent effervescing had stopped and then placed on a hot plate at 60°C for 3-4 hours with additions of H2O2 as required to stop the sample drying out. A few drops of 10% HCl were added to remove carbonates and then the contents of the beaker were transferred to a centrifuge tube. The sample was then centrifuged at 1200 rpm for 5 minutes and the supernatant removed. The pellet was then washed and re-suspended in distilled water and centrifuged again. This process was repeated a total of four times. At this stage microspheres were then added (see below).

Epiphyton and Rope Samples

The first step in preparing the epiphytic and rope samples was to determine the dry weight of the sample. This was done by placing the sample in a pre-weighed 100 ml beaker (weighed to 0.0001 g) and then oven drying overnight at 105°C, followed by re-weighing the beaker plus sample. The preparation technique then followed that used for epilithon. After about 2 hours in H2O2 the sample was sonicated for 5-10 seconds and any remaining plant material (or rope) removed with careful back-washing into the beaker with distilled water. The sample was then left on the hot plate for 1 hour before centrifuging and washing as above.

Epipelic Samples

In the Eaton & Moss (1966) method of lens tissue extraction the tissue was dissolved using chromic acid. The use of chromic acid is no longer permissible and thus an alternative method was required. Hydrogen peroxide did not dissolve the tissue. Furnace ignition of the tissue was tried at 500°C but resulted in noticeable deterioration in some of the more delicate diatom species (e.g. valves of Nitzschia accicularis were seen to severely

deform). The final method adopted for the extraction of the diatoms from the lens tissue was to first dry the tissue in an oven at 105°C and then ignite it in a glass petri dish with a match. This resulted in a small ashy deposit which was transferred into a centrifuge tube and washed with distilled water.

2.4.2 Quantitative Assessment

The addition of external markers has been extensively used for the determination of diatom numbers, with commercially available microspheres being considered the most convenient of these (Battarbee 1986). The microspheres are supplied in a suspension of known concentration and thus a known number of microspheres can be added to each of the samples. The amount added was ideally enough to obtain a 1:1 ratio of microspheres to diatom valves. Slides were only re-made if the ratio dropped below 1:3. From this the number of diatom valves per unit area (or dry weight) can be calculated:

Microspheres introduced x Diatoms counted Microspheres counted

Battarbee (1986) 2.4.3 Slide Preparation

Slides were prepared by allowing a dilute suspension of the diatom sample to settle out and evaporate on a 19 mm, grade zero, circular coverslip until dry. This was then mounted on to a microscope slide using a high refractive index mountant (Naphrax), and heated on a hot plate at 130°C until set. Slides were made up with two cover slips on each with different concentrations to ensure one would be suitable for counting.

2.4.4 Archiving

Each diatom slide was given a unique ECRC slide number and archived in slide drawers. The remaining suspension was placed in a glass vial and the water replaced with methanol to prevent dissolution. This too was labelled with the same number as its corresponding slide and placed in the ECRC diatom suspension archive. Information on the sampling site, slide and suspension location and project to which it is related, were entered into

AMPHORA (Beare 1997), a purpose built computer database for storing and manipulating diatom and water quality data.

2.4.5 Diatom Counting

The techniques used when counting followed those described by Battarbee (1986). Counting was performed on a Leitz Laborlux S microscope at a magnification of xlOOO using predominantly phase contrast with an oil immersion lens. On each slide one or more transects were counted to a total in excess of 350 (±50) valves. Where one taxon occurred at greater than 50% abundance the count was doubled to ensure the inclusion of less common taxa. Care was taken to include equal proportions of central and edge areas of the cover slip to avoid any sorting that may have occurred during settling.

In general the preservation of the siliceous valves was very good, due to the sampling of live diatom communities. It is inevitable, however, that some valves are broken during sampling or preparation and thus it is important to adopt a counting strategy to avoid the over-representation of any species. Any valve fragments that included a distinctive central area were counted as a single valve, and thus any other fragments of the same taxon that could be identified but did not include the central area were not included in the count. Taxa identified in this way included: Navicula spp., Gomphonema spp., Cymbella spp.,

Sellaphora spp. and the raphe valves of Cocconeis spp. Where taxa did not have a distinctive central area the valve ends were counted as half-valves. Taxa counted by the identification of valve ends included: Fragilaria spp., Eunotia spp., Synedra spp.,

Diatoma spp. and the rapheless valves of Cocconeis spp.

2.4.6 Counting LiveiDead Cell Ratios

The epilithon and tile samples were assessed for the proportion of live material prior to preparation. The sample was well mixed by gentle agitation and one drop of the suspension was then placed on a glass slide and covered with a coverslip. 300 cells were then counted under bright field at x500 magnification. A live diatom was taken to be any cell which contained healthy chloroplasts, i.e. not shrivelled or grossly misshapen. Broken cells were judged to have been alive on collection if the cell contents were still visible and

not decayed. A dead diatom was counted as having only one valve or no cellular contents. Any cell with shrivelled and diminished cellular contents was also considered dead. No record of the diatom species was made during this count. Live:dead cell ratios were only recorded for the first stage of this study (Chapter 3).

2.4.7 Diatom Identification

Diatom identification to species level or below was primarily from the floras of Krammer and Lange-Bertalot (1986, 1988, 1991a, 1991b), Patrick and Reimer (1966, 1975), Hustedt (1930-1966) and Germain (1981). Valuable taxonomic advise was also obtained from numerous colleagues within the Environmental Change Research Centre at UCL and from Dr. Eileen Cox at the Natural History Museum, London.

The nomenclature used mainly follows that of Hartley (1986); the exceptions being the splitting of the genera of Sellaphora and Fallacia from Navicula (Round et al. 1990), the use of Ctenophora pulchella (Ralfs ex Kutz.) Williams & Round (1986) rather than its original inclusion in the genus Synedra and Reimeria sinuata (Greg.) Kociolek & Stoermer (1987) rather than Cymbella sinuata.

In the final analyses of species data only those taxa which exceeded 2% abundance in any one sample, or occurred in more than five samples, were included. Of these there were three species which could not be identified. Full descriptions of these taxa are given in Appendix I. In the text they are referred to as: Navicula [small species 1], Navicula

[species 2] and Navicula [pseudogregaria]. Of the rare taxa that could not be identified from the available floras (i.e. those of < 2% abundance and <5 occurrences) a brief description was made, including the co-ordinates on the slide, and a temporary “Z” code assigned rather than the specific “diatcodes” given to the identified taxa. The Z codes consisted of a prefix of ZZZ followed by 999, the number descending for each successive unidentified taxon. A full list of the diatom species found in this study and their authorities is given in Appendix U.

Some difficulties were experienced with the exact identification of Gomphonema parvulum. This taxon is widely reported as being taxonomically indistinct because of

apparent polymorphism (Lowe 1972, Krammer & Lange-Bertalot 1986, Round 1991). An initial attempt was made to separate the ''Gomphonema parvulum complex” into morphs based on stria density, apical width and whether or not the valve ending was rostrate (pinched). This separation was not used for the majority of counts in this study because no ecological differences were observed between the morphs. All morphs were found at both low and high phosphorus concentrations. For the purposes of this study the name

"Gomphonema parvulum^' is used for all the morphs as described in Krammer & Lange- Bertalot (1986).