In this study diatom samples were collected from many different substratum types including three natural and five artificial. These were: cobbles (epilithon), sediment (epipelon), submerged macrophytes (epiphyton), rough clay tile, smooth clay tile.
polyethylene rope, perspex and glass. All collections were made in triplicate for the first part of this study (Chapter 3) to assess sample variation. Later deployments of artificial substrata were restricted to one of each type (rough tile and rope only) per site. All the samples, with the exception of epipelon, were treated on site with acidified Lugol’s iodine to kill and preserve the diatoms and then refrigerated until preparation.
It was initially proposed that the artificial substrata would be held in 35 x 35 cm metal frames, securely anchored to the river bed. This was tried but found to be too susceptible to vandalism, with 8 out of the original 12 frames being removed in the first sampling month and 6 out of 11 in the second month. After this the tile substrata were left, unanchored, on the river bed and the rope either attached to a steel stake driven in to the river bed or attached to a heavy weight (normally a stone or brick).
2.3.1 Natural Substratum Sampling
Cobbles
Strictly speaking the epilithon are those diatoms living attached to or on the surface of rock. It is recommended in the literature, therefore, that when selecting cobbles for diatom sampling, only those free from sediment and other algal or macrophyte growths should be chosen (Round 1993). In lowland rivers this was often not possible and therefore stones which were least contaminated with silt or other algae were chosen. If the cobble had an even surface, a 25 cm^ area was delimited using a plastic frame and the cobble carefully brushed with a toothbrush into a 30 ml sampling vial. For less even cobbles, or those smaller than 25 cm^, the area was calculated approximately using a ruler and the whole upper surface of the stone brushed. Care was taken to clean the sampling equipment thoroughly between each sample and particularly between sites.
Sediment Samples
Diatoms living within the sediment (epipelon) move around in the uppermost surface layers. By simply taking a sediment sample not only the live fraction will be collected but also numerous dead cells. To avoid this a post-collection harvesting technique was used. The sediment was collected by taking a 1 cm deep core from a silty area of the river bed using a 5 cm diameter perspex tube. The sample was then transferred to a sample bottle
and placed in the dark. The method used for “harvesting” the live diatom fraction followed that of Eaton and Moss (1966). This method relies on the live diatoms moving up to the sediment surface as a result of the intrinsic diurnal rhythm. If a piece of lens tissue is placed on the surface the motile diatoms move from the sediment and into the lens tissue, which can then be removed.
In the evening of the day the sample was collected the sediment was thoroughly mixed and evenly spread in a petri dish. After allowing to settle, the surface water was carefully removed with a pipette. A double thickness of Whatman grade 105 lens tissue (5 x 7.5 cm) was placed directly onto the sediment surface and the sample left where it could receive direct daylight. The following morning the lens tissue was removed and treated with Lugol’s iodine. Eaton & Moss (1966) reported the best time of “harvesting” to be between 9.00 am and 10.00 am; prior to this many individuals had not moved into the tissue and by 12.00 noon a net downward movement was observed. Although untested here, Eaton & Moss reported 87.5% recovery of live epipelic diatoms using this method.
Submerged Macrophytes
Samples of submerged macrophytes were taken at each site to assess the epiphytic assemblages. Whenever possible the species of plant selected was kept constant;
Ranunculus sp. was the easiest to find at all sites in the summer. Later in the year, however, this was not found and it was therefore only possible to find species with similar growth forms (e.g. Myriophyllum sp.). At Alton only small, submerged leaves of Berula
sp. were sampled in winter.
The sampling technique used was to select 3-4 fronds of the plant of approximately 10 cm in length. These were placed in sample bags and treated with Lugol’s iodine.
2.3.2 Artificial Substrata Sampling
All artificial substrata were left in the river sites for approximately one month and then removed for sampling. Replacements were put in on the same day for the following month’s samples. The sampled tiles were taken back to the laboratory and thoroughly cleaned by soaking in detergent and scrubbing with a wire brush. These were then reused.
The rope was replaced with a new 25 cm length of 15 mm diameter polyethylene rope. A sketch map was made after placement to aid relocation on the following trip.
Clay Tiles
Both the rough and smooth clay tiles were sampled in the same way as the natural epilithon. A 25 cm^ area of the tile was delimited using a flat perspex frame with a 5 x 5 cm square cut in the centre (Plate 2.1). The inside area of the frame was brushed with a toothbrush and the sample washed into a collecting vial. Care was taken to wash the brush out into the vial and then clean it thoroughly before the next sample was taken.