Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers

BS EN 13946:2003 pdf free.Water quality – Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers.
In general, cobbles are the preferred substratum for sampling, as these balance substratum stability (allowing diatom communities to develop) with manoeuvrability. Pebbles and boulders can also be used. At least five cobbles should be sampled. However, if cobbles are unavailable, then either 5 small boulders or 10 pebbles should be sampled. An area of approximately 10 cm2 or more should be scraped- If fewer suitable substrata are available, then a note should be made to this effect.
The following microhabitat conditions should be fulfilled:
1) areas of heavy shade should be avoided ( it cannot be avoided, then a note should be made to this effect). Areas very close to the bank should also be avoided:
2) the substrata shall be submerged for long enough to ensure that assemblages are in equilibrium with their environment. At least tour weeks is recommended but the period depends upon environmental conditions. The precise depth is unimportant so long as the surfaces have not been exposed to air. All depths that can be easily sampled wearing waders are usually suitable, so long as these remain in the euphotic zone;
3) in general, samples should be collected from within the main flow of the river at the sample site. Zones of very slow current (approx. 20 cm 1) should be avoided as these allow the build-up of loosely attached diatoms, silt and other debris.
Collect a selection of substrata from a variety of locations within the sample site, which fulfil the microhabitat requirements listed above. Where suitable substrata are very abundant, random or stratified sampling strategies may be appropriate within a defined sample site.
Remove any loosely attached surface contamination (e.g. organic debris) by washing the substratum briefly in the stream water. Place the substrata in a tray. along with approximately 50 ml of river water.
Wash a stiff toothbrush in clean river water and rub it on a clean surface in order to minimise any diatom contamination from previous samples. Brush the upper surface of the substratum vigorously to remove the diatom film, rinsing the toothbrush periodically in the water in order to transfer the diatoms.
A knife or other sharp instrument can also be used to remove the diatom film. This will be more effective at removing firmly-attached diatoms, but will be less efficient at penetrating crevices on rough surfaces. may cause more damage to trustules and may lead to more rock particles being transferred to the sample. However, it is unlikely that there will be any quantitative difference in results. The knife should also be rinsed in river water and cleaned before use.
Alternatively, the diatom film can be removed using a toothb-ush or knife and washed directly from the surface of the substratum into a sample bottle. The toothbrush or knife can also be used to remove diatoms from the substratum and then rinsed in some stream water collected directly into a sample bottle, if this is preferred.
If> 75 % of substrata are smothered with filamentous algae, these should be sampled in preference to substrata lacking such growths. Remove as many of the filaments as possible prior to brushing or scraping, as above.
Replace the substratum in the stream, and repeat the process for the other replicate substrata. Transfer the water, which should now be brown and turbid due to the presence of diatoms, from the tray into the sample jar.
Label the sample bottle with details relevant to the sample. Transfer the sample to the laboratory in a cool, dark place. If samples are brought to the laboratory within 24 h and these precautions are followed, it is not necessary to add preservative in the field. If preservatives are necessary, then these should be added immediately after collection, unless there are other reasons (e.g. health and safety) why peservatives cannot be used in the field. All future handlers of preserved samples shall be informed of the nature of any preservatives present.
6.3.2 Method for sampling vertical man-made surfaces In situ
The criteria listed above for microhabitat selection should be followed as far as possible.
As this type of sampling is often necessary in lowland, navigable rivers a sample depth of about 30 cm is recommended to allow for fluctuating water levels and wave action.
Agitate the hoe in the water in front of the area to be sampled to dislodge any loosely attached materials.
Scrape the surface with the sharpened blade of the hoe to remove the attached diatoms. An area of approximately 10 cm2 should be scraped. Then remove the diatom film adhering to the hoe blade and directly place into a sample bottle into which some river water has been placed.
NOTE Specialised apparatus may also be useful under some circumstances. More details are given in reference 141.
This process should be repeated at least three times and the replicates pooled. For details regarding labelling and
transfer to the laboratory see 6.3.1.
6.3.3 Use of introduced (“artificial”) substrata
Substrata with heterogeneous surfaces (e.g. rough tiles, frayed polypropylene rope) are preferred over substrata with smooth surfaces (e.g. glass slides). These should be left in the river for long enough to ensure that assemblages are in equilibrium with their environment. At least four weeks is recommended, but the period depends upon environmental conditions and longer periods of exposure may be appropriate under some circumstances (i.e. very oligotrophic conditions, low temperatures, heavy shade).
Detailed methods will depend upon the type of substratum chosen. If rough tiles are used, then samples can be collected as described in 6.3.1. For frayed ropes, the final 5 cm is removed with a pair of scissors or brushed with a toothbrush and placed in a sample container. Full details of methods are necessary if results from introduced substrata are to be interpreted correctly.
Care should be taken with the design and deployment of introduced substrata to ensure that they do not interfere with the activities of legitimate river users and to minimise risks of vandalism. Extra, replicate substrata should be deployed, to allow for potential losses due to spates or vandalism.
Where introduced substrata are to be used for comparative studies in the same watercourse, it is important that all substrata are exposed to identical conditions. Both the length of exposure and the start date shall be the same (to allow for the impact of hydrological events upon the developing diatom community).
For details regarding labelling and transfer to the laboratory see 6.3.1.
6.3.4 Sample collection from submerged macrophytes and macroalgae
Sample the entire plant (five replicates) and place into a plastic bag for transfer to the laboratory. Stir or agitate the plants vigorously in some distilled or demineralised water in a large beaker to dislodge attached diatoms. Remove the macrophytes from the beaker, allow the diatoms to settle and pour off the supernatant.
Alternatively, cut some lengths at random from submerged plants using a knife or scissors and put the sections into a sampling bottle. These can be fractionated further in the laboratory, if required, and the macrophyte sections plus attached diatoms placed directly in a flask for cleaning (6.4.2).BS EN 13946 pdf free.Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers