Appendix 1 Strains shown to be suitable for the test

• Pseudokirchneriella subcapitata, (formerly known as Selenastrum capricornutum), ATCC 22662, CCAP 278/4, 61.81 SAG

• Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) 86.81 SAG

• Navicula pelliculosa, UTEX 664

• Anabaena flos-aquae, UTEX 1444, ATCC 29413, CCAP 1403/13A

• Synechococcus leopoliensis, UTEX 625, CCAP 1405/1

The strains recommended are available in unialgal cultures from the following collections (in alphabetical order):

• ATCC: American Type Culture Collection

  10801 University Boulevard

  Manassas, Virginia 20110-2209

  UNITED STATES

• CCAP, Culture Collection of Algae and Protozoa

  Institute of Freshwater Ecology,

  Windermere Laboratory

  Far Sawrey, Amblerside

  Cumbria

  LA22 0LP

  UNITED KINGDOM

• SAG: Collection of Algal Cultures

  Inst. Plant Physiology

  University of Göttingen

  Nicholausberger Weg 18

  3400 Göttingen

  GERMANY

• UTEX Culture Collection of Algae

  Section of Molecular, Cellular and Developmental Biology

  School of Biological Sciences

  the University of Texas at Austin

  Austin, Texas 78712

  USA

These green algae are generally easy to maintain in various culture media. Information on suitable media is available from the culture collections. The cells are normally solitary, and cell density measurements can easily be performed using an electronic particle counter or microscope.

Various growth media may be used for keeping a stock culture. It is particularly important to avoid allowing the batch culture to go past log phase growth when renewing, recovery is difficult at this point.

Anabaena flos-aquae develops aggregates of nested chains of cells. The size of these aggregates may vary with culturing conditions. It may be necessary to break up these aggregates when microscope counting or an electronic particle counter is used for determination of biomass.

Sonication of sub-samples may be used to break up chains to reduce count variability. Longer sonication than required for breaking up chains into shorter lengths may destroy the cells. Sonication intensity and duration must be identical for each treatment.

Count enough fields on the hemocytometer (at least 400 cells) to help compensate for variability. This will improve reliability of microscopic density determinations.

An electronic particle counter can be used for determination of total cell volume of Anabaena after breaking up the cell chains by careful sonification. The sonification energy has to be adjusted to avoid disruption of the cells.

Use a vortex mixer or similar appropriate method to make sure the algae suspension used to inoculate test vessels is well mixed and homogeneous.

Test vessels should be placed on an orbital or reciprocate shaker table at about 150 revolutions per minute. Alternatively, intermittent agitation may be used to reduce the tendency of Anabaena to form clumps. If clumping occurs, care must be taken to achieve representative samples for biomass measurements. Vigorous agitation before sampling may be necessary to disintegrate algal clumps.

Various growth media may be used for keeping a stock culture. Information on suitable media is available from the culture collections.

Synechococcus leopoliensis grows as solitary rod-shaped cells. The cells are very small, which complicates the use of microscope counting for biomass measurements. Electronic particle counters equipped for counting particles down to a size of approximately 1 µm are useful. In vitro fluorometric measurements are also applicable.

Various growth media may be used for keeping a stock culture. Information on suitable media is available from the culture collections. Note that silicate is required in the medium.

Navicula pelliculosa may form aggregates under certain growth conditions. Due to production of lipids the algal cells sometimes tend to accumulate in the surface film. Under those circumstances special measures have to be taken when sub-samples are taken for biomass determination in order to obtain representative samples. Vigorous shaking, e.g. using a vortex mixer may be required.