{
  "url": "https://openlegislation.uk/eu-regulation-2009-761/annex-IV/appendix-1",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-761/annex-IV/appendix-1.json",
  "legislation": "eur/2009/761",
  "legislation_title": "Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2009-761",
  "path": "annex/IV/appendix/1",
  "kind": "appendix",
  "number": "1",
  "heading": "Strains shown to be suitable for the test",
  "text": "Green algae\nPseudokirchneriella subcapitata, (formerly known as Selenastrum capricornutum), ATCC 22662, CCAP 278/4, 61.81 SAG\nDesmodesmus subspicatus (formerly known as Scenedesmus subspicatus) 86.81 SAG\nDiatoms\nNavicula pelliculosa, UTEX 664\nCyanobacteria\nAnabaena flos-aquae, UTEX 1444, ATCC 29413, CCAP 1403/13A\nSynechococcus leopoliensis, UTEX 625, CCAP 1405/1\nSources of Strains\nThe strains recommended are available in unialgal cultures from the following collections (in alphabetical order):\nATCC: American Type Culture Collection\n10801 University Boulevard\nManassas, Virginia 20110-2209\nUNITED STATES\nCCAP, Culture Collection of Algae and Protozoa\nInstitute of Freshwater Ecology,\nWindermere Laboratory\nFar Sawrey, Amblerside\nCumbria\nLA22 0LP\nUNITED KINGDOM\nSAG: Collection of Algal Cultures\nInst. Plant Physiology\nUniversity of Göttingen\nNicholausberger Weg 18\n3400 Göttingen\nGERMANY\nUTEX Culture Collection of Algae\nSection of Molecular, Cellular and Developmental Biology\nSchool of Biological Sciences\nthe University of Texas at Austin\nAustin, Texas 78712\nUSA\nAppearance and characteristics of recommended species\nMeasured with electronic particle counter\nCalculated from size\nMost frequently observed growth rate in OECD medium at light intensity approx. 70 µE·m–2·s–1 and 21 °C\nP. subcapitata\nD. subspicatus\nN. pelliculosa\nA. flos-aquae\nS. leopoliensis\nAppearance\nCurved, twisted single cells\nOval, mostly single cells\nRods\nChains of oval cells\nRods\nSize (L × W) µm\n8-14 × 2-3\n7-15 × 3-12\n7,1 × 3,7\n4,5 × 3\n6 × 1\nCell volume (µm3/cell)\n40-60\n60-80\n40-50\n30-40\n2,5\nCell dry weight (mg/cell)\n2-3 × 10–8\n3-4 × 10–8\n3-4 × 10–8\n1-2 × 10–8\n2-3 × 10–9\nGrowth rate day–1)\n1,5-1,7\n1,2-1,5\n1,4\n1,1-1,4\n2,0-2,4\nSpecific Recommendations on Culturing and Handling of Recommended Test Species\nPseudokirchneriella subcapitata and Desmodesmus subspicatus\nThese 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.\nAnabaena flos-aquae\nVarious 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.\nAnabaena 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.\nSonication 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.\nCount enough fields on the hemocytometer (at least 400 cells) to help compensate for variability. This will improve reliability of microscopic density determinations.\nAn 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.\nUse a vortex mixer or similar appropriate method to make sure the algae suspension used to inoculate test vessels is well mixed and homogeneous.\nTest 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.\nSynechococcus leopoliensis.\nVarious growth media may be used for keeping a stock culture. Information on suitable media is available from the culture collections.\nSynechococcus 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.\nNavicula pelliculosa\nVarious 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.\nNavicula 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.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "Measured with electronic particle counter"
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Calculated from size"
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "Most frequently observed growth rate in OECD medium at light intensity approx. 70 µE·m–2·s–1 and 21 °C"
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/761/annex/IV/appendix/1",
    "retrieved_at": "2026-11-01T14:51:19.625001+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}