{
  "url": "https://openlegislation.uk/eu-regulation-2009-761/annex-VI/appendix-3",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-761/annex-VI/appendix-3.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/VI/appendix/3",
  "kind": "appendix",
  "number": "3",
  "heading": "Media",
  "text": "Different growth media are recommended for L. minor and L. gibba. For L. minor, a modified Swedish Standard (SIS) medium is recommended whilst for L. gibba, 20X AAP medium is recommended. Compositions of both media are given below. When preparing these media, reagent or analytical-grade chemicals and deionised water should be used.\nSwedish Standard (SIS) Lemna growth medium\nStock solutions I-V are sterilised by autoclaving (120 °C, 15 minutes) or by membrane filtration (approximately 0,2 μm pore size).\nStock VI (and optionally VII) are sterilised by membrane filtration only; these should not be autoclaved.\nSterile stock solutions should be stored under cool and dark conditions. Stocks I-V should be discarded after six months whilst stocks VI (and optionally VII) have a shelf life of one month.\nStock solution No.\nSubstance\nConcentration in stock solution (g·l–1)\nConcentration in prepared medium (mg·l–1)\nPrepared medium\nElement\nConcentration (mg·l–1)\nI\nNaNO3\nKH2PO4\n8,50\n1,34\n85\n13,4\nNa; N\nK; P\n32; 14\n6,0; 2,4\nII\nMgSO4 · 7H2O\n15\n75\nMg; S\n7,4; 9,8\nIII\nCaCl2 · 2H2O\n7,2\n36\nCa; Cl\n9,8; 17,5\nIV\nNa2CO3\n4,0\n20\nC\n2,3\nV\nH3BO3\nMnCl2 · 4H2O\nNa2MoO4 · 2H2O\nZnSO4 · 7H2O\nCuSO4 · 5H2O\nCo(NO3)2 · 6H2O\n1,0\n0,20\n0,010\n0,050\n0,0050\n0,010\n1,00\n0,20\n0,010\n0,050\n0,0050\n0,010\nB\nMn\nMo\nZn\nCu\nCo\n0,17\n0,056\n0,0040\n0,011\n0,0013\n0,0020\nVI\nFeCl3 · 6H2O\nNa2-EDTA·2H2O\n0,17\n0,28\n0,84\n1,4\nFe\n—\n0,17\n—\nVII\nMOPS (buffer)\n490\n490\n—\n—\nTo prepare one litre of SIS medium, the following are added to 900 ml of deionised water:\n10 ml of stock solution I\n5 ml of stock solution II\n5 ml of stock solution III\n5 ml of stock solution IV\n1 ml of stock solution V\n5 ml of stock solution VI\n1 ml of stock solution VII (optional)\nNote: A further stock solution VII (MOPS buffer) may be needed for certain test substances (see last paragraph in section 1.4).\nThe pH is adjusted to 6,5 ± 0,2 with either 0,1 or 1 mol HCl or NaOH, and the volume adjusted to one litre with deionised water.\n20X AAP growth medium\nStock solutions are prepared in sterile distilled or deionised water.\nSterile stock solutions should be stored under cool and dark conditions. Under these conditions the stock solutions will have a shelf life of at least 6-8 weeks.\nFive nutrient stock solutions (A1, A2, A3, B and C) are prepared for 20X — AAP medium, using reagent-grade chemicals. 20 ml of each nutrient stock solution are added to approximately 850 ml deionised water to produce the growth medium. The pH is adjusted to 7,5 ± 0,1 with either 0,1 or 1 mol HCl or NaOH, and the volume adjusted to one litre with deionised water. The medium is then filtered through a 0,2 μm (approximate) membrane filter into a sterile container.\nThe growth medium intended for testing should be prepared 1-2 days before use to allow the pH to stabilise. The pH of the growth medium should be checked prior to use and readjusted if necessary by addition of 0,1 or 1 M NaOH or HCl as described above.\nUnless noted\nStock solution No.\nSubstance\nConcentration in stock solution (g·l–1)\nConcentration in prepared medium (mg·l–1)\nPrepared medium\nElement\nConcentration (mg·l–1)\nA1\nNaNO3\nMgCl2·6H2O\nCaCl2·2H2O\n26\n12\n4,4\n510\n240\n90\nNa; N\nMg\nCa\n190; 84\n58,08\n24,04\nA2\nMgSO4·7H2O\n15\n290\nS\n38,22\nA3\nK2HPO4·3H2O\n1,4\n30\nK; P\n9,4;3,7\nB\nH3BO3\nMnCl2·4H2O\nFeCl3·6H2O\nNa2EDTA·2H2O\nZnCl2\nCoCl2·6H2O\nNa2MoO4·2H2O\nCuCl2·2H2O\n0,19\n0,42\n0,16\n0,30\n3,3 mg·l–1\n1,4 mg·l–1\n7,3 mg·l–1\n0,012 mg·l–1\n3,7\n8,3\n3,2\n6,0\n66 μg·l–1\n29 μg·l–1\n145 μg·l–1\n0,24 μg·l–1\nB\nMn\nFe\n—\nZn\nCo\nMo\nCu\n0,65\n2,3\n0,66\n—\n31 μg·l–1\n7,1 μg·l–1\n58 μg·l–1\n0,080 μg·l–1\nC\nNaHCO3\n15\n300\nNa; C\n220; 43\nFootnote: The theoretically appropriate final bicarbonate concentration (which will avoid appreciable pH adjustment) is 15 mg/l, not 300 mg/l. However, the historical use of 20X-AAP medium, including the ring test for this method, is based upon 300 mg/l. (I. Sims, P. Whitehouse and R. Lacey. (1999) The OECD Lemna Growth Inhibition Test. Development and Ring-testing of draft OECD Test Guideline. R & D Technical Report EMA 003. WRc plc — Environment Agency.\nSTEINBERG medium (After ISO 20079)\nConcentrations and stock solutions\nThe modified Steinberg medium is used in ISO 20079 for Lemna minor alone (as only Lemna minor is allowed there) but tests showed good results could be reached with Lemna gibba too.\nWhen preparing the medium, reagent- or analytical grade chemicals and deionised water should be used.\nPrepare the nutrient medium from stock solutions or the 10-fold concentrated medium which allows maximum concentration of the medium without precipitation.\nTable 1\npH-stabilised STEINBERG medium (modified acc. to Altenburger)\nSubstance\nNutrient medium\nMacroelements\nmol weight\nmg/l\nmmol/l\nKNO3\n101,12\n350,00\n3,46\nCa(NO3)2 · 4H2O\n236,15\n295,00\n1,25\nKH2PO4\n136,09\n90,00\n0,66\nK2HPO4\n174,18\n12,60\n0,072\nMgSO4 · 7H2O\n246,37\n100,00\n0,41\nMicroelements\nmol weight\nµg/l\nµmol/l\nH3BO3\n61,83\n120,00\n1,94\nZnSO4 · 7H2O\n287,43\n180,00\n0,63\nNa2MoO4 · 2H2O\n241,92\n44,00\n0,18\nMnCl2 · 4H2O\n197,84\n180,00\n0,91\nFeCl3 · 6H2O\n270,21\n760,00\n2,81\nEDTA Disodium-dihydrate\n372,24\n1 500,0\n4,03\nTable 2\nStock solutions (Macroelements)\n1. Macroelements (50-fold concentrated)\ng/l\nStock solution 1:\nKNO3\n17,50\nKH2PO4\n4,5\nK2HPO4\n0,63\nStock solution 2:\nMgSO4 · 7H2O\n5,00\nStock solution 3:\nCa(NO3)2 · 4H2O\n14,75\nTable 3\nStock solutions (Microelements)\n2. Microelements (1 000-fold concentrated)\nmg/l\nStock solution 4:\nH3BO3\n120,0\nStock solution 5:\nZnSO4 · 7H2O\n180,0\nStock solution 6:\nNa2MoO4 · 2H2O\n44,0\nStock solution 7:\nMnCl2 · 4H2O\n180,0\nStock solution 8:\nFeCl3 · 6H2O\n760,00\nEDTA Disodium-dihydrate\n1 500,0\nStock solutions 2 and 3 and separately 4 to 7 may be pooled (taking into account the required concentrations).\nFor longer shelf life, treat stock solutions in an autoclave at 121 °C for 20 min. or alternatively carry out a sterile filtration (0,2 µm). For stock solution 8, sterile filtration (0,2 µm) is strongly recommended.\nPreparation of the final concentration of STEINBERG medium (modified)\nAdd 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 900 ml deionised water to avoid precipitation.\nAdd 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3).\nThe pH should be 5,5 ± 0,2 (adjust by addition of a minimised volume of NaOH solution or HCl).\nAdjust with water to 1 000 ml.\nIf stock solutions are sterilised and appropriate water is used no further sterilisation is necessary. If sterilisation is done with the final medium, stock solution 8 should be added after autoclaving (at 121 °C for 20 min).\nPreparation of 10-fold-concentrated STEINBERG medium (modified) for intermediate storage\nAdd to 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 30 ml water to avoid precipitation.\nAdd 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3). Adjust with water to 100 ml.\nIf stock solutions are sterilised and appropriate water is used no further sterilisation is necessary. If sterilisation is done with the final medium, stock solution 8 should be added after autoclaving (at 121 °C for 20 min).\nThe pH of the medium (final concentration) should be 5,5 ± 0,2.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "Unless noted"
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/761/annex/VI/appendix/3",
    "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."
}