{
  "url": "https://openlegislation.uk/eu-regulation-2009-761/annex-IV/appendix-2",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-761/annex-IV/appendix-2.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/2",
  "kind": "appendix",
  "number": "2",
  "heading": "Growth media",
  "text": "One of the following two growth media may be used:\nOECD medium: Original medium of OECD TG 201, also according to ISO 8692\nUS. EPA medium AAP also according to ASTM.\nWhen preparing these media, reagent or analytical-grade chemicals should be used and deionised water.\nComposition of The AAP-medium (US. EPA) and the OECD TG 201 medium\nThe molar ratio of EDTA to iron slightly exceed unity. This prevents iron precipitation and, at the same time, chelation of heavy metal ions is minimised.\nComponent\nEPA\nOECD\nmg/l\nmM\nmg/l\nmM\nNaHCO3\n15,0\n0,179\n50,0\n0,595\nNaNO3\n25,5\n0,300\nNH4Cl\n15,0\n0,280\nMgCl2·6(H2O)\n12,16\n0,0598\n12,0\n0,0590\nCaCl2·2(H2O)\n4,41\n0,0300\n18,0\n0,122\nMgSO4·7(H2O)\n14,6\n0,0592\n15,0\n0,0609\nK2HPO4\n1,044\n0,00599\nKH2PO4\n1,60\n0,00919\nFeCl3·6(H2O)\n0,160\n0,000591\n0,0640\n0,000237\nNa2EDTA·2(H2O)\n0,300\n0,000806\n0,100\n0,000269\nH3BO3\n0,186\n0,00300\n0,185\n0,00299\nMnCl2·4(H2O)\n0,415\n0,00201\n0,415\n0,00210\nZnCl2\n0,00327\n0,000024\n0,00300\n0,0000220\nCoCl2·6(H2O)\n0,00143\n0,000006\n0,00150\n0,00000630\nNa2MoO4·2(H2O)\n0,00726\n0,000030\n0,00700\n0,0000289\nCuCl2.2(H2O)\n0,000012\n0,00000007\n0,00001\n0,00000006\npH\n7,5\n8,1\nIn the test with the diatom Navicula pelliculosa, both media must be supplemented with Na2SiO3·9H20 to obtain a concentration of 1,4 mg Si/l.\nThe pH of the medium is obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air. An approximate relationship between pH at 25 °C and the molar bicarbonate concentration is:\nPHeq = 11,30 + log [HCO3]\nWith 15 mg NaHCO3, pHeq = 7,5 (U.S. EPA medium) and with 50 mg NaHCO3/l, pHeq = 8,1 (OECD medium).\nElement composition of test media\nElement\nEPA\nOECD\nmg/l\nmg/l\nC\n2,144\n7,148\nN\n4,202\n3,927\nP\n0,186\n0,285\nK\n0,469\n0,459\nNa\n11,044\n13,704\nCa\n1,202\n4,905\nMg\n2,909\n2,913\nFe\n0,033\n0,017\nMn\n0,115\n0,115\nPreparation of OECD medium\nNutrient\nConcentration in stock solution\nStock solution 1: macronutrients\nNH4Cl\nMgCl2·6H2O\nCaCl2·2H2O\nMgSO4·7H2O\nKH2PO4\n1,5 g·l–1\n1,2 g·l–1\n1,8 g·l–1\n1,5 g·l–1\n0,16 g·l–1\nStock solution 2: iron\nFeCl3·6H2O\nNa2EDTA·2H2O\n64 mg·l–1\n100 mg·l–1\nStock solution 3: trace elements\nH3BO3\nMnCl2·4H2O\nZnCl2\nCoCl2·6H2O\nCuCl2·2H2O\nNa2MoO4·2H2O\n185 mg·l–1\n415 mg·l–1\n3 mg·l–1\n1,5 mg·l–1\n0,01 mg·l–1\n7 mg·l–1\nStock solution 4: bicarbonate\nNaHCO3\n50 g·l–1\nNa2SiO3·9H20\nSterilise the stock solutions by membrane filtration (mean pore diameter 0,2 µm) or by autoclaving (120 °C, 15 min). Store the solutions in the dark at 4 °C.\nDo not autoclave stock solutions 2 and 4, but sterilise them by membrane filtration.\nPrepare a growth medium by adding an appropriate volume of the stock solutions 1-4 to water:\nAdd to 500 ml of sterilised water:\n10 ml of stock solution 1\n1 ml of stock solution 2\n1 ml of stock solution 3\n1 ml of stock solution 4\nMake up to 1 000 ml with sterilised water\nAllow sufficient time for equilibrating the medium with the atmospheric CO2, if necessary by bubbling with sterile filtered air for some hours.\nPreparation of AAP medium\nA1.1. Add 1 mL of each stock solution in A1.2.1-A1.2.7 to approximately 900 mL of deionised or distilled water and then dilute to 1 L.\nA1.2. Macronutrient stock solutions are made by dissolving the following into 500 mL of deionised or distilled water. Reagents A1.2.1, A1.2.2, A1.2.3, and A1.2.4 can be combined into one stock solution.\nA1.2.1. NaNO3 —12,750 g.\nA1.2.2. MgCL2·6H2O—6,082 g.\nA1.2.3. CaCl2·2H2O—2,205 g.\nA1.2.4. Micronutrient Stock Solution—(see A1.3).\nA1.2.5. MgSO4·7H2O—7,350 g.\nA1.2.6. K2HPO4 —0,522 g.\nA1.2.7. NaHCO3 —7,500 g.\nA1.2.8. Na2SiO3·9H2O—See Note A1.1.\nNote A1.1 — Use for diatom test species only. May be added directly (202,4 mg) or by way of stock solution to give 20 mg/L Si final concentration in medium.\nA1.3. The micronutrient stock solution is made by dissolving the following into 500 mL of deionised or distilled water:\nA1.3.1. H3BO3 —92,760 mg.\nA1.3.2. MnCl2·4H2O–207,690 mg.\nA1.3.3. ZnCl2 —1,635 mg.\nA1.3.4. FeCl3·6H2O—79,880 mg.\nA1.3.5. CoCl2·6H2O—0,714 mg.\nA1.3.6. Na2MoO4·2H2O—3,630 mg.\nA1.3.7. CuCl2·2H2O—0,006 mg.\nA1.3.8. Na2EDTA·2H2O—150,000 mg.\n[Disodium (Ethylenedinitrilo) tetraacetate].\nA1.3.9. Na2SeO4·5H2O—0,005 mg See Note A1.2.\nNote A1.2 — Use only in medium for stock cultures of diatom species.\nA1.4. Adjust pH to 7,5 ± 0,1 with 0,1 N or 1,0 N NaOH or HCl.\nA1.5. Filter the media into a sterile container through either a 0,22-μm membrane filter if a particle counter is to be used or a 0,45-μm filter if a particle counter is not to be used.\nA1.6. Store medium in the dark at approximately 4 °C until use.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "The molar ratio of EDTA to iron slightly exceed unity. This prevents iron precipitation and, at the same time, chelation of heavy metal ions is minimised."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/761/annex/IV/appendix/2",
    "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."
}