# 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), Appendix 2: Growth media

Extent: E+W+S+N.I.

One of the following two growth media may be used:
OECD medium: Original medium of OECD TG 201, also according to ISO 8692
US. EPA medium AAP also according to ASTM.
When preparing these media, reagent or analytical-grade chemicals should be used and deionised water.
Composition of The AAP-medium (US. EPA) and the OECD TG 201 medium
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.
Component
EPA
OECD
mg/l
mM
mg/l
mM
NaHCO3
15,0
0,179
50,0
0,595
NaNO3
25,5
0,300
NH4Cl
15,0
0,280
MgCl2·6(H2O)
12,16
0,0598
12,0
0,0590
CaCl2·2(H2O)
4,41
0,0300
18,0
0,122
MgSO4·7(H2O)
14,6
0,0592
15,0
0,0609
K2HPO4
1,044
0,00599
KH2PO4
1,60
0,00919
FeCl3·6(H2O)
0,160
0,000591
0,0640
0,000237
Na2EDTA·2(H2O)
0,300
0,000806
0,100
0,000269
H3BO3
0,186
0,00300
0,185
0,00299
MnCl2·4(H2O)
0,415
0,00201
0,415
0,00210
ZnCl2
0,00327
0,000024
0,00300
0,0000220
CoCl2·6(H2O)
0,00143
0,000006
0,00150
0,00000630
Na2MoO4·2(H2O)
0,00726
0,000030
0,00700
0,0000289
CuCl2.2(H2O)
0,000012
0,00000007
0,00001
0,00000006
pH
7,5
8,1
In 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.
The 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:
PHeq = 11,30 + log [HCO3]
With 15 mg NaHCO3, pHeq = 7,5 (U.S. EPA medium) and with 50 mg NaHCO3/l, pHeq = 8,1 (OECD medium).
Element composition of test media
Element
EPA
OECD
mg/l
mg/l
C
2,144
7,148
N
4,202
3,927
P
0,186
0,285
K
0,469
0,459
Na
11,044
13,704
Ca
1,202
4,905
Mg
2,909
2,913
Fe
0,033
0,017
Mn
0,115
0,115
Preparation of OECD medium
Nutrient
Concentration in stock solution
Stock solution 1: macronutrients
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
MgSO4·7H2O
KH2PO4
1,5 g·l–1
1,2 g·l–1
1,8 g·l–1
1,5 g·l–1
0,16 g·l–1
Stock solution 2: iron
FeCl3·6H2O
Na2EDTA·2H2O
64 mg·l–1
100 mg·l–1
Stock solution 3: trace elements
H3BO3
MnCl2·4H2O
ZnCl2
CoCl2·6H2O
CuCl2·2H2O
Na2MoO4·2H2O
185 mg·l–1
415 mg·l–1
3 mg·l–1
1,5 mg·l–1
0,01 mg·l–1
7 mg·l–1
Stock solution 4: bicarbonate
NaHCO3
50 g·l–1
Na2SiO3·9H20
Sterilise 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.
Do not autoclave stock solutions 2 and 4, but sterilise them by membrane filtration.
Prepare a growth medium by adding an appropriate volume of the stock solutions 1-4 to water:
Add to 500 ml of sterilised water:
10 ml of stock solution 1
1 ml of stock solution 2
1 ml of stock solution 3
1 ml of stock solution 4
Make up to 1 000 ml with sterilised water
Allow sufficient time for equilibrating the medium with the atmospheric CO2, if necessary by bubbling with sterile filtered air for some hours.
Preparation of AAP medium
A1.1. 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.
A1.2. 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.
A1.2.1. NaNO3 —12,750 g.
A1.2.2. MgCL2·6H2O—6,082 g.
A1.2.3. CaCl2·2H2O—2,205 g.
A1.2.4. Micronutrient Stock Solution—(see A1.3).
A1.2.5. MgSO4·7H2O—7,350 g.
A1.2.6. K2HPO4 —0,522 g.
A1.2.7. NaHCO3 —7,500 g.
A1.2.8. Na2SiO3·9H2O—See Note A1.1.
Note 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.
A1.3. The micronutrient stock solution is made by dissolving the following into 500 mL of deionised or distilled water:
A1.3.1. H3BO3 —92,760 mg.
A1.3.2. MnCl2·4H2O–207,690 mg.
A1.3.3. ZnCl2 —1,635 mg.
A1.3.4. FeCl3·6H2O—79,880 mg.
A1.3.5. CoCl2·6H2O—0,714 mg.
A1.3.6. Na2MoO4·2H2O—3,630 mg.
A1.3.7. CuCl2·2H2O—0,006 mg.
A1.3.8. Na2EDTA·2H2O—150,000 mg.
[Disodium (Ethylenedinitrilo) tetraacetate].
A1.3.9. Na2SeO4·5H2O—0,005 mg See Note A1.2.
Note A1.2 — Use only in medium for stock cultures of diatom species.
A1.4. Adjust pH to 7,5 ± 0,1 with 0,1 N or 1,0 N NaOH or HCl.
A1.5. 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.
A1.6. Store medium in the dark at approximately 4 °C until use.

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Source: https://www.legislation.gov.uk/eur/2009/761/annex/IV/appendix/2 (the official version)
Page: https://openlegislation.uk/eu-regulation-2009-761/annex-IV/appendix-2
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.