Appendix 2 Growth media
United Kingdom
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 | 0,000269f1 |
| 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.
Annotations
Footnotes
- f1
-
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.