Reference: Environment Canada, Method Development and Applications Section, Environmental Technology Centre. 2007. Biological Test Method: Test for Measuring the Inhibition of Growth Using the Freshwater Macrophyte, Lemna minor. EPS 1/RM/37 Second Edition – January 2007.
| Stock | Substance | Concentration | Amount added to medium |
|---|---|---|---|
| Aa | Ca(NO3)2•4H2O | 59.00 g/L | 20 mL/L |
| KNO3 | 75.76 g/L | ||
| KH2PO4 | 34.00 g/L | ||
| B | Tartaric Acid | 3.00 g/L | 1 mL/L |
| Cb | FeCl3•6H2O | 1.21 g/L | 20 mL/L |
| Na2EDTA•2H2Oc | 3.35 g/L | ||
| D | MgSO4•7H2O | 50.00 g/L | 10 mL/L |
| E | H3BO3 | 2.86 g/L | 1 mL/L |
| ZnSO4•7H2O | 0.22 g/L | ||
| Na2MoO4•2H2O | 0.12 g/L | ||
| CuSO4•5H2O | 0.08 g/L | ||
| MnCl2•4H2O | 3.62 g/L | ||
| – | Sucrose | – | 10 g/L |
| – | Yeast extract | – | 0.10 g/L |
| – |
Tryptone (Bactotryptone) or Peptone (Bactopeptone) |
– | 0.60 g/L |
a Add 6 mL of 6N HCl to stock solution A
b Add 1.2 mL of 6N KOH to stock solution C
c Na4EDTA•2H2O can be used instead of Na2EDTA•2H2O. If Na4EDTA•2H2O is used, the concentration in the stock solution is 3.75 g/L instead of 3.35 g/L, and KOH should not be added to stock solution C.
For one litre of medium start with about 900 ml of purified water and add each stock solution in the order above. The medium is then stirred until all the contents are dissolved. Adjust the pH to within the range 4.40 to 4.80 with NaOH or HCl, bring volume up to 1 L with purified water, and autoclave. Stock solutions should be stored in the dark (i.e., dark amber or aluminum-covered bottles) due to potential photosensitivity. Environment Canada culturing requirements indicate that stock solutions may be stored in the refrigerator for up to one month, provided that they are isolated from solvents or other potential contaminants. Once autoclaved, the prepared medium can be stored in the dark at room temperature for up to one month. However, for routine maintenance of plants that will not be used for Environment Canada regulatory toxicity testing, the stock solutions and medium may be stored for a longer period of time.