Commission Regulation (EC) No 761/2009Show full title

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.

Swedish Standard (SIS) Lemna growth medium

• Stock solutions I-V are sterilised by autoclaving (120 °C, 15 minutes) or by membrane filtration (approximately 0,2 μm pore size).

• Stock VI (and optionally VII) are sterilised by membrane filtration only; these should not be autoclaved.

• Sterile 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.

  Stock solution No.	Substance	Concentration in stock solution (g·l–1)	Concentration in prepared medium (mg·l–1)	Prepared medium
  				Element	Concentration (mg·l–1)
  I	NaNO3 KH2PO4	8,50 1,34	85 13,4	Na; N K; P	32; 14 6,0; 2,4
  II	MgSO4 · 7H2O	15	75	Mg; S	7,4; 9,8
  III	CaCl2 · 2H2O	7,2	36	Ca; Cl	9,8; 17,5
  IV	Na2CO3	4,0	20	C	2,3
  V	H3BO3 MnCl2 · 4H2O Na2MoO4 · 2H2O ZnSO4 · 7H2O CuSO4 · 5H2O Co(NO3)2 · 6H2O	1,0 0,20 0,010 0,050 0,0050 0,010	1,00 0,20 0,010 0,050 0,0050 0,010	B Mn Mo Zn Cu Co	0,17 0,056 0,0040 0,011 0,0013 0,0020
  VI	FeCl3 · 6H2O Na2-EDTA·2H2O	0,17 0,28	0,84 1,4	Fe —	0,17 —
  VII	MOPS (buffer)	490	490	—	—

• To prepare one litre of SIS medium, the following are added to 900 ml of deionised water:

  • 10 ml of stock solution I

  • 5 ml of stock solution II

  • 5 ml of stock solution III

  • 5 ml of stock solution IV

  • 1 ml of stock solution V

  • 5 ml of stock solution VI

  • 1 ml of stock solution VII (optional)

  Note: A further stock solution VII (MOPS buffer) may be needed for certain test substances (see last paragraph in section 1.4).

• The 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.

20X AAP growth medium

Stock solutions are prepared in sterile distilled or deionised water.

Sterile 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.

Five 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.

The 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.

a Unless noted
Stock solution No.	Substance	Concentration in stock solution (g·l–1)a	Concentration in prepared medium (mg·l–1)a	Prepared medium
				Element	Concentration (mg·l–1)a
A1	NaNO3 MgCl2·6H2O CaCl2·2H2O	26 12 4,4	510 240 90	Na; N Mg Ca	190; 84 58,08 24,04
A2	MgSO4·7H2O	15	290	S	38,22
A3	K2HPO4·3H2O	1,4	30	K; P	9,4;3,7
B	H3BO3 MnCl2·4H2O FeCl3·6H2O Na2EDTA·2H2O ZnCl2 CoCl2·6H2O Na2MoO4·2H2O CuCl2·2H2O	0,19 0,42 0,16 0,30 3,3 mg·l–1 1,4 mg·l–1 7,3 mg·l–1 0,012 mg·l–1	3,7 8,3 3,2 6,0 66 μg·l–1 29 μg·l–1 145 μg·l–1 0,24 μg·l–1	B Mn Fe — Zn Co Mo Cu	0,65 2,3 0,66 — 31 μg·l–1 7,1 μg·l–1 58 μg·l–1 0,080 μg·l–1
C	NaHCO3	15	300	Na; C	220; 43

Footnote: 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.

STEINBERG medium (After ISO 20079)

Concentrations and stock solutions

• The 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.

• When preparing the medium, reagent- or analytical grade chemicals and deionised water should be used.

• Prepare the nutrient medium from stock solutions or the 10-fold concentrated medium which allows maximum concentration of the medium without precipitation.

Table 1

pH-stabilised STEINBERG medium (modified acc. to Altenburger)

Substance		Nutrient medium
Macroelements	mol weight	mg/l	mmol/l
KNO3	101,12	350,00	3,46
Ca(NO3)2 · 4H2O	236,15	295,00	1,25
KH2PO4	136,09	90,00	0,66
K2HPO4	174,18	12,60	0,072
MgSO4 · 7H2O	246,37	100,00	0,41
Microelements	mol weight	µg/l	µmol/l
H3BO3	61,83	120,00	1,94
ZnSO4 · 7H2O	287,43	180,00	0,63
Na2MoO4 · 2H2O	241,92	44,00	0,18
MnCl2 · 4H2O	197,84	180,00	0,91
FeCl3 · 6H2O	270,21	760,00	2,81
EDTA Disodium-dihydrate	372,24	1 500,0	4,03

Table 2

Stock solutions (Macroelements)

1. Macroelements (50-fold concentrated)	g/l
Stock solution 1:
KNO3	17,50
KH2PO4	4,5
K2HPO4	0,63
Stock solution 2:
MgSO4 · 7H2O	5,00
Stock solution 3:
Ca(NO3)2 · 4H2O	14,75

Table 3

Stock solutions (Microelements)

2. Microelements (1 000-fold concentrated)	mg/l
Stock solution 4:
H3BO3	120,0
Stock solution 5:
ZnSO4 · 7H2O	180,0
Stock solution 6:
Na2MoO4 · 2H2O	44,0
Stock solution 7:
MnCl2 · 4H2O	180,0
Stock solution 8:
FeCl3 · 6H2O	760,00
EDTA Disodium-dihydrate	1 500,0

• Stock solutions 2 and 3 and separately 4 to 7 may be pooled (taking into account the required concentrations).

• For 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.

Preparation of the final concentration of STEINBERG medium (modified)

• Add 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 900 ml deionised water to avoid precipitation.

• Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3).

• The pH should be 5,5 ± 0,2 (adjust by addition of a minimised volume of NaOH solution or HCl).

• Adjust with water to 1 000 ml.

• If 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).

Preparation of 10-fold-concentrated STEINBERG medium (modified) for intermediate storage

• Add to 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 30 ml water to avoid precipitation.

• Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3). Adjust with water to 100 ml.

• If 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).

• The pH of the medium (final concentration) should be 5,5 ± 0,2.