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Toxicity to aquatic plants other than algae

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toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
according to guideline
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
GLP compliance:
Analytical monitoring:
Details on test solutions:
A stock solution with a concentration of 1.0 g/L of the material presented was prepared. It means that 0.1000 g of the test material has been dissolved in 100 mL of culture medium in a 100 mL volumetric flask, Class A by means of agitation. The stock solution has a concentration of 978.7 mg/L of active ingredient. The different solutions used in the study have been prepared from this stock solution of 978.7 mg/L of active ingredient of the test material.
Test organisms (species):
Lemna minor
Details on test organisms:
- Source : Federal Enviromental Agency (Berlin, Germany)
- Culture conditions:
T: 24+-2ºC
Intensity and quality of lighting: 6500 lux
-Number of fronds at the beginning of the test: 12
Test type:
Water media type:
Limit test:
Total exposure duration:
7 d
Test temperature:
Nominal and measured concentrations:
Concentrations tested (active ingredient): 0.99 mg/L; 3.16 mg/L; 9.86 mg/L; 31.55 mg/L; 98.6 mg/L; 315.5 mg/L; 985.9 mg/L
Actual concentration of the stock solution: 985.9 mg/L
Details on test conditions:
- Incubation chamber used: yes
- Test vessel: 150 ml (beaker)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass with 100 ml of test medium
- Type of cover: not specified
- Agitation: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of concentrations: 7 + control
- Geometric progression factor used: 3.2

- Standard medium used: yes (Steinberg)
- Test medium with the following composition:
— Solution 1: KNO3 17.5 g/L H2O; KH2PO4 4.5 g/L H2O; K2HPO4 0.63 g/L H2O
— Solution 2: MgSO4 7H2O 5 g/L H2O
— Solution 3: Ca(NO3)2 4H2O 14.75 g/L H2O

— Solution 4: H3BO3 120mg/L H2O
— Solution 5: ZnSO4 7H2O 180 mg/L H2O
— Solution 6: Na2MoO4 2H2O 44.0 mg/L H2O
— Solution 7: MnCl2 4H2O 180.0 mg/L H2O
— Solution 8: FeCl3 6H2O 760.0 mg/L H2O
AEDT Na2 2H2O 1500.0 mg/L H2O

Add to 1000 mL of deionized sterile water:
Solution 1 20 mL
Solution 2 20 mL
Solution 3 20 mL
Solution 4 1 mL
Solution 5 1 mL
Solution 6 1 mL
Solution 7 1 mL
Solution 8 1 mL

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: not specified
- Light intensity and quality: 6500 Lux. The surface where the test is performed receives uniform fluorescent lighting and continuous (white-cold type).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: [manual counting/computerized counting] not specified
- Determination of biomass: [dry weight / fresh weight] not specified

Reference substance (positive control):
Key result
7 d
Dose descriptor:
Effect conc.:
1 099 mg/L
Conc. based on:
act. ingr.
Basis for effect:
growth rate
7 d
Dose descriptor:
Effect conc.:
102 mg/L
Conc. based on:
act. ingr.
Basis for effect:
Details on results:
The counting of fronds was done at the beginning and at the end of the trial. From the difference in the number of fronds at the beginning and at the end of the trial, the growth rates for the control and for each one of the concentrations are calculated (table 2 and table 3).
The weight of the fronds is determined at the beginning and at the end of the trial. The initial weight is calculated from the average of 6 repetitions of weighing 12 fronds. Table 4 shows the weights obtained for each of the test concentrations and the% inhibition of the biomass (yield)

The validation criterion considered for this study is:
- The average number of fronds in the control must present a growth rate specific medium of at least 0.275 per day.
- Duplication time of approximately 2.5 days, and multiplication by 7 of the number of fronds, at the end of the study

Results with reference substance (positive control):
The result of the periodic verification of the response of the test organisms to the positivo control is EC50 7d = 2.9 mg/L (validation criterion: the EC50 7d value of 3,5-diclofenol is in the range 2.2 - 3.8 mg/L)

Table 1. pH value thorough the test

  initial pH (day 0) final pH (day 7)
 control 5.70 6.80
 minimum concentration 6.64 6.28
 maximum concentration 6.54 6.61



Table 2. Growth rates

                       concentration (mg/L)
  control 0.99 3.16 9.86 31.6 98.6 315.5 985.9
 growth rate 0.329 0.320 0.301 0.281 0.244 0.213 0.189 0.171



Table 3. % of inhibition of growth rate

 concentration (mg/L) % inhibition of growth rate
 control 0
 0.00 2.6
 3.16 8.4
 9.86 14.7
 31.6 24.7
 98.6 35.2
 315.5 42.6
 985.9 48.0



Table 4. Biomass and % of inhibition of Biomass

 conc (mg/L) inicial biomass (day 0) (mg) final biomass (day 7) (mg) difference in biomass (Bf-Bi) (mg) % inhibition of biomass
 control 2.0 14.3 12.3 0
 0.99 2.0 13.6 11.6 5.7
 3.162.0  12.9 10.9 11.4
 9.86 2.0 11.4 9.4 23.6
 31.6 2.0 9.5 7.5 39.0
 98.6 2.0 8.7 6.7 45.5
 315.5 2.0 6.3 4.3 65.0
 985.9 2.0 5.2 3.2 74.0
Validity criteria fulfilled:
The test item has a value of EC50 (7d ) of inhibition of the growth rate of Lemna minor equal to 1099 mg / L (act.ingr.), and a value of EC50 (7d) of inhibition of the biomass (yield) equal to 102 mg/L (act.ingr.)
Executive summary:

The study of acute toxicity to aquatic plants of the substance was carried out.

For the determination of the inhibition of algal growth, the freshwater aquatic plant Lemna minor has been used and following the operative method described in the OECD Guideline 221 (2006).

The freshwater aquatic plant Lemna minor has been subject to the substance at the test concentrations:

1; 3.2; 10; 32; 100; 320 y 100. The total duration of the trial has been 7 days.

The result obtained in this test indicates that the tested substance has an inhibition value of the growth rate EC50 (7d) equal to 1099 mg / L (act.ingr.) after 7 days of testing and an inhibition value of the biomass (yield) EC50 (7d) equal to 102 mg/L (act. ingr.)

Description of key information

There is one study conducted on the substance Direct Black RBK (CAS 2196165 -14 -5) according to the OECD Guideline 221 to determine the toxicity to aquatic plants Lemna minor. Based on the ECHA guidances, this test method is considered more suitable for testing toxicity of coloured test materials to aquatic plants, than the OECD guideline 201 to test the toxicity to aquatic algae.

The EC50 (7d) value 1099 mg/L active ingr. obtained in this study, shows that the tested substance is not harmful to this type of aquatic plant.

Key value for chemical safety assessment

EC50 for freshwater plants:
1 099 mg/L

Additional information