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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th March 1993 to 4th October 1993
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
Purity: 97.3%
Batch no.: 9236-23

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: Each test medium has been twice sampled (at the start and at the end of the study). One supplementary test flask, at the highest tested concentration but without algae has been run under the same experimental conditions, and sampled the same way.
- Sampling method: Not specified
- Sample storage conditions before analysis: The samples were frozen and transmitted frozen at the end of the test to PHARMAKON EUROPE for analytical determination of the test substance concentration levels.

Test solutions

Details on test solutions:
- Method: Owing to the very low test substance solubility in water (0.0478 mg/L), and in order to prevent any test system perturbations by test substance particles (light quantity, gas exchanges, ...), an extract (saturated solution) of test substance was prepared and particles were removed by filtration :
• dilution water was the same as culture medium used for the study
• a test substance stock suspension (1,000 mg/L) was prepared into dilution water 2 hours before the
start of the study
• this stock solution was submitted to magnetic stirring (1 hour 30 minutes)
• it was then coarsely filtered through paper filter, and the filtrate was used for the preparation of the
test media
• pH of the extract was not readjusted.
To obtain 25, 50, 75, 90 and 100% of test substance extract in test medium, appropriate volumes of saturated solution were mixed into dilution water, to give final volumes of 200 mL per concentration. These volumes were then distributed to give 50 mL test medium in each flask, and.the remaining
volumes (50 mL) were used for analytical sampling.
- Controls: A control group (i.e. without exposure to any substance) was tested under the same conditions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The stock solution used for the preparation of the extract, was recorded to be an heterogeneous suspension. The test substance particles were recorded to adsorb onto glass surfaces, and to float (immediately going up to the water surface when stirring stops). The saturated solution and the test media at the start of the study was a limpid colourless liquid (naked eye observation).

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Scenedesmus subspicatus
- Strain: CHODAT 86-81 SAG
- Source (laboratory, culture collection): Aquarium De Trouville (17 rue de Paris - F - 14360 Trouville)
- Age of inoculum (at test initiation): approximately 72 hours old algal culture
- Method of cultivation: Axenic subcultures were made at regular intervals.

- Acclimation period: Sub-culture was made approximately 72 hours before the study in the same culture medium as that used for the study, and kept under the same conditions as those employed for the study (culture medium, lighting, temperature, stirring).
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: Not specified

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
Not performed

Test conditions

40 mg CaCO3/L
Test temperature:
The test and control flasks were maintained at the room temperature range from 20.7°C to 22.1°C.
pH: 7.06 to 7.86 during the study period
Dissolved oxygen:
Not specified
Not determined
Nominal and measured concentrations:
Nominal concentrations: 25, 50, 75, 90 and 100% (Volume by Volume) test substance extract in test medium
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): Closed - the flasks were provided with a carded cotton plug
- Material, size, headspace, fill volume: The containers used for the pre-culture and study were Erlenmeyer flasks of 250 mL capacity,
provided with a carded cotton plug. The volume of culture medium used for the pre-culture and study was 50 mL per flask.
- Aeration: Not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable - static test system
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: Nominal cell concentrations at the start of the study: 0.99176E+04 cells per millilitre of control and test medium.
- Control end cells density: After 72 hours, the mean cell concentration for the control was 6.63E+05 cells/mL.
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 3 vessels per concentration
- No. of vessels per control (replicates): 6 vessels
- No. of vessels per vehicle control (replicates): Not applicable

- Standard medium used: Not specified
- Detailed composition if non-standard medium was used:
Ca(NO3)2.4H2O 40 mg/L
KNO3 100
MgSO4.7H2O 30
K2HP04 40
CuSO4.5 H2O 0.015
(NH4)6 Mo7O24.4H2O 0.030
ZnSO4.7H2O 0.030
CoCI2.6 H2O 0.030
Mn(NO3)2.4H2O 0.030
C6H8O7.H2O 0.030
H3BO3 0.030
Ammonium iron III citrate (Fe2O3 content: 29 - 32%) 0.8125
FeSO4.7H2O 0.3125
FeCl3.6H20 0.3125

- Source/preparation of dilution water: Not specified
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: The pH of the culture and test medium was 7.2 and was readjusted with HCI 0.1N.
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH measurements were performed in total volumes of control and test media related to each concentration at the start of the study, and then approximately 72 hours later.

- Sterile test conditions: Yes - Axenic algal culture was used for pre-culture inoculation, and steril culture medium and dilution water were used (pre-culture and study). All the test flasks had been cleaned and autoclaved (with plug) for the study.
- Adjustment of pH: No
- Photoperiod: Continous white lighting
- Light intensity and quality: 6,100 lux to 6,250 lux
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 24, 48 and 72 hours after the start of the study, the cell concentration was recorded for each control and test flask by direct counting of cells with a microscope.
- Chlorophyll measurement: Not specified

- Spacing factor for test concentrations: Not specified
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: This study is a range-finding study and was performed using the concentrations of 25, 50, 75, 90 and 100% (v/v) of test substance extract in the test medium. No mortality was recorded during the 72 hour study period, until 100% of test substance extract was used. The range-finding study was then considered as a limit test, and no EC50 evaluation was performed.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
No significant inhibition was recorded until 100% (Volume by Volume) test substance extract in test medium was used during the study period (72 hours). The EC(I)50 after 72 hours, for unicellular green algae, in freshwater medium is therefore higher than the test substance limit of water-solubility (0.0478 mg/L).

- Exponential growth in the control (for algal test): Yes - the cell concentration in the control group increased by a factor of at least 16 within 3 days (it increased by of a factor of 66 during the study period).
- Observation of abnormalities (for algal test): No abnormalities were recorded
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: The water solubility of the test substance is 0.0478 mg/L. No effects were seen up to the highest dose tested; hence, the effect concentration must exceed the limit of solubility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data available

Any other information on results incl. tables

No significant cell growth inhibition was recorded during the 72-hour study period, until the concentration of 100% of test substance extract was used. Thus, no determination of the EC(I)50 after 72 hours was performed.

The test samples were analysed by HPLC. The measured concentrations of test material were <0.04 mg/L i.e. below the limit of solubility in water.

Applicant's summary and conclusion

Validity criteria fulfilled:
The EC(I)50 after 72 hours, for unicellular gren algae, in freshwater medium is higher that the test substance limit of water solubility (i.e. >0.04 mg/L).
Executive summary:

The study was performed to evaluate the acute toxicity of Millad 3988 to freshwater unicellular algae after 72 h exposure and was performed in accordance to OECD Guideline No. 201.

No significant inhibition, by comparison with the control cultures, was recorded at the end of the 72 hour study period, until 100% of the test substance saturated solution was used (measured initial concentration: 0.041 mg of test substance per litre of test medium) i.e. the maximum possible concentration of exposure. Therefore, the EC(I)50 after 72 hours, for unicellular gren algae, in freshwater medium is higher that the test substance limit of water solubility (i.e. >0.04 mg/L).