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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29th June 1993 to 1st July 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 guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
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: The test sample containing 100% v/v of the test substance extract and three samples of the frozen stock solutions (nominal test substance concentration 1000 mg/L) were analysed.
- Sampling method: Each test medium has been twice sampled; at the start and at the end of the study.
- Sample storage conditions before analysis: The samples were immediately frozen and transmitted frozen at the end of the experimental period to PHARMAKON EUROPE for analytical determination of the test substance concentration levels.

Test solutions

Details on test solutions:
Identity and concentration of auxiliary solvent for dispersal: No auxilary substance was used.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Owing to the test substance low water solubility (0.0478 mg/L), and in order to prevent test system perturbation by solid test substance particles (trapping Daphnia, preventing water oxygenation, ...), the study was performed using an extract of test substance. It was prepared as follows :
• dilution water: similar as that used for the study
• a test substance stock suspension (1000 mg/L) was prepared in dilution water
• this stock solution was submitted to magnetic stirring for two hours
• it was then coarsly filtered through paper filter, and the filtrate was used for the preparation of the test media
• pH of the extract was not adjusted.
To obtain the concentrations of 25, 50, 75, 90 and 100% test substance extract in test medium, appropriate volumes of test substance extract were mixed into dilution water to give final volumes of 80 mL per concentration.
These volumes were then distributed to give 10 mL test medium in each test tube and the remaining volumes were used for sampling.
- Controls: The control vessels were treated in an identical manner to the test samples but were not exposed to the test substance.
- Chemical name of vehicle: Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No abnormality was recorded in the test media.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia
- Strain: Daphnia magna
- Source: Société d'Elevage Piscicole Contrôlé (La Grande Rivière - F - 69490 Saint-Forgeux)
- Age at study initiation (mean and range, SD): Between 8 hours and 23 hours
- Method of breeding: Not specified
- Feeding during test: No food was provided during the study.

- Acclimation period: last 7 days of the breeding period.
- Acclimation conditions: Same as test. Containers: covered glass vessels of capacity ranging from 2 to 8 litres; Volume of dilution water used for breeding: varying between 1 to 5 litres, non-aerated; pH of breeding water remained between 7.87 and 8.18; Temperature of breeding water remained between 20.2°C and 21.0°C; Lighting: artificial, 12 hours out of 24 (photoperiod = 7h30 - 19h30).
- Type and amount of food: Algal suspensions of Scenedesmus subspicatus, cultivated in the laboratory in L.C. Oligo medium; Amount not specified
- Feeding frequency: Not specified
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught):
Not applicable

Study design

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

Test conditions

Measured total hardness: 250 to 370 mg of CaCO3 per litre.
Test temperature:
Temperature of the test media: 20.4°C - 20.7°C
pH of the test media: 7.78 - 8.10
Dissolved oxygen:
Dissolved oxygen concentration in test media: not lower than 7.9 mg per litre at the end of the study.
Not specified
Nominal and measured concentrations:
Daphnia were exposed to the test substance at theoretical concentrations of 25, 50, 75, 90 and 100% (Volume by Volume) of test substance extract in the test medium.
Details on test conditions:
- Test vessel: Glass test tubes graduated to 10 mL.
- Type (delete if not applicable): Not specified
- Material, size, headspace, fill volume: Volume of dilution water used for the study: 40 mL per concentration and for the control
- Aeration: Not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable - static study
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5 Daphnia per vessel
- No. of vessels per concentration (replicates): 4 vessels per concentration (20 daphnia per concentration tested)
- No. of vessels per control (replicates): 4 vessels
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: Not specified

- Source/preparation of dilution water: 1 litre of dilution water contained 200 mL distilled water and 800 mL natural water (Volvic).
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: pH 8.03 at 21.1°C
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: No
- Test medium: A mixture of dilution water with the test substance extract at selected concentrations (apart from the control) to form a final volume of 10 mL per tube. The loading rate was 2 mL of test medium per organism.
- Intervals of water quality measurement: Measurements of pH, dissolved oxygen and temperature were performed in total volumes of test media related to each concentration and the control, at the start of the study, and then approximately 48 hours later.

- Adjustment of pH: No
- Photoperiod: The study was conducted in darkness.
- Light intensity: Not applicable

24 and 48 hours after the start of the study, the number of immobilized Daphnia was noted. Those organisms incapable of moving during a 15 second period following slight agitation of the container were considered as immobilized.
Observation of the test media was performed during the study; test medium abnormalities (insolubility marks as films, deposits, ...), if any, were recorded.
For each study group, the number of Daphnia at the start of the study and the cumulated number and percentage of immobilization at each observation period were recorded.

- 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 immobilisation was recorded during these 48 hour study period, until 100% of test substance extract. The range-finding study was then considered as a limit test, and no LC(I)50 evaluation was performed.
Reference substance (positive control):

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
> 0.04 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: EC50 is higher than the limit of solubility in water
Details on results:
No immobilization was recorded during the 48 hour study period, until the concentration of 100% of the test substance extract was reached. Thus, no determination of EC50 was performed.

- Behavioural abnormalities:
- Observations on body length and weight:
- Other biological observations:
- Mortality of control: No immobilization of Daphnia was observed in the control group.
- Other adverse effects control: No Daphnia were trapped at the surface of the water.
- Abnormal responses:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data available

Any other information on results incl. tables

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:
No immobilisation was recorded in the experimental conditions during the 48 hour study period. The EC50 is higher than the limit of solubility.
Executive summary:

Daphnia magna were exposed to a test substance extract of Millad 3988 for 48 h in accordance with OECD Guideline No. 202 and EEC Directive 84/449 Annex V Method C2. No immobilisation was recorded in the experimental conditions during the 48-hour study period, until 100% of the test substance extract in test medium was used (containing less than 0.04 mg of test substance per litre of test medium as determined by analysis) which is the maximum possible concentration of exposure.The 48 h EC50 for Daphnia in test medium is therefore higher than the limit of solubility (i.e. >0.04 mg/L).