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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July from 04 to 29 July, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours, for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/l. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: the test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age of porganisms: less than 24 hours old.
- Feeding during test: the daphnids received no food during exposure.

BREEDING
- Medium: adult daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium.
- Temperature: controlled room maintaining the water temperature at 18 to 22 °C.
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Feeding frequency: each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Reproduction and test organisms: culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
Temperature was maintained at 21 to 22 °C throughout the test.
Nominal and measured concentrations:
Nominal 10, 18, 32, 56 and 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 ml glass beakers containing approximately 100 ml of test preparation were used.
- Type: vessels were covered to reduce evaporation.
- Aeration: the test vessels were not aerated duting the test.
- No. of organisms per vessel: 5 daphnids were placed in each test and control vessel.
- No. of vessels per concentration: 4 replicates.
- No. of vessels per control: 4 replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO medium).
- Composition: CaCl2 x 2H2O 294 mg/l; MgSO4 x 7H2O 123 mg/l; NaHCO3 65 mg/l; KCl 5.8 mg/l.
- pH: 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
- Aeration: medium was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Hardness: the reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED
- Observations: any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.
- Water temperature: recorded daily throughout the test.
- Dissolved oxygen: recorded at the start and termination of the test.
- pH: recorded at the start and termination of the test.
- Light intensity: measured using an ATP Instrumentation Lux meter.

RANGE-FINDING STUDY
- Test solution: a nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 ml to give the 100 mg/l test concentration.
- Test concentrations:0.10, 1.0, 10 and 100 mg/l, nominal. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- No. of organisms per vessel: 10 daphnids were placed in each test and control vessel.
- Temperature: temperature at 18 to 22 °C with a maximum deviation of ±1 °C.
- Photoperiod: 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.
- Test vessels: each 150 ml test and control vessel contained 100 ml of test media and was covered to reduce evaporation.
- Measured concentration: a sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions.
- Results used to determine the conditions for the definitive study: no immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/l, however, immobilization was observed at 100 mg/l.

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- no more than 10 % of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water);
- the dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/l in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The slopes and their standard errors of the response curves at 24 and 48 hours were 6.2 (SE = 0.034) and 3.1 (SE = 0.051) respectively.
After 48 hours exposure the 18 mg/l test concentration exhibited a higher immobilization rate than the 32 mg/l test concentration. Statistical analysis using the Step-down Rao-Scott-Cochran-Armitage test procedure has determined that there is no statistically significant difference between this concentration and the control.

A sub-lethal effect of exposure was observed in both the 56 and 100 mg/l test concentrations. This response was reduced mobility.

MEASURED CONCENTRATIONS
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 108 to 114 % of nominal concentrations. There was no significant change in the measured test concentrations in the analysis of the testpreparations at 48 hours; so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations.
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

TEST CONDITIONS
Water Quality CriteriaTemperature was maintained at 21 to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 691 to 744 Lux.

RANGE FINDING TEST
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/l, however, immobilization was observed at 100 mg/l.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 96 to 103 % of nominal concentrations. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.
Results with reference substance (positive control):
EC50 (48h): 0.64 mg/l
No Observed Effect Concentration (24h): 0.56 mg/l

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/l) Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate) Observations
R1 R2 R3 R4 Total % R1 R2 R3 R4
24 hours
Control 0 0 0 0 0 0 5N 5N 5N 5N
10 0 0 0 0 0 0 5N 5N 5N 5N
18 0 0 0 0 0 0 5N 5N 5N 5N
32 0 0 0 0 0 0 5N 5N 5N 5N
56 3 1 2 3 9 45 1R 1N 2R 2N 3R 2N
100 5 5 3 4 17 85 A/I A/I 1R 1N 1N
48 hours
Control 0 0 0 0 0 0 5N 5N 5N 5N
10 0 0 0 0 0 0 5N 5N 5N 5N
18 0 3 2 1 6 30 5N 2N 3N 4N
32 0 0 0 0 0 0 5N 5N 5N 5N
56 5 3 4 4 16 80 A/I 1R 1N 1N 1N
100 5 5 3 4 17 85 A/I A/I 2R 1N

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

TEST CONDITIONS - main test

Nominal Concentration (mg/l) 0 hrs 24 hrs 48 hrs
pH mg O2/l °C °C pH mg O2/l °C
Control 7.2 7 21 22 7.7 8.5 22
10 7.1 8.1 22 22 7.8 8.5 22
18 7.3 7.8 22 22 7.8 8.5 22
32 7.4 8 22 22 7.8 8.4 22
56 7.4 8.3 22 22 7.8 8.4 22
100 7.2 8.3 22 22 7.8 8.4 22

RANGE FINDING

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration (mg/l) Observations (Initial Population: 10 Per Replicate)
24 hours 48 hours
Cumulative Immobilized Daphnia Observations Cumulative Immobilized Daphnia Observations
Control 0 10N 0 10N
0.1 0 10N 0 10N
1 1 9N 0* 10N
10 0 10N 0 10N
100 7 3R 9 1R+C

N = No sub-lethal effects observed

R = Reduced mobility

C = Covered in debris

*Observation shows no immobilized daphnia however, the 24 hour observations show 1 immobilized daphnia. Debris in the beaker was possibly identified incorrectly as an immobilized daphnid. This is considered not to have affected the study.

Validity criteria fulfilled:
yes
Remarks:
none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/l in the control and test vessels
Conclusions:
EC50 (48h): 44 mg/l (Linear Maximum-Likelihood regression, based on nominal conc.).
Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines 202, referenced as Method C.2 of Commission Regulation (EC) No. 440/2008. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of approximately 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 108 to 115 % of nominal concentration and so the results are based on nominal test concentrations.

After 48 hours exposure the 18 mg/l test concentration exhibited a higher immobilization rate than the 32 mg/l test concentration. Statistical analysis using the Step-down Rao-Scott-Cochran-Armitage test procedure has determined that there is no statistically significant difference between this concentration and the control.

A sub-lethal effect of exposure was observed in both the 56 and 100 mg/l test concentrations.

Conclusion

EC50 (48h): 44 mg/l (Linear Maximum-Likelihood regression, based on nominal conc.).

Description of key information

EC50 (48h): 44 mg/l (Linear Maximum-Likelihood regression, based on nominal conc.).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
44 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna, following the method and procedures as designed into the OECD Guidelines 202. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of approximately 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 108 to 115 % of nominal concentration and so the results are based on nominal test concentrations.

After 48 hours exposure the 18 mg/l test concentration exhibited a higher immobilization rate than the 32 mg/l test concentration. Statistical analysis using the Step-down Rao-Scott-Cochran-Armitage test procedure has determined that there is no statistically significant difference between this concentration and the control.

A sub-lethal effect of exposure was observed in both the 56 and 100 mg/l test concentrations. The EC50 (48h) was calculated to be 44 mg/l by Probit analysis using Linear Maximum-Likelihood regression nominal test concentrations.