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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:
16 March 2010 and 15 April 2010
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding test: An amount of test item (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 25 minutes and the volume adjusted to 500 ml to give the 100 mg/l test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/l. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Definitive test: An amount of test item (200 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 2 litres to give the 100 mg/l test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 25, 32, 18 and 10 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
- Common name: Water flea
- Source: Derived from in house laboratory cultures.
- Age at study initiation: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: No food during exposure

ACCLIMATION
- Acclimation period: Not stated
- Acclimation conditions: Adult Daphnia were maintained in polypropylene vessels containing approximatley 2 litres of reconstituted water in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: Each culture was fed daily with a suspension of algae (Chorella sp.). The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Health during acclimation: No mortality observed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
250 mg/l as CaCO3.
Test temperature:
20 - 21°C
pH:
0 hours: 7.9 - 8.0
48 hours: 7.9 - 8.0
Dissolved oxygen:
0 hours: 9.1 - 9.4 mg O2/l
48 hours: 8.3 - 9.0 mg O2/l
Nominal and measured concentrations:
Range-finding test: 0.10, 1.0, 10 and 100 mg/l
Definitive test: 10, 18, 32, 56 and 100 mg/l
Details on test conditions:
Range-finding test: In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20°C to 22°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded. A single temperature at the start of the test was observed to be in excess of the 20 ± 1°C stated in the Study Plan This was considered not to have affected the outcome or validity of the test as the temperature was within test guideline specification. The control group was maintained under identical conditions but not exposed to the test item.

Definitive test: For the purpose of the definitive test the test item was dissolved directly in water. As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item. The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Positive Control: A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l. An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l. Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity. Exposure conditions for the positive control were similar to those used in the definitive test. The temperature was maintained at approximately 20°C.

Reconstituted water: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals. Stock Solutions: 11.76 g/l CaCl2.2H2O, 4.93 g/l MgSO4.7H2O, 2.59 g/l NaHCO3, 0.23 g/l KCl. An aliquot (25 ml) of each of solutions was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL 21-29mg/l
Remarks:
The measured values were 80-112% of the nominal valuesverified by measured concentration (
Details on results:
- Range-finding Test: No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.
- Definitive Test: Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results: 24h-EC50: 28 mg/l (95% CI: 24-33 mg/l); 48h-EC50: 25 mg/l (95% CI: 21-29 mg/l). The slopes and their standard errors of the response curves at 24 and 48 hours were 6.4 (SE = 1.2) and 6.8 (SE = 1.3) respectively.
- All control and test concentrations were observed to remain clear colourless solutions throughout the duration of the test.
- Analysis of the test preparations at 0 and 48 hours (see Appendix 3) showed measured test concentrations to range from 80% to 112% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.
Results with reference substance (positive control):
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results: 24h-EC50: 0.84 mg/l (95% CI: 0.72-0.97 mg/l); 48h-EC50: 0.65 mg/l (95% CI: 0.58-0.72 mg/l). The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours. The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999). Probit analysis is used where two or more partial responses to exposure are shown. When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Table 2 Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

18

1

2

3

15

2

2

4

20

32

6

5

11

55

8

7

15

75

56

10

10

20

100

10

10

20

100

100

10

10

20

100

10

10

20

100

R1– R2= Replicates 1 and 2

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-hour EC50 value of 25 mg/l with 95% confidence limits of 21 - 29 mg/l, using nominal concentrations while the measured were 80-112% of the nominal values.
Executive summary:

The acute toxicity of Cyclacet towards Daphnia magna was investigated according to OECD Guideline 202 under GLP. Daphnids were exposed to nominal concentrations of 0 (control) 10, 18, 32, 56 and 100 mg/l under static conditions and observed for 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 112% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only. The 48h-EC50 was found to be 25 mg/l with 95% confidence limits of 21-29 mg/l.

Description of key information

The acute toxicity of Cyclacet towards Daphnia magna was investigated according to OECD Guideline 202 under GLP. Daphnids were exposed to nominal concentrations of 0 (control) 10, 18, 32, 56 and 100 mg/l under static conditions and observed for 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 112% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only. The 48h-EC50 was found to be 25 mg/l with 95% confidence limits of 21-29 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
25 mg/L

Additional information