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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:
8 May 2012 - 14 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the 10, 18, 32, 56 and 100 mg/L test groups (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Only samples at the No Observed Effect Concentration and above were analysed.
Vehicle:
no
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 (mean and range, SD): 1st instar. 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.
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding during test: No.

ACCLIMATION
- Acclimation conditions: Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: A mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Feeding frequency: Daily.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Approximate theoretical harness of 250 mg/L as CaCO3 (reconstituted water)
Test temperature:
22 ± 1 °C
pH:
7.7 - 8.0
Dissolved oxygen:
8.3 - 9.3 mgO2/L

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test.
Nominal and measured concentrations:
Nominal concentrations: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
RANGE-FINDING TEST
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. The test item was dissolved directly in reconstituted water.

An amount of test item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/L test concentration from which serial dilutions were prepared in reconstituted water to give the remainder of the test concentrations.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

- Experimental Preparation
For the purpose of the definitive test the test item was dissolved directly in reconstituted water, prepared as follows:
Stock Solutions
CaCl2.2H2O 11.76 g/L
MgSO4.7H2O 4.93 g/L
NaHCO3 2.59 g/L
KCl 0.23 g/L
Preparation
An aliquot (25 mL) of each of the stock solutions was added to each litre (final volume) of deionized 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.


An amount of test item (500 mg) was dissolved in reconstituted water and the volume adjusted to 5 litres to give the 100 mg/L test concentration. Aliquots (100, 180, 320 and 560 mL) of the 100 mg/L test concentration were each separately diluted in a final volume of 1 litre of reconstituted water to give the 10, 18, 32 and 56 mg/L test concentrations respectively.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.


Exposure Conditions
In the definitive test, 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 - 23 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 764 to 787 lux. 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. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

EVALUATION OF DATA
An estimate of the EC50 value at 24 hours was given by inspection of the immobilisation data.

The EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood logit method (Finney, 1971) using the ToxCalc computer software package (ToxCalc, 1999).

Logit analysis is used where two or more partial responses to exposure are shown.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
70 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % Confidence Limits 45 - 170 mg/L
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilisation was observed at the test concentrations of 1.0 and 10 mg/L. However, immobilisation was observed at 100 mg/L.

Chemical analysis of the test preparations at 0 and 48 hours showed measured concentrations to range from 103 % to 108 % of nominal, indicating that the test item was stable under test conditions.

DEFINITIVE TEST
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the logit method (Finney, 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time EC50 95 % Confidence limits
(h) (mg/L) (mg/L)
24 >100 -
48 70 45 - 170
The No Observed Effect Concentration (NOEC) after 24 and 48 hours exposure was 32 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 56 mg/L.

A single immobilised daphnid was observed in the 10 mg/L test group after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that less than 10 % immobilisation was observed, no further immobilisation was observed in the test group and no immobilisation was observed at the higher test concentrations of 18 and 32 mg/L.


Observations on Test Item Solubility
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 97 % 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):
A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilisation data by the geometric mean method 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:

Time EC50 95 % Confidence limits
(h) (mg/L) (mg/L)
24 1.3 1.0 - 1.8
48 1.1 1.0 - 1.3

The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.56 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table 1 Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration

(mg/L)

Cumulative Immobilised Daphnia

(Initial population: 10 per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

0

10

0

0

100

3

10

 

Table 2 Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration

(mg/L)

Cumulative Immobilised Daphnia (Initial population: 5 per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

10

1*

0

0

0

1*

5*

1*

0

0

0

1*

5*

18

0

0

0

0

0

0

0

0

0

0

0

0

32

0

0

0

0

0

0

0

0

0

0

0

0

56

0

0

2

3

5

25

0

2

3

3

8

40

100

3

3

3

1

10

50

4

5

5

1

15

75

R1 - R4 = Replicates 1 to 4

*Considered to be due to natural causes rather than toxic effect given that less than 10 % immobilisation was observed, no further immobilisation was observed in the test group and no immobilisation observed at the higher test concentrations of 18 and 32 mg/L.

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 70 mg/L with 95 % confidence limits of 45 - 170 mg/L. The LOEC was considered to be 56 mg/L. The NOEC at 48 hours was 32 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna in accordance with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" 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 22 ± 1 °C under static test conditions. The number of immobilised Daphnia was recorded after 24 and 48 hours.

 

The 48 -hour EC50 for the test item to Daphnia magna based on nominal test concentrations was 70 mg/L with 95 % confidence limits of 45 - 170 mg/L. The Lowest Observed Effect Concentration was considered to be 56 mg/L. The No Observed Effect Concentration was 32 mg/L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 97 % to 112 % of nominal value and so the results are based on nominal test concentrations only.

Description of key information

48 hour EC50 to Daphnia magna = 70 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
70 mg/L

Additional information

A study was performed to assess the acute toxicity of the substance to Daphnia magna in accordance with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" 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 22 ± 1 °C under static test conditions. The number of immobilised Daphnia was recorded after 24 and 48 hours.

 

The 48 hour EC50 for the substance to Daphnia magna based on nominal test concentrations was 70 mg/L with 95 % confidence limits of 45 - 170 mg/L. The Lowest Observed Effect Concentration was considered to be 56 mg/L. The No Observed Effect Concentration was 32 mg/L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 97 % to 112 % of nominal value and so the results are based on nominal test concentrations only.

The study was conclusive and conducted under GLP conditions. As such the study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable as an accurate reflection of the test material.