Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01/12/2008 to 19/02/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No further information required.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. Standard solutions of test material were prepared in water at a nominal concentration of 5.0 mg/l.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.
The test material concentration in the test samples was determined spectrophotometrically using an external standard.

- Sampling method:
Water samples were taken from the control and each test group at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary. Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

- Sample storage conditions before analysis:
Room temperature in the dark
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

- Sample preparation for analytical method
A volume of test sample was diluted with water to giave a final theoretical concentration of 1.0 mg/l to 5.6 mg/l.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
The test material was dissolved directly in water. An amount of test material (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg/l test concentration (definitive test).

- Controls:
The control group was maintained under identical conditions as the test material but not exposed to the test material.

- Evidence of undissolved material:
The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna
- Source: Derived from in-house laboratory cultures.
- Age at study initiation: Young daphnids less than 24 hours old
- Method of breeding: 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. These young were removed from the cultures and used for testing.
- Feeding during test: The daphnids received no food during exposure

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Kept in a temperature controlled room at approximately 20°C
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI
Dissolved oxygen:
Dissolved oxygen concentration was approximately air-saturation value.
ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation.
The %ASV was kept in the range 96-98%.
Salinity:
nda
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. A stock solution of 100 mg/l was prepared from which the test concentrations were made.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 ml gass jars containing 200 ml of test media.
- Aeration: The test vessels were not aerated. Test vessels were covered to reduce evaporation.
- Renewal rate of test solution: The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution 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:
a) CaCI2.2H20 11.76 g/l
b) MgSO4.7H20 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
An aliquot (25 ml) of each of solutions a-d was added to each litre of deionised water.

- Conductivity: <5 µS cm-1

- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Water samples were taken from the control and each test group at 0 and 48 hours for quantitative analysis.

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED
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.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test material was dissolved directly in water.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 0.10 and 1.0 mg/l. However, immobilisation was observed at 10 and 100 mg/l.
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in the following table:
Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
24 > 100 -
48 > 100 -

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Given the difference in immobilisation observed between the first range-finding test and the definitive test, a second range-finding test was conducted in order to confirm the results of the definitive test. No immobilisation was observed at any of the concentrations tested. Based on this information, the immobilisation observed in the first range-finding test was considered to be due to natural causes and not toxicity associated with the test material.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Details on results:
Observations on test material solubility:

The test media preparations were observed to graduating from pale blue to dark blue clear solutions throughout the duration of the test.

Physico-chemical measurements:

The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Immobilisation data:

There was no significant immobilisation in 20 daphnids exposed to a test concentration of 100 mg/I for a period of 48 hours.
The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/I. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/I.
The immobilisation observed at the test concentrations of 1.0, 5.6, 10 and 32 mg/I was considered to be due to natural causes rather than a toxic effect of the test material as no concentration-dependent pattern in toxicity was observed and that no immobilisation occurred at the highest two test concentrations.

Verification of test concentrations:

Analysis of the test preparations at 0 and 48 hours (see Appendix 3) showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Second Range-finding Test:

Given the difference in immobilisation observed between the first range-finding test and the definitive test, a second range-finding test was conducted in order to confirm the results of the definitive test. No immobilisation was observed at any of the concentrations tested.

Based on this information, the immobilisation observed in the first range-finding test was considered to be due to natural causes and not toxicity associated with the test material.
Results with reference substance (positive control):
- Mortality of control: The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l.

Cumulative Immobilisation Data in the Positive Control:

Nominal Concentration (mg/l) Cumulative Immobilised Daphnia (Initial population: 10 per replicate)
3 Hours 24 Hours 48 Hours
R1 R2 Total % R1 R2 Total % R1 R2 Total %
Control 0 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
0.56 0 0 0 0 1 1 2 10 2 2 4 20
1.0 0 0 0 0 7 8 15 75 9 9 18 90
1.8 0 0 0 0 10 10 20 100 10 10 20 100
3.2 0 0 0 0 10 10 20 100 10 10 20 100

R1 - R2 = Replicates 1 and 2
Reported statistics and error estimates:
Evaluation of data for the positive control:
An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 value and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

Any other information on results incl. tables

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Cumulative Immobilisation Data in the First Range-finding Test:

Nominal

Concentration

(mg/l)

Cumulative Immobilised Daphnia

(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

4

100

0

10

Cumulative Immobilisation Data in the Second 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

0

0

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 % R2 R2 Total %
Control 0 0 0 0 0 0 0 0
1 0 1 1 10 0 1 1 10
1.8 0 0 0 0 0 0 0 0
3.2 0 0 0 0 0 0 0 0
5.6 0 1 1 10 1 1 2 20
10 0 0 0 0 0 1 1 10
18 0 0 0 0 0 0 0 0
32 0 0 0 0 1 0 1 10
56 0 0 0 0 0 0 0 0
100 0 0 0 0 0 0 0 0

R1 & R2 = Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in test medium. A method of analysis was validated and proven to be suitable for use.
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.
Executive summary:

In an Acute Toxicity to Daphnia Magna study (Harlan project number: 0959/0231) the test material was found to have an EC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in 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.

The immobilisation observed at the test concentrations of 1.0, 5.6, 10 and 32 mg/l was considered to be due to natural causes rather than a toxic effect of the test material as no concentration-dependent pattern in toxicity was observed and that no immobilisation occurred at the highest two test concentrations.