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Diss Factsheets

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:
27 March 2007 to 27 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/l

- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l test groups (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.
The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.

- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20 degC for further analysis if necessary.

Test solutions

Vehicle:
yes
Details on test solutions:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.
An amount (100 mg) of test material was dissolved directly in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 deg 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. Each culture was fed daily with a suspension of algae (Chlorella sp.). 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
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.

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21.2 degC to 21.6 degC
pH:
8.0 – 8.1
There was no treatment related differences for pH.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test.
Results: 8.6 mg O2/l
Salinity:
Not data.
Nominal and measured concentrations:
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.
Nominal concentration = 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: glass jars
- Material, size, headspace, fill volume: 250 ml glass jars
- Aeration: Not aerated. Covered to reduce evaporation.
- Renewal rate of test solution (frequency/flow rate):The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4



TEST MEDIUM / WATER PARAMETERS
- Source: Reconstituted Water.


i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d 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.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.





EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

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.

An estimate of the EC50 values was given by inspection of the immobilisation data.


TEST CONCENTRATIONS

- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.1, 1.0, 10, and 100 mg/l.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/l. Based on this information, a single test concentration of four replicates, of 100 mg/l was selected for the definitive test.
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
Remarks on result:
other: -
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: -
Details on results:
- Behavioural abnormalities: None noted
- Observations on body length and weight: No data
- Other biological observations: No data
- Abnormal responses: None observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed

Throughout the duration of the test the test concentrations were observed to be clear, colourless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50/LC50: EC50 at 48hrs = 0.85 mg/l
- Other: 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 0.82 mg/l (sd = 0.22).
Reported statistics and error estimates:
Positive control: The slope and standard error of the response curve at 24 hours was 9.2 (SE = 2.1). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.

Any other information on results incl. tables

Cumulative immobilisation data from the exposure ofDaphnia magnato the test material during the definitive test are given in Table below:

Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

 


R1– R4= Replicates 1 to 4

.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results: EC50 (48hrs) = >100 mg/l.

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. 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/l.
Executive summary:

Introduction. 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 Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

Methods. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of 21.2°C to 21.6°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 20.0°C to 21.1°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results. The 48-Hour EC50for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so the results are based on nominal test concentrations only.

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

The 48-Hour EC50for the reference material to Daphnia magna based on nominal concentrations was 0.85 mg/l with 95% confidence limits of 0.76 – 0.96 mg/l. The No Observed Effect Concentration was 0.56 mg/l.