C-C1

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:

Definitive test between 23 April 2007 and 25 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Remarks:

Date of inspection: 30th August 2005 Date of signature: 21st November 2005

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification :C-C1
Description : dark blue powder
Date received : 5 March 2007
Lot Number : MB-1
Storage conditions :room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Standard solutions of test material were prepared in water* at a nominal concentration of 100 mg/l.

- Sampling method: 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.
The method was based on a procedure supplied by the Sponsor.


- Sample storage conditions before analysis: -20°C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.
An amount (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the test concentration 100 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- EluatStock 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.


- Differential loading: not applicable

- Controls: A (negative) control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable

- Concentration of vehicle in test medium (stock solution and final test solution): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): 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: Water flea
- Strain: Not applicable
- Source: Derived from in-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24 hours
- Weight at study initiation (mean and range, SD): not determined
- Length at study initiation (length definition, mean, range and SD): not determined
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: Parthenogenesis
- Feeding during test Not fed during test

ACCLIMATION
- Not applicable

QUARANTINE (wild caught)
- not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Reconstituted water had a hardness of 250 mg CaCO3/L

Test temperature:

Recorded daily throughout test using a Hanna Instruments HI 93510 digital thermometer.
Maintained at 19.3°C to 21.6°C throughout the test.

pH:

Recorded at start and termination of test using WTW pH/Oxi 340I pH and dissolved oxygen meter.
pH was recorded as 8.0 for the test solution

Dissolved oxygen:

Recorded at start and termination of test using WTW pH/Oxi 340I pH and dissolved oxygen meter
Ranged from 8.6 to 9.0 mg O2/l

Salinity:

Not applicable as freshwater study

Nominal and measured concentrations:

Nominal concentration of 100 mg/l in defintive study

Details on test conditions:

TEST SYSTEM
- Test vessel: 250ml glass jars
- Type (delete if not applicable): closed - covered to reduce evaporation
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 200 ml of test preparation
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5 Daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: not determined

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water

- Intervals of water quality measurement: 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.

- Eluate 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.

OTHER TEST CONDITIONS
- Adjustment of pH: not conducted during the study
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: not stated in report

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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, as 100 mg/l only concentration

- Justification for using less concentrations than requested by guideline: Based on results of range-finding study

- Range finding study:
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l

- Results used to determine the conditions for the definitive study: No iimobilisation observed at any of the test concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

- Behavioural abnormalities: none

- Observations on body length and weight: none. Immobilisation only observed.

- Other biological observations: none

- Mortality of control: immobilisation only observed. Mortality not assessed.

- Other adverse effects control: none stated in report

- Abnormal responses: none stated in report

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- Mortality: Not applicable

- EC50/LC50: The mean 48-Hour EC50 value calculated from all positive controls was 0.85 mg/l (sd = 0.22).

- Other: The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
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.

Reported statistics and error estimates:

None

Any other information on results incl. tables

Verification of test concentrations

Analysis of the test preparations at 0 and 48 hours 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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

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:

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 19.3°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 EC₅₀ for 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 EC₅₀ for 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.