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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-06 - 2012-04-22
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Temperature, oxygen and pH ranges outside of recommended parameters
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Water samples were taken from the control and each test group (replicates R1-R4 pooled) at 0 and 48 h for quantitative analysis.
- Duplicate samples were taken.
- All samples were stored at approximately -20 °C prior to analysis.
Details on test solutions:
- During the range finding test (1.0, 10 and 100 mg/L loading rates), test solutions were stirred for 23 h after addition of test item (preparation of WAFs, Water Accomodated Fractions). In the 100 mg/L loading rate a significant amount of dispersed test item was observed and therefore the test solutions were filtered over a glass wool plug. However, analytical measurement of the test concentrations indicated that the test item was soluble and therefore the test item was dissolved directly in the test medium for the purpose of the definitve test.
- The 100 mg/L test concentrations was prepared directly by adding 500 mg of the test substance 5 L of test medium.
- The other test concentrations for the definitve test were prepared by dilution (not sequential) of the 100 mg/L solution.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Strain: 1st instar Daphnia magna
- Source: in-house laboratory culture
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: parthenogenesis
- Feeding during test: no

- Acclimation period: no specific acclimation period
- Acclimation conditions (same as test or not): same as test
- Feeding during culturing: daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
250 mg CaCO3/L
Test temperature:
19-22 degrees C (> 1°C deviation during test, however, this was not considered to affect integrity of test results, moreover, the deviation in individual test vessels was never > 1°C)
6-9 (pH of 18 and 100 mg/L test solution at 0 h was above the recommended range of 6-9, however, this was not considered to affect the integrity of the test results given that immobilisation was also observed at the test concentration of 10 mg/L which showed a pH within the recommended range)
Dissolved oxygen:
ASV > 100% (most likely due to presence of microscopic air bubbles in the test media, this was not considered to affect integrity of test results)
Nominal and measured concentrations:
Nominal concentrations: control, 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
Measured concentrations ranged from 84-107% of nominal
Details on test conditions:
- Test vessel: jars
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: glass, 250 mL, 200 mL fill volume
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: deionised reverse osmosis water
- Test medium: reconstituted water - Elendt M7 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: batch
- During the test, water temperature and light intensity were recorded daily. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 632-663 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobility, 24 and 48 h

- Spacing factor for test concentrations:~2
- Range finding study: yes
- Test concentrations during range finding study: 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
7.5 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CI = 5.6-10 mg/L
Details on results:
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- 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: none
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference item.
48-h EC50 = 1.1 mg/L (95% CI = 1.0-1.3 mg/L)
Reported statistics and error estimates:
48-EC50 value and 95% CI calculated using the geometric mean method (no partial mortality).

The Lowest Observed Effect Concentration (LOEC) was considered to be 10 mg/L. The No Observed Effect Concentration (NOEC) was 5.6 mg/L.

Validity criteria fulfilled:
The 48-h EC50 of the test substance for Daphnia magna was 7.5 mg/L with 95% confidence limits of 5.6 - 10 mg/L. Analytical verification of test substance concentrations showed measured concentrations to be close to nominal. The results of this test can therefore be considered reliable.

Description of key information

In this study, the acute toxicity of the test substance to Daphnia magna was investigated in a 48-h static study with concentrations between 0 and 100mg/L, resulting in a 48-h EC50 of 7.5 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
7.5 mg/L

Additional information

The acute study of Goodband and Mullee (2012) was used as a key study for PNEC derivation. The 48-h test with Daphnia magna was conducted according to OECD guideline 202. The test concentrations were were 1, 1.8, 3.2, 5.6, 10, 18, 32, 56, 100 mg/L and were within 84 -107% of the nominal range. A 48-h EC50 of 7.5 mg/L for Daphnia magna was obtained in this study. This indicates that the test substance is toxic to aquatic invertebrates.