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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:
07-08-2003 until 09-08-2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study, GLP. Analysis of the freshly prepared test concentrations at 0 and 24 hours showed the measured test concentrations to range from 81% to 107% of the nominal values. However, analysis of the old or expired test preparations sampled at 24 and 48 hours showed a marked decline in the measured test concentrations ranging from 49% to 105% of nominal. These results were in line with the preliminary stability analyses indicating that the test solutions were unstable under the test conditions employed. Although efforts were made to minimize losses due to the suspected volatile nature of the test material, such as using completely-filled, ground-glass, staggered, conical flasks as exposure vessels, losses may also have occurred by this route.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: verified by chemical analysis at 0 (fresh media) 24 (old and fresh media) and 48 hours (old media).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to weighing out the test material was heated to 30 °C to aid dissolution in the diluent.
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/L test concentration. Aliquots (10, 18, 32, 56, 100, 180, 320 and 560 mL) were each separately dispersed in reconstituted water and the volume adjusted to 1 litre to give the remaining test concentrations.
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: Water samples were taken from the control (replicates R1 - R1 pooled) and all the test groups (replicates R1 - R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored frozen (approximately -20 °C) for further analysis if necessary.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: in-house laboratory cultures. Gravid adults were isolated the day before initiation of the test
- Age at initiation of study: <24 h.
- Feeding during test: Not fed during exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L CaCO3
Test temperature:
21 °C
pH:
7.8 +/- 0.2
Dissolved oxygen:
Air-saturation value
Salinity:
not specified
Conductivity:
<5 µS/cm
Nominal and measured concentrations:
1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL ground glass stoppered conical flasks.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 300 mL of media were used due to the suspected volatile nature of the test material.
- Aeration: None.
- No. of organisms per vessel: 10 daphnids were placed in each test and control vessel.

WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water.
- Stock solution: 11.76 g/L CaCl2.2H2O, 4.93 g/L MgSO4.7H2O, 2.59 g/L NaHCO3, 0.23 g/L KCL.
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: 0.2 if necessary with NaOH and HCl.
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minutes dawn and dusk transition periods.

RANGE-FINDING STUDY
- The range-finding test was conducted between 18-07-2003 and 20-07-2003
- Test concentrations: nominal 0.10, 1.0, 10 and 100 mg/L.
- Testing conditions: 10 daphnids placed in each test and control vessel and maintained in a temperature controlled room at ca. 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minutes dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised daphnia were recorded.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: C.I. 95%: 14 - 20 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
5.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 48 hours by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). Probit analysis is used where two or more partial responses to exposure are shown. When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

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.

Given that a decline in measured test concentrations was observed over each 24-Hour renewal period it was considered justifiable to present a 'worst case' analysis of the data by calculating the results based on the time-weighted mean measured test concentrations also.

The time-weighted mean measured test concentrations were calculated as follows:

Nominal Concentration (mg/L) Time-weighted Mean Measured Concentration (mg/L) Percentage of Nominal Concentration
1 0.74 74
1.8 1.5 81
3.2 2.7 83
5.6 5.4 97
10 9.1 91
18 17 95
32 30 94
56 53 95
100 88 88

Analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the time-weighted mean measured test concentrations gave the following results:

Time (h) EC50 (mg/L) 95 % Confidence limits (mg/L)
24 63 58-69
48 17 14-20
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the time-weighted mean measured test concentrations the 48-Hour EC50 was 17 mg/L with 95% confidence limits of 14 - 20 mg/L and the No Observed Effect Concentration was 5 .4 mg/L.
Executive summary:

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 (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67 /548/EEC).

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 21 °C under semi-static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Although efforts were made to minimise losses due to the suspected volatile nature of the test material, such as employing completely filled ground glass stoppered conical flasks as exposure vessels, losses may also have occurred via this route. Given the decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations. The 48-Hour EC50 based on the time-weighted mean measured test concentrations was 17 mg/L with 95% confidence limits of 14 -20 mg/L and the No Observed Effect Concentration was 5.4 mg/L.

Description of key information

EC50 (48h) = 17 mg/L (measured concentration), OECD202, SafePharm Laboratories (2003)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
17 mg/L

Additional information

One study has been selected for assessing the acute toxicity of cyclohexanol to aquatic invertebrates. A semi-static test was performed according to OECD guideline 202 by SafePharm Laboratories (2003). In this test, cyclohexanol exhibited harmful effects to Daphnia magna, resulting in an EC50 (48h) value of 17 mg/L (measured concentration). This data point represents the most sensitive endpoint for acute aquatic effects. Measurements of cyclohexanol concentrations as performed in this study, showed markedly decreased test substance concentrations within the time frame of 24 - 48 hours. In contrast, high test substance recoveries were observed over the first 24 hours of the experiment.