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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:
from 2008-04-04 to 2008-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0 mg/L; 1.8 mg/L; 3.2 mg/L; 5.6 mg/L; 10 mg/L; 18 mg/L; 32 mg/L; 56 mg/L; 100 mg/L in reconstituted water
- Sampling method: aqueous solution of test material
- Sample storage conditions before analysis: -20 °C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
For the purpose of the definitive test the test material was dissolved directly in reconstituted 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. Aliquots (10, 18, 32, 56, 100, 180, 320 and 560 mL) of the 100 mg/L test concentration were each separately dispersed in a final volume of 1 litre of reconstituted water to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg/L test concentrations, respectively. Each stock prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The concentration and stability of the degradants isobutyraldehyde and diethanolamine in the test preparations were verified by chemical analysis at 0 and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory cultures
- Age at study initiation: less than 24 hours
- Method of breeding: Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 °C.
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Feeding during test: no
- Food type: suspension of algae (Chlorella sp.)
- Frequency: daily, except testing period

ACCLIMATION
- Acclimation period: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
230 mg/L CaCO3
Test temperature:
21 °C
pH:
7.9 - 9.0
Dissolved oxygen:
7.7 - 8.7
Nominal and measured concentrations:
The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages. The nominal concentrations were 1 mg/L, 10 mg/L and 100 mg/L.

ISOBUTYRALDEHYDE:
Nominal (mg/L): 0.45
Concentration found after storage in light conditions (mg/L): 0.359

Nominal (mg/L): 4.5
Concentration found after storage in light conditions (mg/L): 4.04

Nominal (mg/L): 45
Concentration found after storage in light conditions (mg/L): 41.9

DIETHANOLAMINE:
Nominal (mg/L): 0.65
Concentration found after storage in light conditions (mg/L): 0.703

Nominal (mg/L): 6.5
Concentration found after storage in light conditions (mg/L): 6.81

Nominal (mg/L): 65
Concentration found after storage in light conditions (mg/L): 68.2

Isobutyraldehyde has been shown to be stable in the test medium. The lowest level stability results were below the acceptance criteria of 80 - 120 %. This is believed to be due to the high recovery result at this concentration having a large impact on the stability value. The unsonicated stability vessels showed no evidence of insolubility or adherence to glass. The open stability vessels showed evidence of volatility. This is believed to account for the slightly low dark stability result at the middle level and unsonicated stability result at the top level.
Diethanolamine has been shown to be stable in the test medium. The unsonicated stability vessel showed no evidence of insolubility or adherence to glass. The open stability vessels showed no evidence of volatility.
Details on test conditions:
TEST SYSTEM
In the definitive test 300 mL glass conical flasks containing approximately 300 mL of test preparation were used. At the start of the study 10 daphnias were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then sealed, with minimal headspace to prevent losses of the degradant isobutyraldehyde due to its volatile nature and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test material. 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 MEDIUM / WATER PARAMETERS:
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

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. The reconstituted water had a pH of 7.8 ± 0.2 adjusted with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

OTHER TEST CONDITIONS:
- Photoperiod: 16 hours light and 8 hours darkness for a period of 48h with 20 minutes dawn and dusk transition period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 0.1; 1.0; 10 and 100 mg/L (range-finding study)
- 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. Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: no
- Any observations that might cause a difference between measured and nominal values:
The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 65.4 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.
Analysis of the test preparations for isobutyraldehyde at 0 hours showed measured concentrations of 83 % to 102 % of the calculated nominal value whilst at 48 hours analysis of the test preparations showed measured test concentrations of 50 % to 81 % of the calculated nominal value. The decline in measured concentration over the 48-Hour period was considered to be due to the volatile nature of the degradant despite all precautions being taken to minimise losses due to volatility.
Analysis of the test preparations for diethanolamine at 0 and 48 hours showed measured concentrations of 99 % to 112 % of the calculated nominal value with the exception of the lowest test concentration which showed a measured concentration of 41 % of the calculated nominal value. However, as this concentration was below the no observed effect concentration (NOEC) and all other values were within the 80 % to 120 % acceptance limits this was considered not to have affected the outcome of the study.
As the test preparations were a mixture of two degradants the results are based on nominal test concentrations only.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Mortality: yes
- EC50 (48-h): 0.47 mg/L
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. The No Observed Effect Concentration (NOEC) was 10 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The test followed the method 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/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 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 21 °C under static test conditions. The number of immobilised Daphnia was 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 21 °C in sealed vessels under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48-hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration NOEC was 10 mg/L.

The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 65.4 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.

Analysis of the test preparations for isobutyraldehyde at 0 hours showed measured concentrations of 83 % to 102 % of the calculated nominal value whilst at 48 hours analysis of the test preparations showed measured test concentrations of 50 % to 81 % of the calculated nominal value. The decline in measured concentration over the 48-hour period was considered to be due to the volatile nature of the degradant despite all precautions being taken to minimise losses due to volatility.

Analysis of the test preparations for diethanolamine at 0 and 48 hours showed measured concentrations of 99 % to 112 % of the calculated nominal value with the exception of the lowest test concentration which showed a measured concentration of 41 % of the calculated nominal value. However, as this concentration was below the no observed effect concentration (NOEC) and all other values were within the 80 % to 120 % acceptance limits this was considered not to have affected the outcome of the study.

As the test preparations were a mixture of two degradants the results are based on nominal test concentrations only.

The 48-hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/L with 95 % confidence limits of 0.43 - 0.53 mg/L. The No Observed Effect Concentration NOEC was 0.32 mg/L.

Description of key information

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. The No Observed Effect Concentration (NOEC) was 10 mg/L.

Key value for chemical safety assessment

Additional information

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The test followed the method 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/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 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 21 °C under static test conditions. The number of immobilised Daphnia was 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 21 °C in sealed vessels under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48-hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration NOEC was 10 mg/L.

The test material was known to degrade rapidly in water to give isobutyraldehyde and diethanolamine. The relative amounts of each degradant were calculated to be 45.3 % isobutyraldehyde and 65.4 % diethanolamine. The test concentrations were monitored by analysis of the test samples for the degradants only. For the calculation of test sample results, the nominal concentration for each degradant was adjusted to account for their relative percentages.

Analysis of the test preparations for isobutyraldehyde at 0 hours showed measured concentrations of 83 % to 102 % of the calculated nominal value whilst at 48 hours analysis of the test preparations showed measured test concentrations of 50 % to 81 % of the calculated nominal value. The decline in measured concentration over the 48-hour period was considered to be due to the volatile nature of the degradant despite all precautions being taken to minimise losses due to volatility.

Analysis of the test preparations for diethanolamine at 0 and 48 hours showed measured concentrations of 99 % to 112 % of the calculated nominal value with the exception of the lowest test concentration which showed a measured concentration of 41 % of the calculated nominal value. However, as this concentration was below the no observed effect concentration (NOEC) and all other values were within the 80 % to 120 % acceptance limits this was considered not to have affected the outcome of the study.

As the test preparations were a mixture of two degradants the results are based on nominal test concentrations only.

The 48-hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/L with 95 % confidence limits of 0.43 - 0.53 mg/L. The No Observed Effect Concentration NOEC was 0.32 mg/L.