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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 June 2010 to 13 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the solvent control and all test groups (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Due to the suspected light sensitive nature of the test item all the sample bottles were shielded from the light.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Initial experiment: An amount of test item (300 mg) was dissolved in acetone and the volume adjusted to 10 ml to give a 300 mg/10 ml solvent stock solution. An aliquot (200 μl) of this was added to reconstituted water and the volume adjusted to 2 litres to give a 3.0 mg/l stock solution. Any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a test concentration of 1.1 mg/l.

Definitive test: An amount of test item (300 mg) was dissolved in acetone and the volume adjusted to 10 ml and stirred using a magnetic stirrer for approximately 10 minutes to give a 300 mg/10 ml solvent stock solution. An aliquot (200 μl) of this was added to reconstituted water and the volume adjusted to 2 litres to give a 3.0 mg/l stock solution. Any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a test concentration of 0.70 mg/l. Aliquots (100, 186, 329 and 571 ml) of the 0.70 mg/l test concentration were each added separately to reconstituted water and the volume adjusted to 1 litre to give the 0.070, 0.13, 0.23 and 0.40 mg/l test concentrations respectively. Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): 24 h old
- 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. 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.
- Feeding during test: No



Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/l as CaCO3
Test temperature:
20-21 ºC
pH:
7.9-8.2
Dissolved oxygen:
8.2-9.5 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: 0.070, 0.13, 0.23, 0.40 and 0.70 mg/l
Measured concentrations: Analysis of the fresh media at 0 hours showed measured concentrations to range from 83% to 101 % of nominal value. Analysis of the old media at 48 hours showed measured concentrations to range from 42% to 69% of nominal value.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jars
- Type (delete if not applicable): The test vessels were covered to reduce evaporation
- No. of organisms per vessel: 10 Daphnids per vessel
- No. of vessels per concentration (replicates): Duplicates
- No. of vessels per control (replicates): Duplicates
- No. of vessels per vehicle control (replicates): Duplicates

TEST MEDIUM / WATER PARAMETERS
- 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
- Photoperiod: The test was performed in the dark as stability analysis performed indicated possible instability of the test item in the light.

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
- Range finding study
Initial range-finding study: No immobilisation was observed at the nominal test concentrations of 0.011, 0.11 and 1.1 mg/l.
Based on this information, a single test concentration of four replicates, of 1.1 mg/l was selected for the initial experiment. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 1.1 mg/l, no immobilisation or adverse reactions to exposure were observed.

Initial experiment: 100% immobilisation was observed at the nominal test concentration of 1.1 mg/l after 24 hours. The toxicity profile obtained during the initial experiment was significantly different to the initial range-finding. Results of the chemical analysis showed a measured concentration of approximately 0.70 mg/l was attained in the initial experiment. This indicated that there was a possible solubility issue. Therefore during the initial range-finding test the highest concentration may have been lower than 0.70 mg/l resulting in no immobilisation. Based on this information, a second rangefinding test was conducted with chemical analysis to determine the concentration range to be used in the definitive test.

Second Range-finding Test: No immobilisation was observed at the nominal test concentrations of 0.011 and 0.11 mg/l. However, immobilisation was observed at the 1.1 mg/l test concentration. Results of the chemical analysis of the highest concentration during the second rangefinding
test showed a measured concentration of 0.68 mg/l was attained. This value was in-line with the results obtained from chemical analysis of the highest test concentration during the initial experiment. This was slightly lower than values obtained during pre-study media preparation trial, however given that the same result was obtained during both the initial experiment and the second range-finding test, the concentration range to be used in the definitive test was refined to 0.070, 0.13, 0.23, 0.40 and 0.70 mg/l.
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits of 0.39 - 0.70 mg/l
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.19).

Analysis of the fresh media at 0 hours showed measured concentrations to range from 83% to 101 % of nominal value. Analysis of the old media at 48 hours showed measured concentrations to range from 42% to 69% of nominal value. This decline in measured concentration was contrary to the stability analysis which showed the test item to be stable in the test medium except when exposed to light. As the test was conducted in the dark, the decline in measured concentration over the 48-Hour test period was considered not to be due to photolysis. lnformation of the Physico-chemical properties for the test item provided by the Sponsor gave a partition co-efficient (log Pow) of 5.1 which indicated a high potential for bioaccumulation and adsorption to organic waste. It was therefore considered that the decline in measured concentration over the test period was possibly due to bioaccumulation and/or adsorption of the test item to organic waste. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in the control was 0%; the dissolved oxygen concentration at the end of the test was greater than 3 mg/l in control and test vessels).
Conclusions:
It was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the geometric mean measured test concentrations was 0.48 mg/L with 95% confidence limits of 0.39 - 0.70 mg/L and the No Observed Effect Concentration was 0.15 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guideline No 202, "Daphnia sp, Acute lmmobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008. lnformation provided by the Sponsor gave a water solubility value for the test item of 1.1 mg/L. Pre-study media preparation trials indicated that the use of a preliminary solution in auxiliary solvent to spike the test medium followed by filtration to remove any undissolved test item was the most suitable method of preparation for the test item. Following preliminary range-finding tests and an initial experiment, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.070, 0.13, 0.23, 0.40 and 0.70 mg/L for 48 hours at a temperature of approximately 20°C under static test conditions in the dark. The number of immobilised Daphnia were recorded after 24 and 48 hours. Due to the possible light

sensitive nature of the test item, the test was performed in the dark. A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 21ºC to 22°C under static test conditions. lmmobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was 0.61 mg/L with 95% confidence limits of 0.51 - 0.82 mg/L. The No Observed Effect Concentration was 0.23 mg/L. Analysis of the fresh media at 0 hours showed measured concentrations to range from 83% to 101 % of nominal value. Analysis of the old media at 48 hours showed measured concentrations to range from 42% to 69% of nominal value. This decline in measured concentration was contrary to the stability analysis which showed the test item to be stable in the test medium over the 48-Hour period except when exposed to light. As the test was conducted in the dark, the decline in measured concentration over the 48-Hour test period was considered not to be due to photolysis. lnformation of the Physicochemical properties for the test item provided by the Sponsor gave a partition co-efficient (log Pow) of 5.1 which indicated a high potential for bio-accumulation and adsorption to organic waste. It is was therefore considered that the decline in measured concentration over the test period was possibly due to bio-accumulation andlor adsorption of the test item to organic waste. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the geometric mean measured test concentrations was 0.48 mg/L with 95% confidence limits of 0.39 - 0.70 mg/L and the No Observed Effect Concentration was 0.15 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
The tests was conducted based on OECD-GLP standard and OECD test guidelines. However, the Ministry of the Environment of Japan suggest confirmation of the results since the test was conducted in 1999 and some of the tests conducted before 2002 were performed using a dispersant.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Daphnia magna
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
48 h
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.84 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
mobility
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 for BHT in Daphnia magna was 0.84 mg/L.
Executive summary:

An acute immobilisation study in Daphnia magna was conducted by the Ministry of the Environment in Japan based on OECD GLP-standards and OECD guideline 202. The 48hour-LC50 of BHT in Daphnia magna was 0.84 mg/L.

Description of key information

Experimental result: OECD 202 and EU C.2. The 48 -h EC50 of BHT on Daphnia magna based the geometric mean measured was 0.48 mg/L and the NOEC was 0.15 mg/L.

Experimental result: OECD guideline 202, GLP. The 48hour-LC50 of BHT in Daphnia magna was 0.84 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.48 mg/L

Additional information

Key study: Experimental results: OECD 202 and EU C.2. It was considered justifiable to base results on the geometric mean measured test concentrations in order to give a worst case analysis of the data. The 48 -h EC50 based the geometric mean measured was 0.48 mg/L and the NOEC was 0.15 mg/L.

Supporting study: Experimental result. OECD guideline 202, GLP. The 48hour-LC50 of BHT in Daphnia magna was 0.84 mg/L.