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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:
April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
test item is unstable in water, therefore the hydrolysis product PMA was tested
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)
Specific details on test material used for the study:
- Description: white powder
- Batch number: 8M30N
- Storage conditions: room temperature in the dark upto 04 April 2008, thereafter at approximately 4 °C, over silica gel, in the dark
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours. Water samples were taken from the control (replicates R1 - R2 pooled) and each test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
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 (50, 90, 160 and 280 ml) of the 100 mg/l test concentration were each separately dispersed in a final volume of 500 ml of reconstituted water to give the 10, 18, 32 and 56 mg/l test concentrations respectively. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.
Test temperature:
The water temperature was controlled at approximately 20°C.
pH:
7.8
Dissolved oxygen:
8.5 to 8.7 mg O2/l
Salinity:
freshwater used
Nominal and measured concentrations:
The test concentrations to be used in the definitive test were determined by a preliminary rangefinding test. In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/l. No immobilisation was observed at the test concentrations of 0.010, 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 103% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.
Details on test conditions:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l. 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 (50, 90, 160 and 280 ml) of the 100 mg/l test concentration were each separately dispersed in a final volume of 500 ml of reconstituted water to give the 10, 18, 32 and 56 mg/l test concentrations respectively. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
As in the range-finding tests 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the study 10 daphnids 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 covered to reduce evaporation and maintained in a temperature controlled room at 21°C to 22°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.
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limit: 58-72 mg/l
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limit: 56-71 mg/l
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 63 mg/1 with 95% confidence limits of 56-71 mg/l. The No Observed Effect Concentration was 32 mg/l.
Results with reference substance (positive control):
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 approximately 20°C 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 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 was 0.32 mg/l.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999). When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
Validity criteria fulfilled:
yes
Conclusions:
The 48-Hour EC50 for the test material to Daphnia magna was 63 mg/l with 95% confidence limits of 56-71 mg/l. The No Observed Effect Concentration was 32 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 No 202, "Daphnia sp, Acute Immobilisation Test". 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 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 21°C to 22°C under static test conditions. The number of imrnobilised Daphnia 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 approximately 20°C 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 63 mg/l with 95% confidence limits of 56- 71 mg/l. The No Observed Effect Concentration was 32 mg/l. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 103% of nominal value and so 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 was 0.32 mg/l.

Description of key information

As the substance to be registered, PMDA, is unstable in water (t1/2 approx. 20 min), the corresponding hydrolysis product Pyromellitic acid (PMA) was tested. The study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines No 202, "Daphnia sp, Acute Immobilisation Test".

The 48-Hour EC50 for the test material to Daphnia magna based on mean measured test concentrations was 63 mg/l with 95% confidence limits of 56- 71 mg/l. The No Observed Effect Concentration was 32 mg/l.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
63 mg/L

Additional information