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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:
1998-05-18 to 1998-05-20 (definitive study); 1997-11-12 to 1997-11-14 (range-finding study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: solvent control and 4.0 mg/L; stock solution
- Sampling method: Water samples were taken at 0 and 48 hours for quatitative analysis.
- Sample storage conditions before analysis: Samples were analyzed immediately after preparation.

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dissolving of test material in dimethylformamide with the aid of ultrasonication for approximately 1 minute and the volume adjusted to 50 mL to give the 2000 mg/50 mL solvent stock solution. An aliquot (200 µL) of this was dispersed in reconstituted water and the volume adjusted to 2 litres to give the 4.0 mg/L test concentration.
- Controls: one control and one solvent control (100 µl/L)
- Chemical name of vehicle: dimethylformamide
- Evidence of undissolved material: precipitation was observed in range-finding study at concentrations in excess of 4.0 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia mangna
- Source: Institut National de Recherche Chimique Appliquée (IRCHA), France
- Age at study initiation: < 24 h young daphnids
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
after 24 and 48 h
Hardness:
250 mg/L
Test temperature:
21°C
pH:
pH 7.6-7.7
Dissolved oxygen:
8.1-8.4 mg O2/L (~ air-saturation value)
Conductivity:
< 5 µS/cm
Nominal and measured concentrations:
Nominal concentration: 4.0 mg/L
Measured concentration at 0 h: 4.56 mg/L (replicates 1, 2) and 2.78 mg/L (replicates 3,4)
Measured concentration at 48 h: < LOQ (0.38 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type: Test vessels were covered
- Material, fill volume: glass, 250 mL
- Aeration: none (during test)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- CaCl2 * 2 H2O: 11.76 g/L
- MgSO4 * 7 H2O: 4.93 g/L
- NaHCO3: 2.59 g/L
- 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 and pH equal to 7.8 +/- 0.2, adjusted (if necessary) with NaOH or HCI.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED:
- Immobilisation was investigated after 24 and 48 hours

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0 and 4.0 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation was observed at all the test concentrations. During preliminary solubility work precipitation of test material was observed (by visual inspection) at concentrations in excess of 4.0 mg/l indicating this to be the highest test concentration that could be prepared under these test conditions. Based on this information, a single test concentration of four replicates, of 4.0 mg/l was selected for the definitive study.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
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 nominal concentration gave a 48-h EC50 of greater than 4.0 mg/L. Correspondingly the No Observed Effect Concentration was greatr than or equal to 4.0 mg/L.
The 48-h EC50 value based on the time-weighted mean measured concentration was greater than 1.2 mg/L. Correspondingly the No Observed Effect Concentration was greater than or equal to 1.2 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.

Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at a concentration of 4.0 mg/L for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48 -hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 4.0 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L.

The test concentration of 4.0 mg/L was the highest attainable test concentration due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Pre-study analysis performed showed the test material to be highly unstable in aqueous media, with hydrolysis occurring virtually instantaneously on contact with water. The degradation product was found to be insoluble in organic and aqueous solvents, negating the possibility of quantitative analysis of the degradant. Therefore, the solvent stock solution and test solutions prepared at 0 hours were extracted and analysed immediately after preparation.

Analysis of the stock solution used to prepare the fresh test media at 0 hours showed a measured concentration of 67% of nominal, the results from analysis of the 4.0 mg/L test concentration at 0 hours showed measured test concentrations of 114% and 70% of nominal for replicates R1 -R2 and R3 -R4 (replicates pooled) respectively. After 48 hours exposure, the measured test concentrations were shown to be less than the limit of quantitation of the analytical method employed.

The results show, that despite immediate extraction and analysis at 0 hours, variation in measured concentrations of the stock solution and test solutions was unavoidable due to rapid hydrolysis of the test material and that further hydrolysis occurred over the study period.

Given the marked and rapid decline in measured test concentrations over the test periods, it was considered justifiable to base the results on the time-weighted mean measured test concentrations also.

The 48 -hour EC50 value based on time-weighted mean measured concentrations was greater than 1.2 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.2 mg/L.

So, even at concentrations exceeding the water solubility of the test item, no effects were noted. Consequently, no effects are expected in the aquatic environment.

Finally, this acute toxicity test in daphnia represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as EC50 refers to a substance concentration which was reduced due to hydrolysis.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
A sample of 100mL was removed from the remainder of the test medium after distribution into the test vessels for each exposure or control group at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours. Immediately the sample was analyzed by HPLC method.
Vehicle:
not specified
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 +/- 1°C
Nominal and measured concentrations:
Nominal concentrations: 2.2, 3.2, 4.6, 6.8, 10, 15, 22, 32, 46, 68 mg/L
Measured concentrations: 1.96, 2.81, 4.32, 6.32, 9.58, 13.9, 20.8, 29.3, 42.8, 60.8 mg/L
Details on test conditions:
TEST SYSTEM
- Fill volume: 100 mL
- No. of organisms per vessel: 5 individuas
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour darkness
- Light intensity: room light



Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Reported statistics and error estimates:
Probit method
Validity criteria fulfilled:
yes
Conclusions:
In this 48-hour acute toxicity test with Daphnia magna the 48-hour EC 50 value was determined to be 4.5 mg/L, and the 48-hour NOEC was determined as 2.2 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the degradation product ( 4,4'-bi-o-toluidine) of TODI to Daphnia magna as requested by ECHA. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202.

Daphnia (5 individuals per vessel) were exposed to the test material over a range of concentrations of 2.2, 3.2, 4.6, 6.8, 10, 15, 22, 32, 46 and 68 mg/L for a period of 48 hours under static test conditions.

 

The sample of 100mL was removed from the remainder of the test medium after distribution into the test vessels for each exposure or control group at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours. Immediately the sample was analyzed by HPLC method. The recovery rate was determined as followed:94.5, 94.5, 96.5% (average 95.2%) at 2 mg/L; 97.2, 97.9, 93.9% (average 96.3%) at 72mg/L.

Temperature of the test solution and dissolved oxygen concentration were measured at 0 and 48 hours.

 

The following results were obtained in this 48-h acute toxicity test with Daphnia magna:

 

24 hours EC50: 11 mg/L (95% Confidential Limit:5.6-19mg/L, Probit method)

48 hours EC50: 4.5 mg/L (95% Confidential Limit:3.2-6.1mg/L, Probit method)

24 hour NOEC: 3.2 mg/L

48 hour NOEC: 2.2 mg/L.

 

Description of key information

 The acute toxicity of the test material TODI to the freshwater invertebrate Daphnia magna has been investigated and based on the nominal concentration gave a 48-hour EC50 of greater than 4.0 mg/L. Correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L. The 48-hour EC50 value based on time-weighted mean measured concentrations was greater than 1.2 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.2 mg/L.

 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.2 mg/L

Additional information

Key study

The purpose of this study was to evaluate the influence of the test item TODI on the mobility respectively survival of Daphnia magna. Young Daphnia were exposed in a static test to the test item for 48 hours.

Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at a concentration of 4.0 mg/L for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 28 hours. The test item was presented in dimethylformamide to allow the formation of a stable dispersion.

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the nominal concentration gave a 48-hour EC50 of greater than 4.0 mg/L. Correspondingly the No Observed Effect Concentration was greater than or equal to 4.0 mg/L. The 48-hour EC50 value based on time-weighted mean measured concentrations was greater than 1.2 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1.2 mg/L. So, even at concentrations exceeding the water solubility of the test item, no effects were noted. Consequently, no effects are expected in the aquatic environment.

Finally, this acute toxicity test in daphnia represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as EC50 refers to a substance concentration which was reduced due to hydrolysis.

 

Supporting study

A study was performed to assess the acute toxicity of the degradation product ( 4,4'-bi-o-toluidine) of TODI to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202.

Daphnia (5 individuals per vessel) were exposed to the test material over a range of concentrations of 2.2, 3.2, 4.6, 6.8, 10, 15, 22, 32, 46 and 68 mg/L for a period of 48 hours under static test conditions.

 

The sample of 100mL was removed from the remainder of the test medium after distribution into the test vessels for each exposure or control group at 0 hour. Equal volume of samples were removed from replicate test vessels and mixed for each exposure or control group to 100 mL at 72 hours. Immediately the sample was analyzed by HPLC method. The recovery rate was determined as followed:94.5, 94.5, 96.5% (average 95.2%) at 2 mg/L; 97.2, 97.9, 93.9% (average 96.3%) at 72 mg/L.

Temperature of the test solution and dissolved oxygen concentration were measured at 0 and 48 hours.

 

The following results were obtained in this 48-h acute toxicity test with Daphnia magna:

 

24 hours EC50: 11 mg/L (95% Confidential Limit:5.6-19mg/L, Probit method)

48 hours EC50: 4.5 mg/L (95% Confidential Limit:3.2-6.1mg/L, Probit method)

24 hour NOEC: 3.2 mg/L

48 hour NOEC: 2.2 mg/L.