4,4'-methylenebis[2,6-diethylaniline]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Molecular formula: C21H30N2
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples were taken from each testing concentrations and from the control at the start and at the end of each water renewal period. Four replicate samples were taken from the test solutions and one control sample on each analytical occasion.

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test item is poorly water soluble material. In order to achieve maximum dissolved concentration, a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.1 g test item into 1000 mL test medium (ISO medium) at least one day before the start of the test. This solution was agitated by orbital shaker (~350 rpm) for a period of at least one day and then the non-dissolved test material was separated by filtration through a fine (0.22 μm) filter in order to obtain the saturated test solution (i.e. 100 % v/v saturated solution). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared just before introduction of the test animals (start of the water renewal periods).

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species and strain: Daphnia magna (Straus)
- Age of animals: Less than 24 h old at the beginning of the test
- Sex: Female
- Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Breeding: The Daphnia are bred in the test lab
- Acclimatization: Test animals were bred under similar conditions as used during exposure period, therefore additional acclimatisation before the test was not necessary
- Animal health: Apparently healthy animals were used in this test with a known history (breeding method, pre-treatment)
- Number of animals: There were 20 animals in each group (concentration and untreated control) divided into 4 replicates.
- Food and Feeding: The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding. Test animals were not fed during the exposure

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

The immobilisation of the test animals was recorded 24 and 48 hours after treatment.

Post exposure observation period:

None.

Hardness:

The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 20.6 – 20.9°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.5 – 21.5°C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.69 – 7.97 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 7.56 – 8.98 mg/L during the test.

Salinity:

Freshwater used

Conductivity:

No data

Nominal and measured concentrations:

Based on the results of the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 1.7 and a concurrent control were investigated in the main study. The following nominal concentrations were tested: 12.0, 20.4, 34.6, 58.8 and 100 % v/v saturated solution.
The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.18, 0.31, 0.57, 0.98, and 1.69 mg/L.
All biological results are based on the geometric mean of the measured concentrations.

Details on test conditions:

- Light: 12 to 16 hours photoperiod daily;
- Temperature: 20.5 – 21.5°C

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 1.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 1.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

MOBILITY:
Immobilisation was not detected either in the control or in the test item treated group.

TOXIC EFFECTS:
Sublethal effects were not detected either in the control or in the test item treated group.

DOC:
The dissolved oxygen concentration in was greater 3 mg/L in each test vessel at the end of the test.

Results with reference substance (positive control):

The date of the last study with reference item Potassium dichromate was: September 24 - 25, 2014. The 24h EC50: 1.40 mg/L, (95 % confidence limits: 1.19 – 1.68 mg/L).

Reported statistics and error estimates:

No toxic effect were observed during the study, therefore using statistical analysis was not necessary to evaluate the biological results. The 48-h EC50, NOEC, LOEC and EC100 values were determined directly from the raw data

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effect on Daphnia magna up to at least the solubility limit of the test item in the test medium (1.69 mg/L measured). The 48-h NOEC was determined to be the saturation concentration; the EC50 results and the LOEC are higher than the saturation concentration (1.69 mg/L measured).

Executive summary:

The acute toxicity of the test item was assessed on Daphnia magna in a 48 -hour Acute Immobilisation Test. The study was performed according to EU testing method C.2 and OECD no. 202. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours at a range of concentrations. Based on the analytical results obtained during the previously performed acute toxicity test of the test item on Daphnia magna, the test item was not expected to be stable for the duration of 48 hours in ISO Medium, therefore a semi-static test was performed using daily water renewal periods. Based on the results of the preliminary experiment, nominal concentrations of 12.0, 20.4, 34.6, 58.8 and 100 % v/v saturated solution were tested in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.18, 0.31, 0.57, 0.98, and 1.69 mg/L. All biological results are based on the geometric mean of the measured concentrations. The immobilisation of the test animals was recorded 24 and 48 hours after treatment. There was no immobilisation observed in daphnids exposed to either the test item concentrations (up to the solubility level of the test item in the test medium) or in the control group. In addition to immobility, no abnormal behaviour or appearance of test animals was noted. The concentrations of the test item remained stable (i.e. within ± 20 per cent of the measured initial concentration) in the three highest test item concentrations during the 48h exposure period. Since there was no immobilisation or any sub-lethal effects observed up to the saturation concentration (1.69 mg/L measured) and the test item was stable at least at saturation for the duration of the test it can be stated that the test item has no toxic effects on Daphnia magna up to at least the solubility limit of the test item in the test medium (1.69 mg/L measured).

Description of key information

Short-term toxicity to aquatic invertebrates

The acute toxicity of the test item was assessed on Daphnia magna in a 48 -hour Acute Immobilisation Test. The study was performed according to EU testing method C.2 and OECD no. 202. Since there was no immobilisation or any sub-lethal effects observed up to the saturation concentration (1.69 mg/L measured) and the test item was stable at least at saturation for the duration of the test it can be stated that the test item has no toxic effects on Daphnia magna up to at least the solubility limit of the test item in the test medium (1.69 mg/L measured).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

1.69 mg/L

Additional information

Source: GLP-report