N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

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:

2022-02-08 to 2022-02-10

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 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

October 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical Name: N,N’-Bis(1,3-dimethylbutylidene)ethylenediamine
- CAS No.: 25707-70-4
- EC No.: 247-196-6

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken from each testing concentration and from the control at
the start and at the end of each water renewal period
- Sample storage conditions before analysis: no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: mechanical dispersion of about 1 hour to obtain a clear solution without using of any solubilising agent; test solutions of subsequent lower concentrations were prepared by appropriate diluting of this stock solution
- Controls: negative control, positive control
- Test concentration separation factor: 2.1
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: less than 24 hours old at the start of the exposure period
- Stage at study initiation: juvenile
- Method of breeding: bred in laboratory
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 48 hours prior to the start of the test
- Acclimation conditions: same as test
- Type and amount of food: daphnids were fed with concentrated algal suspension of
Raphidocelis subcapitata during the holding
- Health during acclimation: apparently healthy animals

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Post expossure observation not performed

Hardness:

The mean hardness of test water was determined to be 42.7 mg/L (as CaCO3) using “VISCOLOR® HE kit total Hardness H 20 F” reagent set (Lot: 0988, Exp. date: 31 May 2022). Due to the chemical properties of the used test medium (OECD medium) the measured values were out of the recommended range of 140 – 250 mg/L, however this deviation was considered to be acceptable and does not affect the results of the test.

Test temperature:

The test temperature was in the range of 19.6 – 20.4°C measured in the test vessels during the test.

pH:

The test was carried out without adjustment of pH. The pH did not vary by more than 1.5 units within the test vessels during the exposure. Measured values of pH was in the range of 6.83 – 8.92 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 8.07 – 8.64 mg/L during the test.

Salinity:

not applicable

Nominal and measured concentrations:

nominal: 5.14, 10.8, 22.7, 47.6 and 100 mg/L; measured: 1.05, 2.64, 4.02 and 26.09 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Material, size, headspace, fill volume: Glass beakers of 50 mL volume filled up with approx. 40 mL test medium.
- Renewal rate of test solution (frequency/flow rate): renewal of the test solution after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Based on the results of preliminary solubility test, the test item could not be completely dissolved at a concentration level of 100 mg/L in ISO medium, therefore OECD medium (reconstituted water, according to OECD 201) was used as dilution water (and for holding the daphnia during acclimatization) in the experiment (in which the test item could be diluted completely at 100 mg/L). This approach has been used previously and showed to have no effect on the daphnia growth. Test animals showed no sign of any stress during acclimatisation or testing in OECD medium.

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of pH. The pH did not vary by more than 1.5 units within the test vessels during the exposure.
- Photoperiod: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.
- Light intensity: Light intensity was measured at the start of the experiment and determined as 540 lux.

EFFECT PARAMETERS MEASURED :
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition to immobility, behaviour or appearance was also observed. The behaviour of the test item in test medium was observed daily and any occurred phenomena (e.g.: precipitation, adsorption) recorded.

RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: After 48 h, 0 animals of the 1 mg/L test concentration and 3 and 10 of the10 and 100 mg/L test group, respectively, were found to be immobilized.

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.15 mg/L

95% CI:

>= 1.66 - <= 2.52

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.44 mg/L

95% CI:

>= 0.84 - <= 1.81

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Immobilisation of control: After 48-h exposure no immobilisation was observed in the control.
- Any observations that might cause a difference between measured and nominal values: No precipitation or any abnormal appearance of test solutions was observed during the test.

Table 1: Immobilisation of the test animals (summary)

Concentration[mg/L]		Number of Daphnia tested	Number of immobilised Daphnia		% of immobilised Daphnia
nominal	exposure		24 h	48 h	24 h	48 h
Control	–	20	0	0	0	0
5.14	1.05	20	0	0	0	0
10.8	2.64	20	5	16	25	80
22.7	4.02	20	6	19	30	95
47.6	9.97	20	12	20	60	100
100	26.26	20	15	20	75	100

 

Table 2: Summary of Biological Endpoints (based on the measured concentrations (i.e. calculated exposure concentrations)

Endpoints	Concentration [mg/L]
48 h-EC10	1.44
95 % conf. limits	0.84 – 1.81
48 h-EC20	1.65
95 % conf. limits	1.07 – 2.01
48 h-EC50	2.15
95 % conf. limits	1.66 – 2.52
48 h-NOEC	1.05
48 h-LOEC	2.64.2

 

Validity of the test:

– The immobilisation rate of the introduced Daphnia did not exceed 10 % in the control. No immobility was observed and no Daphnia were trapped at the water surface or showed any sign of stress.

– The dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels. All validity criteria were met, therefore the study is considered to be fully valid.

Validity criteria fulfilled:

yes

Conclusions:

In the 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item had toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 2.15 mg/L. The 48-h NOEC was determined to be 1.05 mg/L. The results are based on measured test item concentrations.

Executive summary:

The acute toxicity of the test item was investigated in a 48-h immobilization test with Daphnia magna. The study was conducted according to OECD Guideline No. 202 and in compliance with GLP. Young Daphnia were exposed in a semi-static immobilisation test to aqueous test media containing the test item for 48 hours. The test media renewal period was 24 h. Based on the results of a preliminary experiment, nominal concentrations of 5.14, 10.8, 22.7, 47.6 and 100 mg/L were investigated in the main study. The corresponding calculated exposure concentrations (based on analytical measurements) were the following: 1.05, 2.64, 4.02, 9.86 and 26.09 mg/L. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Concentrations were determined using GC-MS method. All validity criteria were met and therefore the study was considered to be valid. As a result, the 48-h EC50 value was determined to be 2.15 mg/L. The 48-h NOEC was determined to be 1.05 mg/L.

Description of key information

In the 48-hour acute toxicity test with Daphnia magna, the obtained results showed that the test item had toxic effects on the mobility of Daphnia. The 48-h EC50 value was determined to be 2.15 mg/L. The 48-h NOEC was determined to be 1.05 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

2.15 mg/L

Additional information

The acute toxicity of the test item was investigated in a 48-h immobilization test with Daphnia magna. The study was conducted according to OECD Guideline No. 202 and in compliance with GLP. Young Daphnia were exposed in a semi-static immobilisation test to aqueous test media containing the test item for 48 hours. The test media renewal period was 24 h. Based on the results of a preliminary experiment, nominal concentrations of 5.14, 10.8, 22.7, 47.6 and 100 mg/L were investigated in the main study. The corresponding calculated exposure concentrations (based on analytical measurements) were the following: 1.05, 2.64, 4.02, 9.86 and 26.09 mg/L. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Concentrations were determined using GC-MS method. All validity criteria were met and therefore the study was considered to be valid. As a result, the 48-h EC50 value was determined to be 2.15 mg/L. The 48-h NOEC was determined to be 1.05 mg/L.