N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-22 to 2018-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure (0 hours), 1 sample (1 retention sample) of the fresh media were taken after preparation of the limit concentration and analyzed. At the end of the exposure (72 hours), samples (1 sample, 1 retention sample) of the old media were taken from the test vessels for analyses.

Vehicle:

yes

Remarks:

Tetrahydrofuran + 0.1% formic acid

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the preparation of the limit concentration of the test item N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a stock solution of 1 g test item/L was prepared in the solvent Tetrahydrofuran containing 0.1% formic acid (THF+FA).
The stock solution was treated with ultrasound for 60 minutes at room temperature.
The limit test item concentration was prepared by adding 0.1 mL/L of the appropriate stock solution to the dilution water.
The solution was agitated and used for testing.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Laboratory culture, origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: A four days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LÜTTGE et al. (1994)
Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23 °C

pH:

7.63 - 8.02

Nominal and measured concentrations:

0.100 (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: 5523 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.


TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: refer to attached document (Range finding study)
- Range finding study: refer to attached document
- Test concentrations: 0.100 mg/L
- Results used to determine the conditions for the definitive study: refer to attached document

Reference substance (positive control):

yes

Remarks:

Results of the most recent of the monthly performed reference tests (potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities.
- Effect concentrations exceeding solubility of substance in test medium:
Different solvents revealed the maximum possible concentration was 1 g/L in the solvent THF. Following the test guideline OECD 201, the maximum solvent use is 0.1 mL/L.
Any modifications allowing for possible higher recoveries of the test item would contradict the conditions given in the guideline (i.e. temperature) or compromise the biological system (i.e. higher solvent doses).
Inside the conditions of the test guideline and the necessary conditions for the biological system, a higher concentration than 0.1 mg test item/L cannot be achieved.
Therefore, the test item concentration of 0.1 mg/L is considered to represent the maximum solubility in test medium under test conditions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 48 hr growth: 0.574 mg/L;
- EC50 48 hr yield: 0.337 mg/L

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item Concentration [mg/L]	Replicate   No.	Growth rate   [d-1]	Growth rate inhibition [%]	Yield   [cells/mL]	Inhibition of yield [%]
0.100	1	1.90	5	1631041	25
	2	1.92	4	1761924	19
	3	1.88	6	1529840	30
	4	1.81	9	1259924	42
	5	1.91	4	1718560	21
	6	1.96	2	1977561	10
	Mean	(+)* 1.90	5	(+) 1646475	25
Solvent control	1	1.99		2150228
	2	2.00		2201890
	3	1.99		2174528
	4*	1.99		2140696
	5	1.98		2109679
	6	2.02		2334453
	Mean	1.99		2185246
Control	1	1.97		2035248
	2	1.98		2080835
	3	2.00		2250539
	4*	1.99		2133139
	5	2.00		2240217
	6	2.00		2239427
	Mean	1.99		2163234

 

 

Validity criteria fulfilled:

yes

Conclusions:

For the test item N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) no effects on growth rate were observed up to the limit of solubility of the test item in the test medium and under the test conditions.

Executive summary:

The toxicity of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3.The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5523 cells/mL. For preparation of the test concentration of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a stock solution was prepared inTHF+0.1% formic acid(solvent). The stock solution was treated with ultrasound for 60 minutes at room temperature.The test concentration was prepared by adding 0.1 mL/L of the appropriate stock solutions to the dilution water.

Sixreplicates were used for the limit concentration, the control and solvent control.

The environmental conditionswere within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of thetest item N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration, the solvent control and control.

The measured concentration in the limit concentration was 106% at the start of the exposure (0 hours) and 37% at the end of the exposure (72 hours), representing 35% of the initially measured test item concentration.

Since the limit of solubility of the test item in test medium under test conditions is considered to be 0.1 mg/L and the initially measured concentration was inside the nominal range, all effect values given are based on the nominal test item concentration of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide).

No effects on growth rate were observedup to the limit of solubility of the test item in the test medium and under the test conditions.

 

NOEC 72 hr.: ≥ 0.100 mg/L

ErC10 72 hr.: > 0.100 mg/L

ErC20 72 hr.: > 0.100 mg/L

ErC50 72 hr.: > 0.100 mg/L

 

The study meets the validity criteria of the guideline.

 

Description of key information

No effects on growth rate of green alga Pseudokirchneriella subcapitata were observed up to the limit of solubility of the test item N,N’-[(methylimino)bis(trimethylene)]bis(stearamide).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.1 mg/L

Additional information

The toxicity of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5523 cells/mL. For preparation of the test concentration of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a stock solution was prepared in THF+0.1% formic acid (solvent). The stock solution was treated with ultrasound for 60 minutes at room temperature. The test concentration was prepared by adding 0.1 mL/L of the appropriate stock solutions to the dilution water.

Six replicates were used for the limit concentration, the control and solvent control.

The environmental conditionswere within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of thetest item N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration, the solvent control and control.

The measured concentration in the limit concentration was 106% at the start of the exposure (0 hours) and 37% at the end of the exposure (72 hours), representing 35% of the initially measured test item concentration.

Since the limit of solubility of the test item in test medium under test conditions is considered to be 0.1 mg/L and the initially measured concentration was inside the nominal range, all effect values given are based on the nominal test item concentration of N,N’-[(methylimino)bis(trimethylene)]bis(stearamide).

No effects on growth rate were observed up to the limit of solubility of the test item in the test medium and under the test conditions.

 

NOEC 72 hr.: ≥ 0.100 mg/L

ErC10 72 hr.: ≥ 0.100 mg/L

ErC20 72 hr.: ≥ 0.100 mg/L

ErC50 72 hr.: ≥ 0.100 mg/L

 

The study meets the validity criteria of the guideline.