Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine

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:

May 2022 to February 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance OECD 201 guideline without deviation. The substance was a multi-constiuent of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

2012, “Water quality-Freshwater algal growth inhibition test with unicellular green algae”

Deviations:

yes

Remarks:

pH increase in control group; temperature increase above recommended in all test due to failure in incubator. These deviations did not affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006 Corrected 2011

Deviations:

yes

Remarks:

pH increase in control group; temperature increase above recommended in all test due to failure in incubator. These deviations did not affect the integrity of the study.

Principles of method if other than guideline:

- Principle of test: Water accomodated fraction (WAF) was used in accordance with the OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Feb-2019).
- Short description of test conditions: Each concentration is prepared separately, whereby the test item is added to the medium at a specific loading rate. The material is left until fully dissolved (or as dissolved as possible), then particulate matter is left to settle out of the water column. The remaining substance in the water column of the sample is transferred to the test vessels. This is done at each concentration. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Tyndall's effect is monitored to ensure minimal/no particulate matter or critical micelles are transferred to the test system. Further, chemical analysis is conducted to confirm that the correct loading has occurred, presence of the test item and to confirm there is a series of varying concentrations.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Main Constituents: N,N,N',N'-TETRABUTYLMETHYLENEDIAMINE (TBMDA) and DIBUTYLAMINE (DBA)
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 July 2022
- Purity test date: ND

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.28, 0.9, 2.9, 9.3, and 29.8 mg/L
- Sampling method: Samples were taken at the start (t=0 h) and every subsequent 24 hours till the end of the experiment. Samples for analysis were taken from all test concentrations (WAFs) and the control. Sampling consisted of single samples taken per treatment.
- Sample storage conditions before analysis:NS

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L (except for the preparation of the lowest loading rate where a 5-L glass bottle was used). A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with minimum headspace). The loading rates of the test item were weighed on weighing boats that afterwards were placed above the mixing vessels and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water and closed. Mixing was initiated with the vortex in the centre extending maximally to around 10% of the vessel depth from the top to the bottom of the vessel. After 22 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand undisturbed for 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into silanised test vessels containing a fixed amount of inoculum (5×103 cells/mL per vessel) that were immediately closed after filling with a minimum headspace. At the start of the test, the WAFs in test vessels were observed to be clear and colourless (Tyndall effect, checked via laser beam, was negative). The test was carried out without adjustment of the pH.

WAFs were prepared at 0.5, 1.4, 4.0, 11.3, and 32.0 mg/L
- Eluate: N/A
- Differential loading: N/A
- Controls: OECD algal medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Test concentration separation factor: 2.83
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None in range finder
- Other relevant information: -

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions, according to the test guidelines.
- Age of inoculum (at test initiation): not relevant
- Method of cultivation: Algae stock cultures are started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
- Acclimation period: Test medium and conditions are the same as breeding medium and conditions, no acclimation required.
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Exposure duration in line with OECD TG 201

Post exposure observation period:

Not required

Hardness:

No data

Test temperature:

23 ± 2 °C. A failure of the air conditioning in the incubation room led to a problem with the temperature control of the incubator, which remained above the high limit (> 24°C) for about 12 hours after the start of the test. However, the temperature returned close to normal values during the rest of the test (average value: 24.98°C).

pH:

0 h: 8.03 - 8.60; 72 h: 8.79 - 10.7 (Increase in pH above the recommended pH levels within the control treatment. The cause of the pH increase in the control was possibly due to the substantial algal growth in conjunction with closed conditions used in the test. This did not sufficiently negatively impact algal growth in the control treatment.)

Dissolved oxygen:

No data

Salinity:

NA

Conductivity:

No data

Nominal and measured concentrations:

Range finder:
TMBDA: 43 - 63 % nominal at the end of the test. Start of the test recoveries were 80 - 120 %.
DBA: within 80 - 120 % nominal for the entire duration of the test.

See Table 1 and 2 in "any other information on methods incl. tables.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Minimal
- Volume of solution: 100 mL
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Not required
- Renewal rate of test solution (frequency/flow rate): Not relevant
- No. of cells per vessel (starting density ): 1 x 10^4 cell/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): No vehicle used, not required.
- Biomass loading rate: 1 x 10^4 cells/mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (OECD algal medium), as prescribed by OECD TG 201
- Total organic carbon: Not measured
- Particulate matter: None
- Metals: None
- Pesticides: None
- Chlorine: None
- Alkalinity: Not measured.
- Ca/mg ratio: <20 ppm
- Conductivity: Not measured.
- Salinity: Not measured.
- Culture medium different from test medium: No
- Intervals of water quality measurement: start and end of the test for pH, light intensity at the start of the test in 5 positions, temperature will be monitored continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity: 60-120 μE.m-2.s-1 (equivalent range: 4440-8880 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 72hour, NOEC, EC10 and EC50 for yield and growth

VEHICLE CONTROL PERFORMED: Not relevant

RANGE-FINDING STUDY
- Test concentrations: 0, 1, 3.2, 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study: See table 3 in "any other information on methods incl. tables".

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.702 mg/L

95% CI:

0.259 - 1.631

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.461 mg/L

95% CI:

2.489 - 4.798

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.191 mg/L

95% CI:

2.372 - 5.805

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.943 mg/L

95% CI:

11.468 - 19.923

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The resulting algal cell densities can be seen in the any other information on results incl. tables section, growth curves can be seen in th over-all remarks, attachments section.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: None
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
- Other: -
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation or micelles as confirmed by eye and tyndalls effect. Substance was.soluble at 100 mg/L in the test medium.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.791 mg/L

Table 4. Algal cell densities during the final test (expressed as density of algal cells/mL×10⁴).

	Replicate	Nominal concentration (loading rate) and corresponding analytically confirmed concentration (geometric mean)  (mg/L)
		Control (0)	0.5 (0.53)	1.4 (1.70)	4.0 (4.13)	11.3 (12.14)	32.0 (30.50)
t=24 h	1	8.4	7.6	11.6	5.6	3.2	0.8
	2	8.8	11.2	10.0	6.4	3.6	0.8
	3	11.6	8.4	7.6	11.2	2.8	0.8
	4	10.0
	5	9.2
	6	9.2
	Mean	9.5	9.1	9.7	7.7	3.2	0.8
	Std. Dev.	1.14	1.89	2.01	3.03	0.40	0.00
t=48 h	1	37.2	26.4	35.2	18.4	3.6	0.4
	2	37.6	23.2	23.6	25.6	8.0	0.8
	3	34.4	26.0	25.2	15.2	4.4	0.4
	4	36.0
	5	30.0
	6	36.8
	Mean	35.3	25.2	28.0	19.7	5.3	0.5
	Std. Dev.	2.85	1.74	6.29	5.33	2.34	0.23
t=72 h	1	102.4	62.4	80.0	35.2	9.2	0.8
	2	104.0	86.4	72.4	63.6	18.4	2.0
	3	98.8	95.6	72.8	32.0	15.2	0.8
	4	107.2
	5	83.2
	6	88.4
	Mean	97.3	81.5	75.1	43.6	14.3	1.2
	Std. Dev.	9.48	17.14	4.28	17.39	4.67	0.69

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test    concentration.

Std. Dev.: standard deviation.

 

Validity criteria fulfilled:

yes

Remarks:

195-fold increase of control algae within 72 hours. Mean coefficient of variation for section-by-section specific growth rates in the control cultures was 8.0% in 72 h. Coefficient of variation of growth rates in replicate control cultures was 1.9%.

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 13.94 mg/L and 3.46 mg/L, respectively. The 72-hour EC10 for growth rate was 4.19 mg/L and for yield was 0.70 mg/L. The 72-hour NOEC for growth rate was 0.53 mg/L.

Executive summary:

A study was performed to assess the test item Reaction mass of N,N,N',N' tetrabutylmethylenediamine and dibutylamine for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (1), referenced as Method C.3 of Commission Regulation No. 440/2008 (2) and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23) (7).

 

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over 72 hours at the required nominal loading rates 0.5, 1.4, 4.0, 11.3 and 32 mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours and thus over several algal generations. Samples taken from the control and all test solutions were analysed at the start and every day thereafter until the end of the test to determine if concentrations of the test item were maintained.

 

The analytical results of this test showed that test item levels, represented by both constituents of the test item, were overall maintained within 80% of the nominal concentrations throughout the duration of the test. Furthermore, test item levels were found to be relatively stable (within ± 20% of the initial concentrations throughout the test), except with TBMDA at 1.4 and 32.0 mg/L where losses of approx. 40 and 30% were observed between the beginning and end of the 72-hour exposure, respectively. The evaluation of the effects on P. subcapitata were therefore based on the geometric means of the analytically determined concentrations: 0.53, 1.70, 4.13, 12.14 and 30.50 mg/L.

The E_rC_x, E_yC_x and NOEC values after 72 hours were as follows:

Parameter	Growth rate in mg/L (and 95% conf. limits)	Yield in mg/L (and 95% conf. limits)
72-hour EC10	4.191 (2.372 – 5.805)	0.702 (0.259 – 1.163)
72-hour EC20	6.332 (4.204 – 8.122)	1.214 (0.593 – 1.794)
72-hour EC50	13.943 (11.468 – 16.923)	3.461 (2.489 – 4.798)
72-hour NOEC	0.530	< 0.530

 

 

 

Description of key information

 

72 h EC50 (growth rate) 13.943 mg/L, 72 h NOEC (growth rate) 0.53 mg/L (Pseudokirchnerella subcapitata). OECD 201

Key value for chemical safety assessment

EC50 for freshwater algae:

13.943 mg/L

EC10 or NOEC for freshwater algae:

0.53 mg/L

Additional information

A study was performed to assess the test item Reaction mass of N,N,N',N' tetrabutylmethylenediamine and dibutylamine for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (1), referenced as Method C.3 of Commission Regulation No. 440/2008 (2) and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23) (7).

 

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over 72 hours at the required nominal loading rates 0.5, 1.4, 4.0, 11.3 and 32 mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours and thus over several algal generations. Samples taken from the control and all test solutions were analysed at the start and every day thereafter until the end of the test to determine if concentrations of the test item were maintained.

 

The analytical results of this test showed that test item levels, represented by both constituents of the test item, were overall maintained within 80% of the nominal concentrations throughout the duration of the test. Furthermore, test item levels were found to be relatively stable (within ± 20% of the initial concentrations throughout the test), except with TBMDA at 1.4 and 32.0 mg/L where losses of approx. 40 and 30% were observed between the beginning and end of the 72-hour exposure, respectively. The evaluation of the effects on P. subcapitata were therefore based on the geometric means of the analytically determined concentrations: 0.53, 1.70, 4.13, 12.14 and 30.50 mg/L.

The E_rC_x, E_yC_x and NOEC values after 72 hours were as follows:

Parameter	Growth rate in mg/L (and 95% conf. limits)	Yield in mg/L (and 95% conf. limits)
72-hour EC10	4.191 (2.372 – 5.805)	0.702 (0.259 – 1.163)
72-hour EC20	6.332 (4.204 – 8.122)	1.214 (0.593 – 1.794)
72-hour EC50	13.943 (11.468 – 16.923)	3.461 (2.489 – 4.798)
72-hour NOEC	0.530	< 0.530