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

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:

3 December 2021 to 14 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:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

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: N/D

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: 26, 36, 51, 71 and 100 mg/L
- Sampling method: Samples will at least be taken at start (t=0 h) and at the end of the test (t=48 h). Samples for analysis will be taken from all test concentrations (WAFs) and the control. Sampling will consist of single samples taken per treatment.
- Sample storage conditions before analysis: TBC

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The study was carried out using WAFs (Water Accommodated Fractions). A WAF was prepared for each concentration separately. The WAFs were prepared under closed conditions and by slow-stirring.
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. A magnetic stirring bar was placed in each vessel and test medium was added. The loading rates of the test item were weighed on weighing boats that afterwards were placed above the mixing vessels and rinsed with test medium. The mixing vessels were then carefully filled with the remaining volume of test medium and closed. Mixing were 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 24 ± 2 hours of gentle stirring in the dark at room temperature, the contents of the vessel were allowed to stand undisturbed for at ≥1 hour before use. The first 100 mL was discarded via the drain port. Then the WAFs were directly added into test vessels and immediately sealed with screw caps after introduction of daphnids. The test was carried out without adjustment of the pH.

WAFs will be prepared at 26, 36, 51, 71 and 100 mg/L
- Eluate: N/A
- Differential loading: N/A
- Controls: Elendt M4 medium only
- 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: 1.4
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Age at study initiation (mean and range, SD): < 24 hours old at the test initiation and were not from first brood progeny
- Weight at study initiation (mean and range, SD): Not required
- Length at study initiation (length definition, mean, range and SD): Not required
- Stage and instar at study initiation: Juvenile, 1st instar
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not relevant
- Peripheral shell growth removed prior to test initiation: Not relevant
- Method of breeding: Daphnids are cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessels containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water is changed at least once a week. These culture conditions allow to maintain the daphnids in the parthenogenetic reproductive stage.
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Age of parental stock (mean and range, SD): Beyond first brood.
- Feeding during test: None, in line with OECD TG 202


ACCLIMATION
- Acclimation period: Test medium and conditions are the same as breeding medium and conditions, no acclimation required
- Acclimation conditions (same as test or not): -
- Type and amount of food: -
- Feeding frequency: -
- Health during acclimation (any mortality observed): -


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: See above

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Standard duration as specified in the OECD 202.

Post exposure observation period:

Not relevant.

Hardness:

140 and 250 mg/L (as CaCO3)

Test temperature:

20 °C ± 2 °C

pH:

6 - 9

Dissolved oxygen:

> 3 mg/L

Salinity:

Not relevant

Conductivity:

Not measured

Nominal and measured concentrations:

Range finder:
TMBDA: 92 - 147 % nominal.
DBA: 92 - 121 % nominal.

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

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: None
- Volume of solution: 60 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 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): No vehicle used, not required.
- Biomass loading rate: Not relevant

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), as prescribed by OECD TG 202
- Total organic carbon: Not measured
- Particulate matter: None
- Metals: None
- Pesticides: None
- Chlorine: None
- Alkalinity: Not measured.
- Ca/mg ratio: Not measured.
- Conductivity: Not measured.
- Salinity: Not measured.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH and dissolved O2 will be measured at the start and end of the test. Temperature will be continuously measured.
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h:8 h (light:dark)
- Light intensity: -

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 48 hour, EC50 for immobility.

VEHICLE CONTROL PERFORMED: Not relevant

RANGE-FINDING STUDY
- Test concentrations: 26, 36, 51, 71 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:

48 h

Dose descriptor:

EC50

Effect conc.:

72.28 mg/L

95% CI:

65.499 - 79.763

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Based on current range finder (main study ongoing)
- Behavioural abnormalities: None
- Observations on body length and weight: not required
- Other biological observations: Not required
- Mortality of control: None
- Other adverse effects control: None
- Immobilisation of control: None
- Abnormal responses: None
- 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: None

Results with reference substance (positive control):

On April 6, 2022 (KD22-001; most recent test), the 24h-EC50 was 1.223 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L).

Table 4. Acute immobilisation of daphnids after 24 and 48 hours in the final test.

 

Nominal concentration (loading rate)(mg/L)	Analytically confirmed concentration (geometric mean) (mg/L)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
				Number	Total %	Number	Total %
Control	Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
25	26.88	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
36	38.39	1 2 3 4	5 5 5 5	0 1 0 0	5	0 1 1 1	15
50	54.51	1 2 3 4	5 5 5 5	1 1 0 0	10	1 1 2 0	20
71	73.72	1 2 3 4	5 5 5 5	1 0 0 0	5	2 0 2 0	20
100	101.71	1 2 3 4	5 5 5 5	0 1 0 1	10	5 5 5 5	100

At t= 24 & 48 h, at loading rates of 36 to 100 mg/L, daphnids were distributed at the bottom of the test vessels, compared to those at 25 mg/L and those in the control (distributed across the height of the test vessels).

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test substance to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test according to OECD guideline 202. Under experimental conditions, the 48-hour EC50 value was estimated to be 72.28 mg/L, based upon analytically determined concentrations.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the EC₅₀ (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over 48 hours at the required nominal loading rates 25, 36, 50, 71 and 100 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h), in order to determine if concentrations of the test item were maintained.

The test item levels, represented by both constituents of the test item (TBMDA and DBA), were found to be stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). The evaluation of the effects on Daphnia magna was based on the geometric means of the analytically confirmed concentrations: 26.88, 38.39, 54.51, 73.72 and 101.71 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 26.88 mg/L, 15% at 38.39 mg/L, 20% at 54.51 and 73.72 mg/L, and 100% at 101.71 mg/L.

The EC₅₀ value at the end of the test was as follows:

Time (h)	EC50 (mg/L)	95% confidence limits (mg/L)
48	72.280	65.499 – 79.763

 

 

 

Description of key information

 

48 h EC50 72.28 mg/L Daphnia magna, OECD 202 (2004). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

72.28 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the EC₅₀ (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over 48 hours at the required nominal loading rates 25, 36, 50, 71 and 100 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h), in order to determine if concentrations of the test item were maintained.

The test item levels, represented by both constituents of the test item (TBMDA and DBA), were found to be stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). The evaluation of the effects on Daphnia magna was based on the geometric means of the analytically confirmed concentrations: 26.88, 38.39, 54.51, 73.72 and 101.71 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 26.88 mg/L, 15% at 38.39 mg/L, 20% at 54.51 and 73.72 mg/L, and 100% at 101.71 mg/L.

The EC₅₀ value at the end of the test was as follows:

Time (h)	EC50 (mg/L)	95% confidence limits (mg/L)
48	72.280	65.499 – 79.763