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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2022 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Principles of method if other than guideline:

- Principle of test: Water accomodated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- 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 particulate matter is transferred to the test system. Further, chemical analysis is conducted to confirm that the correct loading has occurred, presence of the test item, test item stability 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:

Batch No. HOU7520LN-46
Purity: Main constituent 1 (N,N,N',N'-tetrabutylmethylenediamine): 78.10% Main constituent 2 (Dibutylamine): 18.79%
Storage: 2-8 °C - protected from light

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions at the start (t=0h) and every 24 hours thereafter in old and new solutions.

- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

To be confirmed

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: NS
- Source:UAR TEFOR Paris-Saclay - CNRS UMS2010 / INRAE UMS1451 - Université Paris-Saclay - Bat 151, 151 route de la Rotonde - 91400 SACLAY
- Age at study initiation (mean and range, SD): juveniles
- Length at study initiation (length definition, mean, range and SD):
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Maintenance of the brood fish:

ACCLIMATION
- Acclimation period: At least 9 days under similar conditions to test
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: flaked, powdered or live food (such as brine shrimp nauplii) until 24 - 48 hours before the exposure is started.

- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed):


FEEDING DURING TEST (as applicable) NA

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Duration is in line with OECD TG 203

Post exposure observation period:

Not required

Hardness:

Between 40 and 250 mg/L (as CaCO3).

Test temperature:

23°C ± 2°C

pH:

Between 6.0 -8.5 (See "Any other information on materials and methods incl. tables" section.)

Dissolved oxygen:

≥ 60 % (8.15 - 9.02 mg/L)

Salinity:

NA

Conductivity:

< 10 µS/cm

Nominal and measured concentrations:

Nominal:5, 10, 20, 40 and 80 mg/L.

See analytical results for both active substances in "Any other information on materials and methods incl. tables" section.

Details on test conditions:

TEST SYSTEM
- Test vessel:All-glass aquaria, of approx. 3.5 L capacity closed by placing a glass plate on the aquarium and sealing with Vaseline, minimum headspace.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: See above
- Aeration: No aeration.
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): daily renewal (whole aquaria)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): NA
- Biomass loading rate: Maximum loading of 0.8 g wet weight fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, as prescribed by the OECD Guideline 203.
Stock solutions: a) CaCl2.2H2O 11.76 g/L
b) MgSO4.7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L

- Total organic carbon: The Total Organic Carbon (TOC) in the test water was ≤ 0.3 mg/L.
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Culture medium different from test medium:
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8 h dark
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish mortality, 3 and 6 h after start, then every 24 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (approx.)
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: 10, 50 and 100 mg/L of test substance.
- Results used to determine the conditions for the definitive study: In the range-finding test, no mortality was observed in the 10 and 50 mg/L treatments and 100% mortality was observed in the 100 mg/L treatment. In this test, 3 fish were exposed per treatment and pH was adjusted to an optimal level for zebrafish. Following this, a first definitive test was carried out in which 100% mortality was observed in all exposure levels (48 mg/L and above) after 3 hours (7 fish per treatment), in this test the pH was not adjusted and was higher in all exposure treatments than the zebrafish friendly upper limit of 8.5. After this, the current test concentrations were picked.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

78.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

41.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

57.1 mg/L

95% CI:

41.67 - 78.29

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Same result for 24, 48, 72 hours

Details on results:

The evaluation of the effects on Danio rerio was based on the geometric mean of the analytically confirmed concentrations, as recommended by OECD Guideline 203: 5.34, 11.22, 20.26, 41.66 and 78.29 mg/L. After the 96-hour exposure, mortality was 0% at 5.34, 11.22, 20.26 and 41.66 mg/L, and 100% at 78.29 mg/L. Therefore, the highest concentration without effect was 41.66 mg/L and the lowest concentration with 100% mortality was 78.29 mg/L.

Results with reference substance (positive control):

Not relevant

Sublethal observations / clinical signs:

Table 4. Incidence of mortality and total mortality during the final test.

 

	Nominal concentration (loading rate) and corresponding analytically confirmed concentration (geometric mean)  (mg test item/L)
Cumulative mortality	Control (0) M      S	5 (5.34) M      S	10 (11.22) M      S	20 (20.26) M      S	40 (41.66) M      S	80 (78.29) M      S
t=3 h	0       0	0       0	0       0	0       0	0       0	7^a       0
t=6 h	0       0	0       0	0       0	0       0	0       0	7       0
t=24 h	0       0	0       0	0       0	0       0	0       0	7       0
24 h Total mortality (%)	0	0	0	0	0	100
t=48 h	0       0	0       0	0       0	0       0	0       0	7       0
48 h Total mortality (%)	0	0	0	0	0	100
t=72 h	0       0	0       0	0       0	0       0	0       0	7       0
72 h Total mortality (%)	0	0	0	0	0	100
t=96 h	0       0	0       0	0       0	0       0	0       0	7       0
96 h Total mortality (%)	0	0	0	0	0	100

M: Number of dead fish; S: Number of surviving fish but with sublethal effects / visible abnormalities.

The initial number of fish in each test vessel was 7.

^aFish were found dead quickly (within 5-10 min) after introduction into the test vessel.

Validity criteria fulfilled:

yes

Remarks:

In the batch of fish that was used for the test, no mortality during the seven days prior to the start of the test was observed; air saturation was above 60 %; concentrations of the test material were stable throughout the test.

Conclusions:

The toxic effect of Reaction mass of N,N,N',N' tetrabutylmethylenediamine and dibutylamine to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 57.1 mg/L. The NOEC was 41.7 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item Reaction mass of N,N,N',N' tetrabutylmethylenediamine and dibutylamine to the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, groups of seven fish were exposed to Water Accommodated Fractions (WAFs) of the test item over 96 hours at the required nominal loading rates 5, 10, 20, 40 and 80 mg/L in aquaria closed over the study period with a minimum headspace. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 3, 6, 24, 48, 72 and 96 hours. The test medium was renewed every 24 hours to ensure consistent exposure levels. Water samples taken from the control and all WAFs were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

The analytical results of this test showed that test item levels (represented by the two tracer compounds TBMDA and DBA) were overall satisfactorily maintained within 80% of the nominal concentrations throughout the duration of the test. The evaluation of the effects on Danio rerio was based on the geometric mean of the analytically confirmed concentrations, as recommended by OECD Guideline 203 (1): 5.34, 11.22, 20.26, 41.66 and 78.29 mg/L. After the 96-hour exposure, mortality was 0% at 5.34, 11.22, 20.26 and 41.66 mg/L, and 100% at 78.29 mg/L. Therefore, the highest concentration without effect was 41.66 mg/L and the lowest concentration with 100% mortality was 78.29 mg/L.

The 96-hour LC₅₀ value was 57.1 mg/L and the NOEC was 41.7 mg/L.

 

Description of key information

96h LLC50 57.1 mg/L to Danio rerio, OECD 203 (1992) Anon., 2021.

Additional information

A study was performed to assess the acute toxicity of the test item Reaction mass of N,N,N',N' tetrabutylmethylenediamine and dibutylamine to the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, groups of seven fish were exposed to Water Accommodated Fractions (WAFs) of the test item over 96 hours at the required nominal loading rates 5, 10, 20, 40 and 80 mg/L in aquaria closed over the study period with a minimum headspace. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 3, 6, 24, 48, 72 and 96 hours. The test medium was renewed every 24 hours to ensure consistent exposure levels. Water samples taken from the control and all WAFs were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

The analytical results of this test showed that test item levels (represented by the two tracer compounds TBMDA and DBA) were overall satisfactorily maintained within 80% of the nominal concentrations throughout the duration of the test. The evaluation of the effects on Danio rerio was based on the geometric mean of the analytically confirmed concentrations, as recommended by OECD Guideline 203 (1): 5.34, 11.22, 20.26, 41.66 and 78.29 mg/L. After the 96-hour exposure, mortality was 0% at 5.34, 11.22, 20.26 and 41.66 mg/L, and 100% at 78.29 mg/L. Therefore, the highest concentration without effect was 41.66 mg/L and the lowest concentration with 100% mortality was 78.29 mg/L.

The 96-hour LC₅₀ value was 57.1 mg/L and the NOEC was 41.7 mg/L.

A supporting study also exists where full analytical verification was not possible with a derived LL50 > 100 mg/L (Anon., 2023). Other than the analytical verification methods employed were highly similar i.e. WAF method was used.