N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

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:

2021-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

version from 18 June 2019

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of the test item concentrations in the test solutions was performed to determine exposure concentration of the test item during the exposure period. Substance specific analysis was performed at the test site menal GmbH.
Each sample of 4 mL was filled directly into a 12 mL glass vial. To each vial, 4 mL acetonitrile was
added and samples were stored in the freezer (≤ -18 °C). The sampling was conducted according to the following specification:
After 0 h, 48 h and 96 h exposure, NC and A were sampled; 2 samples per treatment group.
Of each sampled treatment, the samples from 0 h, 48 h and 96 h were sent to the analytical
laboratory at the test site menal GmbH for chemical analysis using an insulated box with thermal
packs. Detailed information of the analytical method is given in the analytical phase report (attached).

Vehicle:

yes

Details on test solutions:

The stock solution was prepared by adding 622 μL (corresponds to 522.5 mg based on a specific
gravity of 0.84 g/mL) test item to 8 L test medium and shaking rigorously. This stock solution was
adjusted to pH 8.5 (from pH 9.5) and used as single test item concentration in the test according to
the following scheme. One replicate with seven fish was prepared for the test item treatment as well
as for the negative control (NC).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Source: in-house breeding of Hydrotox GmbH
- Age at study initiation (mean and range, SD): ca. 2.5 months old juveniles
- Length at study initiation (length definition, mean, range and SD): 23.8 mm
- Weight at study initiation (mean and range, SD): mean wet weight of 149.9 mg
- Method of breeding: in-house breeding
- Maintenance of the brood fish: They are kept in glass tanks, equipped with external filters, at ≤ 1 fish/L

ACCLIMATION
- Acclimation period: Seven days
- Acclimation conditions (same as test or not): The fish were kept under test conditions in test medium at 21.8 – 23.3 °C, with 14 h light : 10 h dark and aeration with air pumps
- Type and amount of food during acclimation: commercial fish food (TetraMin) until 24 h before test start, when feeding was stopped.
- Feeding frequency during acclimation: Animals were fed every working day until 24 h before test start. On workdays, they are fed with commercial fish food (TetraMin flake food, Tetra GmbH, Melle, Germany) and two times per week with freshly hatched Artemia salina larvae.
- Health during acclimation (any mortality observed): The vitality of the fish was assessed after 24 h, 48 h, 72 h and 96 h. 96 h exposure.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

The vitality of the fish was assessed after 24 h, 48 h, 72 h and 96 h exposure.

Test temperature:

26 ± 2 °C

pH:

7.5 – 7.7 in the control and 7.9 – 8.5 in the test item treatment

Dissolved oxygen:

The dissolved oxygen concentration was 8.1 – 8.5 mg/L in the control and 8.1 – 8.4 mg/L in the
test item treatment (> 60 % of the air saturation).

Conductivity:

The conductivity was 681 – 691 μS/cm in the control and 733 – 742 μS/cm in the test item
treatment

Nominal and measured concentrations:

In the present study, the Threshold Approach for Acute Fish Toxicity according to OECD Series on Testing and Assessment No. 126 was applied. An Algal Growth Inhibition Test (OECD 201; Report No. 1517, Hydrotox GmbH, 16 September 2020) and a Daphnia magna Acute Immobilisation Test (OECD 202; Report No. 1518, Hydrotox GmbH, 16 September 2020) were performed before the start of this study. The lowest EC50 from these two tests resulted from the Algal Growth Inhibition Test: ErC50 (72 h) = 31.4 mg/L test item (=> threshold test item concentration).
According to the decision of the study monitor, this acute fish limit test was performed applying a nominal concentration of 65.3 mg/L test item which was, therefore, even higher than the threshold test item concentration (ErC50 (72 h) from the Algal Growth Inhibition Test). The absence of any mortality as well as no observable effects on appearance and behavior in the present study indicates that, according to the Threshold Approach for Acute Fish Toxicity, fish is not the most sensitive group of test organisms after short-term exposure and no further testing is required.

Details on test conditions:

Preparation of test treatment and control
The stock solution was prepared by adding 622 μL (corresponds to 522.5 mg based on a specific
gravity of 0.84 g/mL) test item to 8 L test medium and shaking rigorously. This stock solution was
adjusted to pH 8.5 (from pH 9.5) and used as single test item concentration in the test according to the following scheme. One replicate with seven fish was prepared for the test item treatment as wellas for the negative control (NC).

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 65.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

As no fish died until the end of the exposure, the mortality in the control as well as in the test item
treatment was 0 %.
No obvious effect on appearance and behaviour of the fish in the test item treatment was observed.
All test organisms in the control as well as in the test item treatment appeared healthy and lively
throughout the exposure.
According to the decision of the study monitor, this acute fish limit test was performed applying a nominalconcentration of 65.3 mg/L test item which was, therefore, even higher than the threshold test itemconcentration (ErC50 (72 h) from the Algal Growth Inhibition Test). The absence of any mortality as well asno observable effects on appearance and behavior in the present study indicates that, according to the
Threshold Approach for Acute Fish Toxicity, fish is not the most sensitive group of test organisms aftershort-term exposure and no further testing is required.

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria
No mortality of test organisms occurred in the control until the end of the test and was therefore
≤ 1 fish.
 Dissolved oxygen concentration in the control and the test item treatment throughout the test was
≥ 8.1 mg/L and therefore ≥ 60 % of the air saturation.
 Analytical determination of test item concentrations was performed

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 65.3 mg/L