6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec - 27 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 Apr 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany (Karlsruhe, 10 Dec 2015)

Test material

Test material information

Constituent 1

Reference substance name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

EC Number:

254-074-6

EC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Cas Number:

38668-46-1

Molecular formula:

C9H13N7

IUPAC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

- Concentrations: All test item concentrations and the control at test start and after 24 h from fresh and aged solutions and after 48 h from aged solutions.
- Sampling method: Samples were taken from fresh media at 0 h and 24 h and from aged media at 24 and 48 h.
- Sample storage conditions before analysis: All samples were deep frozen (≤ -18 °C) until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 100 mg/L stock solution was prepared by homogenising an appropriate amount of test item with an appropriate amount of test medium by treatment with 10 min ultrasonication. Afterwards, the solution was clear and transparent. Lower test solutions were prepared by dilution of the stock solution with test medium.
- Differential loading: No
- Controls: Untreated test medium
- Evidence of undissolved material: The stock solution was clear and transparent.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone V
- Age of daphnids at test start: Freshly hatched daphnids, less than 24 h old
- Source: In-house culture of testing facility, originally purchased from the Federal Environment Agency in Berlin, Germany.
- Culture conditions: Single culture (1 daphnid per 100 mL) in aerated Elendt M4 medium, at 20 ± 2 °C and pH 6.0 - 9.0. The dissolved oxygen was > 60% saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14 °dH. The daphnids were fed with unicellular green algae (Desmodesumus subspicatus) at least 3x/week. The medium was changed 3x/week.
- Feeding during test: No feeding during test

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

214 mg/L (CaCO3) = 12 °dH (in test medium)

Test temperature:

18.8 - 20.4 °C

pH:

7.60 - 8.44

Dissolved oxygen:

≥ 8.4 mg/L

Nominal and measured concentrations:

Control, 0.01, 0.10, 1.00, 10.0, and 100.0 mg/L (nominal)
Control, -- , --, --, --, and 100 - 114 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers filled with 50 mL test solution and covered with a glass plate
- Aeration: None
- Renewal rate of test solution: After 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1 daphnid/10 mL test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium according to guideline
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Test temperature, pH and oxygen concentrations were measured in one separate replicate of each test item level (without daphnids) in fresh test solutions at t = 0 and 24 h and in aged solutions at t = 24 and 48 h

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobilization: After 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: A semi-static GLP range-finder test was performed in a limit test design with nominal test solution concentrations of 0.01, 0.10, 1.00, 10.0, and 100.0 mg/L and a control.
- Results used to determine the conditions for the definitive study: Yes, no immobilisation was observed after 48 h in the control and at test item concentrations from 0.10 to 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate, tested periodically

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No behavioural changes were observed.
- Other biological observations: After 24 h no immobilization was observed in the control and up to and including the 100 mg/L treatment level. After 48 h no immobilization occurred in the control and at test item concentrations of 0.10 - 100 mg/L. At 0.01 mg/L no immobilization higher than the permitted control immobilization was observed.
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 (24 h): 1.00 mg/L (potassium dichromate)
- Other: Reference test dating 12 Dec 2017

Reported statistics and error estimates:

Due to lack of immobilization, no statistical analysis was performed.

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the guideline (Table 1).

 

Table 1. Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	In this study the control immobilisation was 0 %.	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	In this test, the dissolved oxygen concentration at the end of the test was≥8.4 mg/L.	Yes

 

ANALYTICAL RESULTS

The measured test item concentrations in fresh samples (0 h) ranged between 100 – 109% of nominal concentrations, with a mean initial concentration of 105% of nominal. In aged samples (24 h) the measured test item concentrations ranged from 107 to 114% of nominal, with a mean measured concentration of 111% of nominal (Table 2). Therefore, the effect values were based on nominal test item concentrations.

 

 Table 2. Measured test item concentrations.

nominal test item concentration	sampling time	measured concentration
[mg/L]	[h]	[mg/L]	% of nominal
100	0 h, fresh	100	100
	24 h, fresh	109	109
	24 h aged	107	107
	48 h, aged	114	114

  

BIOLOGICAL RESULTS

After 24 h, no immobilization was observed in all test levels and the control.

After 48 h, no significant immobilization was observed in all test levels and the control. At the 0.01 mg/L treatment level, 5% immobilization (1/20 daphnids) was observed, which is below the permitted control immobilization of 10%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.