4H-1,2,4-triazol-4-ylamine

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:

06.09.2021 to 10.09.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted: April 13, 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

report annex: Test Facility GLP Certificate

Test material

Test material information

Constituent 1

Reference substance name:

4H-1,2,4-triazol-4-ylamine

EC Number:

209-533-5

EC Name:

4H-1,2,4-triazol-4-ylamine

Cas Number:

584-13-4

Molecular formula:

C2H4N4

IUPAC Name:

4H-1,2,4-triazol-4-ylamine

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the highest (100 mg·L-1) and the lowest test concentration (1.0 mg·L-1)

- Sampling method: The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed on the day of delivery.

- Sample storage conditions before analysis: All samples were stored at laboratory temperature.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution of the test item was prepared in the dilution water. 50 mg of the test item was weighed into 500 mL of the dilution water for the preliminary test. The concentrations
of solutions used in the preliminary test were obtained by dilution of the stock solution with dilution water. The exact concentrations are given on tables in Chapter 4.1. of study report.

The stock solution of the test item in dilution water was prepared for the preliminary test. The test was performed in a range of nominal concentrations from 1 mg·L-1 to 100 mg·L-1. The test solutions were prepared by dosing of appropriate volumes of the stock solution into volumetric flasks and replenishment up to mark with dilution water. Control test was performed concurrently with dilution water only. The test was performed in four replicates. The volume of the test solution was 50 mL in each test beaker. 5 animals were put into each beaker. Total number of daphnia used for each test concentration and control was 20. The daphnia immobilisation was observed and recorded after 24 and 48 hours. At the beginning and at the end of the test the oxygen content and pH values were measured. The ambient temperature was recorded throughout the test. The conductivity of deionized water was measured at the beginning of the test. The content of Ca + Mg were measured in dilution water at the beginning of the test.

Using the procedures described in EU method, a limit test may be performed at 100 mg·L-1 of test item in order to demonstrate that the EC50 is greater than this concentration. The limit test should be performed using 20 daphnids with the same number in the control. Because the preliminary test was performed according criteria of limit test, results are in line with EU method requirements, further testing is not needed and the study was terminated.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Strauss
- Source: own laboratory breeding
- Age at study initiation (mean and range, SD): young daphnia born in 24 hours
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no

ACCLIMATION
- Acclimation period: The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: a mixture of algae

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Content of (Ca+Mg) in dilution water: 2.5 mmol.L-1

Test temperature:

in general 20 +/- 2°C, with a variation of +/- 1°C at each individual test;
test range of temperature: 18.9 – 19.8 °C

pH:

8

Dissolved oxygen:

8.0 mg.L-1

Conductivity:

1.94 uS/cm

Nominal and measured concentrations:

Nominal concentrations: 100, 50, 10, 5, 1 mg·L-1
The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (49 mg.L-1) and the lowest (10 mg.L-1) test concentration:
Measured concentration: 1.05 mg.L-1 and 100.3 mg.L-1 (0 hours), 1.02 mg.L-1 and 102.4 mg.L-1 (48 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: open beakers
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: one daphnia per 50 mL of test solution (least 2 mL per individual)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard dilution water was prepared just before the test.
- The dilution water should be of pH 7.8 +/- 0.2. The sum of Ca + Mg ions should be approx. 2.5 mmol.L-1.
- Ca/mg ratio: 2.5 mmol.L-1
- Conductivity: 1.94 uS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The daphnia immobilisation was observed after 24 and 48 hours.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: The test was performed in range nominal concentrations from 1 mg.L-1 to 100 mg.L-1.
Using the procedures described in EU method, a limit test may be performed at 100 mg·L-1 of test item in order to demonstrate that the EC50 is greater than this concentration. The limit test should be performed using 20 daphnids with the same number in the control. Because the preliminary test was performed according criteria of limit test, results are in line with EU method requirements, further testing is not needed and the study was terminated.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? Yes. The immobilisation of test organism by the reference substance (48h – EC50) obtained from last reference test meets the calculated range from the interlaboratory test.

Reference test:
48 hour – EC50 = 0.73 mg.L-1 (95% confidence limit: 0.63 – 0.85 mg.L-1)
Interlaboratory test:
48 hour – EC50 = 0.50 – 1.16 mg.L-1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The experiment with the nominal concentration of 100 mg·L-1 included in the preliminary test has been performed using 20 daphnia, therethrough the conditions of the limit test given
in guidelines have been fulfilled. It was demonstrated that the EC50 is higher than this concentration.
No immobilisation was observed in the test. Therefore exact values of EC50 could not be calculated and the values of EC are given in the form of a range. The EC0 and EC100 values are identified by direct observation.
24 hour – EC50 > 100 mg·L-1 (nominal concentration)
48 hour – EC50 > 100 mg·L-1 (nominal concentration)
24 hour – EC0 = 100 mg·L-1 (nominal concentration)
48 hour – EC0 = 100 mg·L-1 (nominal concentration)
24 hour – EC100 > 100 mg·L-1 (nominal concentration)
48 hour – EC100 > 100 mg·L-1 (nominal concentration)

Executive summary:

The test item, 4H-1,2,4-triazol-4-amine, was tested in acute immobilisation test on Daphnia magna.

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The preliminary test was performed in a range of the test item nominal concentrations 1 – 100 mg·L^-1. The test was performed as static.

Based on no toxicity of the test item found in the preliminary test, the study was terminated. The experiment with the concentration of 100 mg·L^-1 included in the preliminary test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guideline have been fulfilled.

It was demonstrated that the EC₅₀ is higher than this concentration.

There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test item has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values.

In this study concentration of the test item has been satisfactorily maintained within ± 20 percent of the nominal concentration throughout the test.

Further the nominal concentrations were used for all evaluation and results.

Test results:

24 hour – EC₅₀ > 100 mg·L^-1              (nominal concentration)

48 hour – EC₅₀ > 100 mg·L^-1              (nominal concentration)

24 hour – EC₀ = 100 mg·L^-1                (nominal concentration)

48 hour – EC₀ = 100 mg·L^-1                (nominal concentration)

24 hour – EC₁₀₀ > 100 mg·L^-1            (nominal concentration)

48 hour – EC₁₀₀ > 100 mg·L^-1            (nominal concentration)