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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.09.2021 to 23.09.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

other: Study plan

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No. 2016/266.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006 (Annex 5 corrected: 28 July 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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:

Limit test
- Concentrations: The samples for analysis were taken from the stock solution (100 mg·L-1) with and without algae.
- 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 were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed 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 solutions of the test item were prepared in the test medium. 100 mg of the test item was weighed into 1000 mL of the test medium for the preliminary and limit test. The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with test medium.
The exact concentrations are given on tables in Chapter 4.1. of report.

The stock solution of the test item in the test medium was prepared for the preliminary/limit test. The test was performed in a range nominal concentrations from 1 mg·L-1 to 100 mg·L-1. Testing mixtures were prepared by dosing appropriate volumes of stock solution of the test item and inoculum into volumetric flasks. Testing mixtures were incubated in the Erlenmeyers’ flasks. The test contained three (preliminary) or six parallel series (limit test) of the test item concentrations and three (six) controls without the test item. The volume of the test solution was 50 mL in each testing flask. The initial density of 5 000 cells per mL was used in the test. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): The collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21-24°C controlled at +/- 2°C; limit test 22.0 – 22.5 °C

pH:

8

Conductivity:

1.74 µS·cm-1

Nominal and measured concentrations:

Limit test:
nominal 100 mg.L-1, measured 98.1 mg.L-1 (0 hours) and 99.1 mg.L-1 (72 hours);
nominal 100 mg.L-1 w.a. (without algae), measured 98.9 mg.L-1 (0 hours) and 101.2 mg.L-1 (72 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyers flasks with stoppers venting air
- Aeration: no
- Initial cells density: 5000 cells in a 1 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water with conductivity smaller than 5 μS.cm-1 was used for the preparation of solutions. All chemicals used were of analytical-grade.
- According to Method C.3 Algal Inhibition Test were prepared four basic stock solutions. The test medium was prepared by admixing of basic stock solutions. The solutions were kept in the dark at temperature of 4 +/- 1°C in fridge.

OTHER TEST CONDITIONS
- Lighting: continuous white light, 4 440 lux to 8 880 lux; limit test 7 600 – 7 620 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The density of algae culture was evaluated microscopically at 24, 48 and 72 hours.
(At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. )

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 100, 50, 10, 5 and 1 mg.L-1 (nominal concentration)
Based on low toxicity of the test item found in the preliminary test, the limit test was performed subsequently.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOECy

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Results with reference substance (positive control):

- Results with reference substance valid?
Yes. The inhibition of test organisms by the reference substance (72h – ErC50) obtained from last reference test meets the calculated range from the interlaboratory test.

Reference test: 72 hour – ErC50 = 1.01 mg.L-1 (95% confidence limit: 0.66 – 1.53 mg.L-1)
Interlaboratory test: 72 hour – ErC50 = 0.57 – 1.33 mg.L-1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 1.0 % and inhibition of yield was 3.7 % in the limit test. Therefore exact values of ErC50 and EyC50 could not be calculated and the value of EC is given in the form of a range.
The determination of NOEC value was done by evaluation of the results from preliminary test using statistical software ToxRat Professional Version 3.3.0

72 hour – ErC50 > 100 mg·L-1 (nominal concentration)
72 hour – EyC50 ˃ 100 mg·L-1 (nominal concentration)
72 hour – NOECr ˃ 100 mg·L-1 (nominal concentration)
72 hour – NOECy ˃ 100 mg·L-1 (nominal concentration)

Executive summary:

The test item, 4H-1,2,4-triazol-4-amine, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No.2016/266.

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

The inhibition of growth rate was 0.8 % in the highest concentration and the inhibition of yield was 3.0 %  in the highest concentration in the preliminary test. Based on low toxicity of the test item found in the preliminary test, the limit test was performed subsequently.

Samples for analytical determination were taken at the beginning and at the end of the test. The analytical results showed, that the test item was sufficiently stable in test medium in the conditions of the test, therefore the limit test was performed as static one.

The concentration of 100 mg·L^-1 was tested in the limit test. The inhibition of growth rate was 1.0 % and inhibition of yield was 3.7 %.

Samples for analytical determination were taken at the beginning and at the end of the test.

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.

The nominal concentrations were used for all evaluations and results.

Test results:

72 hour – E_rC₅₀ > 100 mg·L^-1 (nominal concentration)

72 hour – E_yC₅₀ ˃ 100 mg·L^-1 (nominal concentration)

72 hour – NOEC_r ˃ 100 mg·L^-1 (nominal concentration)

72 hour – NOEC_y ˃ 100 mg·L^-1 (nominal concentration)