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

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.05. 2021 – 14.07. 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 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

Adopted July 17, Paris 1992.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage.

- Preparation of inoculum for exposure: The fresh collected waste water was filtered through paper filter and aerated till using. The inoculum preparation is in conformity with the recommendations of the test guideline.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

2.5 mg/L

Based on:

test mat.

Remarks:

The stock solution of the test item was prepared at a concentration of 0.1 g·L-1 in deionized water and was diluted with inoculated mineral medium in such a way that the final concentration of the test item was 2.5 mg·L-1.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Inoculated medium
The volume of waste water for the inoculation of mineral medium was chosen 2.1 mL per 1 L of medium. 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 63 mL of modified waste water. The pH value of medium: 7.4.


TEST CONDITIONS
The stock solution of the test item was prepared in concentration 0.1020 g·L-1 of deionized water. From this stock solution 147.0 mL (24.5 mL·L-1) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L. The solution prepared in this way contained 2.5 mg·L-1 of the test item. The pH value of solution: 7.4.
Concentration of stock solution: 102.0 mg·L-1
ThOD of the test/reference item: 3.045 mg·mg-1

- Composition of medium:
Preparation of 1 litre of mineral medium: 1 mL of solutions a), b), c) and d) were mixed with approx. 500 mL of water, the mixture was replenished with water up to 1 000 mL. The solution was prepared from aerated water.

Solution a)
Monopotassium dihydrogen orthophosphate, (KH2PO4) 8.50 g
Dipotassium monohydrogen orthophosphate, (K2HPO4) 21.75 g
Disodium monohydrogen orthophosphate dodekahydrate, (Na2HPO4·12H2O) 67.21 g
Ammonium chloride (NH4Cl) 0.50 g
Water up to the volume of 1 000 mL. The right composition of medium was checked by the determination of pH value, which must be 7.4.

Solution b) 22.50 g Magnesium sulphate heptahydrate, (MgSO4·7H2O) in 1 000 mL of water
Solution c) 27.50 g Calcium chloride, anhydrous, (CaCl2) in 1 000 mL of water
Solution d) 0.25 g Iron (III) chloride hexahydrate, (FeCl3·6H2O) in 1 000 mL of water

- Solubilising agent (type and concentration if used): The deionized water with conductivity less than 5 uS·cm-1 was used for the preparation of all solutions. The same batch of water was used for each set of the experiments. Analytical grade reagents were used.
- Test temperature: 20±1.0 °C
- pH adjusted: The right composition of medium was checked by the determination of pH value, which must be 7.4.
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
Test item series: 2 x 9 bottles with test item and inoculum
Reference item series: 2 x 9 bottles with reference substance and inoculum
Blank series: 2 x 9 bottles with inoculated medium only
Toxicity test series: 2 x 5 bottles with test item, reference item and inoculum for toxicity test
Series for nitrification determination: 1 x 9 bottles with test item and inoculum
The prepared dosing solutions for each series were filled in two parallel bottle replicates (except series for nitrification determination). The bottles were placed into thermostat.

- Method used to create aerobic conditions: the solution was prepared from aerated water
- Measuring equipment: oximeter WTW OXI 730
- Test performed in open system: no

SAMPLING
- Sampling frequency: at the zero-time and on the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test
- Sampling method: the appropriate bottles from each series were taken off
The first two bottles from each series were immediately submitted to analysis for dissolved oxygen (zero-time) and other bottles were placed in the thermostat.
On the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test, two bottles (parallel replicate) from the test item series, from the reference item series and from the blank series were taken off and the same determination was performed.

On the 3rd, 7th, 10th, 14th day of the test, two bottles from the toxicity test series were taken off and the same determination was performed.

On the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test, one bottle from the seriesfor nitrification determination was taken off and stored in freezer. The determinations of nitrite-N concentration and nitrate-N concentration were carried out by ion chromatography at one time at the end of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, the inoculated mineral medium
- Toxicity control: yes.
The solution was prepared by dosing 98 mL (24.5 mL·L-1) of stock solution of the test item and 10 mL (2.5 mL·L-1) of stock solution of the reference item into inoculated medium and completed with this medium to the volume of 4 L. The mixture prepared in this way contained 2.5 mg·L-1 of the test item and 2.5 mg·L-1 of sodium benzoate.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

No deviations.
Degradation is determined by analysis of dissolved oxygen over a 28-day period.
The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

2.6

Sampling time:

28 d

BOD5 / COD results

BOD5 / COD

Parameter:

ThOD

Value:

3.045 other: mg·mg-1

Remarks on result:

other: (with total nitrification)

Results with reference substance:

Sodium benzoate ThOD 1.665 mg·mg-1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In this 28-day study of ready biological degradability the degradation of 2.6 % of the test item, 4H-1,2,4-triazol-4-amine, was attained in the end of study.

Executive summary:

The test item, 4H-1,2,4-triazol-4-amine, was tested for the ready biological degradability in Closed Bottle Test.

Test performance

The test was performed according to:

-        Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008

-        OECD Test Guideline No. 301 D - Closed Bottle Test. Adopted July 17, 1992

The test item was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out.

Sodium benzoate was used as the reference item.

In parallel to the main test the toxicity test was performed.

Because the test item contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out.

The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7 – 8 at the beginning of the test.

The calculations of biological degradation are based on ThOD (theoretical oxygen demand) values of the test and reference item.

 

Validity of the test

The prescribed validity criteria in the test were fulfilled.

The test item was not inhibiting for the used inoculum.

Since all criteria of acceptability were met, this study is considered to be valid.

 

Test results

In this 28-day study of ready biological degradability the degradation of 2.6 % of the test item, 4H-1,2,4-triazol-4-amine, was attained in the end of study.