5-nitro-2,4,6-triaminopyrimidine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according OECD 301B

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-nitro-2,4,6-triaminopyrimidine

EC Number:

213-742-7

EC Name:

5-nitro-2,4,6-triaminopyrimidine

Cas Number:

1006-23-1

Molecular formula:

C4 H6 N6 O

IUPAC Name:

5-nitropyrimidine-2,4,6-triamine

Details on test material:

- Physical state: Solid / pink to red
- Storage condition of test material: Storage at room temperature

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 09 February 2015 from the aeration tank of the plant. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 24 hours at 22 ± 2° C.
- Preparation of inoculum for exposure: At the day of exposure the suspension was washed one time with drinking water. Subsequently the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up
with drinking water and the concentration oft the sludge was adjusted to 6.0 g/L dry weight.
- Concentration of sludge: Aliquots of 7.5 mL were added to the test vessels to obtain an activated sludge concentration of 30 mg/L dry weight.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

other: TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
Solution A: KH2PO4 : 8.50 g; K2HPO4 : 21.75 g; Na2HPO4 × 2 H2O : 33.40 g; NH4Cl : 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.
Solution B: CaCl2 × 2 H2O : 36.40 g
The compound was dissolved with deionized water to 1000 mL
Solution C: MgSO4 × 7 H2O : 22.50 g
The compound was dissolved with deionized water to 1000 mL
Solution D: FeCl3 × 6 H2O : 0.25 g
The compound was dissolved with deionized water to 1000 mL
15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.
- Test temperature: 22 ± 2° C
- pH: 7.4
- pH adjusted: The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2, if necessary

TEST SYSTEM
- Culturing apparatus: 2 L incubation bottles filled up to a volume of 1.5 L.
- Number of culture flasks/concentration: blank control assays (BC), 2 test substance assays (TS), 1 inhibition control test assay (IH), 1 reference substance assay (RS)
- Method used to create aerobic conditions:
- Measuring equipment: The TIC- and DOC-analyses were performed as repeat determination, using a TOCanalyzer equipped with an auto sampler (Shimadzu TOC-5000A and/or TOC-L CSH/CSN). The system works with a combustion/non-disperse infrared gas analysis method. For calibration of the TOC-Analyzer, standard samples were measured before start of measurements to prove the conformity with the calibration curve. The samples for TICanalysis (absorption solution) were measured without further treatment. The samples for the DOC-analysis were centrifuged for about 15 minutes at 4000 rpm. The samples were analyzed on the day of sampling.

EXPERIMENTAL PROCEDURE
The Carbon Dioxide Evolution Test was performed in 2 L incubation bottles filled up to a volume of 1.5 L. The bottles were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes. Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately. The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount. The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.
The test assays were prepared at the day of exposure. First, the required volumes of deionized water and the solutions of mineral salts were dosed to all test vessels. For preparation of the test vessels with test substance, the required amounts of the test substance aliquots for a test concentration of 20 mg/L TOC were weighed onto small plastic cups and completely added / plastic cups to the vessels of the test substance assays and to the vessel of the inhibition control. Because of poor water solubility of test substance these test assays were treated for few minutes in an ultrasonic bath to
ensure an even distribution of test substance in test medium. Finally enough reference substance stock solution was added to reach 20 mg TOC/L in the reference substance assay and 20 mg TOC/L in the inhibition control.
The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2, if necessary. Aliquots of activated sludge suspension were added to all test vessels, to adjust the concentration of activated sludge to 30 mg/L dry weight. Samples for DICmeasurement (validity criterion) from the blank control assays were taken. For determination of the decrease of dissolved organic carbon (DOC) samples were taken from the test vessels of the blank control and from the test vessel of the reference substance control and the DOC content was determined after centrifugation (approx. 15 minutes at 4000 rpm). At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers. At the end of exposure after 28 days, the pH values were measured in the other test vessels. For stripping of carbon dioxide, dissolved in the test medium, each test vessel
was acidified by adding 2 mL of concentrated hydrochloric acid. The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

- Degree of biodegradation of the reference substance after 14 days: > 60 % CO2/ThCO2 (1)
- Degree of biodegradation in the inhibition control after 14 days: 34 % CO2/ThCO2

(1) Remark: Due to a technical disturbance at the test vessel of the reference substance assay the assay was acidified on day 14 and the carbon dioxide, dissolved in the test medium was stripped overnight. For the evaluation of the amount of produced carbon dioxide concerning the required validity criterion the test assays of blank control were not considered. The data of the additional DOC measurement and the evaluation of the carbon dioxide measurement show clearly that the required validity criterion of the degree of biodegradation of the reference substance after 14 days was met.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is not readily biodegradable according OECD criteria.