2,4,6-trinitrobenzene-1,3,5-triamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

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:

guideline study

Remarks:

This study was performed according to OECD Guideline 301F with GLP statement. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of compliance with good laboratory practice according to directives 2004/9/CE and 2004/10/CE

Specific details on test material used for the study:

- Physical state: White powder

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge from a predominantly municipal WWTP.
Decantation of sludge was fast and total (no floating sludge)
Inoculum was kept at room temperature with large aeration and was not pre-adapted to the test substance. Before use, the activated sludge was washed with media after decantation.
Activated sludge were maintained in the laboratory under aeration condition before use. The concentration of suspended matter of the activated sludge used for the study was 3.28 g/L.

Duration of test (contact time):

28 d

Initial conc.:

102.68 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium ventilated until a value close to saturation in air and pH stabilization were obtained. No pH adjustment was needed.
Test media was used the day of preparation
- Test temperature: 21 °C
- Continuous darkness: Yes
- Water: Demineralised water used during the study

TEST SYSTEM
- Culturing apparatus: glass flask SCHOTT Duran
- Number of culture flasks/concentration:
2 with test media and inoculum (blank solutions = control),
2 with test media, inoculum and test item (test item suspensions),
1 with test media, inoculum and reference item (reference suspension / procedure control),
1 with test media, inoculum, test item and reference item (toxicity control),
1 with test media, test item and sterilizing agent (abiotic sterile control).
- Measuring equipment: The respirometers used during the study are Measurement system Oxitop made by WTW
- Test performed in closed vessels: Yes

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Procedure control: Yes with the reference substance
- Toxicity control: Yes

TOXICITY OF THE TEST SUBSTANCE
The toxicity of the test substance for the inoculum was checked. A flask of the volumetric respirometer was filled with mineral medium + test substance + reference substance + inoculum and their respirations were recorded as for the other flasks. If they are lower than those of the flasks containing mineral medium + reference substance + inoculum, the test substance can be assumed to be inhibitory to the inoculum used.

Reference substance:

benzoic acid, sodium salt

Remarks:

104.64 mg/L

Preliminary study:

None

Test performance:

The BOD (Biological Oxygen Demand) values were given by OXITOP OC 110 Controller and depending from the initial parameters (volume of bottle: 610 mL, filling volume: 252 mL ± 1 mL).

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

For the test item solutions abiotic control, BODs after 28 days were lower than in the controls. However, due to the results from the toxicity control this was not considered to suggest an inhibitory impact of the test substance on the inoculum.
The test item solutions and abiotic control showed no significant oxygen consumption after 28 days.

Results with reference substance:

Degradation of sodium benzoate exceeded 40% after 3 days and 65% after 4 days: the activity of the inoculum was thus verified (validity criterion).

Percentage of biodegradation

	Biodegradation rate (%)
Time (days)	Test item solution 1	Test item solution 2	Absolute difference	Reference control	Toxicity control
0	0.0	0.0	-	0.0	0.0
1	0.0	1.3	1.3	16.5	13.2
2	0.0	1.3	1.3	36.4	23.5
3	0.6	1.9	1.3	59.0	28.7
4	-0.6	1.9	2.5	73.6	39.6
5	0.0	0.0	0.0	79.4	41.1
7	-0.6	4.4	5.0	83.4	46.2
10	-1.3	1.3	2.5	89.5	48.9
14	-3.1	3.1	6.3	92.8	48.8
21	-3.1	0.6	3.8	98.8	51.4
28	-3.8	-2.5	1.3	99.3	50.1

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the test conditions, test substance should be regarded as not readily biodegradable according to this test.

Executive summary:

The ready biodegradability of the test substance has been determined by Manometric Respirometry Test according to the OECD Guideline No. 301F, EU Method C.4 -D with GLP compliance.

A nominal concentration of the test substance (102.68 mg/L) introduced in the system.

The curves obtained for the test item solutions abiotic control, BODs after 28 days were lower than in the controls. However, due to the results from the toxicity control this was not considered to suggest an inhibitory impact of the test substance on the inoculum. The test substance underwent 0% biodegradation after 28 days under the test conditions. The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled for the flasks containing test substance. Therefore, the test is considered valid.

Degradation of sodium benzoate exceeded 40 % after 3 days and 65 % after 4 days: the activity of the inoculum is thus verified.

Description of key information

OECD Guideline 301F, EU Method C.4 -D, GLP, key study, validity 1:

0% biodegradabilty after 28 days.

Not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The biodegradability study (SGS, 2018) was assessed as the key study and was performed on the registered substance according to the OECD Guideline No. 301F, EU Method C.4 -D with GLP compliance. The test substance was exposed at a concentration of 102.68 mg/L to activated sewage sludge micro-organisms with culture medium at a temperature of 21°C for 28 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. Control solutions with inoculum and the standard material (Sodium benzoate), together with a toxicity control were used for validation purposes. The curves obtained for the mineralisation of test item solutions after 28 days were lower than in the controls. However, due to the results from the toxicity control this was not considered to suggest an inhibitory impact of the test substance on the inoculum. The test substance underwent 0% biodegradation after 28 days under the test conditions. The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled for the flasks containing test substance. Therefore, the test is considered valid. Degradation of sodium benzoate exceeded 40 % after 3 days and 65 % after 4 days: the activity of the inoculum was thus verified. In conclusion, under the test conditions, test substance should be regarded as not readily biodegradable.