3-[ethyl[4-[(4-nitrophenyl)azo]phenyl]amino]propiononitrile

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

Study period:

4 October 2017 to 1 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Secondary effluent, microorganisms from a domestic waste water treatment plant, was used in the study. The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).

Duration of test (contact time):

28 d

Initial conc.:

2.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Adequate amount of the stock solutions were combined and filled up with deionised water to the appropriate final volume (ratio of the stock solutions in line with guideline). The test medium was aerated for 20 minutes and allowed to stand for 20 hours at test temperature. The dissolved oxygen concentration was 9 mg/L at approximately 22°C.
- Test temperature: 21.4-23.2ºC
- pH: 7.22
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 300 mL BOD bottles with special neck and glass stoppers
- Number of culture flasks/concentration: 10 bottles containing each of (1) the test material and inoculum, (2) the reference item and inoculum, (3) inoculum only and (4) the test material, reference item and inoculum.
- Measuring equipment: The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode
- Other:
Preparation of test flasks:
Test Material (flasks 1a and 1b) - Based on the ThOD of 2.62 mg O2/mg test material, 7.8 mg of test material was thoroughly mixed into 3.4 litres of aqueous test medium (corresponding to 2.2 mg/L test material, with a ThOD of about 6.026 mg O2/L).
Procedure control (sodium benzoate) - Based on the ThOD of Sodium benzoate (1.67 mg O2 per mg), stock solution (concentration 360 mg/L) corresponding to 12.24 mg of Sodium benzoate was mixed into 3.4 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
Inoculum control (flasks 3a and 3b) - Only filtered inoculum was added to 3.4 litres of aqueous test medium.
Toxicity control (flasks 4a and 4b) - The test material (7.8 mg) and the reference item stock solution (34 mL at a concentration of 360 mg/L) were mixed into 3.4 litres of aqueous test medium corresponding to 2.3 mg/L test material (ThOD of 6.026 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).

SAMPLING
- Sampling frequency: days 0, 7, 14, 21 and 28 in duplicate

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

STATISTICAL METHODS:
Calculation of BOD:

(mgO2/L of T.i. and/or R.i. - mg O2/L of i.control) / (mg T.i. and/or R.i./L in flask)

where:
T.i. = test item
R.I. = reference item
i. control = inoculum control

Calculation of biodegradation %

((BOD (mg O2/mg T.i. or R.i.)) / (ThOD (mgO2/mg T.i.) or ThOD (mg O2/mg R.i.))) x 100

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

1.7

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test material reached a mean of 1.7% after 28 days based on the ThOD of the test material. According to the test guidelines the pass level for ready biodegradability is removal of 60% ThOD. Therefore the test material is considered not readily biodegradable.

In the toxicity control containing both the test material and the reference item, a mean of 37.9% biodegradation was noted within 14 days and 40.7% biodegradation after 28 days of incubation. According to the test guidelines the test material can be assumed to be not inhibitory at the applied concentration level of 2.3 mg/L on the secondary effluent microorganisms because degradation was > 25% within 14 days.

Key result

Parameter:

BOD5

Value:

0.045 mg O2/g test mat.

Results with reference substance:

The reference item was sufficiently degraded to a mean of 78.3% after 14 days, and to a mean of 83.3% after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

BOD at different time intervals during the 28 day exposure period

Treatment	Concentration (mg/L)	Flask No.	BOD after n days of exposure
			7	14	21	28
Test material	2.3	1a	-0.07	-0.07	0.15	0.07
		1b	-0.02	-0.02	0.15	0.02
Reference item	3.6	2a	1.18	1.29	1.38	1.38
		2b	1.15	1.32	1.38	1.40
Toxicity control	Test material: 2.3	4a	0.79	0.84	0.87	0.87
	Reference item; 3.6	4b	0.74	0.79	0.86	0.87

Percentage biodegradation at different time intervals during the 28 day exposure period

Treatment	Concentration (mg/L)	Flask No.	Percent biodegradation after n days of exposure
			7	14	21	28
Test material	2.3	1a	-2.5	-2.5	5.8	2.5
		1b	-0.8	-0.8	5.8	0.8
		mean	-1.7	-1.7	5.8	1.7
Reference item	3.6	2a	70.8	77.5	82.5	82.5
		2b	69.2	79.2	82.5	84.2
		mean	70.0	78.3	82.5	83.3
Toxicity control	Test material: 2.3	4a	36.8	39.1	40.7	40.7
	Reference item: 3.6	4b	34.4	36.8	39.9	40.7
		mean	35.6	37.9	40.3	40.7

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of the study, the test material was considered to be not readily biodegradable. The test material was also not considered to have significant inhibitory effects on secondary effluent microorganisms.

Executive summary:

The ready biodegradability of the test material was investigated in a Closed Bottle Test over 28 days in line with standardised guidelines OECD 301D, EPA OPPTS 835.3110 and EU Method C.4 -E, and under GLP conditions. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Sodium benzoate was tested simultaneously as a reference item. The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions, the percentage biodegradation of the test material reached a mean of 1.7% after 28 days based on the ThOD of the test material. As the pass level for ready biodegradability is removal of 60% of ThOD, the test material is considered not readily biodegradable. The reference item was sufficiently degraded to confirm the suitability of the used inoculum. In the toxicity control containing both the test material and the reference item, a mean of 37.9% biodegradation was noted within 14 days and 40.7% biodegradation after 28 days of incubation. The test material can be assumed to be not inhiitory at a concentration of 2.3 mg/L on the secondary effluent microorganisms.

Description of key information

Under the test conditions, the percentage biodegradation of the test material reached a mean of 1.7% after 28 days based on the ThOD of the test material.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in a Closed Bottle Test over 28 days in line with standardised guidelines OECD 301D, EPA OPPTS 835.3110 and EU Method C.4 -E, and under GLP conditions. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. Sodium benzoate was tested simultaneously as a reference item. The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions, the percentage biodegradation of the test material reached a mean of 1.7% after 28 days based on the ThOD of the test material. As the pass level for ready biodegradability is removal of 60% of ThOD, the test material is considered not readily biodegradable. The reference item was sufficiently degraded to confirm the suitability of the used inoculum. In the toxicity control containing both the test material and the reference item, a mean of 37.9% biodegradation was noted within 14 days and 40.7% biodegradation after 28 days of incubation. The test material can be assumed to be not inhiitory at a concentration of 2.3 mg/L on the secondary effluent microorganisms.