2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets requirements of OECD Guideline 301 F and GLP requirements without deviations.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 2 days.
10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.1 x 10e9 CFU/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

55 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: 20.5-21.5 °C
- pH:
pH-Values in the Test Vessels at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.77 1) 7.59
2) 7.59
Functional Control 7.79 7.86
Test Item 7.74* 1) 7.65
2) 7.62
Toxicity Control 7.74* 7.86
* measured in separate replicates

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.



SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
Concerning the OECD-guideline 301 the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No preliminary study

Test performance:

Based on the calculated theoretical oxygen demand the test con-centration of 55 mg/L corresponding to an oxygen demand of
72.6 mg O2/L in the test vessel was selected.

The test solutions were prepared in measuring flasks and given into brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass bottles (test item, toxicity control):

• two for the test item (P1, P2)
• one for the functional control (R1)
• one for the toxicity control (T1)
• two for the inoculum control (C1, C2)

Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:

• one for the test item (P1)
• one for the toxicity control (T1)

The test item was weighed out and was transferred directly into the brown glass bottle with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, deminer-alised water and 10 mL/L inoculum).
Accordingly, the test and reference item for the toxicity control were weighed out and transferred directly into the brown glass
bottle.
For the functional control the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The appropriate volumes of mineral medium stock solutions and 10 mL/L inoculum were added, then the flask was filled up with demineralised water. This test solution was given into the brown glass bottle.
The inoculum control, consisting of mineral medium stock solutions, demineralised water and inoculum, was prepared in a
500 mL measuring flask. This test solution was divided using a 250 mL measuring flask before being filled into the brown glass bottles.
A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.

Key result

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

28 d

Details on results:

Results of the Functional Control
The pass level of a biodegradation > 60 % was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.

Results of the Toxicity Control
In the toxicity control the biodegradation achieved 53 % after 14 days. A maximum of 56 % was reached after 23 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Course and Stages of Biodegradation of Functional Control and Test Item
In case of the functional control, the adaptation phase changed to degradation phase after 1 day (degradation > 10 %). The pass level > 60 % was reached after 2 days. The biodegradation rate reached 97 % on day 14 and a maximum of 99 % on day 18.

The biodegradation of the test item is shown in Table 3 and Figure 1 in comparison to the readily degradable functional control. Both test item replicates did not reach the 10 % level (beginning of biodegradation) until day 28. The mean biodegradation after 28 days was 1 %.

The test item must be regarded as not readily biodegradable in the 10-d-window and after
28 days.


Biodegradation [%] of the Test Item Hansa-Brillantgelb 2GX 70 in
Comparison to the Functional Control and Toxicity Control

Biodegradation [%]
Date [d] Functional
Control
45 mg/L Test Item

55 mg/L
Toxicity
Control
55 + 45 mg/L
R1 P1 P2 T1
15.10.08 0 0 0 0 0
16.10.08 1 59 0 3 27
17.10.08 2 75 0 4 38
18.10.08 3 79 0 4 41
19.10.08 4 82 0 4 43
20.10.08 5 84 0 2 46
21.10.08 6 84 0 2 46
22.10.08 7 87 0 1 47
23.10.08 8 89 0 1 48
24.10.08 9 93 0 1 51
25.10.08 10 94 0 2 50
26.10.08 11 95 0 1 52
27.10.08 12 95 0 1 53
28.10.08 13 96 0 1 53
29.10.08 14 97 0 2 53
30.10.08 15 98 0 3 54
31.10.08 16 97 0 2 54
01.11.08 17 97 0 0 54
02.11.08 18 99 0 2 54
03.11.08 19 99 0 2 55
04.11.08 20 99 0 2 55
05.11.08 21 99 0 4 55
06.11.08 22 97 0 2 55
07.11.08 23 98 0 1 56
08.11.08 24 97 0 2 55
09.11.08 25 98 0 1 56
10.11.08 26 99 0 2 56
11.11.08 27 97 0 1 54
12.11.08 28 96 0 2 53

Results with reference substance:

In the toxicity control the biodegradation achieved 53 % after 14 days. A maximum of 56 % was reached after 23 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control,
Test Item and Toxicity Control

		BOD [mgO2/L]			BOD [mgO2/L]
Date	[d]	Inoculum Control	Functional Control 45 mg/L		Test Item 55 mg/L				Toxicity Control 55 + 45 mg/L
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
16.10.08	1	0.7	45.1	44.4	0.0	0.0	2.8	2.1	40.9	40.2
17.10.08	2	2.8	59.2	56.4	0.0	0.0	5.6	2.8	59.2	56.4
18.10.08	3	5.6	64.8	59.2	4.2	0.0	8.5	2.9	66.2	60.6
19.10.08	4	7.1	69.0	62.0	4.2	0.0	9.9	2.9	70.5	63.5
20.10.08	5	8.5	71.9	63.5	5.6	0.0	9.9	1.5	76.1	67.7
21.10.08	6	9.9	73.3	63.4	7.0	0.0	11.3	1.4	77.5	67.6
22.10.08	7	10.6	76.1	65.5	8.5	0.0	11.3	0.7	80.3	69.7
23.10.08	8	10.6	77.5	66.9	7.0	0.0	11.3	0.7	81.7	71.1
24.10.08	9	9.2	78.9	69.7	8.5	0.0	9.9	0.7	84.5	75.3
25.10.08	10	9.9	80.3	70.4	8.5	0.0	11.3	1.4	84.5	74.6
26.10.08	11	10.6	81.7	71.1	8.5	0.0	11.3	0.7	87.4	76.8
27.10.08	12	10.6	81.7	71.1	8.5	0.0	11.3	0.7	88.8	78.2
28.10.08	13	10.6	83.1	72.5	9.9	0.0	11.3	0.7	88.8	78.2
29.10.08	14	11.3	84.5	73.2	9.9	0.0	12.7	1.4	90.2	78.9
30.10.08	15	10.6	84.5	73.9	9.9	0.0	12.7	2.1	90.2	79.6
31.10.08	16	11.3	84.5	73.2	9.9	0.0	12.7	1.4	91.6	80.3
01.11.08	17	12.7	86.0	73.3	11.3	0.0	12.7	0.0	93.0	80.3
02.11.08	18	12.7	87.4	74.7	12.7	0.0	14.1	1.4	93.0	80.3
03.11.08	19	12.7	87.4	74.7	12.7	0.0	14.1	1.4	94.4	81.7
04.11.08	20	12.7	87.4	74.7	12.7	0.0	14.1	1.4	94.4	81.7
05.11.08	21	12.7	87.4	74.7	12.7	0.0	15.5	2.8	94.4	81.7
06.11.08	22	14.1	87.4	73.3	12.7	0.0	15.5	1.4	95.8	81.7
07.11.08	23	13.4	87.4	74.0	12.7	0.0	14.1	0.7	95.8	82.4
08.11.08	24	14.1	87.4	73.3	14.1	0.0	15.5	1.4	95.8	81.7
09.11.08	25	13.4	87.4	74.0	12.7	0.0	14.1	0.7	95.8	82.4
10.11.08	26	12.7	87.4	74.7	12.7	0.0	14.1	1.4	95.8	83.1
11.11.08	27	16.2	88.8	72.6	12.7	0.0	16.9	0.7	95.8	79.6
12.11.08	28	16.9	88.8	71.9	11.3	0.0	18.3	1.4	95.8	78.9

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item must be regarded as not readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item was determined with a non adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted from 2008-10-15 to 2008-11-12according to OECD guideline 301 F at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. The test item concentration selected as appropriate was 55 mg/L, corresponding to a ThOD of 72.6 mg O₂/L per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 16.9 mg O₂/L on day 28.

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 2 days. The biodegradation rate reached 97 % on day 14 and a maximum of 99 % on day 18.

In the toxicity control containing both test and reference item 53 % degradation occurred within 14 days. A maximum of 56 % was reached after 23 days. Therefore, the degradation of the reference item was not inhibited by the test item.

The biodegradation of the test item is shown in Table 1 and graphically indicated in Figure 1 in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10 % level (beginning of biodegradation) until day 28. The mean biodegradation after 28 days was 1 %.

The validity criteria of the guideline are fulfilled.

The test item must be regarded as

not readily biodegradable in the 10-d-window and after 28 days.

Mean Biodegradation of the Test Item in Comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 55 mg/L	1	0	0	0	0
	2	1	2	4	2
Functional Control 45 mg/L	1	87	97	99	96
Toxicity Control 55 + 45 mg/L	1	47	53	55	53

Description of key information

The test item must be regarded as not readily biodegradable in the 10-d-window and after 28 days.