2,3-Quinoxalinedione, 1,4-dihydro-6-methoxy-7-nitro-

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:

2011-07-20 to 2011-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

EC Number:

614-960-9

Cas Number:

69399-57-1

Molecular formula:

C9H7N3O5

IUPAC Name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nitro-C-Dion (NMQD)
- Physical state: Solid, yellow powder
- Sum formula: C9H7N3O5
- Analytical purity: 91 % (w/w) (7.3 % water, 1.7 % impurities)
- Purity test date: 2011-07-04
- Storage condition of test material: Room temperature, cool, dry

Study design

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: 1.4 x 10e10 CFU/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

50 other: and 70 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

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: 21-22 °C
- pH:
pH-Values at Test Start and Test End
pH-Values at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.72 1) 7.74
2) 7.75
Functional Control 7.73 1) 7.93
2) 7.91
Test Item
(50 mg/L) 7.72 1) 7.71
2) 7.69
Test Item
(70 mg/L) - 1) 7.71
2) 7.64
Toxicity Control 7.72 7.87

- 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

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

No preliminary study

Test performance:

Based on the calculated oxygen demand, the test concentration of 50 and 70 mg/L corresponding to an oxygen demand of 37.0 and 51.8 mg O2/L in the vessel were selected.

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

• two for each test item concentration (P1 50 mg/L, P2 50 mg/L, P3 70 mg/L, P4 70 mg/L)
• two for the functional control (R1, R2)
• one for the toxicity control (T1)
• two for the inoculum control (C1, C2)

Separate replicates of test item (50 mg/L) and toxicity control will be prepared for measurement of the pH-value at test start:

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

The test medium, consisting of the required volumes of mineral medium stock solutions, demineralised water and 10 mL/L inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.
The test item was weighed out and transferred directly into the brown glass bottle with 250 mL test medium.

For the functional control the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The required 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 placed in the brown glass bottle.
For the toxicity control the test item was weighed out and transferred directly into the brown glass bottle with the reference item solution.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with OxiTop measuring heads and the measuring system was activated.

% Degradation

Parameter:

other: O2 consumption at 50 mg/L

Value:

19

Sampling time:

28 d

Details on results:

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 a plateau phase with 67 % degradation in the 1st replicate and 90 % in the 2nd replicate on day 13.

The biodegradation of the test item in both concentrations is shown in Table 1 and graphically presented in Figure 1 in comparison to the readily degradable functional control and the toxicity control. The 1st test item replicate, 50 mg/L, reached the 10 % level (beginning of biodegradation) after 14 days, and the 2nd test item replicate after 9 days. The 1st test item replicate, 70 mg/L, reached the 10 % level after 17 days and the 2nd test item replicate after 8 days. The 60 % pass level was not reached within 28 days in both concentrations. After 28 days the mean biodegradation was 19 % at test item concentration 50 mg/L and 20 % at test item concentration 70 mg/L.
Functional control: The pass level for ready biodegradation (> 60 % degradation) was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.

BOD5 / COD results

Results with reference substance:

In the toxicity control containing both, test item (50 mg/L) and reference item, 39 % degradation occurred within 13 days and a plateau was reached. The degradation of the reference item was not inhibited by the test item. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Any other information on results incl. tables

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

		BOD [mg O2/L]
Date	[d]	Inoculum Control	Functional Control 45 mg/L				Toxicity Control 50 mg/L Test Item+ 45 mg/L Reference Item
			R1		R2		T1
		mean	gross	net	gross	net	gross	net
2011-08-19	1	0.7	36.6	35.9	39.5	38.8	36.6	35.9
2011-08-20	2	1.4	50.7	49.3	55.0	53.6	49.3	47.9
2011-08-21	3	1.4	55.0	53.6	59.2	57.8	52.1	50.7
2011-08-22	4	2.1	56.4	54.3	62.0	59.9	52.1	50.0
2011-08-23	5	2.1	56.4	54.3	63.4	61.3	52.1	50.0
2011-08-24	6	1.4	53.5	52.1	63.4	62.0	45.1	43.7
2011-08-25	7	2.8	57.8	55.0	67.6	64.8	50.7	47.9
2011-08-26	8	2.8	57.8	55.0	69.0	66.2	50.7	47.9
2011-08-27	9	2.8	57.8	55.0	70.5	67.7	50.7	47.9
2011-08-28	10	3.5	57.8	54.3	73.3	69.8	49.3	45.8
2011-08-29	11	2.8	56.4	53.6	70.5	67.7	47.9	45.1
2011-08-30	12	2.8	55.0	52.2	70.5	67.7	46.5	43.7
2011-09-01	13	2.8	53.5	50.7	70.5	67.7	46.5	43.7
2011-09-02	14	7.1	47.9	40.9	84.5	77.5	38.0	31.0
2011-09-03	15	7.1	47.9	40.9	86.0	79.0	35.2	28.2
2011-09-04	16	7.1	47.9	40.9	86.0	79.0	35.2	28.2
2011-09-05	17	7.8	47.9	40.2	87.4	79.7	36.6	28.9
2011-09-06	18	7.8	47.9	40.2	87.4	79.7	36.6	28.9
2011-09-07	19	7.8	46.5	38.8	88.8	81.1	35.2	27.5
2011-09-08	20	7.8	46.5	38.8	88.8	81.1	35.2	27.5
2011-09-09	21	7.8	46.5	38.8	88.8	81.1	35.2	27.5
2011-09-10	22	8.5	47.9	39.5	90.2	81.8	35.2	26.8
2011-09-11	23	8.5	46.5	38.1	90.2	81.8	33.8	25.4
2011-09-12	24	8.5	45.1	36.7	91.6	83.2	33.8	25.4
2011-09-13	25	8.5	45.1	36.7	91.6	83.2	33.8	25.4
2011-09-14	26	8.5	45.1	36.7	93.0	84.6	33.8	25.4
2011-09-15	27	8.5	43.7	35.3	94.4	86.0	32.4	24.0
2011-09-16	28	9.2	43.7	34.6	95.8	86.7	32.4	23.3

Biological Oxygen Demand (BOD) of Test Item Nitro-C-Dion (NMQD)

		BOD [mg O2/L]				BOD [mg O2/L]
Date	[d]	Test Item 50 mg/L				Test Item 70 mg/L
		P1		P2		P3		P4
		gross	net	gross	net	gross	net	gross	net
2011-08-19	1	1.4	0.7	2.8	2.1	1.4	0.7	1.4	0.7
2011-08-20	2	1.4	0.0	2.8	1.4	2.8	1.4	2.8	1.4
2011-08-21	3	2.8	1.4	2.8	1.4	4.2	2.8	4.2	2.8
2011-08-22	4	2.8	0.7	4.2	2.1	4.2	2.1	5.6	3.5
2011-08-23	5	2.8	0.7	4.2	2.1	4.2	2.1	5.6	3.5
2011-08-24	6	0.0	-1.4	0.0	-1.4	0.0	-1.4	2.8	1.4
2011-08-25	7	4.2	1.4	5.6	2.8	5.6	2.8	7.0	4.2
2011-08-26	8	4.2	1.4	5.6	2.8	5.6	2.8	8.5	5.7
2011-08-27	9	5.6	2.8	7.0	4.2	5.6	2.8	8.5	5.7
2011-08-28	10	5.6	2.1	8.5	5.0	7.0	3.5	9.9	6.4
2011-08-29	11	4.2	1.4	7.0	4.2	7.0	4.2	12.7	9.9
2011-08-30	12	4.2	1.4	7.0	4.2	5.6	2.8	12.7	9.9
2011-09-01	13	4.2	1.4	7.0	4.2	5.6	2.8	14.1	11.3
2011-09-02	14	12.7	5.7	9.9	2.9	11.3	4.3	18.3	11.3
2011-09-03	15	14.1	7.1	11.3	4.3	9.9	2.9	16.9	9.9
2011-09-04	16	14.1	7.1	11.3	4.3	9.9	2.9	16.9	9.9
2011-09-05	17	15.5	7.8	12.7	5.0	12.7	5.0	19.7	12.0
2011-09-06	18	15.5	7.8	12.7	5.0	12.7	5.0	19.7	12.0
2011-09-07	19	15.5	7.8	14.1	6.4	12.7	5.0	21.1	13.4
2011-09-08	20	15.5	7.8	12.7	5.0	11.3	3.6	18.3	10.6
2011-09-09	21	15.5	7.8	12.7	5.0	9.9	2.2	18.3	10.6
2011-09-10	22	16.9	8.5	14.1	5.7	11.3	2.9	19.7	11.3
2011-09-11	23	16.9	8.5	14.1	5.7	11.3	2.9	19.7	11.3
2011-09-12	24	16.9	8.5	14.1	5.7	12.7	4.3	19.7	11.3
2011-09-13	25	16.9	8.5	14.1	5.7	14.1	5.7	22.5	14.1
2011-09-14	26	16.9	8.5	14.1	5.7	14.1	5.7	22.5	14.1
2011-09-15	27	16.9	8.5	14.1	5.7	14.1	5.7	22.5	14.1
2011-09-16	28	16.9	7.8	15.5	6.4	14.1	5.0	24.0	14.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.

Executive summary:

The ready biodegradability of the test item Nitro-C-Dion (NMQD) (batch number: 01/05) was determined with non adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted from from 2011-07-20 to 2011-09-10 with the definitive exposure phase from 2011-08-19 to 2011-09-16 according to OECD guideline 301 F atDr.U.Noack-Laboratorien, D-31157 Sarstedt. The test item concentrations selected as appropriate were 50 and 70 mg/L, corresponding to ThODs of 37.0 and 51.8 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 (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 9.2 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 a plateau phase with 67 % degradation in the 1^streplicate and 90 % in the 2^nd replicate on day 13.

In the toxicity control containing both, test item (50 mg/L) and reference item, 39 % degradation occurred within 13 days and a plateau was reached. The degradation of the reference item was not inhibited by the test item.

The biodegradation of the test item in both concentrations is shown in Table 1 and graphically presented in Figure 1 in comparison to the readily degradable functional control and the toxicity control. The 1^sttest item replicate, 50 mg/L, reached the 10 % level (beginning of biodegradation) after 14 days, and the 2^nd test item replicate after 9 days. The 1^st test item replicate, 70 mg/L, reached the 10 % level after 17 days, and the 2^nd test item replicate after 8 days. The 60 % pass level was not reached within 28 days in both concentrations. After 28 days the mean biodegradation was 19 % at test item concentration 50 mg/L and 20 % at test item concentration 70 mg/L.

The validity criteria of the guideline are fulfilled.

The test item is classified as

not readily biodegradable

in the 10-d-window and after 28 days.

Biodegradation of the Test Item Nitro-C-Dion (NMQD) in Comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 50 mg/L	1	4	15	21	21
	2	8	8	13	17
Test Item 70 mg/L	1	5	8	4	10
	2	8	22	20	29
Functional Control 45 mg/L	1	73	*	*	*
	2	86	*	*	*
Toxicity Control 50 mg/L Test Item + 45 mg/L Reference Item		43	*	*	*