4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-07 to 2012-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to 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 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

Instead of the test system generated of a mixed inoculum from different sources, inoculum of the aqueous phase of non-adapted activated sludge was used. This deviation was considered to have no impact on quality and integrity of the study.

Principles of method if other than guideline:

Instead of the test system generated of a mixed inoculum from different sources, inoculum of the aqueous phase of non-adapted activated sludge was used. This deviation was considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

61932-63-6

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): Certificate of Analysis AZ564pc_specd2: Permanent-Rot P-FK
Analytical purity: 45.1 % (w/w) (comp. 1) Pigment Red 170, 53.6 % (w/w) (comp. 2) Pigment Red 266

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):Inoculum of the aqueous phase of non adapted activated sludge. Municipal sewage treatment plant, D-31137 Hildesheim. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was re-suspended in mineral salts medium. 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 4 days.
- Inoculation: 50 mL/L inoculum (functional control: 20 mL/L) corresponding to the final concentration of 98.5 mg/L (functional control: 39.4 mg/L) suspended solids was used to initiate inoculation.
- Initial cell/biomass concentration: 50 mL/L inoculum was used to initiate inoculation
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

30 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 302C
- Test temperature: 25 +/- 2°C
- pH:
pH-Value
Start End
Inoculum Control 7.13 1) 6.56
2) 6.71
Functional Control 7.77 1) 6.78
2) 6.85
Test Item 6.88 1) 6.91
2) 6.67
3) 6.83
Blank 6.20 8.69


- pH adjusted: no
- Aeration of dilution water: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Brown glass bottles, volume 500 mL, test volume: 250 mL
- Number of culture flasks/concentration: Triplicates
- Method used to create aerobic conditions: Continuous stirring
- Measuring equipment: The oxygen consumption was determined in the incubation vessels by the OxiTop¿ measuring system at 360 measuring points during the 28 d incubation period.
- Details of trap for CO2 and volatile organics if used: 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.


SAMPLING
- Sampling frequency: The temperature in the incubator was documented continuously throughout the test with a hygrothermograph.
The colony forming units and concentration of suspended solids of the inoculum were determined at test start.
The pH of the test item solution was determined in a separate replicate at test start. No adjustment of pH was necessary.
The pH of the test solutions was determined at test end.
The oxygen consumption was determined in the incubation vessels by the OxiTop¿ measuring system at 360 measuring points during the 28 d incubation period.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 30 mg/L test item in aqua demin.
- Abiotic sterile control: No
- Functional control: Anilin, 25 mg/L
- Inoculum control: Test medium and inoculum without test and reference item.

Reference substance

Reference substance:

aniline

Results and discussion

Test performance:

Based on the calculated oxygen demand (ThOD), the test con-centration of 30 mg/L corresponding to an oxygen demand of
54.6 mgO2 /L in the 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, blank):
• 3 incubation vessels for the test item (P1, P2, P3)
• 2 incubation vessels for the functional control (R1, R2)
• 1 incubation vessel for the blank (B1)
• 2 incubation vessels for the inoculum control (C1, C2)

Separate replicates for the test item and blank were prepared for pH measurement at test start:
• 1 incubation vessel for the test item (PpH)
• 1 incubation vessel for the blank (BpH)

The test item was weighed out and was transferred directly into the brown glass bottles with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, de-mineralised water and inoculum) for the test item replicates and with 250 mL demineralised water for the blank, respectively.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Colony forming units (CFU) of the inoculum were determined at test start by standard dilution plate count: 2.0 x 10^9 CFU/L.
The concentration of suspended solids of the inoculum at test start was 1.97 g/L, corresponding to 98.5 mg/L in the test vessels of the test item and control replicates and 39.4 mg/L in the vessels of the reference item replicates.
All test item replicates did not reach the 10 % level (beginning of biodegradation) within the test duration of 28 days. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 0 %.

BOD5 / COD results

Results with reference substance:

The adaptation phase changed to degradation phase after 4 days in both replicates. The functional control reached the pass level of > 65 % after 9 days in the 1st replicate and after 11 days in the 2nd replicate. The mean biodegradation rate on day 14 was 78 %. Hence the validity criterion that the degradation should be > 65 % after 14 days was fulfilled. It came to a biodegradation rate of 100 % after 28 days

Any other information on results incl. tables

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

		BOD [mg O2/L]
		Inoculum Control	Functional Control 25 mg/L				Blank 30 mg/L
			R1		R2		B1
Date	[d]	mean	gross	net	gross	net	gross
2012-03-28	1	2.2	0.0	-2.2	0.0	-2.2	-5.6
2012-03-29	2	5.1	0.0	-5.1	0.0	-5.1	-5.6
2012-03-30	3	7.0	7.0	0.0	1.4	-5.6	-5.6
2012-03-31	4	9.0	38.0	29.0	12.7	3.7	-4.2
2012-04-01	5	10.4	42.3	31.9	36.6	26.2	-2.8
2012-04-02	6	11.3	45.1	33.8	43.7	32.4	-2.8
2012-04-03	7	12.1	46.5	34.4	46.5	34.4	-2.8
2012-04-04	8	13.0	49.3	36.3	47.9	34.9	-1.4
2012-04-05	9	13.8	52.1	38.3	49.3	35.5	0.0
2012-04-06	10	14.6	55.0	40.4	52.1	37.5	0.0
2012-04-07	11	15.2	57.8	42.6	53.5	38.3	0.0
2012-04-08	12	15.5	59.2	43.7	55.0	39.5	0.0
2012-04-09	13	16.1	63.4	47.3	56.4	40.3	0.0
2012-04-10	14	16.6	66.2	49.6	57.8	41.2	1.4
2012-04-11	15	17.2	70.5	53.3	59.2	42.0	1.4
2012-04-12	16	17.5	74.7	57.2	60.6	43.1	2.8
2012-04-13	17	17.8	77.5	59.7	63.4	45.6	2.8
2012-04-14	18	18.6	81.7	63.1	64.8	46.2	2.8
2012-04-15	19	19.2	84.5	65.3	67.6	48.4	4.2
2012-04-16	20	19.4	87.4	68.0	70.5	51.1	4.2
2012-04-17	21	20.0	93.0	73.0	74.7	54.7	4.2
2012-04-18	22	20.6	98.6	78.0	77.5	56.9	5.6
2012-04-19	23	20.9	107.0	86.1	81.7	60.8	5.6
2012-04-20	24	21.1	116.0	94.9	86.0	64.9	5.6
2012-04-21	25	21.4	121.0	99.6	90.2	68.8	5.6
2012-04-22	26	22.0	125.0	103.0	95.8	73.8	5.6
2012-04-23	27	22.5	130.0	107.5	101.0	78.5	7.0
2012-04-24	28	22.5	134.0	111.5	106.0	83.5	7.0

Biological Oxygen Demand (BOD) of Test Item

		BOD [mg O2/L]
		Inoculum Control	Test Item
			P1		P2		P2
Date	[d]	mean	gross	net	gross	net	gross	net
2012-03-28	1	5.6	1.4	-4.2	2.8	-2.8	2.8	-2.8
2012-03-29	2	12.7	8.5	-4.2	9.9	-2.8	9.9	-2.8
2012-03-30	3	17.6	14.1	-3.5	14.1	-3.5	15.5	-2.1
2012-03-31	4	22.5	18.3	-4.2	19.7	-2.8	21.1	-1.4
2012-04-01	5	26.1	21.1	-5.0	22.5	-3.6	25.4	-0.7
2012-04-02	6	28.2	24.0	-4.2	25.4	-2.8	26.8	-1.4
2012-04-03	7	30.3	25.4	-4.9	26.8	-3.5	29.6	-0.7
2012-04-04	8	32.4	26.8	-5.6	28.2	-4.2	32.4	0.0
2012-04-05	9	34.5	29.6	-4.9	29.6	-4.9	33.8	-0.7
2012-04-06	10	36.6	31.0	-5.6	31.0	-5.6	35.2	-1.4
2012-04-07	11	38.0	31.0	-7.0	32.4	-5.6	36.6	-1.4
2012-04-08	12	38.8	32.4	-6.4	32.4	-6.4	38.0	-0.8
2012-04-09	13	40.2	32.4	-7.8	33.8	-6.4	39.5	-0.7
2012-04-10	14	41.6	33.8	-7.8	33.8	-7.8	40.9	-0.7
2012-04-11	15	43.0	33.8	-9.2	35.2	-7.8	40.9	-2.1
2012-04-12	16	43.7	35.2	-8.5	36.6	-7.1	43.7	0.0
2012-04-13	17	44.4	36.6	-7.8	38.0	-6.4	43.7	-0.7
2012-04-14	18	46.5	36.6	-9.9	39.5	-7.0	45.1	-1.4
2012-04-15	19	47.9	38.0	-9.9	39.5	-8.4	46.5	-1.4
2012-04-16	20	48.6	38.0	-10.6	40.9	-7.7	47.9	-0.7
2012-04-17	21	50.0	39.5	-10.5	42.3	-7.7	49.3	-0.7
2012-04-18	22	51.4	39.5	-11.9	43.7	-7.7	49.3	-2.1
2012-04-19	23	52.2	40.9	-11.3	45.1	-7.1	50.7	-1.5
2012-04-20	24	52.9	40.9	-12.0	45.1	-7.8	52.1	-0.8
2012-04-21	25	53.6	42.3	-11.3	46.5	-7.1	52.1	-1.5
2012-04-22	26	55.0	42.3	-12.7	46.5	-8.5	53.5	-1.5
2012-04-23	27	56.4	42.3	-14.1	47.9	-8.5	55.0	-1.4
2012-04-24	28	56.4	42.3	-14.1	49.3	-7.1	55.0	-1.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Executive summary:

The inherent biodegradability of the test item was determined in the Modified MITI Test (II) with non adapted activated sludge for a period of 28 days. The study was conducted according to OECD Guideline 302 C and compliant to GLP (reliability category 1). The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 54.6 mgO₂/L. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day

The mean oxygen depletion in the inoculum control was 56.4 mg O₂/L on day 28.

In order to check the activity of the test system Aniline was used as functional control. The functional control reached the pass level of > 65 % after 9 days in the 1^st replicate and after 11 days in the 2^ndreplicate. The mean biodegradation rate on day 14 was 78%. It came to a biodegradation rate of 100% after 28 days.

All test item replicates did not reach the 10 % level (beginning of biodegradation) within the test duration of 28 days. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 0 %.

Biodegradation of the Test Item in Comparison to the Functional Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item	1	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
	mv	0	0	0	0
Functional Control	1	59	85	100	100
	2	59	70	94	100
	mv	59	78	97	100