Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

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:

01 September 2017 to 29 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: The sewage treatment plant of Rossdorf, Germany.
- The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
- Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment, final sludge concentration in test flasks: 28.7 mg sludge/L.

Duration of test (contact time):

28 d

Initial conc.:

103.5 mg/L

Based on:

test mat.

Initial conc.:

171.4 mg/L

Based on:

ThOD

Remarks:

(NH4)

Initial conc.:

225.6 mg/L

Based on:

ThOD

Remarks:

(NO3)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water:
- Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1 000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume.
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume.
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume. In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
- 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5 000 mL with pure water.
- Additional substrate: The amounts of test material and reference material were directly weighed into the test flasks. No emulsifiers or solvents were used.
- The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45 %) of potassium hydroxide.
- Test temperature: 22 ± 1 °C.
- pH: 7.6 to 7.7
- pH adjusted: No
- Suspended solids concentration: Stock suspension of 3.5 g/L on dry matter base (final sludge concentration in test flasks: 28.7 mg sludge/L)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: A Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
- The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide.

SAMPLING
- The change of pressure in the test flasks was measured by means of a manometric method (BSB/BODSensor- System, Aqualytic Dortmund, Germany) each day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flasks containing the activated sludge and test water only.
- Procedural control: 1 flask containing the reference material sodium benzoate, activated sludge and test water. This was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.
- Abiotic sterile control: 1 flask containing the test material, CuSO4 and test water.
- Toxicity control: 1 flask containing the test material, the reference material, activated sludge and test water.

EVALUATION OF THE RESULTS
- Calculation of BOD: The biodegradability (BOD = mg O2 per mg test material or reference material) exerted after each period was calculated as:

BOD = (mg O2 uptake of test material (or reference material) - mg O2 uptake of inoculum control) / mg test material (or reference material) in flask

- For the toxicity control, the BOD of the treatment is the sum of test material and reference material and is calculated as the sum of test material and reference material concentration in the flask.
- The percentage biodegradation of the test material and of the reference material sodium benzoate was calculated as:

% degradation = [ BOD (mg O2/mg test material or reference material) / (ThOD NH4 (mg O2/mg test material or reference material) x correction volume test material flasks) ] x 100

- or in case of nitrification of the test material:

% degradation = [ BOD (mg O2/mg test material or reference material) / (ThOD NO3 (mg O2/mg test material or reference material) x correction volume test material flasks) ] x 100

Reference substance:

benzoic acid, sodium salt

Test performance:

VALIDITY CRITERIA
- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 0 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
- pH-Value: The pH-value of the test material flasks at the end of the test was 7.7 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
- Reference Material: The percentage degradation of the reference material should reach the level for ready biodegradability (>60 %) within 14 days as required by the test guideline. The reference material sodium benzoate was degraded to more than 60 % after 4 days of incubation.
- Test Material: The difference of duplicate values for the degradation of the test material at the end of the test was less than 20 %. The difference of duplicate values at day 28 differed by 3 % (ThODNH4 and ThODNO3). The validity criterion was fulfilled.
- Toxicity Control: If in a toxicity test, containing both the test material and a reference material less than 25 % biodegradation (based on total ThOD) occurred within 14 days, the test material can be assumed to be inhibitory. The biodegradation was 46 % (based on ThODNH4) at day 14; the test material was not inhibitory.

Key result

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Remarks on result:

other: Based on ThODNH4

Key result

Parameter:

% degradation (O2 consumption)

Value:

6

Sampling time:

28 d

Remarks on result:

other: Based on ThODNO3

Details on results:

TEST MATERIAL
- Nitrogen is part of the molecular structure of the test material; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test material of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test material reaches at least 10 % degradation.
- The test material did not reach 10 % degradation during the incubation time of 28 days. The mean biodegradation percentage at the end of the 28-day exposure period was 6 %.
- If no nitrification is considered, the mean biodegradation was 8 % after 28 days of incubation.
- The degradation rate of the test material did not reach 60 % within the 10-day window or after 28 days. Therefore, it is considered to be not readily biodegradable.

TOXICITY CONTROL
- Percentage Biodegradation: In the toxicity control containing both, the test material and the reference material sodium benzoate, 46 % (ThODNH4) biodegradation was noted within 14 days and 50 % (ThODNH4) biodegradation after 28 days of incubation (39 and 43 % based on ThODNO3). According to the test guidelines the test material can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25 % within 14 days.

ABIOTIC CONTROL
- Oxygen Demand: The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test material and toxicity control.

Results with reference substance:

- The percentage degradation of the reference material should reach the level for ready biodegradability (>60 %) within 14 days as required by the test guideline.
- The reference material sodium benzoate was sufficiently degraded to 93 % after 14 days and to 102 % after 28 days of incubation.
- The percentage biodegradation of the reference material confirms the suitability of the used aerobic activated sludge inoculum.

Table 1: Percentage Biodegradation of Test Material, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time (days)	Percentage Biodegradation (%)
	Test Material		Reference Material	Toxicity Control
	Flask 1	Flask 2
1	0	0	23	13
2	0	0	41	21
3	3	0	55	28
4	3	3	64	35
5	3	3	76	37
6	3	3	78	38
7	6	3	81	40
8	6	3	84	41
9	6	3	84	41
10	6	3	87	43
11	6	3	87	44
12	6	3	90	44
13	9	3	90	46
14	9	6	93	46
15	9	6	93	46
16	9	6	96	47
17	9	6	96	47
18	9	6	96	49
19	9	6	96	49
20	9	6	96	49
21	9	6	96	49
22	9	6	99	49
23	9	6	99	50
24	9	6	99	50
25	9	6	99	50
26	9	6	99	50
27	9	6	102	50
28	9	6	102	50

 

Table 2: Percentage Biodegradation of Test Material and of the Toxicity Control based on ThODNO3 and of Sodium Benzoate based on ThODNH4

Time (days)	Percentage Biodegradation (%)
	Test Material		Reference Material	Toxicity Control
	Flask 1	Flask 2
1	0	0	23	11
2	0	0	41	18
3	2	0	55	24
4	2	2	64	30
5	2	2	76	32
6	2	2	78	33
7	4	2	81	34
8	4	2	84	36
9	4	2	84	36
10	4	2	87	37
11	4	2	87	38
12	4	2	90	38
13	7	2	90	39
14	7	4	93	39
15	7	4	93	39
16	7	4	96	41
17	7	4	96	41
18	7	4	96	42
19	7	4	96	42
20	7	4	96	42
21	7	4	96	42
22	7	4	99	42
23	7	4	99	43
24	7	4	99	43
25	7	4	99	43
26	7	4	99	43
27	7	4	102	43
28	7	4	102	43

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material is considered not readily biodegradable based on ThODNH4/ThODNO3.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OCED 301 F and EU Method C.4-D, under GLP conditions.

The test material was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

Nitrogen is part of the molecular structure of the test material; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test material of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test material reaches at least 10 % degradation. The test material did not reach 10 % degradation during the incubation time of 28 days. The mean biodegradation percentage at the end of the 28-day exposure period was 8 and 6 %, based on ThODNH4 and ThODNO3, respectively.

The reference material sodium benzoate was sufficiently degraded to 93 % after 14 days and to 102 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test material and the reference material sodium benzoate, 46 % (ThODNH4) biodegradation was noted within 14 days and 50 % (ThODNH4) biodegradation after 28 days of incubation (39 and 43 % based on ThODNO3). According to the test guidelines, the test material can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25 % within 14 days.

Under the conditions of this study, the test material is considered not readily biodegradable based on ThODNH4/ThODNO3.

Description of key information

Under the conditions of this study, the test material is considered not readily biodegradable based on ThODNH4/ThODNO3.

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 accordance with the standardised guidelines OCED 301 F and EU Method C.4-D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference material sodium benzoate was tested simultaneously under the same conditions as the test material, and functioned as a procedure control.

Nitrogen is part of the molecular structure of the test material; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test material of at least 60 %, reached within a 10-day window; the 10-day window starts when the degradation of the test material reaches at least 10 % degradation. The test material did not reach 10 % degradation during the incubation time of 28 days. The mean biodegradation percentage at the end of the 28- day exposure period was 8 and 6 %, based on ThODNH4 and ThODNO3, respectively.

The reference material sodium benzoate was sufficiently degraded to 93 % after 14 days and to 102 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test material and the reference material sodium benzoate, 46 % (ThODNH4) biodegradation was noted within 14 days and 50 % (ThODNH4) biodegradation after 28 days of incubation (39 and 43 % based on ThODNO3). According to the test guidelines, the test material can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25 % within 14 days.

Under the conditions of this study, the test material is considered not readily biodegradable based on ThODNH4/ThODNO3.