Reaction mass of (disodium 4-[[4-(acetylmethylamino)-2-sulphonatophenyl]amino]1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium sulphate and sodium chloride)

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:

2017 - 03 - 03 - 2017 - 05 - 05

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant 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 re-suspended 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 (see 6.5) and aerated overnight. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Details on study design:

The test item Acid Blue 182 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 item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Test Units:
Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus:
BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
Principle:
The test flasks prepared according paragraph 6.6 were incubated at 22°C * 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
Preparation of Test Flasks:
The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
Incubation:
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 Duration:
28 days
Measurement of Oxygen:
The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
Temperature:
The temperature was recorded by means of the automated software AMR Wincontrol©.
pH-Value:
pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

Reference substance:

benzoic acid, sodium salt

Test performance:

The test item never reached a biodegradation of 10%.
The mean biodegradation at test end after 28 days was 0% (ThODNH4) and 0% (ThODNO3).
Therefore, Acid Blue 182 is considered to be not readily biodegradable based on ThODNH4 / ThODNO3.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Biodegradation of the test item:
The test item contains nitrogen; 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 item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.
The test item never reached a biodegradation of 10%.
The mean biodegradation at test end after 28 days was 0% (ThODNH4) and 0% (ThODNO3).
Therefore, the test item is considered to be not readily biodegradable based on ThODNH4 / ThODNO3.

Results with reference substance:

Percentage Biodegradation:
The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 90% after 28 days of incubation.

The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum

Table2. Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	0	0	0	0	55	0	0
2	0	0	0	0	85	0	70
3	0	0	0	5	100	0	90
4	5	0	5	5	115	0	110
5	5	0	5	5	115	0	120
6	5	0	5	5	125	0	135
7	5	0	10	5	130	0	140
8	5	0	10	5	135	0	140
9	5	0	10	5	140	0	155
10	5	0	15	5	140	0	155
11	5	0	15	5	145	0	155
12	5	0	15	5	145	0	160
13	5	0	15	5	145	0	160
14	5	0	15	5	145	0	160
15	5	0	15	5	145	0	165
16	5	0	15	5	150	0	165
17	5	0	15	5	150	0	165
18	5	0	15	5	150	0	165
19	5	0	15	5	155	0	165
20	5	0	15	5	155	0	165
21	5	0	15	5	155	0	165
22	5	0	15	5	155	0	170
23	5	0	15	5	160	0	170
24	5	0	15	10	160	0	170
25	5	0	15	10	160	0	170
26	5	0	15	10	160	0	170
27	5	0	15	10	165	0	170
28	5	0	15	10	165	0	170

            Flasks 1 and 2: test ite

            Flasks 3 and 4: inoculum control

Flask5: reference(procedure control)

            Flask 6: abiotic control

            Flask 7: toxicity control Table2. Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	0	0	0	0	55	0	0
2	0	0	0	0	85	0	70
3	0	0	0	5	100	0	90
4	5	0	5	5	115	0	110
5	5	0	5	5	115	0	120
6	5	0	5	5	125	0	135
7	5	0	10	5	130	0	140
8	5	0	10	5	135	0	140
9	5	0	10	5	140	0	155
10	5	0	15	5	140	0	155
11	5	0	15	5	145	0	155
12	5	0	15	5	145	0	160
13	5	0	15	5	145	0	160
14	5	0	15	5	145	0	160
15	5	0	15	5	145	0	165
16	5	0	15	5	150	0	165
17	5	0	15	5	150	0	165
18	5	0	15	5	150	0	165
19	5	0	15	5	155	0	165
20	5	0	15	5	155	0	165
21	5	0	15	5	155	0	165
22	5	0	15	5	155	0	170
23	5	0	15	5	160	0	170
24	5	0	15	10	160	0	170
25	5	0	15	10	160	0	170
26	5	0	15	10	160	0	170
27	5	0	15	10	165	0	170
28	5	0	15	10	165	0	170

            Flasks 1 and 2: test item

            Flasks 3 and 4: inoculum control

Flask5: reference(procedure control)

            Flask 6: abiotic control

            Flask 7: toxicity control

Table3.Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time	Percentage Biodegradation^1
(Days)	Test item^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	32	0
2	0	0	50	26
3	0	0	57	32
4	0	0	65	39
5	0	0	65	42
6	0	0	71	48
7	0	0	72	49
8	0	0	75	49
9	0	0	78	54
10	0	0	76	54
11	0	0	79	54
12	0	0	79	55
13	0	0	79	55
14	0	0	79	55
15	0	0	79	57
16	0	0	82	57
17	0	0	82	57
18	0	0	82	57
19	0	0	85	57
20	0	0	85	57
21	0	0	85	57
22	0	0	85	59
23	0	0	88	59
24	0	0	87	58
25	0	0	87	58
26	0	0	87	58
27	0	0	90	58
28	0	0	90	58

               ¹ThOD_NH4of test item: 0.937 mg O₂/mg test item
               ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Table4. Percentage Biodegradation of Test Item and of the Toxicity Control based on ThOD_NO3and of Sodium Benzoate based on ThOD_NH4

Time	Percentage Biodegradation^1
(Days)	^Test item 1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	32	0
2	0	0	50	24
3	0	0	57	30
4	0	0	65	35
5	0	0	65	39
6	0	0	71	44
7	0	0	72	45
8	0	0	75	45
9	0	0	78	50
10	0	0	76	49
11	0	0	79	49
12	0	0	79	51
13	0	0	79	51
14	0	0	79	51
15	0	0	79	52
16	0	0	82	52
17	0	0	82	52
18	0	0	82	52
19	0	0	85	52
20	0	0	85	52
21	0	0	85	52
22	0	0	85	54
23	0	0	88	54
24	0	0	87	53
25	0	0	87	53
26	0	0	87	53
27	0	0	90	53
28	0	0	90	53

               ¹ThOD_NO3of test item: 1.216 mg O₂/mg test item
               ²ThOD_NH4of Sodium Benzoate: 1.666 mg O₂/mg reference item

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of Acid Blue 182 never reached 60% within the 10-day window or after 28 days.
Therefore, Acid Blue 182 is considered to be not readily biodegradable.

The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Executive summary:

 

Title:	Test item: Ready Biodegradability in a Manometric Respirometry Test
Guidelines:	This study was designed to comply with the following methods: -     Commission Regulation 440/2008/EC, Method C.4-D of: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008) -     OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted
Material and Methods:
Test Item:	Test item; Batch No.:CHA0 122490
Test Species:	Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Rossdorf,.
Test Design:	The test item 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 item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
Endpoints:	Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.
Test Item Loading Rate (initial concentration in medium C0):	101.2 mg/L corresponding to an oxygen demand of about 98.5 mg/L (ThODNH4) and 123.1 mg/L (ThODN03)
Reference Item:	Sodium Benzoate
Reference Item Loading Rate:	102.0 mg/L corresponding to an oxygen demand of about 170.0 mg/L (ThODNH4)
Test Conditions:	22°C ± 1°C, darkness
Results:
Biodegradation of Acid Blue 182:	The test item contains nitrogen; therefore the evaluation of biodegradation has to be based on ThODNH4and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation. The test item never reached a biodegradation of 10%. The mean biodegradation at test end after 28 days was 0% (ThODNH4) and 0% (ThODNO3). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4/ ThODNO3.
Biodegradation of Sodium Benzoate:	The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 90% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.
Biodegradation of the Toxicity Control:	In the toxicity control containing both, the test item and the reference item sodium benzoate, 55% (ThODNH4) biodegradation was noted within 14 days and 58% (ThODNH4) biodegradation after 28 days of incubation (51% and 53% based on ThODNO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Description of key information

The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The degradation rate of Acid Blue 182 never reached 60% within the 10-day window or after 28 days. Therefore, Acid Blue 182 is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater