Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

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:

2016-12-21 - 2017-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

EC Number:

220-678-3

EC Name:

Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

Cas Number:

2861-02-1

Molecular formula:

C14H10N2O10S2.2Na

IUPAC Name:

disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2,6-disulfonate

Specific details on test material used for the study:

Theoretical Oxygen Demand:
ThODNH4: 0.877 mg oxygen per mg test item
ThODNO3: 1.147 mg oxygen per mg test item

Study design

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 tap 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.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study 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.
This study is recognized by the OECD and EEC guide-lines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge.

Test Units
Type and Size: 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.
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

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Percentage Biodegradation: The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.
The mean biodegradation from day 1 till the test end at day 28 was 0% (THODNH4, ThODNO3).

The degradation rate of the test item did not reach 60% within the 10-day window and after 28 days.
Therefore, the test item is considered to be not readily biodegradable.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Remarks on result:

not readily biodegradable based on QSAR/QSPR prediction

Details on results:

Percentage Biodegradation: Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of results.
The degradation of the test item did never reach 10% degradation; the criterion of the 10 day window was not passed. The mean biodegradation percentage at the end of the 28-day exposure period was 2%.
If no nitrification is considered, the mean biodegradation was 3% after 28 days of incubation. The criterion of the 10 day window was not fulfilled.

Conclusion: the test item is considered to be not readily biodegradable.

BOD5 / COD results

Results with reference substance:

Percentage Biodegradation: The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 90% after 28 days of incubation.
Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Any other information on results incl. tables

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	45	0	50
2	0	0	5	0	70	0	75
3	0	0	10	5	95	0	90
4	0	0	10	5	120	0	115
5	0	0	10	5	120	0	125
6	0	0	10	5	130	0	130
7	0	0	10	5	135	0	135
8	0	0	10	5	140	0	135
9	0	0	10	5	145	0	140
10	5	0	10	5	150	0	140
11	5	0	10	5	150	0	145
12	5	0	10	5	150	0	145
13	5	5	15	5	155	0	150
14	5	5	15	5	155	0	150
15	10	5	15	5	160	0	150
16	10	5	15	5	160	0	150
17	10	5	15	5	160	0	155
18	10	5	20	5	160	0	155
19	10	5	20	5	160	0	155
20	15	5	20	5	165	0	155
21	15	5	20	5	165	0	160
22	15	5	20	5	165	0	160
23	15	5	25	5	165	0	160
24	20	5	25	5	165	0	165
25	20	5	25	5	165	0	165
26	20	5	25	5	165	0	165
27	20	5	25	5	170	0	165
28	20	5	25	5	170	0	165

Flasks 1 and 2: test item

            Flasks 3 and 4: inoculum control

Flask 5:reference(procedure control)

            Flask 6: abiotic control

            Flask 7: toxicity control

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	26	19
2	0	0	39	28
3	0	0	51	31
4	0	0	66	41
5	0	0	66	45
6	0	0	71	47
7	0	0	74	49
8	0	0	77	49
9	0	0	80	51
10	0	0	83	51
11	0	0	83	52
12	0	0	83	52
13	0	0	85	53
14	0	0	85	53
15	0	0	88	53
16	0	0	88	53
17	0	0	88	55
18	0	0	86	54
19	0	0	86	54
20	3	0	89	54
21	3	0	89	56
22	3	0	89	56
23	0	0	88	55
24	5	0	88	57
25	5	0	88	57
26	5	0	88	57
27	5	0	90	57
28	5	0	90	57

¹ThOD_NH4of test item: 0.877 mg O₂/mg test item
            ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

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	26	19
2	0	0	39	28
3	0	0	51	31
4	0	0	66	41
5	0	0	66	45
6	0	0	71	47
7	0	0	74	49
8	0	0	77	49
9	0	0	80	51
10	0	0	83	51
11	0	0	83	52
12	0	0	83	52
13	0	0	85	53
14	0	0	85	53
15	0	0	88	53
16	0	0	88	53
17	0	0	88	55
18	0	0	86	54
19	0	0	86	54
20	3	0	89	54
21	3	0	89	56
22	3	0	89	56
23	0	0	88	55
24	5	0	88	57
25	5	0	88	57
26	5	0	88	57
27	5	0	90	57

28

5

0

90

57

Time	^1ThODNH4of test item: 0.877 mg O2/mg test item             ^2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item 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	26	19
2	0	0	39	28
3	0	0	51	31
4	0	0	66	41
5	0	0	66	45
6	0	0	71	47
7	0	0	74	49
8	0	0	77	49
9	0	0	80	51
10	0	0	83	51
11	0	0	83	52
12	0	0	83	52
13	0	0	85	53
14	0	0	85	53
15	0	0	88	53
16	0	0	88	53
17	0	0	88	55
18	0	0	86	54
19	0	0	86	54
20	3	0	89	54
21	3	0	89	56
22	3	0	89	56
23	0	0	88	55
24	5	0	88	57
25	5	0	88	57
26	5	0	88	57
27	5	0	90	57
28	5	0	90	57

¹ThOD_NO3of test item: 1.147 mg O₂/mg test item
               ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Percentage Biodegradation of Test Item and of the Toxicity Control based on ThOD_NO3, and 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	26	17
2	0	0	39	25
3	0	0	51	28
4	0	0	66	37
5	0	0	66	41
6	0	0	71	42
7	0	0	74	44
8	0	0	77	44
9	0	0	80	46
10	0	0	83	46
11	0	0	83	47
12	0	0	83	47
13	0	0	85	48
14	0	0	85	48
15	0	0	88	48
16	0	0	88	48
17	0	0	88	50
18	0	0	86	49
19	0	0	86	49
20	2	0	89	49
21	2	0	89	51
22	2	0	89	51
23	0	0	88	50
24	4	0	88	52
25	4	0	88	52
26	4	0	88	52
27	4	0	90	52
28	4	0	90	52

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	26	17
2	0	0	39	25
3	0	0	51	28
4	0	0	66	37
5	0	0	66	41
6	0	0	71	42
7	0	0	74	44
8	0	0	77	44
9	0	0	80	46
10	0	0	83	46
11	0	0	83	47
12	0	0	83	47
13	0	0	85	48
14	0	0	85	48
15	0	0	88	48
16	0	0	88	48
17	0	0	88	50
18	0	0	86	49
19	0	0	86	49
20	2	0	89	49
21	2	0	89	51
22	2	0	89	51
23	0	0	88	50
24	4	0	88	52
25	4	0	88	52
26	4	0	88	52
27	4	0	90	52
28	4	0	90	52

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of the test item did not reach 60 % within the 10-day window and after 28 days.
Therefore, the test item 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:	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:	Batch No.:19.05.2016g
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):	104.1 mg/L corresponding to an oxygen demand of about 91.3 mg/L (ThODNH4) and 119.4 mg/L (ThODN03)
Reference Item:	Sodium Benzoate
Reference Item Loading Rate:	102.9 mg/L corresponding to an oxygen demand of about 171.4 mg/L (ThODNH4)
Test Conditions:	22°C ± 1°C, darkness
Results:
Biodegradation of the test item:	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 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The test item did never reach 10% degradation during incubation time. The mean biodegradation at test end after 28 days was 3% (ThODNH4) and 2% (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 85% 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, 53% (ThODNH4) biodegradation was noted within 14 days and 57% (ThODNH4) biodegradation after 28 days of incubation (48% and 52% 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.

 This study is classified acceptable and satisfies the guideline requirements for ready biodegradability studies.