Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

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:

05 April 2017 - 11 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

EC Number:

274-366-7

EC Name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Cas Number:

70179-77-0

Molecular formula:

C28H30N2O8S.Na

IUPAC Name:

sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Test material form:

solid: bulk

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 Bensheim, 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 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 Acid Violet 126 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.
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:

The degradation rate of Acid Violet 126 did never reach 60% within the 10-day window nor after 28 days.
Therefore, Acid Violet 126 is considered to be not readily biodegradable.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

not readily biodegradable based on QSAR/QSPR prediction

Details on results:

Nitrogen is part of the molecular structure of the test item; 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 mean biodegradation of 10% of Acid Violet 126 was never reached during the incubation time and therefore the 10 day window criterion was not passed. The mean biodegradation at test end after 28 days was 0% (ThODNH4) and 0% (ThODNO3).

BOD5 / COD results

Results with reference substance:

Biodegradation of Sodium Benzoate:

The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 81% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Any other information on results incl. tables

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	5	5	50	0	60
2	5	5	10	10	85	0	80
3	10	5	10	10	105	0	110
4	10	10	10	15	130	0	125
5	10	10	10	15	135	0	130
6	10	15	15	20	145	5	140
7	15	15	15	25	150	5	140
8	15	20	15	25	155	5	150
9	15	20	15	25	160	5	155
10	20	20	20	30	165	5	160
11	20	25	20	30	165	5	160
12	20	25	20	30	165	5	160
13	20	25	20	30	165	5	160
14	20	25	20	30	165	5	165
15	20	25	20	30	165	5	165
16	20	25	20	30	165	5	165
17	25	25	20	30	170	5	165
18	25	30	20	30	170	5	165
19	25	30	25	35	170	5	165
20	25	30	25	35	170	5	165
21	25	30	30	40	175	5	170
22	25	30	30	40	175	5	170
23	25	30	30	40	175	5	170
24	25	30	30	40	175	5	170
25	25	30	30	40	175	5	170
26	25	30	30	40	175	5	170
27	25	30	30	40	175	5	170
28	25	30	30	40	175	5	170

            Flasks 1 and 2: Acid Violet 126

            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)	Acid Violet 126^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	26	16
2	0	0	44	20
3	0	0	55	29
4	0	0	68	32
5	0	0	71	34
6	0	0	74	34
7	0	0	76	33
8	0	0	78	36
9	0	0	81	38
10	0	0	81	38
11	0	0	81	38
12	0	0	81	38
13	0	0	81	38
14	0	0	81	39
15	0	0	81	39
16	0	0	81	39
17	0	0	84	39
18	0	0	84	39
19	0	0	81	38
20	0	0	81	38
21	0	0	81	38
22	0	0	81	38
23	0	0	81	38
24	0	0	81	38
25	0	0	81	38
26	0	0	81	38
27	0	0	81	38
28	0	0	81	38

            ¹ThOD_NH4of Acid Violet 126: 1.748 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)	Acid Violet 126^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	26	15
2	0	0	44	19
3	0	0	55	27
4	0	0	68	31
5	0	0	71	32
6	0	0	74	32
7	0	0	76	31
8	0	0	78	34
9	0	0	81	35
10	0	0	81	35
11	0	0	81	35
12	0	0	81	35
13	0	0	81	35
14	0	0	81	37
15	0	0	81	37
16	0	0	81	37
17	0	0	84	37
18	0	0	84	37
19	0	0	81	35
20	0	0	81	35
21	0	0	81	35
22	0	0	81	35
23	0	0	81	35
24	0	0	81	35
25	0	0	81	35
26	0	0	81	35
27	0	0	81	35
28	0	0	81	35

            ¹ThOD_NO3of Acid Violet 126: 1.970 mg O₂/mg test item
            ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of Acid Violet 126 never reached 60%.
Therefore, Acid Violet 126 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:

1. Summary
Title:	Acid Violet 126: 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:	Acid Violet 126; Batch No.:PQD21/1609023
Test Species:	Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Bensheim,.
Test Design:	The test item Acid Violet 126 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):	103.5 mg/L corresponding to an oxygen demand of about 180.9 mg/L (ThODNH4) and 203.9 mg/L (ThODN03)
Reference Item:	Sodium Benzoate
Reference Item Loading Rate:	103.3 mg/L corresponding to an oxygen demand of about 172.1 mg/L (ThODNH4)
Test Conditions:	22°C ± 1°C, darkness
Results:
Biodegradation of Acid Violet 126:	Nitrogen is part of the molecular structure of the test item; 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 mean biodegradation of 10% of Acid Violet 126 was never reached during the incubation time and therefore the 10 day window criterion was not passed. The mean biodegradation at test end after 28 days was 0% (ThODNH4) and 0% (ThODNO3). Therefore, Acid Violet 126 is considered to be not readily biodegradable based on ThODNH4/ ThODNO3.
Biodegradation of Sodium Benzoate:	The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 81% 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, 39% (ThODNH4) biodegradation was noted within 14 days and 38% (ThODNH4) biodegradation after 28 days of incubation (37% and 35% 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.