Sulfonation products of (N-phenyl-4-[[4-(phenylamino)phenyl][4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline, monohydrochloride and 4,4'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]bis[N-phenylaniline], monohydrochloride), sodium salts

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:

2017 - 01 - 31 - 2017 - 03 - 30

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:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

EC Number:

944-086-6

IUPAC Name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

Test material form:

solid

Details on test material:

UVCB

Specific details on test material used for the study:

Name: Duasyn-Tintenblau SLK
Batch No.: DDIBLN519
Certificate of Analysis Date: November 11, 2014
Aggregate State at room temperature: Solid
Colour: Dark violet
Chemical Oxygen Demand: 1.117 mg O2/mg test item
Expiry Date: November 11, 2024
Storage Conditions at Test Facility: At 20 +- 5 °C, in the dark

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 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.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The purpose of this study was to determine the ready biodegradability of the test item . The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 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 type of study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with aerobic activated sludge.

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.

Course of the Test
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

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

28 d

Remarks on result:

not readily biodegradable based on QSAR/QSPR prediction

Details on results:

Biodegradation of the Test Item
Percentage Biodegradation: Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of results. The test item never reached 10% biodegradation. The mean biodegradation percentage at the end of the 28-day exposure period was 7%.
Conclusion: The degradation rate of Duasyn-Tintenblau SLK never reached 10%.
Therefore, thr test item is considered to be not readily biodegradable.

BOD5 / COD results

BOD5 / COD

Parameter:

ThOD

Value:

7 other: %

Remarks on result:

other: ThOD NO3 after 28 days

Results with reference substance:

Biodegradation of Reference Item Sodium Benzoate
Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item sodium benzoate, 43% (ThODNO3) biodegradation was noted within 14 days and 48% (ThODNO3) biodegradation after 28 days of incubation.
Conclusion: 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.

Any other information on results incl. tables

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

Time	Percentage Biodegradation
(Days)	Duasyn-Tintenblau SLK^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	4	0	23	16
2	4	4	38	25
3	4	4	44	28
4	4	4	50	32
5	2	2	57	36
6	2	2	60	38
7	0	0	64	39
8	4	4	70	39
9	4	4	73	41
10	2	2	74	42
11	4	4	73	41
12	4	4	76	43
13	4	9	76	43
14	4	4	76	43
15	4	4	76	43
16	4	4	79	44
17	4	4	79	44
18	4	4	79	44
19	0	4	79	44
20	4	4	79	44
21	4	4	82	44
22	4	4	82	46
23	4	4	82	46
24	4	4	82	46
25	4	9	82	46
26	4	9	85	48
27	4	9	85	48
28	4	9	85	48

            ¹COD of Duasyn-Tintenblau SLK: 1.117 mg O₂/mg test item
            ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	Test item	7.5
2	Test item	7.5
3	Inoculum control	7.5
4	Inoculum control	7.6
5	Reference item (procedure control)	7.6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Biodegradation of Test Item
The degradation rate of the test item never reached 10%.
Therefore, the test item is considered to be not readily biodegradable.
Biodegradation of Reference Item Sodium Benzoate
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.
Biodegradation in the toxicity control
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:	Test item: Ready Biodegradability in a Manometric Respirometry Test
Guidelines:	This study is designed to comply with the following methods: -     Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: 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 July 17, 1992
Material and Methods:
Test Item:	Batch No.:DDIBLN519
Test Species:	Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
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):	103.7 mg/L corresponding to an oxygen demand of about 1.117 mg O2/mg TS (COD)
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 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 mean biodegradation of 10% of the test item was never reached. The mean biodegradation at test end after 28 days was 7% (ThODNO3). Therefore, the test item is considered to be not readily biodegradable based on ThODNO3.
Biodegradation of Sodium Benzoate:	The reference item sodium benzoate was sufficiently degraded to 76% after 14 days and to 85% 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, 43% (ThODNO3) biodegradation was noted within 14 days and 48% (ThODNO3) biodegradation after 28 days of incubation. 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.