Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

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:

19 July 2018 to 16 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Secondary effluent of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan (Sampling date: July 19, 2018)
- Laboratory culture: The secondary effluent was filtered through a No.2 filter paper and the filtrate was used as inoculum. The inoculum was kept under aerobic conditions until use.

Duration of test (contact time):

28 d

Initial conc.:

4.95 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium (5 L) was prepared at the same proportion as the following method: each 1 mL of stock solutions (a), (b), (c), and (d) prescribed in the OECD 301D test method were made up to 1 L with purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical). After the preparation, it was aerated for 60 minutes and left to stand for 23 hours at test temperature.
- Test temperature: 19.3 – 20.6 °C
- Suspended solids concentration: 0.05 mL/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass bottle (100 mL in volume)
- Number of culture flasks/concentration: 10
- Test solution (inoculum + test material) (n= 10): Each test bottle was filled with 4.95 mg/L test material solution prepared with the inoculated mineral medium. The test material solution of 4.95 mg/L was prepared as follows: the test sample (110 mg i.e. 99 .0 mg as the test material) was accurately weighed with an electronic analytical balance and dissolved in the inoculated mineral medium in order to obtain 495 mg/L solution of the test material (200 mL). The test solution of 4.95 mg/L was then prepared from this solution (15 mL) by dilution with the inoculated mineral medium (1.5 L).
- Measuring equipment: DO of each test solution was measured by diaphragm electrode method, and biochemical oxygen demand (BOD) was calculated (DO meter (No. 00-016): B-100BTi (Iijima Electronics)
- Stationary incubation was performed in sealed bottles

SAMPLING
- Frequency and number of measurement:
Test solution (inoculum + test material): At the beginning of incubation and on Day 7, 14, 21 and 28, n=2.
Test solution (inoculum + sodium benzoate): At the beginning of incubation and on Day 7, 14, 21 and 28, n=2.
Inoculum blank: At the beginning of incubation and on Day 7, 14, 21 and 28, n=2.
- During the incubation, appearances of the test solutions were observed once a day. The incubation temperature was measured and recorded once a day.

CONTROL AND BLANK SYSTEM
- Inoculum blank (n= 10): Each test bottle was filled with the inoculated mineral medium.
- Reference substance control: Test solution (inoculum + sodium benzoate) (n= 10): Each test bottle was filled with 3.00 mg/L sodium benzoate solution prepared with the inoculated mineral medium. The sodium benzoate solution of 3.00 mg/L was prepared as follows: sodium benzoate (150 mg) was accurately weighed with an electronic analytical balance and dissolved in the inoculated mineral medium in order to obtain 1500 mg/L solution of the test material (100 mL). The sodium benzoate solution of 3.00 mg/L was then prepared from this solution (3 mL) by dilution with the inoculated mineral medium (1.5 L).

CALCULCATION OF PERCENTAGE BIODEGRADATION
The percentage biodegradation was calculated by the following equations and rounded off to the whole number. In the calculation of TOD of the test material, nitrogen form was treated as NH3.
Percentage biodegradation (%) = (BODx / TOD) x 100
BODx: BOD in the test solutions (inoculum + test material) or (inoculum + sodium benzoate) on x days (experimental: mg O2/L)
TOD: Theoretical oxygen demand required when the test material or sodium benzoate was completely oxidised (calculated: mg O2/L)
BODx = (mt(0) - mt(x)) - (mb(0) - mb(x))
mb(x): Mean DO in the inoculum blank on x days (mg O2/L)
mb(0): Mean DO in the inoculum blank at the beginning of incubation (mg O2/L)
mt(x): DO in the test solutions (inoculum + test material) or (inoculum + sodium benzoate) on x days (mg O2/L)
mt(0): DO in the test solutions (inoculum + test material) or (inoculum + sodium benzoate) at the beginning of incubation (mg O2/L)

Reference substance:

benzoic acid, sodium salt

Test performance:

Validity of the Test
- Difference between extremes of replicate values of percentage biodegradation of test material by BOD on Day 28 should be < 20 %. The value in this test was 1 %.
- Percentage biodegradation of sodium benzoate by BOD on Day 14 should be > 60 %. The values in this test were 70 and 71 %.
- Amount of oxygen depletion of inoculum blank on Day 28 should be ≤ 1.5 mg O2/L. The value in this test was 0.72 mgO2/L.
- Residual concentration of oxygen in each bottle during the incubation should be ≥ 0.5 mg O2/L. The value in this test was ≥ 4.47 mgO2/L.

Key result

Parameter:

% degradation (O2 consumption)

Value:

-1

Sampling time:

28 d

Details on results:

The average percentage biodegradation by BOD was -1 % on Day 28 under the test conditions of this study. The value did not meet a criterion of ready biodegradability (percentage biodegradation by BOD > 60 %) described in OECD Guidelines for the Testing of Chemicals, No. 301. It showed that the test material can be considered as not a readily biodegradable substance.

Results with reference substance:

Percentage biodegradation values of sodium benzoate by BOD on Day 14 were 70 and 71 %, which met the validity criteria of > 60 %.

Table 1: Percentage Biodegradation

	Inoculum + Test Material (The Value in [ ] is a Mean Value)
	Day 7	Day 14	Day 21	Day 28
Percentage Biodegradation by BOD	-2	-1	0	0
	-2	-2	-2	-1
	[-2]	[-2]	[-1]	[-1]

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the average percentage biodegradation by BOD was -1 % on Day 28. This showed that the test material can be considered as not a readily biodegradable substance.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301D, under GLP conditions.

Filtrate of secondary effluent of a sewage treatment plant treating predominantly domestic sewage was exposed to the test material at 4.95 mg/L for 28 days in darkness. Dissolved oxygen was measured by diaphragm electrode method.

The validity criteria of the test were all met.

Under the conditions of this study, the average percentage biodegradation by BOD was -1 % on Day 28. This showed that the test material can be considered as not a readily biodegradable substance.

Description of key information

Under the conditions of the study, the average percentage biodegradation by BOD was -1 % on Day 28. This showed that the test material can be considered as not a readily biodegradable substance.

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 guideline OECD 301D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Filtrate of secondary effluent of a sewage treatment plant treating predominantly domestic sewage was exposed to the test material at 4.95 mg/L for 28 days in darkness. Dissolved oxygen was measured by diaphragm electrode method.

The validity criteria of the test were all met.

Under the conditions of the study, the average percentage biodegradation by BOD was -1 % on Day 28. This showed that the test material can be considered as not a readily biodegradable substance.