Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-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:

04 July 2016 - 04 August 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test.

Version / remarks:

1999

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium".

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of certificate: 3 Nov 2015

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: the freshly obtained sludge was preconditioned to experimental conditions by continuous aeration until further treatment.
- Storage length: not indicated
- Preparation of inoculum for exposure: before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: the day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each test bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: 3.4 g suspended solids/L in the concentrated sludge (before preperation of inoculum)
- Water used: tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to OECD 301
- Test temperature: 21.4 - 22.9 °C.
- pH: at the start of the test: 7.5 - 7.6 in different test media; at the end of the test: 7.6 - 8.0 in different test media
- pH adjusted: no
- Aeration of dilution water: continuously during the test period
- Suspended solids concentration: 10 mL of supernatant liquid /L of mineral medium (for more details see 'Details on inoculum' section)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre glass brown bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: synthetic air was sparged through the scrubbing solutions at a rate of approx. 1-2 bubbles per second (ca. 30-100 mL/min)
- Measuring equipment: the amount of CO2 produces was determined by titraining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl). Phenolphthalein (1% solution in ethanol) was used as pH-indicator.
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics: CO2 absorbers, i.e. bottles filled with 100 mL 0.0125 M Ba(OH)2

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Positive control: yes, 1 replicate
- Toxicity control: yes, 1 replicate

STATISTICAL METHODS: none

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.1%; 12 mg TOC/L

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

5.5

Sampling time:

28 d

Remarks on result:

other: Mean of 2 bottles with 6% and 5% degradation

Details on results:

- The ThCO2 of the test item was calculated to be 1.93 mg CO2/mg, the ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
- The relative biodegradation values calculated from the measurements performed during the test period revealed no significant (6% and 5%) biodegradation of the test item. In the toxicity control, more than 25% biodegradation occurred within 14 days (34%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see also paragraph 5.7.2).

BOD5 / COD results

Results with reference substance:

73% biodegradation within 14 days.

Any other information on results incl. tables

Table 1 CO₂ production in the blank

Day	HCl (0.05 M) titrated (mL)		Produced CO2	Produced CO2	Cumulative CO2
	Ba(OH)2^1)	Blank (mean)	(mL HCl)	(mg)	(mg)
2	49.51	46.69	2.83	3.1	3.1
5	49.65	45.57	4.08	4.5	7.6
7	48.74	44.79	3.95	4.3	11.9
9	48.77	45.67	3.10	3.4	15.3
14	49.10	44.31	4.80	5.3	20.6
19	49.29	43.62	5.67	6.2	26.9
23	49.26	42.58	6.68	7.3	34.2
27	50.00	43.46	6.55	7.2	41.4
29	49.22	42.79	6.43	7.1	48.5
29	49.62	46.14	3.48	3.8	52.3
29	49.61	48.30	1.31	1.4	53.7

¹⁾: "Strength" of untreated 0.0125 M Ba(OH)₂solution

Table 2 CO₂ production and percentage biodegradation of the positive control item.

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Positive control
2	46.69	45.56	1.13	1.2	1.2	1
5	45.57	23.80	21.77	23.9	25.2	29
7	44.79	30.22	14.57	16.0	41.2	48
9	45.67	36.61	9.06	10.0	51.2	60
14	44.31	33.63	10.68	11.7	62.9	73
^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.9 mg CO2/2L

Table 3 CO₂ production and percentage biodegradation of the test item (bottle A).

Day	HCl (0.05 M)titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle A
2	46.69	47.27	0.00	0.0	0.0	0
5	45.57	45.95	0.00	0.0	0.0	0
7	44.79	44.64	0.15	0.2	0.2	0
9	45.67	45.63	0.04	0.0	0.2	0
14	44.31	44.58	0.00	0.0	0.2	0
19	43.62	43.45	0.16	0.2	0.4	0
23	42.58	42.06	0.52	0.6	1.0	1
27	43.46	42.05	1.41	1.5	2.5	3
29	42.79	41.25	1.54	1.7	4.2	5
29	46.14	46.49	0.00	0.0	4.2	5
29	48.30	47.84	0.45	0.5	4.7	6
^1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 82.9 mg CO2/2L

Table 4 CO₂ production and percentage biodegradation of the test item (bottle B).

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)
	Blank (mean)	Bottle B				Biodegradation 1) (%)
2	46.69	46.70	0.00	0.0	0.0		0
5	45.57	45.15	0.41	0.5	0.5		1
7	44.79	44.98	0.00	0.0	0.5		1
9	45.67	45.39	0.28	0.3	0.8		1
14	44.31	43.43	0.88	1.0	1.7		2
19	43.62	42.78	0.83	0.9	2.6		3
23	42.58	41.97	0.61	0.7	3.3		4
27	43.46	43.06	0.39	0.4	3.8		4
29	42.79	43.25	0.00	0.0	3.8		4
29	46.14	46.72	0.00	0.0	3.8		4
29	48.30	48.05	0.25	0.3	4.0		5

¹⁾: Calculated as the ratio between CO₂ produced (cumulative) and the ThCO₂of the test item: 82.8 mg CO₂/2L 

Table 5 CO₂ production and percentage biodegradation of the toxicity control.

Day	HCl (0.05 M) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Toxicity control
2	46.69	45.36	1.33	1.5	1.5	1
5	45.57	24.24	21.33	23.5	24.9	14
7	44.79	34.16	10.63	11.7	36.6	21
9	45.67	36.71	8.96	9.9	46.5	27
14	44.31	35.35	8.96	9.9	56.3	33

¹⁾: Calculated as the ratio between CO₂ produced (cumulative) and the sum of the ThCO₂ of the test item and positive control: 172.6 mg CO₂/2L (ThCO₂ test item: 86.7 mg CO₂/2L + ThCO₂ sodium acetate: 85.9 mg CO₂/2L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' section for more details on validity criteria.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of the modified Sturm test, according to OECD 301B, Disodium 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonate was found to biodegrade for 5.5% (for both duplicates tested) after 28 days of incubation. Therefore, the test substance is considered to be not readily biodegradable under the experimental conditions described in this report.

Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and GLP, Disodium 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonate did not reach the pass level of 60% for ready biodegradability, neither within the 10 day window, nor after 28 days of incubation. Therefore, the test substance is designated as not readily diodegradable. All the criteria for acceptibility of the test were met and the study is considered valid without restrictions.