Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

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:

28 Sep 2015 to 28 Oct 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Qualifier:

according to guideline

Guideline:

other: International Standard ISO 9439 (Water Quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of released carbon dioxide)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Eukesolar Black ER Liquid - dried
- Batch identification: Dye powder sample 14/103 from Material no. 52629782; batch no. M-R/G
- Purity: 99.11 area-% (HPLC, 260 nm), 99.71 area-% (HPLC, 375 nm)
- Homogeneity: homogeneous
- Expiry date: 09 June 2016
- Date of production: 09 June 2014
- Appearance: solid / black
- Water solubility: not sufficiently soluble
- Storage conditions: storage at room temperature
- Total Organic Carbon: 543 mg/g

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: from the aeration tank of the wastewater treatment plant of Mannheim, Germany
- Collected on: 28 September 2015

Duration of test (contact time):

28 d

Initial conc.:

36.9 mg/L

Based on:

test mat.

Remarks:

replicate 1, corresponding to 20 mg/L TOC

Initial conc.:

36.7 mg/L

Based on:

test mat.

Remarks:

replicate 2, corresponding to 20 mg/L TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

COMPOSITION OF TEST MEDIUM
- Solution A: KH2PO4: 8.50 g; K2HPO4: 21.75 g; Na2HPO4·2H2O: 33.40 g; NH4Cl: 0.50 g. The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.
- Solution B: Solution B: CaCl2·2H2O : 36.40 g, the compound was dissolved with deionized water to1000 mL
- Solution C: MgSO4·7H2O : 22.50 g, the compound was dissolved with deionized water to 1000 mL
- Solution D: FeCl3·6H2O : 0.25 g, the compound was dissolved with deionized water to 1000 mL

TEST CONDITIONS
- Test temperature: 22 ± 2 °C

TEST SYSTEM
- Test assays: 2 blank control assays (BC), 2 test substance assays (TS), 1 inhibition control test assay (IH) and 1 reference substance assay (RS)
- Test vessels: 2 L incubation bottles filled up to a volume of 1.5 L
- Setup: the bottles were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes.

PREPARATION OF INOCULUM
Preparation of inoculum for exposure: A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 48 hours at 22 ± 2° C. At the day of exposure the suspension was washed one time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water and the concentration oft the sludge was adjusted to 6.0 g/L dry weight. The activated sludge concentration per test vessel was 30 mg/L.

PREPARATION OF TEST ASSAYS
- The test assays were prepared at the day of exposure. First, the required volumes of deionized water and the solutions of mineral salts were dosed to all test vessels. For preparation of the test vessels with test substance, the required amounts of the test substance aliquots for a test concentration of 20 mg/L TOC were weighed onto small glass plates (microscope cover slips) and completely added with glass plates to the vessels of the test substance assays and to the vessel of the inhibition control. Because of poor water solubility of test substance these test assays were treated for few minutes in an ultrasonic bath to ensure an even distribution of test substance in test medium. Finally enough reference substance stock solution was added to reach 20 mg TOC/L in the reference substance assay and 20 mg TOC/L in the inhibition control, related to aniline.
- The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2, if necessary.
- Aliquots of activated sludge suspension were added to all test vessels, to adjust the concentration of activated sludge to 30 mg/L dry weight. Samples for DIC-measurement (validity criterion) from the blank control assays were taken. For determination of the decrease of dissolved organic carbon (DOC) samples were taken from the test vessels of the blank control and from the test vessel of the reference substance control and the DOC content was determined after centrifugation (approx. 15 minutes at 4000 rpm).

EXPOSURE
At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers. At the end of exposure, the pH values were measured in each test vessel. For stripping of carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid. The concentration of dissolved organic carbon in the blank controls and reference substance assays were determined. Since the test substance was insufficiently soluble in water, no DOC-measurements could be performed from the test assay of the inhibition control and from the test substance test assays. The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day.

SAMPLING AND DETERMINATION OF TOTAL INORGANIC CARBON (TIC)
- Frequency: usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.
The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount. The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.

Reference substance:

aniline

Key result

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

Details on results:

DEGREE OF BIODEGRADATION
- Test substance: biodegradation increased up to 7% after 7 days, but did not increase any further. For more detail, see 'Any other information on results incl. tables'.
- Degree of biodegradation in the inhibition control after 14 days: 34 % CO2/ThCO2

Results with reference substance:

Degree of biodegradation of the reference substance after 14 days: 73 % CO2/ThCO2

PRODUCED CARBON DIOXIDE AMOUNT IN THE TEST VESSELS:

 

Table: produced carbon dioxide amount in the test vessels

Duration of exposure [days]	[mg CO2/test vessel]					mg CO2added up after subtraction of the mean value of the blank controls
	BC mv	RS	IH	TS1	TS2	RS	IH	TS1	TS2
2	2.7	4.4	7.9	4.4	5.5	1.7	5.2	1.7	2.8
5	5.6	50.9	49.4	9.4	10.4	47.0	49.0	5.5	7.6
7	4.3	17.3	18.1	4.6	5.6	60.0	62.8	5.8	8.9
12	10.2	24.1	16.2	9.3	10.8	73.9	68.8	4.9	9.5
14	4.3	11.2	9.7	4.4	4.9	80.8	74.2	5.0	10.1
16	4.1	9.5	7.0	3.7	4.6	86.2	77.1	4.6	10.6
19	5.3	9.6	7.0	4.4	6.6	90.5	78.8	3.7	11.9
22	4.6	6.9	4.9	5.4	3.6	92.8	79.1	4.5	10.9
26	5.8	8.0	8.0	5.2	5.9	95.0	81.3	3.9	11.0
28	3.5	3.7	4.0	2.8	3.7	94.2	81.2	3.4	11.7
29	5.2	4.2	4.6	5.4	5.7

BC = blank control; RS = reference substance; IH = inhibition control; TS = test substance; mv = mean value

 

CALCULATION OF DEGREE BIODEGRADATION:

 

Table: theoretical carbon dioxide [mg/test vessel]

RS: 110.0

IH: 220.4

TS1: 110.4

TS2: 109.8

 

BC = blank control; RS = reference substance; IH = inhibition control; TS = test substance

 

 Table: degree of biodegradation; [CO₂/ThCO₂]

Test duration [days]	RS	IH	TS1	TS2	TS mv
0	0	0	0	0	0
2	2	2	2	3	3
5	43	22	5	7	6
7	55	28	5	8	7
12	67	31	4	9	7
14	73	34	5	9	7
16	78	35	4	10	7
19	82	36	3	11	7
22	84	36	4	10	7
26	86	37	4	10	7
28	86	37	3	11	7

BC = blank control; RS = reference substance; IH = inhibition control; TS = test substance; mv = mean value

 

 

MEASUREMENT OF DISSOLVED ORGANIC CARBON:

 

Table: measured values [mg/L], DOC - elimination degree [%]

	[mg/L DOC]
	BC1	BC2	BC mv	RS	IH	TS1	TS2	TS mv
DOC at test begin	0.4	0.4	0.4	19.8	-	-	-
	0.4	0.3		19.8	-	-	-
DOC at test end	0.7	0.7	0.7	1.0	-	-	-
	0.7	0.7		1.0	-	-	-
DOC elimination [%]				98	-	-	-	-

BC = blank control; RS = reference substance; IH = inhibition control; TS = test substance; mv = mean value

Remark: Since the test substance was insufficiently soluble in water, no DOC-measurements could be performed from the test assay of the inhibition control and from the test substance test assays.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods'

Interpretation of results:

not readily biodegradable

Description of key information

The test substance was found to be not readily biodegradable in an OECD TG 301B guideline study.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The biodegradation potential of the substance in water was determined in a study according to OECD TG 301B (CO2-Evolution Test) and in compliance with GLP criteria (BASF, 2016). In this study, ca. 36.8 mg/L test substance (corresponding to 20 mg/L TOC) was inoculated with non-adapted activated sludge from predominantly domestic sewage for 28 days under aerobic conditions. At regular intervals throughout the incubation period the evolved CO2, measured as mg total inorganic carbon (TIC), was determined and compared to the ThCO2 as indication of biodegradation. Based on CO2/ThCO2, <10% (7%) of the test substance was biodegraded after the 28-day exposure period. Based on these findings the substance is classified as not readily biodegradable.