Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

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:

13 Sep 2016 to 28 Feb 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Storage at room temperature

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 13 September 2016 from the aeration tank of the plant.
- 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.
- Pretreatment: 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 of the sludge was adjusted to 6.0 g/L dry weight.
- Concentration of sludge: Aliquots of 7.5 mL were added to the test vessels to obtain an activated sludge concentration of 30 mg/L dry weight.

Duration of test (contact time):

28 d

Initial conc.:

ca. 33.4 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SYSTEM
The Carbon Dioxide Evolution Test was performed in 2 L incubation bottles filled up to a volume of 1.5 L. 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.
- Test temperature: 22 ± 2 °C

EXPERIMENTAL PROCEDURE
The following test assays were prepared:
- 2 blank control assays (BC)
- 2 test substance assays (TS)
- 1 inhibition control assay (IH)
- 1 reference substance assay (RS)

COMPOSITION OF TEST MEDIUM
The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
- Solution A: 8.50 g KH2PO4; 21.75 g K2HPO4; 33.40 g Na2HPO4.2H2O; and 0.50 g NH4Cl.
- Solution B: 36.40 g CaCl.2H2O.
- Solution C: 22.50 g MgSO4.7H2O
- Solution D: 0.25 g FeCl3.6H2O
For all solutions, the compounds were dissolved with deionised water to 1000 mL; for solution A, the pH value was adjusted to 7.4. 15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

PREPARATION OF TEST ASSAYS
- The test assays were prepared at the day of exposure. First, the required volumes of deionised 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 the 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.

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 analysed 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.
- Before exposure, 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).

Reference substance:

aniline

Test performance:

VALIDITY CRITERIA
- Measured DIC-concentrations in the blank controls at begin of exposure was 0.7 mg/L (mean value).
- Amount of produced CO2 in the blank controls at the end of exposure was 51.5 mg/L (mean value).
- Deviation of the degree of biodegradation of the test substance in the plateau phase was <20%.
- The degree of biodegradation of the reference substance was >60% CO2/ThCO2 after 14 days.
- The degree of biodegradation in the inhibition control was >25 % CO2/ThCO2 after 14 days.
- The content of DIC in the blank control at start of exposure at the test concentration of 20 mg/L TOC was <1 mg/L.
- The amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure was <70 mg/L (mean value).
The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study. No deviations from the test guidelines or other incidents occurred during the course of the reported test, which may have influenced the result.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

Details on results:

Almost no biodegradation of the test substance was observed: the highest % CO2/ThCO2 reached was 9% at day 11, which decreased to 4% at day 28. A detailed overview of the test results is provided in 'Any other information on restults incl. tables'. The degree of biodegradation in the inhibition control after 14 days was 39 % CO2/ThCO2.

Results with reference substance:

The degree of biodegradation of the reference substance after 14 days was 76 % CO2/ThCO2.

Table: Degree of biodegradability; [% CO2/ ThCO2]

Test duration [days]	RS	IH	TS1	TS2	TS mean value
0	0	0	0	0	0
1	0	0	0	0	0
4	24	14	3	4	4
7	51	29	6	10	8
11	67	37	7	11	9
14	76	39	6	11	9
18	83	41	3	9	6
21	86	42	2	9	6
25	88	43	1	9	5
27	89	44	-2	10	6
28	88	44	-2	9	4

RS: reference substance assay

IH: inhibition control assay

TS: test substance assay

Table: Produced carbon dioxide amount in the test vessels

[mg CO2/test vessel]						mg CO2 added up after subtracting of the mean value of the blank controls
Duration of exposure [days]	BC mv	RS	IH	TS1	TS2	RS	IH	TS1	TS2
1	2.1	2.2	2.3	2.3	2.1	0.1	0.2	0.2	0.0
4	8.9	35.0	40.1	11.9	13.1	26.2	31.4	3.2	4.2
7	7.1	37.7	40.3	11.0	14.0	56.8	64.6	7.1	11.1
11	10.4	27.4	26.4	11.0	12.0	73.8	80.6	7.7	12.7
14	8.8	19.1	15.0	7.4	7.8	84.1	86.8	6.3	11.7
19	12.0	19.3	16.0	8.6	9.9	91.4	90.8	2.9	9.6
22	6.1	9.9	8.9	5.8	6.4	95.2	93.6	2.6	9.9
25	7.0	9.2	9.2	5.8	7.0	97.4	95.8	1.4	9.9
27	4.2	5.5	5.0	4.7	5.2	98.7	96.6	1.9	10.9
28	3.3	3.5	3.5	2.9	3.4	97.3	96.1	-1.7	10.4
29	7.4	5.8	6.7	4.2	6.8

Table: Measurement of Total Inorganic Carbon

Day	Rep.	BC NaOH	BC1	BC2	RS	IH	TS1	TS2
0	a	0.8	-	-	-	-	-	-
	b	0.7
1	a	0.8	6.4	6.5	6.7	7.2	7.0	6.3
	b	0.7	6.5	6.8	6.9	7.0	7.1	6.4
4	a	0.8	24.4	26.0	96.2	110	33.3	36.8
	b	0.9	24.7	25.4	96.1	110	33.2	36.3
7	a	0.8	21.2	19.3	104	111	31.0	39.0
	b	0.9	20.7	19.1	103	110	30.7	38.8
11	a	0.8	31.3	27.5	75.5	73.2	30.9	33.3
	b	0.9	31.1	27.3	75.3	72.3	30.7	33.7
14	a	1.1	25.3	24.3	53.1	41.8	21.1	22.4
	b	1.2	25.3	23.9	52.7	41.9	20.9	22.0
19	a	0.9	32.0	35.2	53.6	44.6	24.3	28.0
	b	0.9	32.3	35.1	53.2	44.4	24.1	27.9
22	a	0.7	17.2	18.0	28.3	25.2	17.2	18.9
	b	0.9	17.7	17.7	28.2	25.4	16.9	18.3
25	a	0.9	19.4	20.9	26.4	26.2	16.8	20.0
	b	0.9	19.4	20.5	25.8	25.6	16.4	20.1
27	a	1.0	11.7	13.0	16.0	14.7	13.9	15.1
	b	1.1	11.4	12.9	15.7	14.4	13.5	14.9
28	a	1.3	10.2	9.8	10.3	10.6	9.0	10.4
	b	1.2	10.1	9.4	10.4	10.3	8.7	10.1
flask 1 29	a		16.9	20.1	14.1	15.3	9.1	16.1
	b		16.9	19.7	14.1	15.4	9.0	15.9
flask 2 29	a		3.8	4.2	4.0	5.3	4.8	5.1
	b		3.8	4.1	4.1	5.2	4.6	4.8

Table: Measured values [mg/L], determination of DOC after centrifugation  

		BC1	BC2	BC mv	RS	IH	TS1	TS2	TS mv
At begin of exposure	a	0.4	0.3	0.3	18.3	-	-	-
	b	0.3	0.2		18.4	-	-	-
At the end of exposure	a	1.0	1.0	0.1	1.0	-	-	-
	b	1.0	0.9		1.1	-	-	-
Degradation of DOC [%]					99	-	-	-	-

Validity criteria fulfilled:

yes

Remarks:

See 'Test performance'.

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.

Description of key information

The test substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.

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 screening study according to OECD TG 301B (CO2 -Evolution Test) and in compliance with GLP criteria (BASF SE 2017). In this study 20 mg/L test substance (expressed as TOC) was inoculated with activated sludge from a municipal sewage plant for 28 days under aerobic conditions. During the incubation period degradation was followed by determining the carbon dioxide produced. After the 28-day incubation period < 10% of the test substance was biodegraded. Correspondingly, the required pass level for ready biodegradability within a ten day window was not reached. Based on these findings, the test substance is classified as not readily biodegradable.