9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

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:

20 Jul 2016 to 18 Aug 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 4

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.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

275-640-9

EC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

71566-55-7

Molecular formula:

C44H50CoN10O10S2.C28H31N2O3

IUPAC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-N-3-(isopropoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

Test material form:

solid

Details on test material:

- Physical state / Appearance: Solid / red
- Total Organic Carbon: 612 mg/g

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: At room temperature

Study design

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.
- Preparation of inoculum: The inoculum was collected on 18 July 2016 from the aeration tank of the plant. 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.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

20 mg/L

Based on:

TOC

Initial test substance concentration 2

Initial conc.:

33.5 mg/L

Based on:

test mat.

Remarks:

replicate 1

Initial test substance concentration 3

Initial conc.:

32.8 mg/L

Based on:

test mat.

Remarks:

replicate 2

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

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.

TEST CONDITIONS
- Composition of medium: 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.
1. Solution A: KH2PO4 : 8.50 g; K2HPO4 : 21.75 g; Na2HPO4 × 2 H2O : 33.40 g; NH4Cl : 0.50 g. The compounds were dissolved with deionised water to 1000 mL; the pH value was adjusted to 7.4.
2. Solution B: CaCl2 × 2 H2O : 36.40 g. The compound was dissolved with deionised water to 1000 mL.
3. Solution C: MgSO4 × 7 H2O : 22.50 g. The compound was dissolved with deionised water to 1000 mL.
4. Solution D: FeCl3 × 6 H2O : 0.25 g. The compound was dissolved with deionised water to 1000 mL.
- Temperature: 22 + 2 °C
- pH adjusted: Yes, to 7.4 ± 0.2, if necessary.
- Aeration: Yes, with carbon dioxide free air 800 mL/h
- Suspended solids concentration: 30 mg/L dry weight.
- Stirring: The test assays were stirred using magnetic stirrers.

TEST SYSTEM
- Culturing apparatus: 2 L incubtion bottles, filled up to a volume of 1.5 L
- Number of culture flasks: 2

SAMPLING
- Sampling frequency: Usually twice a week
- Sampling method: 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. For determination of the decrease of 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). 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 assays (BC)
- Inhibition control: 1 assay (IH)
- Reference substance: 1 assay (RS)

Reference substance

Reference substance:

aniline

Remarks:

20 mg/L TOC

Results and discussion

Test performance:

- Measured DIC-concentrations in the blank controls at begin of exposure (mean value): 1.0 mg/L
- Amount of produced CO2 in the blank controls at the end of exposure (mean value): 31.4 mg/L
- 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 not <1 mg/L
- The amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure (mean value) was <70 mg/L

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

< 10

Sampling time:

28 d

Details on results:

During the contact time of 28 days, no biodegradation of the test substance was observed. For more information, see 'Any other information on results incl. tables'; The degree of biodegradation of the inhibition control was determined to be 30% after 14 days.

BOD5 / COD results

Results with reference substance:

The degree of biodegradation for the reference substance was 84% after 14 days.

Any other information on results incl. tables

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

Test duration [days]	RS	IH	TS1	TS2	TS mv
0	0	0	0	0	0
2	0	-1	-1	-1	-1
5	34	13	-3	-3	-3
7	50	20	-2	-2	-2
14	84	30	-3	-1	-2
19	94	32	-4	0	-2
21	97	33	-5	0	-3
23	98	33	-6	0	-3
26	100	34	-7	0	-4
28	101	35	-9	-1	-5

RS: reference substance assay

IH: inhibition control assay

TS: test substance assay

mv: mean value

Table: Produced carbon dioxide amount in the test vessels

[mg CO2/test vessel]						mg CO2 added up after substraction of the mean value of the blank controls
Duration of exposure [days]	BC mv	RS	IH	TS1	TS2	RS	IH	TS1	TS2
2	4.2	3.7	2.8	2.9	3	-0.5	-1.4	-1.3	-1.2
5	7.2	45.1	37.8	4.6	5.4	37.4	29.2	-3.9	-3
7	3.3	21.1	17.6	4.4	4.4	55.2	43.5	-2.8	-1.9
14	8	45.3	30.1	7.2	9.3	92.5	65.6	-3.6	-0.6
19	6.4	16.8	12	5	7	102.9	71.2	-5	0
21	3.2	6.2	4.5	2.2	3.4	105.9	72.5	-6	0.2
23	3	4.7	3.6	2.3	3.2	107.6	73.1	-6.7	0.4
26	3.8	5.5	5	2.4	3.9	109.3	74.3	-8.1	0.5
28	2.8	4.3	3.9	2.4	2.8	110.4	77.4	-10.6	-0.6
29	5.2	4.8	7.2	3.1	4.1

BC: blank control assay

See table above for the other abbreviations

Measured data: TIC [mg/L]

Day		BC NaOH	BC1	BC2	RS	IH	TS1	TS2
	a	1
0	b	1	-	-	-	-	-	-
	a	0.9	12.8	12.1	11.1	8.7	8.9	9.3
2	b	1	13	12.3	11.3	8.8	9	9.3
	a	0.7	19.7	21.3	124	104	13.6	15.6
5	b	0.7	19.9	21.4	124	104	13.2	15.6
	a	0.7	8.5	11.2	58.4	48.9	13	13.1
7	b	0.9	8.7	11.2	58.6	48.9	12.8	12.9
	a	0.6	28.8	15.7	124	82.9	20.6	26
14	b	0.7	29.1	16.1	124	82.7	20.2	25.9
	a	0.7	23.4	13.2	46.9	33.5	14.2	19.8
19	b	0.6	23.2	12.8	46.5	33.7	14.4	19.9
	a	0.8	10.5	7.9	17.6	13	6.9	9.9
21	b	0.8	10.7	8	17.6	12.6	6.5	9.9
	a	0.7	9.6	8.3	13.6	10.6	6.9	9.3
23	b	0.7	9.5	8.1	13.4	10.5	6.7	9.3
	a	0.8	13.2	9.2	15.8	14.3	7.5	11.3
26	b	0.9	13.2	9.4	15.8	14.3	7.4	11.4
	a	0.6	8.7	8	12.3	11.3	7.2	8.3
28	b	0.7	8.7	8	12.7	11.4	7.4	8.4
flask 1	a		15.7	9.4	11.2	17.1	6.6	9.8
29	b		15.4	9.6	11.1	16.8	6.4	9.6
flask 2	a		3.5	3.1	3.4	4.1	3.5	3
29	b		3.4	3.1	3.4	4	3.5	2.9

See tables above for abbreviations

The NaOH blank value is determined on filling the absorption vessels.

The measured values, for instance applies on day 0 for the first and the second sampling.

The NaOH blank value was subtracted from each test vessel.

Applicant's summary and conclusion

Interpretation of results:

not readily biodegradable

Conclusions:

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