Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

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:

15 Dec 2017 - 12 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

EC Number:

257-479-6

EC Name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

Cas Number:

51858-17-4

Molecular formula:

C25H16N4O12S2.2K

IUPAC Name:

dipotassium dihydrogen 4-{5-[3-carboxylato-5-oxo-1-(4-sulfonatophenyl)-1,5-dihydro-4H-pyrazol-4-ylidene]penta-1,3-dien-1-yl}-5-hydroxy-1-(4-sulfonatophenyl)-1H-pyrazole-3-carboxylate

Test material form:

solid: crystalline

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Aerobic activated sludge from sewage treatment plant Rossdorf, Germany
- Storage conditions: aerated overnight
- Storage length: sludge was stored overnight
- Preparation of inoculum for exposure: Activated sludge was washed by centrifugation and the supernatant liquid phase was decanted; solid material was resuspended in test water and centrifuged again and repeated three times; based on the the ration of wet sludge to its dry weight calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight.
- Pretreatment: An aliquot of sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
- Concentration of sludge: 3.5 g dry material per litre

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

102.9 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

107.1 mg/L

Based on:

ThOD

Remarks:

ThODNH4

Initial test substance concentration 3

Initial conc.:

144.4 mg/L

Based on:

ThOD

Remarks:

ThODNO3

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according the OECD guideline 301
- Test temperature: 22 ± 1 °C
- pH: 7.2 - 7.4 at the end of the test; 8.0 at the beginning (adjusted to 7.6 with hydrochloric acid)
- pH adjusted: Yes
- Continuous darkness: Yes
- Other: Continuous stirring

TEST SYSTEM
- Culturing apparatus and measuring equipment: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany (Manometric Test System, 500 mL fill volume in flasks)
- Number of culture flasks/concentration: 2
- Details of trap for CO2: potassium hydroxide solution (45%)

SAMPLING
- Sampling method: Change of pressure in the test flasks was measured by means of a manometric method each day.

CONTROL AND BLANK SYSTEM
- Inoculum control: Yes, 2 replicates
- Abiotic sterile control: Yes,1 replicate (poisoned with HgCl2)
- Toxicity control: Yes,1 replicate
- Other: procedure control: Yes, 1 replicate

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

28 d

Remarks on result:

other: mean of two replicates

Details on results:

The test item never reached 10% biodegradation. The mean biodegradation percentage at the end of the 28-day exposure period was 1% based on ThODNH4 (1% based on ThODNO3).The degradation rate of the test item never reached 60%. Therefore, Oxonole Blue is considered to be not readily biodegradable.
In the toxicity control 53% and 47% biodegradation was noted within 14 days and 60% as well as 53% biodegradation after 28 days of incubation (based on ThODNH4 and ThODNO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

BOD5 / COD results

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 77% after 14 days and to 85% after 28 days of incubation.

Any other information on results incl. tables

Table 1: Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time	Percentage Biodegradation1
(Days)	Oxonole Blue1		Sodium Benzoate 2	Toxicity control 1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	32	23
2	0	0	41	29
3	0	0	49	34
4	0	0	56	41
5	0	0	64	43
6	0	0	68	48
7	0	0	69	49
8	0	0	72	49
9	0	2	74	50
10	0	0	72	51
11	0	0	75	51
12	0	5	75	52
13	0	5	75	52
14	0	2	77	53
15	0	2	77	55
16	0	2	80	55
17	0	0	78	54
18	0	5	78	54
19	0	2	77	55
20	0	2	77	55
21	0	2	80	57
22	0	2	80	59
23	0	2	80	59
24	0	2	80	59
25	0	2	80	59
26	0	2	82	59
27	0	2	82	59
28	0	2	85	60

1 ThODNH4 of Oxonole Blue: 1.041 mg O2/mg test item

2 ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference item

Table 2: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	2%	yes
Percentage degradation of the reference compound has reached the pass levels by day 14.	>60%	yes
The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.	27.5 mg/L	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See details in section "Any other information on results incl tables"

Interpretation of results:

under test conditions no biodegradation observed