1H-Pyrazolo[3,4-b]pyridine-3-carboxamide, 5-fluoro-1-[(2-fluorophenyl)methyl]-

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:

24 Jul - 21 Aug 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

EC Number:

811-886-0

Cas Number:

1361232-73-6

Molecular formula:

C14 H10 F2 N4 O

IUPAC Name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

Test material form:

solid

Study design

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Species/Origin: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
Conditioning: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water (see 6.5) and aerated overnight. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
Principle: The test flasks prepared according paragraph 6.6 were incubated at 22°C * 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.
Surrounding Type: Climatised chamber
Temperature: 22°C ± 1°C
Light Conditions: Darkness
pH-Value of Test Solutions: 7.6 (measured at the start of the test)
7.4 to 7.9 (measured at the end of the test)
Recording: Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and were not reported.)

Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume

In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Remarks on result:

not readily biodegradable based on QSAR/QSPR prediction

Details on results:

Biodegradation of Test Item
Percentage Biodegradation:
Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

The test item never reached 10% biodegradation.
The mean biodegradation percentage at the end of the 28-day exposure period was 3% (ThODNO3).
If no nitrification is considered, the mean biodegradation was 8% (ThODNH4) after 28 days of incubation.

Conclusion:
The degradation rate of Fluorpyridoamid never reached 10%.
Therefore, Fluorpyridoamid is considered to be not readily biodegradable.

BOD5 / COD results

Results with reference substance:

7.2 Biodegradation of Reference Item Sodium Benzoate
Percentage Biodegradation:
The reference item sodium benzoate was sufficiently degraded to 82% after 14 days and to 88% after 28 days of incubation.


Conclusion:
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Any other information on results incl. tables

Table3.Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time	Percentage Biodegradation^1
(Days)	Fluorpyridoamid^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	28	14
2	0	0	46	27
3	0	0	55	32
4	2	2	64	40
5	0	0	69	42
6	0	0	72	44
7	0	0	75	45
8	0	0	75	47
9	0	0	78	49
10	0	0	78	49
11	0	0	81	49
12	0	0	81	50
13	2	0	82	50
14	5	2	82	51
15	5	2	82	51
16	5	2	82	51
17	5	2	82	51
18	5	2	82	51
19	5	2	85	51
20	5	5	85	51
21	5	5	85	53
22	5	5	85	53
23	5	5	88	53
24	5	5	88	53
25	5	5	88	53
26	5	5	88	53
27	5	5	88	53
28	5	5	88	53

               ¹ThOD_NH4of Fluorpyridoamid: 1.388 mg O₂/mg test item
               ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Table4. Percentage Biodegradation of Test Item and of the Toxicity Control based on ThOD_NO3and of Sodium Benzoate based on ThOD_NH4

Time	Percentage Biodegradation^1
(Days)	Fluorpyridoamid^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	28	11
2	0	0	46	21
3	0	0	55	25
4	1	1	64	31
5	0	0	69	33
6	0	0	72	34
7	0	0	75	35
8	0	0	75	36
9	0	0	78	38
10	0	0	78	38
11	0	0	81	38
12	0	0	81	39
13	1	0	82	38
14	3	1	82	40
15	3	1	82	40
16	3	1	82	40
17	3	1	82	40
18	3	1	82	40
19	3	1	85	40
20	3	3	85	40
21	3	3	85	41
22	3	3	85	41
23	3	3	88	41
24	3	3	88	41
25	3	3	88	41
26	3	3	88	41
27	3	3	88	41
28	3	3	88	41

            ¹ThOD_NO3of Fluorpyridoamid: 2.276 mg O₂/mg test item
            ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Table5.pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	Fluorpyridoamid	7.4
2	Fluorpyridoamid	7.4
3	Inoculum control	7.5
4	Inoculum control	7.5
5	Reference item (procedure control)	7.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Fluorpyridoamid never reached 10% biodegradation (ThODNH4, ThODNO3). The mean biodegradation at test end after 28 days was 5% (ThODNH4) and 3% (ThODNO3).
Therefore, Fluorpyridoamid is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.

Executive summary:

Biodegradation of Fluorpyridoamid: Nitrogen is part of the molecular structure of the test item; therefore the evaluation of biodegradation has to be based on ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation. Fluorpyridoamid never reached 10% biodegradation (ThODNH4, ThODNO3). The mean biodegradation at test end after 28 days was 5% (ThODNH4) and 3% (ThODNO3). Therefore, Fluorpyridoamid is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.