1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

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:

Experimental Starting Date: 13 May 2014 Experimental Completion Date: 16 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

BMS-589152-01

IUPAC Name:

BMS-589152-01

Test material form:

solid: particulate/powder

Details on test material:

Identification: BMS-589152-01
Physical state/Appearance: tan-colored powder
Batch: 2G72631N
Purity: 99.5%
Expiry Date: 19 June 2014
Storage Conditions: room temperature in the dark
Intended use/Application: chemical intermediate

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Test System:
A mixed population of activated sewage sludge micro-organisms was obtained on 12 May 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum:
The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.1 g/L prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SYSTEM:
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

TEST CONDITIONS:
Mineral medium: The mineral medium used in this study was that recommended in the OECD Guidelines.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 to 23 °C, in darkness.

Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 29 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to
100 mL/min per vessel and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

EXPERIMENTAL PREPARATION:
TEST ITEM:
The test item was dispersed directly in mineral medium.

An amount of test item (49.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 16.5 mg/L, equivalent to 10 mg carbon/L. The preparation of the test item dispersion was carried out under laboratory safety lighting.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

CONTROL AND BLANK SYSTEM:
- Reference item:
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

- Toxicity control:
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An amount of test item (49.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 16.5 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

DATA EVALUATION:
Observations: The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

Calculation of Carbon Content:
The test item contains 60.55% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:

No of C atoms x mol wt of C atoms / mol wt of sodium benzoate x 100

(7 x 12.011 / 144.11) x 100 = 58.34%

Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

Percentage degradation:
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1*, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation:
* Table 1 can be found in the any other information on results section.

%ThCo2(=%degradation)= [(mg IC in test flask - mg IC in control) / mg TOC added as test material] x 100.

Total Co2 evolution:
The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control x 100/%C of Co2 x 1/test volume.

= mg IC in control x 100/27.29 x 1/3

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Test performance:

Validation Criteria:
The total CO2 evolution in the inoculum control vessels on Day 28 was 15.73 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3*) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

* all tables can be found in the any other information on results section below.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

61

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1*. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2* and the biodegradation curves are presented in Figure 1**. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3*. Observations made on the contents of the test vessels are given in Table 4*.

* All tables can be found in the any other information on results section (please see below)

** Figure 1 can be found in the illustration section below

Biodegradation values:
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of procedure control R1. This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test item attained 61% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.

The toxicity control attained 46% biodegradation after 14 days and 44% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 79% biodegradation after 14 days and 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Tables

Table 1     Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	0.47	0.35	0.35	0.47	0.58	1.05	0.47	0.35	0.35	0.58	0.47	0.35	0.47	0.58
2	3.83	-	3.83	-	19.25	-	18.21	-	7.66	-	7.42	-	10.67	-
6	8.54	-	8.19	-	29.41	-	24.68	-	15.57	-	14.76	-	14.07	-
8	10.66	-	9.52	-	36.58	-	30.96	-	20.41	-	17.77	-	36.58	-
10	10.15	-	11.29	-	36.25	-	34.54	-	24.17	-	20.18	-	36.71	-
14	11.90	-	11.22	-	37.97	-	32.30	-	27.31	-	22.55	-	38.99	-
21	14.53	-	13.63	-	38.98	-	35.26	-	31.43	-	24.90	-	40.11	-
28	12.66	-	13.10	-	41.66	-	35.73	-	32.15	-	29.23	-	41.77	-
29	14.81	0.81	15.59	1.04	40.86	0.93	36.85	0.70	33.84	0.70	33.07	0.70	41.89	0.70

R₁– R₂          =   Replicates 1 and 2

Abs              =  CO₂absorber vessels

Table 2     Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	50	12	11
6	62	23	10
8	79	30	44
10	82	38	43
14	79	45	46
21	77	47	43
28	86	59	48
29*	79	61	44

*Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Table 3     Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	9.87^**	0.14	1
Test Item 10 mg C/L R2	9.82^**	-1.66	0

R₁– R₂     =  Replicates 1 and 2

*       Corrected for control values. Negative values are due to measured concentrations being less than control values

**    Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Table 4     Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown very slightly cloudy dispersion with fine particles of test item visible on the surface	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible
	R2	Light brown very slightly cloudy dispersion with fine particles of test item visible on the surface	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible	Light brown very slightly cloudy dispersion, no undissolved test item visible
Toxicity Control		Light brown very slightly cloudy dispersion with fine particles of test item visible on the surface, no undissolved reference item visible	Light brown very slightly cloudy dispersion, no undissolved test item or reference item visible	Light brown very slightly cloudy dispersion, no undissolved test item or reference item visible	Light brown very slightly cloudy dispersion, no undissolved test item or reference item visible	Light brown very slightly cloudy dispersion, no undissolved test item or reference item visible

 

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 61% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.

Executive summary:

Introduction:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

 

 Methods:

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 to 23°C for 28 days.The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

  

Results:

The test item attained 61% biodegradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301B the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. However, the test item has exhibited the potential for rapid biodegradation.