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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 31 May 2016 and 01 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

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

Specific details on test material used for the study:

Identification: BMS-589152-01
Physical state/Appearance: beige powder
Batch: AAG8999N
Purity: 100.1% W/W
Retest Date: 27 December 2017
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

As it was not a requirement of the Test Guideline, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement in the original study report.

Test solutions

Vehicle:

no

Details on test solutions:

In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water. The preparation of the test item dispersion was carried out under laboratory safety lighting.
Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 21 °C, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.
The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 1 June 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.6 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.
* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

Test water: The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Synthetic sewage: A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock was 7.4. The pH value was measured using a Hach HQ40d Flexi handheld meter.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

19 - 21 ºC

pH:

7 - 8

Nominal and measured concentrations:

Test substance - Nominal: 10,100 and 1000 mg/l
Reference substance (3,5-dichlorophenol) - Nominal: 3.2, 10 and 32 mg/l

Details on test conditions:

Preparation of Test System:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as previously described. Finally two further control vessels were prepared.
The test was conducted under laboratory safety lighting in a temperature controlled room at measured temperatures of between 19 and 21 ºC.

Assessments
Observations:
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.

pH Measurements:
The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.

Oxygen Concentration:
The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Measurement of the Respiration Rates
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

inhibition of respiration rate

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

inhibition of respiration rate

Details on results:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 3.5% and the specific respiration rate of the controls was 20.89 mg oxygen per gram dry weight of sludge per hour.
The reference item gave a 3-Hour EC50 value of 9.5 mg/L, 95% confidence limits 7.6 to 12 mg/L.
The validation criteria have therefore been satisfied.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (3hr): 9.5 mg/l (95% confidence limits 7.6 - 12 mg/L)

Any other information on results incl. tables

 Inhibition of Respiration Rate

The following results were derived:

	BMS-589152-01		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>1000	-	2.5	-
EC20	>1000	-	3.5	-
EC50	>1000	-	9.5	7.6 - 12
EC80	>1000	-	26	-
NOEC	1000	-	-	-

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O₂/L and 2 mg O₂/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test are displayed in the table below:

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	10	6.0	7.1
	100	6.2	7.0
	1000 R1	6.4	7.3
	1000 R2	6.4	7.2
	1000 R3	6.6	7.3

 R₁– R₃= Replicates 1 to 3

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test are displayed in the table below:

 

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	7.2	81
	R2	7.1	80
	R3	6.4	72
	R4	5.9	66
Test Item	10	6.3	71
	100	6.7	75
	1000 R1	6.5	73
	1000 R2	5.9	66
	1000 R3	6.1	69
3,5-dichlorophenol	3.2	7.3	82
	10	8.3	93
	32	8.7	98

R₁– R₄= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test are displayed in the table below:

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.2	8	2.1	30.75	-
	R2	6.5	8	2.4	30.75	-
	R3	6.6	8	2.2	33.00	-
	R4	5.8	7	2.2	30.86	-
Test Item	10	5.9	7	1.8	35.14	[12]
	100	5.7	6	2.1	36.00	[15]
	1000 R1	6.1	7	1.8	36.86	[18]
	1000 R2	6.6	8	1.9	35.25	[12]
	1000 R3	5.8	7	1.7	35.14	[12]
3,5-dichlorophenol	3.2	6.9	10	2.4	27.00	14
	10	7.9	10	5.7	13.20	58
	32	8.7	10	7.8	5.40	83

[Increase in respiration rate as compared to controls]

R₁– R₄= Replicates 1 to 4

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test are displayed in the table below:

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.8	7.8
	R2	7.8	7.7
	R3	7.8	7.7
	R4	7.8	7.7
Test Item	10	7.8	7.7
	100	7.9	7.7
	1000 R1	7.9	7.7
	1000 R2	7.9	7.8
	1000 R3	7.8	7.7
3,5-dichlorophenol	3.2	7.8	7.9
	10	7.9	8.2
	32	7.8	8.2

R₁– R₄= Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Executive summary:

 Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 19 and 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC50value of 9.5 mg/L, 95% confidence limits 7.6 to 12 mg/L.