1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Sept 2016 - 23 Sept 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate date: 3-Nov-2015

Specific details on test material used for the study:

Solubility in water: 0.0115 g/L

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation was as much as possible performed under low light conditions. Since the test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water, test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) and stirred in closed amber coloured test bottles. Subsequently, synthetic medium (=sewage feed) and sludge were added, resulting in the required loading rates. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period.
- Controls: test medium without test item and treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation of sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids. The batch of sludge was used one day after collection; therefore, 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g SS/L sludge

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

17.3 - 22.9 °C

pH:

Before addition of sludge: 7.6 - 7.7
After 3 hours of exposure: 7.4 - 8.1
(For more details on pH, see table 1 in 'Any other information on materials and methods')

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass, open bottles/vessels, fill volume: 500 mL
- Aeration: the aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension. Aeration was continuous during the 3 hours of testing.
- No. of vessels per concentration (replicates): 1, except for the highest loading rate (1000 mg/L) where 3 replicates were tested
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g SS/L in the final test mixture
- Nutrients provided for bacteria: sewage feed (synthetic medium)
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: milli-RO water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption after a 3-hour exposure period, measured for approximately 10 minutes
- Additional measurements: medium temperature was recorded continuously in a temperature control vessel. pH was determined at the start and at the end of the test for all test item and reference item concentrations and controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: combined limit/range finding study

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

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

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

Details on results:

- No statistically significant inhibition of the respiration rate of sludge was recorded at a loading rate of 1000 mg test item per litre, therefore no EC50-value could be calculated.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes
- Blank controls oxygen uptake rate: 25.56 mg O2/g*h
- Coefficient of variation of oxygen uptake rate in control replicates: 17%
- Other: there was no significant oxygen uptake from abiotic processes.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 5.3 mg/L, 95%-CI: 2.6 - 11.1 mg/L

Reported statistics and error estimates:

Determination of EC50: For the reference item calculation of the EC50 value was based on non-linear regression analyses (3-parameter normal CDF [cumulative distribution function]) with the percentages of respiration inhibition versus the corresponding concentrations of the item.
For the test item no EC50-value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L).

Determination of NOEC: An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller). No statistically significant inhibition of the respiration rate was observed at a loading rate of 1000 mg/L. Therefore, the NOELR was considered to be the highest test concentration.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Respiration rate/inhibition

Replicate		Loading rate (mg/L)	Respiration rate		% Inhibition respiration rate (mean value)
			(mg O2/L.h)	(mg O2/g.h)¹
C 1		0	34.20	22.80
C 2		0	37.50	25.00
C 3		0	34.00	22.67
C 4		0	47.12	31.41
C 5		0	45.85	30.57
C 6		0	31.36	20.91
C Mean			38.34	25.56 (RTB)
SD			6.62	4.41
CV (%)			17	17
R 1		2.0	29.83	19.89	22.19
R 2		4.0	21.40	14.27	44.18
R 3		8.0	15.68	10.45	59.10
R 4		16	7.41	4.94	80.67
T 1		10	33.98	22.65	11.37
T 2		100	43.21	28.81	-12.71
T 3a		1000	41.06	27.37	-7.10
T 3b		1000	30.10	20.07	21.49
T 3c		1000	31.06	20.71	18.98
T3 Mean			34.07	22.72 (RT)	11.12 (IT)
TA		1000	0.31	0.21	99.19

C: Blank control  R: Reference item, 3,5-dichlorophenol T: Test item, BMS-589154-01 TA: Abiotic control of BMS-589154-01 SD: Standard deviation

¹The amount of suspended solids in the final test mixture was 1.5 g/L. RTB: Total respiration blank RT: Total respiration with BMS-589154-01 IT: % inhibition of total respiration relative to RTB CV: Coefficient of variation

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' section for details on the validity criteria.

Conclusions:

Under the conditions of this present test the test substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOEC). The EC50 was above a loading rate of 1000 mg/L.

Executive summary:

This study was performed to assess the influence of the test item on the respiration rate of activated sludge after a contact time of 3 hours. The study procedures were based on OECD guideline No. 209 and performed under GLP conditions.

Because the test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water, test concentrations were prepared separately in Milli-RO water and stirred in the test bottle. Subsequently, synthetic medium (=sewage feed) and sludge were added, resulting in the required loading rates. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

A combined range finding/limit test was performed with loading rates of 10 , 100 and 1000 mg/L. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the blank controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg test substance per litre.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The test item was shown to be not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOEC). EC50 could not be determined (> 1000 mg/L).

Description of key information

Under the conditions of this present test the test substance was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOEC). The EC50 was above a loading rate of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L