tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

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:

Feb - April 2014

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))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

recorded for a period of 10 to 15 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared to the control vessels. A temperature control vessel with a REES (temperature) sensor was placed in each fume cupboard of the climate room.

Vehicle:

yes

Details on test solutions:

The batch of LEE011-A3 tested was a beige powder. No correction was made for the purity/composition of the test substance.
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates (see also paragraph). Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.9 - 22.7°C

pH:

The pH in all test vessels, before addition of sludge was 7.5. After the 3 hour exposure period the pH was between 7.3 and 8.2.

Details on test conditions:

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) was included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.

Reference substance (positive control):

no

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (initial)

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:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg LEE011-A3 per litre (Student-t test for homogeneous variances, α=0.05, one-sided smaller).
There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic respiration rate was not inhibited. Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be 9%.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test LEE011-A3 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).
The EC50 exceeded a loading rate of 1000 mg/l.

Description of key information

Under the conditions of this present test LEE011-A3 was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

The EC50 exceeded a loading rate of 1000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L