2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Feb - 21 Feb 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no dose-response relationship; EC10/NOEC could not be determined

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Cas Number:

1211443-61-6

Molecular formula:

C14 H17 Cl N4 O

IUPAC Name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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 (= sewage feed), 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
- Eluate: no
- Differential loading: yes
- Controls: blank control, reference control, nitrification control, abiotic control prepared with synthetic medium (sewage feed) according to OECD guideline 209
- Evidence of undissolved material: not reported

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: 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 (= 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20.9 - 22.7

pH:

7.4 - 8.1

Nominal and measured concentrations:

Nominal loading rates: Control, 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass 1 L open bottles/vessels with 500 mL fill volume
- Aeration: Clean, oil-free air. 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.
- No. of vessels per concentration (replicates): 1 each for 10 and 100 mg/L loading rates; 3 for 1000 mg/L loading rate
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- No. of vessels per nitrification control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nitrification inhibitor used: A 2.32 g/l solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/l).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic medium (sewage feed) according to OECD guideline 209

OTHER TEST CONDITIONS
- Details on termination of incubation: After the 3-hour contact time, the oxygen consumption was 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 and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
EFFECT PARAMETERS MEASURED: Determination of oxygen was performed with oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
- Other: The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES (temperature) sensor was placed in each fume cupboard of the climate room.

TEST CONCENTRATIONS
- Range finding study : Loading rates of 10, 100 and 1000 mg/L. Controls: blank control, nitrification control, abiotic control, nitrification inhibitor (only at the highest loading rate). Responses were compared to the blank and nitrification controls.
- Results used to determine the conditions for the definitive study: Definitive test was designed based on the results of the range finding study. However no evaluation of the results of the range finding study is reported.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

The respiration rates of 10 and 100 mg/L were considered less reliable because the oxygen decrease was not consistent. No explanation could be given for this finding. The inhibition observed at 1000 mg/l was statistically significant (average value 43%). There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be 44%. A NOEC value could not be determined.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h) = 5.6 mg/L

Reported statistics and error estimates:

Calculation of EC50 values was based on probit analysis using linear max. likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test substance.

Any other information on results incl. tables

Table 1: Study results

Flask	Concentration/Loading rate (mg/L)	pH before addition of sludge	pH after 3 h contact time	Respiration rate (mg O2/L per h	Respiration rate (mg O2/g per h (1), (A)	% Inhibition respiration relative to the blank control (mean value)
C1	0	7.5	7.7	63	42
C2	0	7.5	7.7	55	37
C3	0	7.5	7.7	58	39
C4	0	7.5	7.5	65	43
C5	0	7.5	7.5	60	40
C6	0	7.5	7.5	58	39
C Mean				60 (=RTB)	40
SD				4	2
CV (%)				6	6
CN1	0	7.5	8.0	35	23	42
CN2	0	7.5	8.1	28	19	53
CN Mean				32 (=RHB)	21
R1	2.0	7.5	7.9	44	29	26
R2	5	7.5	7.9	34	23	43
R3	12	7.5	8.1	17	11	72
T1	10	7.5	7.5	107	71	-79#
T2	100	7.5	7.6	65	43	-9#
T3a	1000	7.5	7.6	40	27	33*
T3b	1000	7.5	7.6	34	23	43*
T3c	1000	7.4	7.7	29	19	52
T3 Mean				34 (=RT)	23	43 (=IT)
	1000	7.5	8.0	21	14	33
	1000	7.5	8.1	17	11	46
	1000	7.5	8.1	17	11	46
TN Mean				18 (=RH)	12	42 (=IH)(2)
TA	1000	7.4	7.3	0	0	100

C: Blank control

CN: Nitrification control

R: Reference substance, 3,5-dichlorophenol

SD: Standard deviation

CV: Coefficient of variation

(A) The amount of suspended solids in the final test mixture was 1.5 g/l.

T: Test substance

TA: Abiotic control of test substance.

TN: Test substance with N-allylthiourea.

(1) The amount of suspended solids in the final test mixture was 1.5 g/l.

(2) Relative to CN

* Statistically significant different compared to C (Student-t test for Homogeneous Variances: α=0.05)

# Unreliable value: The respiration rates of 10 and 100 mg/l were considered less reliable because the oxygen decrease was not consistent. No explanation could be given for this finding.

Table 2: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	40 mg O2 per g of activated sludge in 1 h	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	< 30%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The EC50 (3 h) of the substance is > 1000 mg/L.