1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d] pyrimidin-5-yl)phenylsulphonyl]-4-methylpiperazine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-03 to 2005-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid

Cas Number:

171599-83-0

Molecular formula:

C28H38N6O11S

IUPAC Name:

5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch: November 1, 2005

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient
- Stability under test conditions: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The contents of the first control vessel were transferred to a BOD bottle and dissolved oxygen concentrations were measured every 10 seconds over a 10 minute period, or until dissolved oxygen concentrations fell below 1.0 mg/L, using a YSI Model 50B Dissolved Oxygen Meter. The respiration rate in subsequent vessels was determined in an identical manner at 15 minute intervals.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weight addition
- Controls: A positive control with a known respiration inhibitor was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L.
The test substance was tested at nominal concentrations of 1, 3, 10, 30, 100, 300, and 1000 mg/L.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test substance dissolved poorly in the water.

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: Denton Wastewater Treatment Plant, Denton, Maryland on June 27, 2005.
- Preparation of inoculum for exposure: The sludge was sieved using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed.
- Pretreatment: Total suspended solids in the settled sludge were adjusted to by dilution with municipal water. 50 mL of synthetic sewage was added to each liter of adjusted sludge.
- Initial biomass concentration: A nominal concentration of approximately 4000 mg/L

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Control, reference, and treatment test mixtures were incubated at 20 ± 2ºC and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension.

Post exposure observation period:

No post exposure observation period was conducted.

Test conditions

Test temperature:

20 ± 2ºC

pH:

The measured initial pH of the sludge on the day of testing was 8.3 and was adjusted to 7.6 prior to use in the test system.

Nominal and measured concentrations:

Nominal concentrations:
- Concentration of Inncoulum: 4000 mg/L
- Concentrations of Positive Control: 3, 15 and 50 mg/L
- Concentrations of Test Substance: 1, 3, 10, 30, 100, 300, and 1000 mg/L
Measured concentrations:
- Concentration of Inncoulum: 4393 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml plastic Erlenmeyer flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 300 mL was filed
- Aeration: Yes, at a rate sufficient to provide aerobic conditions and maintain solids in suspension.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4393 mg/L
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Municipal water
OTHER TEST CONDITIONS
- Adjustment of pH: The measured initial pH of the sludge on the day of testing was 8.3 and was adjusted to 7.6 prior to use in the test system
- Details on termination of incubation: Upon terminitation, smaples weretransferred into 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements
EFFECT PARAMETERS MEASURED:
DO measurements were measured every 10 seconds over a 10 minute period for the first control vessel.
The respiration rate in subsequent vessels was determined in an identical manner at 15 minute intervals.

TEST CONCENTRATIONS
- Concentrations of Positive Control: 3, 15 and 50 mg/L
- Concentrations of Test Substance: 1, 3, 10, 30, 100, 300, and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol at 99.1% purity at concentration of 3, 15 and 50 mg/L.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Details on results:

The observed percent inhibitions for sildenafil citrate ranged from –53.6 to 15.7% and were not concentration dependent.

Results with reference substance (positive control):

The positive control EC50 had 95 percent confidence limits of 23 and 29 and was within the 5 to 30 mg/L range considered acceptable for the test. This makes the result of this test valid.

Reported statistics and error estimates:

The EC50 was calculated using the moving average method.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study accurately assesed the EC50 and NOEC for respiration inhibition in activated sludge per guidelines.