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Ecotoxicological information

Toxicity to microorganisms

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 concentrationsopen allclose all
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
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.