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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-10-12 to 2007-06-29
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) and EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test) without deviations.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
The testing facility indicated that the protocol was followed without deviation.
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
The testing facility indicated that the protocol was followed without deviation.
Principles of method if other than guideline:
Not applicable
GLP compliance:
Department of Health of the Government of the United Kingdom

Test material

Details on test material:
- Name of test material (as cited in study report): VRT-126016
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: white to off-white powder
- Analytical purity: 99.8 area % by High Performance Liquid Chromatography (HPLC)
- Impurities: chloride: <0.1%
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-02-02
- Lot/batch No.: 25719
- Expiration date of the lot/batch: February 2008
- Stability under test conditions: no data
- Storage condition of test material: at room temperature
- Other: no data
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: blank and 2.5 g/L
- Sampling method: two samples of the test substance were prepared in RO water at nominal concentrations of 2.5 g/L. The sample was further prcessed for analysis by HPLC, along with a blank control. The second sample was processed and analysed a day later. These were not directly for the test. The purpose of these samples was to investigate the stability of the test substance at preparation, and after one day.
- Sample storage conditions before analysis: not data

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate weights were added to one L test beakers, 16 mL synthetic sewage feed, 284 mL water, and 200 mL microbial inoculum.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: 16 mL synthetic sewage feed, 284 mL water, and 200 mL microbial inoculum, no test substance
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Results of preliminary test showed the test substance insufficiently soluble to allow a suitable stock solution.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Sample of activated sludge was obtained the day before the start of the test from the aeration basin Worlington Sewage Treatment Works, which treats predominantly domestic sewage.
- Method of cultivation: In the laboratory, the samples were maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: The suspended solids concentration in a homogenized sample was determined on the day of collection immediately before the start of the test. 25 mL of the activated sludge were filtered through dried and preweighed Whatman GF/C filter papers which were then dried again at approximately 105 deg C for at least 1 h, allowed to cool in a dessicator, and re-weighed. The mixed-liquor suspended solids content of the activated sludge was then calculated. In the definitive test, the MLSS was adjusted to 4 g/L by removal of the supernatant.
- Pretreatment: Synthetic sewage feed (50 mL/L) was added to each stock of activated sludge, and these were aerated overnight. MLSS was determined , and adjusted with the addition of dechlorinated tap water.
- Initial biomass concentration: 4 g/L

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:

Test conditions

no data
Test temperature:
initial-19.8-20.0 deg C
final-19.5-19.7 deg C
initial-7.3-7.5 SU
final-7.8-8.0 SU
Dissolved oxygen:
initial- 6.5-8.8 mg/L
final-2.5-7.9 mg/L
Not applicable
Nominal and measured concentrations:
Nominal- Control, 10, 100, 1000, 1000, 1000 mg/L; 3,5-DCP- 2 Control, 3, 10, 32 mg/L
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 L glass
- Aeration: yes, with oil-free compressed air.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1 in definitive test, 2 for reference substance
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 4 g/L

- Source/preparation of dilution water: Dechlorinated and purified tap water
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: not applicable
- Intervals of water quality measurement: not applicable

- Adjustment of pH: no
- Photoperiod: not applicable
- Light intensity: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : not applicable

- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: no range finding study
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Concentrations were not measured.
- Effect concentrations exceeding solubility of substance in test medium: not applicable
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3 h EC50 8.3 mg/L
- Other: Acceptable range 5-30 mg/L
Reported statistics and error estimates:
The EC50 and confidence limits were calculated using the SAFEStat curvefit program.

Any other information on results incl. tables

Table 1. Definitive test respiration rate and % inhibition.

 Test Mixture (mg/L) Respiration Rate (mg O2/g/h)  % Inhibition


 19.7  --


19.0 4


 23.1  0


 21.1  0


 21.4  0


21.4   0

 3,5 -DCP



15.8  20


9.2  54


 3.9 80
 Control (2)



Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
The test substance was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge, at any of the concentrations used in the test. Therefore, the EC50 must be > 1000 mg/L.
Executive summary:

Not applicable

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