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Diss Factsheets

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th January 2008 to 21st January 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Date of inspection: 21st August 2007 Date of inspection: 15th October 2007

Test material

Details on test material:
Sponsor's identification :C-C1
Description : dark blue powder
Batch number : MB-1
Date received : 5 March 2007
Storage conditions :room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:
Details on sampling:
Not applicable, as no samples taken for chemical analysis

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Test material preparation
For the purpose of the definitive test, the test material was dissolved directly in water.
An amount of test material (2000 mg) was dissolved in water with the aid of shaking by hand for approximately 1 minute. As the test material had not completely dissolved the stock was further subjected to ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. An aliquot (250 ml) of this stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 1000 mg/l. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the stock solution.
This method of preparation was performed for the three separate replicate test vessels.
The control group was maintained under identical conditions but not exposed to the test material.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/l prior to use.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
Not applicable

Test conditions

140 mg/l as CaCO3
Test temperature:
Approximately 21°C +/- 1°C
7.8 at 0 hours
8 at 3 hours
Dissolved oxygen:
6.2, 5.9 and 6.1 mg O2/l at 0 hours
3.5, 2.8 and 2.9 mg O2/l at 3 hours
Not applicable, as freshwater study
Nominal and measured concentrations:
Range finder: 100 and 1000 mg/l
Definitive test: 1000 mg/l
Details on test conditions:
- Test vessel:
- Type (delete if not applicable):closed
- Material, size, headspace, fill volume: 500 ml conical flask
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate):not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): Two
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not applicable

-The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in the attached Appendix.

- Adjustment of pH: not conducted
- Photoperiod: normal laboratory lighting
- Light intensity: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate. The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 (after 3 hours) values determined from the equation for the fitted line.

- Spacing factor for test concentrations: not applicable, as only one concentration used
- Justification for using less concentrations than requested by guideline: deciion based on results of range finding study

- Range finding study
- Test concentrations:100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: Yesd
Reference substance (positive control):
3,5 dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
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
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: none
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (3-hrs) = 9.5 mg/l
- Other: 95% confidence limits: 7.5-12
Reported statistics and error estimates:
No statistics used

Any other information on results incl. tables

Range-finding Test

No significant effect on respiration was observed at either test concentration employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.

It was not possible to obtain an EC80 value after 30 minutes contact time for the reference material as no concentration tested resulted in greater than 80% inhibition. It was also not possible to obtain 95% confidence limits for the reference material EC50value after 30 minutes contact time as the data generated did not fit the models available for calculation.

Applicant's summary and conclusion

Validity criteria fulfilled:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.
Executive summary:

Introduction. A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC50of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC50value of 9.5 mg/l, 95% confidence limits
7.5 - 12 mg/l.