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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Range-finder: 5th May 2006 Definitive test: 8th May 2006
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
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to guideline
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes (incl. QA statement)
Date of inspection: 30th August 2005 Date of signature: 21st November 2005

Test material

Constituent 1
Reference substance name:
Cas Number:
Details on test material:
Sponsor's identification: MAHP
Description: clear colourless slightly viscous liquid
Batch number: L508708-A
Date received: 29th March 2006
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Not applicable, as no analysis conducted therefore no samples obtained.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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 ultrasonication for approximately 30 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which a dilution was made to give a 500 mg/l stock solution. Aliquots (100 and 180 ml) of the 500 mg/l stock solution were each separately dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentrations of 100 and 180 mg/I. Similarly, aliquots (80, 140 and 250 ml) of the 2000 mg/l stock solution were used to prepare the test concentrations of 320, 560 and 1000 mg/I.

The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

- Differential loading: not applicable

- Controls: Maintained under identical conditions but not exposed to the test material.

- 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): None

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 8 May 2006 from the aeration stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

- Laboratory culture: not applicable

- Method of cultivation: not applicable as used on day of collection

- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was 7.8, measured using a WTW pH/Oxi 3401 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 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

- Pretreatment: as above

- Initial biomass concentration: The suspended solids was equal to 3.9 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

~ 100 mg/l CaCO3
Test temperature:
8.3 for controls
8.2 - 6.8 for test material
8.4 - 8.6 for reference substance
Dissolved oxygen:
Initial readings: 4.9 - 7.1 mg O2/l
Final readings: 1.9 - 4.8 mg O2/l
Not applicable, as freshwater study
Nominal and measured concentrations:
Range finder: 100 and 1000 mg/l

Main study: 100, 180, 320, 560 and 1000 mg/l
Details on test conditions:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.

As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg 0 2/1 and 2.5 mg 0 2/1). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. After measurement the contents of the BOD bottle were returned to the test vessel.

This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21 °C.

- Source/preparation of dilution water: 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 100 mg/l as CaC03. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

- Adjustment of pH: not required

- Photoperiod: test conducted under normal laboratory lighting

- Light intensity: not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.

% inhibition = [1- (2RS/(RC1 + RC2))] x 100


RS = oxygen consumption rate for test or reference sample
RC 1 + RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit3 software package (IDBS 2002) and the ECso values determined from the equation for the fitted line.

The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC5o (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.

- Spacing factor for test concentrations: 100, 180, 320, 560 and 1000 mg/l

- Justification for using less concentrations than requested by guideline: not applicable

- Range finding study
- Test concentrations: 100 and 1000 mg/l

- Results used to determine the conditions for the definitive study: Significant. inhibition of respiration was observed at 1000 mg/I. However, there were no significant effects on respiration at 100 mg/I. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/l were selected for the definitive test.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
750 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
3 h
Dose descriptor:
Effect conc.:
180 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
Percentage inhibition is plotted against concentration for the test and reference materials.
The following results were derived:

MAHP ECso (30 minutes): 960 mg/l
3,5-dichlorophenol ECso (30 minutes): 15 mg/l
Variation in respiration rates of controls 1 and 2 after 30 minutes: ± 1%.

MAHP ECso (3 hours): 750 mg/l
3,5-dichlorophenol ECso (3 hours): 9.9 mg/l
Variation in respiration rates of controls 1 and 2 after 3 hours: ± 2%.

- 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: ECso (3 hours): 9.9 mg/l

Any other information on results incl. tables

The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg 02/l and 2.5 mg 02/1 respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Relatively low initial oxygen readings were observed in the test vessels. This was considered to be due to the response of the activated sewage sludge on the day and was considered not to have had a significant effect on the results of the test given that the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Values obtained for the pH of the test preparations at the end of the exposure period, showed a concentration dependent decrease in the pH values when compared to the control vessels. This may be considered to be a contributing factor to the toxic nature of the test material but is considered not to affect the integrity of the study given that the purpose of the study was to determine the effect that the test material would have on a waste treatment facility where pH adjustment of the incoming effluent would not occur.

Observations made throughout the test period showed that at all test concentrations employed there was no undissolved test material visible at 0 hours, 30 minutes and 3 hours contact time.

Applicant's summary and conclusion

Validity criteria fulfilled:
The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour ECso of 750 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/I.

The reference material gave a 3-Hour ECso value of 9.9 mg/I.
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", EEC Commission Directive 87/302/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 concentrations of 100, 180, 320, 560 and 1000 mg/l for a period of 3 hours at a temperature of 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 EC50 of 750 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/l. The reference material gave a 3-Hour EC50 value of 9.9 mg/l.