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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2006 to Feb 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Hill Empirical Formula: C16H16O3S CAS Empirical Formula: C16H16O3S
Test material form:
solid: particulate/powder
Details on test material:
Lot no: 151105Purity - 101.39%

Sampling and analysis

Analytical monitoring:

Test solutions

not specified
Details on test solutions:
Nominal concentrations of 1, 10 and 100mg/L of Beta Ketosulfide were used in duplicate

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was collected from one of the sludge return lines at Burley Menston sewage treatment works (West Yorkshire, UK), a treatment plant with a waste-water catclunent that is pred ominantly domestic. The activated sludge used in this study was not acclimatised or adapted to Beta Ketosulfide in the labcratory before exposure to the test substance under test conditions.

Study design

Test type:
other: aerobic
Water media type:
other: dechlorinated tap water
Total exposure duration:
3 h

Test conditions

Details on test conditions:
All mixtures comprised, 16 mL synthetic sewage (to provide a uniform respiration substrate), the te st or reference substance as required, and sufficient volumes of dechlorinated tap water to achieve a volume of 300 mL. Vessels containing the test substance were treated with ultrasound for 10 minutes after these additions had been made. The purpose of the ultrasound treatment was to disper se as much of the test substance as possible into the culture. Exposure to the remainder of the test substance on the inner surface of the vessel is achieved by the constant agitation of the test mixture duc to the vigorous aeration employed during the incubation period. Inoculation entailed addition of 200 mL activated sludge, giving a final volume of 500 mL. The test mixtures were inoculatd sequenttally at timed intervals, and aerated with a supply of compressed air delvered to each vessel by a Pasteur pipette for 3 hours. At the end of the tncubation, dissolved oxygen (DO) measurements were made from a sub-sample of each preparation usmg a DO meter and probe. The pH of each test mixture was measured at the start and end of the incubation.
Reference substance (positive control):
Reference inhibitor 3,5-dichlorophenol (3,5-DCP)

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
3 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Details on results:
Confirmatory Analysis of Dosing Solutions: Analytical procedure (HPLC) was used to analyse the dosing stock solutions . The mean recoveries of the dosing stock as a percentage of the nominal concentrations ranged from 102.73 to 104.36%.Range-finder: Following ultrasound treatment the test mixtures at 100 mg/L appeared hazy relative to the blank controls. This is considered to be due to the presence of dispersed test substance. The measured pH of the mixtures ranged from 6.73 to 6.99 at the start of incubation and from 7.52 to 7.77 at the end of incubation. In the range-finder test, at a nominal test concentration of 1mg/L a slight (2.5%) enhancement in the respiration rate relative to the blank control vessels was observed. At nominal test concentrations of 10 and 100 mg/L, inhibition in respiration relative to the blank controls of 1.5% and 3.8% was observed, respectively. The threshold of significance in studies of this type is set at 10%. Consequently, these results are not considered to be biologically significant and a definitive test was not triggered.Statistical treatment of the data was not considered appropriate. Consequently, the EC50 for Beta Ketosulfide could not be determined, but must be greater than 100 mg/L the highest concentration tested. The observed effect of Beta Ketosulfide on the respiration rate of activated sludge was not considered to be biologically significant. Consequently, the no effect concentration of Beta Keto sulfide was estimated to be greater than or equal to the highest concentration tested. The mean respiration rate of the solvent controls was 47.50 mg02!/L/hr. This was equivalent to 101 .9% of the mean respiration rate in the blank control (46.63 mg02/L/hr) illustrating that the solvent dosing procedure did not influence the respiration rate of the activated sludge.
Results with reference substance (positive control):
The applied reference inhibitor concentrations were plotted against the resulting percentage inhibition observed. The EC50 estimate for 3,5·DCP was estimated from this plot to be 5.94 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
Samples of activated sludge were exposed to Beta Ketosulfide at nominal concentrations ranging from 1 to 100 mg/l and their respiration rates measured after 2 hours contact time. The test substance, Beta Ketosulfide did not inhibit the respiration rate of activated sludge at concentrations upto and including 100mg/l. Consequently, the EC50 could not be determined, but must be greater than100mg/l, the highest concentration tested.
Executive summary:

The impact of Beta Ketosulfide on the respiration rate of activated sludge was assessed according to Guideline 209 of the Organisation for Economic Co-operation and Development (OECD). The method is identifical to that described in method C.11 of Annex V of EU Directive 67/548/EEC and US EPA OPPTS method 850.6800.


EC50 > 100mg/l