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Diss Factsheets

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference Type:
study report

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:
C35H31N8O18S3.xNa.yK, where x+y=3
dipotassium sodium 3-[(1E)-2-{4-[(1E)-2-{4-[(1E)-2-(5-carbamoyl-1-ethyl-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazen-1-yl]-2-sulfonatophenyl}diazen-1-yl]-2,5-bis(2-hydroxyethoxy)phenyl}diazen-1-yl]-4,5-dihydroxynaphthalene-2,7-disulfonate

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
A nominal 500 mg/1 stock solution of test substance was prepared in deionised water. This stock was observed to be a deep blue coloured solution and the pH was measured as 7.8. Test solutions were prepared by the addition of a known volume of this stock solution to a total volume of 500 ml aerated tapwater, synthetic sewage and activated sludge.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was obtained from Buckland Sewage Treatment Works, Newton Abbot, Devon, UK, 3 days prior to the exposure of test and reference substances. This works treats sewage of predominantly domestic origin.

On return to the laboratory, the activated sludge was settled and the concentrated sludge washed with tapwater. The washed settled sludge was fed with 50 ml of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test. The total filterable solids concentration was determined on the day of the test and was found to be 4563 mg/l. The pH was measured as 8.6, and was subsequently adjusted to 6.6 with 2M hydrochloric acid prior to use.

Study design

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

Test conditions

Test temperature:
The temperatures of the flask contents after the 3 hours aeration were all within the range 20 ± 2°C.
Nominal and measured concentrations:
The following nominal concentrations of the test substance were prepared: 1.0, 3.2, 10, 32 and 100 mg/l
Details on test conditions:
Nominal 1.0, 3.2, 10, 32 and 100 mg/l concentratlons of the test susbtance were prepared together with two control culture flasks. Five flasks containing the reference substance, 3,5-dichlorophenol, at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were also prepared.

Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/1, an appropriate quantity of either test substance or 3,5-dichlorophenol stock solution and aerated tap water to give a final flask contents volume of 500 ml. The pH of each flask was measured before the start of the test. The synthetic sewage mixture was as described by the OECD guideline and contained approximately the following constituents per litre of deionised water: 15.2 g of peptone, 10.5 g of meat extract, 2.9 g of urea, 0.7 g of NaCI, 0.4 g ofCaCl2.2H2O, 0.2 g of MgS04.7H2O and 2.8 g of K2HP04.

Flasks were set up at 10 minute intervals and aerated at 20 ± 2°C for 3 hours. The temperatures of the flask contents were measured at the end of the 3 hours aeration using a mercury-in-glass thermometer. The respiration rate of each culture was measured after 3 hours and compared with the mean respiration rate of the two control cultures. The rate of oxygen uptake was measured in a confined perspex cell into which a polarographic oxygen electrode was inserted. The electrode was connected to a meter whose output was recorded on a potentiometric chart recorder. The dissolved oxygen concentration after the 3 hour aeration period was at least
6.5 mg O2 per litre.

The respiration rate was measured over the linear part ofthe curve for five to ten minutes. The rates of oxygen uptake were expressed as mg O2 1itre per hour.

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Basis for effect:
inhibition of total respiration
respiration rate
3 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
The reference substance 3,5-dichlorophenol cuased a substantial inhibition of the respiration rate of the activated sludge. From the results obtained the 3 hour EC50 value was estimated to be 17 mg/l. This is within the expected normal range of 5 to 30 mg/l indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other.

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance is not toxic to activated sludge under the conditions of the test.