Registration Dossier

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: Guideline study conducted under GLP

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:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
EC Number:
EC Name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
Cas Number:
Molecular formula:

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Details on test solutions:
An amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.The pH of the test item stock solutions was measured and adjusted to between pH 7.0 to pH 8.0.

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 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage. The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage sludge (500 mL). The pH of the sample on the day of the test was 7.4 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 deionized 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Study design

Total exposure duration:
3 h

Test conditions

Test temperature:
20±2 °C
Nominal and measured concentrations:
10, 100 and 1000 mg/L nominal
Details on test conditions:
After perparation of the inoculated controls, reference substance, and test substance and as each vessel reached 3 hours 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 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 7.0 mg O2/L and 2.0 mg O2/L). 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. The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph.
Reference substance (positive control):

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
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 9.3 mg/L, 95% confidence limits 7.3- 12 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No 209 "Activated Sludge, Respiration Inhibition Test" (Carbon and Ammonium Oxidation). Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol. The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3-Hour EC50 value of 9.3 mg/L, 95% confidence limits 7.3- 12 mg/L.