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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-22 to 2011-08-22
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Study performed according to internationally accepted test guidelines for toxicity to microorganisms (OECD 209) and conform CLP requirements.
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: PFW100113
- Expiration date of the lot/batch: 2012-12-01
- Analytical purity: 99.3 %
- Impurities (identity and concentrations): not reported

- Storage condition of test material: room temperature in the dark
- Stability under test conditions: not reported

- Name of test material (as cited in study report): 2,2'-dimorpholinodiethyl ether (DMDEE)
- Substance type: pale yellow liquid
- Physical state: liquid
Analytical monitoring:
not required
Details on sampling:
Not applicable.
Details on test solutions:
- Preparation of stock solutions of 2500 mg/L and (through dilution) 250 and 25 mg/L.
- Preparation of test solutions: aliquot of stock solution + synthetic sewage (standard composition) + activated sludge sample + deionized reverse osmosis water to a total volume of 500 mL.
- Volumetric flasks were inverted several times to ensure homogeneity of stock solutions
- pH adjusted to pH 7.0-8.0.
- The 1000 mg/L test concentration was prepared in triplicate.
- 2+2 controls were prepared.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source microorganisms: activated sewage sludge was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Sludge was used on the day of collection.
- Maintainance conditions: continuous aeration, 21 °C
- pH of sample was 7.6
- Suspended solids concentration of sample: 2.8 g/L. Determination of suspended solids: filtration of 100-mL sample by suction through pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL deionised reverse osmosis water. Filtration continued for 3 minutes. Prior to use, the filter paper was rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven. After filtration, the filter paper was dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. The process was repeated until a constant weight was attained.
- 250 mL of inoculum added to 500 mL test flasks.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
After 3 h contact time, measurement of oxygen consumption in BOD bottle (approximately 10 min or less, depending on how fast oxygen consumption occurs).
Not reported.
Test temperature:
20 ± 2 °C
minimum and maximum pH values determined at start and end of exposure in all test vessels:
- control: 7.6-7.8
- treatment: 7.7-7.9
- reference substance: 7.6-8.2
Dissolved oxygen:
minimum and maximum dissolved oxygen concentrations (mg O2/L) after 30 minutes of contact time:
- control: 6.1-6.2
- treatment: 6.0-6.5
- reference substance: 6.1-6.4
not applicable
Nominal and measured concentrations:
Nominal concentrations: control, and 10, 100, 1000 mg/L
Details on test conditions:
- Test vessel: 500-mL conical glass flasks, measurement of oxygen consumption in 250-mL darkened glass BOD bottles
- Type (delete if not applicable): BOD bottles for measurement of oxygen consumption were closed
- Aeration: during exposure with clean, oil-free compressed air via narrow bore glass tubes at a rate of apprixmately 0.5 to 1 L/min; no aeration during measurement of oxygen consumption
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L, 3 for 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per reference control (replicates): one replicate per reference control concentration
- After 3 h of exposure a 250-mL aliquot was removed from the conical flask into the measuring vessel. The content of the measuring vessel was constantly stirred by a magnetic stirrer.
- The rate of respiration was measured using a Yellow Springs oxygen meter fitted with a BOD probe, 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 low oxygen consumption, the rate was determined over an approximate 10 minute period.

- Spacing factor for test concentrations: 10
- Range finding study: the current study was a range finding study
- Test concentrations: control + 10, 100, 1000 mg/L
- Based on the results of the range finding test it was decided not to perform a definitive test as significant toxic effects were not shown.

- Test was performed under normal laboratory lighting, BOD bottles for measurement of oxygen consumption however were from darkened glass.
- Test performed in temperature controlled room.

- Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
- pH of the control, reference item and test item preparations was measured using a WTW pH/Oxi 340l pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
- Oxygen concentrations in all vessels were measured after 30 minutes contact time.
Reference substance (positive control):
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:
The oxygen consumption rate in the 10 and 100 mg/L treatments were statistically different from the control, but this was due to the oxygen consumption values in the replicate control vessels being very similar with little variation. Therefore it was concluded that no significant differences were observed and that a definitive test was not needed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3-h EC50 = 7.4 mg/L
Reported statistics and error estimates:
- EC15, EC20, EC50 and EC80 values were determined by linear regression (% inhibition of respiration rate versus concentration, line fitted using Xlfit software package (IDBS)).
- 95% confidence limits calculated using method of Litchfield and Wilcoxon.
- For comparing treatments with control: one-way ANOVA incorporating Bartlett's test for homegeneity of variance and Dunnett's multiple comparison procedure.
- SAS software was used.
Validity criteria fulfilled:
In this study, activated sludge from a predominantly domestic WWTP was exposed to 10, 100 and 1000 mg/L test substance for 3 h, after which the oxygen consumption rate was measured in closed BOD bottles. No significant differences in oxygen consumption rate were observed compared to the control. Therefore, the EC50, NOEC and LOEC were concluded to be > 1000, >= 1000 and > 1000 mg/L, respectively. No further testing was considered necessary.

Description of key information

The key value for acute toxicity to microorganism was obtained from a study investigating the inhibitory effect on respiration of activated sewage sludge under GLP conditions and according to OECD 209.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A study investigating the toxicity to aquatic microorganisms was available. This study of Clarke (2011), performed by Harlan Laboratories, exposed activated sludge of predominantly domestic sewage to a series of test substance concentrations (10, 100 and 1000 mg/l) during 3 hours in a static test design. The EC50, NOEC and LOEC were concluded to be > 1000, >= 1000 and > 1000 mg/L resp.