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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-01-2015 to 23-01-2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: test according to the guideline under GLP. Slight deviations of the oxygen concentration during the test did not affect the results
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Principles of method if other than guideline:
only the range-finding study was performed
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct addition to reversed osmosis water, followed by ultrasonication for 15 min. The solution was stirred magnetically for 24 hours at 21-22 °C (shielded from light)
- Differential loading: 0, 10, 100 and 1000 mg/L (at 1000 mg/L 3 replicates)
- Controls: no loading (4 replicates) and positive control 3,5 dichlorophenol ( 1 replicate at 3.2, 10 and 32 mg/L)
- Evidence of undissolved material: at 1000 mg/L visible dispersed test substance throughout and adhered to the side of flask
- pH adjustment: at 100 and 1000 mg/L
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant Loughborough, Leicesterhire, UK
- Storage conditions: under aeration at 21 °C
- Storage length: used on the day of collection
- Preparation of inoculum for exposure: washed once by settlemnt and resuspended
- Concentration of sludge: suspended solids 3.0 g/L on day of the test
- pH: 7.2
Test type:
Water media type:
Total exposure duration:
3 h
Post exposure observation period:
Test temperature:
20-21 °C
at 30 min 7.6-7.8
at 3 hours 7.5-8.0
Dissolved oxygen:
measured at 30 minutes
in controls: 62-94% of saturation
in test solutions: 55-58% of saturation
Details on test conditions:
- Test vessel: 500 mL
- Type: conical (open)
- Aeration: 0.1-0.5 L/min
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L, 3 at 1000 mg/L
- No. of vessels per control (replicates): 4

- Test water: deionized reverse osmosis water
- Concentration of sludge: suspended solids 3.0 g/L on day of the test
- pH: 7.6-7.8 (initial after adjustment)
- respiratory substrate (dissolved in 1 liter of water with the aid of ultrasonication):
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
- Adjustment of pH: yes at 100 and 1000 mg/L
- Test temperature: 20-21 ºC

EFFECT PARAMETERS MEASURED : oxygen consumption at t=30 min and t=3 h (over linear part of the respiration curve)

TEST CONCENTRATIONS: 10, 100 and 1000 mg/L
Reference substance (positive control):
3,5 dichlorophenol
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:
oxygen respiration rate controls: 26.65. mg O2/g dry weight sludge/h
coefficient of variation replicates at 1000 mg/L : 4.0%
Results with reference substance (positive control):
EC50 for 3,5 dichlorophenol 7.1 mg/L (oxygen concentration at initial reading at 10 and 32 mg/l was > 7.0 mg/L)
Reported statistics and error estimates:
Bartlett test for homogeneity of variance
Dunnett test for statistically differences between test groups and controls
Validity criteria fulfilled:
it was concluded that the deviations of the oxigen concentrations did not affect the study outcome
In a respiration inhibition test the NOEC of the test substance was 1000 mg/L
Executive summary:

The test substance was tested in a respiration inhibition test at concentrations of 10, 100 and 1000 mg/L. No effects on the respiratory rate of the inoculum were reported at any of the concentrations tested. The NOEC is therefore 1000 mg/L.

Description of key information

The EC50 and NOEC were determined to be above the nominal tested concentration of 1000 mg/L (Harlan 2015)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information