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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Jul - 01 Aug 2000
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate weight of the test substance were added directly to the test beakers. Dechlorinated tap water (284 mL) was added and the mixtures were treated with ultrasound for 10 min. Then synthetic sewage and inoculum were added.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Activated sludge was obtained on the day before test start from Oakley Sewage Treatment Works, UK, which treats predominantly domestic waste.
- Laboratory culture: The sample was maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: Aliquots (25 mL) of the sludge were filtered through Whatman GFC filter papers. Synthetic sewage (50 mL/L) was added and the mixture aerated overnight.
- Initial biomass concentration: 4 g/L mixed liquor suspended solids
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
19.3 - 20.3 at test start
18.6 - 19.2 at test end
7.5 - 7.7 at test start
7.9 - 8.1 at test end
Dissolved oxygen:
5.0 - 6.5 mg/L at test start
2.5 mg/L at test end
Nominal and measured concentrations:
Nominal: 0, 1, 10 and 100 mg/L
Details on test conditions:
- Test vessel: Respiration rate was measured in BOD bottles of 270 mL capacity.
- Aeration: The prepared mixtures were aerated for 3 h.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

- Source/preparation of dilution water: Dechlorinated tap water
- Hardness: 200 - 250 mg/L as CaCO3
- Intervals of water quality measurement: Temperature and pH of the samples were measured at test start and test end.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Respiration rate was measured after 3 h of incubation.

- Spacing factor for test concentrations: 10
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
- Control: The specific respiration rate of the control at test end (27.8 mg O2/g*h) was 104% of the rate at test start (26.8 mg O2/g*h).
- Test samples: Respiration rate was 27.3, 28.3 and 30.0 mg O2/g*h at 1, 10 and 100 mg test item/L, respectively.
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (3h) = 11.5 mg/L (95% CL = 9.2 - 14.8 mg/L)

Description of key information

No adverse effects on aquatic micro-organisms were observed at the highest test concentration (100 mg/L)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The toxicity of 2-ethylhexyl benzoate to micro-organisms was investigated in a respiration inhibition test according to OECD guideline 209 and GLP (Barnes, 2000). The substance had no inhibitory effect on the respiration rate of activated sludge following three hours of exposure at 1, 10 and 100 mg/L. Therefore, the three-hour EC50 could not be calculated, but is thus considered to be greater than 100 mg/L, the highest level tested.