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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep - 11 Sep 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 2010
GLP compliance:
yes (incl. QA statement)
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (5, 50 and 500 mg) were each separately dispersed in approx. 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approx. 15 min followed by magnetic stirring for 24 h in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Source of inoculum: A mixed population of activated sewage sludge micro-organisms was obtained on 10 Sep 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, UK, which treats predominantly domestic sewage.
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approx. 21 °C overnight prior to the test. On the day of collection the sludge (8 L) was fed synthetic sewage sludge (400 mL).
- Initial suspended solids concentration: 3.0 g/L prior to use
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
7.8 - 7.9 at test start
8.3 - 8.5 at test end
Dissolved oxygen:
5.3 - 5.9 (controls), 4.1 - 6.8 mg/L (test flasks), 5.5 - 7.5 (reference flasks)
Nominal and measured concentrations:
Nominal: 0, 10, 100 and 1000 mg/L
Details on test conditions:
- Test vessel: 500 mL conical flasks containing 500 mL of test culture for incubation; 250 mL darkened glass BOD bottles for respiration rate measurements
- Aeration: yes, with clean, oil-free air at 0.5 - 1 L/min
- No. of vessels per concentration (replicates): 10 and 100 mg/L 1 flask, 1000 mg/L 3 flasks
- No. of vessels per control (replicates): 1

- Source/preparation of dilution water: Deionised reverse osmosis water containing < 1 mg/L dissolved organic carbon (DOC) was used in the test.
- Intervals of water quality measurement: Observations were made on the test preparations throughout the test period. The pH of all vessels was measured at 0 and 3 h, the oxygen concentrations in all vessels were measured after 30 min of contact time.

- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- After 3 h of contact time, an aliquot from each conical flask was poured into a BOD bottle and the rate of respiration measured over the linear portion of the oxygen consumption trace. The contents of the BOD bottles were stirred constantly by magnetic stirrer. The respiration rates were determined over periods of 4 - 10 min.

- Spacing factor for test concentrations: 10
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
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:
- All validation criteria were fulfilled.
- No statistically significant toxic effects of the test item were shown at all of the test concentrations employed. The inhibition of respiration ranged from -3% to +1% for the test item and from 24% to 78% for 3,5-dichlorophenol.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All flasks contained pale/dark brown dispersions. In the test cultures test item was visible floating on the surface. In the reference control cultures no undissolved reference item was visible.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (3 h) = 10 mg/L, 95% CL = 8 - 13mg/L

Description of key information

EC50 (3 h) > 1000 mg/L (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

The toxicity of (Z)-N-Octadec-9-enylhexadecan-1-amide (CAS No. 16260-09-6) to aquatic microorganisms was investigated in one respiration inhibition test according to OECD guideline 209 under GLP conditions (Harlan, 2012). Predominantly domestic activated sludge was incubated with test item concentrations of 10, 100 and 1000 mg/L. The highest test concentration was prepared in triplicate while the two lower concentrations were single preparations. No statistically significant toxic effects of the test item were shown at all test concentrations employed. Therefore, an EC50 (3 h) > 1000 mg/L and a NOEC ≥ 1000 mg/L were assumed.