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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 December 2011 to 24 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
- Concentrations: 10, 30, 100, 300 and 1000 mg/L.
- Sampling method: Their rates of oxygen consumption were determined using oxygen electrodes and were compared with those of four replicate controls that contained activated sludge and synthetic sewage alone.
- Sample storage conditions before analysis: sampled in situ
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: On the day of collection of the activated sludge, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
- Eluate: Reverse osmosis water
- Controls: The method of preparation used during the definitive test was based on the results of a formulation trial. Volumes of the test substance (0.0049, 0.0147, 0.049, 0.147 and 0.490 mL, corrected for a specific gravity of 1.02) were added to test bottles (500 mL), made up to volume as appropriate, to give four replicates of test solutions, with nominal concentrations of 10, 30, 100, 300 and 1000 mg/L.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Method of cultivation: On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed.
The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
- Preparation of inoculum for exposure: On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
No data. The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.
Test temperature:
20 ± 2°C.
pH:
7.5 ± 0.5
Dissolved oxygen:
No data. The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.
Salinity:
No data. The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.
Nominal and measured concentrations:
Test substance nominal concentrations: 10, 30, 100, 300 and 1000 mg/L.
Reference substance: 3, 10, 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL bottles
- Type: closed
- Material, size, headspace, fill volume: no data
- Aeration: no
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per reference (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis water

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3
- Range finding study: no
- Test concentrations: 10, 30, 100, 300 and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Reference substance; 3,5-DCP
Details on results:
The no observed effect concentration (NOEC) for TCD was 100 mg/L. The EC20, EC50 and EC80 values could not be calculated, although there was significant inhibition of respiration rate at test concentrations of 300 (26%) and 1000 mg/L (40%).
Results with reference substance (positive control):
- Results with reference substance valid: Yes, the three-hour EC50 for 3,5-DCP (2.22 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L).
Reported statistics and error estimates:
Where possible, the EC20, EC50 and EC80 (and 95% confidence intervals) and no observed effect concentration (NOEC) of the test substance were calculated using SAFEstat programme SAS Proc NLIN (SAS Institute, 2002).

For the 3,5-dichlorophenol reference substance, only the EC50 was calculated.

The EC50 (and EC20 and EC80) was the concentration of the test or reference material at which the respiration rate is 50% of mean control value (or 20% and 80% of the mean control value).

For substances that do not significantly inhibit the activity of the sludge, the no observed effect concentration (NOEC) will be taken as the highest concentration tested.

TCD and 3,5-Dichlorophenol mixtures were analysed separately.

Inhibition for TCD

10 mg/L       8%

30 mg/L        12%      

100 mg/L      18%

300 mg/L      26%

1000 mg/L    40%

Inhibition for 3,5 -DCP

3 mg/L        57%

10 mg/L       55%

32 mg/L       80%

Validity criteria fulfilled:
yes
Conclusions:
The no observed effect concentration (NOEC) for TCD was 100 mg/L. The EC20, EC50 and EC80 values could not be calculated, although there was significant inhibition of respiration rate at test concentrations of 300 (26%) and 1000 mg/L (40%).

Description of key information

The no observed effect concentration (NOEC) for TCD was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

In a test carried out in accordance with OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (carbon and ammonium oxidation), the test substance showed significant inhibition of respiration rate at test concentrations of 300 (26%) and 1000 mg/L (40%). However EC values could not be determined as there was not enough evidence to identify the concentration-response relationship.

The ‘no observed effect concentration’ (NOEC) for TCD was determined as 100 mg/L, and the ‘lowest observed effect concentration’ (LOEC) was 300 mg/L.