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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 5 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD & EU guidelines and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Method of cultivation:
- Preparation of inoculum for exposure: The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was
weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre, were made up with tap water. To this mixture, 50 mL synthetic sewage feed per litre was added one day
prior to use, and the sludge was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was determined, and adjusted to 4 g/L with tap water. The pH of the activated sludge was determined to be pH 7.8.
- Pretreatment:
- Initial biomass concentration:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
10 minutes
Test temperature:
19-20°C
pH:
pH measurements given in Tables 2 and 3 in results section
Dissolved oxygen:
Measurements given in Tables 2 and 3 in results section
Nominal and measured concentrations:
Nominal concentrations are presented in Table 1.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks 1L and Karlsruher flasks 250 mL
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:
- Aeration: Compressed air (0.6 L/minute)
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 10, 32, 100, 320, 1000 mg/L
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3-hour EC50 of reference substance 6.7 mg/L
- Other:

Table 2 below gives results for the reference substance, table 3 gives results for dimethylcarbonate.

Table 2. Influence of 3,5-Dichlorophenol on oxygen consumption of activated sludge

Flask No.

Treatment

Concentration (mg/L)

Oxygen Consumption (mg O2/L min)

Inhibition %

pH Values

Oxygen concentration (mg O2/L)

start*

end*

start*

end*

1

Control

-

0.506

 

7.4

8.3

8.0

7.7

10

Control

-

0.470

 

7.4

8.2

8.2

7.6

Mean

% deviation

0.488

 

7.1

2

3,5-DCP

32

0.060

87.7

7.5

8.4

7.8

8.3

3

3,5-DCP

10

0.170

65.2

7.5

8.4

7.7

8.3

4

3,5-DCP

3.2

0.360

26.2

7.4

8.4

7.8

8.0

 

 

 

 

 

 

 

 

 

*: start and end of 3-hour aeration

3,5-DCP: 3,5-Dichlorophenol

Table 3. Influence of dimethylcarbonate (DMC) on oxygen consumption of activated sludge

Flask No.

Treatment

Concentration (mg/L)

Oxygen Consumption (mg O2/L min)

Inhibition %

pH Values

Oxygen concentration (mg O2/L)

Start*

End*

Start*

End*

1

Control

-

0.506

 

7.4

8.3

8.0

7.7

10

Control

-

0.470

 

7.4

8.2

8.2

7.6

Mean

% deviation

0.488

 

7.1

5

DMC

1000

0.310

36.5

7.4

8.3

7.8

8.2

6

DMC

320

0.320

34.4

7.4

8.3

8.0

7.8

7

DMC

100

0.380

22.1

7.4

8.3

8.1

7.9

8

DMC

32

0.440

9.8

7.5

8.3

8.1

7.8

9

DMC

10

0.500

-2.5

7.4

8.2

8.0

7.1

* start and end of 3-hour aeration

Validity criteria fulfilled:
yes
Conclusions:
Based on measured inhibition rates, the 3-hour EC 50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 50 % inhibition was noted after three hours incubation. Nevertheless, the 3 hour EC 50 is clearly higher than 1000 mg/L nominal under the present test conditions. The 3-hour EC 20 was calculated to be 92 mg test item/L. The lowest test concentration of 10 mg/L (nominal) can be regarded as the NOEC.

Description of key information

Based on measured inhibition rates, the 3-hour EC 50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 50 % inhibition was noted after three hours incubation. Nevertheless, the 3 hour EC 50 is clearly higher than 1000 mg/L nominal under the present test conditions. The 3-hour EC 20 was calculated to be 92 mg test item/L. The lowest test concentration of 10 mg/L (nominal) can be regarded as the NOEC.

Key value for chemical safety assessment

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

Additional information

The study was performed by Institut für Biologische Analytik und Consulting IBACON GmbH, Arheilger Weg l7, 64380 Rossdorf Germany. Study conducted to GLP according to OECD Guideline 209 and EU Method C.11.

Based on measured inhibition rates, the 3-hour EC 50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 50 % inhibition was noted after three hours incubation. Nevertheless, the 3 hour EC 50 is clearly higher than 1000 mg/L nominal under the present test conditions. The 3-hour EC 20 was calculated to be 92 mg test item/L.