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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-29 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to accepted international guidelines and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
Information provided by the Sponsor indicated that the test substance was a liquid and was insufficiently soluble in water to prepare a stock solution. Therefore at test initiation, concentrations of the test substance were achieved by the addition of appropriate volumes to 284 mL of reverse osmosis water. See table 1 in "Any other information on materials and methods" below.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Method of cultivation:
- Preparation of inoculum for exposure: 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 and concentrated by the removal of supernatant. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight. 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.
- 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:
ca 20°C
pH:
7.4-8.1
Dissolved oxygen:
See table 3 in results section.
Nominal and measured concentrations:
Nominal concentrations 0, 10, 30, 100, 300, 1000 mg/L
Details on test conditions:
See table 1 below for the preparation of the test mixtures.
The prepared mixtures were aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity, 270 mL). The rate of oxygen consumption was measured, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below ca. 2 mg O2/L using a Yellow Springs Instrument (YSI) dissolved oxygen meter with a self stirring bottle probe connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.
Reference substance (positive control):
yes
Remarks:
3,5- Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
824 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL was 355 to 2680 mg/L
Duration:
72 h
Dose descriptor:
other: EC20
Effect conc.:
39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL was 5.1 to 162 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 3-hour EC50 for 3,5-dichlorophenol was 6.9 mg/L.

Table 3 below gives the measurements of dissolved oxygen in the study.

Table 3 Dissolved oxygen (DO) concentrations, measurement times, respiration rates and percentage inhibition

 

Test mixture

Initial DO concentration in the culture(mgO2/L)

Initial measured DO concentration(mgO2/L)

Final measured DO concentration(mgO2/L)

Measurement time(minutes)

Specific respiration rate(SRR mgO2/g/h)

% inhibition

Control (1)

7.7

6.5

2.5

5.9

25.4

 

Softanol 30

(mg/L)

 

 

 

 

 

 

10

7.4

6.0

2.0

6.1

25.6

0

30

6.9

6.0

2.0

6.9

21.7

12

100

7.9

7.0

3.7

8.1

15.3

38

300

7.6

7.0

4.0

8.1

13.9

44

1000

7.6

7.0

4.2

8.1

13.0

47

1000

7.9

7.0

4.5

7.3

12.8

48

1000

8.2

7.4

5.0

7.3

12.3

50

3,5-DCP (mg/L)

 

 

 

 

 

 

3

8.7

8.0

4.0

8.1

18.5

25

10

9.4

8.7

6.7

7.8

9.6

61

32

9.8

9.5

8.7

8.7

3.4

86

Control (2)

7.4

6.5

2.5

6.3

23.8

 

Validity criteria fulfilled:
yes
Conclusions:
The three-hour EC50 for Softanol 30 was calculated to be 824 mg/L (95% confidence limits, 355 to 2680 mg/L). The EC20 was calculated to be 39 mg/L (95% confidence limits, 5.1 to 162 mg/L). The EC80 could not be calculated.

Description of key information

The effect of Softanol 30 on the respiration rate of activated sludge was assessed by the methods detailed in OECD Test Guideline 209, ‘Activated Sludge, Respiration Inhibition Test’ and EC Directive 88/302, Method C.11, ‘Biodegradation - Activated Sludge Respiration Inhibition Test’ (Huntingdon Life Sciences Ltd, 2010, PLZ0027)
Samples of activated sludge, fed with synthetic sewage, were exposed to dilutions of the test substance (concentrations of 10, 30, 100, 300 and 1000 mg/L. for three hours.
The three-hour EC50 for Softanol 30 was calculated to be 824 mg/L (95% confidence limits, 355 to 2680 mg/L). The EC20 was calculated to be 39 mg/L (95% confidence limits, 5.1 to 162 mg/L). The EC80 could not be calculated.

Key value for chemical safety assessment

EC50 for microorganisms:
824 mg/L

Additional information