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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 24 to July 6, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to international guidelines, with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on April 12, 2005/ signed on June 01, 2005)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In a preliminary solubility trial, test item was insufficiently soluble in dechlorinated tap water to prepare a stock solution at 500 mg/L. However the results of a preliminary formulation trial showed that aliquots (50 mg) of the test substance could be dissolved in 284 mL of dechlorinated tap water followed by overnight stirring. Therefore, the day before test initiation, a subsample of the test substance was ground with an agate pestle and mortar and appropriate weights were established in one-litre test beakers and dechlorinated tap water was added with a magnetic stirrer bar. The mixtures were capped with aluminium foil, the water level was marked on the beaker and the mixtures stirred overnight on a magnetic stirrer. On the day of the test, the level of water in each beaker was checked and any loss by evaporation made up using dechlorinated water as required. Control and reference mixtures were treated similarly. The pH of the test mixtures was determined and adjusted to the range of 6.5 - 8.5 with 5N HCI as required.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A sample of activated sludge was obtained the day before the start of the tests from Worlingworth Sewage Treatment Works, which treats predominantly domestic waste. In the laboratory, the samples were maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: Aliquots of the activated sludge were filtered and dried. Synthetic sewage was added to each stock of activated sludge and these were aerated overnight.
- Initial biomass concentration: 4 g/L
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
200-250 mg/L as CaCo3
Test temperature:
19.4-20 °C
pH:
7.5-8.1
Dissolved oxygen:
Initial dissolved oxygen concentration in the culture: 6.2 – 8.6 mgO2/L
Initial measured dissolved oxygen concentration: 6.0 – 8.5 mgO2/L
Final measured dissolved oxygen concentration: 2.5 – 7.9 mgO2/L
Salinity:
None
Nominal and measured concentrations:
Preliminary range finding test, nominal concentration: 1, 10 and 100 mg/L
Definitive test, nominal concentration: 76, 152, 304, 608 and 1216 mg/L
Details on test conditions:
The day before test initiation, the tested substance was weighted and mixed with dechlorinated tap water. Control and reference mixtures were treated similarly. The pH of the test mixtures was determined and adjusted to the range of 6.5 -8.5 with 5N HCl as required.

Additions of synthetic sewage and microbial inoculum were made at fifteen minute-intervals to give the needed final volume. See Table 6.1.7/1

The prepared mixtures were capped with aluminium foil and aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory supply of oil-free compressed air (ca. one litre/minute).

Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (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 2 mgO2/L, using a Yellow Springs Instrument (YSI) dissolved oxygen meter; with temperature probe and 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 (3,5-DCP), nominal concentration: 3, 10 and 32 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
270 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% confidence limits 206 – 353 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
120 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% confidence limits 71 – 183 mg/L
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
605 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% confidence limits 425 – 932 mg/L
Details on results:
The specific respiration rates of control cultures established at the end of the two test series (25.0 and 27.8 mgO2/g/h) were 88% and 115% of the rates of those established at the start (28.3 and 24.2 mgO2/g/h).

These results show that the tests were valid and that the sample of activated sludge employed was sensitive to inhibition.

Concentration-related inhibition of respiration rates was seen at each tested substance concentration. The highest tested concentration of the tested substance in the definitive test (1216 mg/L) caused 91% inhibition.

Results with reference substance (positive control):
EC50 (3h, preliminary test): 6.6 mg/L (95% confidence limits 3.9 - 10.3 mg/L)
EC50 (3h, definitive test): 11.7 mg/L (95% confidence limits 7.4 – 19.1 mg/L)
Reported statistics and error estimates:
No data

Table 6.1.7/2: Definitive test: temperature, pH and measurements of respiration rate

 

Test mixture

Temperature (°C)

pH

Specific

respiration rate

mgO2/g/h

% inhibition

Initial

Final

Initial

Final

Control (1)

20.0

19.8

7.6

8.2

24.2

-

Test item (mg/L)

76

20.3

19.8

7.4

8.0

22.6

13

152

20.0

19.7

7.4

8.0

20.2

22

304

19.9

19.6

7.5

8.0

10.5

60

608

20.0

19.6

7.4

8.0

5.5

79

1216

19.6

19.6

7.4

8.1

2.4

91

3,5-DCP (mg/L)

3

19.7

19.6

7.5

8.1

20.8

20

10

19.8

19.6

7.5

8.1

14.0

46

32

19.8

19.6

7.5

8.2

6.9

74

Control (2)

19.7

19.6

7.5

8.0

27.8

-

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the EC50 of the test substance was 270 mg/L (95% confidence limits 206 - 353 mg/L). The EC20 and EC80 values were calculated to be 120 and 605 mg/L, with 95% confidence limits of 71 - 183 and 425 - 932 mg/L, respectively.
Executive summary:

The effect of test item on the respiration rate of activated sludge was assessed according to OECD Guideline 209 / EU Method C.11 / EPA OPPTS 850.6800 with GLP statement.

 

Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed for three hours to dilutions of the test substance. Their rates of oxygen consumption were determined using an oxygen electrode and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of each of the tests. Two tests were conducted, the first of which comprised a preliminary range finding test at nominal concentrations of 1, 10 and 100 mg/L. In the second (definitive) test, test item was employed at nominal concentrations of 76, 152, 304, 608 and 1216 mg/L. The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed in both tests at 3, 10 and 32 mg/L, as a positive control.

 

The specific respiration rates of control cultures established at the end of the two test series (25.0 and 27.8 mgO2/g/h) were 88% and 115% of the rates of those established at the start (28.3 and 24.2 mgO2/g/h). The three-hour 50% effect concentration (EC50) for 3,5-DCP in the preliminary test was calculated to be 6.6 mg/L (95% confidence limits 3.9 -10.3 mg/L) and in the definitive test, 11.7 mg/L (95% confidence limits 7.4 - 19.1 mg/L). These results show that the tests were valid and that the samples of activated sludge employed were sensitive to inhibition.

 

Concentration-related inhibition of respiration rates was seen at and above a nominal test concentration of 76 mg/L in the definitive test. The highest tested concentration (1216 mg/L) caused 91% inhibition.

 

Under the test conditions, the EC50 of the test substance was 270 mg/L (95% confidence limits 206 - 353 mg/L). The EC20 and EC80 values were calculated to be 120 and 605 mg/L, with 95% confidence limits of 71 - 183 and 425 - 932 mg/L, respectively.

Description of key information

OECD Guideline 209, GLP, key study, validity 1:

3h-EC50 (activated sludge) = 270 mg/L based on nominal concentrations.

Key value for chemical safety assessment

EC50 for microorganisms:
270 mg/L

Additional information

The effect of test item on the respiration rate of activated sludge was assessedaccording to OECD Guideline 209 / EU Method C.11 / EPA OPPTS 850.6800 with GLP statement.

 

Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed for three hours to dilutions of the test substance. Their rates of oxygen consumption were determined using an oxygen electrode and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of each of the tests. Two tests were conducted, the first of which comprised a preliminary range finding test at nominal concentrations of 1, 10 and 100 mg/L. In the second (definitive) test, test item was employed at nominal concentrations of 76, 152, 304, 608 and 1216 mg/L. The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed in both tests at 3, 10 and 32 mg/L, as a positive control.

 

The specific respiration rates of control cultures established at the end of the two test series (25.0 and27.8 mgO2/g/h) were 88% and 115% of the rates of those established at the start (28.3 and 24.2 mgO2/g/h). The three-hour 50% effect concentration (EC50) for 3,5-DCP in the preliminary test was calculated to be 6.6 mg/L (95% confidence limits 3.9 -10.3 mg/L) and in the definitive test, 11.7 mg/L (95%confidence limits 7.4 - 19.1 mg/L). These results show that the tests were valid and that the samples of activated sludge employed were sensitive to inhibition.

 

Concentration-related inhibition of respiration rates was seen at and above a nominal test concentration of 76 mg/L in the definitive test. The highest tested concentration (1216 mg/L) caused 91% inhibition.

 

Under the test conditions, the EC50 of the test substance was 270 mg/L (95% confidence limits 206 - 353 mg/L). The EC20 and EC80 values were calculated to be 120 and 605 mg/L, with 95% confidence limits of 71 - 183 and 425 - 932 mg/L, respectively.