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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 August 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled. A significant inhibition of the respiration rate (21% relative to mean of the controls) was noted only at the highest test concentration (1000 mg/L), far greater than the limit of solubility.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None
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)

Each test solution was prepared by direct addition of the test item in the test flask with an appropriate micropipette (the volume added was based on the density = 920 Kg/m3 at 20°C) and vigorous agitation of the solution for a few minutes before the beginning of the test (i.e. just before addition of the inoculum).

Groups of test and reference solutions and controls were set up as follows:
- two controls containing incoulum at 1.6 g/L;
- five test concentrations containing the test item at 10, 31.6, 100, 316 and 1000 mg/L and inoculum at 1.6 g/L;
- three reference concentrations containing the reference item 3,5-dichlorophenol at 4, 12 and 36 mg/L and inoculum at 1.6 g/L.
These solutions were prepared in test flasks and then immediately aerated for 3 hours before being transfered to an oxygen measuring apparatus.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: The inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particles) and washed with dechlorinated water. Four samples of 50 mL were taken to determine the content in suspended matter (dry weight). The concentratio of sludge (6.22 g/L) was then adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water. The inoculum was maintained under agitation for two days before the test. For every day the sludge was not used, 50 mL of sewage feed were added, per liter. Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.
Each concentration was prepared separately by adding 16 mL of sewage feed to each of the flasks which were made up to 300 mL with water only in the case of the controls or a nominal mixture of water and test item in the case of the test solutions. Each mixture was then made up to 500 mL by adding 200 mL of inoculum. As 200 mL of inoculum were added in a final volume of 500 mL, the organic material concentration from inoculum was 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
No
Hardness:
280 +/- 20 mg/L as CaCO3
Test temperature:
20°C +/- 2°C
pH:
The pH of the reference stock solution was 7.92 after agitation. No other informations on pH values.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 10, 31.6, 100, 316 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: test flasks
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: no data
- Aeration: air was bubbled through each solution at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements.
- No. of organisms per vessel: inoculum at 1.6 g/L
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1 (but 2 controls. In order to verify the quality of the inoculum, the controls were prepared at the beginning (first control) and the end (second control) of the test).
- No. of vessels per vehicle control (replicates): not applicable

Solutions were prepared approximately every 15 minutes for test times of 3 hours.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

OTHER TEST CONDITIONS
- Adjustment of pH: The inoculum was buffered, if necessary, to pH 6.0 - 8.0 using sodium bicarbonate solution.
- Photoperiod: the stock vessel was kept away from light by wrapping it in opaque material.
- Light intensity: no special precautions were taken.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 3 hours of aeration/contact time, the content of the test flask was poured into the measuring apparatus and the oxygen concentration was determined for a period of approximately 10 minutes.Measurement of oxygen concentration of the test solutions was determined using an oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test or reference solution which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter and thence to a chart recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Test concentrations: 10, 31.6, 100, 316 and 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Inhibitions of the respiration rate at 10, 31.6, 100, 316 and 1000 mg/L were 0%, 0%, 9%, 11% and 21%, respectively, relative to mean of the controls. Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted only at 1000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3h EC50 = 11.8 mg/L (95% CL 9.73-14.2 mg/L)
- Other: none
Reported statistics and error estimates:
No data

Table 6.1.7/1: Respiration rate in mg O2/L/h

 

Concentration (mg/L)

Respiratory rate (mg O2/L/h)

Inhibition (%)

Controls

Control 1

42.0

 -

Control 2

44.0

Reference item

4

36.0

16

12

19.2

55

36

8.0

81

Test item

10

44.0

0

31.6

43.2

0

100

39.0

9

316

38.4

11

1000

34.0

21

Validity criteria fulfilled:
yes
Conclusions:
Under experimental conditions, the 3h-NOEC and the 3h-EC50 of the test item is 31.6 mg/L and >1000 mg/L for activated sludge, respectively.
Executive summary:

The effect of the test item on the respiration of activated sewage sludge was evaluated using a 3h static test according to OECD guideline 209 with GLP statement.

The test solutions were prepared as follows:

- two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/L,

- five test item solutions: activated sludge in dechlorinated water (at 1.6 g/L) and test item (to obtain final concentrations of 10, 31.6, 100, 316 and 1000 mg/L),

- three reference item (3,5 -dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/L) and reference item (to obtain final concentrations of 4, 12 and 36 mg/L).

The oxygen consumption of these solutions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours. The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and the reference item was determined considering the oxygen consumption of the controls as 100%.

As the validity criteria were met (the difference between the two controls was below 15% and the EC50 of the reference item was between 5 and 30 mg/L), the test was considered valid.

Inhibitions of the respiration rate at 10, 31.6, 100, 316 and 1000 mg/L were 0%, 0%, 9%, 11% and 21%, respectively, relative to mean of the controls. Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted only at 1000 mg/L.

Under experimental conditions, the 3h-NOEC and the 3h-EC50 of the test item is 31.6 mg/L and >1000 mg/L for activated sludge, respectively.

Description of key information

GLP study performed according to OECD 209 guideline:
3h-NOEC = 31.6 mg/L.
3h-EC50 >1000 mg/L.

Key value for chemical safety assessment

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

Additional information

One key study is available to assess the effect of the test item on the respiration of activated sewage sludge, using a 3h static test according to OECD guideline 209 with GLP statement.

The test solutions were prepared as follows:

- two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/L,

- five test item solutions: activated sludge in dechlorinated water (at 1.6 g/L) and test item (to obtain final concentrations of 10, 31.6, 100, 316 and 1000 mg/L),

- three reference item (3,5 -dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/L) and reference item (to obtain final concentrations of 4, 12 and 36 mg/L).

The oxygen consumption of these solutions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours. The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and the reference item was determined considering the oxygen consumption of the controls as 100%.

As the validity criteria were met (the difference between the two controls was below 15% and the EC50 of the reference item was between 5 and 30 mg/L), the test was considered valid.

Inhibitions of the respiration rate at 10, 31.6, 100, 316 and 1000 mg/L were 0%, 0%, 9%, 11% and 21%, respectively, relative to mean of the controls. Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted only at 1000 mg/L.

Under experimental conditions, the 3h-NOEC and the 3h-EC50 of the test item is 31.6 mg/L and >1000 mg/L for activated sludge, respectively.