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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 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled. However, restrictions should be considered because the respiration rate was evaluated only on the highest concentration (100 mg/L).
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 (incl. certificate)
Remarks:
2000-01-18
Specific details on test material used for the study:
- Molecular weight: 270.3 g/mol
- Vapour pressure: 0.36 Pa/25°C (calculated)
- Water solubility (under test conditions): 10.9 mg/L
- log Pow: 4.74-4.79
- specific gravity: 1.0091
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)
- Method: As the solubility of the test substance in water is 10.9 mg/L, test solutions at 10, 31.6 and 100 mg/L were prepared by direct addition of the test substance in the test vessels with a micropipette. Test solutions at 1 and 3.16 mg/L were prepared by means of a stock solution at 6 mg/L in dechlorinated water. The stock solution was agitated for 5 minutes (protected from light) and test solutions (1 and 3.16 mg/L) were prepared immediately by further dilution of this stock solution with dechlorinated water.
- Eluate: dechlorinated tap water
- Controls: yes with 2 replicates
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Type: activated sludge collected from a water treatment works containing effluent from a predominantly domestic origin (the water treatment plant of Evreux, France).
- Preparation: 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 concentration of sludge was not adjusted because the quantity of suspended matter was 4 g/L +/- 10%.
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:
between 20 and 21°C.
pH:
No data
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 1, 3.16, 10, 31.6 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: test flasks
- Material, size, headspace, fill volume: no data
- Aeration: yes. The solutions were aerated with between 0.5 to 1 litre of air per minute using a Pasteur pipette as an aeration device.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2 (in order to verify the quality of the inoculum, the controles were prepared at the beginning (first control) and at the end (second control) of the test).
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 1.6 g/L

TEST MEDIUM / WATER PARAMETERS
Dechlorinated tap water, with a hardness of 280 +/- 20 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: no data
- Illumination: no special precautions were taken.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate was determined from the recorder trace in terms of mg O2/L/h. Measurement of oxygen concentration of the test solutions was determined using an oxygen probe placed into an opaque BOD 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
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no
- Test concentrations: 1, 3.16, 10, 31.6 and 100 mg/L
- Results used to determine the conditions for the definitive study: no data
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (three reference concentrations: 4, 12 and 36 mg/L)
Key result
Duration:
3 h
Dose descriptor:
EC50
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:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
See table in "Any other information on results incl. tables".
The respiration rate of the test solution of highest concentration (100 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test solutions (1 to 31.6 mg/L) was not determined.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3h-EC50 = 16.6 mg/L (95% confidence limits = 13.2-21.8 mg/L).
Reported statistics and error estimates:
The EC50 is calculated according to Probit analysis. The confidence interval limits are calculated statistically according to Fieller's method.

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

 

Concentration (mg/L)

Respiration rate (mg O2/L/h)

Percent inhibition (%)

Control 1

Control 2

0

0

40.0

42.0

 

Reference substance

4

12

36

32.0

28.0

10.8

22

32

74

Test substance

1

3.16

10

31.6

100

ND

ND

ND

ND

44.0

 

 

 

 

0

Validity criteria fulfilled:
yes
Conclusions:
Under our experimental conditions, the 3-hour NOEC of the test substance is >= 100 mg/L for activated sludge. The test substance should be considered as non-toxic for the microorganisms of a water treatment plant.
Executive summary:

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

The test solutions were prepared as follows:

- two control solutions: In order to verify the quality of the inoculum, the controles were prepared at the beginning (first control) and at the end (second control) of the test. Activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/L.

- five test substance solutions: activated sludge in dechlorinated water (at 1.6 g/L) and test substance to obtain final concentrations of 1, 3.16, 10, 31.6 and 100 mg/L.

- three reference substance (3,5 -dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/L) and reference substance to obtain final concentration 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 of the test substance and the reference substance 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 3,5 -dichlorophenol was between 5 and 30 mg/L (16.6 mg/L with 95% CL = 13.2 - 21.8 mg/L), the test was considered valid.

The respiration rate of the test solution of highest concentration (100 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test solutions (1 to 31.6 mg/L) was not determined.

Under our experimental conditions, the 3-hour NOEC of the test substance is >= 100 mg/L for activated sludge. The test substance should be considered as non-toxic for the microorganisms of a water treatment plant.

Description of key information

OECD Guideline 209, GLP, key study, validity 2:

3h-NOEC >= 100 mg/L (nominal concentration), based on the respiration rate of activated sludge.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

One key study, performed by CIT laboratory, according to the OECD Guideline 209 with GLP statement, is available on the respiration of activated sewage sludge, using a 3h static test.

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 substance solutions: activated sludge in dechlorinated water (at 1.6 g/L) and test substance to obtain final concentrations of 1, 3.16, 10, 31.6 and 100 mg/L.

- three reference substance (3,5 -dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/L) and reference substance to obtain final concentration 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.

As the validity criteria were met: the difference between the two controls was below 15% and the EC50 of 3,5 -dichlorophenol was between 5 and 30 mg/L (16.6 mg/L with 95% CL = 13.2 - 21.8 mg/L), the test was considered valid.

The respiration rate of the test solution of highest concentration (100 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test solutions (1 to 31.6 mg/L) was not determined.

Under our experimental conditions, the 3-hour NOEC of the test substance is >= 100 mg/L for activated sludge. The test substance should be considered as non-toxic for the microorganisms of a water treatment plant.