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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 June 2009 to 6 July 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to the old version of the OECD Guideline 209 (1984), EU Method C.11 and US EPA Guideline OPPTS 850.6800 with GLP statement. All validity criteria were fulfilled but the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines. This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. This study is considered reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(1984)
Deviations:
yes
Remarks:
the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
No. 440/2008
Deviations:
yes
Remarks:
the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
yes
Remarks:
the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2009-03-04
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: In order to aid weighing the test material was warmed to approximately 35°C prior to an amount of test material (2000 mg) being dispersed in water with the aid of high shear mixing at approximately 7500 rpm for 30 minutes and the volume adjusting to 1L to give a 2000 mg/L stock dispersion from which serial dilutions were made to give 200 and 20 mg/L stock dispersions. An aliquot (250 mL) of the 20 mg/L stock dispersion was dispersed with synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (80 and 250 mL) of the 200 and 2000 mg/L stock dispersions were used to preparer the test concentrations of 32, 100, 320 and 1000 mg/L. The volumetric flasks containing the stock dispersions were inverted several times to ensure homogeneity of the stock dispersions.
- Eluate: water
- Control: yes
- 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): See table 6.1.7/2 in "Any other information on results incl. tables".
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample was 7.3.
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.7 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
140 mg/L as CaCO3
Test temperature:
21 +/- 1 °C
pH:
7.5-8.1
Dissolved oxygen:
See table 6.1.7/1 in "Any other information on results incl. tables".
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: no data
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable

At time "0", 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 L/min. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.
As each vezssel reached 3h contact time, an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (DOB) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace.

TEST MEDIUM / WATER PARAMETERS
The test water used was laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened giving water with a total harness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

OTHER TEST CONDITIONS
- Temperature controlled room: 21 +/- 1°C
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate and observations on the test preparation throughout the test period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
390 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: 320-470 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Variation in respiration rates of controls 1 and 2 after 3 hours contact time was +/- 5%.
- See tables 6.1.7/1 and 6.1.7/2 in "Any other information on results incl. tables".
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 3h-EC50 = 5.5 mg/L (95% CL: 3.3-9.1 mg/L).
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the NOEC, EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% confidence limits were calculated for the EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
The NOEC was taken as being the EC15 value.

Table 6.1.7/1: Oxygen consumption rates and percentage inhibition values after 3 hours contact time

Nominal concentrations (mg/L)

Initial O2 reading (mg O2/L)

Measurement period (minutes)

Final O2 reading (mg O2/L)

O2 consumption rates (mg O2/L/min)

% inhibition

Control

R1

6.9

10

2.7

0.42

-

R2

5.9

7

2.7

0.46

-

Test material

10

6.4

10

1.8

0.46

[5]

32

6.4

8

2.8

0.45

[2]

100

6.4

8

2.9

0.44

0

320

7.0

10

4.3

0.27

39

1000

8.3

10

7.8

0.05

89

3,5-dichlorophenol

3.2

7.7

10

5.1

0.26

41

10

6.8

4*

6.1

0.18

59

32

8.4

10

7.7

0.07

84

* Due to a malfunction with the chart recorder it was only possible to calculate the oxygen consumption rate over a four minute period.

Table 6.1.7/2: Observations on the test preparations throughout the test period

Nominal concentrations (mg/L)

Observations on test preparations

0h

30min contact time

3h

contact time

Control

R1

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

R2

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

Test material

10

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

32

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

100

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

320

Light amber dispersion throughout*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

1000

Light amber dispersion throughout*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

* Observations prior to addition of activated sewage sludge and synthetic sewage.

Validity criteria fulfilled:
yes
Remarks:
The two control respiration rates are within 15% of each other (+/- 5%) and 3h-EC50 for 3,5-dichlorophenol lies within the range 5-30 mg/l (5.5 mg/L).
Conclusions:
The effect of the test substance on the respiration of activated sewage sludge gave a 3h-EC50 of 390 mg/L (95% CL: 320 -470 mg/L). The 3h-NOEC was 190 mg/L.
Executive summary:

This study was performed according to the old version of the OECD Guideline 209 (1984), EU Method C.11 and US EPA Guideline OPPTS 850.6800 with GLP statement, to assess the effect of the test substance on the respiration of activated sewage sludge.

Activated sewage sludge was exposed to an aqueous dispersion of the test substance at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of approximately 21°C within the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3h contact time and compared to data for the control and a reference material 3,5 -dichlorophenol.

All validity criteria were fulfilled (the two control respiration rates are within 15% of each other (+/- 5%) and 3h-EC50 for 3,5-dichlorophenol lies within the range 5-30 mg/l (5.5 mg/L)) but the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines. This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The effect of the test substance on the respiration of activated sewage sludge gave a 3h-EC50 of 390 mg/L (95% CL: 320 -470 mg/L). The 3h-NOEC was 190 mg/L.

Description of key information

OECD Guideline 209 (1984), EU Method C.11, US EPA Guideline 850.6800, GLP, key study, validity 2:
3h-EC50 = 390 mg/L (95% CL: 320 -470 mg/L).
3h-NOEC = 190 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
390 mg/L
EC10 or NOEC for microorganisms:
190 mg/L

Additional information

One valid key study is available to assess the effect of the substance on the respiration of activated sewage sludge. This study was performed according to the old version of the OECD Guideline 209 (1984), EU Method C.11 and US EPA Guideline OPPTS 850.6800 with GLP statement.

Activated sewage sludge was exposed to an aqueous dispersion of the substance at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of approximately 21°C within the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3h contact time and compared to data for the control and a reference material 3,5 -dichlorophenol.

All validity criteria were fulfilled (the two control respiration rates are within 15% of each other (+/- 5%) and 3h-EC50 for 3,5-dichlorophenol lies within the range 5-30 mg/l (5.5 mg/L)) but the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines. This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The effect of the substance on the respiration of activated sewage sludge gave a 3h-EC50 of 390 mg/L (95% CL: 320 -470 mg/L). The 3h-NOEC was 190 mg/L.