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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-06 to 2004-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
NA
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
For the pre-test, a stock solution containing 200 mg test item/L in deionized water was prepared. After 10 minutes in the ultrasonic bath the emulsion didn’t show any undissolved particles. The stock solution then was used to prepare solutions at the concentrations of 100, 10, and 1 mg/L. Because of the low water solubility, the treatment of 1000 mg/L was prepared by adding the appropriate volume of the test item directly.
In the main study, the test item was used directly.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
- Pretreatment: The sludge was washed with tap water three times, then centrifuged and resuspended in tap water. 200 ml of the sludge were added to each test vessel.
- Initial biomass concentration: 4.1 g suspended solids/litre
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
NA
Hardness:
NA
Test temperature:
20 +/- 2 °C
pH:
7.4 (control)
Dissolved oxygen:
NA
Salinity:
NA
Nominal and measured concentrations:
Pre-test: Nominal concentrations: 1, 10, 100, 1000 mg/L of test item;
Main test: 1000 mg/L of test item;
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers as test vessels; narrow-neck glass with flat bottom as measuring flasks
- fill volume: 500 mL
- Aeration: purified air, using Pasteur pipettes, flow approx. 0.75 L/min.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): two replicates before, two between and two after measuring O2-concentrations in the reference and test item replicates
- Sludge concentration: 1.64 g suspended solids/L in the main test
- Biomass loading rate:

EFFECT PARAMETERS MEASURED:
- O2-consumption
- pH value at the beginning of the O2-measurement

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No inhibition was observed at 1000 mg/L.

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Two valid experiments were performed.
In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 4.0 g suspended solids/L, giving a concentration of 1.6 g sus-pended solids/L in the test. As the pre-test was performed for range-finding only, no reference item was tested.
No inhibition of the respiration of the activated sludge was detected in the treatments.
The main study was performed as a limit-test with a nominal concentration of 1000 mg/L. Conduct of the study was the same as in the pre-test. The dry matter of the activated sludge was determined as 4.1 g suspended solids/L, giving a concentration of 1.64 g suspended solids/L in the test. No inhibition was observed.
Results with reference substance (positive control):
3,5-Dichlorophenole was used as reference item. The EC50 of the reference item was determined as 8.2 mg/L (95% confidence interval: 6.4 – 10.2 mg/L), which lies within the recommended range of 5 – 30 mg/L.
Reported statistics and error estimates:
NA

Pre-Test

O2-Consumption

No.

Content

Nominal Concentration in mg/L

O2Consumption in mg/(L*min.)

O2consumption in mg/(L*h)

1

Control

0

0.4119

24.71

2

Test Item

1000

0.4352

26.11

3

Test Item

100

0.4268

25.61

4

Test Item

10

0.3861

23.17

5

Test Item

1

0.3704

22.22

6

Control

0

0.4486

26.92

 Inhibition in %

Item

Nominal Concentration (mg/L)

Inhibition in %

Test Item

1000

-1.2

Test Item

100

0.8

Test Item

10

10.3

Test Item

1

13.9

Main Study

O2-Consumption

No.

Content

Nominal Concentration (mg/L)

O2consumption in mg/(L*min.)

Mean O2cons. in mg/(L*min.)

Mean O2cons. in mg/(L*h)

1

Control

0

0.9702

0.9292

55.7534

2

Control

0

0.8883

3

Reference

2.5

0.7419

0.6991

41.9471

4

Reference

2.5

0.6563

5

Reference

5

0.5867

0.6125

36.7471

6

Reference

5

0.6383

7

Reference

10

0.4191

0.4363

26.1800

8

Reference

10

0.4536

9

Reference

20

0.3127

0.3257

19.5416

10

Reference

20

0.3386

11

Reference

40

0.1634

0.1599

9.5416

12

Reference

40

0.1556

13

Control

0

0.7520

0.7271

43.6274

14

Control

0

0.7022

15

Test Item

1000

0.9058

1.0110

60.6569

16

Test Item

1000

1.1161

17

Control

0

1.0116

0.9983

59.8994

18

Control

0

0.9850

The control vessels no. 13 and 14 were stated as outliers and were not used for the determination of the results, since the O2-consumption was much lower than in the other control vessels.

Inhibition of the O2consumption was calculated by comparison with the controls and is given in the following table:

Item

Nominal Concentration (mg/L)

Inhibition in %

Test Item

1000

-4.9

Reference

2.5

27.5

Reference

5

36.5

Reference

10

54.7

Reference

20

66.2

Reference

40

83.4

Validity criteria fulfilled:
yes
Conclusions:
Sika HardenerLH was tested for toxicity to aquatic micro-organisms in a 3 h-activated sludge respiration inhibition test according to EU method C.11.
The following results could be determined for the test item Sika Hardener LH (VP)(species: activated sludge from a domestic sewage treatment plant):
3h NOEC = 1000 mg/L
3h EC20 > 1000 mg/L
3h EC50 > 1000 mg/L
Executive summary:

Sika Hardener LH was assessed in an activated sludge respiration inhibition test, according to the EU-method C. 11 and OECD guideline 209. Two valid experiments were performed. In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 4.0 g suspended solids/L, giving a concentration of 1.6 g suspended solids/L in the test. As the pre-test was perforned for range-finding only, no reference item was tested.

No inhibition of the respiration of the activated sludge was detected in the treatments.

The main study was performed as a limit test with a nominal concentration of 1000 mg/L. Conduct of the study was the same as in the pre-test. The dry matter of the activated sludge was determined as 4.1 g suspended solids/L, giving a concentration of 1.64 g suspended solids/L in the test.

3,5 -Dichlorophenole was used as reference item. The EC50 of the reference item was determined as 8.2 mg/L (95% CL: 6.4 - 10.2 mg/L), which lies within the recommended range of 5 - 30 mg/L.

The following results could be determined for the test item Sika Hardener LH (VP)(species: activated sludge from a domestic sewage treatment plant):

3h NOEC = 1000 mg/L

3h EC20 > 1000 mg/L

3h EC50 > 1000 mg/L

Description of key information

Sika Hardener LH was assessed in an activated sludge respiration inhibition test, according to the EU-method C. 11 and OECD guideline 209. The 3-hour EC50 was determined to be greater than 1000 mg/L and the 3-hour NOEC determined at 1000 mg/L.

 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Sika Hardener LH was assessed in an activated sludge respiration inhibition test, according to the EU-method C. 11 and OECD guideline 209. Two valid experiments were performed. In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 4.0 g suspended solids/L, giving a concentration of 1.6 g suspended solids/L in the test. As the pre-test was perforned for range-finding only, no reference item was tested.

No inhibition of the respiration of the activated sludge was detected in the treatments.

The main study was performed as a limit test with a nominal concentration of 1000 mg/L. Conduct of the study was the same as in the pre-test. The dry matter of the activated sludge was determined as 4.1 g suspended solids/L, giving a concentration of 1.64 g suspended solids/L in the test.

3,5 -Dichlorophenole was used as reference item. The EC50 of the reference item was determined as 8.2 mg/L (95% CL: 6.4 - 10.2 mg/L), which lies within the recommended range of 5 - 30 mg/L.

The following results could be determined for the test item Sika Härter LH (VP)(species: activated sludge from a domestic sewage treatment plant):

3h NOEC = 1000 mg/L

3h EC20 > 1000 mg/L

3h EC50 > 1000 mg/L