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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989/06/01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
This study was performed according to a method developed in the laboratory, without GLP statement. This method is well documented in the report. The purity of the test substance is not mentionned in the report.
Principles of method if other than guideline:
This study is an oxygen consumption test, method developed at the HULS AG. Bacterial suspension of Pseudomonas putida was added to the test substance in sealed erlenmeyer flasks and incubated for 5-6 hours at 25 +/- 2°C in a brood cabinet. The difference between the oxygen content of the vessels containing solutions at time zero and after the incubation period results in the bacterial oxygen consumption. This comparison gives information about the concentration of the test substance influencing the oxygen consumption.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Test organisms (species):
Pseudomonas putida
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
6 h
Remarks on exposure duration:
5-6 hours
Key result
Duration:
6 h
Dose descriptor:
EC10
Effect conc.:
1.15 other: mL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

BSVn (%) = [(BSVo - BSVn)/BSVo]*100

Where,

BSVn (%): Indication of the percentage of inhibition (positive values) or promotion (negative values) of oxygen consumption.

BSVo: Biochemical oxygen demand of the controls within the incubation period

BSVn: Biochemical oxygen demand of the test substance concentrations within the incubation period.

Table 6.1.7/1: Oxygen concentration and calculation of the inhibitory concentration

 

Control

173 µL/L

432 µL/L

860 µL/L

1693 µL/L

With HgCl2

O2- initial values

8.28

8.27

8.24

8.20

 

 

8.26

8.21

 

 

8.13

8.25

 

 

8.25

8.23

 

 

8.19

8.20

Average

8.25

8.24

8.19

8.24

8.20

Difference

-

0.01

0.06

0.01

0.05

Without HgCl2

O2- final values

3.31

3.34

3.45

3.32

 

 

3.49

3.34

 

 

3.66

3.57

 

 

3.85

3.71

 

 

3.90

3.84

Average

3.36

3.42

3.62

3.78

3.87

O2- consumption

4.89

4.82

4.58

4.46

4.33

Difference

-

0.07

0.32

0.43

0.57

% effect

-

1.5

6.5

8.8

11.6

Validity criteria fulfilled:
not applicable
Conclusions:
The EC10 value based on the inhibition of oxygen consumption was calculated at 1.15 mL/L.
Executive summary:

This study, an oxygen consumption test, was performed according to a method developed in the laboratory HULS AG, without GLP statement. Bacterial suspension of Pseudomonas putida was added to the test substance (with a vehicle, Nonylphenolethoxypropoxylate) in sealed erlenmeyer flasks and incubated for 5-6 hours at 25 +/- 2°C in a brood cabinet. The difference between the oxygen content of the vessels containing solutions at time zero and after the incubation period results in the bacterial oxygen consumption. This comparison gives information about the concentration of the test substance influencing the oxygen consumption.

14 flasks were prepared: 4 flasks per test substance concentrations (173; 432; 860 and 1693 µL/L) - 2 of them were incubated with HgCl2; 4 flasks without test substance but with HgCl2 and 6 flasks without test substance and without HgCl2. HgCl2 solution was added to stop the biochemical reaction. After an incubation period of 5 -6 hours at 25 +/- 2°C, the oxygen concentration measurements were carried out.

The percentage of inhibition of oxygen consumption were 1.5% at 173 µL/L, 6.5% at 432 µL/L, 8.8% at 860 µL/L and 11.6% at 1693 µL/L. Based on these results, the EC10 was calculated (probit analysis) at 1151 µL/L, equivalent to 1.15 mL/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-02-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to the EC Guideline 88/302/EC (similar to EU Method C.11) with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
other: EC Guideline 88/302/EC (30.5.1988)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
The oxygen consumption of activated sludge, which was charged with a standardized amount of synthetic wastewater is measured after a contact time of 3 hours. Similarly, the oxygen consumption of the same activated sludge with synthetic wastewater and with different concentrations of the test substance is measured. The inhibitory effect of the test substance at a given concentration is expressed as a percentage of the average oxygen consumption of 2 controls. From tests with different test substance concentrations, the EC50 is calculated. Furthermore, the non-inhibitory concentrations of test substances can be used in biodegradability tests.
GLP compliance:
yes (incl. certificate)
Remarks:
1997-02-18
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: 250 µL, 50 µL and 25 μL of test substance was placed directly into the 100 mL of deionized water, and magnetic stirring was performed.
- Controls: 2 controls without test substance and without reference substance (initial and final determination) + 1 control with reference substance.
- 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): no data
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation: Activated sludge (4-5 L) was, the day before the test, settle once and the supernatant was aspirated. The sludge was aerated for 10 minutes and stirring, allow to settle and washed with 2 L of drinking water and aerated and allowed to settle again briefly and filled with 200 mL of synthetic wastewater and drinking water to 4 L and aerated and stirred.
On the test day was the activated sludge can settle, the supernatant aspirated scum, washed with 2 L of drinking water, allow to settle again and refilled with potable water to 4L. After the quick-dry weight determination, the volume was concentrated to 3 L.
- Content of suspended solids: 3.22 g/L (activated sludge). This level gives a concentration of 1287 mg/L in the test medum.
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:
No data
Test temperature:
18-22°C were hold by cooling the water in the incubation vessel with ice.
At the beginning of incubation: 18.5-19.5°C.
During the determination of oxygen content: 19.5-20.5°C.
pH:
See table 6.1.7/2 in "Any other information on results incl. tables".
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 140.6, 234.3, 328.0, 468.5 and 702.8 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: no data
- Material, size, headspace, fill volume: 500 mL
- Aeration: continuously (air flow = 0.5 to 1 L/min).
- 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
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
Drinking water.

OTHER TEST CONDITIONS
No data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.40-1.67
- Range finding study: No
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
302 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:
EC10
Effect conc.:
122 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 6.1.7/2 in "Any other information on results incl. tables".
- To calculate EC50 and EC10, 3 dose-effect pairs were evaluated in the range of concentration from 140.6, 234.4 and 328.0 mg/L. Increased concentration of 468.5 and 702.8 mg/L did not indicate a significant increased inhibition effect.
- The difference between oxygen consumption of control 1 and control 2 does not exceed 15%, here 3.5%.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 5.5 mg/L.
Reported statistics and error estimates:
Probit analysis was performed to determine EC50 values.

Table 6.1.7/2: Oxygen depletion and percentage of inhibition

 

Initial value (mg O2/L)

Final value (mg O2/L)

Respiration rate (mg O2/L*h)

Inhibition (%)

pH value

Test substance

702.8 mg/L

8.02

6.11

13.48

51.5

7.9

468.5 mg/L

8.22

6.31

12.73

54.2

7.9

328.0 mg/L

7.90

6.10

12.71

54.3

7.8

234.3 mg/L

7.83

5.48

17.63

36.6

7.9

140.6 mg/L

7.41

4.20

24.08

13.4

8.1

Reference substance

5 mg/L

7.72

6.29

14.30

48.6

8.3

15 mg/L

8.34

7.08

9.45

66

8.3

30 mg/L

8.56

7.80

6.51

76.6

8.4

Control 1

7.53

4.80

27.30

-

8.2

Control 2

7.05

3.75

28.29

-

8.2

Validity criteria fulfilled:
yes
Conclusions:
The EC50 and EC10 were determined to be 302 mg/L and 122 mg/L, respectivel.
Executive summary:

This study was performed, according to the EC Guideline 88/302/EC (similar to EU Method C.11) with GLP statement, to determine the inhibition effect of the test substance on the respiration of activated sludge after 3 hours of exposure at 20 +/- 2°C.

The activated sludge was obtained from the sewage treatment plant Marl-West (Lippeverband), treating predominantly domestic sewage. The content of suspended solids was 3.22 g/L (activated sludge), equivalent to a concentration of 1287 mg/L in the test medium.

10 vessels were prepared: 2 controls vessels (without test substance and reference substance), 3 positive controls (with 3,5 -DCP at 5, 15 and 30 mg/L) and 5 test substance vessels (at 140.6, 234.3, 328.0, 468.5 and 702.8 mg/L). 16 mL of synthetic sewage and 200 mL of inoculum were added to each vessels and filled with water up to 500 mL.

The oxygen consumption in the controls vessels were 27.30 and 28.29 mg O2/L*h. The difference between these controls does not exceed 15%, here 3.5%.

The EC50 value of the reference substance (3,5 -DCP) was determined to be 5.5 mg/L.

For the test substance, the EC50 and EC10 values were determined at 302 mg/L and 122 mg/L, respectively. 3 dose-effect pairs were evaluated in the range of concentration from 140.6, 234.3 and 328.0 mg/L. Increased concentration of 468.5 and 702.8 mg/L did not indicate a significant increased inhibition effect.

Description of key information

EC Guideline 88/302/EC, similar to EU Method C.11, GLP, key study, validity 1:

3h-EC50 = 302 mg/L;

3h-EC10 = 122 mg/L;

Key value for chemical safety assessment

EC50 for microorganisms:
302 mg/L
EC10 or NOEC for microorganisms:
122 mg/L

Additional information

Two studies are available to assess the toxicity of the registered substance to microorganisms.

The first study, assessed as a key study, was performed according to the EC Guideline 88/302/EC similar to the EU Method C.11 with GLP statement, to determine the inhibition effect of the test substance on the respiration of activated sludge after 3 hours of exposure at 20 +/- 2°C. The percentage of inhibition of oxygen consumption were 13.4% at 140.6 mg/L, 36.6% at 234.3 mg/L and 54.3% at 328.0 mg/L. Increased concentration of 468.5 and 702.8 mg/L did not indicate a significant increased inhibition effect. The EC50 and EC10 values were determined at 302 mg/L and 122 mg/L, respectively, for the test substance.

The second study, assessed as a supporting study, was performed according to a method developed in the laboratory HULS AG without GLP statement, to determine the inhibition of oxygen consumption of Pseudomonas putida after 5-6 hours of exposure at 25 +/- 2°C. The percentage of inhibition of oxygen consumption were 1.5% at 173 µL/L, 6.5% at 432 µL/L, 8.8% at 860 µL/L and 11.6% at 1693 µL/L. Based on these results, the EC10 value was calculated at 1.15 mL/L.

The result of the key study, performed similar to international guideline, is taken into account for chemical safety assessment.