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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-01-28 to 2004-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
May 1988
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Just before the start of the test defined amounts of the test item were directly weighed into each test flask. The test concentrations (10, 32, 100, 320 and 1000 mg/L) were chosen to permit the determination of the EC 50. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Vehicle:
no
Details on test solutions:
Just before the start of the test defined amounts of the test item were directly weighed into each test flask. The test concentrations (10, 32, 100, 320 and 1000 mg/L) were chosen to permit the determination of the EC 50. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Origin
The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.

Conditioning
The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature until use. Before use, the dry weight of the activated sludge was measured again, and the measured value of the dry weight was adequate. The pH of the activated sludge inoculum was determined to be pH 7.88.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure period
Test temperature:
19.3 – 20.3 °C (during the incubation) and 19.1 – 20.5 °C (during oxygen measurement)
pH:
The pH of the activated sludge inoculum was determined to be pH 7.88.
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 6.5 mg O2/L.
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
Test solutions:
Just before the start of the test defined amounts of the test item were directly weighed into each test flask.
The test concentrations (10, 32, 100, 320 and 1000 mg/L) were chosen to permit the determination of the EC 50. Concentrations in excess of nominal 1000 mg test item/L were not tested.

Control
Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.

Ratio of test compounds:
One test solution with a final volume of 500 mL is tested per treatment in a glass flask. 16 mL synthetic sewage and an adequate amount of the test item or an adequate volume of the stock solutions of the reference item is filled up with deionised water to 300 mL before the start of the test. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 4 g/L (dry weight) is added, first to control 1, then in time intervals of approximately 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item and the test item and finally to an additional control.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
79.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
LiOH
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: calculated by linear regression (not part of the study report)
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
114.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
LiOH
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 98.9 - 132.0 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
180.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
LiOH
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 160.5 - 203.7 mg/L
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
286.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
LiOH
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95%CL: 248.5 - 329.5 mg/L
Details on results:
Inhibition of respiration rate:
In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by –1.8 % at the lowest nominal concentration of 10.0 mg/L.
At the nominal concentrations of 32 and 100 mg/L, the respiration rate was inhibited by 1.8 % and 7.1 %, respectively.
At the nominal concentrations of 320 and 1000 mg test item/L, the respiration rate was inhibited by 94.6 % and 98.2 %, respectively.
Results with reference substance (positive control):
Inhibition of respiration rate:
The following nominal concentrations of the positive control 3,5-dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L.
In comparison to the controls the respiration rate of the activated sludge was inhibited by 23.2 % at the lowest nominal concentration of 3.2 mg/L.
At the nominal concentrations of 10 and 32 mg/L, the respiration rate was inhibited by 64.3 % and 85.7 %, respectively.
3-hour EC 20, EC 50 and EC 80 of reference item:
The 3-hour EC 50 of 3,5-dichlorophenol was calculated to be 7.5 mg/L with 95 % confidence limits of 6.1 to 9.2 mg/L.
Reported statistics and error estimates:
Based on measured inhibition rates, the 3-hour EC 20, EC 50 and EC 80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

Influence of test item on oxygen consumption of activated sludge:

 

Flask
No.

Identification

Concentration

Oxygen consumption

Inhibition

pH-values

Oxygen concentration
(mg O2/L)

 

 

(mg/L)

(mg O2/L/min)

%

start *

end *

start *

end *

1

Control

-

0.570

-

7.74

7.89

7.5

8.3

10

Control

-

0.550

-

7.70

7.86

7.6

8.7

 

mean

-

0.560

-

-

-

-

-

 

% deviation

-

3.5

-

-

-

-

-

5

Test item

10

0.570

-1.8

8.38

8.54

7.5

8.4

6

Test item

32

0.550

1.8

8.76

8.97

7.6

8.4

7

Test item

100

0.520

7.1

9.62

9.79

7.7

8.3

8

Test item

320

0.030

94.6

11.53

11.72

7.8

9.3

9

Test item

1000

0.010

98.2

12.30

12.51

7.6

9.4

*   start and end of 3-hour aeration

Influence of reference item on oxygen consumption of activated sludge:

 

Flask
No.

Identification

Concentration

Oxygen consumption

Inhibition

pH-values

Oxygen concentration
(mg O2/L)

 

 

(mg/L)

(mg O2/L/min)

%

start *

end *

start *

end *

1

Control

-

0.570

-

7.74

7.89

7.5

8.3

10

Control

-

0.550

-

7.70

7.86

7.6

8.7

 

mean

-

0.560

-

-

-

-

-

 

% deviation

-

3.5

-

-

-

-

-

2

3,5-DCP

3.2

0.430

23.2

7.79

7.92

7.6

8.3

3

3,5-DCP

10.0

0.200

64.3

7.89

8.07

7.6

8.7

4

3,5-DCP

32.0

0.080

85.7

7.92

8.16

7.6

9.2

*   start and end of 3-hour aeration

3,5-DCP: 3,5-Dichlorophenol

Validity criteria fulfilled:
yes
Conclusions:
The 3 hours EC20, EC50, and EC80 values for the test substance lithium hydroxide in the activated sludge respiration inhibition test were 114.3, 180.8, and 286.1 mg/L (based on measured inhibition rates), respectively. The EC10 value was calculated by linear regression to be 79.2 mg/L.
Based on this data, 3 hours EC10, EC20, EC50, and EC80 values can be calculated for lithium carbonate as 122.2, 176.3, 278, and 441.4 mg/L, respectively.
Executive summary:

A microorganism toxicity test with lithium carbonate is not available. Consequently, read-across was applied using study results obtained from lithium hydroxide.

The influence of the test item lithium hydroxide on the activity of activated sludge by measuring the respiration rate was evaluated according to OECD Guideline 209 and EU method C.11. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls. Following test concentrations were used: 10, 32, 100, 320 and 1000 mg lithium hydroxide/L; 3.2, 10 and 32 mg 3,5 -dichlorophenol/L and two inoculum controls. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between –1.8% and 98.2 % up to the highest nominal test concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. The 3-hour EC 50 for the positive control 3,5 -dichlorophenol, which was tested in the same way as the test item, was found to be 7.5 mg/L and is within the range of 5 – 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. The 3 hours EC20, EC50, and EC80 values for the test substance lithium hydroxide in the activated sludge respiration inhibition test were 114.3, 180.8, and 286.1 mg/L (based on measured inhibition rates), respectively. The EC10 value was calculated be linear regression to be 79.2 mg/L.

Based on this data 3 hours EC10, EC20, EC50, and EC80 values can be calculated for lithium carbonate as 122.2, 176.3, 278, and 441.4 mg/L, respectively (calculated EC10 for lithium ion: 22.95 mg/L).

Description of key information

Read-across from lithium hydroxide anhydrous:

The 3 hours EC20, EC50, and EC80 values for the test substance lithium hydroxide in the activated sludge respiration inhibition test were 114.3, 180.8, and 286.1 mg/L (based on measured inhibition rates), respectively. The EC10 value was calculated be linear regression to be 79.2 mg/L. Based on this data 3 hours EC10, EC20, EC50, and EC80 values can be calculated for lithium carbonate as 122.2, 176.3, 278, and 441.4 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:
278 mg/L

Additional information

A microorganism toxicity test with lithium carbonate is not available. Consequently, read-across was applied using study results obtained from lithium hydroxide.

The influence of the test item lithium hydroxide on the activity of activated sludge by measuring the respiration rate was evaluated according to OECD Guideline 209 and EU method C.11. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls. Following test concentrations were used: 10, 32, 100, 320 and 1000 mg lithium hydroxide/L; 3.2, 10 and 32 mg 3,5 -dichlorophenol/L and two inoculum controls. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between –1.8% and 98.2 % up to the highest nominal test concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. The 3-hour EC 50 for the positive control 3,5 -dichlorophenol, which was tested in the same way as the test item, was found to be 7.5 mg/L and is within the range of 5 – 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. The 3 hours EC20, EC50, and EC80 values for the test substance lithium hydroxide in the activated sludge respiration inhibition test were 114.3, 180.8, and 286.1 mg/L (based on measured inhibition rates), respectively. The EC10 value was calculated be linear regression to be 79.2 mg/L.

Based on this data 3 hours EC10, EC20, EC50, and EC80 values can be calculated for lithium carbonate as 122.2, 176.3, 278, and 441.4 mg/L, respectively.

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