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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-13
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:100 - 180 - 320 - 560 - 1000 mg/L were weighed out directly in Erlenmeyer flasks
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 100 - 180 - 320 - 560 - 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.09 g/L, corresponding to 1.55 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
19.9 °C
pH:
pH-value of the activated sludge 7.70
pH-value of the synthetic waste water 7.40
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.


- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- Composition of test medium:

Table for the Test Item Concentrations
Test Item Test concentration [mg/L]
100 180 320 560 1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Demineralised water [mL] filled up to 250 mL




EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests

Nominal Test pH pH pH Inhibition
Item (test item in (adjusted to) (test item [%]
Concentration demin. water) in medium)
[mg/L]

1st preliminary test –with pH adjustment, direct aeration
10 10.2 7.15*1 7.30 -4
10.1 - 7.37 -5
100 11.1 6.42*2 7.28 -12
11.2 - 8.01 -4
1000 11.9 5.19*3 6.89 0
11.8 - 10.9 84
2nd preliminary test –with pH adjustment, direct aeration
10 10.3 7.45*1 7.46 -2
10.2 - 7.45 2
100 11.1 6.97*2 7.54 -2
11.3 - 7.88 -5
1000 11.9 # 9.09 2
12.0 - 10.6 90


#) not adjustable
*1) adjusted with 0,05 mol/L HCL
*2) adjusted with 0,5 mol/L HCL
*3) adjusted with 2 mol/L HCL






Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
560 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:
EC10
Effect conc.:
690 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 624 - 750 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
825 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 773 - 867 mg/L
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: EC80
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:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item was not completely dissolved in the test solutions. The powder was partly found on the bottom of the test vessels. The test solutions with 1000 mg/L test item were brighter than the other test solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 106 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.



From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Test Item Concentrations

 

Test Item
Concentration
[mg/L]

Repl.

Oxygen Uptake Rate R
[mg O2/L
×h]

Specific Respiration Rate Rs
[mg O2/g
×h]

Inhibition

[%]

Mean Inhibition
[%]

 

Control

1

26.0

16.8

2

25.6

16.5

3

26.8

17.3

4

25.6

16.5

5

24.0

15.5

6

23.6

15.2

Test Item

100

1

26.8

 

-6

-3

2

25.6

-1

3

25.6

-1

4

25.6

-1

5

27.2

-8

180

1

25.2

 

0

-4

2

26.0

-3

3

26.8

-6

4

25.6

-1

5

27.6

-9

320

1

26.4

 

-4

-6

2

27.6

-9

3

27.2

-8

4

26.4

-4

5

26.8

-6

560

1

24.8

 

2

4

2

24.8

2

3

24.4

4

4

25.6

-1

5

22.4

11

1000

1

17.2

 

32

35

2

16.4

35

3

16.8

34

4

17.2

32

5

14.8

42

Mean specific respiration rate of the control: 16.3 mg O2/g×h

Coefficient of variation of oxygen uptake rates of the control: 4.8%

Suspended solids in the test vessel: 1.55 g/L

Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Concentrations

 

Reference Item
Concentration
[mg/L]

Repl.

Oxygen Uptake

Rate R
[mg O2/L
×h]

 

Inhibition

 

[%]

Mean Inhibition

[%]

Reference Item

58

1

20.4

 

19

22

2

18.8

26

3

20.0

21

100

1

14.0

 

45

46

2

13.6

46

3

13.6

46

180

1

5.6

 

78

76

2

6.4

75

3

6.0

76

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of Celitement is 560 mg/L. The test item is not toxic up to the concentration of 560 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 690 mg/L, the EC20 is 825 mg/L. The EC50 and EC80 are > 1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Celitement (batch number:not specified) on2014-05-13 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations100 - 180 - 320 - 560 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -6 % to 35 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 106 mg/L.

NOEC and EC-Values with Confidence Interval of Celitement

 

EC-values

[mg/L]

Confidence interval
P = 95 [%]
[mg/L]

NOEC*

560

 -

EC10

690

624 – 750

EC20

825

773 – 867

EC50

> 1000

 -

EC80

> 1000

 -

*) No statistically significant inhibition (P < 0.05), ANOVA, Dunnett`s Method

 

 

 

The NOEC of Celitement is 560mg/L.

The test item is not toxic up to the concentration of 560 mg/L to activated sludge of a municipal sewage treatment plant. The EC10is 690 mg/L, the EC20is 825 mg/L.

The EC50and EC80are > 1000 mg/L.

Description of key information

Short-term toxicity testing to microorganisms was carried out due to GLP and thus resulting in a Klimish 1 score.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
640 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test itemCelitement (batch number:not specified) on2014-05-13 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations100 - 180 - 320 - 560 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -6 % to 35 %.