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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:
2012-11-20 to 2012-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies
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: Directly weighing of the stock solution (5 g/L), 30 min ultrasonic treatment at room temperature
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 10 - 32 - 100 - 320 - 1000 mg/L corresponding to 6.10 - 19.5 - 61.0 - 195 - 610 mg a.i./L
- Controls: 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: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 3.21 g/L corresponding to 1.60 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:
19.5 °C
pH:
pH-value of the activated sludge 7.58
pH-value of the synthetic waste water 7.50
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain
the sludge flakes suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 3
- Composition of test medium:

Test item concentration [mg/L]
10 32 100 320 1000
Inoculum 250
Synthetic waste water 16
Stock solution [g/L] 5
Volume stock solution [mL] 1 3.2 10 32 100
Demineralised water [mL] 233 230.8 224 202 134




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
- Spacing factor for test concentrations:
- Range finding study

Test item concentration pH (test item in water) Inhibition [%]

[mg/L] [mg a.i./L]
10 6.10 - 1
100 61.0 * *
1000 610 * *
100 61.0 6.81 17
1000 610 7.45 38




Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
is UVCB means 100% act.ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
is UVCB means 100% act.ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CI 53-95 mg/L
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: In higher test item concentrations foam was formed on the surface.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 94.4 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 were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-values was 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

Repl.

Oxygen Uptake Rate
R

Specific Respiration Rate
Rs

Inhibition

Mean Inhibition

[mg/L]

[mg a.i./L]

[mg O2/L×h]

[mg O2/g×h]

[%]

[%]

Control

1

23.6

14.8

2

26.0

16.3

3

30.4

19.0

4

30.0

18.8

5

29.2

18.3

6

28.4

17.8

10

6.10

1

26.4

5

3

2

26.8

4

3

27.6

1

4

24.0

14

5

25.6

8

32

19.5 

1

28.0

0

3

2

27.6

1

3

26.0

7

4

26.4

5

5

26.8

4

100

61.0 

1

22.8

18

15

2

23.6

15

3

24.4

13

4

24.0

14

5

24.4

13

320

195   

1

17.6

37

36

2

18.8

33

3

19.2

31

4

16.8

40

5

16.4

41

1000

610   

1

14.8

47

47

2

14.8

47

3

15.2

46

4

15.2

46

5

14.0

50

Repl. = Replicate

Mean specific respiration rate of the control: 17.5 mg O2/g×h

Coefficient of variation of oxygen uptake rates of the control: 9.4 %

Suspended solids in the test vessel: 1.60 g/L

Validity criteria fulfilled:
yes
Conclusions:
The EC10 of the test item Sodium methyl oleyl taurate is 72.2 mg/L, corresponding to 44.0 mg a.i./L. The test item is not toxic up to the concentration of 72.2 mg/L, corresponding to 44.0 mg a.i./L, to activated sludge of a municipal sewage treatment plant.
The EC50 is > 1000 mg/L, corresponding to > 610 mg a.i./L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Sodium methyl oleyl taurate (batch number: DEB4003485) from 2012-11-20 to 2012-11-21, with the definitive exposure on 2012-11-21 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 substance concentrations 10 - 32 - 100 - 320 - 1000 mg/L, corresponding to 6.10 - 19.5 - 61.0 - 195 - 610 mg a.i./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 3 % to 47 %.

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 94.4 mg/L.

IMPORTANT NOTE TO AVOID CONFUSION:

As the registration substance is a UVCB this means that the test item is 100% active ingredient.

NOEC and EC-Values with Confidence Intervals of Sodium methyl oleyl taurate

EC-values

[mg/L]

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

NOEC*

32  

EC10

72.2

53.2 - 95.4

EC20

142  

118 - 168

EC50

> 1000  

  

EC80

> 1000  

The EC10of the test item Sodium methyloleyl taurate is 72.2 mg/L.

The test item is not toxic up to the concentration of 72.2 mg/L to activated sludge of a municipal sewage treatment plant.
The EC50 of the test item is
> 1000 mg/L.

Description of key information

The EC10 of the test item Sodium methyl oleyl taurate is 72.2 mg/L, corresponding to 44.0 mg a.i./L. The test item is not toxic up to the concentration of 72.2 mg/L, corresponding to 44.0 mg a.i./L, to activated sludge of a municipal sewage treatment plant.

The EC50 is > 1000 mg/L, corresponding to > 610 mg a.i./L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information