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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-16 to 2012-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance
Qualifier:
according to guideline
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: No vehicle, directly weighing, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 6.50 - 13.0 - 26.0 - 52.0 - 104.0 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %
- Dry sludge concentration: 3.12 g/L (corresponding to 1.56 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
21 - 22 °C
pH:
7.99 (activated sludge)
7.51 (synthetic waste water)
Salinity:
None, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS

- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item Concentration Pretreatment Inhibition [%]
[mg/L]
10 Stirring overnight 6
100 93
1000 97








Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
35.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (31.2-40.0)
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 93.3 (89.3 - 97.3) 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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.


The EC-values of the test item was calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC-values were carried out using standard procedures.

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

Reference 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

30.0

19.2

–

–

2

31.6

20.3

3

30.4

19.5

4

30.4

19.5

5

30.4

19.5

6

29.2

18.7

58

1

20.4

33

30

2

20.8

31

3

22.0

27

100

1

15.2

50

52

2

14.8

51

3

14.0

54

180

1

6.4

79

79

2

6.4

79

3

6.8

78

Mean specific respiration rate of the control: 19.4 mg O2/g×h

Coefficient of variation of oxygen uptake rates of the control: 2.6 %

Suspended solids in the test vessel: 1.56 g/L

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


Test item Concentration
[mg a.i./L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L×h]


Inhibition

[%]

Mean Inhibition

[%]

6.25

1

25.2

17

9

2

28.4

6

3

27.2

10

4

28.0

8

5

29.2

4

12.5

1

26.4

13

17

2

28.0

8

3

23.2

23

4

25.6

16

5

22.4

26

25

1

21.6

29

27

2

32.4

-7

3

17.6

42

4

19.2

37

5

19.6

35

50

1

6.8

78

75

2

7.2

76

3

8.4

72

4

8.0

74

5

7.6

75

100

1

2.8

91

94

2

1.6

95

3

2.0

93

4

1.6

95

5

1.6

95
Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item 3-(Isodecyloxy)propylamine, acetate is 6.25 mg a.i./L. The test item is not toxic at the concentration of 6.25 mg a.i./L to activated sludge of a municipal sewage treatment plant. The EC50 of the test item is 35.3 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 3-(Isodecyloxy)propylamine, acetate (batch number: 110727JC) from 2012-04-16 to 2012-04-17, with the experimental phase on 2012-04-17 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the substance concentrations 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L (a.i.), corresponding to the nominal test item concentrations 6.50 – 13.0 – 26.0 – 52.0 – 104 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 9 % to 94 %.

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 93.3 mg/L.

EC-Values with Confidence Interval of 3-(Isodecyloxy)propylamine, acetate

EC-values

Confidence interval

[mg a.i./L]

P = 95 [%]

[mg a.i./L]

NOEC*

6.25

–

EC20

20.0

14.3 - 23.9

EC50

35.3

31.2 - 40.0

EC80

55.3

48.2 - 65.0

                                               *) no statistically significant effect (p 0.001) , ANOVA, Dunnett`s Method

                                              

The NOEC of the test item 3-(Isodecyloxy)propylamine, acetate is 6.25 mg a.i./L.

The test item is not toxic at the concentration of 6.25 mg a.i./L to activated sludge
of a municipal sewage treatment plant. The EC50 of the test item is 35.3 mg a.i./L.

Description of key information

3-(Isodecyloxy)propylamine, acetate (CAS no 28701 -67 -9) is not toxic at the concentration of 6.25 mg a.i./L to activated sludge of a municipal sewage treatment plant.

The EC50 is 35.3 mg a.i./L.

3-(Isodecyloxy)propylamine (CAS no 30113 -45 -2) is not toxic (EC10) at the concentration of 13 mg a.i./L to activated sludge of a municipal sewage treatment plant.

The EC50 is 23.6 mg a.i./L.

For riks assessment the lowest NOEC value of 6.25 mg a.i./L will be used as a worst-case.

Key value for chemical safety assessment

EC50 for microorganisms:
35.3 mg/L
EC10 or NOEC for microorganisms:
6.25 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with 3-(Isodecyloxy)propylamine, acetate and also with 3-(Isodecyloxy)propylamine

The lowest NOEC/EC10 is found for 3-(Isodecyloxy)propylamine, acetate of 6.25 mg a.i./L.

The substance is not toxic at the concentration of 6.25 mg a.i./L to activated sludge of a municipal sewage treatment plant. The EC50 of the test item is 35.3 mg a.i./L.