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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 november 2012 - 6 february 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preparation of test item solutions: a stock solution with a nominal concentration of 14 mg test item/L was prepared by dissolving 14045 mg of the test item in 1000 mL water and thoroughly mixed by intense stirring for 10 minutes. In this way a clear solution was obtained. The pH of the stock solution was 7.7. Adequate volumes of this stock solution were diluted with water to prepare the test media with the lower test item concentrations. The exact amounts used are given in the table 1. Then, 16 mL of synthetic sewage feed and 250 mL of activated sludge inoculum were added.
- Preparation of reference substance (3,5-dichlorophenol) solutions: a stock solution of 3,5-dichlorophenol was prepared by dissolving 0.5g of 3,5-dichlorophenol directly in about 950 mL water with the aid of ultrasonification and intense stirring. The 3,5-dichlorophenol solution was adjusted with NaOH to pH 7.7 and make up to one liter. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with water, synthetic sewage feed and inoculum in the respective vesssels to obtain the desired test concentrations (3.2, 10 and 32 mg/L).
- Preparation of test system: at the start of the test, 16 ml of synthetic sewage were made up to 250 mL with water and 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to the first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage were added to the appropriate amounts of reference item or test item in water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum. The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation period.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of the inoculum: aerobic sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.
- Pretreatment: no.
- Method of cultivation: no data.
- Preparation of inoculum for exposure: the sludge was washed twice with the chlorine free tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and reajusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge was 7.1.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
The temperature in the test media, measured in one control, was 20°C at the start and at the end of the incubation period.
pH:
7.2 - 8.2 (test substance)
7.3 - 8.0 (reference substance)
Dissolved oxygen:
8.0 mgO2/L - 8.3 mgO2/l (test substance)
8.1 mgO2/L - 8.2 mgO2/l (reference substance)
Nominal and measured concentrations:
Nominal concentrations: 72, 180, 449, 1124 and 2809 mg test item/L (corresponding to 33.1, 82.8, 206.5, 517.0 and 1292.1 mg solid content/L).
Reference substance: 3.2, 10 and 32 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel:
-- Type: open;
-- Material, size, headspace, fill volume: the test was performed in 2000 mL glass beakers containing 500 mL of incubation mixture;
-- Aeration: during the incubation period of 3 hours, all vessels were aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item;
-- No. of vessels per concentration (replicates): three flasks per concentration (test item) - one flask per concentration (reference item) - six blank controls (containing an equal volume of activated sludge and synthetic medium but no test or reference item);
-- The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), two further blank controls, the test item group (with ascending concentrations) and then two blank control vessels.

TEST MEDIUM / WATER PARAMETERS : activated sludge with the addition of a synthetic sewage.
- Source / preparation of dilution water: distilled or deionized water, containing less than 1 mg/L dissolved organic carbon was used except where chlorine free tap water is specified.
- Synthetic sewage feed composition:
Peptone : 16 g
Meat extract : 11 g
Urea : 3 g
NaCl : 0.7 g
CaCl2.2H2O : 0.4 g
MgSO4.7H2O : 0.2 g
K2HPO4 : 2.8 g
dissolved in 1 litre of deionized water. The pH of this solution was 7.2.

EFFECT PARAMETERS MEASURED
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the oxygen consumption rate (in mg O2 L-1 minute-1) was measured with an oxygene electrode and was continuously recording. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate was determined from the linear part of the respiration curve normally in the range 7-2.5 mg O2/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Acros Organics BVBA; Batch : A0257431) - purity : 99% (GC).Expiration date : 13 march 2013
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 571 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 2336-2881 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 182.7 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
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: 95% CL = 636-986 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
379.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
449 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:
NOEC
Effect conc.:
206.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solid content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The mean inhibition of the respiration rates for all tested concentrations was in the range -13.6 to 53.6% compared to the control, i.e. a stimulatory effect was observed for some concentrations tested. Up to and including the concentration of 449 mg test item/L, corresponding to 206.5 mg solid content/L, the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. At the next higher test concentrations of 1124 mg test item/L, corresponding to 517 mg solid content/L, the inhibitory effect reached 18.7%. At the highest tested concentration of 2809 mg test item/L, corresponding to 1292.1 mg solid content/L, the inhibition of the respiration increased to 53.6%. Concentrations exceeding 2809 mg test item/L were not tested.

The following validity criteria were fulfilled:
- The specific respiration rate of the blank controls was in the range 35 to 41 mg oxygen per gram dry weight of sludge per hour and therefore, higher than 20 mg O2/g/h;
- The coefficient of variation of oxygen uptake rate in control replicates was 6% and therefore, less than 30%;
- The EC50 (3 -hour) of the reference item 3,5 -dichlorophenol was 3 mg/L and therefore in the range 2 to 25 mg/L.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 3 mg/L. The 3-hour EC50 is within the Guideline-recommended range of 2-25 mg/l, confirming suitability of the activated sludge used.

Table 2 - Influence of Lauramidopropylhydroxysultaine (test item) and 3 -5 DCP (reference item) on the Oxygen Consumption of Activated Sludge – Range finding test

Vessel

No.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate

Inhibition

 

 

%

Ph values

Oxygen concentration (mgO2/L)

Respiration rate

(R)

(mg O2/Lh)

Specific respiration rate (Rs) (mg O2/gh)

start*

end*

start*

end*

                    Control

1

2

11

12

0

0

0

0

47.04

44.72

60.72

51.93

31.36

29.81

40.48

34.62

 

7.9

7.8

7.5

7.5

8.1

8.1

8.0

8.0

8.0

8.0

8.2

8.3

8.4

8.4

8.4

8.5

Mean

Standard Deviation

Coefficient of Variation

51.10

7.08

13.86%

34.07

4.72

13.86%

 

 

                    Test item                                                                              

6

28.1

49.28

32.85

3.6

7.6

8.1

8.3

8.4

7

281

53.64

35.76

-5.0

7.6

8.0

8.4

8.2

8

2809

24.63

16.42

51.8

7.6

8.2

8.6

8.5

9

2809

22.61

15.07

55.8

7.6

8.2

8.6

8.5

10

2809

23.34

15.56

54.3

7.6

8.2

8.7

8.5

                Reference item

3

4

5

3.2

10

32

23.54

19.42

5.94

15.69

12.95

3.96

53.9

62.0

88.4

7.6

7.6

7.6

8.3

8.3

8.3

8.0

8.1

8.3

8.5

8.4

8.7

*: Start and end of the 3 -hour incubation period

% inhibition: Increased oxygen consumption rate relative to control

 

Table 3 - Influence of Lauramidopropylhydroxysultaine (test item) and 3 -5 DCP (reference item) on the Oxygen Consumption of Activated Sludge – Definitive test

Vessel

No.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate

Inhibition

 

 

%

Ph values

Oxygen concentration (mgO2/L)

Respiration rate

(R)

(mg O2/Lh)

Specific respiration rate (Rs) (mg O2/gh)

start*

end*

start*

end*

                    Control

1

2

6

7

23

24

0

0

0

0

0

0

61.80

58.80

54.76

52.56

56.18

56.20

41.20

39.20

36.51

35.04

37.45

37.47

 

7.3

7.2

7.2

7.2

7.5

7.4

7.9

7.8

7.7

7.8

8.0

8.0

8.0

8.1

8.2

8.1

8.0

8.1

8.5

8.3

7.5

8.2

8.2

8.2

Mean

Standard Deviation

Coefficient of Variation

56.72

3.22

5.68%

37.81

32.15

5.68%

 

 

                    Test item                                                                               Mean

8

72

60.27

59.47

-6.3

 

-3.9

7.2

7.2

7.2

7.8

8.0

8.0

8.1

8.1

8.2

8.4

8.5

8.5

9

72

83.70

58.00

-2.3

10

72

86.40

58.53

-3.2

11

180

87.80

39.47

-4.4

 

-2.7

7.2

7.4

7.4

8.0

7.9

7.9

8.0

8.0

8.2

8.5

8.3

8.4

12

180

100.80

36.95

2.3

13

180

93.36

40.10

-6.1

14

449

130.50

40.80

-7.9

 

-13.6

7.3

7.3

7.4

7.9

8.0

7.9

8.4

8.3

8.4

8.5

8.5

8.5

15

449

137.10

41.78

-10.5

16

449

112.08

46.29

-22.4

17

1124

93.09

29.40

22.2

 

18.7

7.4

7.4

7.4

8.0

8.0

8.0

8.6

8.6

8.6

8.5

8.5

8.5

18

1124

95.83

37.60

0.6

19

1124

83.52

25.26

33.2

20

2809

95.49

16.68

55.9

 

53.6

7.3

7.5

7.5

8.0

8.0

8.2

8.7

8.7

8.6

8.4

8.3

8.3

21

2809

67.60

18.00

52.4

22

2809

76.35

17.94

52.6

                Reference item

3

4

5

3.2

10

32

70.40

58.91

19.02

46.93

39.27

12.68

12.8

27.1

76.5

 

7.2

7.2

7.2

7.9

7.8

7.7

8.2

8.3

8.4

8.5

8.3

8.6

*: Start and end of the 3 -hour incubation period

% inhibition: Increased oxygen consumption rate relative to control

Validity criteria fulfilled:
yes
Conclusions:
The test item had no inhibitory effect on the respiration rate of activated sludge bacteria at a concentration of 449 mg test item/L (corresponding to 206.5 mg solid content/L). A 50%-inhibition of the respiration rate was observed at 2571 mg test item/L (corresponding to 1182.7 mg solid content/L).
Executive summary:

The aquatic toxicity of the test item (= (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide / EC 242-893-1) was investigated in a GLP- compliant study performed with activated sludge microorganisms in accordance with OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test). The 3-hour NOEC and ECx-values were as follows:

- NOEC = 449 mg test item/L (206.5 mg solid content/L);

- EC10 = 825 mg test item/L (379.5 mg solid content/L);

- EC50 = 2571 mg test item/L (1182.7 mg solid content/L).

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to 449 mg test item/L (corresponding to 206.5 mg solid content/L).

Description of key information

The aquatic toxicity of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a GLP-compliant study (Eisner, 2013) performed with activated sludge microorganisms in accordance with OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test). The 3-hour NOEC and ECx-values are as follows:

- NOEC = 449 mg test item/L (206.5 mg solid content/L);

- EC10 = 825 mg test item/L (379.5 mg solid content/L);

- EC50 = 2571 mg test item/L (1182.7 mg solid content/L).

After the incubation period of three hours, the test item had no inhibitory effect on the respiration rate of activated sludge up to 449 mg test item/L (corresponding to 206.5 mg solid content/L).

Key value for chemical safety assessment

EC50 for microorganisms:
1 182.7 mg/L
EC10 or NOEC for microorganisms:
379.5 mg/L

Additional information

The toxicity to activated sludge microrganisms of the substance EC 242-893-1 was investigated in a GLP-compliant study (Eisner, 2013) performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and was selected as key study for the endpoint.