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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- the test item was directly pipetted in Erlenmeyer flasks, no adjustment of pH was made
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.5°C
pH:
7.28
Dissolved oxygen:
6.62 - 8.81 mg/L
Nominal and measured concentrations:
nominal 10, 32, 100, 320, and 1000 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks, 500 mL fill volume
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70% saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 3 at the beginning of the test, 3 at the end of the test
- No. of vessels per positive control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3.21 g/L, corresponding to 1.61 g/L in the test vessel

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic waste water

OTHER TEST CONDITIONS
- Adjustment of pH: not required (within prescribed range)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
oxygen uptake rate, 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)
- Range finding study
- Test concentrations:10, 100, 1000
- Results used to determine the conditions for the definitive study: -2.5% inhibition at 10 mg a.i./L, 41.5% inhibition at 100 mg a.i./L, 96% inhibition at 1000 mg a.i./L
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
28.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% c.i. 24.9 - 32.3 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
127 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% c.i. 120 - 135
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
- Adsorption (e.g. of test material to the walls of the test container):
- Blank controls oxygen uptake rate:
- Coefficient of variation of oxygen uptake rate in control replicates:
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: EC50 = 108 mg/L, 95% Confidence Interval = 103 - 113 mg/L
- valid range: 53 - 155 mg/L

The mean specific oxygen uptake rate of the control replicates was 18.9 mg O2/q-h (validity criterion: should be >/= 20 mg O2/g·h). The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc.

The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC50-value of the reference item was in the required range. Therefore, no impact on the results of the test item can be expected through lower respiration.

The coefficient of variation of the oxygen uptake rates in the control replicates was 3.6 % (validity criterion: < 30 %).

The EC50 of the reference item was 108 mg/L and in the range of 53 - 155 mg/L.

Oxygen Uptake Rates and Inhibition of the Respiration

 

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

Replicate

Oxygen Uptake Rate [mg O2/L·h]

Inhibition [%]

Mean inhibition [%]

Control

1

2

3

4

5

6

31.6

31.6

30.4

28.8

29.6

30.4

---

---

10

1

2

3

4

5

30.4

30.4

30.0

30.4

29.6

0

0

1

0

3

 

32

1

2

3

4

5

26.4

26.4

25.2

26.0

26.0

13

13

17

14

14

14

100

1

2

3

4

5

18.0

18.4

18.8

18.8

18.0

41

39

38

38

41

39

320

1

2

3

4

5

6.0

5.6

5.6

6.4

7.2

80

82

82

79

76

80

1000

1

2

3

4

5

2.8

2.4

2.8

2.4

2.4

91

92

91

92

92

92

 

Validity criteria fulfilled:
yes
Conclusions:
The 3 h NOEC of Di-C12-18 alkyldimethyl ammonium chloride is 10 mg a.i./L. The 3 h EC10 is 28.5 mg a.i./L, and the EC50 is 127 mg a.i./L.
Executive summary:

In a 3 hour toxicity study conducted according to OECD Guideline 209 (2010), the cultures of activated sludge of a predominantly domestic sewage treatment plant were exposed to Di-C12-18 alkyldimethyl ammonium chloride (68.79% a.i.) at nominal concentrations of 0 (control), 10, 32, 100, 320, and 1000 mg a.i./L mg/L under static conditions.

The batch of activated sludge was checked for sensitivity by testing the reference substance Copper (II) sulphate pentahydrate.

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 controI respiration rates. The mean inhibition of respiration for the test item replicates ranged from 1 % to 92 %

The 3 h NOEC of C12-18 alkyldimethyl ammonium chloride is 10 mg a.i./L. The 3 h EC10 is 28.5 mg a.i./L, and the EC50 is 127 mg a.i./L.

 

Results Synopsis

Test Organism: activated sludge from a sewage treatment plant treating predominantly domestic sewage

Test Type: Static 

3 h NOEC: 10 mg/L

3 h EC10: 28.5 mg/L, 95% c.i. 24.9 - 32.3 mg/L

3 h EC50: 127 mg/L, 95% c.i. 120 – 135 mg/L

Endpoint effected: Inhibition of respiration

Description of key information

3 h EC10 = 28.5 mg a.i./L, EC50 = 127 mg a.i./L (OECD TG 209; RL1; GLP)

Key value for chemical safety assessment

EC50 for microorganisms:
127 mg/L
EC10 or NOEC for microorganisms:
28.5 mg/L

Additional information

In a 3 hour toxicity study conducted according to OECD Guideline 209 (2010), the cultures of activated sludge of a predominantly domestic sewage treatment plant were exposed to Di-C12-18 alkyldimethyl ammonium chloride (68.79% a.i.) at nominal concentrations of 0 (control), 10, 32, 100, 320, and 1000 mg a.i./L mg/L under static conditions.

The batch of activated sludge was checked for sensitivity by testing the reference substance Copper (II) sulphate pentahydrate.

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 controI respiration rates. The mean inhibition of respiration for the test item replicates ranged from 1 % to 92 %

The 3 h NOEC of Di-C12-18 alkyldimethyl ammonium chloride is 10 mg a.i./L. The 3 h EC10 is 28.5 mg a.i./L, and the EC50 is 127 mg a.i./L.