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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
The effect of the test substance on the inhibition of respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. The test organisms were exposed to a series of test concentrations of the test substance for 3 hours and the effects on oxygen up take were measured. Results are reported relative to the reference standard controls: copper (II) sulphate pentahydrate.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2013; signature: February 2016
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: aerobic activated sludge from 31137 Hildesheim, Germany municipal sewage treatment plant
- Method of cultivation:
- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %.
- Pretreatment: The sludge was used within 24 h after sampling. The dry sludge concentration was 3.27 g/L, corresponding to 1.64 g/L in the test vessel. The test item was not pretreated. The reference item was ultrasonicated.
- Other: Synthetic waste water was prepared according to OECD 209. The pH value of the activated sludge was determined prior to test
start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
- Initial biomass concentration: suspended solids 1.64 g per liter (aerobic sludge per litre water).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
nominal 18 - 22 or 20 ± 2°C; actual: 20 °C
pH:
The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
pH value measured during the study was pH = 7.57 at final (test vessel 1 at 1000 mg/L nominal test item)
Dissolved oxygen:
Dissolved oxygen concentrations: 100 mg/L nominal test item: 41.88 - 48.03 mg O2 /L before incubation and aeration and 73.66 - 74.07 mg O2/L after 3 hours incubation and aeration.
Aeration was employed by shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
Nominal and measured concentrations:
Preliminary test: 10, 100 and 1000 mg/L nominal (test item)
The nominal concentrations tested were: 0 (control), 0.75, 5.6, 32, 180 and 1000 mg/L (in five replicates per concentration)
The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (single replicates per concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 ml Erlenmeyer flasks
- Type (delete if not applicable): Open, vessel continuously aerated
- Material, size, headspace, fill volume: See table 1.
- Aeration: Continuously aerated with compressed air at 1L per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Five (negative) and one (reference item)
- Biomass loading rate: See table 1.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage water was in accordance with OECD TG 209
- Culture medium different from test medium: Yes, chlorine free tap water used for culture medium; synthetic wastewater and RO used for test medium. Replaced during inoculum pretreatment stage and media preparation.
- Intervals of water quality measurement: pH and temperature were determined in all test media and controls; prior to and at the end of the 3 hour incubation period. and dissolved oxygen values was determined in the 100 mg/L test item vessels only and or within preliminary test.

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase. The oximeter instrument measured the oxygen depletion for at least 3 minutes.

TEST CONCENTRATIONS
- Test concentrations: See table 1.
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
0.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No statistically significant inhibition, ANOVA, Dunnett's Method
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
11.6 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% C.I.: 8.65- 14.8
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
21.4 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% C.I.: 18.4- 24.4
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
52.5 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% C.I.: 47.2-59.7
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
142 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% C.I.: 112-175
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: EC50 96 (95% C.I. 88 -104) mg/L
- Other: Yes the reference substance results were valid, the EC50 for copper (II) sulphate was between 88 and 104 mg/L (total respiration) and was within the expected range: 53 to 155 mg/L.

Table 3. Oxygen uptake rates and Inhibition of the respiration of the control and test item and reference items

Test concentration [mg/L]

Replicate

Oxygen uptake Rate R [mg O2 / L h-1]

Inhibition [%]

Mean inhibition [%]

Control

1

33.6

-

-

2

33.6

 

3

34.0

-

4

34.4

-

5

34.4

-

6

34.4

-

0.75

1

34.4

-1

0

2

34.4

3

3

33.2

-2

4

34.0

0

5

33.6

1

5.6

1

30.0

12

9

2

32.0

6

3

32.0

6

4

32.0

6

5

28.4

17

32

1

23.6

31

31

2

24.0

30

3

24.4

28

4

23.2

32

5

22.8

33

180

1

5.2

85

85

2

5.6

84

3

5.2

85

4

5.2

85

5

5.2

85

1000

1

3.2

91

91

2

2.4

93

3

3.6

89

4

3.2

91

5

3.2

91

Reference item concentration [mg/L]

 

 

 

 

58

1

24.4

28

29

2

23.6

31

3

24.8

27

100

1

16.8

51

49

2

17.6

48

3

17.6

48

180

1

4.0

88

80

2

8.4

75

3

7.6

78

 

 

 

 

 

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 1.2%

Suspended solids in the test vessel: 1.64 g/L.

 

Table 4.0 Specific respiration rates of the control replicates

 

Replicate

 Specific Respiration rate Rs [mg O2/gh)

Control

1

20.5

2

20.5

3

20.7

4

21.0

5

21.0

6

21.0

Mean

 

20.8

Repl. = Replicate

Suspended solids in the test vessel: 1.64 g/L.

 

The mean specific respiration rate (Rs) of the control cultures was > 20 mg O2/gh (actual 20.8 mg O2/gh) and the oxygen consumption rates of the two negative (blank; inoculum) controls differed by 1.2 % therefore meeting guideline the validity criteria (30%). The reference substance results were valid, the EC50 for copper (II) sulphate was (total respiration): actual 96 mg/L and was within the expected range: 53 to 155 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
A 3 hour EC50 of 52.5 mg/L (nominal) was reported. The NOEC was 0.75 mg/L (nominal) with no significant inhibition of respiration rates below this concentration determined under the conditions of the study.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Samples of activated sludge (suspended solids 1.64 g/L, were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 0.75, 5.6, 32, 180 and 1000 mg/L. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The nominal test substance concentrations used in the study were 0.75, 5.6, 32, 180, and 1000 mg/L; five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 20.8 mg O2/gh. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 96 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from 0 % to 91 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 0.75 mg/L. The EC10 was 11.6 mg/L and the EC20 was 21.4 mg/L. The EC50 was 52.5 mg/L.

Description of key information

EC50 = 52.5 (C.I 47.2 - 59.7) mg/L (nominal), 20 °C, OECD TG 209, 2016

EC10 = 11.6 (C.I. 8.65-14.8) mg/L (nominal), 20°C, OECD TG 209, 2016

NOEC = 0.75 mg/L (nominal), 20 °C, OECD TG 209, 2016

Key value for chemical safety assessment

EC50 for microorganisms:
52.5 mg/L
EC10 or NOEC for microorganisms:
0.75 mg/L

Additional information

Key study: OECD TG 209, 2016 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Samples of activated sludge (suspended solids 1.64 g/L, were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 0.75, 5.6, 32, 180 and 1000 mg/L. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. The nominal test substance concentrations used in the study were 0.75, 5.6, 32, 180, and 1000 mg/L; five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 20.8 mg O2/gh. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 96 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of respiration for the test item replicates ranged from 0 % to 91 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the concentration of 0.75 mg/L. The EC10 was 11.6 mg/L and the EC20 was 21.4 mg/L. The EC50 was 52.5 mg/L.