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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-01-2015 to 17-06-2015
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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
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.
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
See above
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: June 2013; signature: November 2013
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was dispersed directly in water.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: Three stock solutions of 58, 100 and 180 mg/L were prepared by dissolving the reference material directly in water with the aid of ultrasonic pretreatment. A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported.
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 2.83 g/L, corresponding to 1.42 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 1.42 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: 20.6 °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 preliminary study was pH = 7.57 - 7.63 (in medium) at final.
Dissolved oxygen:
Dissolved oxygen concentrations: 100 mg/L nominal test item: 4.42 - 4.50 mg O2 /L before incubation and aeration and 7.41 - 7.43 mg O2/L after 3 hours incubation and aeration in the preliminary study.
In the definitive test. 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: 9.35, 93.5 and 935 mg/L nominal (test item)
plus two replicates 1000 mg/L nominal with ATU (11.6 mg/L) to measure nitrification inhibition
plus two replicates 100 mg/L to measure loss of test item during incubation/aeration
Definitive test:
The nominal concentrations tested were: 0 (control), 10, 32, 100. 320 and 1000 mg/L (in five replicates per concentration, six for control)
The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (three 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): Six (negative) and three (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.:
32 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.:
25.2 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.: 20.0 - 31.3
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
55.8 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.: 45.0- 70.0
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 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.: -
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 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.: -
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: EC50 103 (95% C.I. 100 -106) mg/L
- Other: Yes the reference substance results were valid, the EC50 for copper (II) sulphate was between 100 and 106 mg/L (total respiration) and was within the expected range: 53 to 155 mg/L.

Table 3.0 : 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

34.8

-

-

2

33.2

 

3

30.2

-

4

24.8

-

5

22.0

-

6

24.0

-

10.0

1

30.8

-9

-4

2

28.4

-1

3

29.2

-4

4

29.2

-4

5

28.8

-2

32.0

1

23.6

16

13

2

25.2

11

3

25.2

11

4

25.6

9

5

23.2

18

100.0

1

21.6

23

26

2

21.2

25

3

20.4

28

4

20.0

29

5

20.8

26

320.0

1

19.2

32

31

2

18.4

35

3

19.2

32

4

20.0

29

5

20.0

29

1000

1

20.0

29

36

2

18.4

35

3

17.2

39

4

17.6

38

5

17.6

38

Reference item concentration [mg/L]

 

 

 

 

58

1

20.0

29

27

2

20.8

26

3

20.8

26

100

1

14.8

48

48

2

15.2

46

3

14.4

49

180

1

7.6

73

73

2

7.6

73

3

7.2

74

 

 

 

 

 

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 18.8%

Suspended solids in the test vessel: 1.42 g/L.

 

Table 4.0 : Specific respiration rates of the control replicates

 

Replicate

 Specific Respiration rate Rs [mg O2/gh)

Control

1

24.5

2

23.4

3

21.4

4

17.5

5

15.5

6

16.9

Mean

 

19.9

Repl. = Replicate

Suspended solids in the test vessel: 1.42 g/L.

 

The mean specific respiration rate (Rs) of the control cultures was slightly less than 20 mg O2/gh (actual 19.9 mg O2/gh). The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal variations. The lower uptake reflects the range of common uptake rates of activated sludge and was considered to have no impact on the validity of the study. The reference item had an 3 -hour EC50 in the required range despite the lower blank respiration. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 18.8 % therefore meeting guideline the validity criteria (30%). The reference substance results were valid, the EC50 for copper (II) sulphate was (total respiration): actual 103 mg/L and was within the expected range: 53 to 155 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3-hour EC50 for total inhibition was > 1000 mg/L. The EC20 was 55.8 (C.I. 45.0 – 70.0) mg/L, the EC10 was 25.2 (C.I. 20.0 – 31.3) mg/L. The NOEC was 32 mg/L with no significant inhibition of respiration rates below this concentration determined under the conditions of the study. All effect levels were based on nominal test item concentrations.
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. In a preliminary range finding test Samples of activated sludge suspended solids 1.42 g/L, were exposed to dilutions of the test item for three hours under static conditions with the concentrations: 10, 100 and 1000 mg/L (nominal). Further checks for nitrification inhibition were also performed. No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed. The rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone in a control. In the definitive test: nominal test item concentrations used in the study were 0 (control), 10, 32, 100. 320 and 1000 mg/L with 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 19.9 mg O2/gh. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal variations. The lower uptake reflects the range of common uptake rates of activated sludge and was considered to have no impact on the validity of the study. The reference item had an 3 -hour EC50 in the required range despite the lower blank respiration. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 18.8 % therefore meeting guideline the validity criteria (30%). The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 103 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 -4 % to 36 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the NOEC concentration of 32mg/L. The EC10 was 25.2 (C.I. 20.0 – 31.3) mg/L and the EC20 was 55.8 (C.I. 45.0 – 70.0) mg/L. The EC50 was > 1000 mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

EC50 = > 1000 (C.I - ) mg/L (nominal), 20 °C, OECD TG 209, 2015

EC10 = 25.2 (C.I. 20.0 - 31.3) mg/L (nominal), 20°C, OECD TG 209, 2015

NOEC = 32 mg/L (nominal), 20 °C, OECD TG 209, 2015

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
25.2 mg/L

Additional information

Key Study : OECD TG 209, 2015 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. In a preliminary range finding test Samples of activated sludge suspended solids 1.42 g/L, were exposed to dilutions of the test item for three hours under static conditions with the concentrations: 10, 100 and 1000 mg/L (nominal). Further checks for nitrification inhibition were also performed. No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed. The rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone in a control. In the definitive test: nominal test item concentrations used in the study were 0 (control), 10, 32, 100. 320 and 1000 mg/L with 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 19.9 mg O2/gh. The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal variations. The lower uptake reflects the range of common uptake rates of activated sludge and was considered to have no impact on the validity of the study. The reference item had an 3 -hour EC50 in the required range despite the lower blank respiration. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 18.8 % therefore meeting guideline the validity criteria (30%). The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 103 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 -4 % to 36 %. The test item was not toxic to activated sludge of a municipal sewage treatment plant at the NOEC concentration of 32mg/L. The EC10 was 25.2 (C.I. 20.0 – 31.3) mg/L and the EC20 was 55.8 (C.I. 45.0 – 70.0) mg/L. The EC50 was > 1000 mg/L. All effect levels were based on nominal test item concentrations.

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