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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
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
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: June 2012; signature: September 2012
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: WWTP culture: aerobic activated sludge from ARA Ergolz II, Fullinsdorf, Switzerland waste water treatment plant
- Method of cultivation:
- Preparation of inoculum for exposure: Aerobic activated sludge, washed twice and supernatant liquid phase decanted. Homogenised final sludge solution weighed for wet and dry weight determination.
- Pretreatment: For holding period of three days prior to use, sludge fed daily with 50mL synthetic waste water per litre, at room temperature and under aeration. Dry weight was determined again prior to use with pH adjustment from 8.1 to 7.4 was completed with dilute sulfuric acid.
- Other: Synthetic waste water (50mL per day) had following composition; 16g peptone, 11g meat extract, 3g urea, 0.7g NaCl, 0.4g CaCl2.2H2O, 0.2g MgSO4.7H2O, 2.8g K2HPO4 within 1L deionised water.
- Initial biomass concentration: suspended solids 1.6 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:
20 ± 2°C
pH:
pH value was 7.4 ± 0.1 prior to study initiation and 7.5 ± 0.5 at final (minimum 7.71 and maximum 7.90 in test bottles: all controls were 7.5± 0.5)
Dissolved oxygen:
Dissolved oxygen concentrations was 4.58 - 6.28 mg/L at test initation and 2.08 - 4.32 at test finish - determined before and after determination of respiration rates in the test system. Tes mixtures were aerated and stirred for three hours in a thermostatically controlled water bath, using an aerator connected to a supply of oil-free compressed air (one litre/minute)
Nominal and measured concentrations:
The nominal concentrations tested were 10, 100 and 1000 mg/L (in five replicates per concentration)
The nominal concentrations of reference inhibitor 3,5-dichlorophenol (3,5-DCP) were 3, 10 and 32 mg/L (single replicates per concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500mL glass bottles fitted with oxygen probe and sinter (for gas nozzle delivery)
- Type (delete if not applicable): Open, vessel continuously aerated with compressed air.
- 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 had following composition; 16 g/L peptone, 11 g/L meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.4 g/L CaCl2.2H2O, 0.2 g/L MgSO4.7H2O, 2.8 g/L K2HPO4 within 1litre deionised water.
- Culture medium different from test medium: Yes, 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 and dissolved oxygen values were determined in all test media and controls; prior to and at the end of the 3 hour incubation period.

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. Measurements were made over a 15-minute period. The respirometer instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes.

TEST CONCENTRATIONS
- Test concentrations: See table 1.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
Duration:
3 h
Dose descriptor:
NOEC
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: NOEC was determined based on the absence of significant inhibition of respiration rates below this concentration.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 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:
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
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
Results with reference substance (positive control):
- Results with reference substance valid?:
- Relevant effect levels: 12.5 (95% C.I. 5.01 – 31.2) mg/L
- Other: Yes the reference substance results were valid, the EC50 for 3,5-dichlorophenol was between 2 and 25 mg/L (total respiration): actual 12.5 mg/L and was within the expected range: 2 to 25 mg/L.

Table 2.0 - Dissolved oxygen concentrations, measurement times and specific respiration rates

Group

Nominal concentration / mL

Bottle

Initial DO / mg O2/L

Final DO / mg O2/L

Measurement time / minutes

Specific respiration rate / Rs mg O2/gh

Mean Rs / mg O2/gh

% inhibition

Mean inhibition

Control

-

1

6.22

2.70

6

22.0

 

 

 

 

 

2

5.87

2.28

5

26.9

 

 

 

 

 

3

5.37

2.60

4

26.0

 

 

 

 

 

4

5.67

2.69

4

27.9

 

 

 

 

 

5

5.34

3.43

4

17.9

24.1

-

-

 

 

 

 

 

 

 

 

 

 

Test item

10

6

4.58

2.38

4

20.6

 

15

 

 

 

7

5.80

3.08

6

17.0

 

30

 

 

 

8

5.46

2.33

4

29.3

 

-22

 

 

 

9

5.89

2.34

5

26.6

 

-10

 

 

 

10

5.94

2.50

5

25.8

23.9

-7

1

 

 

 

 

 

 

 

 

 

 

 

100

11

5.58

3.47

4

19.8

 

18

 

 

 

12

6.00

2.73

5

24.5

 

-2

 

 

 

13

5.26

2.82

3

30.5

 

-26

 

 

 

14

4.87

2.11

3

34.5

 

-43

 

 

 

15

6.14

2.42

6

23.3

26.5

4

-10

 

 

 

 

 

 

 

 

 

 

 

1000

16

5.16

2.22

4

27.6

 

-14

 

 

 

17

6.22

2.60

6

22.6

 

6

 

 

 

18

6.45

4.32

5

16.0

 

34

 

 

 

19

6.31

2.91

7

18.2

 

25

 

 

 

20

6.28

2.69

9

15.0

19.9

38

18

 

 

 

 

 

 

 

 

 

 

Reference Item

3

21

6.15

2.91

6

20.3

 

16

 

 

10

22

6.11

2.45

9

15.3

 

37

 

 

32

23

7.02

6.57

5

3.4

-

86

-

 

 

 

 

 

 

 

 

 

 

DO = Dissolved Oxygen

The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may be observed in the respiration rate calculation if rounded values are used to calculate the data. This minor discrepancy is not considered to be significant

The Coefficient of variation for the control group was 17.2%

Negative inhibition indicates a respiration rate greater than that of the mean control respiration rate

 

The mean specific respiration rate (Rs) of the control cultures was > 20 mg O2/gh (actual 24.1 mg O2/gh) and the oxygen consumption rates of the two negative (blank; inoculum) controls differed by 17.2 % therefore meeting guideline the validity criteria (30%). The reference substance results were valid, the EC50 for 3,5-dichlorophenol was between 2 and 25 mg/L (total respiration): actual 12.5 mg/L and was within the expected range: 2 to 25 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
A 3 hour EC50 was > 1000 mg/L (nominal) was reported. The NOEC was 1000 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 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test substance for three hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with controls that contained activated sludge and synthetic sewage alone. The nominal test substance concentrations used in the study were 10, 100 and 1000 mg/L; five replicates were prepared for each concentration including the control. Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 24.1 mg O2/gh with a coefficient of variation (CV) of 17.2%. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 12.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The test substance did not significantly inhibit the respiration rates of the samples of activated sludge, with a maximum reduction in respiration of 18% at 1000 mg/L. Consequently, the 20, 50 and 80% effect concentrations (EC20/50/80) for inhibition could not be calculated but must be greater than 1000 mg/L (highest concentration employed in this study) and the no observed effect concentration (NOEC) for the test substance was 1000 mg/L (the highest concentration tested).

Description of key information

EC50 = > 1000 mg/L (nominal), 20 ± 2°C °C, OECD TG 209, 2014

EC20 = > 1000 mg/L (nominal), 20 ± 2°C °C, OECD TG 209, 2014

NOEC = 1000 mg/L (nominal), 20 ± 2°C °C, OECD TG 209, 2014

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

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 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test substance for three hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with controls that contained activated sludge and synthetic sewage alone. The nominal test substance concentrations used in the study were 10, 100 and 1000 mg/L; five replicates were prepared for each concentration including the control. Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 24.1 mg O2/gh with a coefficient of variation (CV) of 17.2%. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 12.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The test substance did not significantly inhibit the respiration rates of the samples of activated sludge, with a maximum reduction in respiration of 18% at 1000 mg/L. Consequently, the 20, 50 and 80% effect concentrations (EC20/50/80) for inhibition could not be calculated but must be greater than 1000 mg/L (highest concentration employed in this study) and the no observed effect concentration (NOEC) for the test substance was 1000 mg/L (the highest concentration tested).

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