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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully documented study under GLP and according to international guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
The temperature in the pre-test was in a range of 21.2 – 23.1 °C instead of 20 ±2 °C. The oxygen uptake rate of the activated sludge was lower than stated in the guideline. These deviations were stated as uncritical.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
The temperature in the pre-test was in a range of 21.2 – 23.1 °C instead of 20 ±2 °C. The oxygen uptake rate of the activated sludge was lower than stated in the guideline. These deviations were stated as uncritical.
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)
- Method: Because of high volatility, the nominal load of test item was directly injected into the vessels with a suitable syringe. Then the vessels were immediately closed. The injected volume based on a mean density of test item of 0.75 g/mL.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: Upon arrival in the test facility, the sludge was washed with tap water three times, then centrifuged and resuspended in tap water.
- Pretreatment: No
- Initial biomass concentration: 3.40 g/L, corresponding to a concentration of 1.70 g/L suspended solids in the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21.2 – 23.1 °C measured in vessels
pH:
pH in the activated sludge was measured as 7.3. pH at the end of the test: see table in "any other information on results".
Nominal and measured concentrations:
Nominal concentrations of test item (mg/L): 10000, 3200, 1000, 320, 100, 32, 10, 3.2, 1
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000 mL Schott flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: material: glass, fill volume: 500 mL
- Aeration: none, as the test item is volatile; in deviation from the guideline, the treatments were not aerated but stirred strongly to ensure oxygen maintenance
- No. of vessels per concentration (replicates): one replicate/treatment (positive control), one replicate/treatment (test item)
- No. of vessels per control (replicates): two replicates before and two after measuring positive control and test item, respectively


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: <1 mg/L
- Particulate matter: no data
- Metals:
antimony < 0.001 mg/L
arsenic < 0.001 mg/L
lead < 0.001 mg/L
cadmium < 0.0005 mg/L
chromium < 0.002 mg/L
copper 0.001 mg/L
nickel < 0.001 mg/L
mercury < 0.0001 mg/L
selenium < 0.001 mg/L
bor 0.017 mg/L

- Pesticides: < limit of detection
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: 189 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen concentration
Reference substance (positive control):
yes
Remarks:
3.5-Dichlorophenol
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
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
400 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% confid. interv.: 120 - 970 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 400 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% confid. interv.: 1000 - 7300 mg/L
Results with reference substance (positive control):
The calculated EC50 value for the positive control 3,5-dichlorophenole is 10 mg/L which lies in the recommended range of 2 – 25 mg/L.
Reported statistics and error estimates:
The estimation of the EC50 was accomplished using the software OriginTM. The data were evaluated using linear fit on a probability-logarithmic scale.

Study Performance – Main Study

Time Schedule

The time schedule is presented in the following table.

Time Schedule

No.

Start of Test

Begin of O2-Measurement

End of O2-Measurement

1

10:25

13:25:36

13:29:06

2

10:30

13:29:16

13:35:11

3

10:35

13:35:46

13:39:47

4

10:40

13:40:17

13:45:05

5

10:45

13:45:40

13:50:04

6

10:50

13:50:42

13:55:00

7

10:55

13:55:18

13:59:58

8

11:00

14:00:36

14:04:59

 

 

 

 

18

11:50

14:50:11

14:54:46

19

11:55

14:54:24

14:59:59

20

12:00

15:00:29

15:04:52

21

12:05

15:05:17

15:09:23

22

12:10

15:11:11

15:14:53

23

12:15

15:15:11

15:19:59

24

12:20

15:20:42

15:24:59

25

12:25

15:25:22

15:29:40

26

12:30

15:30:35

15:34:48

27

12:35

15:35:10

15:39:40

28

12:40

15:40:30

15:44:40

29

12:45

15:45:10

15:49:48

30

12:50

15:50:11

15:55:28

Annotation: Other test item was measured in vessels 9 – 17.

 


1.1.3      O2-Consumption, Inhibition

The values of the O2 consumption (which is a measure for the viability of the bacteria) of controls, test and positive control and the calculated inhibition are presented in the following table. The measured pH at the end of the test is also stated.

 O2-Consumption, Inhibition

Vessel No.

Content

Concentration in mg/L

O2consumption in mg/(L*min.)

O2consumption in mg/(L*h)

Inhibition in %

pH

1

Control

0

0.4050

24.302

--

7.7

2

Control

0

0.4064

24.382

--

7.7

3

Positive Control

5

0.3143

18.858

28.6

7.5

4

Positive Control

10

0.2029

12.174

53.9

7.5

5

Positive Control

20

0.1205

7.233

72.6

7.5

6

Positive Control

40

0.0922

5.532

79.0

7.6

7

Control

0

0.4177

25.060

--

7.7

8

Control

0

0.4603

27.615

--

7.7

 

 

 

 

 

 

 

18

Control

0

0.4632

27.789

--

7.7

19

Control

0

0.4454

26.724

--

7.7

20

Test Item

10000

0.0982

5.894

77.7

7.6

21

Test Item

3200

0.2103

12.619

52.2

7.8

22

Test Item

1000

0.3129

18.776

28.9

7.8

23

Test Item

320

0.4425

26.553

-0.6

7.7

24

Test Item

100

0.4035

24.211

8.3

7.7

25

Test Item

32

0.4616

27.696

-4.9

7.7

26

Test Item

10

0.4591

27.545

-4.4

7.7

27

Test Item

3.2

0.4351

26.106

1.1

7.8

28

Test Item

1

0.4579

27.475

-4.1

7.7

29

Control

0

0.4420

26.517

--

7.8

30

Control

0

0.4797

28.780

--

7.8

 

Annotation: Other test item was measured in vessels 9 – 17.

The inhibition in treatment 320 mg/L was unexpectedly low. This value was considered as not valid and therefore not used for evaluation of the results.

In treatments below 100 mg/L, no inhibition could be observed. Therefore, treatments 10; 3.2 and 1 mg/L were not required for evaluation of the results.

Validity criteria fulfilled:
yes
Remarks:
Oxygen uptake rate was too low for the blank control.
Conclusions:
The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:
3h NOEC = 32 mg/L
3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)
3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)
Executive summary:

The Inhibition of the Respiration of Activated Sludge when exposed to "2-propanol and 2-butanol production, distn. residues" was determined according to OECD 209 resp. EU C.11

One valid experiment was performed and in the main study nine concentrations, ranging from 10000 to 1 mg/L (nominal concentrations) were tested. Due to the volatility of the test item, the study was performed in a closed system without aeration.

The dry matter of the activated sludge was determined as 3.4 g suspended solids/L, giving a concentration of 1.7 g suspended solids/L in the test. 3.5-Dichloro-phenole was used as positive control and four concentrations were tested; an EC50 of 10 mg/L (95% confidence interval: 3 – 19 mg/L) was determined, which lies within the demanded range of 2 – 25 mg/L.

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

3h NOEC = 32 mg/L

3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

Description of key information

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

3h NOEC = 32 mg/L

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:
2 400 mg/L
EC10 or NOEC for microorganisms:
32 mg/L

Additional information

The EC20 value, EC50 value and the NOEC for the inhibition of the respiration in activated sludge of "2-propanol and 2-butanol production, distn. residues" was measured according to OECD 209, respectively EU C.11 guideline under GLP. The following results were obtained:

3h NOEC = 32 mg/L

3h EC20 = 400 mg/L (95% confid. interv.: 120 - 970 mg/L)

3h EC50 = 2400 mg/L (95% confid. interv.: 1000 - 7300 mg/L)

These values are used for the calculation of the PNEC for microorganisms in sewage treatment plants (PNEC STP).