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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 03-05 June 2013 / signed on 05 November 2013
Specific details on test material used for the study:
- Storage condition of test material: Keep in the dark at 0-10 °C, under nitrogen
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
See table 6.1.7/1: Dilution table for the test item

- Control: Test medium without test or reference item
Test organisms (species):
activated sludge
Details on inoculum:
- Source: Municipal sewage treatment plant of 31137 Hildesheim, Germany.
- Pretreatment: The sludge was washed twice with tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.76 g/L corresponding to 1.47 g/L in the test vessel
- Dry solids concentration of sludge: 2.94 g/L; Dry solids concentration in test solution: 1.38 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
No data
Test temperature:
Nominal: 18 - 22 °C, actual: 21.6 °C
pH:
- pH value of the activated sludge: 7.95
- pH value of the synthetic waste water: 7.34
Dissolved oxygen:
Oxygen uptake: 14.0-29.2 mg O2/L.h
Salinity:
None
Nominal and measured concentrations:
46, 100, 220, 460 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Aeration: Permanent to keep the dissolved oxygen concentration above 60-70% saturation and to maintain the sludge flasks in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test)
- Dry solids concentration of sludge: 2.76 g/L
- Dry solids concentration in test solution: 1.38 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD Guideline 209
- Volume of the test medium: 500 mL

EFFECT PARAMETERS MEASURED
- 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 control respiration rates.
- 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.
- Oxygen: After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.
- Temperature: The temperature was determined in the measuring cell once during the measurement. The room temperature during the test was recorded continuously by a hygrothermograph.

TEST CONCENTRATIONS
- Preliminary test
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate carbohydrate p.a.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
213 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration
Remarks on result:
other: 95% CI: 194-232 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
288 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration
Details on results:
The mean inhibition of respiration for the test item replicates were 0, 1, 11, 37 and 47% at 46, 100, 220, 460 and 1000 mg/L, respectively.
Results with reference substance (positive control):
EC50 value for the reference item was 103 mg/L (95% CI: 97.3-108 mg/L). This is in the recommended range of validity of 53-155 mg/L.
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and Dunnett's test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism, Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Table 6.1.7/3: Inhibitions in the Preliminary test

Nominal test item concentration (mg/L)

 

pH (test item in demin. water)

pH (adjusted to)

pH (test item in medium)

Inhibition (%)

1st Preliminary test – direct aeration

10

5.71

-

7.13

7

5.48

-

7.33

3

100

5.45

-

7.41

5

5.71

-

7.46

4

1000

5.59

-

7.55

35

5.40

-

7.59

31

2nd Preliminary test – shaking aeration

10

6.12

-

7.57

1

 

6.99

-

7.54

5

100

6.91

-

7.61

8

 

9.23

-

7.60

10

1000

6.71

-

7.62

43

6.79

-

7.62

41

 

Additionally, one preliminary test was carried out focusing on the loss of test item during the incubation / aeration. A final concentration of 100 mg/L test item was pipetted in Erlenmeyer flasks and filled up to 500 mL with mineralized water. Sampling was done before and after aeration (direct aeration and aeration by shaking, respectively).

 

Table 6.1.7/4: DOC development during aeration

Nominal test item concentration (mg/L)

DOC before incubation and aeration (mg C/L)

DOC after 3 h incubation and aeration (mg C/L)

Preliminary test – direct aeration

100

31.90

7.39

Preliminary test – shaking aeration

100

43.61

51.85

50.22

50.00

 

The results of the DOC measurements indicate that with aeration by shaking, the DOC remains unchanged. Compared to the preliminary tests with direct aeration, the DOC as well as the inhibition is higher with shaking aeration. Therefore, the test item was considered to be suitable for testing with shaking aeration.

 

Table 6.1.7/5 Inhibitions in the Preliminary test (with / without ATU)

 

Nominal test item concentration (mg/L)

Inhibition of total respiration (%)

Inhibition of heterotrophic respiration (%)

Inhibition of nitrification (%)

1000

38

14

52

37

14

50

 No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

 

Table 6.1.7/6: Oxygen uptake rates and inhibition of the respiration of the control and test item concentrations

 

Test concentration (mg/L)

Replicates

Oxygen uptake rate R (mg O2/L.h)

Inhibition (%)

Mean Inhibition (%)

Control

1

29.2

-

-

2

28.0

3

28.4

4

25.6

5

24.0

6

26.0

Test item

46

1

26.4

2

0

2

26.0

3

3

26.4

2

4

26.8

0

5

28.4

-6

100

1

27.2

-1

1

2

26.4

2

3

26.8

0

4

27.6

-3

5

25.6

5

220

1

23.6

12

11

2

25.2

6

3

23.6

12

4

23.2

14

5

24.0

11

460

1

17.6

35

37

2

16.8

38

3

16.8

38

4

17.2

36

5

17.2

36

1000

1

14.0

48

47

2

14.4

46

3

14.4

46

4

14.8

48

5

14.8

45

 

 

Coefficient of variation of oxygen uptake rates of the control: 7.4%

Suspended solids in the test vessel: 1.38 g/L

 

Table 6.1.7/7: Specific respiration rates of the control replicates

 

Control

Replicates

Specific respiration rate Rs(mg O2/g.h)

1

21

2

20

3

21

4

19

5

17

6

19

Mean value

20

 

Suspended solids in the test vessel: 1.38 g/L

 

Validity criteria:

- The mean specific oxygen uptake rate of the control replicates was 20 mg O2/g.h and therefore not meeting the criterion (validity criterion: should be ≥ 20 mg 02/g.h).

- The coefficient of variation of the oxygen uptake rates in the control replicates was 7.4% (validity criterion: < 30 %).

- The EC50 of the reference item was in the range of 53 - 155 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the NOEC of test item is 100 mg/L. The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The EC10, EC20 and EC50 were 213, 288 and > 1000 mg/L.
Executive summary:
The effect of test item on the respiration rate of activated sludge was assessed according to OECD Guideline 209 with GLP statement.

 

The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 46, 100, 220, 460 and 1000 mg/L. 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 control respiration rates.

 

The mean inhibition of respiration for the test item replicates were 0, 1, 11, 37 and 47% at 46, 100, 220, 460 and 1000 mg/L, respectively.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 103 mg/L. This is in the recommended range of validity of 53-155 mg/L.

 

Under the test conditions, the NOEC of test item is 100 mg/L. The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The EC10, EC20 and EC50 were 213, 288 and > 1000 mg/L, respectively.

Description of key information

OECD Guideline 209, GLP, key study, validity 1:

3h-EC50 (activated sludge of a municipal sewage treatment plant) > 1000 mg/L based on nominal concentration

3h-EC10 (activated sludge of a municipal sewage treatment plant) = 213 mg/L based on nominal concentration

3h-NOEC (activated sludge of a municipal sewage treatment plant) = 100 mg/L based on nominal concentration

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

One key study is available (NOACK, 2014) to assess the toxicity of the registered substance to microorganisms. In this study, the effect of the registered substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement. The test was carried out under static conditions with the test substance concentrations 46, 100, 220, 460 and 1000 mg/L. The inhibitory effects of the test and reference substances were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test substance replicates were 0, 1, 11, 37 and 47% at 46, 100, 220, 460 and 1000 mg/L, respectively. In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 103 mg/L. This is in the recommended range of validity of 53-155 mg/L.

Under the test conditions, the NOEC of test substance is 100 mg/L. The test substance is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The EC10, EC20 and EC50 were 213, 288 and > 1000 mg/L, respectively.