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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Feb 2012 to 16 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Application of test substance: Direct addition by initial weight into the test flasks. The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL demineralized water.

Reference substance: 500.0 mg of the reference substance was added to about 800 mL of demineralized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.2 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with demineralized water. The stock solution appeared yellowish and no undissolved reference substance was visible.

Aliquots of the reference substance stock solution were dosed to the test vessels and made up with demineralized water to a volume 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of demineralized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked and adjusted.

After addition of 250 mL of inoculum suspension the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant of Mannheim, Germany was collected on 15 Feb 2012 from the aeration tank of the plant. The activated sludge suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with tap water and the suspension was adjusted to 3 g/L dry matter.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Test temperature:
19.8 to 20.3 °C
pH:
blanc and reference assays: pH 7.1 to 7.2
test item assays: initial: pH 8.5 to 10.8; adjusted: pH 7.2; after incubation: pH 7.7 to 8.2
Dissolved oxygen:
The content of oxygen at the start of the measurements was > 7 mg/L.
Nominal and measured concentrations:
1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity or composition and blank controls.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL glass beakers (nominal volume 1 L)
- No. of vessels per blank control (replicates): 4
- No. of vessels per reference substance concentration (replicates): 2
- No. of vessels per test substance concentration (replicates): 2
- Oxygen concentration during aeration: > 2 mg/L
- Oxygen concentration immediately before measurement: > 7.0 mg/L
- Duration of the measurement of oxygenconsumption: 5 - 10 min
- Sludge concentration in the test vessel: 1.5 g/L
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The total oxygen consumption of the two blank controls (BC1+2) were measured at first. The total oxygen consumption of the reference substance assays (RS) were measured in the sequence RS1+2, RS3+4 and RS5+6. Then the total oxygen consumption of the test substance assays were measured in the sequence TS1+2, TS3+4, TS5+6, TS7+8 and TS9+10. The oxygen consumption of the blank control BC3+4 were measured at last. No abiotic control was tested. The oxygen uptake was measured for a period of 5 to 10 minutes or until the oxygen concentration fell below 2 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (1, 10, 100 mg/L)
Duration:
180 min
Dose descriptor:
EC10
Effect conc.:
34 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% CL (lower/upper): 10.7/110
Duration:
180 min
Dose descriptor:
other: EC20
Effect conc.:
98 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% CL (lower/upper): 47.1/201.7
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
720 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% CL (lower/upper): 415.2/1245.9
Duration:
180 min
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: no CL95%
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 6.5 mg/L. These results indicate that the microorganisms are responding normally to toxicant stress.
Reported statistics and error estimates:
The consumption rate (mg O2/L*6 minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.
The coefficient of variation of the four replicates of blank control was 17.8 % O2 consumption.

The consumption rates were used for the determination of the ECx by the probit method based on Finney [1] with the software TOXRAT Professional 2.10 [2]. The effect concentrations were given with an accuracy of one respectively two significant digits.

Results for controls and reference substance

Oxygen consumption

Treatment

BC1

BC2

BC3

BC4

RS1

RS2

RS3

RS4

RS5

RS6

Concentration of test substance

-

-

-

-

1

10

100

(mg/L)

O2 concentration at test start

7.8

7.9

6.5

7.4

6.8

7.4

7.4

7.6

8.3

8.0

(mg O2/L)

O2 concentration after 6 min

4.0

4.0

2.5

2.5

3.0

3.5

6.0

6.0

8.0

7.7

(mg O2/L)

O2 consumption rate

38

39

40

49

38

39

14

16

3

3

(mg O2/L*h)

Specific O2 consumption rate

25

26

27

33

25

26

9

11

2

2

(mg O2/g*h)

Inhibition of respiration

-

-

-

-

8

6

66

61

93

93

compared to control (%)

Results for test substance

Oxygen consumption

Treatment

TS1

TS2

TS3

TS4

TS5

TS6

TS7

TS8

TS9

TS10

Concentration of test substance

62.5

125

250

500

1000

(mg/L)

O2 concentration at test start

7.2

6.9

7.3

7.7

6.4

7.2

7.2

7.3

6.8

7.3

(mg O2/L)

O2 concentration after 6 min

3.5

2.5

4.5

4.5

4.0

4.5

5.0

5.0

5.0

5.0

(mg O2/L)

O2 consumption rate

37

44

28

32

24

27

22

23

18

23

(mg O2/L*h)

Specific O2 consumption rate

25

29

19

21

16

18

15

15

12

15

(mg O2/g*h)

Inhibition of respiration

11

-6

33

23

42

35

47

45

57

45

compared to control (%)

pH values

Treatment

TS1

TS2

TS3

TS4

TS5

TS6

TS7

TS8

TS9

TS10

Concentration of test substance

(mg/L)

62.5

125

250

500

1000

pH values measured

before correction

8.5

8.5

9.0

9.0

9.6

9.6

10.2

10.2

10.8

10.8

pH values corrected

before addition of inoculum

7.3

7.2

7.2

7.2

7.3

7.3

7.2

7.2

7.2

7.2

pH values measured

after incubation time

7.7

7.7

7.8

7.8

8.1

8.1

8.2

8.2

8.2

8.2

Validity criteria fulfilled:
yes

Description of key information

3-h EC10 =  34 mg/L (OECD 209; domestic activated sludge; BASF SE, 2012, report no. 08G0402/04G008)

Key value for chemical safety assessment

EC50 for microorganisms:
720 mg/L
EC10 or NOEC for microorganisms:
34 mg/L

Additional information

The microbial toxicity of N,N'-bis(3 -aminopropyl)ethylenediamine (CAS 10563 -26 -5) was studied in two tests: 1) GLP guideline study, respiration inhibition test with activated sludge, OECD 209; 2) non-GLP guideline study, Pseudomonas putida growth inhibition according to Bringmann-Kühn, German Industrial Standards DIN 38412, part 8.

The respiration inhibition test used activated sludge as inoculum taken from a wwtp treating predominantly domestic sewage. The 3 -h EC10 was determined to be 34 mg/L, the 3 -h EC50 was 720 mg/L (BASF SE, 2012; report no. 08G0402/04G008).

In the growth inhibition test, the test species was Pseudomonas putida. The test concentrations were not analytically verified. The 17 -h EC10 was determined to be 3.97 mg/L, the 17 -h EC50 was 8.8 mg/L (BASF AG, 1990; report no. 1/89/0502). This result will be used as supporting study.

The OECD 209 study was selected as key study since this result was determined under GLP according to an international guideline. The PNECs derived from the EC10 values of both studies are very similar (OECD 209: 3.4 mg/L; P.putida: 4.0 mg/L).