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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.04.04.05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: directly weighted
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: ESN (Stadtentsorgung Neustadt) D-67435 NW-Lachen-Speyerdorf
- Method of cultivation:
- Preparation of inoculum for exposure: washing 3x tap water, re-suspension in tap water
- Pretreatment: aeration, feeding daily 50 mL synthetic sewage/L
- Initial biomass concentration: Test 1: dry matter sludge: 3.14 g suspended solids/L; dry matter in test: 1.57 g suspended solids/L
Test 2: dry matter sludge: 2.60 g suspended solids/L; dry matter in test: 1.30 g suspended solids/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Test 1 (range finding): 19.2 - 20.3 °C
Test 2: 19.2 - 20.6 °C
pH:
check tables on results
Nominal and measured concentrations:
Test 1 (range finding): 1000, 100, 10, 1 mg/L (nominal)
Test 2: 3200, 1000, 320, 100, 32 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass,
- Aeration: purified air via Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1 (positive), 2 (system)
- No. of vessels per vehicle control (replicates): -
- No. of vessels per abiotic control (replicates):
- Sludge concentration (weight of dry solids per volume): Test 1 (range finding): 1.57 g/L; Test 2: 1.30 g/L
- Weight of dry solids per volume of reaction mixture per unit of time:-
- Nutrients provided for bacteria: 16 mL standard nutrient solution / vessel
- Biomass loading rate:
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Particulate matter:
OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline:-
- Range finding study
- Test concentrations: see above
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5-DCP
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
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
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: Test 1 (range finding): EC50 8.8 mg/L (95 % c.i. 7.0-11 mg/L); Test 2: EC50 6.9 mg/L (95 % c.i. 5.2-8.4 mg/L)
- Other:

O2 consumption, first experiment (range finding)

Vessel No.

Content

Concentration in mg/L
nominal

O2consumption in mg/(L*min)

O2consumption in mg/(L*h)

Inhibition in %

pH

1

Control

0

0.7222

43.330

0.0

7.7

2

Control

0

0.7091

42.545

0.0

7.7

3

Positive

Control

5

0.5043

30.261

29.8

7.9

4

Positive

Control

10

0.3257

19.541

54.6

7.9

5

Positive

Control

20

0.1462

8.775

79.6

7.9

6

Positive

Control

40

0.0674

4.044

90.6

7.9

7

Control

0

0.7104

42.624

0.0

7.7

8

Control

0

0.7014

42.083

0.0

7.7

9

Test Item

1000

0.4564

27.382

36.5

7.8

10

Test Item

1000

0.4582

27.493

36.2

7.8

11

Test Item

1000

0.4855

29.127

32.4

7.8

12

Test Item

1000

0.4636

27.816

35.4

7.8

13

Test Item

1000

0.4587

27.525

36.1

7.8

14

Test Item

100

0.6546

39.277

8.8

7.7

15

Test Item

10

0.7055

42.331

1.8

7.7

16

Test Item

1

0.6971

41.827

2.9

7.6

17

Control

0

0.6954

41.724

0.0

7.7

18

Control

0

0.7704

46.222

0.0

7.7

O2 consumption, second experiment

Vessel No.

Content

Concentration in mg/L
nominal

O2consumption in mg/(L*min)

O2consumption in mg/(L*h)

Inhibition in %

pH

1

Control

0

0.5135

30.810

0.0

8.0

2

Control

0

0.4884

29.305

0.0

8.0

3

Positive Control

5

0.3139

18.833

40.6

8.1

4

Positive Control

10

0.2104

12.625

60.2

8.1

5

Positive Control

20

0.1205

7.228

77.2

8.2

6

Positive Control

40

0.0713

4.277

86.5

8.2

7

Control

0

0.5302

31.813

0.0

8.0

8

Control

0

0.5161

30.968

0.0

8.0

9

Test Item

3200

0.3208

19.248

39.3

8.1

10

Test Item

3200

0.2644

15.863

50.0

8.1

11

Test Item

3200

0.2727

16.359

48.4

8.1

12

Test Item

3200

0.2664

15.984

49.6

8.1

13

Test Item

3200

0.2710

16.262

48.7

8.1

14

Test Item

1000

0.2927

17.561

44.6

8.1

15

Test Item

1000

0.3017

18.101

42.9

8.1

16

Test Item

1000

0.3052

18.313

42.3

8.1

17

Test Item

1000

0.3067

18.403

42.0

8.1

18

Test Item

1000

0.3002

18.011

43.2

8.1

19

Test Item

320

0.4007

24.044

24.2

8.0

20

Test Item

320

0.4062

24.374

23.2

8.0

21

Test Item

320

0.4031

24.186

23.7

8.0

22

Test Item

320

0.4073

24.439

23.0

8.0

23

Test Item

320

0.3908

23.449

26.1

8.0

24

Test Item

100

0.4854

29.126

8.2

8.0

25

Test Item

100

0.4838

29.029

8.5

8.0

26

Test Item

100

0.4975

29.850

5.9

7.9

27

Test Item

100

0.4880

29.280

7.7

7.9

28

Test Item

100

0.4922

29.533

6.9

7.9

29

Test Item

32

0.5300

31.799

-0.3

7.9

30

Test Item

32

0.5814

34.884

-10.0

7.9

31

Test Item

32

0.5611

33.668

-6.1

7.9

32

Test Item

32

0.5491

32.945

-3.9

7.9

33

Test Item

32

0.5507

33.044

-4.2

7.9

34

Control

0

0.5640

33.842

0.0

7.9

35

Control

0

0.5596

33.575

0.0

7.9

Validity criteria fulfilled:
yes
Conclusions:
In the first experiment, which was performed as the range finding test, the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Because signifi-cant inhibition was observed, an additional experiment was performed under the same test conditions.
In the second experiment, the test item was tested using 5 concentrations ranging from 3200 to 32 mg/L nominal concentration. The inhibition in the highest concentrated treatment 3200 mg/L was only marginal higher than in the treatment 1000 mg/L. For the estimation of the EC50 the fits showed acceptable statistical correspondence of the data with the dose-response-equation. For the test item an EC50 of 2500 mg/L was calculated. The difference between trea-tment 32 mg/L and the control can be considered as not significant. Therefore, the concentrati-on 32 mg/L is stated as NOEC.

The pre-test and the main test show good correlation of the inhibitory effects of the test item. The result of the test can be considered valid.
No observations were made which might cause doubts concerning the validity of the study outcome.
Executive summary:

Two valid experiments were performed.

In the first experiment, the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.14 g suspended solids/L, giving a concentration of 1.57 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 8.8 mg/L (95% confidence interval: 7.0 – 11 mg/L) was determined, which lies within the range of 2 – 25 mg/L.

Because significant inhibition was observed, an additional experiment was performed under the same test conditions.

 

In the second experiment, the test item was tested using 5 concentrations ranging from 3200 to 32 mg/L nominal concentration.

The dry matter of the inoculum was determined as 2.60 g suspended solids/L, giving a concentration of 1.30 g suspended solids/L in the test.

For the positive control an EC50 of 6.9 mg/L was determined. The value lies within the recommended range.

The following results for the test item2,2'-Azobis (4-methoxy-2,4-dimethyl valeronitrile)were determined:

 

3h NOEC = 32 mg/L
3h EC50=2500 mg/L

3h EC10=110 mg/L

Description of key information

The key values for the chemical safety assessment are determined based on the findings in an OECD 209 study exposing activated sludge for 3 hours with the test item.

Key value for chemical safety assessment

EC50 for microorganisms:
2 500 mg/L
EC10 or NOEC for microorganisms:
110 mg/L

Additional information