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Reference
Endpoint:
toxicity to microorganisms, other
Remarks:
activated sludge respiration inhibition testing and activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jan - 09 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Treatments were prepared by direct weighing of the test item into each test vessel (with five-fold replicates in the definitive test) and then filled up with 142 ml tap water and 8 ml of the synthetic sewage. The reaction was started by the addition of 100 mL of the prepared inoculum. The final volume in each test vessel was 250 mL. In parallel, identical treatments were prepared containing the specific nitrification inhibitor allylthiourea at a concentration of 12.05 mg/L. Analogous to these treatments non-affected controls were prepared in parallel with and without allylthiourea which were used for the calculation of the inhibition values.
- Controls: yes
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the effluent of the aeration tank of the municipal sewage treatment plant at Taunusstein-Bleidenstadt, Germany
- Preparation of inoculum for exposure: The washed sludge was fed with the synthetic sewage. The feeding procedure was repeated daily.
- Pretreatment: activated sludge was washed twice with mineral nutrient medium acc. OECD 301B and aerated thereafter until test start.
- Initial biomass concentration: 1.45 g/L of dry mass in the test treatments
- Others: The dry mass of the activated sludge determined one day before start of the definitive main test was 5.26 g/L. By the addition of 2808 mL tap water to 7 L of sludge a theoretical dry matter content of 3.75 g/L should be achieved. As the sludge was still fed with 350 mL of synthetic sewage medium the theoretical dry matter content was slightly lower as being 3.626 g/L resulting in a final concentration of 1.45 g/L of dry mass.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18.8 - 19.5 (Control without ATU)
18.8 - 19.6 (Control with ATU)
18.3 - 19.8 (Test item concentration without ATU)
18.9 - 19.7 (Test item concentration with ATU)
pH:
7.07 - 8.18 (Control without ATU)
7.07 - 8.35 (Control with ATU)
7.07 - 8.23 (Test item concentration without ATU)
7.08 - 8.33 (Test item concentration with ATU)
Nominal and measured concentrations:
Control, 93.9, 207, 455, 1000 and 2200 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 250 mL test vessels with 250 ml test volume
- Aeration: wetted compressed air from central system equipped with a systemof tubings and single-use Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): was tested within the screening test
- Sludge concentration (weight of dry solids per volume): 1.45 g/L dry mass
- Nutrients provided for bacteria: synhetic sewage feed: For each liter of water - 16.0 g peptone, 11.0 g meat extract , 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O , 0.2 g MgSO4 x 7H2O, and 2.8 g K2HPO4
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water

OTHER TEST CONDITIONS
- Details on termination of incubation:
In order to get the same incubation period of 3 hr for each test solution, a new test solution was started every 5 min. The first and the last treatment were without test item and served as controls (further ones intermittently). For testing sensitivity, three positive controls with 5, 15 und 30 mg/L 3,5-dichlorophenol were prepared and incubated for 3 h. A treatment for determination of autoxidation (approximately 1000 mg/L final concentration, performed within the screening test) of the test item was prepared without inoculum the amount of activated sludge being replaced by 100 ml tap water. As there was no larger autoxidation in the treatment with 1000 mg/l (only 0.02 mg O2/min) no further test on autoxidation was done.
After each incubation, the aeration was stopped and the oxygen consumption was recorded individually in order to determine the respiration rates using an oxygen electrode equipped with a recorder. Then, pH was measured anew and documented.


EFFECT PARAMETERS MEASURED:
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study
- Test concentrations: Control, 10, 100, 1000 mg/L (screening study)
- Results used to determine the conditions for the definitive study: Since statistically significant effects were determined for the screening study
Reference substance (positive control):
no
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
379 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other: 95% confidential limits
Remarks:
159 - 902 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
663 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other: 95% confidential limits
Remarks:
277 - 1591 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 935 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other: 95% confidential limits
Remarks:
581 - 5949 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
411 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
530 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95% confidential limits
Remarks:
276 - 1019 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
947 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95% confidential limits
Remarks:
479 - 1878 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 873 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 95% confidential limits
Remarks:
1033 - 7582 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 85.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
480 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidential limits
Remarks:
334 - 688 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
850 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95/ confidential limits
Remarks:
587 - 1232 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 540 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidential limits
Remarks:
1474 - 4255 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 85.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 21.33 mg O2/g DM*h (total)
- Coefficient of variation of oxygen uptake rate in control replicates: 3.0%
Reported statistics and error estimates:
Statistical significance was evaluated using the ToxRat Program ToxRatPro Version 3.2.1

Results:

Test Item: Respiration Rates and Inhibition Values within the Definitive Test

Concentration set [mg/L]

Respiration Rate Total (- ATU) mg O2/g DM * h

Respiration Rate (+ ATU) mg O2/g DM * h heterotrophic

Calculated Respiration Rate due to Nitrification mg O2/g DM * h

% Inhibition of the Total Respiration Rate

% Inhibition of the heterotr. Respiration Rate 1)

% Inhibition of the Nitrification Rate 1)

Control

21.3

15.6

5.7

-

-

-

 

22.2

16.6

4.7

-

-

-

 

20.5

13.9

7.4

-

-

-

 

20.8

14.2

7.1

-

-

-

 

21.9

15.8

5.5

-

-

-

mean

21.3

14.9

6.5

-

-

-

93.9

18.3

12.3

7.3

14.2

17.4

-12.6 1)

 

19.0

11.5

8.1

10.9

22.7

-25.0

 

20.9

12.8

6.8

2.0

14.0

-4.8 1)

 

19.9

12.3

7.3

6.7

17.4

-12.6 1)

 

19.7

20.8

-1.2 3)

7.7

-39.8

119.2 3)

2072)

20.0

13.9

6.3

6.2

6.6

2.0

 

20.3

13.6

6.6

4.8

8.6

-2.6 1)

 

19.5

13.7

6.5

8.6

8.0

-1.1 1)

 

21.1

13.6

6.6

1.1

8.6

-2.6 1)

 

20.2

13.2

7.0

5.3

11.3

-8.8 1)

4552)

19.2

13.6

6.1

10.0

8.6

5.7

 

19.7

13.9

5.8

7.7

6.6

10.4

 

19.3

15.5

4.2

9.5

-4.1

35.2

 

19.3

13.4

6.3

9.5

10.0

2.6

 

20.9

11.6

8.1

2.0

22.1

-25.3

10002)

14.5

11.9

3.9

32.0

20.0

39.8

 

15.8

10.2

5.6

25.9

31.5

13.5

 

15.2

11.4

4.4

28.8

23.4

32.1

 

16.1

10.7

5.1

24.5

28.1

21.2

 

17.3

10.6

5.2

18.9

28.8

19.7

2200

13.1

9.1

2.9

38.6

38.9

54.7

 

12.1

7.9

4.1

43.3

46.9

36.1

 

13.1

9.1

2.9

38.6

38.9

54.7

 

10.4

8.9

3.1

51.3

40.2

51.6

 

11.4

7.9

4.1

46.6

46.9

36.1

DM     = Dry mass;

ATU = allylthiourea;

Negative value indicates an activation (increase of the respiration rate);

Exact concentrations have to be indicated for each individual treatment as the test item was weighed individually into the treatments separately.

Obviously, ATU was omitted in that treatment, and thus, this calculated inhibition value is not valid

Table 2: Results Calculated Individual Concentrations in the Treatments 

Nominal Conc. set

93.9 mg/L

207 mg/L

455 mg/l

1000 mg/L

2200 mg/L

 

Weight in mg/250 mL

Weight in mg/250 mL

Weight in mg/250 mL

Weight in mg/250 mL

Weight in mg/250 mL

-ATU (a)

+ ATU (b)

-ATU (a)

+ ATU (b)

-ATU (a)

+ ATU (b)

-ATU (a)

+ ATU (b)

-ATU (a)

+ ATU (b)

 

23.8

23.9

52.9

52.0

114.1

114.8

249.3

251.8

552.5

553.2

 

23.3

24.1

51.9

52.7

114.7

115.0

249.2

252.2

554.2

553.2

 

23.6

23.6

52.2

52.1

113.6

113.6

253.2

250.6

550.6

556.9

 

24.2

24.5

51.9

52.0

113.3

114.8

250.5

251.3

551.3

553.2

 

24.0

23.4

52.0

52.5

114.1

114.4

251.7

250.6

556.1

555.1

Mean:

23.78

23.90

52.18

52.26

113.96

114.52

250.78

251.39

552.94

554.32

Final mean Conc.[mg/l]:

95.12

95.60

208,72

209.04

455.84

458.08

1003.12

1004.16

2211.76

2217.28

Mean of (a) and (b):

95.36 mg/l

208.88 mg/l

456.96 mg/l

1011.92 mg/l

2214.52 mg/L

On basis of active substance (90%)

85.8 mg/l

188.0 mg/l

411.3 mg/l

910.7 mg/l

1993.1 mg/L

 

 

 

Table 1: Validity criteria for OECD 209

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

Oxygen uptake was >20 mg/g dry mass of suspended solids/h

yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

<30%

yes

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test the following EC-values were determined on basis of the mean active substance concentrations in the treatments:
Inhibition of the total respiration: EC10= 480 mg/L; EC50=2540 mg/L
Inhibition of the heterotrophic respiration: EC10= 530 mg/L; EC50= 2873 mg/L
Inhibition of the respiration due to nitrification: EC10= 379 mg/L; EC50= 1935 mg/L

Description of key information

EC50 (3 h) 1935 mg/L (respiration due to nitrification, activated sludge, OECD 209)

EC10 (3 h) 379 mg/L (respiration due to nitrification, activated sludge, OECD 209)

NOEC (3 h) 411 mg/L (respiration due to nitrification, activated sludge, OECD 209)

EC50 (3 h) 2540 mg/L (total respiration, activated sludge, OECD 209)

EC10 (3 h) 480 mg/L (total respiration, activated sludge, OECD 209)

NOEC (3 h) < 85.8 mg/L (total respiration, activated sludge, OECD 209)

EC50 (3 h) 2873 mg/L (heterotrophic respiration, activated sludge, OECD 209)

EC10 (3 h) 530 mg/L (heterotrophic respiration, activated sludge, OECD 209)

NOEC (3 h) < 85.8 mg/L (heterotrophic respiration, activated sludge, OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
1 935 mg/L
EC10 or NOEC for microorganisms:
379 mg/L

Additional information

One experimental study was conducted in accordance with OECD Guideline 209 and under GLP conditions (Lebertz, 2017d). The activated sludge was exposed to the test item concentrations of 0, 93.9, 207, 455, 1000 and 2200 mg/L (nominal) together with and without the specific nitrification inhibitor allylthiourea. After 3 hours of incubation period the inhibition of the respiration rates of the activated sludge were determined in comparison to the respective set of test solutions without any test item. All conditions of validity of the test were kept within this study. Under the conditions of the test the following EC-values were determined on basis of the mean active substance concentrations in the treatments. The EC50 based of the inhibition of total respiration was determined to be at 25410 mg/L. The EC50 based of the inhibition of the heterotrophic respiration was determined to be at 2873 mg/L. The EC50 based of the inhibition of the respiration based on nitrification was determined to be at 1935 mg/L.