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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-08 to 2016-06-07
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
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: The test substance concentration (1000 mg/L) was pipetted directly in the flasks, 200 mL demineralised water was added and stirred over night.
- Eluate: 490 µl test substance, 16 ml/L synthetic waste water, filled up to 250 mL with demineralised water, 250 mL inocolum is added
- Controls: test medium without test or reference item, triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: Activated sludge
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of 31137 Hildesheim, Germany
- Method of cultivation:
- Preparation of inoculum for exposure/ Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration: Dry sludge concentration 3.21 g/L (corresponding to 1.6 g/L suspended solids in the test vessels).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
nominal 18 - 22 °C, actually measured 19.5°C
pH:
7.5 ± 0.5
Dissolved oxygen:
Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
Nominal and measured concentrations:
In a preliminary test 10, 100, and 1000 mg/L with two replicates has been tested. Additionally two replicates with 1000 mg/L test substance and Allylthiourea has been used to study the inhibition of the nitrification in the preliminary test. Based on the results of the preliminary test it was concluded that the definitive study should be carried out with the concentration of 1000 mg/L in triplicates (limit test to determine the NOEC) as the heterotrophic respiration and nitrification has not been inhibited in the preliminary test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL flasks
- Material, size, headspace, fill volume: volume 500 mL, erlmeyer flask
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 at the beginning of the test + 3 at the end of the test
- Sludge concentration (weight of dry solids per volume): 3.21 g/L (corresponding to 1.6 g/L suspended solids in the test vessels)
- Nitrification inhibitor used: N-allylthiourea

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

OTHER TEST CONDITIONS
- Adjustment of pH: In the preliminary test, the pH was adjusted in one replicate with the concentration of 1000 mg/L and 1000 mg/L with Allylthiourea. 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 and adjustment to 7.5 ± 0.5 was not necessary.
- Details on termination of incubation: The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: preliminary test: factor 10. The reference substance has been tested with an dilution factor of 1.8. Definitve test: 1000 mg/L
- Justification for using fewer concentrations than requested by guideline:mBased on the results of the preliminary test it was concluded that the definitive study should be carried out with the concentration of 1000 mg/L in triplicates (limit test to determine the NOEC) as the heterotrophic respiration and nitrification has not been inhibited in the preliminary test.

- Range finding study: yes
- Test concentrations: In a preliminary test 10, 100, and 1000 mg/L with two replicates has been tested. Additionally two replicates with 1000 mg/L test substance and Allylthiourea has been used to study the inhibition of the nitrification in the preliminary test.
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test it could be concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in triplicates (limit test to determine the NOEC).
To study the inhibition of the nitrification Allylthiourea was used. In the preliminary test with 1000 mg/L test substance and N-Allylthiourea no inhibiting effect was observed.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
- Blank controls oxygen uptake rate: 20 mg O2 / g * h
- Coefficient of variation of oxygen uptake rate in control replicates: 3.4%
- The mean inhibition of respiration for the test item replicates was -13%.
Results with reference substance (positive control):
- Results with reference substance valid: Yes, in the range of 53-155 mg/L
- Relevant effect levels: EC50 of the reference item was 124 mg/L

Oxygen Uptake Rates, Inhibition of the Respiration of Control, Test and Reference Substance Concentrations

 

Test and Reference Item Concentration [mg/L]

Replicate

 

Oxygen Uptake Rate R

[mg O2/L·h]

Inhibition

[%]

Mean Inhibition

[%]

Control

1

2

3

4

5

6

1 32.0

2 32.0

3 33.6

4 30.4

5 31.2

6 31.6

 

-

 

Test item

1000

 

1

2

3

35.2

36.4

36.0

 

-11

-14

-13

-13

 

Reference item

58

 

1

2

3

27.2

26.0

26.4

 

14

18

17

16

 

100

1

2

3

19.2

20.0

19.6

 

40

37

38

38

 

180

1

2

3

10.4

10.4

10.0

 

67

67

69

68

 

 

Coefficient of variation of oxygen uptake rates of the control: 3.4 %

Suspended solids in the test vessel: 1.61 g/L

 

Validity criteria fulfilled:
yes
Remarks:
- mean specific oxygen uptake rate of the control replicates >=20 mg 02 / g * h. - Coefficient of variation of the oxygen uptake rates in the control replicates was < 30 %. - EC50 of the reference item is in the range of 53 - 155 mg/L.
Conclusions:
The NOEC of the test item Di(iso)-pentylterephthalate is >= 1000 mg/L. Di(iso)pentylterephthalate caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Description of key information

The NOEC of the test substance is >= 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test substance concentration of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A respiration inhibition test with activated sludge has been performed according to OECD guideline No. 209 (2010) in compliance with GLP. The study has been conducted with activated sludge of a municipal treatment plant under static conditions. In a limit test, the activated sludge has been exposed to 1000 mg/L test substance for 3 hours. The mean inhibition of the respiration for the test substances replicates was -13 %. The NOEC of the test substance is >=1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test substance concentration of 1000 mg/L.