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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
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:
Adopted 22 July 2010
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
TEST SOLUTIONS
The test was performed at three concentrations of the test item, used 5 parallels: 10 (TI11, TI12, TI13, TI14, TI15), 100 (TI21, TI22, TI23, TI24, TI25) and 1000 (TI31, TI32, TI33, TI34, TI35) mg/L were used.
The purity of the test item, was not be taken in consideration.
Because of the good water solubility, a stock solution (2500 mg/L concentration) of the test item was prepared.
The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.
The pH of the solution was checked.

CONTROLS
Abiotic Control (A)
In parallel to the study, abiotic control flasks (A1, A2, A3, A4, and A5) were tested without inoculum, but with the highest concentration of the test item, under identical test conditions.
Untreated Control (B)
Five replicates (B1, B2, B3, B4, and B5) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the test item) were tested. The study sample measurements were made in blocks of 6 samples, there was always one blank control in each set of 6 (one blank and 5 treated or positive control samples per series, in 5 series, hence 5 blank samples).
Reference Control (REF)
In parallel with the test item, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3) was tested (the nominal test concentrations of 40; 4 and 0.4 mg/L) under otherwise identical test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Veszprém, Hungary.
- Preparation of inoculum for exposure: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge was suspended in chlorine-free tap water to yield a concentration equivalent to 2 g per litre (on dry weight basis).
The pH of the activated sludge inoculum was determined to be pH 7.80. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.2 - 21.8°C
Nominal and measured concentrations:
10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Infusion bottles of approximately 500 mL volume, and BOD bottles with special neck of approximately 330 mL volume.
- Aeration: compressed air (approx. 1 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5 (B); 3 (REF)
- No. of vessels per abiotic control (replicates): 5
- Sludge concentration (weight of dry solids per volume): 2 g per litre
- Nutrients provided for bacteria: Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL); Peptone 16.0 g, Meat extract 11.0 g, Urea 3.0 g, NaCl 0.7 g, CaCl2 x 2H2O 0.4 g, MgSO4 x 7H2O 0.2 g, K2HPO4 2.8 g, Deionised water add 1000.0 ml

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Respiration rate (Dissolved oxygen concentration) recorded every 30 seconds for 15 minutes after incubation time.
- Water Temperature: Start and end of incubation period
- pH: Start and end of incubation period

TEST CONCENTRATIONS
- Range finding study : No
- Test concentrations: 10,100 and 1000mg/L (nominal)
- Results used to determine the conditions for the definitive study: Not applicable
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 25.12 mg O2/L/h/g solid.
- Coefficient of variation of oxygen uptake rate in control replicates: 3.41 %.
Results with reference substance (positive control):
- Results with reference substance valid? Yes - The EC50 value of the 3,5-DCP is lies in the range of the international ring test (in a 2004 international ring test organized by ISO (4)) using activated sludge derived from domestic sewage.
- Relevant effect levels: The EC50 of the positive control substance 3,5-DCP was 3.98 mg/L (in the range 2 mg/L to 25 mg/L).
Reported statistics and error estimates:
The 3-hour EC50 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software, for the reference item.

Table 2: Results

Test Group

Conc. Of test item in test mix. (mg/L)

Total O2consumption rate (RT) (mg O2/L/hr)

Specific respiration rate (RS) (mg O2/L/h/g solid)

Inhib

(%)

pH values

Temp (°C)

Variation coefficient of O2 consumption rate (%)

ID

Name

start

end

(Min./Max.)

A1

Abiotic

1000

0.84

-

-

7.29

7.09

19.2/21.8

-

A2

1000

0.83

-

-

7.31

7.08

-

A3

1000

0.89

-

-

7.33

7.07

-

A4

1000

1.11

-

-

7.31

7.07

-

A5

1000

0.56

-

-

7.31

7.07

-

 

B1

Blank

0.0

25.86

25.86

-

7.75

7.91

19.2/21.8

3.41

B2

0.0

23.83

23.83

-

7.77

7.93

B3

0.0

25.93

25.93

-

7.73

7.94

B4

0.0

25.07

25.07

-

7.76

7.94

B5

0.0

24.90

24.90

-

7.71

7.89

 

REF1

Ref. Item

40.0

3.99

3.99

86.83

7.77

7.93

19.2/21.8

-

REF2

4.0

12.94

12.94

49.24

7.77

7.92

-

REF3

0.4

21.40

21.40

13.74

7.78

7.95

-

 

TI1

Test Item

10

24.30

-

9.56

7.75

7.95

19.2/21.8

-

T12

100

24.35

-

6.26

7.73

7.96

-

T13

1000

20.67

-

20.38

7.51

7.87

-

Validity criteria fulfilled:
yes
Remarks:
The reference control EC50 and other validation criteria were met in this study.
Conclusions:
The test item (LiPO2F2) had no significant inhibitory effect on the test system with three concentrations (10, 100 and 1000 mg/L) of the test item. The EC50 value was not calculated due to insignificant inhibition.

The EC50 of the test item in this study was higher than 1000 mg/L hence no further testing with this test item is required.

Description of key information

In an OECD guideline, GLP-compliant study, activated sludge respiration inhibition was determined. The 3h EC50 was greater than the highest concentration tested (1000mg/L).

The key value used for the Chemical Safety Assessment was conservatively set at 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The NOEC was not determined in the study.