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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 16, 2012 - May 25, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to guideline and GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Lemonile
- Physical state: Liquid
- Anlytical purity: 99.4% (sum of components)
- Lot/batch No.: VE00101509
- Expiration date of the lot/batch: September 09, 2012
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance is not soluble enough in water to allow the preparation of an aqueous stock solution in tap water.

Different solutions were prepared by dissolution of Lemonile in ethanol in order to add always the same volume (ie 5 ml) of ethanol in each cylinder.


The following solutions were prepared:

A1: 10.00 g/l; prepared by dissolving of 1000 mg of Lemonile in 100 ml ethanol

A2: 3.16 g/l; prepared by dissolving of 316 mg of Lemonile in 100 ml ethanol

A3: 1.00 g/l; prepared by diluting 2 ml stock solution A1 to a total volume of 20 ml with ethanol

A4: 0.32 g/l; prepared by diluting 2 ml of stock solution A2 to a total volume of 20 ml with ethanol

A5: 0.10 g/l; prepared by diluting 2 ml of stock solution A3 to a total volume of 20 ml with ethanol
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was kept aerobic until being used on the same day. The pH was between 7 and 8.

The suspended solids content was determined by drying and weighing 50 ml of the sludge: dry weight of suspended solids: 3.939 g/l

By adding 200 ml of sludge to the 500 ml test vessels, the final concentration of suspended solids was 1.58 g/l, ie in the range 0.8 to 1.6 g/l as recommended in ISO 8192-1986
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
Refer to Table 1 test results
Dissolved oxygen:
Refer to Table 1 test results
Nominal and measured concentrations:
1.0, 3.2, 10.0, 31.6 and 100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml scaled cylinders
- Aeration: Yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 90 mg/L
Results with reference substance (positive control):
- Results with reference substance are valid. The EC50 (3 hours) of 3,5-dichlorophenol is between 5 and 30 mg/l

Table 1: Test results

vessel

pH

∆O2 [mg/l]

Time [min]

rate

% inhibition

Conc.finale [mg/l]

Log (conc)

C1

8.03

2.80

5.00

0.56

-

-

-

Tr

8.02

2.55

5.00

0.51

-

-

-

R1

8.05

2.30

5.00

0.46

14.0

5.0

0.70

R2

8.06

1.15

5.00

0.23

57.0

30.0

1.48

S1

8.05

1.20

5.00

0.24

55.1

100.0

2.00

S2

8.03

2.80

5.00

0.56

-4.7

31.6

1.50

S3

8.02

2.90

5.00

0.58

-8.4

10.0

1.00

S4

8.03

2.75

5.00

0.55

-2.8

3.2

0.51

S5

8.01

2.75

5.00

0.55

-2.8

1.0

0.00

Only at a test concentration of 100 mg/l a significant inhibition of the respiration was observed. For estimating the EC50 (3 hours) a linear relationship between % inhibition and the common logarithm of the test substance concentration was assumed for concentrations ranging from 31.6 mg/l (test vessel S2) to 100 mg/l (test vessel S1). A linear regression was performed (least squares method) and parameters a and b were obtained:

 

% inhibition = a * log conc. + b

 

a = 119.3  b = -184.0

 

Using the equation and parameters above, the concentration corresponding to an inhibition of 50% was calculated EC50 = ~ 0.9 . 102mg/l 

Validity criteria fulfilled:
yes
Conclusions:
The EC50 ( 3 hours) of Lemonile is ~ 90 mg/l

Description of key information

EC50 (3  hours) ~ 90 mg/L (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
90 mg/L

Additional information

A reliable study was performed in accordance with GLP and OECD Guideline No. 209 to assess the effect of Lemonile in an activated sludge respiration inhibition test. Five concentrations were tested in the range 1 to 100 mg/L. The 3 hour EC50 of Lemonile was found to be approximately 90 mg/L.