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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
An abiotic Control for the reference item was performed during this study although it was not planned in the study plan.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 20180329 (Source Lead)
- Expiration date of the lot/batch: March 2019
- Purity test date: March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: romm temperature 20 +/-10°C
- Stability under test conditions: stable
- Solubility in water : 0.19 to 0.26 g/L
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: directly diluted
- Controls: Yes
- Chemical name of vehicle : osmosed water
- Concentration of vehicle in test medium : 10 - 40 - 160 - 640 and 2560 mg/L
- Evidence of undissolved material: the test item is poorly soluble in water
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: FCBA
- Name and location of sewage treatment plant where inoculum was collected: STEP de Lille, 33290 BLANQUEFORT - FRANCE
- Method of cultivation: A filtration through a fine sieve (around 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in osmosed water and test medium ( 50/50 (v/v) to adjust concentration of dry extract around 3 g/L. The activated sludge was continuously aerated until usage in the test and fed daily with 50 mL/L synthetic sewage feed for one or two additional days.
- Test medium : mineral mediium (Na2HPO4, 2H2O, K2HPO4, NH4Cl, KH2PO4, CCl2, 2H2O, MGSO4, 7H2O, FeCl3, 6H20)
- Initial biomass concentration: 1.45 g suspended solids/L in this test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 d
Hardness:
No data
Test temperature:
20 +/-2°C
pH:
End of the test : 8.2 to 8.6
Salinity:
Not relevant
Conductivity:
Not relevant
Nominal and measured concentrations:
Nominal : 10 - 40 - 160 - 640 and 2560 mg/L
Details on test conditions:
The test was carried out into 1000 mL glass flasks containing 500 mL as below:
- 16.0 ml synthetic sewage feed (Peptone, Meat Extract, Ura, NCl, CaCl2-2H2O, MgSO4-7H20, K2HPO4, ultrapure water)
- Adequate weighing of test item
- osmosed water (final volume: 500 mL)
- 250 mL of sludge suspension

Replicates: Six replicates for controls (Control a to Control f)
Two replicates for abiotic controls
Three replicates (a, b and c) per treatment (1 to 5) for the test item
One replicate per treatment for the reference item (3,5 DCP)

The concentrations for the 3,5 DCP were : 0.5 – 1.6 – 5.1 – 16.40 and 52.4 mg/L

At the start of the test, the first control (Control a) was prepared and covered with parafilm. Then every 10 -15 minutes the others flasks were prepared in turn in the same conditions. Finally, the last control (Control f) was prepared.
All suspensions were aerated with purified air (0.5 to 1 L/min), and stirred during 3 hours at 20 ± 2°C. Then, each suspension was poured into Karlsruher flask (following the order preparation). The Oxygen was measured every minute, during 10 minutes, with a Dissolved Oxygen Sensor (WTW StirrOx G) integrated Stirrer, inserted into the Karlsruher flask

EFFECT PARAMETERS MEASURED : Activated sludge respiration inhibition, measured as oxygen consumption rate (dissolved oxygen concentration)
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 43.2 - < 296.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 165.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
ca. 40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
-
Results with reference substance (positive control):
- Results with reference substance valid : Yes
- Relevant effects levels : EC50 : 13.7 mg/L ; oxygen uptake in the control : 38.8 mg O2/ g sludge/h ; Coefficient of varation of O2 : 0.0 to 14.6%

  O2 consumption and inhibition

Treatment

Name

O2consumption in

mg O2/(L*h)

% inhibition

pH (end of the test)

Inoculum Control

Control a

51.00

/

8.4

Control b

52.50

/

8.4

Control c

61.50

/

8.3

Control d

60.00

/

8.2

Control e

57.60

/

8.2

Control f

55.20

/

8.3

Abiotic Control reference item

Abiotic Control 3,5 DCP

/

/

7.0

Reference Item

3,5 DCP Conc 1

55.5

1.42

8.3

3,5 DCP Conc 2

54.0

4.09

8.4

3,5 DCP Conc 3

48.0

14.74

8.4

3,5 DCP Conc 4

22.7

59.68

8.4

3,5 DCP Conc 5

7.5

85.79

8.4

Abiotic Control Test item

Abiotic Control Test item a

/

/

7.0

Test Item

1a

62.00

- 10.1

8.2

1b

62.00

- 10.1

8.2

1c

54.00

4.1

8.3

2a

42.00

25.4

8.3

2b

43.00

23.6

8.3

2c

36.00

36.1

8.4

3a

8.00

85.8

8.4

3b

3.43

93.9

8.5

3c

3.33

94.1

8.4

4a

0.00

100.0

8.6

4b

0.67

98.8

8.6

4c

0.00

100.0

8.6

5a

0.67

98.8

8.6

5b

0.00

100.0

8.6

5c

0.67

98.8

8.6

Abiotic Test item

Abiotic Test item b

/

/

7.0

Validity criteria fulfilled:
yes
Conclusions:
This study was performed using five concentrations, ranging from 10 to 2560 mg/L nominal concentrations. The dry matter of the activated sludge was determined as 2.9 g suspended solids/L, giving a concentration of 1.45 g suspended solids/L in this test. The validity criteria for OECD 209 were successful. And the nominal concentrations of 4-PhenoxyPhenol are :
- CE50-3h = 165.4 mg/L [43.2 - 296.6]
- NOEC = 10 mg/L
- LOEC = 40 mg/L
Executive summary:

An OECD 209 was performed on 4 -PhenoxyPhenol using five concentrations, ranging from 10 to 2560 mg/L nominal concentrations. The dry matter of the activated sludge was determined as 2.9 g suspended solids/L, giving a concentration of 1.45 g suspended solids/L in this test. The validity criteria for OECD 209 were successful. And the nominal concentrations of 4-PhenoxyPhenol are :

  • CE50-3h = 165.4 mg/L [43.2 - 296.6]
  • NOEC = 10 mg/L
  • LOEC = 40 mg/L

Description of key information

An OECD 209 was performed on 4 -PhenoxyPhenol using five concentrations, ranging from 10 to 2560 mg/L nominal concentrations. The dry matter of the activated sludge was determined as 2.9 g suspended solids/L, giving a concentration of 1.45 g suspended solids/L in this test. The validity criteria for OECD 209 were successful. And the nominal concentrations of 4-PhenoxyPhenol are :

  • CE50-3h = 165.4 mg/L [43.2 - 296.6]
  • NOEC = 10 mg/L
  • LOEC = 40 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
165.4 mg/L
EC10 or NOEC for microorganisms:
10 mg/L

Additional information