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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 2: Effects on biotic systems, Guideline no. 209, "Activated Sludge, Respiration Inhibition Test", adopted April 4, 1984.
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
European Economic Community (EEC), EEC Directive 67/548 amended November 18, 1987 (87/302), Part C: Methods for the determination of ecotoxicity, Publication No. L 133, "Biodegradation: Activated sludge respiration inhibition test', adopted May 30, 1988.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether
EC Number:
Cas Number:
Molecular formula:
1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether
Test material form:
Specific details on test material used for the study:
Molecular formula: C4H3F7O
Molecular weight: 200.07
CAS Number: 406-78-0
Description: Clear colourless liquid
Batch: 50407070
Purity: 99.985%
Test substance storage: In refrigerator in the dark
Stability under storage conditions: Stable
Expiry date: 07 July 2005
Specific Gravity: 1.47
Stability in vehicle: Water, Not indicated

Sampling and analysis

Analytical monitoring:
Details on sampling:
1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was known to be extremely volatile and the test concentration was not confirmed analytically (not a requirement of the test guideline), the stability of the test substance in the test system could not be shown.

Test solutions

Details on test solutions:
Test substance - preparation and test concentrations
1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was a clear colourless liquid with a purity of 99.985%. The water solubility of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether at 20.1 ± 0.5°C was determined to be 0.805 g/1, using the flask method. Since 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was extremely volatile, no stock solution of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was prepared, but the test substance was quantitatively added to the test vessels. A nominal concentration of 100 mg/I was tested in duplicate. Since 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was known to be extremely volatile and the test concentration was not confirmed analytically (not a requirement of the test guideline), the stability of the test substance in the test system could not be shown. However, every possible effort was made to prevent the test substance from evaporating from the test vessels (low air flow, narrow flasks with a minimum of headspace and direct addition of the test substance). Results were based on nominal concentrations.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test System: Micro-organisms in activated sludge.

Source: Municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage.

Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.

Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with tap-water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (3.0 g/1 of sludge, as used for the test). The pH was 7.2 on the day of sampling and 7.9 on the day of testing. The batch of sludge was used on the day after collection, therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the day. The sludge was kept aerated at test temperature until use.

Rationale: Recognized by international guidelines as the recommended test system.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
30 min

Test conditions

Test temperature:
19.0 and 20.5°C
Nominal and measured concentrations:
Nominal: 0, 100 mg/l.
Details on test conditions:
Contact time: 30 minutes, during which aeration and stirring took place.

Vessels: All glass, approximately 300 ml oxygen bottles and 1l test bottles.

Milli-RO/Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion­exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7g NaCl
0.4 g CaCl2.2H20
0.2 g MgSO4.7H20
2.8 g K2HPO
Dissolved in Milli-Q water, made up to 1 I and filtered.
The pH was 7.1.

Air supply: Clean, oil-free air.

Oxygen meter: WTW inolab Oxi Level 2 supplied with a WTW CellOx 325 oxygen electrode, electrolyte type EL Y/G.

Recorder: Flatbed recorder SE 102 (Kipp & Zonen).

Performance of the test: The synthetic sewage feed (16 ml) and an adequate amount of the test substance were mixed and made up to 300 ml with Milli-RO water. Activated sludge (200 ml) was added to provide a final volume of 500 ml. The mixture was then aerated in a 1 I bottle during the contact time, using a pipette as an aeration device.

Then a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption
was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the
remaining part of the reaction mixture.

This procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and one at the end.

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol.

Acceptability of the test:
1. The mean respiration rate of control 1 and 2 was 32 mg 02/1/hr. The difference between the controls was within 15% of each other (3%). The mean respiration rate of control 3 and 4 was 36 mg
02/1/hr. The difference between the controls was within 15% of each other (3%).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/I (14.9 mg/I, regression line: Y = 47.05 X -5.21, Y = % inhibition and X log concentration (mg/I)).

Results and discussion

Effect concentrations
Key result
30 min
Dose descriptor:
Respiration inhibition
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
No significant inhibition of respiration rate of the sludge was recorded at nominal 100 mg 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed.

Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/I.

Any other information on results incl. tables

Controls (C) and 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether (T): pH, oxygen concentration, oxygen consumption and percentage Inhibition

 Flask  Concentration (mg/I)  O2 concentration at start (mg O2/l)  O2 consumption mg O2/l/hr % inhibition pH 
C3 0 6.2  35 8.0 





Mean C3 + C4


36 (3%) 













Controls (C) and reference substance (R; 3,5-dichlorophenol): pH, oxygen concentration, oxygen consumption and percentage inhibition


 Concentration (mg/I)

 O2 concentration at start (mg O2/l)

 O2 consumption mg O2/l/hr % inhibition pH 
C1 0 6.2  32  8.1 
C2 0 6.6 31 8.1 
Mean C1 +C2   32 (3%)     
R1 3.2 6.7  25  21  8.2 
R2 10  6.3  20  37  8.0 
R3 32 6.8  10 68  7.9 

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the conditions of this present test, 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/I.
Executive summary:

The toxicity of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether to activated sludge was determined in a respiration inhibition study conducted in accordance with OECD TG 209. The study determined a 30 minute EC50 (respiration) of >100 mg/l (nominal).