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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Carried out using OECD guideline and under GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
not required
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
Preparation of the test and reference solutions
Each test solution was prepared by direct addition of the test item in the test flask with an appropriate micropipette (the volume added was based on the density = 920 kg/m3 at 25°C) and vigorous agitation of the solution for a few minutes before the beginning of the test (i.e. just before addition of the inoculum).

The reference stock solution was prepared by dissolving 0.5 g of the reference item, 3,5-dichlorophenol, in 10 mL of NaOH at 1 N. The obtained preparation was then diluted to 30 mL with purified water. Aliquots of H2S04 at 1 N were added while stirring until the point of incipient precipitation was obtained (approximately 8 mL of H2S04 at 1 N were required). The solution was made up to 1000 mL using purified water. The reference stock solution was agitated for 19 hours. The pH of this solution was 7.87 after agitation.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Type:
activated sludge collected from a water treatment works containing effluent from a predominantly domestic origin.

Origin:
the water treatment plant Emeraude (SIARR, 76141 Petit-Quevilly, France).

Preparation:
The inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particles) and washed with dechlorinated water. Four samples of 50 mL were taken to detennine the content in suspended matter (dry weight). The concentration of sludge (5.8 gIL) was then adjusted to 4 gIL by addition of the appropriate volume of dechlorinated water.

Environmental conditions during culture
The inoculum was maintained under agitation for one day before the test. As the sludge was not used immediately, 50 mL of sewage feed were added, per liter, just before agitation. Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.

Temperature: 20°C ± 2°C. The temperature in the culture room was recorded continuously and records retained.
Illumination: the stock vessel was kept away from light by wrapping it in opaque material.
Aeration: clean air was passed through the inoculum at the rate of 0.5 to 1 L/min until use.
pH: was checked before use. The inoculum was buffered, if necessary, to pH 6.0 -8.0 using sodium bicarbonate solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not relevant
Hardness:
no data
Test temperature:
20-22 oC
pH:
6.0 -8.0 (inoculum)
Dissolved oxygen:
air passed in inoculum at rate of 0.5 to 1 L/min until use
Salinity:
not applicable
Nominal and measured concentrations:
nominal - 10, 31.6, 100, 316, 1000
Details on test conditions:
Test system
Groups of test and reference solutions and controls were set up as follows:
-two controls containing inoculum at 1.6 g/L,
- three reference concentrations containing the reference item 3,5-dichlorophenol at 4, 12 and 36 mg/L and inoculum at 1.6 g/L.
-five concentrations containing the test item 2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane at 10,31.6, 100,316 and 1000 mg/L and inoculum at 1.6 g/L,
These solutions were prepared in test flasks and then immediately aerated for 3 hours before being transferred to an oxygen measuring apparatus.

Measurement of oxygen concentration of the above test solutions was determined using an oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test or reference solution which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter and thence to a chart recorder.

Test method
Each concentration was prepared separately by adding 16mLof sewage feed to each of the flasks which were made up to 300 mL with water only in the case of the controls or a nominal mixture of water and test item (added directly using a micropipette) in the case of the test solutions. Each mixture was then made up to 500 mL by adding 200 mL of inoculum. As 200 mL of inoculum were added in a final volume of 500 mL, the organic material concentration from inoculum was 1.6 g/L. The solutions were aerated at between 0.5 to 1 liter of air per minute using a Pasteur-pipette connected with a flexible tube to an air pump as an aeration device.

Solutions were prepared approximately every 15 minutes for test times of 3 hours.

In order to verify the quality of the inoculum, the controls were prepared at the beginning (first control) and the end (second control) of the test.

After 3 hours of aeration/contact time, the content of the test flask was poured into the measuring apparatus and the oxygen concentration was determined for a period of approximately 10 minutes.

Reference substance (positive control):
yes
Remarks:
2,5 dichlorophenol
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
Remarks:
respiration rate
Details on results:
The respiration rate of the test solution of highest concentration (1000 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test solutions (10 to 316 mg/L) was not determined.
Results with reference substance (positive control):
3hr EC50 = 10.9 mg/L 95% C.I. 8.86 - 13.4. The EC50 of 3,5-dichlorophenol is between 5 and 30 mg/L, the test is therefore considered valid with respect to the positive control.
Reported statistics and error estimates:
see above for reference substance

Respiration rate in O2/L/hr

 

Conc (mg/L)

Resp. rate (mg O2/L/hr

Inhibition (%)

Control 1

0

37.2

 

Control 2

0

36.0

 

 

 

 

 

Reference item

4

28.8

21

 

12

16.8

55

 

36

6.9

81

 

 

 

 

2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane

 

10

Not determined

 

 

31.6

Not determined

 

 

100

Not determined

 

 

316

Not determined

 

 

1000

32.0

13

Validity criteria fulfilled:
yes
Remarks:
As the difference between the two controls was below 15% and the EC50 of 3,5-dichlorophenol was between 5 and 30 mg/L, the test was therefore considered valid.
Conclusions:
Under the experimental conditions, the 3-hour EC50 of the test item 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYL PEROXY)HEXANE is > 1000 mg/L for activated sludge.
Executive summary:

Summary

At the request of ATOFINA, Paris-la-Defense, France, the effect of the test item, 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYL PEROXY)HEXANE, on the respiration of activated sewage sludge was evaluated using a 3-hour static test according to OECD guideline (No. 209, 4th April 1984).

 

Methods

The test solutions were prepared as follows:

Two control solutions: activated sludgeindechlorinated water to obtain a final sludge concentration of 1.6 g/L,

Five test item solutions: activated sludge in dechlorinated water (at 1.6 g/L) and test item (to obtain final concentrations of 10, 31.6, 100, 316 and 1000 mg/L),

 Three reference items (3,5-dichlorophenol) solutions: activated sludge in dechlorinated water (at1.6 g/L)and reference item (to obtain final concentrations of 4, 12 and 36mg/L). .

 

The oxygen consumption of these solutions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours.

 

The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and the reference item was determined considering the oxygen consumption of the controls as 100

 

Results

As the validity criteria were met (the difference between the two controls was below 15% and the EC50 of3,5-dichlorophenol was between 5 and 30mg/L),the test was considered valid.

 The respiration rate of the test solution of highest concentration (1000mg/L)was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test solutions (10 to 316mg/L)was not determine  EC50s, based on nominal concentrations, were as follows:Test item (3 hr EC50) > 1000 mg/LReference item (3 hr EC50) = 10.9 mg/L

Conclusion

Under the experimental conditions, the 3-hour EC50 of the test item 2,5-DIMETHYL-2,5­DI(2-ETHYLHEXANOYL PEROXY)HEXANE is > 1000 mg/L for activated sludge. The test item should be considered as non-toxic for the micro-organisms of a water treatment plant


Description of key information

One OECD 209 study is available and the 3 h  EC50 of the test item 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYL PEROXY)HEXANE  is > 1000 mg/L for activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

There is one OECD 209 study available carried out under GLP. Under the experimental conditions, the 3-hour EC50 of the test item 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYL PEROXY)HEXANE is > 1000 mg/L for activated sludge.