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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-02-22 to 2000-08-21
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:
no
GLP compliance:
yes
Specific details on test material used for the study:
batch number: protocol and labelling: 860-9806-703
description: translucent yellow liquid
composition: peroxide content: approximately 70%
isododecane content: approximately 30%
density: 0.921 at 7°C
container: one plastic flask
date of receipt: 1 December 1999
storage conditions: at -20°C.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Each concentration was prepared separately by adding 16 mL of 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 substance (added directly using a micropipette or a syringue for microinjections) in the case of the test solutions. Each mixture was then made up to 500 mL by adding 200 mL of inoculum. The solutions were aerated at between 0.5 to 1 litre of air per minute using a Pasteur-pipette 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 during a few minutes.

Groups of test and reference solutions and controls were set up as follows:
- two controls containing inoculum at 1.6 g/L,
- three test concentrations containing CUMYLPEROXYNEODECANOATE at 2, 10 and 20 mg/L and inoculum at 1.6 g/L,
- three reference concentrations containing the reference substance (3,5-Dichlorophenol) at 4,12 and 36 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 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 organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Emeraude (SIARR) (76141 Petit-Quevilly, France).
- Method of cultivation: The inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particules) and washed with dechlorinated water.
- Pre-treatment: 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 litre, just before agitation. Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.
Temperature: between 22 °C and 23 °C.
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 to 8.0 using sodium bicarbonate solution.
- Initial biomass concentration: Four samples of 50 mL were taken to determine the content in suspended matter (dry weight). The concentration of sludge (6.74 g/L) was then adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
280 ± 20 mg/L as CaCO3.
Test temperature:
22 - 23 °C
pH:
6.0 - 8.0
Dissolved oxygen:
Not reported in detail: Air was bubbled through each solution at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements.
Nominal and measured concentrations:
Nominal concentrations: 2, 10, 20 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: BOD flask
- Aeration: yes, air was bubbled through each solution at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2
- No. of vessels per reference control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was checked before use. The inoculum was buffered, if necessary, to pH 6.0 to 8.0 using sodium bicarbonate solution.
- Illumination: the stock vessel was kept away from light by wrapping it in opaque material

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Respiration rate
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The respiration rate of the test solution at the highest concentration in cumylperoxyneodecanoate (20 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 two other test solutions was not determined.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the difference between the two controls was below 15% and the EC50 of 3,5-dichlorophenol was between 5 and 30 mg/L
- Relevant effect levels: EC50 of 3,5-dichlorophenol: 10 mg/L (95% C.I.: 9-12)
Reported statistics and error estimates:
When for at least two concentrations, inhibition is > 0 % and < 100 %, the EC50 is calculated according to Probit analysis (i.e. Finney’s method, published by E. Weber, combined with Bliss’s method). The confidence interval limits are calculated statistically according to Fieller’s method.
When at only one concentration, inhibition is > 0 % and < 100 %, the EC50 is also calculated by Probit analysis. In this case, the highest concentration causing no inhibition and the lowest concentration producing 100 % inhibition are used as confidence limits.
If at all concentrations inhibition is 0 % or 100 %, the EC50 corresponds to the geometric mean of the highest concentration causing no inhibition and the lowest concentration producing 100 % inhibition.

The respiration rate of the test solution at the highest concentration in cumylperoxyneodecanoate (20 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 two other test solutions was not determined.

EC50s, based on nominal concentrations, were as follows:

 Substance  3-hour EC50 (mg/L)  95% confidence limits (mg/L)
 Test substance  > 20  
 Reference substance 10   9 - 12

Based on the inhibition percentage of the respiration rate obtained in the test solution of highest concentration, the 3-hour EC10 of the test substance was also > 20 mg/L

Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition test according to OECD 209 resulted in an EC50 > 20 mg/L. The test substance did not inhibit the respiration of the activated sludge. Cumylperoxyneodecanoate (70 % in isododecane) is not toxic to aerobic microorganisms in sewage sludge.
Executive summary:

The effect of cumylperoxyneodecanoate (70 % in isododecane) on respiration of activated sewage sludge was evaluated using a 3 -hour static test according OECD guideline 209. No inhibition (0 %) of the respiration rate was noted at 2, 10 and 20 mg/L of cumylperoxyneodecanoate, relative to mean of the controls. The 3-hour EC50 of the test item for activated sludge is > 20 mg/L. As the validity criteria were met (the difference between the two controls was below 15 % and the EC50 of the reference substance 3,5-dichlorophenol was between 5 and 30 mg/L. The test is considered valid.

Description of key information

A respiration inhibition test with activated sludge according OECD guideline 209 was conducted by L´Haridon (2000) to determine the toxicity of 1-methyl-1-phenylethyl peroxyneodecanoate (70% in isododecane) towards microorganisms. The respiration rate of the test solution at the highest test concentration of 20 mg/L (nominal) was equivalent to the respiration rate of the control.

This result can be used for the registration of pure 1-methyl-1-phenylethyl peroxyneodecanoate after backcaluclation to the content of 1-methyl-1-phenylethyl peroxyneodecanoate in isododecane. The NOEC for pure 1-methyl-1-phenylethyl peroxyneodecanoate is 14 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
14 mg/L

Additional information