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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-11-2011 -22-11-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP according the guideline meeting all validity criteria
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
minor deviation
Principles of method if other than guideline:
The average oxygen uptake rate for the control replicates was slightly lower than prescribed in the guideline
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (21-11-2011) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge
plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The dry weight of the
homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry
weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature of the reaction mixtures after the incubation period ranged from 20.1 to 20.8°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.1.
Dissolved oxygen:
aerobic during test
Nominal and measured concentrations:
The nominal concentrations of 3,5-Dichlorophenol tested were: 1.3, 2.5, 5.1, 10.2, 20.3 and 40.6 mg/L
The nominal concentrations of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate tested were: 38, 74, 152, 316, 644 and 1280 mg/L.
Details on test conditions:
Test conditions:
The test was performed in 0.30 liter Erlenmeyer flasks with a total working volume of 50 ml. The homogenized activated sludge was incubated in a
shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test compound and synthetic sewage

Synthetic sewage feed and stocks:
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water:
16 g peptone,
11 g meat extract,
3 g urea,
0.7 g NaCl,
0.3 g CaCl2,
0.2 g MgSO4.7H2O,
2.8 g K2HPO4.
In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL.

O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was weighed directly in the incubation vessels.
3,5-Dichlorophenol was added to the incubation vessels using a stock solution of approximately 1.0 g/L.
The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1046 g 3,5-dichlorophenol in
approximately 40 mL of deionized water with the addition of a few drops of 3 M NaOH.
Finally the pH was corrected with H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.




Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 280 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.1. Temperature varied from 20.1 to 20.8°C. These conditions allow respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated by the following criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was
3%, which is smaller than the maximum prescribed variation of <30%. Secondly, the EC50 of the reference compound was 8 mg/L which is within the
prescribed range of 2 to 25 mg/L (Table I and Figure 1). The third criterion, an oxygen uptake rate of >20 mg O2/g dry weight/hour for the control
measurements, was not achieved. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and therefore
has a natural lower oxygen uptake rate. The average oxygen uptake rate for the control replicates was 15 mg O2/g dry weight/hour, which is slightly lower than the prescribed rate. Accurate oxygen uptake rate measurements were very well possible, as shown by the very low coefficient of variation
between the control replicates and therefore this is a minor deviation which does not invalidate the test.

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate are presented in Table II and the
results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate at a particular
concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The EC50 of
O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate for activated sludge after 3 hours contact time is > 1280 mg/L. O-(2-ethylhexyl)
O,O-tert-pentyl peroxycarbonate is therefore considered harmless to activated sludge.
Inhibition of the activated sludge respiration ranged from 14% to 24% over the measured concentration range of 38 to 1280 mg/L (Figure 2). The reason for this divergent concentration-effect relationship may be explained by the low water solubility of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate. Poorly water soluble substance may not be bioavialable at higher concentrations
Results with reference substance (positive control):
The EC50 of the reference compound (3,5-dichlorophenol) was 8 mg/L which is within the prescribed range of 2 to 25 mg/L
Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol (8 mg/L) and the coefficient of variation of the replicates of the
control oxygen uptake rates (3%). The average oxygen uptake rate for the control replicates is slightly lower (15 mg O2/g dry weight/hour) than the
prescribed rate. The accuracy of the oxygen uptake rate measurements is not affected by the slightly lower oxygen uptake rates, as shown by the low
coefficient of variation for the replicates of the control oxygen uptake rates, and herewith does not invalidate the test.
The toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to activated sludge was determined at a contact time of 3 hours, using various
concentrations of the test substance. The inhibitory effect of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate at a particular concentration is expressed as a percentage of the two controls. The EC50 of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate for activated sludge after 3 hours contact time
is > 1280 mg/L. O-(2-ethylhexyl) O,O-tertpentyl peroxycarbonate is therefore considered harmless to activated sludge.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference compound, 3,5- dichlorophenol (8 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (3%). The average oxygen uptake rate for the control replicates is slightly lower (15 mg O2/g dry weight/hour) than the prescribed rate. The accuracy of the oxygen uptake rate measurements is not affected by the slightly lower oxygen uptake rates, as shown by the low coefficient of variation for the replicates of the control oxygen uptake rates, and herewith does not invalidate the test. The toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate at a particular concentration is expressed as a percentage of the two controls. The EC50 of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate for activated sludge after 3 hours contact time is > 1280 mg/L. O-(2-ethylhexyl) O,O-tertpentyl peroxycarbonate is therefore considered harmless to activated sludge.

Description of key information

One OECD 209 test has been conducted under GLP conditions. The EC50 of for activated sludge after 3 hours contact time is > 1280 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
1 280 mg/L

Additional information

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference compound, 3,5- dichlorophenol (8 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (3%). The average oxygen uptake rate for the control replicates is slightly lower (15 mg O2/g dry weight/hour) than the prescribed rate. The accuracy of the oxygen uptake rate measurements is not affected by the slightly lower oxygen uptake rates, as shown by the low coefficient of variation for the replicates of the control oxygen uptake rates, and herewith does not invalidate the test. The toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate at a particular concentration is expressed as a percentage of the two controls. The EC50 of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate for activated sludge after 3 hours contact time is > 1280 mg/L. O-(2-ethylhexyl) O,O-tertpentyl peroxycarbonate is therefore considered harmless to activated sludge.