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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 7.3.
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 dihexadecyl peroxodicarbonate tested were: 38, 76, 153, 305, 611 and 1220 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.

Accurate administration of the water-insoluble dihexadecyl peroxydicarbonate was accomplished by dissolving the test substance in
dichloromethane. Stock solutions of approximately 1.0 and 2.0 g/L were prepared by dissolving 0.0954 g and 0.2047 g in 100 mL dichloromethane,
respectively. The test substance was added with the dichloromethane to the incubation vessels and the dichloromethane was removed by placing the bottles in a ventilated hood for 24 hours.
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.:
> 1 220 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 7.3. Temperature varied from 20.1 to 20.8°C. These conditions allow
respiration of the activated sludge used.

Validity of the test
The reference substance and test substance were measured on two subsequent days. The validity of the test is demonstrated by three criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 2% on the first day and 3% on the second day, which fulfils 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 on the first day (15 mg O2/g dry weight/hour) and was met on the second day (26 mg O2/g dry weight/hour). 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. According the guideline activated sludge used should be fed with synthetic sewage feed, for a maximum of two additional days, when used after the day of collection. Adding synthetic sewage feed leads to an increase of the oxygen uptake rate of the activated sludge, but does not affect the sensitivity of the activated sludge significantly (AkzoNobel, 2012).
Accurate oxygen uptake rate measurements were very well possible, with the slightly lower oxygen uptake rate on the first day, as shown by the very low coefficient of variation. 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 dihexadecyl peroxodicarbonate are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of dihexadecyl peroxodicarbonate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of dihexadecyl peroxodicarbonate for activated sludge after 3 hours contact time is > 1220 mg/L. Dihexadecyl peroxodicarbonate is therefore considered harmless to activated sludge.
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 reference substance and test substance were measured on two subsequent days. The test is valid as shown by the EC50 of the reference
compound, 3,5-dichlorophenol (8 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (2% on first day and 3% on second day) and the average oxygen uptake rate for the control replicates of 15 mg O2/g dry weight/hour on the first day and 26 mg O2/g dry weight/hour on the
second day. The oxygen uptake rate on the first day is slightly lower than prescribed. Accurate oxygen uptake rate measurements were very well possible
as shown by the very low coefficient of variation. Therefore this is a minor deviation which does not invalidate the test.
The toxicity of dihexadecyl peroxodicarbonate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test
substance. The inhibitory effect of dihexadecyl peroxodicarbonate at a particular concentration is expressed as a percentage of the two controls.
The EC50 of dihexadecyl peroxydicarbonate for activated sludge after 3 hours contact time is > 1220 mg/L. Dihexadecyl peroxodicarbonate 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 reference substance and test substance were measured on two subsequent days. The test is valid as shown by the EC50 of the reference compound, 3,5- dichlorophenol (8 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (2% on first day and 3% on second day) and the average oxygen uptake rate for the control replicates of 15 mg O2/g dry weight/hour on the first day and 26 mg O2/g dry weight/hour on the second day. The oxygen uptake rate on the first day is slightly lower than prescribed. Accurate oxygen uptake rate measurements were very well possible as shown by the very low coefficient of variation. Therefore this is a minor deviation which does not invalidate the test. The toxicity of dihexadecyl peroxodicarbonate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of dihexadecyl peroxodicarbonate at a particular concentration is expressed as a percentage of the two controls. The EC50 of dihexadecyl peroxodicarbonate for activated sludge after 3 hours contact time is > 1220 mg/L. Dihexadecyl peroxodicarbonate is therefore considered harmless to activated sludge.

Description of key information

The EC50 of dihexadecyl peroxodicarbonate for activated sludge after 3 hours contact time is > 1220 mg/L. Dihexadecyl peroxodicarbonate is therefore considered harmless to activated sludge.

Key value for chemical safety assessment

Additional information