Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A sample of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (see annex 2) was received from AkzoNobel Functional Chemicals Deventer, The Netherlands
and archived on 16-06-2011. The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
Chemical name: 1,1,3,3-tetramethylbutyl peroxyneodecanoate
Product name: Trigonox® 423-C70
CAS reg. No.: 51240-95-0
Purity: 70.5%
Batch/lot No.: 1102442046
Appearance: colourless clear liquid
Solubility in water: insoluble in water
Volatile: No (<0.006 kPa, 20°C)
Log Kow : >6.5
Stability: unstable under test conditions, reaction with organics in test
Storage: in the freezer (<-20°C) in the dark

The concentrations cited in this report refer to the as-received sample of 1,1,3,3-tetramethylbutyl peroxyneodecanoate

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

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 was 7.0
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 1,1,3,3-tetramethylbutyl peroxyneodecanoate tested were: 36, 74, 152, 320, 638 and 1276 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.

1,1,3,3-tetramethylbutyl peroxyneodecanoate 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

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 276 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 was 7.0. 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 1,1,3,3-tetramethylbutyl peroxyneodecanoate are presented in Table II and the results of
the graphical probit analysis are shown in Figure 2. The inhibitory effect of 1,1,3,3-tetramethylbutyl peroxyneodecanoate at a particular concentration is
expressed as a percentage of the two controls. From the results EC values were calculated. The EC10 and EC50 of
1,1,3,3-tetramethylbutyl peroxyneodecanoate for activated sludge after 3 hours contact time are >1276 mg/L.
1,1,3,3-tetramethylbutyl peroxyneodecanoate 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

Applicant's summary and conclusion

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 1,1,3,3-tetramethylbutyl peroxyneodecanoate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of 1,1,3,3-tetramethylbutyl peroxyneodecanoate at a particular concentration is expressed as a percentage of
the two controls. The EC10 and EC50 of 1,1,3,3-tetramethylbutyl peroxyneodecanoate for activated sludge after 3 hours contact time are > 1276 mg/L.
1,1,3,3-tetramethylbutyl peroxyneodecanoate is therefore considered harmless to activated sludge.
Executive summary:

In order to predict effects of chemicals in the environment and in biological wastewater 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 1,1,3,3-tetramethylbutyl peroxyneodecanoate to activated sludge

was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of 1,1,3,3-tetramethylbutyl peroxyneodecanoate at a particular concentration is expressed as a percentage of

the two controls. The EC10 and EC50 of 1,1,3,3-tetramethylbutyl peroxyneodecanoate for activated

sludge after 3 hours contact time are > 1276 mg/L. 1,1,3,3-tetramethylbutyl peroxyneodecanoate is therefore considered harmless to activated sludge.