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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-12-2011 -07-12-2011
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 (05-12-2011) 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 19.9 to 20.9°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 6.9 to 7.5.
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 2,4-pentanedione, peroxide, 30% in solvent mixture tested were: 36, 80, 160, 316 , 638 and 1282 mg/L
Details on test conditions:
Test conditions:
The test was performed in 300 mL Erlenmeyer flasks with a total working volumes 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.

2,4-pentanedione, peroxide, 30% in solvent mixture 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.:
614 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits of 321 mg/L and 2450 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
182 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits of 95 mg/L and 728 mg/L
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 6.9 to 7.5. Temperature varied from 19.9 to 20.9°C. These conditions allow
respiration of the activated sludge used.

Validity of the test
The test was performed on two subsequent days after collection of the activated sludge. 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 5% on the first day and 6% on the second day, which fulfils the maximum
prescribed variation of <30%. Secondly, an oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements, was not achieved on the
first day (18 mg O2/g dry weight/hour) and was met on the second day (36 mg O2/g dry weight/hour). Oxygen uptake rate measurements were very
accurate 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.
The inoculum used, was obtained from a treatment plant which is operated at a low organic sludge load which is common. Low organic loads result in
low oxygen uptake of the sludge. 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. The toxicity of the test substance was assessed two days after collection of the activated sludge. To determine if the sensitivity of the activated sludge was changed due to the increased oxygen uptake rate the reference substance was measured again. The third criterion was met as shown by the EC50 of the reference
compound of 12 mg/L on the first day and 15 mg/L on the second day, which is within the prescribed range of 2 to 25 mg/L (Table I-II and Figure 1-2).
From these EC50 values it can also be concluded that the sensitivity of the activated does not significantly change due to the increased oxygen uptake rate.

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of 2,4-pentanedione, peroxide, 30% in solvent mixture are presented in Table III and the results of the graphical probit analysis are shown in Figure 3. The inhibitory effect of 2,4- pentanedione, peroxide, 30% in solvent mixture at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of 2,4-pentanedione, peroxide, 30% in solvent mixture for activated sludge after 3 hours contact time is 614 mg/L with 95% confidence limits of 321 and 2450 mg/L. The EC10, EC20 and EC80 are 210, 303 and 1245 mg/L, respectively. The active ingredient concentration of 2,4-pentanedione, peroxide, 30% in solvent mixture, (a mixture of cis-2,4-pentanedione peroxide
and trans-2,4-pentanedione peroxide) is 29.7 % w/w. Recalculated to the active ingredient concentration, the EC50 of a mixture of cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide for activated sludge after 3 hours contact time is 182 mg/L with 95% confidence limits of 95 and 728 mg/L. The EC10, EC20 and EC80 are 62, 90 and 370 mg/L, respectively.
Results with reference substance (positive control):
The EC50 of the reference compound (3,5-dichlorophenol) was 12 mg/L on the first day and 15 mg/L on the second day which is both within the prescribed range of 2 to 25 mg/L
Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The test was performed on two subsequent days after collection of the activated sludge. The test is valid as shown by the coefficient of variation of
the replicates of the control oxygen uptake rates (5% on the first day and 6% on the second day), the EC50 of the reference compound 3,5-dichlorophenol (12 mg/L on the first day and 15 mg/L on the second day) and the average control oxygen uptake rate of 18 mg O2/g dry weight/hour on the first day and 36 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 increase in the oxygen uptake rates on the second day compared to the first day does not significantly influence the sensitivity of the activated sludge
as shown by the EC50 values of the reference substance.

The EC50 of 2,4-pentanedione, peroxide, 30% in solvent mixture for activated sludge after 3 hours contact time is 614 mg/L with 95% confidence
limits of 321 and 2450 mg/L. The EC10, EC20 and EC80 are 210, 303 and 1245 mg/L, respectively. The active ingredient concentration of 2,4-pentanedione,
peroxide, 30% in solvent mixture, (a mixture of cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide) is 29.7 % w/w. Recalculated to the active
ingredient concentration, the EC50 of a mixture of cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide for activated sludge after 3 hours contact time is 182 mg/L with 95% confidence limits of 95 and 728 mg/L. The EC10, EC20 and EC80 are 62, 90 and 370 mg/L, respectively.
2,4-pentanedione, peroxide, 30% in solvent mixture is therefore considered not harmful to activated sludge and neither is the active ingredient a mixture of
cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide.


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 was performed on two subsequent days after collection of the activated sludge. The test is valid as shown by the coefficient of variation of the replicates of the control oxygen uptake rates (5% on the first day and 6% on the second day), the EC50 of the reference compound 3,5-dichlorophenol (12 mg/L on the first day and 15 mg/L on the second day) and the average control oxygen uptake rate of 18 mg O2/g dry weight/hour on the first day and 36 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 increase in the oxygen uptake rates on the second day compared to the first day does not significantly influence the sensitivity of the activated sludge as shown by the EC50 values of the reference substance. The toxicity of 2,4-pentanedione, peroxide, 30% in solvent mixture to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of 2,4-pentanedione, peroxide, 30% in solvent mixture at a particular concentration is expressed as a percentage of the two controls. The EC50 of 2,4-pentanedione, peroxide, 30% in solvent mixture for activated sludge after 3 hours contact time is 614 mg/L with 95% confidence limits of 321 and 2450 mg/L. The EC10, EC20 and EC80 are 210, 303 and 1245 mg/L, respectively. The active ingredient concentration of 2,4-pentanedione, peroxide, 30% in solvent mixture, (a mixture of cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide) is 29.7 % w/w. Recalculated to the active ingredient concentration, the EC50 of a mixture of cis-2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide for activated sludge after 3 hours contact time is 182 mg/L with 95% confidence limits of 95 and 728 mg/L. The EC10, EC20 and EC80 are 62, 90 and 370 mg/L, respectively. 2,4-pentanedione, peroxide, 30% in solvent mixture is therefore considered not harmful to activated sludge and neither is the active ingredient a mixture of cis- 2,4-pentanedione peroxide and trans-2,4-pentanedione peroxide.

Description of key information

The EC10 and EC50 (3h) of the substance for activated sludge are 62 mg a.i./L and 182 mg a.i./L, respectively. The substance is therefore considered harmless to activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
182 mg/L
EC10 or NOEC for microorganisms:
62 mg/L

Additional information