O,O-tert-butyl isopropyl monoperoxycarbonate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 21-10-2014 Experimental completion date: 22-10-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name Tert-Butylperoxy isopropyl carbonate
EC name O,O-tert-butyl isopropyl monoperoxycarbonate
Batch/Lot no. 1203442069
Appearance Clear liquid
Solubility Miscible in water (water solubility 2.2 g/L)
Volatile No (vapour pressure 200 Pa, 38°C)
Stability Unstable under test conditions
Storage -20°C in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance was pipetted into the incubation vessels and the added weight was directly measured on an analytical mass balance. The weight of the test substance volumes pipetted in the 1 L incubation vessels could not be directly determined. The average weight of four measurements of the same pipetted volume, determined in a 100 mL beaker glass, was used.
Since the test substance was weighed directly into the incubation vessel and was considered unlikely to be air-stripped during incubation, no specific analyses were performed.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (21-10-2014) 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 suspended solids (dry weight) content of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The activated sludge was aerated and fed overnight with OECD synthetic sewage concentrate at 50 mL/L prior to use on the day following collection. The suspended solids concentration of the activated sludge in the incubation vessels was 1.5 g dry weight/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.1 to 20.7°C.

pH:

6.9 to 7.1

Nominal and measured concentrations:

nominal concentrations: 18.0, 36.0, 70.0, 144.0, 284.0, 564.0 and 1148.0 mg/L

Details on test conditions:

Test conditions
The test was performed in 300 mL or 1 L Erlenmeyer flasks with total working volumes of 50 and 200 mL, respectively. 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, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4. In the incubation vessels 1.6 or 6.4 mL of the synthetic sewage feed is added in a total volume of 50 and 200 mL, respectively. O,O-tert-butyl isopropyl monoperoxycarbonate was weighed directly in the incubation vessels. 3,5-Dichlorophenol was added to the incubation vessels using a stock solution of 1.0 g/L. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1003 g of 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Finally the pH was corrected with 1M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.

Test procedures
The activated sludge respiration inhibition test was performed according to the study plan. The study plan was developed from an OECD Test Guideline (OECD 209, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the respiration by micro-organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:
• Prior to use the activated sludge was homogenized with a syringe.
• The activated sludge with test substances was incubated in a shaking bath (100 rpm) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two. The tested nominal concentrations of the reference substance were 1.25, 2.5, 5, 10, 20 and 40 mg/L and 18.0, 36.0, 70.0, 144.0, 284.0, 564.0 and 1148.0 mg/L for the test substance.
An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

530 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Activated sludge respiration inhibition test
The inhibitory effect of O,O-tert-butyl isopropyl monoperoxycarbonate at a particular nominal concentration is expressed as a percentage of the two controls. From these results EC values were calculated. Computation of the EC confidence limits, on the basis of Fieller's theorem (Zerbe,1987), was not possible for the obtained dose response curve. The EC50 of O,O-tert-butyl isopropyl monoperoxycarbonate for activated sludge after 3 hours contact time is 530 mg/L. The EC10, EC20 and EC80 are 18, 58 and 4861 mg/L, respectively.

Quality criteria
The validity of the test is demonstrated by three validity criteria which were met according the guideline. First, the average oxygen uptake rate for the control replicates was 20.2 mg O2/g dry weight/hour, which is just meeting the criterion of >20 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 as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. A lower oxygen uptake rate does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel, 2012). A lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 4%, which shows a high precision and fulfills the maximum prescribed variation of <30%.

Results with reference substance (positive control):

The EC50 of the reference compound for activated sludge after 3 hours contact time is 13 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of O,O-tert-butyl isopropyl monoperoxycarbonate after 3 hours contact time

Weighed test substance (mg)	Concentration (mg/L)	Activity (mg O2/g/min)	Inhibition (%)	pH at start test	pH at end test	Temperature at end test (°C)
-	Control	0.3429	-	7.0	6.9	20.7
-	Control	0.3381	-	6.9	7.0	20.5
3.6# (0.00)	18.0	0.2917	14	6.9	7.0	20.5
7.2# (0.05)	36.0	0.3000	12	6.9	7.0	20.3
3.5	70.0	0.2667	22	6.9	6.9	20.1
7.2	144.0	0.2417	29	7.0	7.1	20.4
14.2	284.0	0.2286	33	7.0	7.1	20.4
28.2	564.0	0.1852	46	7.0	7.1	20.1
57.4	1148.0	0.0917	73	7.0	7.1	20.4

# = Test substance pipetted in 1 L incubation vessels, average weighed and between brackets the standard deviation of four measurements of a pipetted volume of the test substance.

Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of 3,5-dichlorophenol after 3 hours contact time

Concentration (mg/L)	Activity (mg O2/g/min)	Inhibition (%)	pH at start test	pH at end test	Temperature at end test (°C)
Control	0.3167	-	7.0	7.1	20.3
Control	0.3286	-	7.0	7.0	20.4
Control*	0.3381	-	6.9	7.0	20.5
Control*	0.3524	-	7.0	7.1	20.3
1.25*	0.3083	11	7.0	7.0	20.3
2.5	0.2952	8	7.0	7.1	20.5
5.0	0.2875	11	7.1	7.0	20.2
10.0	0.2630	18	7.1	7.0	20.1
20.0	0.0815	75	7.1	7.0	20.4
40.0	0.0407	87	7.1	7.0	20.5

Concentration stock solution 3,5-dichlorophenol = 1000 mg/L pH stock solution 3,5-dichlorophenol = 7.7

* = corresponding control and reference substance concentration

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of O,O-tert-butyl isopropyl monoperoxycarbonate for activated sludge after 3 hours contact time is 530 mg/L. The EC10, EC20 and EC80 are 18, 58 and 4861 mg/L, respectively.

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 substance, 3,5-dichlorophenol (13 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (4%). The average oxygen uptake rate for the control replicates is 20.2 mg O2/g dry weight/hour this is just above the prescribed rate (>20 mg O2/g dry weight /hour). The toxicity of O,O-tert-butyl isopropyl monoperoxycarbonate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of O,O-tert-butyl isopropyl

monoperoxycarbonate at a particular nominal concentration is expressed as a percentage of the two controls. The EC50 of O,O-tert-butyl isopropyl monoperoxycarbonate for activated sludge after 3 hours contact time is 530 mg/L. The EC10, EC20 and EC80 are 18, 58 and 4861 mg/L, respectively.

Description of key information

Toxicity to microorganisms was determined in an activated sludge respiration inhibition test was performed according to OECD 209 under GLP.

The EC50 of O,O-tert-butyl isopropyl monoperoxycarbonate for activated sludge after 3 hours contact time is 530 mg/L. The EC10, EC20 and EC80 are 18, 58 and 4861 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

530 mg/L

EC10 or NOEC for microorganisms:

18 mg/L