Di-tert-butyl sec-butylidene diperoxide

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: 15 February 2000 Experimental completion date: 15 February 2000.

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

protocol: 2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE
- labelling: LUPEROX 220EM50
- Elf Atochem trade name: LUPEROX 220 M50
All denominations correspond to the same test substance.
. batch number:
- protocol and labelling: 1 1579-99 10- 102
. description: colourless liquid
. composition:
- peroxide content: approximately 50%
- isododecane content: approximately 50%
density: 0.8 10 at 20°C
container: one plastic flask
date of receipt: 1 December 1999
storage conditions: at room temperature.
Data relating to the characterization of the test substance are documented in an analytical certificate (presented in appendix 1) provided by the Sponsor.

Analytical monitoring:

no

Details on sampling:

two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/l.
three test substance solutions: activated sludge in dechlorinated water (at 1.6 g/I) and test substance (to obtain final concentrations of 2, 10 and 20 mg/I in 2,2-DI-(T-BUTYLPEROXY)BUTANE).
three reference substance (3,5-dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/l) and reference substance (to obtain final concentrations of 4, 12 and 36 mg/l).

Vehicle:

no

Details on test solutions:

two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/l.
three test substance solutions: activated sludge in dechlorinated water (at 1.6 g/I) and test substance (to obtain final concentrations of 2, 10 and 20 mg/I in 2,2-DI-(T-BUTYLPEROXY)BUTANE).
three reference substance (3,5-dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/l) and reference substance (to obtain final concentrations of 4, 12 and 36 mg/l).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Type: activated sludge collected from a water treatment works containing effluent from a predominantly domestic origin.
Origin: the water treatment plant Emeraude (SIARR) (76141 Petit-Quevilly, France).
Reason for this choice: activated sludge is recommended in OECD guideline for this test.
Preparation: the inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particules) and washed with dechlorinated water. Four samples of 50 ml were taken to determine the content in suspended matter (dry weight). The concentration of sludge (8.80 g/l) was then adjusted to 4 g/1 by addition of the appropriate volume of dechlorinated water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

280 ± 20 mg/I as CaCO3

Test temperature:

22°C

pH:

6.0-8.0

Nominal and measured concentrations:

two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/l.
three test substance solutions: activated sludge in dechlorinated water (at 1.6 g/I) and test substance (to obtain final concentrations of 2, 10 and 20 mg/I in 2,2-DI-(T-BUTYLPEROXY)BUTANE).
three reference substance (3,5-dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/l) and reference substance (to obtain final concentrations of 4, 12 and 36 mg/l).

Details on test conditions:

Reference substance
The reference substance 3,5-dichlorophenol used in the study was supplied by Sigma-Aldrich Chimie, 38297 Saint-Quentin-Fallavier, France.
The reference substance was identified as follows:
. name:
- protocol and labelling: 3,5-Dichlorophenol, 97%
. batch number:
- protocol and labelling: 00410-047
- analytical certificate: 00410
Both batch numbers correspond to the same substance according to a statement dated
2 1 October 1998 supplied by Sigma-Aldrich Chimie.
description: white crystalline powder
quantity and container: 10 g in one smoked glass flask
date of receipt: 22 September 1998
storage conditions: at room temperature and protected from light
purity: 99.4%.
Data relating to the characterization of the reference substance are documented in an analytical
certificate (presented in appendix 1) provided by Sigma-Aldrich Chimie.

Other substances
To prepare the reference substance:
sodium hydroxyde, batch No. 99064 (Prolabo, 94126 Fontenay-sous-Bois, France),
. sulfuric acid, batch No. 9819 1 (Prolabo, 94126 Fontenay-sous-Bois, France).

Preparation
Each test solution was prepared by direct addition of the test substance in the test flask with an appropriate micropipette (the volume added was based on the density) immediately before the start of the test.
The reference solution was prepared by dissolving 0.5 g of the reference substance, 3,5-Dichlorophenol, in 10 ml of NaOH at 1 N. The obtained preparation was then diluted to 30 ml with purified water. Aliquots of H2S04 at 1 N were added while stirring until the point of incipient precipitation was obtained. The solution was made up to 1000 ml using purified water.
The stock solution was agitated for 18 hours. The pH was 7.9 after agitation.

Environmental conditions during culture
The inoculum was maintained under agitation for one day before the test. As the sludge was not used immediately, 50 ml of sewage feed were added, per litre, just before agitation.
Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.
Temperature: between 20 ± 2°C. The temperature in the culture room was recorded continuously and records retained.
Illumination: the stock vessel was kept away from light by wrapping it in opaque material.
Aeration: clean air was passed through the inoculum at the rate of 0.5 to 1 I/min until use.
pH: was checked before use. The inoculum was buffered, if necessary, to pH 6.0 to 8.0 using sodium bicarbonate solution.

Environmental conditions during the test
Temperature: 22°C.
Illumination: no special precautions were taken.
Dilution water: dechlorinated tap water was used, with a hardness of 280 ± 20 mg/I as CaCO3
Duration of test: 3 hours.
Aeration: air was bubbled through each solution at the rate of 0.5 to 1 I/min until the start of oxygen concentration measurements.

Test system
Groups of test and reference solutions and controls were set up as follows:
two controls containing inoculum at 1.6 g/l,
three test concentrations containing 2,2-DI-(T-BUTYLPEROXY)BUTANE at 2, 10 and 20 mg/I and inoculum at 1.6 g/l,
three reference concentrations containing the reference substance (3,5-Dichlorophenol) at 4, 12 and 36 mg/I and inoculum at 1.6 g/1.
The two control solutions and the three reference concentrations were also those of the CIT Study No. 18957 EAS but were attached to this study.
These solutions were prepared in test flasks and then immediately aerated for 3 hours before being transferred to an oxygen measuring apparatus.
Measurement of oxygen concentration of the above test solutions was determined using an oxygen probe placed into an opaque BOD flask filled with the test or reference solution which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter and thence to a chart recorder.

Test method
Each concentration was prepared separately by adding 16 ml of sewage feed to each of the flasks which were made up to 300 ml with water only in the case of the controls or a nominal mixture of water and test substance (added directly using a micropipette) in the case of the test solutions.
Each mixture was then made up to 500 ml by adding 200 ml of inoculum. The solutions were aerated at between 0.5 to 1 litre of air per minute using a Pasteur-pipette as an aeration device.
Solutions were prepared approximately every 15 minutes for test times of 3 hours.
In order to verify the quality of the inoculum, the controls were prepared at the beginning (first control) and the end (second control) of the test.
After 3 hours of aeration/contact time, the content of the test flask was poured into the measuring apparatus and the oxygen concentration was determined during a few minutes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Respiration rates of the test and reference solutions are presented in table 1.
The respiration rate of the test solution at the highest concentration in 2,2-DI(T-BUTYLPEROXY)BUTANE (20 mg/1) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the two other test solutions was not determined.
The EC50 calculation of the reference substance is presented in appendix 2.
Based on the inhibition percentage of the respiration rate obtained in the test solution of highest concentration, the 3-hour EC10 of the test substance was also> 20 mg/l (1).

Results with reference substance (positive control):

As the difference between the two controls was below 15% and the EC50 of 3,5-dichlorophenol was between 5 and 30 mg/I, the test was considered valid.

EC₅₀s, based on nominal concentrations, were as follows:

Substance	3-hour EC50 (mg/l)	95% confidence limits (mg/l)
Test substance	>20 (1)	-
Reference substance	14	12-17

Table 1: Respiration rate in mg O₂.l^-1.h^-1

	Concentration (mg/l)	Respiratory rate (mg O2.l^-1.h^-1)	Percent inhibition (%)
Control 1	0	33.6
Control 2	0	33.0
Reference substance	4	30.9	7
	12	16.0	52
	36	6.9	79
Test substance (1)	2	ND
	10	ND
	20	33.0	1

(1) expressed as 2,2-DI-(T-BUTYLPEROXY)BUTANE

ND: not determined

REFERENCE SUBSTANCE: 3-HOUR EC₅₀ ESTIMATION

Concentration (mg/l)	Percentage inhibition
4	7
12	52
36	79

 

Slope: 1.002 Equation: Y = 1.002 x X + 2.354

 

3-hour EC50	Lower limit		Upper limit
14	(12	-	17)

mg/l

Significance: 95%

Validity criteria fulfilled:

yes

Conclusions:

Under our experimental conditions, the 3-hour EC50 of the test substance, expressed as 2,2-DI-(T-BUTYLPEROXY)BUTANE, is >20 mg/l for activated sludge.

Executive summary:

At the request of Elf Atochem SA, Paris-la-Defense, France, the acute toxicity of the test substance, 2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE, on micro-organisms present in activated sludge was evaluated using a 3-hour static test according to OECD guideline (No. 209, 4 April 1984).

Methods

The test solutions were prepared as follows:

two control solutions: activated sludge in dechlorinated water to obtain a final sludge concentration of 1.6 g/l,

three test substance solutions: activated sludge in dechlorinated water (at 1.6 g/I) and test substance (to obtain final concentrations of 2, 10 and 20 mg/I in 2,2-DI-(T-BUTYLPEROXY)BUTANE),

three reference substance (3,5-dichlorophenol) solutions: activated sludge in dechlorinated water (at 1.6 g/l) and reference substance (to obtain final concentrations of 4, 12 and 36 mg/l).

The oxygen consumption of these solutions was measured for a few minutes, with a chart recorder, after an aeration of 3 hours.

The EC₅₀ (concentration expected to cause 50% inhibition of the respiration rate) of the test substance and the reference substance was determined considering the oxygen consumption of the controls as 100%.

Results

As the validity criteria were met (the difference between the two controls was below 15% and the EC₅₀ of 3,5-dichlorophenol was between 5 and 30 mg/l), the test was considered valid.

The respiration rate of the test solution at the highest concentration in 2,2-DI(T-BUTYLPEROXY)BUTANE (20 mg/l) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the two other test solutions was not determined.

EC₅₀s, based on nominal concentrations, were as follows:

Substance	3-hour EC50 (mg/l)	95% confidence limits (mg/l)
Test substance	>20 (1)	-
Reference substance	14	12-17

The 3-hour EC₁₀ of the test substance was also> 20 mg/l ( 1).

(1) Expressed as 2,2-DI-(T-BUTYLPEROXY)BUTANE

Conclusion

Under our experimental conditions, the 3-hour EC₅₀ of the test substance, expressed as 2,2-DI-(T-BUTYLPEROXY)BUTANE, is > 20 mg/l for activated sludge.

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 2,2-di(tert-butylperoxy)butane for activated sludge after 3 hours contact time is greater than 20 mg/L. The EC10 is also greater than 20 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

20 mg/L