Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 23 January 2017 Experimental completion date 20 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

EC Number:

229-782-3

EC Name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Cas Number:

6731-36-8

Molecular formula:

C17H34O4

IUPAC Name:

1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane

Constituent 2

Reference substance name:

Trigonox® 29

IUPAC Name:

Trigonox® 29

Constituent 3

Reference substance name:

0905137109

IUPAC Name:

0905137109

Details on test material:

Identification: Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide
CAS Number: 6731-36-8
Trade Name: Trigonox® 29
Batch Number: 0905137109

Specific details on test material used for the study:

Identification: Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide
CAS Number: 6731-36-8
Physical state/Appearance: clear colorless viscous liquid
Batch: 0905137109
Purity: 93.4%
Expiry Date: 01 June 2019
Storage Conditions: approximately 4 ºC in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The samples were transferred to polyethylene bottles and an aliquot (250 mL) of potassium chloride (0.1M) added. The mixtures were then shaken (200 rpm, for between 33 and
35 minutes) using an INFORS TR-225 orbital platform shaker prior to removal of the aqueous phase by filtration (0.45 µm AcroCap filter).

Test substrate

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

The test item was prepared in a preliminary solvent stock solution. All test concentrations were corrected for a purity value of 93.4%.

Nominal amounts of test item (1713 and 5353 mg) were separately dissolved in acetone and the volumes adjusted to 10 mL to give 1713 mg/10 mL and 5353 mg/10 mL solvent stock solutions respectively equivalent to 1600 mg ai/10 mL and 5000 mg ai/10 mL solvent stock solutions. Serial dilutions from the 5000 mg ai/10 mL solvent stock solution were made to give 500 and 50 mg ai/10 mL stock solutions.

Additionally, a serial dilution from the 1600 mg ai/10 mL solvent stock solution was made to give a 160 mg ai/10 mL solvent stock solution.

Aliquots (1 mL) of each of the solvent stock solutions were dispensed separately onto dry quartz sand (5 g) and the solvent allowed to evaporate to dryness. The test item/sand combination and an amount of powdered Lucerne-green-grass-meal*** (2.5 g) was then added to a final bulk soil weight of 500 g* and thoroughly mixed (Kenwood Chef Mixer) for 2 minutes prior to the addition of an aliquot (50.4 mL) of water and further mixing for 10 minutes to give the required test concentrations of 10, 32, 100, 320, 1000 mg ai/Kg with a nominal moisture content of 45% of the Water Holding Capacity (WHC). Three 50 g* aliquots were then taken for Day 0 analysis. The remaining soil bulk was incubated in amber glass jars.

The test item was prepared in a preliminary solvent stock solution. All test concentrations were corrected for a purity value of 93.4%.

Nominal amounts of test item (1713 and 5353 mg) were separately dissolved in acetone and the volumes adjusted to 10 mL to give 1713 mg/10 mL and 5353 mg/10 mL solvent stock solutions respectively equivalent to 1600 mg ai/10 mL and 5000 mg ai/10 mL solvent stock solutions. Serial dilutions from the 5000 mg ai/10 mL solvent stock solution were made to give 500 and 50 mg ai/10 mL stock solutions. Additionally, a serial dilution from the 1600 mg ai/10 mL solvent stock solution was made to give a 160 mg ai/10 mL solvent stock solution.

Aliquots (1 mL) of each of the solvent stock solutions were dispensed separately onto dry quartz sand (5 g) and the solvent allowed to evaporate to dryness. The test item/sand combination and an amount of powdered Lucerne-green-grass-meal*** (2.5 g) was then added to a final bulk soil weight of 500 g* and thoroughly mixed (Kenwood Chef Mixer) for 2 minutes prior to the addition of an aliquot (50.4 mL) of water and further mixing for 10 minutes to give the required test concentrations of 10, 32, 100, 320, 1000 mg ai/Kg with a nominal moisture content of 45% of the Water Holding Capacity (WHC). Three 50 g* aliquots were then taken for Day 0 analysis. The remaining soil bulk was incubated in amber glass jars.

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

Room temperature was recorded daily throughout the test. Temperature was maintained at temperatures of between 21 and 22 ºC throughout the test

Moisture:

Each individual test vessel was weighed on Day 0 and deionized reverse osmosis water added on a weight basis on Days 7, 14, 21 and 28 in order to maintain the moisture content within ±5% of the initial moisture content.
The moisture content of each control, solvent control and test vessel was adjusted to 45% of the WHC (± 5%) of the soil on days 7, 14, 21 and 28

Details on test conditions:

The soil for the study was obtained on 15 December 2016 from LUFA Speyer, Germany. The sampling site had not been treated with crop protection products or organic fertilizer for at least 3 years prior to sampling (data from supplier).
The pH of the soil in water was 7.6 (information supplied by LUFA Speyer). The organic carbon content was determined to be 0.67 ± 0.03% whilst the sand content of the soil was determined to be 57.5% (information supplied by LUFA Speyer.
The microbial biomass of the soil was shown to be 16.19 C/g (ISO 14240-2, 1997). This was equivalent to 2% of the total soil organic carbon content.
The total nitrogen content of the soil was 76 mg/100 g (information supplied by LUFA Speyer, see Annex 2) and the initial nitrate content of the soil determined on the day the test was initiated was 6.4 mg/100 g.

Experimental Design and Study Conduct
Preparation of Soil
On arrival at Envigo Research Limited, Shardlow, UK the soil was stored at temperatures of between 4 and 12 ºC prior to use. Prior to the start of the test, an amount of soil (approximately 11 kg) was removed and stored at temperatures of between 20 and 21 ºC for 12 days and shielded from light.

The moisture content of the soil was determined prior to the start of the test by drying an amount of soil (100 g) at 105 C (± 10%) until a constant weight was obtained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 6%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.6 g/100 g ± 1.4 and hence 50.4 mL of deionized reverse osmosis water per 0.5 kg* of soil was added. This gave a final water content of 15.5 g/100 g**, i.e. 45% of the WHC as recommended by the Test Guidelines.

6 replicates per concentration

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg ai/kg

Reference substance (positive control):

yes

Remarks:

A positive control (Envig: SD86WH) used 2-Chloro-6-(trichlormethyl)pyridine as the test item at concentrations of 0.10, 1.0 and 10 mg/kg. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Effect concentrations 2

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Details on results:

Validation Criteria
The variation between replicate solvent control nitrification rates was less than 15% and therefore satisfied the validation criterion given in the Test Guidelines.

Environmental Measurements
Temperature was maintained at temperatures of between 21 and 22 ºC throughout the test and the moisture content of each control, solvent control and test vessel was adjusted to 45% of the WHC (± 5%) of the soil on days 7, 14, 21 and 28.

Nitrogen Transformation Activity
The effect of the test item on the nitrogen transformation activity of the soil microorganisms gave an EC50 of greater than 1000 mg ai/Kg. The No Observed Effect Concentration (NOEC) after 28 days exposure was 1000 mg ai/Kg.

Results with reference substance (positive control):

Positive Control
A positive control (Envigo Research Limited, Study Number: SD86WH) used 2-Chloro-6-(trichlormethyl)pyridine as the test item at concentrations of 0.10, 1.0 and 10 mg/kg.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of the soil to the test item gave the following results:

EC10 : 1.2 mg/kg
EC16 : 1.7 mg/kg
EC50 : 3.9 mg/kg, 95% confidence limits 3.5 – 4.4 mg/kg
EC80 : 7.0 mg/kg

The results from the positive control with 2-Chloro-6-(trichlormethyl)pyridine were within the normal ranges for this test item.

Any other information on results incl. tables

Absorbance Values and Nitrate Concentration Values

Nominal Concentration (mg ai/kg)		Day 0			Nominal Concentration (mg ai/kg)		Day 0
		Absorbance*(324 nm)	Nitrate Concentration (mg NO3/kg)				Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.2738	93.60		320	R1	0.2912	99.50
	R2	0.2751	94.05			R2	0.2988	102.05
	R3	0.2800	95.70			R3	0.2930	100.10
	Mean	-	94.45			Mean	-	100.55
	SD	-	1.11			SD	-	1.33
	CV%	-	1.18			CV%	-	1.32
Solvent Control	R1	0.2829	96.70		1000	R1	0.2903	99.20
	R2	0.2919	99.75			R2	0.2928	100.05
	R3	0.2717	92.90			R3	0.3025	103.30
	Mean	-	96.45			Mean	-	100.85
	SD	-	3.43			SD	-	2.16
	CV%	-	3.56			CV%	-	2.14
10	R1	0.2992	102.20
	R2	0.3048	104.10
	R3	0.3010	102.80
	Mean	-	103.03
	SD	-	0.97
	CV%	-	0.94
32	R1	0.3119	106.50
	R2	0.3098	105.75
	R3	0.3179	108.50
	Mean	-	106.92
	SD	-	1.42
	CV%	-	1.33
100	R1	0.3137	107.10
	R2	0.3182	108.60
	R3	0.3054	104.30
	Mean	-	106.67
	SD	-	2.18
	CV%	-	2.04

Table 1 (continued)  Absorbance Values and Nitrate Concentration Values

Nominal Concentration (mg ai/kg)		Day 28			Nominal Concentration (mg ai/kg)		Day 28
		Absorbance*(324 nm)	Nitrate Concentration (mg NO3/kg)				Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R4	0.6972	236.70		320	R4	0.6251	212.35
	R5	0.6475	219.90			R5	0.6033	204.95
	R6	0.5964	202.65			R6	0.6500	220.75
	Mean	-	219.75			Mean	-	212.68
	SD	-	17.03			SD	-	7.91
	CV%	-	7.75			CV%	-	3.72
Solvent Control	R4	0.6738	228.80		1000	R4	0.6262	212.70
	R5	0.6599	224.10			R5	0.6154	209.05
	R6	0.5907	200.70			R6	0.6692	227.25
	Mean	-	217.87			Mean	-	216.33
	SD	-	15.05			SD	-	9.63
	CV%	-	6.91			CV%	-	4.45
10	R4	0.6829	231.85
	R5	0.6870	233.25
	R6	0.6305	214.15
	Mean	-	226.42
	SD	-	10.65
	CV%	-	4.70
32	R4	0.6563	222.85
	R5	0.6546	222.30
	R6	0.6478	220.00
	Mean	-	221.72
	SD	-	1.51
	CV%	-	0.68
100	R4	0.6841	232.25
	R5	0.6340	215.35
	R6	0.6535	221.90
	Mean	-	223.17
	SD	-	8.52
	CV%	-	3.82

 

Table2            Inhibition of Nitrogen Transformation Activity

Nominal Concentration (mg ai/kg soil)	Day 28
	Nitrate Formation Rate (mg ai NO3/kg/d)	% Inhibition
Control	4.48	-
Solvent Control	4.34	-
10	4.41	[2]
32	4.10	6
100	4.16	4
320	4.00	8
1000	4.12	5

*Corrected for blank absorbance value

R₁-R₃= Replicates 1 to 3

SD = Standard Deviation

CV = Coefficient of Variation

*Corrected for blank absorbance value

 R₄– R₆= Replicates 4 to 6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

Introduction

A study was performed to assess the long-term effect of the test item, after a single exposure, on the nitrogen transformation activity of soil microorganisms. 

Methods

Soil microorganisms were exposed to the test item at concentrations of 10, 32, 100, 320 and 1000 mg active ingredient(ai)/kg for 28 days at temperatures of between 21 and 22 °C in the dark with the addition of powdered Lucerne-green-grass meal to act as a respiratory substrate.

The inhibitory effect of the test item on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples on Days 0 and 28 and compared to data obtained from solvent control soil samples.

Results

The effect of the test item on the nitrogen transformation activity of the soil microorganisms gave an EC₅₀of greater than 1000 mg ai/kg. The No Observed Effect Concentration (NOEC) was1000 mg ai/kg.