Oxirane, mono[(C12-14-alkyloxy)methyl] derivs.

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 07 November 2016 and 05 December 2016

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)

Version / remarks:

2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Alkyl (C12-C14) glycidyl ether
Label: Epoxide 8
51% C12 Glycidyl ether
21% C14 Glycidyl ether
5% C16 Glycidyl ether
13% N +1's
4% alpha addition Products
94% total active (label claim)
- Physical state: Clear colorless liquid
- Storage condition of test material: ambient

Specific details on test material used for the study:

Identification: Oxirane, mono[(C12-14-alkyloxy) methyl] derivs
Physical state/Appearance: clear colorless liquid
Batch: AAD1326100
Purity: not applicable as is a UVCB
Expiry Date: 29 August 2019
Storage Conditions: approximately 4 ºC in the dark

Sampling and analysis

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This exception with regard to GLP has been reflected in the GLP compliance statement.

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

Test Item Preparation
The test item was prepared in a preliminary solvent stock solution.
Nominal amounts of test item (1600 and 5000 mg) were separately dissolved in acetone and the volumes adjusted to 10 mL to give 1600 mg/10 mL and 5000 mg/10 mL solvent stock solutions respectively. Serial dilutions from the 5000 mg/10 mL were made to give 500 and 50 mg/10 mL stock solutions. Additionally, a serial dilution from the 1600 mg/10 mL solvent stock solution was made to give a 160 mg/10 mL solvent stock solution.
Aliquots (1 mL) 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) prior to the addition of an aliquot (57 mL) of water to give the required test concentrations of 10, 32, 100, 320,
1000 mg/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 glass bottles.
The control soil was prepared in an identical manner but not exposed to the test item or solvent. The solvent control was prepared as the test item vessels but exposed to solvent and not test item. Three replicate 50 g* aliquots were removed for Day 0 analysis from the control and solvent control preparations and the remaining soil bulk was incubated.


* Dry Weight
** Wet Weight
*** Carbon/Nitrogen ratio calculated to be 16.3. This work was performed outside the slope of the study plan for this project by Warwick Analytical Service

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

20 ± 2”C

Moisture:

18.9g/100g i.e. 45% of the Water Holding Capacity (WHC)

Details on test conditions:

Test System and Supporting Information
The soil for the study was obtained on 21 October 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 7.3 (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) and so satisfied the recommendations of the Test Guidelines.
The microbial biomass of the soil was shown to be 17.60 C/g (ISO 14240-2, 1997). This was equivalent to 2.6% of the total soil organic carbon content.
The total nitrogen content of the soil was 56 mg/100 g (information supplied by LUFA Speyer, see Annex 1) and the initial nitrate content of the soil determined on the day the test was initiated was 2.2 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 3 and 4 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 4%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.6 g/100 g± 1.4 and hence 57 mL of deionized reverse osmosis water per 0.5 kg* of soil was added. This gave a final water content of 15.9 g/100 g**. i.e. 45% of the WHC as recommended by the Test Guidelines.
A positive control (Envigo Research Limited, Study Number: SD86WH) used 2-Chloro-6-(trichlormethyl)pyridine as the test item.

Preliminary Solubility Work
Preliminary solubility work showed that the test item was insoluble in water. The test item was soluble in acetone at 5000 mg/10 mL. Therefore, for the purposes of the test, the test concentrations were prepared by dissolving the test item in acetone and adding an aliquot of this to sand prior to dispersal in soil.

Test Item Preparation
The test item was prepared in a preliminary solvent stock solution.
Nominal amounts of test item (1600 and 5000 mg) were separately dissolved in acetone and the volumes adjusted to 10 mL to give 1600 mg/10 mL and 5000 mg/10 mL solvent stock solutions respectively. Serial dilutions from the 5000 mg/10 mL were made to give 500 and 50 mg/10 mL stock solutions. Additionally, a serial dilution from the 1600 mg/10 mL solvent stock solution was made to give a 160 mg/10 mL solvent stock solution.
Aliquots (1 mL) 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) prior to the addition of an aliquot (57 mL) of water to give the required test concentrations of 10, 32, 100, 320,
1000 mg/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 glass bottles.
The control soil was prepared in an identical manner but not exposed to the test item or solvent. The solvent control was prepared as the test item vessels but exposed to solvent and not test item. Three replicate 50 g aliquots were removed for Day 0 analysis from the control and solvent control preparations and the remaining soil bulk was incubated.

Exposure Conditions
The soil samples were incubated in glass jars. The test vessels were sealed with polyurethane foam stoppers in order to minimize moisture loss by evaporation and maintained in a temperature controlled room at 20 ± 2 C in darkness.

Assessments
Environmental Measurements
Room temperature was recorded daily throughout the test. 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.

Sampling
On Days 0 and 28 three aliquots (50 g*) of soil were removed from one of the control, solvent control and test item vessels for nitrate analysis.

Nitrate Analysis
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, 30 minutes) using an INFORS TR-225 orbital platform shaker prior to removal of the aqueous phase by filtration (0.45 µm AcroCap filter).
To an aliquot (35 mL) of acid mixture was added 5 mL of the soil filtrate followed by 5 mL of 2,6-dimethylphenol solution. The mixture was thoroughly mixed and allowed to stand for between 15 and 25 minutes prior to determination of the absorbance at 324 nm.
A calibration curve was prepared outside the scope of this study by measuring absorbance values at 324 nm of standard solutions of potassium nitrate at the following concentrations: 1.0, 5.0, 10, 15, 20 and 25 mg NO3- N/L. The standard solutions were treated in the same manner as the test samples. Linear regression analysis of the standard curve data produced an equation for the best-fit line into which the test sample extract absorbance values were substituted to determine the nitrate nitrogen concentration.
The concentration of nitrate (mg/L) in the test sample extracts was obtained by multiplying the nitrate nitrogen concentration (mg/L) by a factor of 4.427.

Nominal and measured concentrations:

Nominal

Reference substance (positive control):

yes

Remarks:

2-Chloro-6-(trichlormethyl)pyridine

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:

test mat.

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:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Absorbance readings and nitrate concentrations determined on each sampling day are given in Table 1. The nitrate formation rates and percentage inhibition of nitrogen transformation activity on Day 28 are given in Table 2. (Below)

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 20 ± 2 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.

Results with reference substance (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

Table 1       Absorbance Values and Nitrate Concentration Values

Day 0:

 

Nominal Concentration (mg/kg)		Absorbance (324nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.1541	53.15
	R2	0.1479	51.05
	R3	0.1584	54.60
	MEAN	-	52.93
	SD	-	52.93
	CV%	-	2
Solvent Control	R1	0.1602	55.20
	R2	0.1545	53.20
	R3	0.1586	54.70
	MEAN	-	54.40
	SD	-	0.98
	CV%	-	2
10	R1	0.1636	56.35
	R2	0.1534	52.90
	R3	0.1566	54.00
	MEAN	-	54.42
	SD	-	1.76
	CV%	-	3
32	R1	0.1404	58.65
	R2	0.1667	57.40
	R3	0.1655	57.00
	MEAN	-	57.68
	SD	-	0.86
	CV%	-	1
100	R1	0.1531	52.80
	R2	0.1558	53.75
	R3	0.1469	50.70
	MEAN	-	52.42
	SD	-	1.56
	CV%	-	3
320	R1	0.1613	55.60
	R2	0.1612	55.55
	R3	0.1777	60.90
	MEAN	-	57.35
	SD	-	3.07
	CV%	-	5
1000	R1	0.1738	59.80
	R2	0.1706	58.75
	R3	0.1655	57.00
	MEAN	-	58.52
	SD	-	1.41
	CV%	-	2

Day 28:

Nominal Concentration (mg/kg)		Absorbance (324nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.4761	164.95
	R2	0.4326	157.40
	R3	0.4651	158.25
	MEAN	-	159.20
	SD	-	2.42
	CV%	-	2
Solvent Control	R1	0.4914	167.15
	R2	0.4740	161.25
	R3	0.4563	155.30
	MEAN	-	161.23
	SD	-	5.93
	CV%	-	4
10	R1	0.4327	147.30
	R2	0.4482	152.55
	R3	0.4688	159.50
	MEAN	-	153.12
	SD	-	6.12
	CV%	-	4
32	R1	0.4768	162.20
	R2	0.4636	157.75
	R3	0.4979	169.35
	MEAN	-	163.10
	SD	-	5.85
	CV%	-	4
100	R1	0.5251	178.55
	R2	0.4852	165.05
	R3	0.4606	156.75
	MEAN	-	166.78
	SD	-	11.00
	CV%	-	7
320	R1	0.4448	151.40
	R2	0.4654	158.35
	R3	0.4851	165.00
	MEAN	-	158.25
	SD	-	6.80
	CV%	-	4
1000	R1	0.4517	153.75
	R2	0.4877	165.90
	R3	0.4740	161.25
	MEAN	-	160.30
	SD	-	6.13
	CV%	-	4

Table 2: Inhibition of Nitrogen Transformation Activity Day 28

Nominal Concentration (mg/kg soil)	Nitrate Formation Rate (mg NO3/kg/d)	% Inhibition
Control	3.80	-
Solvent Control	3.82	+
10	3.53	8
32	3.77	1
100	4.08	[7]
320	3.60	6
1000	3.64	5

[Increasing nitrate formation rate as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

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

Methods

Soil microorganisms were exposed to the test item at concentrations of 10, 32, 100, 320 and 1000 mg/kg for 28 days at temperatures of between 18 and 21 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 EC50 of greater than 1000 mg/kg.  The No Observed Effect Concentration (NOEC) was 1000 mg/kg.