A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 January 2015 to 13 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The soil was stored in the dark at temperatures of between 5 to 8 °C prior to use in the study for 21 days which was outside the range of 4 ± 2 °C. After storage the soil was equilibrated at temperatures of between 21 to 23 °C for 6 days before use in the study.

This equilibration temperature was outside the range of 20 ± 2 °C and the temperature that the soil aliquots were incubated at ranged between 20 to 23 °C which was also outside the range of 20 ± 2 °C as stated in the study plan

This deviation is considered to have had no effect on the integrity of the study given that the temperatures were only 1 °C outside of the accepted range.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not specified

Details on sampling:

On Days 0 and 28 three aliquots (52.90 g wet weight equivalent to 50 g dry weight) of soil were removed from the control and test item vessels prepared for nitrate analysis.

Vehicle:

yes

Details on preparation and application of test substrate:

Test Item Preparation
The test item was dispersed directly in water.

Nominal amounts of test item (640 and 2000 mg) were separately dissolved in deionized reverse osmosis water with the aid of ultrasonication for approximately 3 and 5 minutes respectively and the volumes adjusted to 200 mL to give 3200 and 10000 mg/L stock solutions. A serial dilution was performed on the 3200 mg/L stock solution to give stock solution of 320 mg/L. A further serial dilution was performed on the 10000 mg/L stock solution to give a 1000 mg/L stock solution from which a further serial dilution was prepared to obtain a stock concentration of
100 mg/L.

Aliquots (50 mL) of the 100, 320, 1000, 3200 and 10000 mg/L stock solutions were sprinkled separately over the surface of 0.5 kg (dry weight) of soil and thoroughly mixed (Kenwood Chef Mixer) prior to the addition of an amount of powdered Lucerne-green-grass-meal (2.5 g) and an aliquot (0.8 mL) of water to give the required test concentrations of 10, 32, 100, 320 and 1000 mg/kg with a nominal moisture content of 45% of the Water Holding Capacity (WHC). The soil for each test concentration was then mixed again to ensure homogeneity prior to removing three
50 g* aliquots for Day 0 analysis. The remaining soil bulk was incubated in 500 mL glass jars.

The control soil was prepared in an identical manner but not exposed to the test item with the addition of 50.8 mL of water instead of 0.8 mL as added to the test item vessels. Three replicate 50 g (dry weight) aliquots were removed for Day 0 analysis from the control preparation and the remaining soil bulk was incubated.

Exposure Conditions
The soil samples were incubated in 500 mL 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 temperatures of between 20 and 23 C in darkness. This was a deviation to the study plan which states that the soil will be equilibrated at 20 ± 2 °C but was considered to have had no effect on the study given that it was only 1 °C outside the accepted range.

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.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Temperature was maintained at between 20 to 23 ºC throughout the test.

Moisture:

The moisture content of each control and test vessel was adjusted to 45% of the WHC (± 5%) of the soil on Days 7, 14, 21 and 28.

Reference substance (positive control):

no

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

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

RESULTS

Absorbance readings and nitrate concentrations determined on each sampling day are given in the following table. 

Nominal Concentration (mg/kg)		Day 0		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.297	89.45	0.515	173.4
	R2	0.299	90.25	0.524	176.85
	R3	0.302	91.4	0.519	174.9
	Mean	-	90.37	-	175.05
	SD	-	0.98	-	1.73
	CV%	-	0.01	-	0.01
10	R1	0.329	101.8	0.537	181.85
	R2	0.285	84.85	0.513	172.6
	R3	0.308	93.7	0.524	176.85
	Mean	-	93.45	-	177.1
	SD	-	8.48	-	4.63
	CV%	-	0.09	-	0.03
32	R1	0.301	91	0.528	178.4
	R2	0.303	91.75	0.515	173.4
	R3	0.285	84.85	0.515	173.4
	Mean	-	89.2	-	175.07
	SD	-	3.79	-	2.89
	CV%	-	0.04	-	0.02
100	R1	0.316	96.8	0.518	174.55
	R2	0.32	98.3	0.545	184.95
	R3	0.306	92.95	0.531	179.55
	Mean	-	96.02	-	179.68
	SD	-	2.76	-	5.2
	CV%	-	0.03	-	0.03
320	R1	0.318	97.55	0.541	183.4
	R2	0.308	93.7	0.522	176.1
	R3	0.32	98.3	0.539	182.6
	Mean	-	96.52	-	180.7
	SD	-	2.47	-	4
	CV%	-	0.03	-	0.02
1000	R1	0.315	96.4	0.546	185.3
	R2	0.297	89.45	0.531	179.55
	R3	0.332	102.95	0.539	182.6
	Mean	-	96.27	-	182.48
	SD	-	6.75	-	2.88
	CV%	-	0.07	-	0.02

* Corrected for blank absorbance value

R1-R6 = Replicates 1 to 6

SD = Standard Deviation

CV = Coefficient of Variation

 

The nitrate formation rates and percentage inhibition of nitrogen transformation activity on Day 28 are given in the following table.  

Nominal Concentration (mg/kg soil)	Day 28
	Nitrate Formation Rate (mg NO3/kg/d)	% Inhibition
Control	3.02	-
10	2.99	1
32	3.07	[2]
100	2.99	1
320	3.01	0
1000	3.08	[2]

Validation Criteria

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

 

Nitrogen Transformation Activity

Statistical analysis of the nitrate concentration values was carried out for the control and test item groups using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). The test item groups were shown to have similar or not significantly different nitrate concentrations as the control group (P0.05). 

 

It was therefore considered that the test item exhibited no adverse effect on the nitrogen transformation rate and therefore the "No Observed Effect Concentration" (NOEC) was

1000 mg/kg soil dry weight.

 

Environmental Measurements

Temperature was maintained at between 20 to 23 ºC throughout the test. The moisture content of each control and test vessel was adjusted to 45% of the WHC (± 5%) of the soil on Days 7, 14, 21 and 28.

 

CONCLUSION

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 test item showed no significant effect on the nitrogen transformation activity of soil microorganisms at a test concentration of 1000 mg/kg soil dry weight over a 28-Day period and therefore can be considered to have no long-term effect on nitrogen transformation in soil. Therefore the No Observed Effect Concentration (NOEC) was 1000 mg/kg.

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 test item showed no significant effect on the nitrogen transformation activity of soil microorganisms at a test concentration of 1000 mg/kg soil dry weight over a 28-Day period and therefore can be considered to have no long-term effect on nitrogen transformation in soil. Therefore the No Observed Effect Concentration (NOEC) was 1000 mg/kg.

Executive summary:

Soil microoganisms were exposed to the test item at concentrations of 10, 32, 100, 320 and 1000 mg/kg for 28 days at temperatures between 20 and 23 °C in the dark. Lucerne-green-grass meal was added to the soil 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 control soil samples.

 

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 test item showed no significant effect on the nitrogen transformation activity of soil microorganisms at a test concentration of 1000 mg/kg soil dry weight over a 28-Day period and therefore can be considered to have no long-term effect on nitrogen transformation in soil. Therefore the No Observed Effect Concentration (NOEC) was 1000 mg/kg.

Description of key information

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 test item showed no significant effect on the nitrogen transformation activity of soil microorganisms at a test concentration of 1000 mg/kg soil dry weight over a 28-Day period and therefore can be considered to have no long-term effect on nitrogen transformation in soil. Therefore the No Observed Effect Concentration (NOEC) was 1000 mg/kg.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw