Reaction mass of decamethyltetrasiloxane; dodecamethylpentasiloxane; hexamethyldisiloxane; octamethyltrisiloxane

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 October to 2 November 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study, with some modifications, with analysis, under GLP. However test concentrations were not maintained, a solvent was used with no solvent control test and the test was performed in bulk without using individual subsamples.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Principles of method if other than guideline:

The following measures were taken to prevent loss of test item, while allowing for air exchange:
- The test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture. 
- All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil.
- Soil was dosed at ~30% above the calculated maximum sorption concentration to allow for some losses through volatility.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg
- Sampling method: Soil samples were collected with a spatula from the test vessels. Two sets of samples (one for analysis and the other for archiving) of the soil from each test substance treatment level and the control were removed on test days 0, 1, 2, 3, 4, 14, and 28 for determination of test substance concentration. In addition, at each interval, two sets of three quality control (QC) samples were prepared, one for analysis and the other for archiving. These QC samples were prepared in test soil at relevant test substance concentrations. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Determination of NO3- Ions to Assess Influences on the Nitrification Process:
At each sampling interval, 20 gram dry weight samples were taken from the control; 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg treatment levels; and reference substance dosed soils and placed in separate containers. The samples were extracted with 100 mL of 0.1 M potassium chloride (KCl) and shaken for 1 hour on a shaker table set at 150 rpm. After the extraction, the samples were centrifuged for 10 minutes at 1000 rpm. An aliquot of approximately 1.5 mL of the supernatant was removed, centrifuged at 10,000 rpm for five minutes, and transferred to a high performance liquid chromatography (HPLC) vial. The KCl extracts were analysed with an Agilent HPLC system using UV detection.

- Sample storage conditions before analysis: Not reported

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Natural soil: The soil used for the test was collected on 15 September 2015, sieved (2 mm), and characterized by Lufa-Speyer (SP) of Germany. No pesticides have been applied for at least four years. The soil was taken from the top 20 cm and freshly-collected samples shipped to Smithers Viscient in Wareham, Massachusetts, USA. Additional characterisation was conducted by Agvise Laboratories in Northwood, North Dakota. The soil was stored refrigerated (i.e. at approximately 4 ºC) initially and then allowed to acclimate at test temperature for 5 days before it was used for this test.

The water holding capacity of the soil was 35.4%. The moisture content used for the definitive test was 15.93% which is equivalent to 45% of the water holding capacity. The starting moisture content of the soil was 4.78% (as dry weight basis), therefore, 111.5 mL of purified reagent water was added per kilogram of soil during dosing to bring the moisture content to 45% of the water holding capacity.

The alfalfa (also known as Lucerne meal) used to amend the test soil was obtained from Hanson Grain in Hanson, Massachusetts on 29 September 2011 and was determined to have a nitrogen content of 2.7% and a carbon content of 41.2% (equalling a ratio of approximately 1:15) by Agvise Laboratories in Northwood, North Dakota. The alfalfa was amended to the soil at an amount of 5 grams of alfalfa per 1000 grams of soil (dry weight).

- Other: Selection of nominal L2 concentrations was based on the maximum sorption concentration for L2 in the soil. The maximum sorption concentration for L2 in the soil used for this study (based on the solubility of L2 in organic carbon and the organic content of the soil) was approximately 6.2 mg L2/kg of dry soil. Based on anticipated volatile losses, the soil was dosed at approximately 30% above the maximum sorption concentration. Therefore, the test was performed at 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The hexamethyldisiloxane stock solution was prepared by dissolving 0.0200 g in 20.0 mL of acetonitrile. The final concentration of this stock solution was 1.00 mg/mL. This stock solution was used in dosing higher concentrations and in preparing secondary stock solutions to dose remaining concentrations. Samples were fortified using a solvent based stock solution, due to the volatile nature and poor water solubility of the test substance. The solvent concentration used was < 1% by volume in all cases, which is typically acceptable for most soil, sludge and water/sediment studies conducted under OECD Guidelines. There was no negative impact of the solvent on the soil, since there was no decline in nitrate formation during 28 days of incubation.

A 10 g batch of quartz sand was dosed with approximately 3.33 mL of the 10 mg/mL reference substance stock solution to achieve a concentration of 33.3 mg/kg in the soil. Seven 1 kg (dry weight) batches of SP 2.3 soil were weighed out into 1-L beakers. One at a time, each 1 kg batch of soil was mixed with 5 g of alfalfa meal, 111.5 mL of purified reagent water, and (reference treatment group only) the dosed batch of quartz sand in a Hobart mixer. Each of the mixed 1 kg (dry weight) batches of soil was then divided into three approximately 333 g (dry weight) replicates. Each replicate was weighed out into 1-L glass bottles (with approximately 2/3 of the test vessel as headspace) and the weights recorded. Control and reference samples did not contain any test substance. All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil. Once dosed, each replicate was hand shaken to homogenise then subsampled for extraction. Soil samples were collected from the test vessels (after the dosing/homogenisation step and prior to being sealed with parafilm.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetonitrile
- Concentration of vehicle in test medium (stock solution and final test solution): The hexamethyldisiloxane stock solution was prepared by dissolving 0.0200 g in 20.0 mL of acetonitrile. The final concentration of this stock solution was 1.00 mg/mL.
The solvent concentration used was < 1% by volume in all cases.
- Evaporation of vehicle before use: No

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

maintained at 45% of the water holding capacity.

Details on test conditions:

TEST SYSTEM
- Testing facility: Smithers Viscient
- Test container (type, material, size): 1 l glass bottle beaker filled with 333g (dry weight) soil, with approximately 2/3 of the test vessel as headspace. Test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture.
- Amount of soil: 333g (dry weight) soil
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle control performed

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Collected by Lufa-Speyer, Germany/Rheinland-Pfalz/Offenbach “Rechts der Landauer Str.”, Nr. 826/7
- History of site: Not reported
- Vegetation cover: Not reported
- Treatments with pesticides or fertilizers: No pesticides have been applied for at least four years
- Accidental contamination: None reported
- Other:
- Depth of sampling: top 20 cm
- Soil texture
- % sand: 65
- % silt: 25
- % clay: 10
- Soil taxonomic classification: USDA
- Soil classification system: Sandy loam
- pH (in water): 6.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 82.8 (dry or wet not specified).
- Maximum water holding capacity (in % dry weight): 35.4%.
- Cation exchange capacity (mmol/kg): 5.9
- Pretreatment of soil: Not reported
- Storage (condition, duration): The soil was stored refrigerated (i.e., at approximately 4 ºC) initially and then allowed to acclimate at test temperature for 5 days before it was used for this test.
- Initial microbial biomass as % of total organic C: Biomass as mg C/ 100 g soil (% of total organic carbon)
Prior to test start: 5.1 mg C/100 g soil (0.85%)
At study termination: 14.4mg C/100 g soil (2.4%)


DETAILS OF PREINCUBATION OF SOIL (if any): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Nitrate concentration at day 0 and day 28.
Nitrate transformation rate at day 28.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY - None reported
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:

Nominal: Control, 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg.
Measured: All measurements

Reference substance (positive control):

yes

Remarks:

dinoseb acetate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 8.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: analytical measurements show test concs were all

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 8.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: analytical measurements show test concs were all

Results with reference substance (positive control):

average inhibition of nitrate formation as compared to the control soil by -229% on test day 28

Reported statistics and error estimates:

The difference in results of the 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg treated soils at day 28 were not statistically significant, due to the reproducibility of the replicates, when compared to the control soil (ANOVA, p < 0.05) when using Dunnett’s Multiple Comparison Test (U.S. EPA, 2002), a statistical program in CETIS™ (Ives, 2013), where p = 0.2301, 0.9961, 0.9685, 0.9966, and 0.9989 for 0.53, 1.1, 2.1, 4.3, and 8.5 mg/kg, respectively.

Table 1. Nitrate concentrations measured during the soil microflora study with hexamethyldisiloxane.

Treatment Group	Day 0 (mg NO3^-/kg dry soil)	Day 28 (mg NO3^-/kg dry soil)
Control
	82.4	141
	83.5	140
	82.4	141
Mean ± SD	82.8 ± 0.642	141 ± 0.506
hexamethyldisiloxane
0.53 mg/kg	82.0	140
	87.1	141
	83.6	140
Mean ± SD	84.2 ± 2.60	140 ± 0.340
hexamethyldisiloxane
1.1 mg/kg	81.4	141
	80.4	141
	80.1	140
Mean ± SD	80.6 ± 0.684	140 ± 0.496
hexamethyldisiloxane
2.1 mg/kg	80.3	136
	80.7	141
	79.9	141
Mean ± SD	80.3 ± 0.400	139 ± 2.46
hexamethyldisiloxane
4.3 mg/kg	81.5	140
	81.1	141
	79.8	141
Mean ± SD	80.8 ± 0.898	141 ± 0.325
hexamethyldisiloxane
8.5 mg/kg	79.8	141
	80.2	141
	81.1	141
Mean ± SD	80.3 ± 0.682	141 ± 0.130
Reference Substance
	81.2	5.07
	79.4	5.00
	79.8	5.00
Mean ± SD	80.1 ± 0.945	5.02 ± 0.431

SD = Standard Deviation.

NA = Not Applicable.

Note: All mean and standard deviation values presented in the table were calculated with unrounded numbers.

Table 2. Nitrate transformation rates measured during the soil microflora study with hextamethyldisiloxane.

Treatment Group	Day 28 (mg NO3^-/kg dry soil/day)
Control
	2.10
	2.03
	2.09
Mean ± SD	2.07 ± 0.0404
hexamethyldisiloxane
0.53 mg/kg	2.08
	1.92
	2.02
Mean ± SD	2.00 ± 0.0809
% difference from control	-3.28%
hexamethyldisiloxane
1.1 mg/kg	2.12
	2.15
	2.13
Mean ± SD	2.13 ± 0.0.0153
% difference from control	2.99%
hexamethyldisiloxane
2.1 mg/kg	2.00
	2.14
	2.17
Mean ± SD	2.10 ± 0.0890
% difference from control	1.41%
hexamethyldisiloxane
4.3 mg/kg	2.10
	2.14
	2.17
Mean ± SD	2.14 ± 0.0351
% difference from control	3.08%
hexamethyldisiloxane
8.5 mg/kg	2.18
	2.15
	2.12
Mean ± SD	2.15 ± 0.0277
% difference from control	3.85%
Reference Substance
	-2.72
	-2.66
	-2.67
Mean ± SD	-2.68 ± 0.0322
% difference from control	-229%

SD = Standard Deviation.

NA = Not Applicable.

Note: All mean and standard deviation values presented in the table were calculated with unrounded numbers.

Table 3. Concentrations of test substance measured in sediment samples during the soil microflora study with hexamethyldisiloxane.

Nominal Concentration (mg/kg)	Measured Concentration mg/kg (Percent of Nominal)^a
	Day 0	Day 1	Day 2	Day 3	Day 4	Day 14	Day 28
Control^b	< 0.0211 (NA^d)	< 0.0196 (NA)	< 0.0205 (NA)	< 0.0195 (NA)	< 0.0195 (NA)	< 0.0211 (NA)	< 0.0222 (NA)
0.53^e	0.0243 (4.58)	< 0.0196 (< 3.70)	< 0.0205 (< 3.86)	< 0.0195 (< 3.69)	< 0.0195 (< 3.69)	< 0.0211 (< 3.99)	< 0.0222 (< 4.19)
1.1^f	0.0191^g (1.74)	0.0139^g (1.27)	< 0.0205 (< 1.86)	< 0.0195 (< 1.78)	< 0.0195 (< 1.78)	< 0.0211 (< 1.92)	< 0.0222 (< 2.02)
2.1^h	0.0465 (2.21)	< 0.0196 (< 0.933)	< 0.0205 (< 0.974)	< 0.0195 (< 0.931)	< 0.0195 (< 0.931)	< 0.0211 (< 1.01)	< 0.0222 (< 1.06)
4.3^i	0.0737 (1.71)	0.0481 (1.12)	0.0147^g (0.341)	< 0.0195 (< 0.455)	< 0.0195 (< 0.455)	< 0.0211 (< 0.492)	< 0.0222 (< 0.516)
8.5^j	0.139 (1.64)	0.0375 (0.441)	0.0111^g (0.130)	< 0.0195 (< 0.230)	< 0.0195 (< 0.230)	< 0.0211 (< 0.249)	< 0.0222 (< 0.261)
QC^k#1 0.100	0.0623 (62.3)^l	0.0795 (79.5)	0.0839 (83.9)	0.0711 (71.1)	0.0889 (88.9)	< 0.0211 (NA)^l[JR(1]	0.0822 (82.2)
QC#2 2.00	1.78 (88.9)	1.57 (78.7)	1.72 (86.0)	1.24 (62.2)^l	1.63 (81.3)	1.86 (92.9)	1.71 (85.4)
QC#3 10.0	9.20 (92.0)	9.28 (92.8)	9.18 (91.8)	7.60 (76.0)	8.53 (85.3)	8.94 (89.4)	8.95 (89.5)

NB: LOQ was approximately 0.02 mg/kg (actual values ranged from 0.0195 to 0.0222 mg/kg)

^a      Mean measured and percent of nominal values were calculated using the actual analytical results and not the rounded values (two significant figures) presented in this table.

^b      Analytical samples were removed from test vessel F1015-1.

^c      Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

^d      NA = Not Applicable.

^e      Analytical samples were removed from test vessel F1015-4.

^f      Analytical samples were removed from test vessel F1015-7.

^g      Response ratio is below that of the lowest standard, therefore, the measured concentration is extrapolated. Even though this value is < the curve, it is within 20% of the low standard response factor. As a result, the measured concentration is considered an accurate representation of the concentration in the exposure.

^h      Analytical samples were removed from test vessel F1015-10.

ⁱ      Analytical samples were removed from test vessel F1015-13.

^j      Analytical samples were removed from test vessel F1015-16.

^k      QC = Quality Control sample.

^l      QC sample outside of the acceptable range (i.e., 70 to 120 %). QC samples can be outside of acceptable range due to a number of factors, some of which are spiking, handling, or instrument errors.

Validity criteria fulfilled:

yes

Conclusions:

A 28 day EC50 value of >8.5 mg/kg soil dw and an EC10 value of >8.5 mg/kg soil dw (nominal) (highest concentrations tested) have been reported for the effects of hexamethyldisiloxane on nitrate formation rate of soil microflora.
The test indicates that there were no effects on the soil micro-organisms. However, analysis of the test substance concentrations show that test material was lost within the first three days of the test, therefore the micro-organisms will have been exposed to unquantifiable, minimal amounts of the test substance. No dose-response relationship was apparent, and in view of the

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 October to 9 November 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study, with some modifications, with analysis, under GLP. However test concentrations were not maintained, a solvent was used with no solvent control test and the test was performed in bulk without using individual subsamples.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Principles of method if other than guideline:

The following measures were taken to prevent loss of test item, while allowing for air exchange:
- The test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture. 
- All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil.
- Soil was dosed at ~30% above the calculated maximum sorption concentration to allow for some losses through volatility.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.41, 0.81, 1.6, 3.3, and 6.5 mg/kg
- Sampling method: Soil samples were collected with a spatula from the test vessels. Two sets of samples (one for analysis and the other for archiving) of the soil from each test substance treatment level and the control were removed on test days 0, 1, 2, 3, 4, 14, and 28 for determination of test substance concentration. In addition, at each interval, two sets of three quality control (QC) samples were prepared, one for analysis and the other for archiving. These QC samples were prepared in test soil at relevant test substance concentrations. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Determination of NO3- Ions to Assess Influences on the Nitrification Process:
At each sampling interval, 20 gram dry weight samples were taken from the control; 0.41, 0.81, 1.6, 3.3, and 6.5 mg/kg treatment levels; and reference substance dosed soils and placed in separate containers. The samples were extracted with 100 mL of 0.1 M potassium chloride (KCl) and shaken for 1 hour on a shaker table set at 150 rpm. After the extraction, the samples were centrifuged for 10 minutes at 1000 rpm. An aliquot of approximately 1.5 mL of the supernatant was removed, centrifuged at 10,000 rpm for five minutes, and transferred to a high performance liquid chromatography (HPLC) vial.

- Sample storage conditions before analysis: Not reported

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Natural soil: The soil used for the test was collected on 15 September 2015, sieved (2 mm), and characterized by Lufa-Speyer (SP) of Germany. No pesticides have been applied for at least four years. The soil was taken from the top 20 cm and freshly-collected samples shipped to Smithers Viscient in Wareham, Massachusetts, USA. Additional characterization was conducted by Agvise Laboratories in Northwood, North Dakota. The soil was stored refrigerated (i.e., at approximately 4 ºC) initially and then allowed to acclimate at test temperature for 12 days before it was used for this test.

The water holding capacity of the soil was 35.4%. The moisture content used for the definitive test was 15.93% which is equivalent to 45% of the water holding capacity. The starting moisture content of the soil was 5.01% (as dry weight basis), therefore, 109.2 mL of purified reagent water was added per kilogram of soil during dosing to bring the moisture content to 45% of the water holding capacity.

The alfalfa (also known as Lucerne meal) used to amend the test soil was obtained from Hanson Grain in Hanson, Massachusetts on 29 September 2011 and was determined to have a nitrogen content of 2.7% and a carbon content of 41.2% (equalling a ratio of approximately 1:15) by Agvise Laboratories in Northwood, North Dakota. The alfalfa was amended to the soil at an amount of 5 grams of alfalfa per 1000 grams of soil (dry weight).

- Other: Selection of nominal L3 concentrations was based on the maximum sorption concentration for L3 in the soil. The maximum sorption concentration for L3 in the soil used for this study (based on the solubility of L3 in organic carbon and the organic content of the soil) was approximately 4.5 mg L3/kg of dry soil. Based on anticipated volatile losses, the soil was dosed at approximately 30% above the maximum sorption concentration. Therefore, the test was performed at 0.41, 0.81, 1.6, 3.3, and 6.5 mg/kg.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The octamethyltrisiloxane stock solution was prepared by dissolving 0.0225 g in 30.0 mL of acetonitrile. The final concentration of this stock solution was 0.750 mg/mL (7414C). This stock solution was used in dosing higher concentrations and in preparing secondary stock solutions to dose remaining concentrations. Samples were fortified using a solvent based stock solution, due to the volatile nature and poor water solubility of the test substance. The solvent concentration used was < 1% by volume in all cases, which is typically acceptable for most soil, sludge and water/sediment studies conducted under OECD Guidelines. There was no negative impact of the solvent on the soil, since there was no decline in nitrate formation during 28 days of incubation.

A 10-g batch of quartz sand was dosed with approximately 3.33 mL of the 10 mg/mL reference substance stock solution to achieve a concentration of 33.3 mg/kg in the soil. Seven 1 kg (dry weight) batches of SP 2.3 soil were weighed out into 1-L beakers. One at a time, each 1 kg batch of soil was mixed with 5 g of alfalfa meal, 109.2 mL of purified reagent water, and (reference treatment group only) the dosed batch of quartz sand in a Hobart mixer. Each of the mixed 1 kg (dry weight) batches of soil was then divided into three approximately 333 g (dry weight) replicates. Control and reference samples did not contain any test substance. All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil. Once dosed, each replicate was hand shaken to homogenize then subsampled for extraction.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetonitrile
- Concentration of vehicle in test medium (stock solution and final test solution): The octamethyltrisiloxane stock solution was prepared by dissolving 0.0225 g in 30.0 mL of acetonitrile. The final concentration of this stock solution was 0.750 mg/mL (7414C)
The solvent concentration used was < 1% by volume in all cases
- Evaporation of vehicle before use: No

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20-23.3

Moisture:

15.93% which is equivalent to 45% of the water holding capacity.

Details on test conditions:

TEST SYSTEM
- Testing facility: Smithers Viscient
- Test container (type, material, size): 1 l glass bottle beaker filled with 333g (dry weight) soil, with approximately 2/3 of the test vessel as headspace. Test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture.
- Amount of soil: 333g (dry weight) soil
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: n/a

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Collected by Lufa-Speyer, Germany/Rheinland-Pfalz/Offenbach “Rechts der Landauer Str.”, Nr. 826/7
- History of site: Not reported
- Vegetation cover: Not reported
- Treatments with pesticides or fertilizers: No pesticides have been applied for at least four years
- Accidental contamination: None reported
- Other:
- Depth of sampling: top 20 cm
- Soil texture
- % sand: 65
- % silt: 25
- % clay: 10
- Soil taxonomic classification: USDA
- Soil classification system: Sandy loam
- pH (in water): 6.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 90.8 (dry or wet not specified).
- Maximum water holding capacity (in % dry weight): 35.4%. The starting moisture content of the soil was 5.01% (as dry weight basis).
- Cation exchange capacity (mmol/kg): 5.9
- Pretreatment of soil: Not reported
- Storage (condition, duration): The soil was stored refrigerated (i.e., at approximately 4 ºC) initially and then allowed to acclimate at test temperature for 12 days before it was used for this test.
- Initial microbial biomass as % of total organic C: Biomass as mg C/ 100 g soil (% of total organic carbon)
Prior to test start: 5.1 mg C/100 g soil (0.85%)
At study termination: 17.8 mg C/100 g soil (2.97%)


DETAILS OF PREINCUBATION OF SOIL (if any): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Nitrate concentration at day 0 and day 28.
Nitrate transformation rate at day 28.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY - None reported
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:

Nominal: Control, 0.41, 0.81, 1.6, 3.3, and 6.5 mg/kg
Measured: All measurements

Reference substance (positive control):

yes

Remarks:

dinoseb acetate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 6.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: analytical measurements show test concs were all

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 6.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: analytical measurements show test concs were all

Results with reference substance (positive control):

average inhibition of nitrate formation as compared to the control soil by -228% on test day 28

Reported statistics and error estimates:

Differences between the control and the treated soils were tested for significance using Dunnett’s Multiple Comparison Test (U.S. EPA, 2002), a statistical program in CETIS™ (Ives, 2013). All significance levels were set at 5% (p ≥ 0.05 indicates no significant difference). The difference in results of the 0.41, 0.81, 1.6, and 3.3 mg/kg treated soils at day 28 were statistically significant, due to the reproducibility of the replicates, when compared to the control soil (ANOVA, p < 0.05) when using Dunnett’s Multiple Comparison Test”. The 6.5 mg/l result did not show significant difference when compared to the control.

Table 1. Nitrate concentrations measured during the soil microflora study with octamethyltrisiloxane.

Treatment Group	Day 0 (mg NO3^-/kg dry soil)	Day 28 (mg NO3^-/kg dry soil)
Control
	87.3	146
	94.1	168
	91.1	147
Mean ± SD	90.8 ± 3.41	154 ± 12.2
octamethyltrisiloxane
0.41 mg/kg	92.1	235
	91.4	261
	90.0	252
Mean ± SD	91.1 ± 1.07	249 ± 12.9
octamethyltrisiloxane
0.81 mg/kg	90.4	207
	90.7	216
	101	211
Mean ± SD	94.0 ± 5.95	211 ± 4.55
octamethyltrisiloxane
1.6 mg/kg	92.7	240
	91.5	255
	89.4	245
Mean ± SD	91.2 ± 1.68	246 ± 7.75
octamethyltrisiloxane
3.3 mg/kg	83.5	339
	83.2	344
	84.3	321
Mean ± SD	83.7 ± 0.555	335 ± 12.2
octamethyltrisiloxane
6.5 mg/kg	91.2	132
	93.1	191
	93.2	171
Mean ± SD	92.5 ± 1.12	165 ± 30.0
Reference Substance
	85.1	7.02
	81.6	4.84
	87.6	5.34
Mean ± SD	84.8 ± 3.00	5.73 ± 1.14

SD = Standard Deviation.

Note: All mean and standard deviation values presented in the table were calculated with unrounded numbers.

Table 2. Nitrate transformation rates measured during the soil microflora study with octamethyltrisiloxane.

Treatment Group	Day 28 (mg NO3^-/kg dry soil/day)
Control
	2.10
	2.62
	2.00
Mean ± SD	2.24 ± 0.336
octamethyltrisiloxane
0.41 mg/kg	5.12
	6.05
	5.79
Mean ± SD	5.65 ± 0.480
% difference from control	155%
octamethyltrisiloxane
0.81 mg/kg	4.16
	4.47
	3.94
Mean ± SD	4.19 ± 0.267
% difference from control	88.6%
octamethyltrisiloxane
1.6 mg/kg	5.24
	5.83
	5.55
Mean ± SD	5.54 ± 0.295
% difference from control	150%
octamethyltrisiloxane
3.3 mg/kg	9.14
	9.32
	8.45
Mean ± SD	8.97 ± 0.456
% difference from control	305%
octamethyltrisiloxane
6.5 mg/kg	1.46
	3.50
	2.79
Mean ± SD	2.58 ± 1.04
% difference from control	14.3%
Reference Substance
	-2.79
	-2.74
	-2.94
Mean ± SD	-2.82 ± 0.102
% difference from control	-228%

SD = Standard Deviation.

Note: All mean and standard deviation values presented in the table were calculated with unrounded numbers.

Table 3. Concentrations of test substance measured in sediment samples during the soil microflora study with octamethyltrisiloxane.

Nominal Concentration (mg/kg)	Measured Concentration mg/kg (Percent of Nominal)^a
	Day 0	Day 1	Day 2	Day 3	Day 4	Day 14	Day 28
Control^b	< 0.199^c (NA^d)	< 0.198 (NA)	< 0.198 (NA)	< 0.201 (NA)	< 0.200 (NA)	< 0.201 (NA)	< 0.202 (NA)
0.41^e	< 0.199 (< 48.6)	< 0.198  (< 48.4)	< 0.198  (< 48.2)	< 0.201  (< 48.9)	< 0.200  (< 48.7)	< 0.201  (< 48.9)	< 0.202  (< 49.2)
0.81^f	< 0.199 (< 24.6)	< 0.198  (< 24.5)	< 0.198  (< 24.4)	< 0.201  (< 24.8)	< 0.200  (< 24.7)	< 0.201  (< 24.8)	< 0.202  (< 24.9)
1.6^g	< 0.199 (< 12.5)	< 0.198  (< 12.4)	< 0.198  (< 12.3)	< 0.201  (< 12.5)	< 0.200  (< 12.5)	< 0.201  (< 12.5)	< 0.202  (< 12.6)
3.3^h	< 0.199 (< 6.04)	< 0.198  (< 6.01)	< 0.198  (< 5.99)	< 0.201  (< 6.08)	< 0.200  (< 6.06)	< 0.201  (< 6.08)	< 0.201  (< 6.12)
6.5^i	< 0.198 (< 3.05)^j	< 0.198  (< 3.05)	< 0.198  (< 3.04)	< 0.201  (< 3.09)	< 0.200  (< 3.07)	< 0.201  (< 3.09)	< 0.201  (< 3.11)
QC^k#1 1.00	1.03 (103)	1.05 (105)	1.03 (103)	1.02 (102)	0.974 (97.4)	1.01 (101)	1.06 (106)
QC#2 3.00	3.07 (102)	3.10 (104)	3.11 (104)	3.10 (103)	3.01 (100)	2.98 (99.3)	3.13 (104)
QC#3 10.0	9.70 (97.0)	9.47 (94.7)	9.48 (94.8)	9.62 (96.2)	9.59 (95.9)	9.19 (91.9)	1.03 (103)

^a      Mean measured and percent of nominal values were calculated using the actual analytical results and not the rounded values (two significant figures) presented in this table.

^b      Analytical samples were removed from test vessel F1015-142.

^c      Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

^d      NA = Not Applicable.

^e      Analytical samples were removed from test vessel F1015-145.

^f      Analytical samples were removed from test vessel F1015-148.

^g      Analytical samples were removed from test vessel F1015-151.

^h      Analytical samples were removed from test vessel F1015-154.

ⁱ      Analytical samples were removed from test vessel F1015-157.

^j      Reanalysis at lowest validated dilution factor.

^k      QC = Quality Control sample.

Validity criteria fulfilled:

yes

Conclusions:

A 28 day EC50 value of >6.5 mg/kg soil dw and an EC10 value of >6.5 mg/kg soil dw (nominal) (highest concentrations tested) have been reported for the effects of octamethyltrisiloxane on nitrate formation rate of soil microflora.
The test indicates that there were no effects on the soil micro-organisms. However, analysis of the test substance concentrations show that test material was lost at the very beginning of the test, therefore the micro-organisms will have been exposed to unquantifiable, minimal amounts of the test substance, if any at all. Apparent significant effects (positive and negative) on nitrate concentration and nitrate transformation rate were observed in this study in the 0.41, 0.81, 1.6, and 3.3 mg/kg treated soils at day 28, and replicates at equivalent test concentration showed good repeatability. No dose-response relationship was apparent, and in view of the

Description of key information

HMDS: Toxicity to soil micro-organisms: HMDS could not be maintained in the test system (refer to Discussion); no dose-response relationship was apparent. (OECD TG 216).

L3: Toxicity to soil micro-organisms: L3 could not be maintained in test system (refer to Discussion); no dose-response relationship was apparent. (OECD TG 216).

L4: An OECD TG 216 study is proposed with L4.

L5: An OECD TG 216 study is proposed with L5.

Additional information

Constituent HMDS

A 28-day EC₅₀ value of >8.5 mg/kg soil dw and an EC₁₀ value of >8.5 mg/kg soil dw (highest nominal concentrations tested) have been reported in an OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test) for the effects of hexamethyldisiloxane (HMDS) on nitrate formation rate of soil microflora (Smithers Viscient, 2016).

The test indicates that there were no inhibitory effects on the soil micro-organisms. However, analysis of the test substance concentrations show that test material was lost by day three of the test, with all concentrations below the limit of detection, therefore the micro-organisms would have been exposed to unquantifiable, minimal amounts of the test substance. No dose-response relationship was apparent, and in view of the <LOQ recoveries of test material, it is not appropriate to ascribe the absence of effects to a lack of toxicity of HMDS. The results of this test cannot be used for derivation of PNECs and risk assessment.

HMDS has a very high Henry's Law Constant and losses through volatilisation during test media preparation and within the test system were anticipated. Because of this, measures were taken during the soil micro-organism test to minimise test substance losses.

 

An initial test was conducted using a closed bottle system. During this test, nitrate levels decreased significantly in all concentrations, including the control. The report authors explained that this was most likely due to the test vessels being sealed air tight, thus not supplying enough oxygen to allow the nitrifying bacteria to convert the alfalfa nitrogen source to NO₃. Thus the study was repeated under a separate protocol, using a different test design.

 

During the definitive test, the following measures were taken to prevent loss of test item, while allowing for air exchange:

-The test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture. 

- All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil.

- Soil was dosed at ~30% above the calculated maximum sorption concentration to allow for some losses through volatility.

 

Despite these measures, significant loss of test material was observed by day 3. It may therefore be concluded that the soil micro-organisms were not exposed to adequate concentrations of test substance, and the EC50 and EC10 values determined are not representative of actual exposure of the test organisms to the test substance. It is therefore not possible to use these test results for calculation of terrestrial PNECs and risk assessment.

 

Constituent L3

A 28-day EC₅₀ value of >6.5 mg/kg soil dw and an EC₁₀ value of >6.5 mg/kg soil dw (highest nominal concentrations tested) have been reported in an OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test) for the effects of octamethyltrisiloxane (L3) on nitrate formation rate of soil microflora (Smithers Viscient, 2016).

The test indicates that there were no inhibitory effects on the soil micro-organisms. However, analysis of the test substance concentrations show that test material was lost at the very beginning of the test, with all concentrations below the limit of detection, therefore the micro-organisms would have been exposed to unquantifiable, minimal amounts of the test substance, if any at all. Apparent significant effects (positive, non-inhibitory) on nitrate concentration and nitrate transformation rate were observed in this study in the 0.41, 0.81, 1.6, and 3.3 mg/kg treated soils at day 28: replicates at equivalent test concentrations showed good repeatability. No dose-response relationship was apparent, and in view of the <LOQ recoveries of test material, it is not appropriate to ascribe the observed effects to L3. The results of this test cannot be used for derivation of PNECs and risk assessment.

L3 has a very high Henry's Law Constant and losses through volatilisation during test media preparation and within the test system were anticipated. Because of this, measures were taken during the soil micro-organism test to minimise test substance losses.

An initial test was conducted using a closed bottle system. During this test, nitrate levels decreased significantly in all concentrations, including the control. The report authors explained that this was most likely due to the test vessels being sealed air tight, thus not supplying enough oxygen to allow the nitrifying bacteria to convert the alfalfa nitrogen source to NO^₃.

During the definitive test, the following measures were taken to prevent loss of test item, while allowing for air exchange:

- The test vessels were sealed with perforated parafilm, which allowed for air exchange and avoided loss in moisture. 

- All test substance concentrations were dosed using gastight Hamilton syringes in multiple spots underneath the soil.

- Soil was dosed at ~30% above the calculated maximum sorption concentration to allow for some losses through volatility.

Despite these measures, significant loss of test material was observed by day 0. It may therefore be concluded that the soil micro-organisms were not exposed to adequate concentrations of test substance, and the EC₅₀ and EC₁₀ values determined are not representative of actual exposure of the test organisms to the test substance. It is therefore not possible to use these test results for calculation of terrestrial PNECs and risk assessment.

 

Constituent L4

No toxicity to soil microorganisms data are currently available with L4. An OECD TG 216 study is proposed with L4.

 

Constituent L5

No toxicity to soil microorganisms data are currently available with L5. An OECD TG 216 study is proposed with L5.