Triethoxyoctylsilane

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:

Experimental start date: 29 October 2021. Experimental completion date: 26 November 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Information provided by the sponsor showed the test substance was not suitable for dosing using a water stock (water solubility <0.13 mg/L). Additionally, the test substance was not suitable for dosing using a sand carrier coated with a solvent stock because the test substance is a liquid. To use this method, the solvent stock is applied to the sand and then the solvent is evaporated off leaving test substance coated sand, however because the test substance is a liquid, it cannot be confirmed if the remaining wet sand (wet because the test substance was a liquid) was still containing residues of solvent.
For these reasons, the test was dosed by adding neat test substance directly to the soil and then thoroughly mixing.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.3-22.2 ºC

Moisture:

The moisture content of the test substrate was adjusted gravimetrically by the addition of RO water every 7 days, where required, and prior to sampling.

Organic carbon content (% dry weight):

0.66

Nitrogen content (% dry weight):

0.08

Details on test conditions:

TEST SYSTEM
- Testing facility: Smithers ERS Limited, 108 Woodfield Drive, Harrogate, North Yorkshire, HG1 4LS, UK
- Test container (type, material, size): 500-mL amber glass jars. After filling, the test vessels were sealed with a punctured plastic sheeting lid to allow for air exchange.
- Amount of soil: 100g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle used.

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): “rechts der Landauer Str”, Nr. 826/7
- History of site: no organic fertilisation used in 2017-2021. No pesticides used in sampling year or previous four years.
- Vegetation cover: not reported
- Treatments with pesticides or fertilizers:
- Accidental contamination: none reported
- Other: Lucern meal added: alfalfa 20/001. Total nitrogen: 3.61%, total carbon 43.42% and carbon/nitrogen ratio 12:1.
- Depth of sampling: approx. 0 - 20 cm
- Soil texture: not reported
- Soil taxonomic classification: Lufa 21/004
- Soil classification system: not reported
- pH (in water): 5.97
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 0.08 ± 0.02 as % N
- Maximum water holding capacity (in % dry weight): 35.7 ± 2.3 %
- Cation exchange capacity (mmol/kg): 6.0 ± 0.9 meq/100g
- Pretreatment of soil: Test soil equivalent to 400 g dry weight was weighed out for each test group. The test substance for each treatment level was weighed out onto glass slides. The appropriate glass slide was held over its respective soil group and, using the reverse osmosis (RO) water due to be added to the soil, the test substance was washed off the slide directly onto the soil. The volume of RO water (28 mL) added to the soil in this way was used to increase the moisture content to the required level (45 % of maximum water holding capacity (MWHC)). A 2 g aliquot of powdered lucerne (alfalfa) was also added. The batch of powdered lucerne used was received on 17 December 2020. It was analysed singularly and had an organic carbon: nitrogen ratio of 12:1 as required by the guideline. Once all components had been added, 60 seconds were allowed for the test substance to soak into the soil. The soil was then mixed using an electric hand-mixer. Mixing of test soil was undertaken until evenly distributed, then approximately 100 g of soil (dry weight) was weighed into each replicate vessel from each bulk group of test soil.
- Storage (condition, duration): 7 days.
- Initial microbial biomass as % of total organic C: 1.5 %

DETAILS OF PREINCUBATION OF SOIL (if any): The test soil was acclimatised for 7 days under test environmental conditions.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Nitrate Extraction: After treatment application on Day 0, and after moisture adjustment on Day 28 of incubation, soil samples were collected and nitrogen extraction was performed. At Day 0, samples were collected from the remaining bulk soil and at Day 28 a sub-sample of soil was collected from each replicate vessel. For each sample approximately 7 g dry weight was collected. Potassium chloride (0.1M KCl, 35 mL) was added to the sample and shaken (ca 150 RPM) for approximately 60 minutes on a shaker table. The extracted samples were then centrifuged at ca 3400 RPM for 10 minutes. The supernatant extract was then filtered using 0.45-µm filters. The Day 0 extracts were frozen after extraction and analysed after 20 days of storage (samples can be stored frozen prior to analysis for up to 6 months). The Day 28 extracts were analysed on the day of sampling. The extracts were stored frozen post-analysis; frozen samples will be destroyed after issuing the final report.

Nitrate Analysis: The 0.1 M potassium chloride extracts were analysed for NO3-N, using a Hach Lange DR 3900 Spectrophotometer.
For each extract, 1.0 mL of sample was added to a Hach LCK 339 cuvette with 0.2 mL of Hach LCK 339 Solution A. This was then shaken vigorously and allowed to stand for 15 minutes to allow the reaction to take place. Following this, the cuvette was placed on the spectrophotometer and the NO3-N reading presented in mg/L was displayed, which was recorded in the raw data. This procedure was repeated for each sample. The Limit of Detection (LOD) for this technique is 0.23 mg/L NO3-N.


VEHICLE CONTROL PERFORMED: not applicable, no vehicle used.

RANGE-FINDING STUDY - not performed.

Nominal and measured concentrations:

Nominal: 0, 10, 32, 100, 320 and 1000 mg/kg dry soil.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

320 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

779 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% C.I. 321 mg/kg dw - upper limit not calculated

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: none
- Other: The 10 – 320 mg/kg dry soil concentrations had inhibition levels that fluctuated ±12% compared to the controls. These were not deemed significant effects as there was no dose-response related trend. Given this variation and the effect it had on the 95% confidence limits for the EC10 (26.8 mg/kg (95% C.I. 321 - upper limit not calculated)) and EC25 (435 mg/kg dw (95% C.I. 140-775 mg/kg dw)), the EC10 and EC25 values were not deemed reliable.

The 1000 mg/kg dry soil concentration had a clear and strong effect (58% inhibition), as such it is possible to calculate accurate NOEC and EC50 values.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v1.8.6.8.
Linear interpolation analysis was performed in order to estimate the EC10, EC25 and EC50 values for test substance. The 95 % confidence limits have also been included.
CETIS deemed the data non-monotonic so a Dunnett multiple comparison test was used to estimate the test substance no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) (the data had equal variance and normal distribution).

Table 1. Amount of nitrate produced over 28 days in test soil: Nitrate-Nitrogen NO₃-N

Treatment (mg/kg dry soil)	Nitrate NO3-N produced over 28 days  (mg N/kg dry soil)	Nitrate NO3-produced over 28 days (mg N/kg dry soil)	Mean Nitrate NO3-N produced over 28 days (mg N/kg dry soil)	Mean Nitrate NO3- produced over 28 days (mg N/kg dry soil)	Difference Compared to the Control (%)
Control (0)	26.5	116.6	25.3	111.2	NA
	27.5	121.0
	21.8	95.9
10	26.1	114.7	28.3	124.7	12.2
	30.5	134.1
	28.5	125.3
32	20.2	88.9	22.3	98.1	-11.7
	24.7	108.7
	22.0	96.8
100	21.5	94.6	23.1	101.5	-8.71
	18.7	82.2
	29.0	127.6
320	28.8	126.7	25.5	112.3	1.06
	19.7	86.6
	28.1	123.6
1000	3.2	14.1	10.5	46.4	-58.3
	11.2	49.3
	17.2	75.7

Day 0 Control Coefficient of Variation = 6.11 %

Day 28 Control Coefficient of Variation = 8.51 %

Table 2. Amount of nitrate produced over 28 days in test soil: Nitrate NO₃^-

Nominal Concentration (mg/kg dry soil)	Vessel	NitrateNO3^- (mg N/kg dry soil)
		Day 0	Mean	Day 28	Mean
Control (0)	1A	46.6	46.2	162.8	157.5
	1B	48.4		167.2
	1C	42.9		142.1
10	2A	38.4	40.6	155.3	165.4
	2B	40.7		174.7
	2C	42.7		165.9
32	3A	52.4	54.1	143.0	152.2
	3B	54.6		162.8
	3C	55.0		150.9
100	4A	57.2	54.6	149.2	156.2
	4B	57.6		136.8
	4C	48.8		182.2
320	5A	58.1	54.6	181.3	166.8
	5B	48.8		141.2
	5C	57.2		178.2
1000	6A	53.7	51.0	65.1	97.2
	6B	51.9		100.3
	6C	47.5		126.7

Validity criteria fulfilled:

yes

Remarks:

Relative standard deviation between control replicate samples was <15% (6.11% and 8.51%)

Conclusions:

28-day EC50 and NOEC values of 779 mg/kg soil dry weight and 320 mg/kg soil dry weight have been determined for the effects of the test substance on nitrate transformation rate of soil microorganisms. The result is expressed as nominal concentrations of the test substance over the test period.

Description of key information

Toxicity to soil microorganisms: 28-day NOEC 320 mg/kg dry weight (nominal), effects of triethoxy(octyl)silane on the nitrogen transformation rate of terrestrial microorganisms.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

779 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

320 mg/kg soil dw

Additional information

A toxicity to soil microorganisms study according to the OECD test guideline 216 and in compliance with GLP has been conducted with the registered substance, triethoxy(octyl)silane (CAS 2943-75-1, EC No. 220-941-2) (Smithers 2022b).

The draft study report only is currently available, which reports the following effects on inhibition of nitrogen transformation:

28-day EC₅₀ value of 779 mg/kg soil dry weight and a 28-day NOEC value of 320 mg/kg soil dry weight have been determined for the effects of the test substance on nitrate transformation rate of soil microorganisms. The result is expressed as nominal concentrations of the test substance over the test period.

The test was conducted at nominal concentrations of 10, 32, 100, 320 and 1000 mg/kg dry soil. The 10 – 320 mg/kg dry soil concentrations had inhibition levels that fluctuated ±12% compared to the controls. These were not deemed significant effects as there was no dose-response related trend. Given this variation and the effect it had on the 95% confidence limits for the EC₁₀ and EC₂₅, the EC₁₀ and EC₂₅ values were not deemed reliable, therefore no conclusions could be drawn on EC₁₀ and EC₂₅.

The 1000 mg/kg dry soil concentration had a clear and strong effect (58% inhibition), therefore it was possible to calculate accurate NOEC and EC₅₀ values, which are used as key values for chemical safety assessment of toxicity of the substance to soil microorganisms.

The hydrolysis half-life of triethoxy(octyl)silane at 20-25°C is approximately 30 hours at pH 7, 0.7 hours at pH 4 and 0.4 hours at pH 9 (QSAR) to form octylsilanetriol and ethanol. However, the hydrolysis rate of the substance in soil is not known and is expected to be slower once adsorbed to organic matter.

It is therefore likely that test organisms were initially exposed predominantly to the parent substance, with hydrolysis occurring throughout the duration of the test resulting in increasing amounts of exposure to the hydrolysis products.

Chemical analysis of test substance concentrations in the soil was not conducted. Neither the substance nor its silanol hydrolysis product are volatile and the silanol is not biodegradable. Therefore, although triethoxy(octyl)silane is expected to transform to its hydrolysis products in contact with moisture in the soil, losses of test substances are not expected through other means. Consequently, the nominal concentrations of the test substance applied to the soil are expected to have remained in the soil throughout the duration of the study, either in the form of the parent substance or the silanol hydrolysis product. The non-silanol hydrolysis product, ethanol, is likely to have volatilised out of the soil. The effects on soil microorganisms observed in the study are therefore attributed to the toxicity of the substance and its hydrolysis products, although it is not possible to assess which chemical species is causing the effects.