Reaction mass of 1,1,1,5,5,5-hexamethyl-3-phenyl-3-((trimethylsilyl)oxy)trisiloxane and 1,1,1,7,7,7-hexamethyl-3,5-diphenyl-3,5-bis[(trimethylsilyl)oxy]tetrasiloxane and 1,1,1,9,9,9-hexamethyl-3,5,7-triphenyl-3,5,7-tris((trimethylsilyl)oxy)pentasiloxane

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:

19 December 2019 - 16 January 2020

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:

yes

Remarks:

EC10 value could not be accurately determined due to a non-monotonic dose response curve.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Natural Speyer 2.3 soil
- Other:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The control sample was treated the same way as the dosed samples but without being dosed. Six 1.0-kg (dry weight equivalent) batches of soil were weighed out into beakers. Starting with the control, each batch of soil was dosed and mixed as follows. Approximately half of the soil batch was transferred to a 4-L jar. An appropriate amount of the test substance was dosed dropwise across the soil surface using a pipette. The remaining half of the soil batch was then added to the 4-L jar. The soil was mixed by rolling the capped jar for approximately 3 hours at approximately 15 rpm on a rolling mill. The fortified soil was transferred to the bowl of a food mixer and mechanically mixed for 10 minutes at low speed. During this mechanical mixing, the soil was amended with 5.0 g of lucerne meal (i.e. alfalfa). Also, during this mechanical mixing, a volume of purified reagent water was added to the soil in order to bring the moisture content to 45 ± 5% of the soil’s maximum water holding capacity. After dosing and mixing, each batch of soil was divided equally into three replicate test samples in 1-L glass bottle test vessels.

VEHICLE: no vehicle used.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

18.6 - 21.2 degrees C

Moisture:

approximately 45% of the maximum water holding capacity

Organic carbon content (% dry weight):

0.5

Details on test conditions:

TEST SYSTEM
- Testing facility: Smithers, 790 Main Street, Wareham, Massachusetts 02571-1037
- Test container (type, material, size): 1L glass bottles covered with perforated parafilm to allow for air exchange while reducing water loss
- Amount of soil: Approximately 333g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: not applicable

SOIL INCUBATION
- Method: bulk. Test samples were incubated in the dark in an environmental chamber.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Rheinland-Pfalz, Germany, "rechts der Landauer Str." No. 826/7
- History of site: No pesticides or organic fertilizers were used within 4 years before collection
- Vegetation cover: Not reported
- Treatments with pesticides or fertilizers: No pesticides or organic fertilizers were used within 4 years before collection
- Accidental contamination: Not reported
- Other:
- Depth of sampling: top 20 cm
- Soil texture
- % sand: 61
- % silt: 26
- % clay: 13
- Soil taxonomic classification: Not reported
- Soil classification system: not reported
- pH (in water): 6.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Mean across all treatments = 107.6
- Maximum water holding capacity (in % dry weight): 28.6%
- Cation exchange capacity (mmol/kg): 6.1%
- Pretreatment of soil: Lucerne meal (alfalfa) was added to the test soil. The alfalfa had a carbon content of 39.9% and nitrogen content of 3.01%.
- Storage (condition, duration): Before use, the soil was transferred to an environmental chamber set to maintain a temperature of 20 degrees C and allowed to equilibrate to incubation conditions for 2-28 days (3 days actual).
- Initial microbial biomass as % of total organic C: 1.75%

DETAILS OF PREINCUBATION OF SOIL (if any):Before use, the soil was transferred to an environmental chamber set to maintain a temperature of 20 degrees C and allowed to equilibrate to incubation conditions for 2-28 days (3 days actual).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : A subsample from each test sample was analyzed for the determination of the concentration of nitrate after 0 and 28 days of incubation.

VEHICLE CONTROL PERFORMED: not necessary as no vehicle used.

Nominal and measured concentrations:

Nominal: untreated control, 1.2, 3.7, 11, 33, 100 mg/kg

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

1.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: All test concentrations had significantly reduced nitrogen formation rates

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: None reported
- Other: Due to the lowest treatment exhibiting a >10% reduction in NO3 formation rates, an EC10 could not be accurately determined. It is not possible to extrapolate an EC10 due to the non-monotonic (flat) dose response curve and this response suggests the effects to be due to a non-toxic mechanism. In addition, no long-term influence on nitrogen transformation in soils is anticipated as the difference in the rates of nitrate formation between the treatments and the controls were below 25%. It is expected that phenyl silsesquioxanes has no long-term effects on the nitrogen transformation rates of soil microflora at environmentally relevant concentrations.

Reported statistics and error estimates:

Inhibition between the untreated control and the treated soils were tested for significance using the ANOVA and Dunnett’s Multiple Comparison Test statistical programs.
The nitrate formation rates were analyzed using the linear interpolation (ICPIN) statistical program in CETIS to determine EC10, EC25, and EC50 values (the concentrations at which nitrate formation is affected by 10, 25, and 50% relative to the untreated control, respectively). The results were investigated for outliers using the Grubbs Extreme Value Test statistical program in CETIS.

Table 1. Nitrogen formation rates of soil microorganisms after exposure of phenyl silsesquioxanes

Nominal concentration (mg/kg)	Mean Day 0 nitrate concentration (mg/kg)	Mean day 28 nitrate concentration (mg/kg)	Mean nitrate formation rate (mg/kg/day)	Rate difference from untreated control
Untreated control	107	275	6.03	N/a
1.2	105	233	4.56	-24.3
3.7	111	248	4.90	-18.76
11	110	252	5.05	-16.26
33	107	242	4.83	-19.86
100	106	244	4.94	-18.05

Validity criteria fulfilled:

yes

Remarks:

Relative standard deviation for the untreated control group ranged from 0.85-4.47% which is less than the 15% outlined in the OECD TG 216 validity criteria

Conclusions:

28-day EC25 and EC50 values of >100 mg/kg were reported for the effects of the test substance on the nitrate formation rate of soil microorganisms, based on nominal concentrations. Nitrate formation rates were significantly reduced in all treatment groups compared to the control, which gives a 28-day LOEC of 1.2 mg/kg dwt. However, a dose response relationship was not observed, suggesting the effects to be due to a non-toxic mechanism. The EC25, EC50 and LOEC values are equivalent to >400 , >400 and 4.8 mg/kg dwt when normalised to 2% organic carbon.

Description of key information

EC₂₅ and EC₅₀ (28-day) >100 mg/kg dwt (nominal), nitrate formation rate, soil microorganisms

These values are equivalent to >400 mg/kg dwt when normalised to 2% organic carbon.

Additional information

An OECD TG 216 soil microorganisms (nitrogen transformation) is available for the registration substances.

28-day EC₂₅ and EC₅₀ values of >100 mg/kg dwt were reported for the effects on the nitrate formation rate of soil microorganisms, based on nominal concentrations. Nitrate formation rates were significantly reduced in all treatment groups compared to the control, which gives a 28-day LOEC of 1.2 mg/kg dwt. However, a dose response relationship was not observed, suggesting the effects to be due to a non-toxic mechanism. The EC₂₅, EC₅₀ and LOEC values are equivalent to >400, >400 and 4.8 mg/kg dwt when normalised to 2% organic carbon.