N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The light intensities measured during the test were in three of nine places in the experimental area not within the range of 4440-8880 Lux stated in the study plan (actual: 7220-8980 Lux). This deviation has no impact on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

EC Number:

810-490-5

Cas Number:

35501-23-6

Molecular formula:

C15H35NO3Si

IUPAC Name:

N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Constituent 2

Reference substance name:

N,N-Dibutylaminomethyl-triethoxysilan

IUPAC Name:

N,N-Dibutylaminomethyl-triethoxysilan

Test material form:

other: liquid

Details on test material:

Identification: N,N-Dibutylaminomethyl-triethoxysilane
CAS Number: 35501-23-6
Batch: 310113
Purity: 96.6%
Molecular Formula: C15H35NO3Si
Molecular Weight: 305.53
Expiry Date: 06 February 2016
Storage Conditions: At room temperature, at 20 ± 5 °C, under N2
Water solubility: Rapid hydrolysis at contact with water
Hydrolysis: Wacker Chemie AG Report (*1): “At all examined pH values only signals of N,N-dibutylaminomethylsilanetriol and ethanol could be observed after the first 1H-NMR measurements. There was no signal of ethoxysilane detectable. Therefore it was concluded that hydrolysis was already completed when the first
spectrum of these pH series (pH 4.0, pH 7.0, pH 9.0 respectively) was measured. Because hydrolysis was finished after the first 1H-NMR measurements (about 2 minutes after mixing the test sample) no exact halflife time or reaction rate could be calculated.” It can be concluded that the hydrolysis half-life of the test item at pH 7 is below two minutes. The water solubility of the hydrolysis product N,N-dibutylaminomethylsilanetriol is > 300 mg/L (*2) at 25 °C. However, the silanetriol tends to form condensation products, which are much less soluble in water. The concentration limit for condensation must be determined prior to testing to achieve stable solutions.

(*1) Final Report Determination of Physico-chemical Properties of SILAN DBA-TEO (CAS No. 35501-23-6) Wacker Chemie AG Study number: N-0002109725-P, see IUCLID chapter 5.1.2 Hydrolysis
(*2) QSAR calculation using WSKOWWIN V 1.42 (US EPA)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:

In consultation with the Sponsor, the test concentrations and the test media preparation steps were chosen based on the results of GLP pre-experiments to determine the solubility of the test item in the test water. It was the aim of the procedure to achieve the maximum technically feasible concentration of the test item or its hydrolysis products in the test solution.

- Controls: Test water without addition of test item or solvent, Solvent control was prepared by adding 100 μL THF to 1.0 L test water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): (THF, HPLC grade, 99.99%)
- Concentration of vehicle in test medium (stock solution and final test solution): 100 μL THF to 1.0 L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test media were observed to be clear and colorless with no visible undissolved test substance following preparation with the exception of the test medium with a nominal concentration of 4.0 mg/L, which was observed to contain scattered small particles on the water surface.

FOR DETAILS OF PREPARATION OF TEST SOLUTION SEE ALSO: Any other information on materials and methods

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG (*1)
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: An inoculum culture was set up four days before the start of the exposure.
- Culturing media and conditions (same as test or not): The algae were cultivated under the test conditions.

*1 Nygaard et al. [Nygaard, 1986] recommended describing the taxa within the Genus Raphidocelis
HINDAK as:
Raphidocelis subcapitata (KORSHIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSHIKOV
Syn.: Kirchneriella subcapitata KORSHIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Hardness:

The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C.

pH:

The pH was 7.5.

Nominal and measured concentrations:

No analytical work was performed, with respect to the hydrolytic instability of the test item. A measurement of total organic carbon (TOC) was not reasonable due to the low test item concentrations and the use of the solvent tetrahydrofuran (THF). For this reason, the reported biological results were based on the nominal concentrations of the test item N,N-Dibutylaminomethyltriethoxysilane.

The following nominal concentrations of N,N-Dibutylaminomethyl-triethoxysilane were tested:
0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L test water.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with a capacity of 50 mL
- Type (delete if not applicable): Each test flask was covered with a glass dish.
- Material, size, headspace, fill volume: 15 mL of test solution.
- Renewal rate of test solution (frequency/flow rate): A static test design was applied.
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): Three replicates per test concentration
- No. of vessels per control (replicates): Three replicates
- No. of vessels per vehicle control (replicates): Six replicates of the solvent control.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:

Macro-nutrients:
Ingredients Concentration
NaHCO3 15.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace elements:
Ingredients Concentration
H3BO3 186.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
ZnCl2 3.27 μg/L
CoCl2 × 6 H2O 1.43 μg/L
CuCl2 × 2 H2O 0.012 μg/L
Na2MoO4 × 2 H2O 7.26 μg/L
FeCl3 × 6 H2O 160.0 μg/L
Na2EDTA × 2 H2O 300.0 μg/L

The pH was 7.5. The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

OTHER TEST CONDITIONS
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 8100 Lux (range: 7220 to 8980 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15%-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: A small volume (2 x 100 μL) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader,
Model FLx800, wavelength: excitation 440 nm, emission 680 nm; Software: KC4, version 3.4 rev. 21). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the solvent control and from the test concentration with the nominal concentration of 4.0 mg/L to determine a potential influence of the test item on the morphology of the algal cells. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline: In consultation with the Sponsor, the test concentrations and the test media preparation steps were chosen based on the results of GLP – pre-experiments.
- Range finding study: GLP – pre-experiments to determine the solubility of the test item in the test water
- Test concentrations: The following nominal concentrations of N,N-Dibutylaminomethyl-triethoxysilane were tested:
0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L test water.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): In the solvent control, the biomass increased by a factor of 165 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the solvent control (section-bysection growth rates) during 72 hours was 5.7%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the solvent control after 72 hours was 2.9%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. All validity criteria were also fulfilled for the control.

- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal concentration of 4.0 mg/L and the algal cells in the solvent control. The shape and size of the algal cells were obviously not affected by the test item up to this concentration.

- Effect concentrations exceeding solubility of substance in test medium: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. Only prior to filtration, scattered particles were observed in the test medium with the nominal concentration of 4.0 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes!
- EC50: 1.1 mg/L
- Other: The result of the latest positive control test performed in August 2014 (72-hour EC50 for the growth rate: 1.1 mg/L , Harlan Study Number D94966) showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate from 2000 to 2014: 0.71-1.7 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the test item N,N-Dibutylaminomethyl-triethoxysilane had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to the maximum technically feasible concentration of the test item or its hydrolysis products in test water under the test conditions, which was determined to be below 4.0 mg/L based on visual inspection (EC50 > 4.0 mg/L).

Executive summary:

SUMMARY

The impact of the test item N,N-Dibutylaminomethyl-triethoxysilane on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011) and the Commission Regulation (EC) No 761/2009, C.3.

The nominal test item concentrations tested were 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg test item/L test water. Additionally, a control and a solvent control group were tested in parallel. Due to the low solubility (See also: Details on test substance) of the test item in water, the organic solvent tetrahydrofuran (THF) was used to dose the test item. The solvent was chosen based on information provided by the Sponsor.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. It was the aim of the procedure to achieve the maximum technically feasible concentration of the test item or its hydrolysis products in the test solution.

After dosing the test substance into the test water, small scattered particles of undissolved test substance were observed in the test medium with the highest nominal concentration of 4.0 mg/L. To avoid a possible secondary effect on the algal cells, all test media were filtered through a membrane filter prior to the start of the exposure.

All required validity criteria were fulfilled in this test.

Since in agreement with the Sponsor no analytical work was performed, the reported biological results were based on the nominal concentrations (See also: Any other information on materials and methods incl. tables) of the test item N,N-Dibutylaminomethyltriethoxysilane:

Parameter (0-72 h):	Growth rate	Yield
EC50 [mg/L]	>4.0	>4.0
EC20 [mg/L]	>4.0	>4.0
EC10 [mg/L]	>4.0	>4.0
NOEC [mg/L]	4.0	4.0
LOEC [mg/L]	>4.0	>4.0

In conclusion, the test item N,N-Dibutylaminomethyl-triethoxysilane had no toxic effects on Pseudokirchneriella subcapitata during the 72-hour test period up to the maximum technically feasible concentration of the test item or its hydrolysis products in test water under the test conditions, which was determined to be below 4.0 mg/L based on visual inspection.