N,N',N''-tributyl-1-methylsilanetriamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analysis of test substance concentrations was carried out

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

No analysis of test substance concentrations was carried out

GLP compliance:

yes

Specific details on test material used for the study:

See test material information

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Tetrahydrofuran

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Pre-testing
The study started with the determination of the test item solubility in tetrahydrofuran (performed under Project 512078). Thereafter, the solubility of test item stock solutions in test medium was determined. Based on the results of this test, the test concentrations for the full fresh water algal growth inhibition test were determined.
The test item undergoes rapid hydrolysis in contact with water. The onset of condensation of the silanetriol product limited the highest soluble test concentration (“technical feasible maximum test concentration”) and was therefore experimentally determined. In this pre-test, tetrahydrofuran THF was used as a solubilising agent to avoid concentration peaks during sample preparation and allow observing the onset of condensation of the silanetriol by visual inspection of the test medium. The determined solubility limit in test medium was applied to justify the selected dose range in the full test.

Solubility in tetrahydrofuran
The test item appeared to be completely soluble in tetrahydrofuran (THF) at a concentration of 1000 mg/mL (Project 512078).

Determination of solubility in test medium
To determine the solubility of a test item stock solution in test medium (M2), two experiments were performed. Preparation of stock solutions started with a concentration of 100 mg/mL. No special treatment other than vigorous shaking was required to fully dissolve the test item in THF. Lower concentrations (i.e. 1.56, 3.13, 6.3, 12.5, 25 and 50 mg/mL) were obtained by subsequent dilutions of the highest concentration in THF with a factor of 2. All stock solutions were clear and colourless.
After preparation, volumes of 100 µL were spiked into 1 L of test medium to achieve the concentrations 5, 2.5, 1.25, 0.63, 0.313 and 0.156 mg/l. Spiking was done slowly by adding the respective stock solution in steps of 20 µL below the test medium surface over a period of six minutes. Throughout, the test medium was magnetically stirred without turbulence at approximately 200 rpm. The pH values of these solutions were in a range of 7.9 to 8.7 and correspondingly adjusted to 7.0 ± 0.2 using 1 M HCl. Thereafter, a 24-hour period of magnetic stirring at 200 rpm was applied. Solutions spiked with stocks of 6.3 mg/mL and lower were clear and colourless at the end of the preparation period. Solutions spiked with stocks of 12.5 mg/mL and higher were colourless but contained undissolved material. The dissolution of the test item was determined visually using a laser pointer.
Consequently, the concentration at which the condensation products were visually completely soluble in ISO medium was considered to be the limit concentration (i.e. 0.63 mg/L).

Preparation of stock and test solutions
The batch of N,N’,N’’-Tributyl-1-methylsilanetriamine tested was a clear slightly yellow liquid with a purity of 86%. No correction was made for the purity/composition of the test item.

Preparation of stock and test solutions for the full test
The highest concentration of the selected range was intended to slightly exceed the determined solubility limit to avoid a potential gap between the highest test concentration and the possible maximum test concentration.
Based on the results of the pre-test, a stock solution of 15 mg test item per mL THF was prepared. No special treatment other than vigorous shaking was required to fully dissolve the test item in THF. Lower concentrations (i.e. 0.94, 1.9, 3.8, and 7.5 mg/mL) were obtained by subsequent dilutions of the highest stock in THF. All stock solutions were clear and colourless.
Thereafter, test solutions were prepared individually by spiking volumes of 100 µL stock solution into 1 L of test medium in steps of 20 µL below the test medium surface over a period of six minutes. Throughout, the test media were magnetically stirred without turbulence at approximately 200 rpm. The pH values of the resulting solutions as well as the solvent control were in a range of 8.2 to 8.3 and correspondingly adjusted to 7.0 using 1 M HCl. Afterwards, the obtained solutions were magnetically stirred for 24 hours at 200 rpm. At the end of the stirring period, pH values were measured again. The pH values of the resulting test solutions were in a range of 7.0 to 7.2. The blank control was not stirred for 24 hours prior to the full test. However, the pH was adjusted from 8.0 to 7.0 using 1 M HCl. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Preparation of stock and test solutions for the limit test
In contrast to the pre-test, the onset of condensation was not observed in the highest test concentration in the full test. An additional limit test was performed to assure that no effects would occur up to the solubility limit of the test item. For this test, three higher test concentrations were prepared. The limit test was conducted with the lowest concentration out of these three concentrations at which undissolved test item/condensation products were observed.
A stock solution of 50 mg test item per mL THF was prepared. No special treatment other than vigorous shaking was required to fully dissolve the test item in THF. Lower concentrations (i.e. 37.5 and 25 mg/mL) were obtained by subsequent dilutions of the highest stock in THF. All stock solutions were clear and colourless.
Thereafter, test solutions were prepared individually by spiking volumes of 100 µL stock solution into 1 L of test medium in steps of 20 µL below the test medium surface over a period of six minutes. Throughout, the test media were magnetically stirred without turbulence at approximately 200 rpm. The pH values of the resulting solutions as well as the solvent control were in a range of 8.0 to 8.5 and correspondingly adjusted to 7.2 using 1 M HCl. Afterwards, the obtained solutions were magnetically stirred for 24 hours at 200 rpm. At the end of the stirring period, pH values were measured again. The pH values of the resulting test solutions were in a range of 7.0 to 7.5. The solutions exceeding a pH of 7.2 were adjusted to 7.1-7.2 using 1 M HCl. The blank control was not stirred for 24 hours prior to the limit test. However, the pH was adjusted from 7.9 to 7.1 using 1 M HCl.
The lowest test concentration as well as the solvent- and the blank-control were clear and colourless, while the two highest concentrations contained undissolved test item. The lower of these concentrations, i.e. 3.75 mg/L, was selected for the limit test. To remove the undissolved fraction, the test solution was siphoned off after a brief settlement period. The resulting solution was clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.


- Controls: Test medium without test item or other additives (blank-control) and one control containing the additive used in the treatment of the stock solutions (solvent-control).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tetrahydrofuran (THF).
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
Full test: stock solution of 15 mg test item per mL THF. Lower concentrations (i.e. 0.94, 1.9, 3.8, and 7.5 mg/mL) were obtained by subsequent dilutions of the highest stock in THF.
Limit test: stock solution of 50 mg test item per mL THF. Lower concentrations (i.e. 37.5 and 25 mg/mL) were obtained by subsequent dilutions of the highest stock in THF.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10 to the power of 4 cells/mL.
- Method of cultivation: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

Full test: 22 - 23°C
Limit test: 22 - 23°C

pH:

Full test: 7.1 - 7.7
Limit test: 7.1 - 7.8

Dissolved oxygen:

Not reported

Salinity:

n/a

Conductivity:

not reported

Nominal and measured concentrations:

Full test: Nominal: blank-control and a solvent-control, 0.094, 0.19, 0.38, 0.75 and 1.5 mg/L.
Limit test: Nominal: blank-control and a solvent-control, 3.75 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel.
- Type (delete if not applicable): capped.
- Material, size, headspace, fill volume: Glass, 100 ml, 50 ml, 50 ml.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- Initial cells density: 1 x 10 to the power of 4 cells/mL.
- Control end cells density: Full test: ; Limit test:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): Full test: 3; Limit test: 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Pre-culture:
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

- Culture medium different from test medium: No
- Intervals of water quality measurement:
pH: At the beginning and at the end of the test in all test groups.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: Full test: At the end of the test, algae in the blank- and the solvent-control as well as the highest test concentration were observed to verify a normal and healthy appearance.
Limit test: At the end of the limit test, microscopic observations were performed on the blank- and the solvent-control as well as the limit concentration to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: not reported.
- Adjustment of pH: Yes
- Photoperiod: Continuous light
- Light intensity and quality: TLD-lamps with a light intensity within the range of 84 to 86 µE/m2/s.
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions.
- Chlorophyll measurement: No
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration of 3.75 mg/L tested was considered higher than the maximum soluble concentration in test medium

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

growth rate

Remarks on result:

other: nominal concentration of 3.75 mg/L tested was considered higher than the maximum soluble concentration in test medium

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

other: yield

Remarks on result:

other: nominal concentration of 3.75 mg/L tested was considered higher than the maximum soluble concentration in test medium

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

other: yield

Remarks on result:

other: nominal concentration of 3.75 mg/L tested was considered higher than the maximum soluble concentration in test medium

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Other:
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 1 mg/l
- Other:

Reported statistics and error estimates:

STUDENT-t test for Homogeneous Variances
Shapiro-Wilk's Test on Normal Distribution
Levene's Test on Variance Homogeneity (with Residuals)
Two-sample t-test Procedure

Table: Percentage inhibition of growth rate (total test period) during the full test

N,N’,N’’-Tributyl-1-methylsilanetriamine Nominal concentration (mg/l)	n	0 - 24 h % inhibition	24 - 48 h % inhibition	48 - 72 h % inhibition
Pooled control	12
0.094	3	-4.7	3.4	2.9
0.19	3	7.0	-5.3	1.9
0.38	3	1.7	-4.6	2.6
0.75	3	4.3	-5.3	2.1
1.5	3	4.2	-2.1	3.0

Table: Percentage inhibition of yield (total test period) during the full test

N,N’,N’’-Tributyl-1-methylsilanetriamine Nominal concentration (mg/l)	n	% inhibition
Pooled control	12
0.094	3	3.8
0.19	3	5.0
0.38	3	-1.0
0.75	3	1.6
1.5	3	7.7

Percentage inhibition of growth rate at different time intervals during the limit test

N,N’,N’’-Tributyl-1-methylsilanetriamine Nominal concentration (mg/l)	n	0 - 24 h % inhibition	24 - 48 h % inhibition	48 - 72 h % inhibition
Pooled control	12
3.75	6	-9.3	11.1	1.4

Percentage inhibition of yield (total test period) during the limit test

N,N’,N’’-Tributyl-1-methylsilanetriamine Nominal concentration (mg/l)	n	% inhibition
Pooled control	12
3.75	6	13.6 (effect is statistically significant)

Validity criteria fulfilled:

yes

Conclusions:

A 72 hour ErC50 value of >3.75 mg/l and a NOEC 3.75 of mg/l (nominal) (highest concentration tested) have been determined for effects of the test substance on growth rate of Pseudokirchneriella subcapitata. An EyC50 value of >3.75 mg/l and a NOEC of 1.5 mg/l have also been determined for effects on yield of Pseudokirchneriella subcapitata. In view of the rapid hydrolysis rate of the test substance and the test preparation methods, it is likely that the algae were exposed to the hydrolysis products of the test substance.

Description of key information

72 hour EC₅₀: >3.75 mg/l and NOEC 3.75 mg/l (nominal) (highest concentration tested) have been determined for effects of the test substance on growth rate of Pseudokirchneriella subcapitata, in compliance with OECD guideline 201. An EC₅₀ value of >3.75 mg/l and a NOEC of 1.5 mg/l have also been determined for effects on yield of Pseudokirchneriella subcapitata.

Additional information

A 72 hour EC₅₀ value of >3.75 mg/l and a NOEC of 3.75 mg/l (nominal) (highest concentration tested) have been determined for effects of the test substance on growth rate of Pseudokirchneriella subcapitata. In view of the rapid hydrolysis rate of the test substance and the test preparation methods, it is likely that the algae were exposed to the hydrolysis products of the test substance.

It should be noted that the nominal concentration of 3.75 mg/l tested was considered higher than the maximum soluble concentration in test medium, therefore there is no gap between maximum soluble concentration and the highest test concentration.