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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2016 to 07 November 2016
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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No analysis of test substance concentrations was carried out
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
See test material information
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Tetrahydrofuran (THF)
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 512076). 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 hydrolysis products 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 solubilizing agent to avoid concentration peaks during sample preparation and allow observing the onset of condensation of the hydrolysis product 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 512076).

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. 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 7.8 to 8.8 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. A 24 hour stabilization period did not change the appearance of the solutions containing 6.3, 12.5 and 25 mg/L. The two higher concentrations contained floating droplets aside from solid, undissolved material at the end of the stirring period. The floating droplets were not observed anymore after 24 hours of stabilization, while solid, undissolved material was still present.

Preparation of stock and test solutions
The batch of 1-Methyl-N,N’,N’’-tris(1-methylpropyl)silantriamine tested was a clear colourless to slightly yellow liquid with a purity of 98%. 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 resulting 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. 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 200 rpm. The pH of the resulting test solutions as well as the solvent control were 8.0 and subsequently adjusted to 6.9-7.0 using 1 M HCl (Merck, Germany). 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 6.9 to 7.2. The pH of the solvent and the blank control were 7.3 and 8.0, respectively and correspondingly adjusted to 7.2 and 6.9 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 8.3 to 8.4 and correspondingly adjusted to 6.9- 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 7.2 to 7.5. The solutions exceeding a pH of 7.2 were adjusted to 7.0 using 1 M HCl. The blank control was not stirred for 24 hours prior to the limit test. However, the pH was adjusted from 8.3 to 6.9 using 1 M HCl.
The solvent- and the blank-control were clear and colourless, while the three prepared concentrations contained undissolved test item. The lowest of these concentrations, i.e. 2.5 mg/L, was selected for the limit test. To remove the undissolved fraction, the test solution was siphoned off after a settlement period of 10 minutes. 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)): Preparation of stock solutions started with a concentration of
100 mg/mL.
- 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 Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:

ACCLIMATION
- Acclimation period: 3 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.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22-23°C
pH:
7.1-7.9
Dissolved oxygen:
Not reported
Salinity:
n/a
Conductivity:
Not reported
Nominal and measured concentrations:
Full Test: blank control, solvent control, 0.094, 0.19, 0.38, 0.75 and 1.5 mg/L (nominal).
Limit Test: blank control, solvent control, 2.5 mg/L (nominal).
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): open but capped vessels
- 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: 131.7 x10 to the power of 4 cells/mL; Limit test: 146.2 x10 to the power of 4 cells/mL.
- 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: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
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: 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: not reported
- Adjustment of pH: Yes
- Photoperiod: Continuous light
- Light intensity and quality: TLD-lamps with a light intensity within the range of 80 to 82 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: Not reported
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
A pre-test to determine the solubility of the test substance in THF was performed. Based on the solubility results the test concentrations for the full acute toxicity test in Daphnia magna were determined.
- Test concentrations: Full test: 0.094, 0.19, 0.38, 0.75, 1.5 mg/l
- Results used to determine the conditions for the definitive study: No immobilisation occurred in the full test. An additional limit test was performed with the lowest concentration at which undissolved material was observed during preparation of the test solutions in order to assure that no effects would occur up to the solubility limit of the test item.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure will have been to the hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: no statistically significant effects on growth rate were observed at the highest concentration which corresponded with the limit of solubility of the test substance
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure will have been to the hydrolysis products
Basis for effect:
growth rate
Remarks on result:
other: no statistically significant effects on growth rate were observed at the highest concentration which corresponded with the limit of solubility of the test substance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure will have been to the hydrolysis products
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure will have been to the hydrolysis products
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- 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: n/a
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: 1 mg/l growth rate
- Other: 0.43 mg/l yield
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)
Williams Multiple Sequential t-test Procedure

Table: Growth rates (day-1) during the full test

 

1-Methyl-N,N’,N’’-tris(1-methylpropyl)silantriamine

Nominal concentration (mg/L)

 
           
 Time  Blank control  Solvent control  0.094  0.19  0.38  0.75  1.5

 0 -24 h

Mean

 1.555  1.815  1.681  1.699  1.56  1.347  1.489

 0 -48 h

Mean

 1.579  1.653  1.622  1.645  1.522  1.52  1.593

 0 -72 h

Mean

 1.596  1.623  1.599  1.613  1.528  1.532  1.581

Table: growth rates (day-1) during the limit test

 

1-Methyl-N,N’,N’’-tris(1-methylpropyl)silantriamine

Nominal concentration (mg/L)

 
   
 Time  Blank control  Solvent control  2.5

 0 -24 h

Mean

 1.759  2.133  1.338

 0 -48 h

Mean

 1.728  1.822  1.604

 0 -72 h

Mean

 1.66  1.715  1.572

Table: Yields during the limit test

 

1-Methyl-N,N’,N’’-tris(1-methylpropyl)silantriamine

Nominal concentration (mg/L)

 
   
 Time  Blank control  Solvent control  2.5

 24 h

Mean

 1.15  0.59  0.63

 48 h

Mean

4.84  3.01  2.68

 72 h

Mean

 145.2  171.3  111.4
Validity criteria fulfilled:
yes
Conclusions:
A 72 hour EC50 value of >2.5 mg/l and a NOEC of 2.5 mg/l (nominal) (highest concentration tested) have been determined for effects of the test substance on growth rate of Pseudokirchneriella subcapitata. An EC50 value of >2.5 mg/l and a NOEC of 0.19 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 would have been exposed to the hydrolysis products of the test substance.

Description of key information

72-hour EC50: >2.5 mg/l and NOEC 2.5 mg/l (nominal) (highest concentration tested) have been determined for effects of 1-methyl-N,N',N''-tris(1-methylpropyl)silanetriamine (CAS 37697-65-7) on growth rate of Pseudokirchneriella subcapitata, in compliance with OECD guideline 201. An EC50 value of >2.5 mg/l and a NOEC of 0.19 mg/l have also been determined for effects on yield of Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

Additional information

A 72-hour EC50 value of >2.5 mg/l and a NOEC of 2.5 mg/l (nominal) (highest concentration tested) have been determined for effects of the read-across substance 1-methyl-N,N',N''-tris(1-methylpropyl)silanetriamine (CAS 37697-65-7) on growth rate of Pseudokirchneriella subcapitata. An EC50 value of >2.5 mg/l and a NOEC of 0.19 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 would have been exposed to the hydrolysis products of the test substance.

It should be noted that the nominal concentration of 2.5 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.

See the ecotoxicological discussion for further details on the test substance and justification of read-across.