1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

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

Study period:

Exposure period: 2018-09-11 to 2018-09-14, total test time 2018-09-10 to 2018-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Substance analysis determined concentration of parent, not hydrolysis product

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analysis of the parent, not hydrolysis product

Details on sampling:

- Concentrations:
- Sampling method: At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of each loading and analyzed. At the end of the exposure (72 hours), samples of the old media were taken from the pooled test solutions from the test vessels.

- Sample storage conditions before analysis: All samples were stored at room temperature until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Five saturated solutions were freshly prepared with nominal loadings of the test item of 1.00 – 3.16 – 10.0 – 31.6 - 100 mg/L (spacing factor v10) with demineralized water one day prior to the start of the exposure (day -1). An appropriate amount of the test item was pipetted under the surface of the demineralized water. For this purpose, the density, but not purity, was taken into account. The stock solutions were stirred for 24 ± 1 hour at approx. 1100 rpm at room temperature. After a separation phase of 1 hour, the saturated solutions were removed by siphoning (from the approximate middle, 2/3 of height, of the glass flask). The resulting saturated solutions were once again stirred for 2 hours at approx. 1100 rpm at room temperature. The saturated solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Prior to testing, the nutrient salts for the medium were added to the saturated solutions.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): No vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No - The saturated solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Not reported, synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus
- Strain: HINDÁK CCAP 278/4
- Source (laboratory, culture collection): laboratory cultures. Original source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day, in Nutrient medium Z according to LÜTTGE et al. (1994)

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions (same as test or not): No, test media (dilution water) was prepared according to OECD TG 201.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Remarks:

OECD TG 201 dilution water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

The test medium has a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

21.5-22.5°C

pH:

7.67-8.80

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: control, 1.00, 3.16, 10.0, 31.6, 100 mg/l
Measured concentrations: all measured concentrations were below the limit of quantification (LOQ = 0.015 mg/L) of the test item at both 0 and 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Material, size, headspace, fill volume: 100 mL
- Aeration: Not reported
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL, Actual: 6442 cells/mL
- Control end cells density: after 72h, mean cell density = 1044458 cells/ml
- No. of organisms per vessel: Nominal: approximately 5 x 103 - 104 cells/mL, Actual: 6442 cells/mL
- No. of vessels per concentration (replicates): Three replicates per loading level
- No. of vessels per control (replicates): Six replicates (without test item) were tested under the same test conditions as the test item replicates.
- No. of vessels per vehicle control (replicates): No vehicle control used

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water was prepared according to OECD TG 201
- Total organic carbon: Not reported
- Metals: trace metals were added to complete the algae growth medium, as according to OECD 201
- Particulate matter: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Conductivity: not reported
- Salinity: not reported
- Ca/mg ratio: This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
- Culture medium different from test medium: Yes, culture medium was Nutrient medium Z according to LÜTTGE et al. (1994), test medium was according to OECD TG 201
- Intervals of water quality measurement: measurements were taken at 0h and 72h

OTHER TEST CONDITIONS
- Sterile test conditions: test vessels were sterile but sterile test conditions were not reported
- Adjustment of pH: not reported
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1. Mean value: 6287 lux (n=23)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Fluorometer: Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH) for determination of the cell density
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the definitive test at the saturated solution with the nominal loading of 100 mg/L.
- Other: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Five loading levels were tested in a geometrical series with a dilution factor of v10
- Range finding study: two range finding tests were conducted: nominal test concentrations of 0.50, 5.00 and 50mg/l for the first test, 1.00, 10.0 and 100mg/l for the second test (both 72h)
- Test concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

exposure is to hydrolysis products

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and at the end revealed no morphological abnormalities
- Unusual cell shape: Microscopic evaluation of the cells at the start and at the end revealed no morphological abnormalities
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported

- Any stimulation of growth found in any treatment: yes, at 10mg/l inhibition of growth rate was -2% after 72h
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The solution was checked for any visual observations (i.e. undissolved material, formation of precipitates/polymers) after 2, 24 and 48 hours. No visual observations were documented.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes, was within the valid range
- EC50: growth rate 72h EC50 (with 95% confidence intervals)=1.09mg/l (0.843-1.31mg/l)
- Other: The toxicity of potassium dichromate to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-03-26 to 2018-03-29

Reported statistics and error estimates:

Statistical Significance (NOEL/LOEL) – Growth Rate was determined using a one way analysis of variance after passing both a normality (Shapiro-Wilk) and equal cariance test. The program states no significant difference for all nominal test item loadings compared to the control.
ELx-Calculation - Yield (Nominal Test Item Loading) - Third order polynomial (cubic) Y=B0 + B1*X +B2*X^2 + B3*X^3 with x= log(x) transformation.

Nominal test item loading (mg/l)	Replicate No.	Growth rate (d-1)	Inhibition of growth rate (%)	Yield (cells/ml)	Inhibition of Yield (%)
100	1	1.65	3	898516	13
	2	1.64	3	883820	15
	3	1.66	2	921454	11
	Mean	(-) 1.65	3	(-) 901263	13
31.6	1	1.69	0	1016087	2
	2	1.66	2	932402	10
	3	1.65	3	905616	13
	Mean	(-) 1.67	2	(-) 951368	8
10.0	1	1.70	-1	1062858	-2
	2	1.75	-3	1231047	-19
	3	1.71	-1	1067227	-3
	Mean	(-) 1.72	-2	(-) 1120377	-8
3.16	1	1.66	2	922001	11
	2	1.62	5	815476	21
	3	1.71	-1	1093963	-5
	Mean	(-) 1.66	2	(-) 943813	9
1.00	1	1.69	0	1011519	3
	2	1.67	1	969441	3
	3	1.61	5	793531	7
	Mean	(-) 1.66	2	(-) 924830	24
Control	1	1.71		1093665
	2	1.65		893849
	3	1.69		1024751
	4	1.70		1063081
	5	1.73		1163101
	6	1.68		989649
	Mean	1.70		1038016

 

Validity criteria fulfilled:

yes

Conclusions:

Pseudokirchneriella subcapitata 72h growth rate NOELR = 100mg/l and EL10 > 100mg/l of 1,3,5-Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione (CAS No. 26115-70-8, EC No. 247-465-8).
Test was performed in accordance with OECD 201 (2011).

Description of key information

Short-term toxicity to algae: 72 hr NOELR = 100 mg/L and EL₁₀ >100 mg/L (nominal, highest concentration tested) 1,3,5-Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione (CAS 26115-70-8, EC No. 247-465-8) (OECD TG 201). The observations in this study are attributed to the exposure of test organisms to the hydrolysis products in the test system. The NOELR is equivalent to 80 mg/L and the EL₁₀ is equivalent to >80 mg/L when expressed in terms of the silanol hydrolysis product,tris[3-(trihydroxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A 72 h NOELR value of 100 mg/L and a 72 h EL₁₀ of >100 mg/L (nominal, highest concentration tested) have been determined for the effects of 1,3,5-Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione (CAS 26115-70-8, EC No. 247-465-8) on growth rate of Pseudokirchneriella subcapitata. In view of the test media preparation method and exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of the tested substance. The study was supported by LC MS/MS analysis of the parent substance and concentrations were below the limit of quantification (LOQ = 0.015 mg/L) at 0 and 48h, which indicated the parent had fully hydrolysed prior to the start of the test. The results may be expressed in terms of concentration of the hydrolysis product, tris[3-(trihydroxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, by applying a molecular weight correction: (MW of silanol=489.62 / MW of parent=615.86) * [CONCENTRATION OF PARENT=100] = >80 mg/L.