3-(triethoxysilyl)propyl methacrylate

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:

2018-07-17 to 2018-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 266/2016

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Test concentrations (nominal):
Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L corresponding to the geometric mean measured test item concentration of 2.04 mg/L, 4.32 mg/L, 13.9 mg/L, 26.4 mg/L and 54.7 mg/L. The concentrations are based on the results of a preliminary range finding test.

Preparation of the test item solutions:
All test item solutions were freshly prepared by direct addition to dilution water. An appropriate volume of the test item (density: 0.989 g/cm3 was taken into account) was placed by pipette under the water surface and the bottle was closed. The test item solutions were stirred with a magnetic stirrer at approximately 1100 rpm for 4 hours at room temperature. At start and end of the stirring period, the pH of the solutions were measured and adjusted to 7.6±0.2 if necessary. The test item solutions were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative for all test item concentrations.

Control: Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Test organisms (species):

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

Details on test organisms:

Test organism: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture of Algae at the University of Goettingen 2016)
Origin: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universitat Gottingen Nikolausberger Weg 18, D-37073 Gottingen
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours/day
Culture medium: Nutrient medium Z according to Luttge et al (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

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

Test temperature:

Temperature (target) nominal range: 21-24°C, controlled at ±2°C

pH:

7.6 ± 0.2

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100 mg/L
Measured geometric mean: 2.04, 4.32, 13.9, 26.4 and 54.7 mg/L

Rang-finding test
Nominal; 1, 10 , 100 mg/l
Measured (GC-MS): Start (0 h): 0.372, 3.91, 39.1 mg/l
End (72 h): Measured (TOC) Start (0 h): End (72 h):

Details on test conditions:

Replicates: Three replicates per concentration and six for the control
Test container: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
Test volume: 100 mL
Dilution water: According to the guidelines, pH-adjusted due to hydrolytic instability of the test item
Preculture: A four days old preculture, prepared in dilution water was used as inoculum
Initial cell density: Nominal: approximately 5000-50000 cells/mL; Current: 5523 cells/mL
Application:
Application was carried out by adding appropriate volumes of the test solutions to the replicate test vessels.
Incubation: The flasks were positioned randomly and repositioned daily
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
Light intensity (target): approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
Light regime: 24 hours/day light
Light homogeneity (target): within ±15% over incubation area

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (34.1-38.3)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

13.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CI (7.96 - 11.8)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

13.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: Growth rate (0.574 mg/L); Yield inhibition (0.337 mg/L)

Biological data

The growth inhibition effects of the test item are summarised in Table 1. The cell densities are listed in Table 2. The evaluation of growth rate and yield inhibition are in Table 3 (calculated based on cell density values).

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are reported based on the geometric mean measured test item concentrations.

Table 1: NOEC, LOEC, ECx-values and 95% confidence intervals (0-72 hours) based on the geometric mean measured test item concentrations (mg/L)

	Growth rate inhibition
NOEC	4.3
LOEC	13.9
ErC10	9.39 (6.03 – 11.7)
ErC20	16.4 (14.4 – 18.5)
ErC50	36.2 (34.1 – 38.3)
	Yield inhibition
NOEC	4.32
LOEC	13.9
ErC10	3.02 (<2.04 – 4.89)
ErC20	5.02 (3.78 – 6.95)
ErC50	10.0 (7.96 – 11.8)

 

Table 2: Cell densities

Geometric mean measured test item concentration	Replicate	Cell densities (cells/mL)
(mg/L)	No.	0 hours	24 hours	48 hours	72 hours
54.7	1 2 3 Mean	5523 5523 5523 5523	7304 6978 7607 7296	10035 9884 12546 10822	20456 20629 25615 22233
26.4	1 2 3 Mean	5523 5523 5523 5523	11176 11319 10460 10985	39708 31361 33238 34769	330381 299858 286226 305488
13.9	1 2 3 Mean	5523 5523 5523 5523	13628 12912 16033 14191	63551 54370 72392 63438	586122 574837 710980 623980
4.32	1 2 3 Mean	5523 5523 5523 5523	26425 19394 28489 24769	229907 163319 235932 209719	1896760 1524557 1751948 1724422
2.04	1 2 3 Mean	5523 5523 5523 5523	24185 25588 20313 23362	199008 221007 192904 204306	2037126 1856063 1653267 1848819
Control	1 2 3 4 5 6 Mean	5523 5523 5523 5523 5523 5523 5523	35698 29840 29739 30260 30998 27156 30615	288444 278921 271108 262850 266406 267690 272570	1970055 1995836 2155662 2020729 1967141 1954793 2010703

 

Table 3: Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Geometric mean measured test item concentration	Replicate	Growth rate	Inhibition growth rate	Yield	Inhibition of yield
(mg/L)	No.	(d^-1)	(%)	(cells/mL)	(%)
54.7	1 2 3 Mean	0.436 0.439 0.511 (+) 0.462	78 78 74 76	14933 15106 20092 (+) 16710	99 99 99 99
26.4	1 2 3 Mean	1.36 1.33 1.32 (+) 1.34	31 32 33 33	324858 294335 280703 (+) 299965	84 85 86 85
13.9	1 2 3 Mean	1.55 1.55 1.62 (+) 1.57	21 21 18 20	580599 569314 705457 (+) 618457	71 72 65 69
4.32	1 2 3 Mean	1.95 1.87 1.92 (-) 1.91	1 5 2 3	1891237 1519034 1746425 (-) 1718899	6 24 13 14
2.04	1 2 3 Mean	1.97 1.94 1.90 (-) 1.94	0 1 3 1	2031603 1850540 1647744 (-) 1843296	-1 8 18 8
Control	1 2 3 4 5 6 Mean	1.96 1.96 1.99 1.97 1.96 1.96 1.97		1964532 1990313 2150139 2015206 1961618 1949270 2005180

 

Table 4: Section-by-Section and Average Specific Growth Rates of the Control Group (0-72 hours)

	Replicate	Specific growth rate (d-1) section-by-section			Mean (0-72 hours)	SD±	CV (%)	Mean CV (%)
	No.	0-24 hours	24-48 hours	48-72 hours
Control	1	1.87	2.09	1.92	1.96	0.116	5.9	12.4
	2	1.69	2.24	1.97	1.96	0.274	14.0
	3	1.68	2.21	2.07	1.99	0.273	13.7
	4	1.70	2.16	2.04	1.97	0.239	12.1
	5	1.73	2.15	2.00	1.96	0.216	11.0
	6	1.59	2.29	1.99	1.96	0.349	17.8
				Mean SD±CV (%0	1.97 0.01 0.62

 

Physicochemical data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits, Table 5. The test media were clear throughout the exposure phase.

Table 5: Environmental conditions

Geometric mean measured test item concentration	pH-values
(mg/L)	Start; 0 hours	End; 72 hours
54.7	7.43	7.53
26.4	7.45	7.80
13.9	7.47	7.98
4.32	7.49	8.32
2.04	7.53	8.40
Control	7.60	8.57
Room temperature (°C): min 21.5	max: 22.5	mean value: 22.0
Light intensity (lux) n =28	mean value: 5957 range of the measured values: 5130 to 6726 equalling -13.9 to 12.9%	CV (%): 6.52

 

Measured concentrations of total organic carbon during the definitive test

The total organic carbon (TOC) was determined at the start and end of the exposure. The results are in Table 6

Table 6: TOC content of the test media at the start and end of exposure

Sampling date	2018-07-17 0 hour Start of exposure		2018-07-20 72 hours End of exposure
Date of analysis	2018-07-17		2018-07-20
Geometric mean measured test item concentration (mg/L)	TOC (mg C/L)
	Meas. TOC (mg C/L)	Calc. TOC (mg/L)	Meas. TOC (mg C/L)	Calc. TOC (mg/L)
54.7	46.5	43.9	33.7	32.7
26.4	23.2	20.6	16.6	15.6
13.9	13.1	10.5	7.78	6.78
4.32	5.96	<LOQ	4.30	3.30
2.04	3.18	<LOQ	2.14	<LOQ
Control (Rep. 1,2,3)	3.01 3.11 1.65	-	1.03 1.01 0.97	-
Control (Mean)	2.59	-	1.00	-
Control (±SD)	0.816	-	0.031	-
LOQ	9.93	-	1.28	-

Meas. TOC = Measured TOC concentration, mean value of 2 injections

Calc. TOC = Calculated TOC (attributed to the test item) = measured TOC – background (mean value for the control)

The limit of quantification (LOQ) was calculated as follows: LOQ = mean value (control) + 9 x standard deviation

Measured exposure concentrations during definitive test

The concentrations of the test item were determined at the start of the exposure (0 hour) and at the end of the exposure (72 hours) of all tested concentrations and the control by GC-MS.

The measured concentrations of the test item at the start of exposure were in the range 72 to 95% of the nominal concentrations of the test item. At the end of the exposure, the measured concentrations were between 15 and 35% of the nominal concentrations of the test item. The geometric mean measured concentrations were calculated. Results are in Table 7

Table 7: Measured concentrations of the test item concentration in fresh medium (0 hour) and old medium (72 hours) with algae

 

Sampling date	Fresh media, 0 hour		Old media, 72 hours		Geometric mean measured concentrations
Nominal test item concentration (mg/L)	Meas. conc. (mg/L)	(%)	Meas. conc. (mg/L)	(%)	(mg/L)
100	84.6	85	35.3	35	54.7
50.0	43.7	87	16.0	32	26.4
25.0	23.7	95	8.13	33	13.9
12.5	10.3	82	1.82	15	4.32
6.25	4.52	72	0.924	15	2.04
Control	<LOQ		<LOQ		<LOQ

Meas. conc. = Measured concentration of the test item (enrichment and dilution factor taken into account)

% = Percent of the nominal concentration of the test item

LOQ = Limit of Quantification (0.008 mg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

A 72 h ErC50 value of 36.2 mg/L, ErC10 value of 9.39 mg/l and NOEC value of 4.32 mg/l have been determined for the effects of the test substance on growth rate inhibition of Pseudokirchneriella subcapitata, based on geometric mean measured concentrations. The result is considered to be reliable.

Description of key information

Algal inhibition: 72 hour EC₅₀ 36.2 mg/l and ErC₁₀ 9.39 mg/l, effects of parent substance on growth rate of Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:

36.2 mg/L

EC10 or NOEC for freshwater algae:

9.39 mg/L

Additional information

A 72-hour ErC₅₀ value of 36.2 mg/l, ErC₁₀ value of 9.39 mg/l and NOEC value of 4.32 mg/l have been determined for the effects of the test substance on growth rate inhibition of Pseudokirchneriella subcapitata, based on geometric mean measured concentrations (Noack, 2018).

Degradation due to hydrolysis in the test media was observed. The adjustment of pH to as close to neutral as reasonably possible was designed to maintain maximum exposure to the parent test substance. However, due to the static regime of the algal test, and the fact that the growth of algae can increase test media pH, loss of parent test substance by degradation was significant. An average of 84% of nominal test substance concentration was determined at the start of the test, but this decreased to 26% in the old media at 72 hours. The test organisms were therefore exposed predominantly to the parent substance at the beginning of the test, but by the end of the test the algae were exposed predominantly to the hydrolysis products.

To support the measured data, a 96-hour EC₅₀ value of 4.1 mg/l has been estimated for the effects of 3-(triethoxysilyl)propyl methacrylate on freshwater algae. It can be seen that the EC₅₀ predicted by QSAR seems to be conservative in comparison to the experimental data.

Reliable existing test results are available for the silanol hydrolysis product, based on studies conducted with the analogous substance 3-trimethoxysilylpropyl methacrylate (CAS 2530-85-0), and are included as supporting read-across evidence. The results may be expressed in terms of concentration of the hydrolysis product, 3-(trihydroxysilyl)propyl methacrylate, by applying a molecular weight correction: (MW of silanol = 206.27 / MW of parent = 248.35) * [Conc. of parent] = [conc. of silanol]. Using this method, the results are as follows: Algae: EC₅₀ (72 h): >83 mg/l and >445 mg/l; NOEC: ≥83 mg/l and 267 mg/l (results re-calculated with molecular weight adjustment and expressed as concentration of 3-(trihydroxysilyl)propyl methacrylate).