Amino functional alkoxysilanes

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 - 22 Nov 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The dissolved organic carbon (DOC) concentration was determined in nominal concentrations of 30, 147.4 and 426 mg/L at 0, 24, 48 and 72 h during the test in media that did not contain algae but was otherwise kept under test conditions. The mean concentrations in the three media were used to estimate the mean concentrations in all the media by linear interpolation.
- Sampling method: not reported
- Sample storage conditions before analysis: Retained samples were stored at -20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was dissolved in algal growth medium to produce a stock solution with a nominal concentration of 473.4 mg/L. The solution was stirred for 1 hour and then diluted with algal growth medium to produce the test concentrations.

Test organisms (species):

Phaeodactylum tricornutum

Details on test organisms:

TEST ORGANISM
- Strain: 1090-1a SAG obtained from the University Institute of Plant Physiology, Göttingen, Germany
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 3 days

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

19.8 - 21.4 °C

pH:

8.0 - 8.8 at start of test
8.0 - 8.2 at end of test

Dissolved oxygen:

not reported

Salinity:

34 ppt

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 30, 51, 86.7, 147.4, 250.6 and 426 mg/L
Calculated effective concentrations: 0 (Control), 42, 58, 87, 135, 217 and 356 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with pulp bung
- Material, size, fill volume: 100 ml containing 50 ml of medium
- Aeration: none
- Initial cells density: 10000
- Control end cells density: 550000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH determined at start and end of test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 8100+/-8% Lux (RSD)
- Salinity (for marine algae): 34 ppt

EFFECT PARAMETERS MEASURED: cell concentrations at the start of the test and after 24, 48 and 72 hours incubation.

- Determination of cell concentrations: counting chamber and microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study: yes
- Test concentrations: concentrations tested and results not reported

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 106.9-146.1 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

118.3 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 104.7-134.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 42.1-84.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

71.1 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 49.4-84.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

58.5 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

58.5 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

91.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 81.9-101.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

90.9 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 82.3-101.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

56.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 39.9-65.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

61.8 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 44.5-71 mg/L

Details on results:

- Exponential growth in the control: yes
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- 72-h EC50 for growth rate: 25 mg/L

Reported statistics and error estimates:

The area under the growth curve (biomass) and growth rate parameters were calculated from the cell concentration data in accordance with the description of the test method.

The EC50 and NOEC values were calculated using the Statistics Software ToxRat® Standard Version 2.09. The software calculates ECx values by Probit analysis using linear, maximum liklihood regression. NOECs were calcultaed using Dunnett's multipley-test procedure.

Table 1. Test results

Nominal test substance concentration (mg/L)	Mean cell concentration at start of test (cells/mL)	Mean cell concentration after 24 h (cells/mL)	Mean cell concentration after 48 hours (cells/mL)	Mean cell concentration after 72 hours (cells/mL)
0 (control)	10000	30000	160000	550000
30	10000	20000	150000	570000
51	10000	30000	160000	550000
86.7	10000	40000	120000	190000
147.4	10000	20000	40000	50000
250.6	10000	10000	0	0
426.0	10000	10000	0	0

Table 2. Results of analysis of test media - samples analysed immediately after collection

Nominal test substance concentration (mg/L)	Nominal TOC concentration (mg/L)	Measured DOC concentration at start of test (mg/L)*	Measured DOC concentration after 24 hours (mg/L)*	Measured DOC concentration after 48 hours (mg/L)*	Measured DOC concentration after 72 hours (mg/L)*
0 (Control)	0	20.0	19.0	14.5	12.5
30.0	12.5	18.6	20.4	29.7	20.0
147.4	61.5	70.0	34.3	65.3	28.4
426.0	177.6	198.2	142.4	146.2	114.2

*corrected for Control DOC

Table 3. Results of analysis of test media - samples analysed following 20 days storage under refridgeration

Nominal test substance concentration (mg/L)	Nominal TOC concentration (mg/L)	Measured DOC concentration at start of test (mg/L)	Measured DOC concentration after 24 hours (mg/L)	Measured DOC concentration after 48 hours (mg/L)	Measured DOC concentration after 72 hours (mg/L)
0 (Control)	0	13.0	14.0	6.4	7.0
30.0	12.5	no data	no data	no data	3.4
147.4	61.5	34.8	15.9	18.9	15.3
426.0	177.6	123.0	75.1	94.9	67.9

The results of the analyses performed immediately after collection of the samples were used as the basis for determining the exposure concentrations that were used as the basis for calculating the effective concentrations.

Validity criteria fulfilled:

yes

Conclusions:

A 72-h EC50 value of 125 mg/L based on nominal concentration (118.3 mg/L based on estimated concentration from DOC measurement) has been determined in the study testing the effect of the test substance on the growth rate of the marine alga Phaeodactylus tricornutum. The 72-h EC10 determined for the same endpoint was 68 mg/L (71.1 mg/L based on estimated concentration from DOC measurement).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 - 23 Aug 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

other: Algal Growth Inhibition Test stipulated in the Testing Methods for New Chemical Substances of Japan

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and the control were sampled at the start and the end of exposure. Algae were removed by centrifugation (3000 rpm, 10 min) prior to sampling at the end of the exposure period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: test item and OECD medium were mixed and stirred for 2 h. The suspension was filtered with a glass fiber filter. The theoretical concentration of methanol in the prepared stock solution was 56 mg/L. Test solutions were prepared mixing the stock solution with the appropriate amount of dilution water.

- Other: in order to ignore the additional effect of methanol on the organisms in the test solutions and to determine only the toxicity of silanol form, the definitive test condition was designed appropriately so that test solution in all exposure levels contained same methanol concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM

- Common name: green algae

- Strain: ATCC 22662

- Source (laboratory, culture collection): laboratory culture (initial source: American Type Culture Collection)

- Age of inoculum (at test initiation): 3 d

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

pH unadjusted: 21.8 - 22.0 °C
pH adjusted: 22.8 - 22.9 °C

pH:

pH unadjusted: 7.8 - 9.0
pH adjusted: 7.9 - 8.0

Nominal and measured concentrations:

0 (control), 0.31, 0.93, 2.8, 8.3, 25 and 75 mg/L (nominal, as silanol form, pH unadjusted)
0 (control), 0.31, 0.93, 2.8, 8.3, 25 and 75 mg/L (nominal, as silanol form, pH adjusted)

Details on test conditions:

TEST SYSTE

- Test vessel: sterilized 500 mL Erlenmeyer flasks (with gas-permeable Silicosen)

- Fill volume: 100 mL

- Initial cells density: 0.75 x 10^4 cells/mL

- Control end cells density: 85 x 10^4 cells/mL (pH unadjusted), 110 x 10^4 cells/mL (pH adjusted)

- Methanol end cells density: 95 x 10^4 cells/mL (pH unadjusted), 100 x 10^4 cells/mL (pH adjusted)

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 3

- No. of vessels per methanol control (replicates): 6

GROWTH MEDIUM

- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: according to OECD guideline

- Intervals of water quality measurement: pH was measured at the start and at the end of the exposure period. Temperature and light intensity were measured daily.

OTHER TEST CONDITIONS

- Adjustment of pH: as the pH in the highest test concentration exceeded pH 9, a parallel was set up for all test concentration in which the pH was adjusted to the pH of the medium, in order to examine wether effects are caused by the high pH values.

- Photoperiod: continuous illumination provided by fluorescent lights with wavelength range of 400 - 700 nm

- Light intensity and quality: 83 - 85 µE*m^-2*s^-1

- Aggitation: continuous rotary shaking at approx. 100 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell biomass was measured every 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3

- Preliminary study: was conducted in order to follow hydrolysis of the the test item. 156 mg/L (as test item concentration) were prepared in ultra pure water. Methanol was measured after 0, 0.5, 1 and 2 hours in order to follow the state of hydrolysis. 94.3, 95.2, 98.5 and 102% of the theoretical formed concentration (87.8 mg/L) was measured after 0, 0.5, 1 and 2 h, respectively.

- Range finding study: yes, 2-3 replicates, 2 parallels were set up with and without pH adjustment, silanol form and methanol were measured with and without algae at the beginning and at the end of the exposure

- Test concentrations: 1.2, 2.6, 5.6, 12, 27 and 60 mg/L (nominal, as silanol form, pH unadjusted), 0.6, 1, 10, 60 and 100 mg/L (nominal, silanol form, pH adjusted)

- Results used to determine the conditions for the definitive study: growth inhibition based on growth rate was 0.033, 3.9, 8.1, 19, 64 and 123% for 1.2, 2.6, 5.6, 12, 27 and 60 mg silanol form/L, respectively in the experiment with no pH adjustment. 2.1, 1.2, 6, 92 and 135% growth rate inhibition was observed at 0.6, 1, 10, 60 and 100 mg/L, respectively in the preliminary experiment with pH adjustment. Based on these results concentrations between 0.31 - 75 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH unadjusted, 95% CI: 26 - 36 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH adjusted, 95% CI: 33-40 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH unadjusted

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH adjusted

Details on results:

- Exponential growth in the control when pH was unadjusted: yes (109-fold increase in the control and 120-fold increase in the methanol control)

- Exponential growth in the control when pH was adjusted: yes (147-fold increase in the control and 127-fold increase in the methanol control)

Results with reference substance (positive control):

- Results with reference substance valid: yes

- EC50: 1 mg/L

Reported statistics and error estimates:

For the estimation of NOEC, Bartlett´s test was applied to determine the homogeneity of variance of the data. Then one-way analysis of variance and Dunnett´s multiple comparison test was used to determine the significant difference between the methanol control and the exposure levels.

Measured test item concentrations at the beginning and at the end of the test:

- pH unadjusted: measured silanol form concentrations at test start: 97 – 108% of nominal concentrations, silanol concentrations at test end: 96 - 108% of nominal concentrations. Measured methanol concentrations at test start: 98 – 112% of nominal concentration, methanol concentration at test end: 104 – 112% of the theoretical formed concentration.

- pH adjusted: measured silanol form concentrations at test start: 106 - 114% of nominal concentrations, measured concentrations of silanol form at test end: 98 – 120% of nominal. Measured methanol in the test solutions at the beginning of the test: 104 – 111% of the nominal concentration, methanol determined at test end: 100 – 112% of the theoretical formed concentration.

Table 1: Growth inhibition in %.

Nominal concentrations [mg/L]	pH unadjusted	pH adjusted
	Inhibition rate %	Inhibition rate %
Control	2.3	-1.1
Methanol control	-	-
0.31	0.16	0.88
0.93	1.6	-0.67
2.8	3.2	-1.2
8.3	4	1.2
25	32	23
75	129	102

Validity criteria fulfilled:

yes

Conclusions:

An EC50 (72 h) value of 31 mg/L and a NOEC of 2.8 mg/L have been determined based on the growth rate of Pseudokirchnerella subcapitata when the pH of the solution was not adjusted. An EC50 (72 h) of 36 mg/L and a NOEC (72 h) of 8.3 mg/L were determined based on the growth rate of the test organism when the pH was adjusted to the pH of the dilution water.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-05-10 to 1994-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 500 mg/l nominal treatment

- Sampling method: Samples of test media were taken at the start and end of the test

- Sample storage conditions before analysis: Refrigerated

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 500 mg/l nominal concentration of the test substance was prepared by stirring overnight.

- Controls: Dilution water (algal growth medium)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM

- Strain: CCAP 278/4

- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ambleside, Cumbria, U.K.

- Age of inoculum (at test initiation): Cultures in exponential growth

- Method of cultivation: Axenic cultures maitained in liquid medium.


ACCLIMATION

- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

24ºC

pH:

Control: 7.9 - 9.6

500 mg/l treatment: 7.9 - 9.3

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentration: 500 mg/l

Measured concentration at start of test: 486 mg/l

Measured concentration at end of test: 508 mg/l

Mean measured concentration: 497 mg/l

Mean measured concentration as a % of nominal: 99.4

The test results are presented and interpreted with reference to nominal concentrations.

Details on test conditions:

TEST SYSTEM

- Test vessel: Erlenmeyer flasks

- Type: closed

- Material, size, headspace, fill volume: Glass, 250 ml, no headspace, full

- Aeration: no

- Renewal rate of test solution (frequency/flow rate): no

- Initial cells density: 10000 cells/ml

- Control end cells density: ≈250000 cells/ml

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 4


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Standard algal growth medium in accordance with EU test method

- Culture medium different from test medium: no


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: Continuous

- Light intensity and quality: 12527 - 11745 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Determination of cell concentrations: electronic particle counter

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

but exposure is to hydrolysis product

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

but exposure is to hydrolysis products

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

but exposure is to the hydrolysis product

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

but exposure is to the hydrolysis product

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes

- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

No toxic effects were observed in the test media, the test results were therefore not subject to statistical analysis.

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of >500 mg/l and NOEC of ≥500 mg/l have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata (tested as: Selenastrum capricornutum) based on nominal concentration of the substance. However it is likely that the test organisms were primarily exposed to the hydrolysis products of the substance.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-04-16 to 2007-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Test Concentrations were measured in each  treatment at test initiation and at test termination

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:   Stock solutions were prepared by direct addition of test article to algal  assay medium

Test organisms (species):

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

Details on test organisms:

- Species/strain # and source: Pseudokirchneriella subcapitata/Strain UTEX 1648,  from in-house cultures initiated from cultures originally supplied by the University of Texas at Austin.

- Initial Density 10,000 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24.0 to 24.4ºC

pH:

7.6-7.7 at start of test

7.8-9.0 at end of test

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: Negative Control, 7.5, 15, 30, 60 and 120 mg/L

Mean measured concentrations:
The large difference between nominal and mean measured exposure concentrations is attributed to the hydrolysis of the test substance under the static test conditions. The hydrolysis half-life of  methyltrimethoxysilane is approximately 2.2 hours.  All measured  concentrations on Day 3 were less than the limit of quantitation (LOQ =  0.27 mg/L).  Due to the decline in test concentrations during the test,  mean measured concentrations were estimated as the geometric mean of Day  0 and Day 3 measured concentrations.  The geometric mean concentrations  were calculated using ½  LOQ for all Day 3 measurements (0.135 mg./L).

Details on test conditions:

TEST SYSTEM

- Exposure vessel type:  250-mL Erlenmeyer flasks covered with a sponge  closure containing 100 mL of test solution.

- Test Design:  Three replicates for each treatment group.  Five test  concentrations and a negative control.

- Element basis (i.e. number of cells/ml, area under the curve, growth  rate, etc.): Yield (Biomass) and Growth Rate "  

- Growth/test medium chemistry:  Algal Assay Medium (ASTM E 1218-04), pH  7.6

- Dilution water source:  Distilled Water

- Water chemistry in test:  Temperature was measured in the environmental  chamber daily.  Measurements of pH were made in each treatment group at  test initiation and test termination.

- Light levels and quality during exposure:  490 to 626 foot-candles,  continuous cool-white fluorescent lighting

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth Rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (Biomass) and Growth Rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth Rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.6

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield (Biomass) and Growth Rate

Details on results:

- Exponential growth in the control (for algal test): yes

- Observations:  Visual examination of algal cells at test termination showed no concentration-dependent changes in shape, color or size.

Reported statistics and error estimates:

Statistical methods: Dunnett's test was used to determine if there were differences between Control and treatments.

Table 1. Test results

Nominal concentration (mg/L)	Geometric mean measured concentration (mg/L)	Mean yield after 72 hours (cells/mL)	Percentage inhibition of yield	Mean growth rate 0 -72 hours	Percentage inhibition of growth rate
Control	Control	1098489	-	1.5692	-
7.5	0.77	1116978	-1.7	1.5749	-0.4
15	1.2	1112844	-1.3	1.5737	-0.3
30	1.7	1048378	4.6	1.5532	1.0
60	2.4	1080444	1.6	1.5635	0.4
120	3.6	1092800	0.5	1.5676	0.1

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of >3.6 mg/L and NOEC of ≥3.6 mg/L have been determined for the effects of the test substance on yield and growth rate of Pseudokirchnerella subcapitata based on geometric mean measured exposure concentrations of the test substance. The test substance is known to hydrolyse rapidly in water and 3.6 mg/L was the concentration measured in the highest nominal test concentration of 120 mg/L. It is therefore likely that the test organisms were predominantly exposed to the hydrolysis products of the substance.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993-08-10 to 1993-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Cited as Directive 92/69/EEC, C.3

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The TOC concentration of the filtered stock solutions used to prepare the test media were determined and used as the basis for calculating the test cincentrations that were prepared by dilution of the stock solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The test substance was added to dilution water and stirred for 18 hours. After mixing it was filtered and used to prepare the other test media by dilution.

The TOC content was determined in the filtered initial solutions.  The TOC content (mg/L) and the substance content (mg/L) in the hydrous, filtered initial solution in Main Test 2 were reported to be 543 mg/L and 1111 mg/L, respectively. The concentrations used in Main Test 2 were calculated from TOC content and included 33, 67, 133, 278, 556, and 1000 mg/L.  The TOC content (mg/L) and the substance content (mg/L) in the hydrous, filtered initial solution in Main Test 4 were reported to be 309 mg/L and 632 mg/L, respectively.  The concentrations used in Main Test 4 were calculated from TOC content and included 1.3, 2.5, 5.1, and 10.1 mg/L

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM

- Strain: 86.81.SAG

- Source (laboratory, culture collection): Institute for Water, Ground and Air Hygiene, Berlin (Germany)

- Age of inoculum (at test initiation): 3 days

- Method of cultivation: A pre-culture is produced from an original culture by super-inoculation three days before the test begins. From this culture the test cultures are inoculated at a density of about 20000 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

24+/-2ºC

pH:

7.9-9.2 in Test 2

7.5-9.0 in Test 4

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations in mg/L in Main test 2: 0, 33, 67, 133, 278, 556, and 1000 mg/L.  

Nominal concentrations in mg/L in Main test 4: 1.3, 2.5, 5.1, and 10.1.

Details on test conditions:

TEST SYSTEM

- Test vessel:

- Type: open Erlenmeyer flasks

- Material: glass

- Aeration: none

- Initial cells density: 20000 cells/ml

- Control end cells density:1150000 cells/ml (Test 2), 970000 cells/ml (Test 4)

- No. of vessels per concentration (replicates): 5

- No. of vessels per control (replicates): 8

GROWTH MEDIUM

- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS

- Culture medium different from test medium: no

- Intervals of water quality measurement: start and end of test

OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 8000 Lux


EFFECT PARAMETERS MEASURED: Determination of cell concentrations after 24, 48 and 72 hours by spectrophotometer (absorption at 685 nm)


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 1.8-2.0

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

321 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

603 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

other

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

no separate NOEC defined for growth rate effects

Reported statistics and error estimates:

ECx values were determined by probit analysis.

NOEC values were determined by comparing Control and treatment means using a Student's t-test.

               
Table 1. Biological observations
Cell density at each flask at each measuring point:

(a) Main Test 2

Cell Count (*104 cells/ml)
Concentration (mg/L)	Test Interval (h)
	0 h *	24 h	48 h	72 h
Control	2	7	35	115
33	2	6	27	106
67	2	6	27	111
133	2	6	25	105
278	2	5	20	82
556	2	5	15	51
1000	2	5	16	50

*No median values.  All concentrations are with respect to the material.  Five each and eight each parallels, respectively, were examined.  (After 0 h, the theoretical cell concentration was evaluated).

(b) Main Test 4

Cell Count (*104 cells/ml)
Concentration (mg/L)	Test Interval (h)
	0 h	24 h	48 h	72 h
Control	2	6	23	97
1.3	2	5	23	89
2.5	2	4	18	85
5.1	2	5	19	87
10.1	2	5	19	88

                     

*No median values.  All concentrations are with respect to the material.

Growth curves:  The EC values are calculated by regression
analysis based on [Probit] transformation of the percentage
suppression values.  These values then serve as the basis
for the subsequent [Probit] analysis in accordance with
Cavalli-Sforza (1972).  The results are presented below.

Effective Concentrations on the Basis of Cell Growth (EbC)

Parameter	Mg/L (substance)
(72 h) EbC50	603
(72 h) EbC10	38
(72 h) EbC90	*

* lies above the highest tested concentration
 

Effective Concentrations on the Basis of Specific Growth Rate µ(ErC)

Parameter	Mg/L (substance)
(72 h) ErC50	*
(72 h) ErC10	321
(72 h) ErC90	*

* lies above the highest tested concentration

Table 2. Percent biomass/growth rate inhibition per concentration

(a) Main Test 2
Areas under growth curves, growth rates, corresponding
percentage suppression rates, and pH values with respect to test  concentrations. (All concentrations are with respect to the material.)

Method		Control	33 mg/L	67 mg/L	133 mg/L	278 mg/L	556 mg/L	1000 mg/L
Area Under Growth Curve	Area	94.5	81	83.5	78.5	61	40.5	41
	% inhibition		14.3	11.6	16.9	35.4	57.1	56.6
Growth Rate µ (0 -72 h)	µ	1.351	1.323	1.339	1.32	1.238	1.08	1.073
	% inhibition		2.1	0.9	2.3	8.4	20.1	20.6
pH Value	After 0 h	7.9	8.3	8.4	8.7	8.9	9.1	9.2
	After 72 h	8.6	8.8	9.1	8.8	8.5	8.3	8.4

(b) Main Test 4
Areas under growth curves, growth rates, corresponding
percentage suppression rates, and pH values with respect to test  concentrations. (All concentrations are with respect to the material.)

Method		Control	1.3 mg/L	2.5 mg/L	5.1 mg/L	10.1 mg/L
Area under growth curve	Area	72.5	67.5	59.5	52.5	63
	% inhibition		6.9	17.9	13.8	13.1
Growth rate µ (0 -72 h)	µ	1.294	1.265	1.25	1.258	1.261
	% inhibition		2.2	3.4	2.8	2.6
pH values	After 0 h	7.5	7.8	7.8	7.9	8.1
	After 72 h	9.0	8.8	8.7	8.7	8.6

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour ErC50 value of >1000 mg/L and a ErC10 of 321 mg/L have been determined for the effects of the test substance on growth rate of Scenedesmus subspicatus. A 72-hour EbC50 of 603 mg/L and EbC50 of 38 mg/Lhave been determined for effects on biomass. The NOEC in the study was 1.3 mg/L and it can be inferred that the biomass effects were most sensitive. It is likely that the test organisms were exposed to the hydrolysis products of the substance.

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-04-28 to 2005-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, conducted in compliance with GLP

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test solutions were prepared individually by stirring appropriate amounts of the test substance in app. 500 ml of seawater. the solutions were stirred with a spin-bar for 20 hours at a speed which formed a vortex two-thirds of the depth of the fluid content, followed by standstill for 4 hours. Samples for testing were taken from the mid of the water phase with a peristalic pump.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM

- Strain: NIVA-BAC 1
- Source (laboratory, culture collection): originally isolated from the Oslo fiord (1962), Norsk Institute for Vannforskning (NIVA), Oslo
- Age of inoculum (at test initiation): 3 days, the cell density of the inoculum was 1.6 million cells/ml determined by microscopic counting using a Palmer-Malony counting slide.
- Method of cultivation: The pre-cultures used in this test was incubated at 20+-2°C under conditions light (>50µE x m² x secE-1) from fluorescent tubes of universal natral white type on an orbital shaker.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

no data

Test temperature:

19.0 - 20.0C

pH:

at start: 8.2 (control) - 9.7 (1549 mg/l test substance)
at end: 8.3 (control) - 8.7 (1549 mg/l test substance

Dissolved oxygen:

not applicable

Salinity:

the used natural seawater has a salinity of 34.5%

Nominal and measured concentrations:

nominal: 6.9; 16.3; 40; 100; 308; 623; 1549 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml test culture in 100 ml flat-bottomed flasks on an orbital shaker
- Initial cells density: 1.6 million cells/ml
- Control end cells density: more than a factor (growth rate: 0.9 per day)

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
Natural seawater from unpolluted site was collected from a tap, after filtration through sand filter. The seawater was filtered through a GF/C filter, and brifly heated to about 75°C prior to use. The seawater was enriched with nutrient according to ISO 10253, and recommendations from the Paris Commission.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: (> 50µE/m²sec) from fluorescent tubes of universal natural white type
- Salinity (for marine algae): 34%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: by fluorescence on a Turner Designs, Model 10 fluorimeter

- Other:

TEST CONCENTRATIONS
- Test concentrations: 6.9; 16.3; 40; 100; 308; 623; 1549 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol 97%, CAS: 591-35-5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Remarks on result:

other: 95%CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

863 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL

Details on results:

One of the test concentrations (100 mg/l) was excluded from further calculations because after the solution was prepared there was an unusual precipitation in this concentration. All the other concentrations were clear.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 35%
The EC50 result of the 72h test with 1.5 mg/l 3.5-dichlorophenol should be within 20-80%.

Reported statistics and error estimates:

In the current study the responses were such that insufficient data are available for statistic evaluation of the response concentration using the computer programm TOXEDO and the EL50 value was estimated by interpolation.
Calculation of NOELR was done by single-factor ANOVA followed by Dunnets procedure for comparing each concentration mean with the control mean.

 Table 1. Responses obtained with Dynaslylan AMEO / Experimental data

Concentration in mg / l	Growth rate	Inhibition in percent
control 1	1.87	-
control 2	1.85	-
control 3	1.94	-
control 4	1.89	-
control 5	1.91	-
control 6	1.94	-
control mean	1.90	0
6.90	2.00	0.0
6.90	2.00	0.0
6.90	1.96	0.0
16.30	1.71	10.0
16.30	1.78	6.3
16.30	1.84	3.2
40.00	1.78	6.3
40.00	1.90	0.0
40.00	1.77	6.8
100.00	0.64	66.3
100.00	0.87	54.2
100.00	0.88	53.7
308.00	1.21	36.3
308.00	1.40	26.3
308.00	1.54	18.9
623.00	1.25	34.2
623.00	1.52	20.0
623.00	1.67	12.1
1549.00	0.00	100.00
1549.00	0.00	100.00
1549.00	0.00	100.00

 

Validity criteria fulfilled:

yes

Conclusions:

A 72 hour EC50 value of 863 mg/l and a NOEC of 40 mg/l have been determined for the effect of the test subsatnce on growth rate of Skeletonema costatum. It is likely that the test organisms were exposed to the hydrolysis products of the substance.

Description of key information

Silanol HP-X - read-across from triethoxy(methyl)silane (CAS 2031-67-6): 72-hour ErC₅₀ >500 mg/l and NOEC ≥500 mg/l, growth rate of Pseudokirchneriella subcapitata (Hazleton, 1994).

Silanol HP-X - read-across from trimethoxy(methylsilane) (CAS 1185-55-3): 72-hour ErC₅₀ >120 mg/l and NOEC ≥120 mg/l, growth rate of Pseudokirchneriella subcapitata (Dow Corning, 2007).

Silanol HP-W and HP-Y - read-across from N,N-bis(3-triethoxysilylpropyl)amine (CAS 13497-18-2): 72-hour ErC₅₀ 125 mg/l, ErC₁₀ 68 mg/l and NOEC 51 mg/l, growth rate of Phaeodactylum tricornutum (Hydrotox, 2004).

Silanol HP-W and HP-Y - read-across from 3-(trimethoxysilyl)-N-[3-(trimethoxysilyl)propyl]-1-propanamine (CAS 82985-35-1): 72-hour ErC₅₀ 36 mg/l, NOEC 8.3 mg/l, growth rate of Pseudokirchneriella subcapitata (Yoshikawa, CERI, 2014).

Silanol HP-Z - read across from 3-aminopropyl(triethoxy)silane (CAS 919-30-2):

72-hour ErC₅₀ >1000 mg/l and ErC₁₀ 321 mg/l, growth rate of Desmodesmus subspicatus (Hüls, 1993).

72-hour ErC₅₀ 863 mg/l and NOEC 40 mg/l, growth rate of Skeletonema costatum (M-Lab, 2005).

Additional information

There are no measured data for the registration substance as a whole. However, according to REACH Annex XI the algal study does not need to be conducted because the data requirements for this endpoint have been addressed using measured or read-across toxicity data for the constituents of the substance and their hydrolysis products.

The majority of the constituents of the substance are expected to hydrolyse rapidly, therefore the environmental aspects of chemical safety assessment are based on the hydrolysis products, which are grouped into four silanol assessment entities Silanol HP-W, Silanol HP-X, Silanol HP-Y and Silanol HP-Z. However, hydrolysis rates of some of the constituents of the substance are uncertain, therefore risk assessment has also been performed on the parent constituents which are divided into separate Blocks. The same data are used to derive PNEC for the parent Block, depending on the hydrolysis product(s) of the Block. This is discussed further in the ecotoxicological general discussion.

Data for each of the four silanol assessment entities are presented below:

 

Silanol HP-X: A 72-hour ErC₅₀ value of >500 mg/l and NOErC of ≥500 mg/l (nominal) have been read-across from a structural analogue, triethoxy(methyl)silane (CAS 2031-67-6) for effects on growth rate of Pseudokirchneriella subcapitata (tested as: Selenastrum capricornutum) (Hazleton, 1994). The study was conducted according to a standard guideline (EU Method C.3) and GLP. It is likely that the test organisms were exposed predominantly to the hydrolysis products of the substance, methylsilanetriol (1 mole) and ethanol (3 moles). Ethanol is not likely to have contributed to toxicity at the tested concentrations. The ErC₅₀ value expressed in terms of methylsilanetriol is >500 x (94.14/178.31) = >264 mg/l.

A 72-hour ErC₅₀ value of >120 mg/l and NOErC ≥120 mg/l (nominal) have been read across from a structural analogue, trimethoxy(methyl)silane (CAS 1185-55-3), for effects on growth rate of Pseudokirchneriella subcapitata (Dow Corning, 2007). The study was conducted according to a standard guideline (OECD 201) and GLP. It is likely that the test organisms were exposed predominantly to the hydrolysis products of the substance, methylsilanetriol and methanol. Methanol is not likely to have contributed to toxicity at the tested concentrations. The ErC₅₀ value expressed in terms of methylsilanetriol is >120 x (94.14/136.22) = >83 mg/l.

 

Silanol HP-Z: A 72-hour ErC₅₀ value of >1000 mg/l and ErC₁₀ of 321 mg/l (nominal) have been determined for the effects of the read-across substance, 3-aminopropyl(triethoxy)silane (CAS 919-30-2), on growth rate of the freshwater algae Desmodesmus subspicatus (Hüls, 1993). A 72-hour EbC₅₀ of 603 mg/l and EbC₁₀ of 38 mg/l have been determined for effects on biomass. The NOEC in the study was 1.3 mg/l (for effects on growth rate and/or biomass; no growth rate-specific NOEC is derived) (derived in an extension to the above study and under consistent conditions).

In a separate test, a 72-hour ErC₅₀ value of 863 mg/l and NOEC of 40 mg/l have also been determined for the effect of 3-aminopropyl(triethoxy)silane (CAS 919-30-2) on growth rate of the marine algae Skeletonema costatum (M-Lab, 2005). It is likely that the test organisms were exposed to the hydrolysis products of the substance. 

The NOEC for Desmodesmus subspicatus is not representative of the inhibition of growth rate, as the EC₁₀ values for growth rate and biomass make clear. It is not appropriate to derive PNEC based on biomass effects in the absence of effects on growth rate. Excluding this result, the NOEC in the Skeletonema costatum study is the most appropriate starting value for the derivation of PNEC. It is likely that the test organisms were exposed predominantly to the hydrolysis products of the substance, 3-aminopropylsilanetriol (1 mole) and ethanol (3 moles). Ethanol is not likely to have contributed to toxicity at the tested concentrations. The ErC₅₀ value expressed in terms of 3-aminopropylsilanetriol is >1000 x (137.21/221.37) = >620 mg/l and the NOEC is 25 mg/l.

 

Silanol HP-W and HP-Y: A 72-hour ErC₅₀ value of 125 mg/l (nominal) and ErC₁₀ of 68 mg/l (nominal); have been read across from a structural analogue,   N,N-bis(3-triethoxysilylpropyl)amine (CAS 13497-18-2), for the effects of the test substance on growth rate of the marine algae Phaeodactylus tricornutum (Hydrotox, 2004). The study was conducted according using a standard method (ISO standard 10235, similar to OECD TG 201) and GLP. It is likely that the test organisms were exposed predominantly to the hydrolysis products of the substance, bis(trihydroxysilylpropyl)amine (1 mole) and ethanol (6 moles). Ethanol is not likely to have contributed to toxicity at the tested concentrations. The ErC₅₀ value expressed in terms of bis(trihydroxysilylpropyl)amine is 125 x (257.39/425.72) = 75 mg/l.

A 72-hour ErC₅₀ value of 36 mg/l and NOErC of 8.3 mg/l (nominal) have been read across from a structural analogue 3-(trimethoxysilyl)-N-[3-(trimethoxysilyl)propyl]-1-propanamine (CAS 82985-35-1), for effects on growth rate of Pseudokirchneriella subcapitata under conditions where pH is controlled at around 8.0 (Yoshikawa, CERI, 2014). The ErC₅₀ was 31 mg/l (nominal) under conditions where pH variation in the test media was not corrected. This value would not be representative of natural waters and so the ErC₅₀ with pH correction is taken forward in the chemical safety assessment. It is likely that the test organisms were exposed predominantly to the hydrolysis products of the substance, bis(trihydroxysilylpropyl)amine (1 mole) and methanol (6 moles). Methanol is not likely to have contributed to toxicity at the tested concentrations. The ErC₅₀ value expressed in terms of bis(trihydroxysilylpropyl)amine is 36 x (257.39/341.56) = 27 mg/l.

 

The basis for read-across is discussed further in the Ecotoxicological information endpoint summary and in the RAAF for ecotoxicity attached in Section 13 of IUCLID and Annex 4 of the CSR.