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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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

- Acclimation period:

- 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
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
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.
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004-11-2 to 2004-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ISO 10235
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 -20ºC.
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:
19.8-21.4ºC
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

- Test concentrations: concentrations tested and results not reported.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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
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
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
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
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
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
58.5 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
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
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
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
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
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 for the effect of the test substance on growth rate of the marine alga Phaeodactylus tricornutum. The 72-h NOEC determined for the same endpoint was 51 mg/l (58.5 mg/l based on estimated concentration from DOC measurement).
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
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
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
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
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)
Conc.(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)
Conc.(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.)

                                          (mg/L)
Method             Control   33    67  133  278  556  1000

Area Under  Area   94.5    81   83.5 78.5 61   40.5  41
Growth     % suppr.       14.3  11.6 16.9 35.4 57.1 56.6
Curve        
                                                        
Growth      µ     1.351  1.323 1.339 1.32 1.238 1.08 1.073
Rate µ    % suppr.        2.1   0.9   2.3  8.4  20.1  20.6
(0-72h)

pH      After 0 h  7.9    8.3   8.4   8.7  8.9   9.1  9.2
values  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.)

                                     (mg/L)
Method            Control   1.3      2.5     5.1    10.1

Area Undern Area  72.5    67.5    59.5    52.5     63
Growth    % suppr.         6.9    17.9    13.8    13.1
Curve

Growth      µ    1.294   1.265    1.25    1.258   1.261
Rate µ    % suppr.        2.2      3.4     2.8     2.6
(0-72 h)

pH     After 0 h   7.5    7.8      7.8     7.9     8.1
values After 72h   9.0    8.8      8.7     8.7     8.6

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >1000 mg/L and a NOEC of 1.3 mg/L have been determined for the effects of the test substance on growth rate of Scenedesmus subspicatus. A 72-hour EC50 of 603 mg/L and NOEC of 1.3 mg/L have been determined for effects on biomass. 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) and trimethoxy(methylsilane) (CAS 1185-55-3):

72 hour ErC50 >500 mg/l and NOEC ≥500 mg/l, growth rate of Pseudokirchneriella subcapitata;

72 hour ErC50 >120 mg/l and NOEC ≥120 mg/l, growth rate of Pseudokirchneriella subcapitata, respectively;

Silanol 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;

Silanol HP-Y - read-across from3-(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;

Silanol HP-Z - read across from 3-aminopropyl(triethoxy)silane (CAS 919-30-2): 72 hour ErC50>1000 mg/l, growth rate, and NOEC 1.3 mg/l, growth rate and biomass, of Desmodesmus subspicatus.

Key value for chemical safety assessment

Additional information

There are no measured data for the registration substance as a whole. However according to REACH Annex XI the short-term algal study does not need to be conducted because the data requirement for this endpoint have been addressed using measured or read-across toxicity data for the constituents of the substance and their hydrolysis products in combination with partitioning theory and the toxic units approach to determine whether a test medium prepared at a nominal loading rate of 100 mg/l would be toxic following short-term exposure. The results of applying this approach indicate that a test medium prepared at a nominal loading rate of 100 mg/l would not be toxic to algae. A more detailed description of the method used is given in the Aquatic toxicity End point summary.

The constituents of the substance hydrolyse rapidly, therefore the environmental aspects of chemical safety assessment are based on the hydrolysis products, which are grouped into three silanol assessment entities, Silanol HP-X, Silanol HP-Y and Silanol HP-Z. This is discussed further in the ecotoxicological general discussion. Supporting data for each of the three silanol assessment entities are sourced from:

Silanol HP-X - read-across from triethoxy(methyl)silane (CAS 2031-67-6) and trimethoxy(methylsilane) (CAS 1185-55-3):

72 hour ErC50 >500 mg/l and NOEC ≥500 mg/l, growth rate of Pseudokirchneriella subcapitata (Dow Corning, 2007);

72 hour ErC50 >120 mg/l and NOEC ≥120 mg/l, growth rate of Pseudokirchneriella subcapitata (Wyness, 1994);

Silanol 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-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, 2014);

Silanol HP-Z - read across from 3-aminopropyl(triethoxy)silane (CAS 919-30-2): 72 hour ErC50 >1000 mg/l, growth rate, and NOEC 1.3 mg/l, growth rate and biomass, of Desmodesmus subspicatus (Hüls, 1994).

The short-term EC50 values for these substances are all greater than 100 mg/l, with the exception of one test read across from 3-(trimethoxysilyl)-N-[3-(trimethoxysilyl)propyl]-1-propanamine (CAS 82985-35-1) where an EC50 of 36 mg/l was reported. In the case of Silanol HP-X and Silanol HP-Z, the EC50 values are limit values. The basis for read-across is discussed further in the Ecotoxicological information endpoint summary.