Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
04 Dec - 07 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is virtually insoluble/immiscible in water. Additionally, the test substance is reported to undergo rapid hydrolysis (half-life of approximately 2.5 hours at pH 7 and 25 °C) to primary hydrolysis products of chloromethylsilanetriol and ethanol. Therefore, test solutions were prepared from a stock solution of (chloromethyl)triethoxysilane which was mixed overnight prior to testing to fully hydrolyze the test substance. Based on the rapid hydrolysis of (chloromethyl)triethoxysilane, no attempt was made to determine the toxicity of parent substance. Test solutions were prepared using tetrahydrofuran (THF) as a carrier to minimize the risk of polymerization and in accordance with OECD recommendation (OECD 2000, Series on Testing and Assessment Number 23. Guidance Document on Aquatic Testing of Difficult Substances and Mixtures. ENV/JM/MONO(2000)6. Organization for Economic Cooperation and Development. Paris, France) and to aid in the delivery of the test substance during test solution preparation.
In detail the test solution preparation was done as follows: A 100 mg/L primary stock solution was prepared prior to test initiation by placing 0.2058 g of (chloromethyl)triethoxysilane (0.2000 g as active ingredient) in a 2000-mL volumetric flask, adding 0.20 mL tetrahydrofuran (THF) directly to the substance and then diluting to a final volume of 2000 mL with AAP medium. The stock solution was initially observed to be clear with undissolved clear bubbles of test substance. Following mixing overnight with a magnetic stir plate and Teflon®-coated stir bar, the stock solution was observed to be clear and colorless with no visible undissolved test substance present in the solution. Exposure solutions were prepared from dilutions of the 100 mg/L primary stock solution. Following mixing by shaking and inversion of the volumetric flask, the test solutions were observed to be clear and colorless with no visible undissolved test substance and remained the same for the duration of the exposure. A solvent control solution was prepared in AAP medium, containing THF at a concentration of 0.10 mL/L, equivalent to the concentration of solvent in the treatment levels. Six flasks for the
solvent control, as required by the OECD guideline, were maintained under the same conditions as the treatment levels but contained no (chloromethyl)triethoxysilane. Additional untreated AAP medium was used for the control. In order to estimate the impact that the presence of algal biomass had on total organic carbon concentration, an additional replicate flask (D) of the 25 mg/L (nominal) test solution was prepared. This flask, which was not inoculated with algae, was analyzed at 72 hours of exposure for total organic carbon concentration. The result of this analysis was compared with the result obtained for the 25 mg/L solution containing algae.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tetrahydrofuran (THF)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: 1648
- Source (laboratory, culture collection): University of Texas, Austin, Texas and maintained in stock culture at Smithers Viscient culture facility
- Method of cultivation: Stock cultures were grown in 250-mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange. The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 °C and continuous illumination at the surface of the medium with an intensity range of 4500 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test with (chloromethyl)triethoxysilane was taken from a stock culture that had been transferred to fresh medium four days before testing.


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 - 24 °C
pH:
7.0 - 9.4
Nominal and measured concentrations:
control, 6.3, 13, 25, 50, and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks, filled with 100 mL test solution, fitted with stainless steel caps which permit gas exchange.
- Initial cells density: 177 x 10^4 cells/mL
- Control end cells density: 91.50 x 10^4 cells/mL (control), 99.83 x 10^4 cells/mL (solvent control)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes; Algal Assay Procedure (AAP) medium prepared with sterile, deionized water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium. Several liters of AAP medium were prepared using sterile, deionized water and were equilibrated to test temperature. The initial pH of this medium was adjusted, if necessary, to 7.5 ± 0.1 prior to use.
- Conductivity: 73 - 80 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 420 to 510 footcandles (4500 - 5500 lux); photosynthetically-active radiation (PAR) range of 64 to 73 pE/m/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At each subsequent 24-hour interval, cell counts were conducted on each replicate solution of the treatment levels and the control. Observations of the health of the algal cells and observations of the physical characteristics of the test solutions (e.g., presence of precipitate, film on the solution's surface) were also made and recorded at each 24-hour interval.
- Determination of cell concentrations: hemacytometer (Neubauer Improved) and compound microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2
- Results used to determine the conditions for the definitive study: Based on the results of solubility trials, preliminary testing and consultation with the Study Sponsor, nominal loading rates of 6.3, 13, 25, 50 and 100 mg/L were selected for the definitive exposure.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
Shapiro-Wilks' and Bartlett's Tests for normality and homogeneity. Bonferroni's Adjusted t-Test to determine treatment-related effects.

Exposure Solution Analyses - TOC Measurements:

An attempt was made to develop a chemical-specific analytical method for (chloromethyl)triethoxysilane and/or the monomer hydrolysis product, chloromethylsilanetriol. Preliminary analytical development was performed using degraded aqueous samples of the

parent substance (chloromethyl)triethoxysilane. Fortified aqueous samples showed no parent material present after 24 hours, with the only peaks present in the chromatogram also present in an iso-octane blank sample. The absence of peaks after 24 hours may have been due to a number of causes including that the degradate could not be extracted from the aqueous solution, elution of the degradate in the solvent front due to a very short retention time, or that the degradate was not amenable to GC-MS analysis. The exact cause of this could not be definitively determined since no analytical method could be developed for either the parent substance or its monomer hydrolysis product. Based on the nature of the test substance including rapid hydrolysis into a mixture of degradation products, exposure solutions were analyzed for total organic carbon (TOC) concentration. At test initiation the average measured TOC for the solvent control and treatment solutions ranged from 26 to 46 mg C/L, increasing slightly with increasing test substance concentrations. The control solution had an average measured TOC concentration of 1.0 mg C/L. These results indicate the contribution of TOC from the solvent, present at a concentration of 0.10 mL/L in all treatment solutions and the solvent control, exceeded the contribution from the test substance. In consultation with the Study Sponsor, the relatively low TOC contribution from the test substance may have been due to volatilization of the hydrolysis products during the TOC analysis. However, despite the large solvent contribution, the increase in TOC relative to test substance concentration did approximate the concentration gradient (dilution factor = 2) based on the actual 0 hour TOC results. At 72 hours of exposure the average TOC concentration in the treatment levels had declined from that at 0-hour, ranging from 19 to 31 mg C/L. The control and solvent control solutions had average TOC values of 1.1 and 17 mg C/L, respectively, at test termination, with the solvent control also showing a slight decline from the 0 hour results. Based on these results it appears the decline in TOC concentration after 72 hours (approximately 35% from the 0 hour results) is related to the presence of THF in the solvent control and treatment samples, as the TOC results for the control solutions were stable over 72 hours and there was a similar decline regardless of loading rate. Generally, TOC concentrations may be impacted by several factors including the uptake of carbon dioxide by algae during photosynthesis, and so are not intended to directly correspond to test substance (parent or monomer hydrolysis product) concentrations during the exposure. The average TOC concentration in the 72-hour sample from the 25 mg/L nominal treatment, with algae present, was 18 mg C/L. The equivalent test solution without algae present resulted in an average TOC concentration of 24 mg C/L and demonstrated that the presence of algae had a slight impact on TOC concentration during the study.

Table: Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to (chloromethyl)triethoxysilane

Nominal Loading Rate (mg/L)

 

Cell Density (x 10^4 cells/mL)

Observation Interval (Hours)

24

48

72

Control

A

6.25

15.25

87.75

B

4.50

18.75

105.50

C

4.75

17.50

81.25

Mean(SD)(a)

5.17 (0.95)

17.17 (1.77)

91.50 (12.55)

Solvent Control

A

5.25

18.00

96.25

B

4.75

17.25

110.50

C

4.50

10.25

89.00

D

4.25

15.00

80.00

E

4.50

15.25

128.75

F

3.25

27.00

94.50

Mean(SD)

4.42 (0.66)

17.13 (5.54)

99.83 (17.33)

6.3

A

4.25

12.50

67.25

B

4.75

20.00

110.00

C

3.50

22.25

72.50

Mean(SD)

4.17 (0.63)

18.25 (5.11)

83.25 (23.31)

13

A

4.75

20.00

89.00

B

2.50

24.00

101.50

C

3.75

23.50

85.75

Mean(SD)

3.67 (1.13)

22.50 (2.18)

92.08 (8.32)

25

A

4.00

25.75

87.75

B

4.75

18.50

87.00

C

4.00

13.25

73.25

Mean(SD)

4.25 (0.43)

19.17 (6.28)

82.67 (8.16)

50

A

4.50

11.25

75.00

B

7.50

18.25

111.50

C

5.25

12.75

81.25

Mean(SD)

5.75 (1.56)

14.08 (3.69)

89.25 (19.52)

100

A

4.50

9.75

62.00

B

3.50

12.25

84.25

C

1.75

12.00

60.50

Mean(SD)

3.25 (1.39)

11.33 (1.38)

68.92 (13.30)

(a) Mean and standard deviation (SD) are calculated from original raw data, not from the rounded values presented in this table

Table: Yield of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to (chloromethyl)triethoxysilane

Nominal Loading Rate (mg/L)

 

Yield (x 10^4 cells/mL)

Observation Interval (Hours)

0-24

0-48

0-72

Percent Inhibition(ab)

Control

A

5.25

14.25

86.75

 

B

3.50

17.75

104.50

C

3.75

16.50

80.25

Mean(SD)(b)

4.17 (0.95)

16.17 (1.77)

90.50 (12.55)

NA(c)

Solvent Control

A

4.25

17.00

95.25

 

B

3.75

16.25

109.50

C

3.50

9.25

88.00

D

3.25

14.00

79.00

E

3.50

14.25

127.75

F

2.25

26.00

93.50

Mean(SD)

3.42 (0.66)

16.13 (5.54)

98.83 (17.33)

NA

6.3

A

3.25

11.50

66.25

 

B

3.75

19.00

109.00

C

2.50

21.25

71.50

Mean(SD)

3.17 (0.63)

17.25 (5.11)

82.25 (23.31)

17

13

A

3.75

19.00

88.00

 

B

1.50

23.00

100.50

C

2.75

22.50

84.75

Mean(SD)

2.67 (1.13)

21.50 (2.18)

91.08 (8.32)

8

25

A

3.00

24.75

86.75

 

B

3.75

17.50

86.00

C

3.00

12.25

72.25

Mean(SD)

3.25 (0.43)

18.17 (6.28)

81.67 (8.16)

17

50

A

3.50

10.25

74.00

 

B

6.50

17.25

110.50

C

4.25

11.75

80.25

Mean(SD)

4.75 (1.56)

13.08 (3.69)

88.25 (19.52)

11

100

A

3.50

8.75

61.00

 

B

2.50

11.25

83.25

C

0.75

11.00

59.50

Mean(SD)

2.25 (1.39)

10.33 (1.38)

67.92 (13.30)(d)

31

(a)   Percent inhibition relative to the solvent control.

(b)  Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from the rounded values presented in this table.

(c) NA = Not Applicable.

(d)   Significantly reduced compared to the solvent control, based on Bonferroni's Adjusted t-Test.

Table: Calculated growth rates of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to (chloromethyl)triethoxysilane

Nominal Loading Rate (mg/L)

 

Growth Rate/days

Observation Interval (Hours)

0-24

0-48

0-72

72 Hour Percent Inhibition(ab)

Control

A

1.99

1.43

1.46

 

B

1.63

1.54

1.52

C

1.69

1.51

1.43

Mean(SD)(b)

1.77 (0.19)

1.49 (0.06)

1.47 (0.04)

NA(c)

Solvent Control

A

1.80

1.52

1.49

 

B

1.69

1.50

1.53

C

1.63

1.23

1.46

D

1.57

1.43

1.43

E

1.63

1.43

1.58

F

1.28

1.74

1.48

Mean(SD)

1.60 (0.18)

1.47 (0.17)

1.49 (0.05)

NA

6.3

A

1.57

1.33

1.37

 

B

1.69

1.58

1.53

C

1.36

1.63

1.39

Mean(SD)

1.54 (0.17)

1.51 (0.16)

1.43 (0.09)

4

13

A

1.69

1.58

1.46

 

B

1.00

1.67

1.50

C

1.44

1.66

1.45

Mean(SD)

1.38 (0.35)

1.64 (0.05)

1.47 (0.03)

1

25

A

1.51

1.71

1.46

 

B

1.69

1.54

1.45

C

1.51

1.36

1.40

Mean(SD)

1.57 (0.11)

1.54 (0.17)

1.44 (0.03)

3

50

A

1.63

1.27

1.41

 

B

2.19

1.53

1.53

C

1.80

1.34

1.43

Mean(SD)

1.88 (0.28)

1.38 (0.13)

1.46 (0.07)

2

100

A

1.63

1.20

1.34

 

B

1.36

1.32

1.44

C

0.61

1.31

1.34

Mean(SD)

1.20 (0.53)

1.28 (0.07)

1.37 (0.06)(d)

8

(a)   Percent inhibition relative to the solvent control.

(b)  Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from the rounded values presented in this table.

(c) NA = Not Applicable.

(d)   Significantly reduced compared to the solvent control, based on Bonferroni's Adjusted t-Test.

Validity criteria fulfilled:
yes
Conclusions:
An EC10 of 4 mg/L, an EC20 of 64 mg/L, an EC50 of >100 mg/L, and a NOEC of 50 mg/L based 72-Hour-Yield and an EC10 of >100 mg/L, an EC20 of >100 mg/L, an EC50 of >100 mg/L, and a NOEC of 50 mg/L based on 72-Hour-Average Growth Rate was determined in a reliable study on the toxicity to algae conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information

A 72-hour ErC50of >100 mg/l and a NOEC of 50 mg/l, algae, RL1

Key value for chemical safety assessment

Additional information

A 72-hour ErC50 value of > 100 mg/l and NOEC value of 50 mg/l have been determined for the effects of the test substance on Pseudokirchneriella subcapitata based on nominal concentration of the substance. The study was conducted according to a standard guideline (OECD Method 201, Smithers Viscient, 2013b) and was selected as key study.

As the substance is subject to rapid hydrolysis, it is therefore likely that the test organisms were primarily exposed to the silanol hydrolysis product, retained in the test media.