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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

- 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.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04/2010-09/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Total Organic Carbon (TOC) analyses occured at test initiation and test termination. Volatile organic analysis vials with Teflon®-lined screw caps were used rather than traditional caps and vessels which allow air exchange to minimize any potential volatilization. At 72 hours, samples were removed from the composite of replicate vessels for each treatment and control.
Vehicle:
no
Details on test solutions:
A 100 mg a.i./L primary stock solution was prepared prior to test initiation by placing 0.2004 g (0.2000 g as active ingredient) of the test substance in a 2000-mL volumetric flask and bringing it to volume with AAP medium. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. The stock solution was mixed using a Teflon®- coated stir bar and magnetic plate and was allowed to hydrolyze for 2 days prior to test initiation. Nominal test concentrations were prepared from dilutions of the 100 mg a.i./L primary stock solution as follows:
Eleven replicates, approximately 60-mL, volatile organic analysis vials for the 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L treatment levels, and 20 for the control were conditioned prior to use by rinsing with untreated algal medium. Each vial was filled with a portion of the final volume of algal medium prior to inoculation. Additional untreated AAP medium was used for the control. Algal inoculum was then added to yield an initial cell density of approximately 1.0 x 10E4 cells/mL. Additional test solution was added to fill the vial to capacity (i.e., zero headspace) and then shaken, after which all solutions were observed to be clear and colorless with no visible undissolved test substance. After each vial was completely filled, the total volume was 65 mL. Twenty replicate vials for the control were maintained under the same conditions as the treatment level vials but contained no test substance. Each test vial was labeled with the test concentration, replicate, test species and study number. One additional replicate for each treatment level and control, for a total of 12 treatment replicates and 21 controls, was established in the same manner as described above to serve as a replacement in the event that any of the vessels were cracked or broken during exposure due to increased pressure in the test vessels. All test vials were covered with Teflon®-lined screw-caps to minimize the potential for volatilization of the test substance. Three replicates per treatment level and six replicates for the control were opened and discarded after daily observation intervals. The remaining two replicates per treatment level and control were used for TOC analysis at test initiation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain 1648, Class Chlorophyceae.
Inoculum - three days since previous transfer. Maintained stock culture at laboratory test facility.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature: 23 ºC.
pH:
Test initiation: pH of the test and control solutions ranged from 7.8 to 8.1.
Test termination: pH of the test and control solutions ranged from 9.6 to 9.8.
Salinity:
Algal Assay Procedure (AAP) medium
Nominal and measured concentrations:
Nominal test concentrations: 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L
Details on test conditions:
Conductivity of the exposure and control solutions measured at test initiation and termination ranged from 370 to 410 μmhos/cm.
Light intensity of the test area ranged from 4500 to 5800 lux.
Photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 55 to 71 μE/m2/s.
Shaking rate maintained at a constant rate of 100 ± 10 rpm.
Reference substance (positive control):
not required
Remarks:
not included during this study
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Definitive Test:
- At test termination, cells exposed to all treatment levels tested and the control were observed to be normal. The 72-hour cell density in the control averaged 100.67 x 10E4 cells/mL. Cell densities in the 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L nominal treatment levels averaged 110.25, 94.92, 105.00, 93.33, 94.08 and 105.25 x 10E4 cells/mL, respectively.
- For cell biomass, the 0- to 72-hour yield in the control averaged 99.67 x 10E4 cells/mL. The 0- to 72-hour yield in the 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L nominal concentration levels averaged 109.25, 93.92, 104.00, 92.33, 93.08 and 104.25 x 10E4 cells/mL, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. Based on Williams’ Test, no significant reduction in yield in any of the nominal concentration levels tested compared to the control. Therefore the NOEC was determined to be 100 mg a.i./L. Since no concentration tested resulted in ≥ 10% inhibition of yield, the 72-hour EyC10, EyC20 and EyC50 values were all empirically estimated to be > 100 mg a.i./L the highest nominal concentration level tested.
- For cell growth rate, the 0- to 72-hour growth rate in the control averaged 1.54 days-1. The 0- to 72-hour growth rate in the 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L nominal treatment levels averaged 1.57, 1.52, 1.56, 1.52, 1.52 and 1.56 days-1, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. Based on Williams’ Test, no significant reduction in growth rate was detected in any of the nominal concentration levels tested compared to the control. Therefore the NOEC was determined to be 100 mg a.i./L. Since no concentration tested resulted in ≥ 10% inhibition of growth, 72-hour EyC10, EyC20 and EyC50 values were all empirically estimated to be > 100 mg a.i./L the highest nominal concentration level tested.
- The following acceptance criteria were required by the protocol: the cell growth in the control should increase from the initial density (1.0 x 10E4 cells/mL) by more than 16 times after 72 hours of growth. Additionally, the mean coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates should not exceed 35%. The CV for the average growth rate of the control for the entire test period (0- to 72-hour growth rate) should not exceed 7%.
- During this study, the 72-hour cell density in the control was 100.67 x 10E4 cells/mL, which meets the above criterion. The CV for 0- to 72-hour growth rate was 3.3%, which meets the above criterion, while the mean daily CV for growth rates was 52%. Although the acceptance criterion suggests that this value should not exceed 35%, this study was conducted using a non-standard exposure system (i.e., closed system), rather than the standard algal test design. Therefore, this study is considered acceptable and the results accurately reflect the sensitivity of P. subcapitata to the test substance.
- Additional testing to further define the EC values was not performed since the highest nominal concentration tested represents the upper testing limit required by the OECD 201 guideline.
Results with reference substance (positive control):
Positive controls are routinely performed by the testing laboratory but not conducted during this study.
Reported statistics and error estimates:
Based on the results of statistical analysis performed for 72-hour total yield and average growth rate, the No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p ≤ 0.05) when compared to the control data, was determined. The data were first checked for normality using Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the tests for homogeneity and normality, then Williams’ Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis' Test was used to determine the NOEC.
All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99% level of certainty was applied. TOXSTAT® version 3.5 was used to perform these calculations.
At test termination, the EC values were calculated for average growth rate (ErC10, ErC20 and ErC50 values) and yield (EyC10, EyC20 and EyC50 values) by linear interpolation of response (percent reduction of yield and growth rate as compared with the control) versus the nominal test substance concentration. The EC10, EC20 and EC50 values are defined as the concentration of test substance which caused a 10%, 20% or 50% reduction, respectively, in average growth rate or yield, compared to the control data. TOXSTAT® version 3.5 was used to perform both the statistical (NOEC determinations) and EC10, EC20 and EC50 calculations. If less than the required response was observed (i.e., < 10, < 20 and < 50% response), EC values were empirically estimated to be greater than the highest concentration tested.

Preliminary Test:

- Preliminary exposure was conducted at nominal test substance concentrations of 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L, a control and solvent (acetone) control. Test solutions were prepared immediately prior to test initiation with no hydrolysis mixing period prior to test initiation. The exposure was conducted in a closed system with 65-mL volatile organic analysis vials covered with Teflon®-lined screw caps with septa, autoclaved. Following preparation, exposure solutions were observed to be clear and colorless with no visible undissolved test substance following several swirls with a syringe. Two exposure vessels were established for each concentration, control and solvent control. Following 72 hours of exposure, cells exposed to all treatment levels tested, the control and the solvent control were observed to be normal. Cell density in the control and solvent control averaged 27 and 31 x 10E4 cells/mL. Cell density in the 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L treatment levels averaged 25, 36, 37, 23 and 26 x 10E4 cells/mL, respectively. Based on these results and in consultation with the study sponsor, treatment levels of 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L were selected for the definitive exposure. At the request of the study sponsor, the stock solution was hydrolyzed prior to initiation of the definitive test. Additionally, based on this change and the observations made during the preliminary test, no solvent was needed in the definitive exposure.

TOC measurements during definitive test:

-Average TOC concentrations in the control, 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L (nominal) solutions at 0 hour were 0.42, 0.38, 0.42, 0.61, 0.69, 0.53 and 0.93 mg C/L, respectively. Measured TOC concentrations at these treatment levels increased between sampling intervals (i.e., 0 and 72 hours). Prior to analysis at the 72 hour interval, samples were allowed to settle before removing a sub-sample for transfer into new TOC vials and analysis. The increase in TOC concentration measured during the exposure is due to the uptake of carbon dioxide by the algae during photosynthesis. Average TOC concentrations in the control, 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L (nominal) solutions at 72 hours were 1.2, 1.4, 1.1, 1.5, 1.1, 1.2, and 1.8 mg C/L, respectively. The 16 mg a.i./L nominal test solution with no algae present had a 72-hour TOC concentration of 0.39 mg C/L. The 16 mg a.i./L test solution with algae had a 72-hour TOC concentration of 1.1 mg C/L. Based on these results it was determined the presence of algae had a significant impact on the TOC concentration in the test solutions.

Validity criteria fulfilled:
yes
Conclusions:
At test termination cells exposed to all treatment levels and the control were observed to be normal.
The results were determined to be:
EC50 (72h) biomass >100 mg a.i./L
NOEC biomass = 100 mg a.i./L
EC50 (72h) growth rate >100 mg a.i./L
NOEC growth rate = 100 mg a.i./L
Executive summary:

The study determined the effect of 2,4,6,8-tetramethylcyclotetrasiloxane on the growth of the freshwater green alga Pseudokirchneriella subcapitata following OECD Guideline 201.Due to the rapid hydrolysis of 2,4,6,8-tetramethylcyclotetrasiloxane, the substance was hydrolyzed for a period of approximately 48 hours prior to test initiation to generate hydrolysis products for use in the toxicity test. The study was conducted in a closed system in an attempt to minimize volatilization of the test substance during the 72 -hour exposure. Volatile organic analysis vials with Teflon®-lined screw caps were used rather than traditional caps and vessels which allow air exchange to minimize any potential volatilization.

Based on the results of the preliminary test, treatment levels of nominal concentrations 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L were selected for the definitive exposure.

At test termination cells exposed to all treatment levels and the control were observed to be normal.

The results were determined to be:

EC50 (72h) biomass >100 mg a.i./L

NOEC biomass = 100 mg a.i./L

EC50 (72h) growth rate >100 mg a.i./L

NOEC growth rate = 100 mg a.i./L

Description of key information

Short-term toxicity to algae: 72 hour EC50 >100 mg/l and NOEC≥100 mg/l (nominal, highest concentration tested) in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test);

72 hour EC50 >500 mg/l and NOEC ≥500 mg/l (nominal, highest concentration tested) (EU Method C.3 (Algal Inhibition Test)), read across from triethoxy(methyl)silane (CAS 2031-67-6). The observations in this study are attributed to the exposure of test organisms to the hydrolysis products in the test system. The EC50 and NOEC are equivalent to >264 mg/l and ≥264 respectively, when expressed in terms of the silanol hydrolysis product, methylsilanetriol.

Key value for chemical safety assessment

Additional information

Toxicity to aquatic plants was studied in a valid algal growth inhibition study with Pseudokirchneriella subcapitata according to OECD 201 under GLP. Test concentrations ranged from 1 to 100 mg/l (nominal concentrations). Because of rapid hydrolysis of the test substance, the stock solution was allowed to hydrolyse for 48 hours prior to test initiation under conditions that prevented evaporation of the test substance. No effect on algal growth parameters could be observed. Under the conditions of the test, the hydrolysis products of the test substance did not induce toxicity to algae. A 48 hour EC50 value of >100 mg/l and NOEC value of 100 mg/l were determined.

 

To further assess the toxicity of the hydrolysis product methylsilanetriol, good quality data have been read across from triethoxy(methyl)silane (CAS 2031-67-6). HD4 and triethoxy(methyl)silane rapidly hydrolyse to the same hydrolysis product; methylsilanetriol. The other hydrolysis products are hydrogen gas and ethanol, respectively. In the test with triethoxy(methyl)silane (CAS 2031-67-7), the observations are attributed to the exposure of test organisms to methylsilanetriol in the test system. There is no reason to expect that ethanol significantly influenced the results of the tests. The toxicity of ethanol is discussed further in the ecotoxicological RAAF document attached in Section 13 of IUCLID.

A 72 hour EC50 value of >500 mg/l and NOEC of ≥500 mg/l (nominal, highest concentration tested) have been determined for the effects of triethoxy(methyl)silane (CAS 2031-67-6) on growth rate of Pseudokirchneriella subcapitata (Hazleton, 1994). In view of the test media preparation method and exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of the tested substance. The results may be expressed in terms of concentration of the hydrolysis product, methylsilanetriol, by applying a molecular weight correction to the nominal test substance concentration: (MW of silanol = 94.14 / MW of parent = 178.31) * EC50 >500 mg/l and NOEC ≥500 mg/l = EC50 >264 mg/l and NOEC ≥264 mg/l. These values are read across to methylsilanetriol without further correction.