Tetramethylsilane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-15 to 2009-09-18

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and single treatment

- Sampling method: At test initiation (0 hour), one sample was removed as a composite from three replicates of the treatment (A, B and C) and six replicates of the control (A through F) and analyzed for tetramethylsilane (TMS). The 0-hour composite samples (10 mL for the control and treatment) were removed using a glass pipette, placed directly into a vial and then prepared for analysis. At 24, 48 and 72 hours (test termination) of exposure, prior to performing cell counts, triplicate samples were removed from the treatment and control replicates used for biological observations at the respective interval and analyzed for tetramethylsilane (TMS). Since the measured concentration of tetramethylsilane (TMS) was expected to decline during the exposure due to the volatilization of the test substance, triplicate samples were analyzed after 24, 48 and 72 hours to better monitor any slight variations in sample recovery. Samples at 24, 48 and 72 hours were removed from a composite of three replicates for the treatment and six for the control established for analysis and observations at each interval. Test vessels were inverted several times prior to compositing solutions and removing samples (30 mL volume for the control and treatment) with a disposable pipette. The extraction solvent, iso-octane, was immediately added to each analytical sample which minimized losses of the test substance due to volatilization, adsorption and degradation. Samples were processed immediately after sampling.

At 24, 48 and 72 hours of exposure, triplicate samples were removed from the 100% WAF prepared from a 2.0 mL/L stock solution test concentration which did not contain algae. The results of these analyses were compared with that obtained for the 24-, 48- and 72-hour analyses of the 100% WAF prepared from a 2.0 mL/L stock solution concentration containing algae to assess the impact that algae had on the test substance concentration.

Three quality control (QC) samples were prepared at 0- and 24-hour sampling intervals, and six QC samples were prepared at the 48- and 72-hour sampling intervals at nominal concentrations bracketing the test concentration and remained with the exposure solution samples throughout the analytical process. The number of QC samples was increased at the later intervals due to the difficulties associated with the analysis of this extremely volatile material. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.

Vehicle:

no

Details on test solutions:

A single concentration limit test at 100% of a water accommodated fraction (WAF) prepared from a saturated solution (2.0 mL/L, equivalent to 1300 mg a.i./L) was conducted.

A 2.0 mL/L stock solution was prepared prior to test initiation by adding 8.0 mL of tetramethylsilane (TMS) to a 4-L Marriotte bottle with a drain in the lower side wall containing 4 L of AAP medium. Due to the known rapid volatilization and low boiling point (approximately 27 °C) of the test substance, the test substance was added to the AAP medium volumetrically using a gas-tight syringe. The algal medium was stirred during addition of the test substance using a magnetic stir plate and Teflon®-coated stir bar. The resulting solution was observed to be clear and colorless with a large amount of undissolved test substance floating on the surface. The stock solution was mixed overnight using a magnetic stir plate and Teflon®-coated stir bar and was observed to be clear and colorless with a small amount of undissolved test substance floating on the surface following mixing. The water accommodated fraction (WAF) of the stock solution was removed through the lower side wall drain and passed through filter floss. The WAF was observed to be clear and colorless with no visible undissolved test substance present. Additional untreated AAP medium was used for the control.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Source of algal culture: The alga was originally obtained from University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.

Culture medium: The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.

pH adjustment of culture medium: If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with either 0.1 N hydrochloric acid or 1.0 N sodium hydroxide.

Culture vessels: Stock cultures were grown in 250-mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel air exchange caps which permitted gas exchange.

Lighting: Continuous illumination at the surface of the medium with an intensity range of 4400 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux® fluorescent bulbs.

Agitation: Culture flasks were agitated continuously on an orbital shaker ( 100 ± 10 rpm).

Temperature control: Temperature (23 ± 2 ºC) was controlled using an environmental chamber.

Inoculum: The inoculum used to initiate the toxicity test with tetramethylsilane (TMS) was taken from a stock culture that had been transferred to fresh medium four days before testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

22 to 23 ºC

pH:

8.2-8.3 at start of test

9.6-9.9 at end of test

The increase in pH during the exposure is common in static algal cultures and is due to photosynthesis by the algae.

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

A single concentration limit test at 100% of a water accommodated fraction (WAF) prepared from a saturated solution (2.0 mL/L, equivalent to 1300 mg a.i./L) was conducted.

The geometric mean measured concentration for the treatment level was 0.0079 mg/L.

Details on test conditions:

Test vessels: Replicate approximately 100-mL clear, glass serum bottles secured with PTFE septa and crimptop caps, 24 for the control and 12 for the treatment level, were conditioned prior to use by rinsing with the appropriate test solution. Each test vessel was labeled with the test concentration, replicate, test species and study number. Three additional replicates for the control, six additional replicates for the treatment for a total of 18 treatment and 27 control vessels, were established to serve as replacements in the event that any of the vessels were cracked or broken during exposure due to increased pressure in the test vessels.

In order to estimate the impact that the presence of algal biomass had on the test substance concentration, nine additional replicates were initiated for the treatment level. These flasks were not inoculated with algae. One solution at the 24-, 48- and 72-hour sampling interval was analyzed for tetramethylsilane (TMS) concentration. The results of these analyses were compared with the equivalent solution containing algae. The remaining six treatment replicates without algae present served as replacements as described above for the treatment vessels with algae present. Test solution samples at each interval were stabilized after collection by addition of the extraction solvent, iso-octane, which stopped hydrolysis of the test substance.

A single replicate control vessel was filled with 110 mL of AAP medium to yield minimal headspace in the vessel. Then, a 1.1-mL inoculum of Scenedesmus subspicatus cells, at a density of 101 x 104 cells/mL, was aseptically introduced, and the vessel was secured with a rubber septum and crimp-top cap. The inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10E4 cells/mL. This process was repeated for the remaining control vessels. Treatment vessels were filled and inoculated using the same process, except that the WAF removed from the 4-L Marriotte bottle was used for the exposure solution.

Cell counting: At each subsequent 24-hour interval, a single cell count was conducted on three replicate solutions of the treatment level and six of the control using a hemacytometer (Neubauer Improved) and a compound microscope.

Temperature: Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test vessels in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shaker were recorded daily.

Lighting: Light intensity was measured at four locations around the perimeter of the shaker table with a VWR Traceable light meter at 0 hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189 photometer with sensor probe LI-190SA. Test vessels were randomly placed on the shaker tables at test initiation based on computer-generated random numbers. Following each observation interval, the test vessels were assigned new random positions based on computer-generated random numbers.

Water quality: Water quality parameters (pH and conductivity) were measured in a composite sample of the solutions at test initiation and at the termination of the 72-hour exposure period.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

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

- Effect concentrations exceeding solubility of substance in test medium: Test media were prepared as an undiluted water-accommodated fraction

Reported statistics and error estimates:

There were no effects on growth; the No-Observed-Effect Concentration (NOEC) was determined to be equal to the concentration tested.

Table 1. Results of analysis of test media

Nominal concentration (% WAF)	Measured concentration at 0 hours (mg/L)	Measured concentration at 24 hours (mg/L)	Measured concentration at 48 hours (mg/L)	Measured concentration at 72 hours (mg/L)	Geometric mean measured concentration (mg/L)	Percentage of nominal
0 (Control)	<0.021	<0.049	<0.0020	<0.00077	Not applicable	Not applicable
100 (with algae)	0.0078	0.034	0.0014	0.0010	0.0079	0.00061
100 (without algae)	0.0078	<0.049	<0.0020	0.0014	0.0072	0.00055

The measured concentrations of tetramethylsilane (TMS) for the treatment at test initiation was 0.078 mg a.i./L. At each subsequent interval, the measured concentrations of TMS declined steadily from the initial (0-hour) measured concentration. At 24, 48 and 72 hours, the measured concentrations of the triplicate samples for the treatment closely approximated the limit of quantitation (LOQ) established for the respective intervals. Likely due to this close proximity, two of the three treatment samples at 24 and 48 hours had

measured values that were below the LOQ, while one sample was measured slightly above the LOQ. For samples that were below the LOQ at an interval, one-half the LOQ was used as a conservative estimate to determine the average measured recovery for that sampling interval. The average measured concentrations at 24, 48 and 72 hours, were 0.034, 0.0014 and 0.0010 mg a.i./L, respectively. The mean measured concentration for this study was calculated as a geometric mean of the 0-, 24-, 48- and 72-hour measured concentrations. The geometric mean measured concentration for the treatment level was 0.0079 mg a.i./L.

Table 2. Test results

Geometric mean measured concentration (mg/L)	Mean cell density at 24 hours (cells/mL)	Mean cell density at 48 hours (cells/mL)	Mean cell density at 72 hours (cells/mL)
Control	41700	155000	315000
0.0079	38300	164200	383300

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of >0.0079 mg/L and NOEC of ≥0.0079 mg/L have been determined for the effects of the test substance on growth rate of Scenedesmus subspicatus.

Description of key information

Toxicity to aquatic algae: 72-hour EC₅₀ >0.0079 mg/l and NOEC ≥0.0079 mg/l, effects on growth rate of Scenedesmus subspicatus (geometric mean measured concentrations).

Additional information

Two toxicity to freshwater algae studies are available with the registered substance, tetramethylsilane (TMS, CAS 75-76-3; EC No. 200-899-1).

In the first study, a 72-hour EC₅₀ value of >78 mg/l and a NOEC value of ≥78 mg/l (arithmetic mean measured concentration) (highest concentration tested) were reported for the effects of tetramethylsilane on growth rate of Scenedesmus subspicatus (Hüls, 1998), conducted in accordance with OECD TG 201 and in compliance with GLP.

 

The substance is highly volatile therefore the study was conducted in headspace containers sealed with a septum in an attempt to minimise volatilisation of tetramethylsilane during the 72-hour exposure.

The test substance solution was made by preparing an initial concentration of approximately 2 ml of test substance in 1 litre of water (equivalent to a concentration of approximately 1300 mg/l), followed by agitation and draining of 500 ml of the solution. The measured concentrations indicate that test substance concentrations were stable over the course of the test. However, the study report contains no details on the analytical method and, as such, the reliability of the measurements cannot be assessed. The water solubility of the substance is estimated to be 19.6 mg/l and data from the fish and algal studies (Springborn Smithers 2010) indicate that the maximum achievable concentrations under ecotoxicity testing conditions may be much lower. Therefore, the measured concentrations were well above the expected limit of water solubility. As no effects were observed and the measured concentrations of test substance were above the limit of solubility of the substance, the EC₅₀ and NOEC values should therefore be reported as not toxic at the limit of solubility (EC₅₀ and NOEC >LoS).

 

In a second study, a 72-hour EC₅₀ value of >0.0079 mg/l and NOEC value of ≥0.0079 mg/l were reported for the effects of the test substance on growth rate of Scenedesmus subspicatus based on geometric mean measured concentrations (Springborn Smithers, 2010). The test was conducted in accordance with OECD TG 201 and in compliance with GLP. The test substance solution was prepared as a WAF from a saturated solution (2.0 mL/L, equivalent to 1300 mg a.i./L) and included overnight stirring. The measured concentration of TMS at test initiation was 0.078 mg a.i./L. Test vessels were secured with rubber septa and crimp-top caps to minimise volatilisation of the test substance. Despite this, losses of test substance occurred throughout the duration of the study so that the measured concentration at 72 hours was 0.001 mg a.i./l.

 

Because both studies attempted to minimise losses through volatilisation and both studies reported no effects at the highest concentrations tested, the second study (Springborn Smithers, 2010) has been selected as key.

This is because the analytical measurements are thought to be more reliable as they do not exceed the expected water solubility of the test substance and the analytical methods are well described in the study report.

Refer to IUCLID Section 6 endpoint summary, Section 7.0 of CSR, for further discussion of the approach to chemical safety assessment for this registered substance.