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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-03-31 to 2008-04-03
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 (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test initiation and at 24 hours of exposure, one sample was removed from the test solution and each control and analysed for triethoxy(octyl)silane. Samples analysed on day 0 were removed from the test solutions in the volumetric flasks prior to inoculation with algae and filling the individual replicate test flasks. Samples analysed at 24 hours of exposure were removed from the approximate midpoint of the test vessel and composited. Since the 24-hour measured concentrations were below the limit of quantitation, no additional analytical samples were collected or analysed.

- Sample storage conditions before analysis: The extraction solvent, iso-octane, was immediately added to each analytical sample which stopped hydrolysis of the test substance.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 1.3 mg a.i./ml stock solution was prepared prior to test initiation by placing 0.0328 g of triethoxy(octyl)silane (0.0325 g as active ingredient) in a 25-ml volumetric flask and bringing it to volume with dimethylformamide (DMF, CAS No. 68-12-2). The resulting stock solution was observed to be clear and colourless with no visible undissolved test substance. Duplicate 0.13 mg a.i./l test solutions were prepared by bringing 0.10 ml of the 1.3 mg a.i./ml primary stock solution to a final volume of 1000 ml with AAP medium. Due to the known rapid hydrolysis of the test substance, the test solutions were prepared in individual volumetric flasks and mixed for approximately one minute.

- Controls: Dilution water control and Dilution water + solvent (DMF; 0.1 ml/l) control.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): The alga was 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.

- Stock culture: 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 ± 1 ºC and 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. 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 triethoxy(octyl)silane was taken from a stock culture that had been transferred to fresh medium three days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Conductivity: 80 μmhos/cm
Test temperature:
24ºC
pH:
7.2 - 7.3 at start of test

8.6 - 9.2 at end of test
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control) and 0.13 mg/l. The nominal concentration of 0.13 mg/l was based on the results of a preliminary study of the solubility of the substance.

At 0 hour, the measured concentration of triethoxy(octyl)silane in the test solution, 0.11 mg a.i./l, closely approximated the desired nominal concentration. The concentration of triethoxy(octyl)silane decreased to below detectable limits (<0.020 mg a.i./l) at 24 hours of exposure. The test substance is known to rapidly hydrolyse in water (e.g., at pH 7.0 the half-life time has been determined to between 0.3 and 0.6 hours at 25 ºC).
Details on test conditions:
TEST SYSTEM

- Test vessel: Replicate 250-ml flasks, three for the control and six for the treatment level and solvent control, were conditioned prior to use by rinsing with the appropriate test solution. One hundred milliliters of the appropriate test solution was then placed in each replicate flask. Each test flask was labelled with the test concentration, replicate, test species and study number. All test vessels were fitted with stainless steel caps which permitted gas exchange.

- Inoculation of test vessels: Each flask was provided with the required cell density of approximately 1.0 x 10E4 cells/ml.

- Culture medium: Algal Assay Procedure (AAP) medium (Miller et al., 1978) used to prepare the exposure solutions. The initial pH of this medium was adjusted, if necessary, to 7.5 ± 0.1 prior to use.

- Test conditions: The test was conducted in an environmental chamber designed to maintain the test conditions: a temperature of 23 ± 2 ºC, continuous light intensity of 4400 to 5900 lux (420 to 550 footcandles) and photosynthetically-active radiation (PAR) range of 60 to 120 μE/m2/s. An orbital shaker table provided a shaking rate of 100 ± 10 rpm. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers.

- Water quality: Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 72-hour exposure period. Test solution pH was measured with a Jenco Model 60 pH meter, and conductivity was measured with a Yellow Springs Instrument (YSI) Model No. 33 salinity-conductivity-temperature meter.

_ Selection of test concentrations: Based on the reported solubility of the test substance (< 0.13 mg/l) and in consultation with the Study Sponsor, nominal concentrations of 0 (control and solvent control) and 0.13 mg a.i./l were selected for the definitive limit test.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test substance hydrolysis products
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test substance hydrolysis products
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test substance hydrolysis products
Basis for effect:
biomass
Remarks:
(expressed as yield)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test substance hydrolysis products
Basis for effect:
biomass
Remarks:
(expressed as yield)
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Since this test was conducted as a 72-hour limit test (one concentration) and the concentration tested did not produce > 10% inhibition in yield, growth rate or biomass, the ECx values were empirically estimated to be greater than the nominal concentration tested.

Table 1. Results of analysis of test media

 

Nominal concentration (mg/l)

Measured concentration at start of test (mg/l)

Measured concentration after 24 hours (mg/l)

0 (Control)

<0.020

<0.020

0 (Solvent control)

<0.020

<0.020

0.13

0.11/0.11*

<0.020/<0.020*

*Analysis performed of media that did not contain algae

 

Table 2. Test results

 

Nominal concentration (mg/l)

Measured cell density at start of test (cells/ml)

Measured cell density after 24 hours (cells/ml)

Measured cell density after 48 hours (cells/ml)

Measured cell density after 72 hours (cells/ml)

Inhibition of growth rate (%)**

Inhibition of biomass (%)**

0 (Control)

10000

69200

380000

1900000

Not applic.

Not applic.

0 (Solvent control)

10000

63800

348000

1770000

-

-

0.13

10000

62900

400000

2020000

-2

-14**

**Compared with solvent control. A negative value indicates that growth was higher than in the Controls

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >0.13 mg/l and NOEC of ≥0.13 mg/l have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. The results are expressed relative to nominal test substance concentration because measured concentrations had declined to below the limit of quantification after 24 hours. The test substance is subject to hydrolysis and it is therefore likely that, under the static test conditions, the test organisms were exposed to the hydrolysis products of the substance.

Description of key information

Short-term toxicity to algae: 72-hour EC50 >0.13 mg/l and 72-hour NOEC ≥0.13 mg/l (nominal, highest concentration tested) (OECD TG 201), in terms of the substance as tested. The observations in this study are attributed to the exposure of test organisms to the hydrolysis products in the test system.

Key value for chemical safety assessment

Additional information

A 72-hour EC50 value of >0.13 mg/l and NOEC of ≥0.13 mg/l have been determined for the effects of the registered substance on growth rate of Pseudokirchneriella subcapitata. The results are expressed relative to nominal test substance concentration because measured concentrations had declined to below the limit of quantification after 24 hours. The test substance is subject to hydrolysis and it is therefore likely that, under the static test conditions, the test organisms were exposed to the hydrolysis products of the substance.

Supporting data are available for a structurally analogous read-across substance; triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435 -21 -3). A 72-hour EL50 of >100 mg/l and NOELR of 32 mg/l have been determined for the effects of triethoxy(2,4,4-trimethylpentyl)silane on growth rate of Pseudokirchneriella subcapitata.

The read-across to the registered substance is considered scientifically justified.