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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:
key study
Study period:
2009-09-29 to 2009-10-02
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:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Prior to test solution preparation, a dosing stock solution was prepared by mixing 1.52 mL of Trichloro(phenyl)silane (corresponding to 2 g) with 2 mL of tetrahydrofuran (THF) using Hamilton syringes. The solution was shaken by hand. A 100 mg test item/L stock solution
was prepared prior to test initiation by adding 176 μL of the dosing stock solution to 800 mL algal medium using a Hamilton syringe. Prior to addition of the dosing stock solution, the measurement flask containing the algal medium was placed on a magnetic stirrer. The spiked solution was stirred continuously over night. The pH of the solution was then adjusted to 7.0 with 0.1 N sodium hydroxide (NaOH). Thereafter, the stock solution was further diluted to a final volume of 1000 mL with algal medium, resulting in a solvent (THF) concentration of 0.10 mL/L. Nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were prepared by dilution of the stock solution and addition of THF to a final concentration of 0.1 mL/L. All resulting test solutions were shaken by hand for 30 seconds and observed to be clear and colorless, with no visible undissolved test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): The alga was originally obtained from Albrecht-v.-Haller-Institut, Göttingen, Germany, and was maintained in stock culture at Springborn Smithers Laboratories (Europe).

- Culture medium: The culture medium used was a synthetic algal assay growth medium (OECD, 2006), prepared by adding appropriate amounts of nutrient stock solutions to sterile, deionized water. Representative samples of the dilution water source used in the preparation of the culture medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals conducted at Bachema, Schlieren, and Interlabor Belp AG, Belp, Switzerland. None of these compounds have been detected at concentrations that are considered toxic to algae in any of the water samples analyzed in agreement with ASTM guidelines.

- Preparation of starter culture: Four days prior to test start, the culture was maintained within the following conditions: a shaking rate of 120 rpm, a temperature of 23ºC and continuous illumination of 7091 to 7123 lux. Lighting was supplied by fluorescent bulbs. Culture flasks were shaken
continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test 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:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.4 to 23.7ºC
pH:
7.30 to 7.65 at test initiation

6.08 to 9.86 at end of test
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 0 (Control), 0 (Solvent control), 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L
Details on test conditions:
TEST SYSTEM

Test vessels: Three sterile 250-mL Erlenmeyer flasks (A to C) for the controls and the treatment levels were prepared. An aliquot of 100 mL of the appropriate test solution was placed in each replicate flask. All test vessels were fitted with stainless steel caps which permitted gas exchange.

- Inoculum: An aliquot of 0.25 mL of the preculture of Pseudokirchneriella subcapitata, at a density of 394 x 104 cells/mL, was then inoculated aseptically in each 250 mL volumetric flask. The resulting slight dilution from the inoculum was considered to be negligible. These volumes of inocula provided the required cell density of 1.0 x 104 cells/mL.

- Algal growth medium: The algal medium used to prepare the exposure solutions was the same as the culture medium. The medium was equilibrated to test temperature. The pH of the medium was 8.03.

- Test conditions: The test was conducted in a temperature-controlled room to maintain the test conditions specified in the study plan: a temperature of 24 ± 2ºC and continuous light intensity of 4400 to 8800 lux. An orbital shaker table provided a shaking rate of 100 rpm. Test flasks were randomly placed on the shaker table at test initiation. Following each observation interval the test flasks were assigned new random positions.

- Algal growth measurement: At each subsequent 24-hour interval, cell counts were conducted on each replicate vessel of the treatment levels and the control using a hemocytometer (Neubauer Improved) and a Leica DMLS microscope. One sample was removed from each flask for counting. One or more hemocytometer field(s), each 0.1 cm x 0.1 cm in size and 0.01 cm deep, containing 0.0001 mL of culture, were examined for each sample until at least 400 cells or a total of four fields were counted. Observations of the health of the algal cells were also made at each 24- hour interval.

- Monitoring culture conditions: Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers (Gerhardt, 450625 R05) were recorded daily. Light intensity was measured with a Pancontrol LX 1308 at hour 0 and at each 24-hour interval during the exposure period. The pH of the test solutions and control was measured at test initiation and at the termination of the 72-hour exposure period. Test solution pH was measured using a Metrohm 691 pH-meter.
Reference substance (positive control):
no
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.:
>= 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:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Definitive EC50 values could not be determined because they were above the highest treatment level.

ANOVA and William’s Test were used to determine the NOEC and LOEC values

Table 1. Test results

 Nominal test concentration (mg/L)  Initial cell density (cells/mL)  Mean cell density after 24 hours (cell/mL)   Mean cell density after 48 hours (cell/mL)  Mean cell density after 72 hours (cell/mL)  Perecntage inhibition in cell density*  Percentage inhibition in growth rate* Percentage inhibition in biomass*
 0 (Control)  10000  48000  247000  1070000  9.0  1.4  9.0
 0 (Solvent control)  10000  59000  228000  1180000  -  -  -
 6.25  10000  65000  279000  1240000  -5.3  -1.9  -5.4
 12.5  10000  49000  254000  1140000  2.7  -0.03  2.7
 25.0  10000  43000  240000  1050000  10.5  1.9  10.6
 50.0  10000 33000   203000  1030000  12.6  2.0  12.7
 100  10000  45000  248000  1030000  12.1  2.2  12.2

*Relative to solvent control. A negative (-) value indicates that growth was higher than in the solvent control.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >100 mg/L and a NOEC of ≥100 mg/L have been determined for the effects of the test substance on growth rate and biomass of Pseudokirchnerella subcapitata. 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification for grouping of substances provided in IUCLID Section 13.
Reason / purpose:
read-across source
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.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Description of key information

72-h EC50 > 100 mg/l and NOEC ≥ 100 mg/l; biomass and growth rate of Pseudokirchnerella subcapitata (OECD 201), RL1

Key value for chemical safety assessment

Additional information

Measured data was not available for trimethoxy(2,4,4-trimethylpentyl)silane (CAS 034396-03-7) data was therefore read across from the comparable substance, trichloro(phenyl)silane (CAS 98-13-5).

Both hydrolyse very rapidly to similar hydrolysis products, (2,4,4-trimethylpentyl)silanetriol and phenylsilanetriol, respectively. The other hydrolysis product is hydrogen chloride; the properties of this substances is well characterised. Effects of hydrogen chloride on aquatic organisms are limited to those that result from changes to pH in unbuffered media.

The influence of the test item on the biomass and growth rate of Pseudokirchneriella subcapitata was investigated in a 21-d static test according to the OECD 201 and GLP (Springborn Smithers, 2009).

A 72-hour EC50 value of >100 mg/l and a NOEC of ≥100 mg/l have been determined for the effects of the test substance on growth rate and biomass of Pseudokirchnerella subcapitata. It is likely that the test organisms were primarily exposed to the hydrolysis products of the substance.