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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-04-07 to 2008-04-11
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Sampling method: Prior to the start of the definitive exposure, water samples were removed from the treatment level and the controls and analysed for triethoxy(octyl)silane concentration. Results of these pretest analyses were used to judge whether sufficient quantities of triethoxy(octyl)silane were being delivered to the test vessels and whether the appropriate test concentrations were being maintained in order to initiate the definitive exposure.

During the in-life phase of the definitive study, two water samples from the treatment level and the controls were collected and analysed for triethoxy(octyl)silane at 0 hour (test initiation) and 48 hours (test termination). The replicates used for sampling were alternated between intervals. Samples were collected from the approximate midpoint of the test vessel by pipet.

Three quality control (QC) samples each were prepared at each sampling interval and remained with the appropriate set of exposure solution samples throughout the analytical process. These QC samples were prepared in dilution water at nominal concentrations similar to the exposure concentration range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Details on test solutions:

A 1.3 mg a.i./ml stock solution was prepared by placing approximately 0.0325 g of triethoxy(octyl)silane in a 25-ml volumetric flask and bringing it to volume with DMF (CAS No. 68-12-2). The resulting stock solution was observed to be clear and colourless with no visible undissolved test substance.

A 0.52 mL/ml solvent stock solution was prepared by bringing 52 ml of DMF to a final volume of 100 ml with deionized water. The resulting stock solution was observed to be clear and colourless.

Prior to test initiation, a Glenco® 20-ml gas-tight syringe in conjunction with a Harvard Syringe Pump was calibrated to deliver 0.0386 ml/cycle of the 1.3 mg a.i./ml triethoxy(octyl)silane stock solution into the diluter system's chemical mixing chamber which also received 0.386 litres of dilution water per cycle. The mixing chamber was positioned over a water-driven magnetic stirrer and was partially submerged within a water bath which continuously mixed the contents of the mixing chamber. The constant mixing aided in the solubilization of the test substance in the dilution water. The concentration of triethoxy(octyl)silane in the solution contained within the mixing chamber was equivalent to that of the nominal test concentration (0.13 mg a.i./l).

The solvent control solution was prepared utilizing a similar system, calibrated to deliver 0.0386 ml/cycle of the solvent control stock solution (0.52 ml/ml) to 0.20 litres of dilution water per cycle which was subsequently delivered to the solvent control vessels. The concentration of DMF in the solvent control vessels was equivalent to the concentration of solvent present in the treatment level solution (0.10 ml/l).
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers. Juvenile daphnids (< 24 hours old) were obtained from the laboratory cultures by removing all immature daphnids from the culture vessels 24 hours prior to test
initiation, thereby isolating all mature gravid adult daphnids in the culture. Daphnids produced by these adult organisms were removed from the cultures and used as test organisms.

- Culture water: The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. This water had a total hardness and total alkalinity as calcium carbonate (CaCO3) of 160 mg/l and 100 mg/l, respectively, a pH range of 7.8 to 8.1, a dissolved oxygen concentration range of 7.1 to 9.0 mg/l, a temperature range of 19 to 21°C and a specific conductance of 500 micromhos per centimeter (μmhos/cm). All water quality ranges presented here were measured during the two weeks prior to testing.

- Culture vessels: Daphnids were cultured in 1.0-litre glass vessels containing 0.80 litres of water.

- Lighting: The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light intensity of 63 to 75 footcandles (680 to 810 lux) at the surface of the culture solutions was provided by fluorescent bulbs.

- Feeding: Daphnids were fed a unicellular green algae (Ankistrodesmus falcatus, 4 x 10E7 cells/ml) at a rate of 1.0 to 2.0 ml per vessel daily depending on the age of the adult organisms in the culture vessel and 0.5 ml of a combination of yeast, cereal leaves and flaked fish food (YCT) daily. Daphnids were not fed during the exposure.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
160-170 mg/l as CaCO3
Test temperature:
Dissolved oxygen:
6.9-9.3 mg/l
Not applicable
Nominal and measured concentrations:
Nominal concentrations: ) (Control), 0 (Solvent control) and 0.13 mg/l.

The mean measured concentration in the 0.13 mg/l nominal treatment ranged from 36 to 40% of nominal and was defined as 0.049 mg a.i./l.

The results of the test are interpreted with reference to the mean measured concentration.
Details on test conditions:
- Exposure system: The diluter system was calibrated prior to test initiation by measuring delivery volumes of test substance and dilution water. The function of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check of the system’s function was performed twice daily. In addition, analysis of the exposure solutions for triethoxy(octyl)silane concentration was used to verify proper operation of the diluter system. The exposure system was in proper operation for two days prior to test initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels.

Four glass tubes with an approximate length of 5 centimeters (cm) and an inside diameter of 2 millimeter (mm) were inserted through silicone stoppers in the flow-splitting chambers, entering individual glass delivery tubes. Delivery tubes were positioned to deliver solution to each replicate. At the end of each delivery tube, 1 mm (I.D.) glass capillary tubes were attached. These glass capillary tubes served to restrict the flow of the test solution and minimized potentially stressful turbulence in the exposure vessels. A total of 50 ml (i.e., one delivery tube per vessel) of test solution was delivered per cycle.

Each test vessel (1600-mL square glass battery jars) had two 2-cm holes drilled in the sides, 15 cm from the bottom, which were covered with Nitex® 40-mesh screen for drainage. The total test solution volume was maintained at 1400 ml. At least 2 ml of test solution was provided for each daphnid. Each replicate vessel received an average of 6.0 solution volume replacements per day in order to provide a 90% test solution volume replacement rate of approximately 9 hours. Exposure vessels were labeled to identify the nominal test substance concentration and designated replicate.

- Test Initiation: Daphnids were added impartially to intermediate beakers by adding no more than two daphnids at a time to each vessel until all beakers contained two daphnids. This process was repeated until each of the intermediate beakers contained 10 daphnids. The test was initiated when 10 daphnids (< 24 hours old) were introduced to each replicate exposure vessel. A total of 40 organisms were exposed to the treatment level and each control solution.

- Observations: The number of immobilized daphnids observed in each replicate test vessel was recorded at test initiation, 24 and 48 hours of exposure. Daphnids were determined immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessel. Biological observations (e.g., abnormal behaviour or appearance of the test organisms) and observations of the physical characteristics of the test solutions (e.g., precipitate, film on the surface of the test solution) were also made and recorded at 0, 24 and 48 hours of exposure.

- Water quality: The pH, dissolved oxygen concentration and temperature were measured once daily in each replicate vessel of each treatment level and the control throughout the exposure period. Temperature was also continuously monitored in replicate B of the 0.13 mg a.i./l nominal treatment throughout the study.

- Preliminary test: A preliminary range-finding exposure was conducted at nominal triethoxy(octyl)silane concentrations of 6.3, 13, 25, 50 and 100 mg a.i./l, and a control. The test substance was delivered directly into the diluter’s mixing chamber, submerged within the water bath of an ultrasonicator. The solution within the mixing chamber was the same as the highest test concentration (i.e., 100 mg a.i./l). Due to the presence of large amounts of visible undissolved material, it was determined during this exposure that the 100 mg a.i./l concentration greatly exceeded the functional limit of this test substance under testing conditions. Toxicity observed in this exposure (i.e., 100% mortality at all test concentrations) was likely due to the presence of a large amount of undissolved material in the test vessels and the fact that ultrasonication may have emulsified the test substance. Therefore, no ultrasonication was provided during the definitive testing.

A second preliminary exposure was conducted under flow-through conditions at nominal concentrations of 0.19, 0.38, 0.75, 1.5 and 3.0 mg a.i./l. Test material was delivered directly into the diluter’s mixing chamber, resulting in a high test concentration of 3.0 mg a.i./l. No ultrasonication was provided during mixing. It was determined during this exposure that this concentration also exceeded the functional limit of solubility. Toxicity that was observed in this preliminary exposure was likely due to the presence of undissolved material in the test vessels.
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
> 0.049 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
>= 0.049 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Immobilisation of control: 0

- Other adverse effects control: None
Reported statistics and error estimates:
The EC50 value was empirically estimated to be greater than the test concentration.

The NOEC was empirically determined and is defined as the highest concentration tested at which there were no toxicant-related immobilization or physical and behavioural abnormalities (e.g., lethargy), with respect to the control organisms.

Table 1. Results of analysis of test media




(mg a.i./l)

Mean measured concentration (mg/l)

Mean measured concentration as percentage of nominal

0 (Control)

Not applicable

Not applicable

0 (Solvent control)

Not applicable

Not applicable





Table 2. Test results


Mean measured


(mg a.i./l)

Percentage immobilisation at end of test

0 (Control)


0 (Solvent control)





Validity criteria fulfilled:
A 48-hour EC50 value of >0.049 mg/l and a NOEC of ≥0.049 mg/l have been determined for the effects of the test substance on mobility of Daphnia magna under flow-through test conditions. The result is expressed as mean measured concentration of the test substance over the test period.

Description of key information

Short-term toxicity to invertebrates: 48-hour EC50 >0.049 mg/l (arithmetic mean measured, highest concentration tested) (OECD TG 202), in terms of the substance as tested. The observations in this study are attributed to the exposure of test organisms to the parent substance in the test system.

Key value for chemical safety assessment

Additional information

A 48-hour EC50 value of >0.049 mg/l has been determined for the effects of the registered substance on mobility of Daphnia magna under flow-through test conditions. The result is expressed as mean measured concentration of the test substance over the test period. The flow-through design of the test maximised exposure of the test organisms to the parent substance. However, analytical recoveries of test substance concentrations showed losses of parent test substance, therefore it is likely that the Daphnia were exposed to a mixture of the parent substance and its hydrolysis products.

Supporting data are available for a structurally analogous read-across substance; triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3, EC No. 252-558-1). A 48-hour EL50 of <100 mg/l has been determined for the effects of triethoxy(2,4,4-trimethylpentyl)silane on mobility of Daphnia magna. The data reflect exposure to a mixture of the parent substance and its hydrolysis products.