2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June - 13 November, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

slightly increased water temperature, method validation

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.3, 13, 25, 50, 100 mg/l
- Sampling method: Prior to the start of the definitive exposure one water sample was removed from Replicate A of the 6.3, 25 and 100 mg/L treatment levels and the control and analysed for test substance concentration. Results of this pretest analysis were used to judge whether sufficient quantities of test substance 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, one water sample (alternating between replicates A and B) from each treatment level and the control was collected, processed, then analysed for the test substance at 0 hour (exposure initiation), 24 hours and 48 hours (exposure termination). 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.
- Sample storage conditions before analysis: Not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior to exposure initiation, a 20-mL Glenco~ gas-tight syringe in conjunction with a Harvard Apparatus Syringe Pump was calibrated to deliver 0.00338 mL/cycle of the test substance, with a density of 1.200 g/cm3 (neat test substance) into the diluter system's chemical mixing chamber which also received 0.393 L of dilution water per cycle. The mixing chamber was positioned over a water-driven magnetic stirrer and was partially submerged within an ultrasonic water bath which continuously mixed the contents of the mixing chamber. The solution contained in the mixing chamber constituted the highest nominal treatment level (100 mg/l) and was subsequently proportionately diluted (50%) to produce the remaining nominal test concentrations 50, 25, 13 and 6.3 mg/l.
- Controls: : Dilution water with no test substance.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None in definitive test.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): An oily sheen was observed on the surface of the solution in the mixing chamber. No undissolved test substance (e.g., precipitate or oily sheen) was observed in the exposure solutions, and all exposure solutions were observed to be clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: Laboratory cultures maintained at Smithers Viscient.
- Age at study initiation (mean and range, SD): <24 hours
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): not reported
- Valve height at study initiation, for shell deposition study (mean and range, SD): not reported
- Peripheral shell growth removed prior to test initiation: not reported
- Method of breeding: not reported
- Feeding during test: none
- Food type: n/a
- Amount: n/a
- Frequency: n/a

ACCLIMATION
- Acclimation period: Two weeks
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: unicellular green algae (4 x 107 cells/mL) in addition to yeast~ cereal leaves and flaked fish food (YCT) daily.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No mortality

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

170 mg/l

Test temperature:

20-22 °C

pH:

8.2-8.3

Dissolved oxygen:

6.9-8.4 mg/l

Nominal and measured concentrations:

Nominal: control, 6.3, 13, 25, 50 and 100 mg/l
Measured: control, 1.1, 2.6, 4.9, 12 and 26 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 1600-mL square glass battery jars with two 2-cm holes drilled in the sides, 15 cm from the bottom, which were covered with Nitex® 40-mesh screen for drainage.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: test solution volume was maintained at 1400 ml
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): intermittent-flow proportional diluter
- Renewal rate of test solution (frequency/flow rate): 10 solution volume replacements per day: 90% test solution replacement rate of approximately 6 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water was prepared by fortifying well water based on the formula for hard water (US EPA 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
- Total organic carbon: 0.29 mg/l
- Alkalinity: as CaC03: 88-90 mg/l
- Conductivity: 660-680 µs/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen concentration, temperature and pH measured once daily in both replicate vessels of each treatment level and control.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light, 8-hour dark
- Light intensity: light intensity range of 30 to 39 footcandles (320 to 420 lux) using fluorescent bulbs

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility at exposure initiation, 24 and 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.9
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0.63, 1.3,2.5,5.0 and 10 mg/l
- Results used to determine the conditions for the definitive study: Following 48 hours of exposure, 5% immobilization was observed among daphnids exposed to the 0.63 mg/L treatment level. No immobilization or adverse effects were observed among daphnids exposed to the remaining treatment levels tested (1.3, 5.0 and 10 mg/L) or the controls.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 26 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 26 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: See table 1
- Observations on body length and weight: not reported
- Other biological observations: none reported
- Mortality of control: no mortality
- Other adverse effects control: See table 1
- Abnormal responses: See table 1
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The EC50 value was empirically estimated to be greater than the highest mean measured concentration tested.

The mean measured concentrations tested and the corresponding immobilization data derived from the definitive toxicity test were used to estimate the 24- and 48-hour median effective concentrations (EC₅₀). The EC₅₀ is defined as the concentration of the test substance in dilution water which produces 50% immobilization of the test organism population at the stated time interval. If at least one test concentration caused immobilization of: 2: 50% of the test population, then a computer program ( Ives. 20 II ) was used to calculate the EC₅₀ values and 95% confidence intervals. During this study, no concentration tested resulted in approx. 50%

immobilization; therefore, the EC₅₀ value was empirically estimated to be greater than the highest mean measured concentration tested.

The No-Observed-Effect Concentration (NOEC) during the 48-hour exposure period was also determined. The NOEC is defined as the highest concentration tested at and below which there were no toxicant-related immobilization or physical and behavioral abnormalities (e.g., lethargy), with respect to the control organisms.

Evaluation of Test Conditions

The pH, dissolved oxygen, temperature measured were unaffected by the concentrations of the substance tested and remained within acceptable levels for the survival and growth of daphnids. The dissolved oxygen concentrations in all replicate vessels of each treatment and the control ranged from 6.9 to 8.4 mg/L and were all safely above the minimum criteria of 3 mg/L (33% saturation at 20°C). Measurements of pH ranged from 8.2 to 8.3 in all replicate vessels of each treatment and the control. Temperature measured in all replicate vessels of each treatment and control ranged from 20 to 21 °C. Continuous temperature monitoring established that the exposure solution temperature ranged from 20 to 22°C during the exposure period.

Analytical Results

The diluter system which prepared and delivered the test solutions to the exposure aquaria functioned properly during the pretest period and throughout the definitive exposure. An oily sheen was observed on the surface of the solution in the mixing chamber. No undissolved test substance (e.g., precipitate or oily sheen) was observed in the exposure solutions, and all exposure solutions were observed to be clear and colorless.

Measured concentrations during the exposure period resulted in mean measured concentrations which ranged from 18 to 26% of the nominal levels and defined the treatment levels tested as 1.1, 2.6, 4.9,12 and 26 mg/L. Since this test substance is known to rapidly hydrolyse and its stability is highly pH dependent, the diluter system was calibrated to the maximum turnover rate allowable in an effort to maintain test solution concentration. Since a visible oily sheen was observed on the surface of the mixing chamber, the high test concentration is considered to approximate the functional limit of solubility under testing conditions. Therefore, due to the hydrolysis and solubility, these concentrations are considered to be the maximum concentrations attainable under testing conditions.

Analysis of QC samples associated with the test samples resulted in measured concentrations that were consistent with the predetermined recovery range and ranged from 90.5 to 107% (N = 9) of the nominal fortified levels (3.00, 50.0 and 100 mg/L). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis

of the exposure solutions.

Biological Results

Following 48 hours of exposure, 5 and 30% immobilization was observed among daphnids exposed to the 2.6 and 26 mg/L treatment levels, respectively. All surviving daphnids exposed to the 26 mg/L treatment level were observed to be lethargic. Immobilization of 5% was observed among daphnids in the control group. No immobilization or adverse effects were observed among daphnids exposed to the remaining treatment levels tested (1.1, 4.9 and 12 mg/L).

Since no concentration tested resulted in >= 50% immobilization, the 48-hour EC₅₀ value was empirically estimated to be > 26 mg/L, the highest mean measured concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be 12 mg/L. Additional testing to further define the EC₅₀ value was not performed since the highest concentration tested is believed to approximate the functional limit of solubility for this test substance under testing conditions.

Validity criteria fulfilled:

yes

Conclusions:

A 48-hour EC50 value of >26 mg/l and a NOEC value of 12 mg/l has been determined for the effects of 3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate on mobility of Daphnia magna, based on arithmetic mean measured concentrations.

Executive summary:

The objective of the study was to determine the 48-hour median effective concentration (EC₅₀) by conducting an acute immobilization test of 2,4 -hexadienoic acid, 3-(trimethoxysilyl) propyl ester on Daphnia magna. Results were based on mean measured concentrations. The results of a 48-hour preliminary range finding exposure determined the test concentrations. Nominal test concentrations were 6.3, 13, 25, 50 and 100 mg/L, a dilution water control was tested concurrently. Each treatment level consisted of two replicate test vessels containing 10 daphnids for a total of 20 daphnia per treatment level. The test was conducted using an exposure system consisting of an intermittent-flow proportional diluter. Since this test substance is known to rapidly hydrolyze and its stability is highly pH dependent, the diluter system was calibrated to the maximum turnover rate allowable in an effort to maintain test solution concentrations. During the study, one water sample from each treatment level and the control was collected, extracted then analyzed for the test substance at 0 hour, 24 hours and 48 hours using high performance liquid chromatography with ultraviolet detection (HPLC/UV). Measured concentrations during the exposure period resulted in mean measured concentrations which ranged from 18 to 26% of the nominal levels and defined the treatment levels as 1.1, 2.6, 4.9, 12 and 26 mg/L. Due to the hydrolysis and solubility of the test substance, these test concentrations are considered to be the maximum concentrations attainable under the testing conditions. Environmental conditions during the test remained within acceptable limits. Irradiation conditions consisted of 16 hours light at an intensity of 30 to 39 footcandles and 8 hours dark. Measurements of dissolved oxygen, temperature and pH of the test solutions during the exposure ranged from 6.9 to 8.4 mg/L, 20 to 21 degrees C and 8.2 to 8.3. Following 48 hours of exposure, 5 and 30% immobilization was observed among daphnids exposed to the 2.6 and 26 mg/L treatment levels, respectively. All surviving daphnids exposed to the 26 mg/L treatment level were observed to be lethargic. Immobilization of 5% was observed among daphnids in the control group. No immobilization or adverse effects were observed among daphnids exposed to the remaining treatment levels tested. Since no concentration tested resulted in > 50% immobilization, the 48-hour EC₅₀ value was empirically estimated to be >26 mg/L, the highest mean measured concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be 12 mg/L.

Description of key information

Short-term toxicity to invertebrates: 48-h EC₅₀ >26 mg/l (highest concentration tested), NOEC 12 mg/l (arithmetic mean measured concentration) (OECD 202). The EC₅₀ is equivalent to >22 mg/l when expressed in terms of the hydrolysis product, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

Additional information

A 48 hour EC₅₀ value of >26 mg/l (highest concentration tested) and a NOEC value of 12 mg/l (arithmetic mean measured concentrations) have been determined for the effects of 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate on mobility of Daphnia magna.

In view of the flow-through exposure regime, the renewal rate of test solution, rapid hydrolysis of the test substance and pH of around 8, it is likely that the test organisms were exposed primarily (75 -87%) to the hydrolysis products of the test substance.

The results may be expressed in terms of concentration of the hydrolysis product, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate, by applying a molecular weight correction: (MW of silanol = 232.31 / MW of parent = 274.37) * >26 = >22 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

The measured concentration may be classed as a conservative result because analysis of exposure concentrations was specific to the parent substance. It may be appropriate to present the results in terms of the nominal concentration for assessment of the hydrolysis product:

The 48 h EC₅₀ value for mobility of Daphnia magna, based on nominal concentrations, is >100 mg/l.

In terms of concentration of the hydrolysis product this is 232.31/274.37 * >100 = >85 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.