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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-01 to 2013-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.1, 6.3, 13, 25 and 50 mg/l.
- Sampling method: Prior to the start of the definitive exposure a pretest analysis was conducted. During the pretest, samples were removed from two replicates of the low, middle and high treatment levels and control and analysed for test substance concentration. Results of the pretest analysis were used to judge whether sufficient quantities of test substance were being delivered to the test vessels, and whether the concentrations were being maintained consistently in order to initiate the definitive exposure. During the in-life phase of the definitive study, water samples were removed from two replicates of each treatment level and control (alternating between replicates A, B, C or D), then analysed for the test substance on test days 0, 7, 14 and 21. Samples were collected from the approximate midpoint of the test vessel by pipet. Three quality control (QC) samples 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 50-mL Glenco~ gas-tight syringe in conjunction with a Harvard Apparatus Syringe Pump was calibrated to deliver 0.0161 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 0.387 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 (50 mg/l) and was subsequently proportionately diluted (50%) to produce the remaining nominal test concentrations 25, 13, 6.3 and 3.1 mg/l.
- Controls: Dilution water without test substance.
- 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/clone:
- Source: laboratory cultures maintained at Smithers Viscient.
- Age of parental stock (mean and range, SD): <24 h
- Feeding during test
- Food type: Algal suspension (Ankistrodesmus falcatus) and a yeast, cereal leaves and digested flaked fish food (YCT) suspension.
- Amount: 3.0 ml algal suspension, 1.0 ml yeast, cereal leaves and digested flaked fish food (YCT) suspension
- Frequency: three times daily

ACCLIMATION
- Acclimation period: Culture conditions same as test conditions
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Algal suspension (Ankistrodesmus falcatus) and a yeast, cereal leaves and digested flaked fish food (YCT) suspension. Various amounts depending on age.
- Feeding frequency: Various depending on age.
- Health during acclimation (any mortality observed): No mortality

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The daphnids used for this test were < 24 hours old, were not from the first brood of the culture daphnids and were not used in any portion of a previous test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

160-180 as CaCO3

Test temperature:

19-21 °C

pH:

8-8.7

Dissolved oxygen:

6.1-10 mg/l

Nominal and measured concentrations:

Nominal: control, 3.1, 6.3, 13, 25 and 50 mg/l.
Measured: control, 0.58, 1.3, 4.3, 6.9 and 15 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 1600 ml 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 and to prevent loss of daphnids.
- Type (delete if not applicable): open / closed: not reported
- Material, size, headspace, fill volume: Glass, 1.6 litre, 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 with a 50% dilution factor.
- Renewal rate of test solution (frequency/flow rate): 10 test vessel volumes per day. Based on analytical recoveries at initiation, the turnover rate was changed to optimise the mixing time allowed for each cycle by decreasing the turnover rate on day 2 to nine test vessel volumes per day. This provided a 90% solution replacement rate of approximately 6 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- 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: 0.53 mg/l
- Alkalinity: 86-92 mg/l
- Conductivity: 660 to 740 microsiemens per centimeter (µS/cm).
- Culture medium different from test medium: no
- Intervals of water quality measurement: The dissolved oxygen concentrations, pH and temperature were measured and recorded in each
test vessel at exposure initiation and weekly thereafter until exposure termination (day 21). In addition, the pH, dissolved oxygen concentration and temperature were measured daily in alternating vessels of each test concentration and the controls. The temperature was also continuously monitored in replicate C of the 13 mg/l nominal treatment level throughout the study using a VWR minimum/maximum thermometer. Total hardness, alkalinity and conductivity were monitored at exposure initiation and weekly thereafter in alternating replicates of the highest treatment level and the control during the exposure. Total hardness and alkalinity of the test solutions were determined according to APHA et al. (1995).


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: 66 to 72 footcandles at the surface of the culture solution, provided by fluorescent bulbs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of immobilized adult daphnids and observations of abnormal behavior were recorded daily.
Observations brood release in any vessel were made (day 8) and numbers of offspring were determined three a week throughout the
remainder of the test.
At exposure termination (day 21), the total body length of each surviving adult female daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the shell spine using an Olympus SZ-STS dissecting scope in combination with a calibrated ocular.

VEHICLE CONTROL PERFORMED: yes/no: n/a

RANGE-FINDING STUDY
- Test concentrations:0.63, 1.3, 2.5, 5.0 and 10 mg/L, a control and a solvent (dimethyl)formamide. (CAS No. 68-12-2) control.
- Results used to determine the conditions for the definitive study: Following 19 days of exposure, 93 and 95% survival was observed in the control and solvent control, respectively. Survival of 90, 93, 90, 98 and 85% was observed in the 0.63, 1.3, 2.5, 5.0 and 10 mg/L treatment levels respectively.
Reproduction among daphnids exposed to the 0.63, 1.3, 2.5, 5.0 and 10 mg/L treatment levels averaged 162, 180, 166, 174 and 167 offspring per female, respectively. During same period, reproduction among daphnids exposed to the control and solvent control both averaged 143 and 153 offspring per female, respectively.
Total length of daphnids exposed to 0.63, 1.3, 2.5, 5.0 and 10 mg/L treatment levels averaged 4.68, 4.68, 4.68, 4.67 and 4.72 mm, respectively. Total length of control and solvent control daphnids averaged 4.63 and 4.66 mm, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction and growth

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival

Remarks on result:

other: lower 95% confidence interval of 4.4 mg/l. An upper 95% confidence interval could not be determined

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 1.7-4.9

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

7.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: length

Remarks on result:

other: 95% CL 2.1-8.7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 2.4 mg/l

Reported statistics and error estimates:

Shapiro-Wilks Test (U.S. EPA 2002) and Bartlett's Test (U.S. EPA 2002). CETIS version 1.8 (Ives 2013) was used to calculate the ECx values and 95% CI.

Validity criteria fulfilled:

yes

Conclusions:

A 21-d EC10 of 2.3 mg/l has been determined for the effects of the test substance on reproduction of Daphnia magna. A NOEC of 6.9 mg/l has been determined for the effects of the test substance on reproduction and growth of Daphnia magna. Although a flow-through test was performed, due to rapid hydrolysis of the test substance and the pH of the test solution being greater than 8, it is likely that the test organisms were exposed primarily to the hydrolysis products of the test substance 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate and methanol.

Overall reproduction and growth in the control met the acceptance criteria; however, control survival did not meet the acceptance criteria established by OECD guidelines. Indeed, after 21 days of exposure, survival among the control organisms averaged 75%. After extensive investigations, the cause of control mortality could not be determined, no apparent source was causing negative impact on the exposure and microscopic observations on culture daphnids did not show any physical abnormalities. Therefore, the test was not deemed to be invalidated. However, these unexplained results among the controls survival makes it more difficult to confidently identify the lowest effect concentration, and hence the value of NOEC. The reviewers consider that as EC10 value is derived based on the interpretation of the dose response across the whole concentration range it is therefore less significantly affected.

Description of key information

Long-term toxicity to invertebrates: 21-d EC₁₀ 2.3 mg/l, NOEC 6.9 mg/l (arithmetic mean measured concentration) (OECD 211). The EC₁₀ is equivalent to 1.9 mg/l when expressed in terms of the hydrolysis product, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.9 mg/L

Additional information

A 21-d EC₁₀ value of 2.3 mg/l (arithmetic mean measured concentration) has been determined for the effects of the test substance on reproduction of Daphnia magna. A NOEC of 6.9 mg/l (arithmetic mean measured concentration) has been determined for the effects of the test substance on reproduction and growth of Daphnia magna.

Although a flow-through test was performed, 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 - 8.7, it is likely that the test organisms were exposed primarily (75-87%) to the hydrolysis products of the test substance 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate and methanol.

Overall reproduction and growth in the control met the acceptance criteria; however, control survival did not meet the acceptance criteria established by OECD guidelines. Indeed, after 21 days of exposure, survival among the control organisms averaged 75%. After extensive investigations, the cause of control mortality could not be determined, no apparent source was causing negative impact on the exposure and microscopic observations on culture daphnids did not show any physical abnormalities. Therefore, the test was not deemed to be invalidated. However, these unexplained results among the controls survival makes it more difficult to confidently identify the lowest effect concentration, and hence the value of NOEC. The reviewers consider that as EC10 value is derived based on the interpretation of the dose response across the whole concentration range it is therefore less significantly affected.

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) * 2.3 = 1.9 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 EC₁₀ value for effects on reproduction of Daphnia magna, based on nominal concentrations lies between 6.3 mg/l and 13 mg/l.

In terms of concentration of the hydrolysis product this is 232.31/274.37 * 6.3, and * 13 = EC₁₀ values lying between 5.3 mg/l to 11 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

The NOEC value for effects on reproduction and growth of Daphnia magna, based on nominal concentrations is 25 mg/l . In terms of concentration of the hydrolysis product this is 232.31/274.37 * 25 = NOEC value of 21 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.