2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of the early life-stage exposure was conducted from 17 February to 20 March 2020 at Smithers, located in Wareham, Massachusetts. The final day of data collection was 21 March 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

Version 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

EPA, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM E: 1241-05 – Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes

Version / remarks:

Version 2013

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Prior to the start of the definitive exposure, samples from one replicate of each treatment level and the control solutions were collected and analysed for Vi4D4. In addition, a sample of the diluter stock solution was also analysed. Results of the pretest analyses were used to judge whether sufficient quantities of Vi4D4 were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure.

During the in-life phase of the definitive study, water samples were removed from a single replicate of each treatment and control, with the sampled replicate alternating at each successive sampling (replicate A, then B, then C, etc.). Samples were removed on day 0, 4 (hatch), 11, 17, 24, and 32. All exposure samples were removed from the approximate midpoint of each aquarium using a pipette. In addition, a sample of the stock solution was analysed at each sampling interval during the exposure period. Additionally, subsamples of the exposure solutions were also collected at each sampling interval and stored frozen as archive samples.

Water samples were extracted immediately after sampling and analysed.

Analysis of the stock solutions and test substance concentrations as well as visual observation of the diluter system indicated that the diluter test system was dosed properly throughout the exposure.

Three quality control (QC) samples were prepared at each sampling interval and stored and analysed with the set of study samples. The QC samples were prepared in dilution water at test substance concentrations similar to the treatment level range. Results of these analyses indicated the accuracy of the analytical method for measuring test substance concentration at each sampling period. The test substance concentration in the exposure solutions were tested by GC/MS and validated at the expected nominal concentration range prior to exposure initiation.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

The test substance was introduced into the exposure system for a minimum of 24 hours before exposure initiation in order to allow the test solution to reach an equilibrium concentration of test substance in the exposure system. The exposure did not start until the diluter and test substance delivery device had been observed to be properly functioning for at least 24 hours. The exposure began when the embryos had been impartially placed in the exposure aquaria and were terminated at 28 days post-hatch.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 5.0 mg/mL diluter stock solution was prepared prior to exposure initiation and as needed thereafter throughout the definitive exposure by adding, for example, 0.1271 g of Vi4D4 (0.1258 g as active ingredient) to a 25-mL volumetric flask and then bringing it to a total volume of 25 mL with dimethylformamide (DMF, CAS No. 68-12-2). The resulting stock solutions were observed to be clear and colorless with no visible undissolved test substance following mixing by inversions of the flask.

A 77 μL/mL solvent stock solution was prepared prior to exposure initiation and as needed throughout the definitive exposure thereafter by diluting 77 mL of DMF to 1000 mL with deionised water. The resultant stock solutions were observed to be clear and colourless following mixing by inversions of the flask.

The exposure was conducted using a system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 28 exposure aquaria. The exposure system provided five concentrations of the test substance, a control, and a solvent control to four replicate exposure aquaria.

Prior to exposure initiation, a Harvard Apparatus pump, in conjunction with a 25.0-mL Hamilton gas-tight syringe, was calibrated to deliver 0.0145 mL/cycle of the 5.0 mg/mL diluter stock solution into the diluter system’s chemical mixing chamber, which also received 1.450 L of dilution water per cycle. The mixing chamber was positioned over a 2Mag eMotion submersible magnetic stir plate and was partially submerged within an ultrasonic water bath. The continuous stirring (with a Teflon coated stir bar) and ultrasonication aided the solubilization of the test substance in the dilution water. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration (0.050 mg/L) and was proportionally diluted by a constant factor of approximately 3.2 to provide the remaining nominal exposure concentrations (0.016, 0.0049, 0.0015, and 0.0048 mg/L).

The concentration of DMF in the solution in the mixing chamber, also the high test concentration, constituted the highest DMF concentration (10 μL/L). A Fluid Metering (FMI) pump was calibrated to deliver 0.72 mL/cycle of the 77 μL/mL solvent stock solution to 5.4 L of dilution water per cycle which was subsequently delivered to the solvent control and remaining treatment vessels. The DMF concentration in the solvent control and the treatment levels was 10 μL/L, which was equal to that of the high test concentration.

A set of control aquaria was also established which contained the same dilution water and was maintained under the same conditions as the treatment aquaria, but contained no Vi4D4 or DMF.

The exposure was conducted using a system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 28 exposure aquaria. The exposure system provided five concentrations of the test substance, a control, and a solvent control to four replicate exposure aquaria. Flow splitting cells between the diluter cells and the exposure aquaria were employed to promote mixing for the test substance solution and diluent water. In each cell, four separate standpipes were employed to equally distribute the solutions to the four replicate aquaria at a rate of approximately 250 mL of test solution per aquaria per cycle. Flow splitting accuracy of the diluter cells was within 5% of the nominal value.

The diluter system was calibrated prior to exposure initiation and calibration was confirmed at exposure termination by measuring delivery volumes of toxicant and dilution water. The function of the diluter system (e.g., cycle rate and stock solution flow rates) was monitored daily and a visual check of the system’s operation was performed twice daily. In addition, analysis of the exposure solutions for Vi4D4 concentration was also used to verify proper operation of the diluter system. The exposure system was functioning properly for 28 days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure aquaria and to monitor the behavior of the test substance over time. The exposure aquaria were labeled to identify the nominal test substance concentration/control designation and designated replicate (A, B, C, and D).
- Eluate: No
- Differential loading: No
- Controls: A set of control aquaria was also established which contained the same dilution water and was maintained under the same conditions as the treatment aquaria, but contained no Vi4D4 or DMF.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 10 μL/L (approximately 10 mg/l)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow (Pimephales promelas)
Fathead minnow is suitable for this type of early life-stage toxicity test due to their ease of handling, their known sensitivity to a variety of toxicants, the ready availability of embryos and the extensive existing data for this common fish species.

Fathead minnow embryos (SMV Lot No. 20A025) used during this testing were obtained from brood stocks maintained at Smithers. The brood stocks (SMV Lot Nos. 19A329 and 19A266) used were approximately 24 to 29 weeks old. No mortality was observed among the brood stock during the 7 days prior to testing. The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure. During the 14 days prior to test initiation, the culture temperature ranged from 23 to 25 °C and the dissolved oxygen ranged from 85.6 to 101% of saturation.

At exposure initiation, a subsample of embryos (N = 30) was collected to determine embryonic stage of development. The embryo subsample was preserved in Stockard’s solution (85/6/5/4 mixture of deionised water, glycerin, formaldehyde, and glacial acetic acid, v/v/v/v) for at least 24 hours prior to assessment. Embryonic stage was determined on each individual embryo within the subsample using guidance in the Prehatching Development of the Fathead Minnow Pimephales promelas Rafinesque (U.S. EPA, 1996).

Spawning substrates were introduced to the brood stock aquaria at the end of the working day the evening prior to exposure initiation (day 0). Therefore, some embryos were likely between 12 and 24 hours old. However, fathead minnows typically spawn in the early morning hours after dawn, or when the controlled light cycle begins, and based on the developmental stages of the embryo subsample collected at exposure initiation, the majority of the embryos used for this exposure were likely less than 6 hours post-fertilisation. The exposure of fathead minnow embryos and larvae to Vi4D4 was initiated when the embryo incubation cups were distributed to each of the exposure aquaria. The embryos were impartially distributed to the embryo incubation cups in the following manner: 28 labeled incubation cups were impartially placed in a water bath containing dilution water maintained at approximately 25 °C. A dish containing the embryos (confirmed to be fertilised) in dilution water was placed in the same water bath. The embryos were impartially placed in the incubation cups five at a time until all cups contained five embryos. This process was repeated until all cups contained 30 embryos. To initiate the exposure, the incubation cups were then placed in the respective exposure aquaria (one cup per replicate aquaria).

Following exposure initiation, dead and live embryos were counted daily until the day of hatch (day 3); dead embryos were removed when observed. Day of completion of hatch was considered to be exposure day 6, when all viable embryos in all embryo incubation cups were hatched. Calculations of hatching success of organisms were based on the number of live, dead, or deformed larvae per incubation cup after hatching was complete (day 6) compared to the number of embryos per cup on day 0. If one or more embryos were unaccounted for after the first day of embryo exposure, then the actual number of embryos on the first day was used as the denominator when computing percent hatch.

The 28-day post-hatch larval exposure was initiated once 90% of viable embryos had hatched (day 4). On day 4, the surviving larvae present in each incubation cup were thinned to 20 organisms per replicate/80 organisms per treatment level or control and placed into each respective exposure aquarium. Egg cups that still contained viable embryos on the day of release remained in their respective aquaria until hatch for that replicate was completed, at which time those larvae were discarded. During the post-hatch exposure period, dead larvae were removed when observed and behavior and appearance of the larval fish were observed and recorded daily. Larval survival was estimated daily.

Beginning on day 5 (day 1 post-hatch), the larvae were fed live brine shrimp nauplii (Artemia salina) three times daily. At each feeding, larvae were fed ad libitum such that all larvae were afforded equal access to food. Larvae were not fed during the 24 hours prior to study termination.

Prior to use of a new batch of food, a representative sample of the food source was analysed for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected in the food samples at concentrations considered toxic to the test organisms. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern in agreement with ASTM (2007). This analysis is presented in Appendix 3.

At 28 days post-hatch, the exposure was terminated. The surviving larvae in each replicate aquarium were euthanised with a buffered solution of MS-222 (tricaine methanesulfonate), counted to determine larval survival, and measured individually to determine total length and wet weight. The larvae were measured and weighed to the nearest 0.01 mm and 0.1 mg, respectively. The mean and standard deviation were calculated using individual measurements (total length and wet weight) for each replicate aquarium.

During the 28-day post-hatch exposure period, biomass loading based on control organism wet weight at test termination, did not exceed 0.056 g/L per day under the exposure’s flow-through conditions or 0.40 g/L at any time in any replicate exposure aquarium.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

64 - 76 CaCO3

Test temperature:

24-26 °C

pH:

6.9-7.8

Dissolved oxygen:

5.10 - 8.76 mg/l
% Saturation 61.5 - 106%

Salinity:

n/a

Conductivity:

430 - 630 µS/cm

Nominal and measured concentrations:

Nominal: 0.00048, 0.0015, 0.0049, 0.016, and 0.050 mg/L
Mean measured: 0.00034, 0.0010, 0.0031, 0.012, and 0.036 mg/L

Details on test conditions:

Embryo Loading – Exposure Initiation:
The exposure was initiated with fertilised embryos. The embryos were ≤24 hours old when obtained from the brood unit facility of Smithers Viscient. Embryos available for initiating a test were combined in a Carolina dish filled with dilution water. Embryos were assessed for fertilisation (e.g., early cell cleavage, blastodisc cleavage, etcetera) under appropriate magnification prior to loading. For the fertilisation assessment, fertilised embryos were maintained and unfertilised embryos were discarded. Embryo cups and the dish containing the embryos were placed in a container of dilution water maintained at approximately 25 ºC. After the fertilisation assessment, viable embryos were distributed five at a time by stratified random assignment to each of the labeled incubation cups (one cup per test chamber) using a serological pipette until each cup contained five embryos. This process was repeated until each cup contained 30 embryos.

Evaluation of Embryonic Development:
At exposure initiation, a subsample of embryos was collected (n = 30) to determine the embryonic stage of development. The subsample was preserved in Stockard solution for at least 24 hours prior to assessment (table 1). Stockard solution aided in distinguishing developmental stages through the chorion.

Embryonic stage was determined with the Pre-Hatching Development of the Fathead Minnow Pimephales promelas Rafinesque. The embryonic stage was determined on each individual embryo within the subsample.

Chemical cleaning of the test diluter and aquaria occurred prior to exposure initiation. At a minimum, all aquaria were scraped and siphoned weekly during the test. The diluter mixing chamber, chemical cells and splitters were cleaned at least weekly and the diluter delivery tubing was cleaned as needed.

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Embryo incubation cups were 5-cm diameter, 8-cm high round glass jars with 475-micron nylon screen bottoms. A rocker arm apparatus, driven by a 1-rpm electric motor was used to gently oscillate the incubation cups in the test solutions.
- Test vessel: The exposure was conducted using a system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 28 exposure aquaria. The exposure system provided five concentrations of the test substance, a control, and a solvent control to four replicate exposure aquaria.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: The diluter system and exposure aquaria were fabricated of glass and silicone sealant. Each 2.5 gallon exposure aquarium measured 30 × 14.5 × 20 cm (L × W × H) with an approximate 12.5 cm high side drain that maintained a constant exposure solution volume of approximately 5.5 L.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Intermittent-flow proportional diluter
- Renewal rate of test solution (frequency/flow rate): The diluter system delivered the control and exposure solutions to the exposure aquaria at a rate sufficient to provide approximately 7.1 aquarium volumes per 24 hour period, with a 90% replacement time of approximately 7 hours.
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: During the 28-day post-hatch exposure period, biomass loading based on control organism wet weight at test termination, did not exceed 0.056 g/L per day under the exposure’s flow-through conditions or 0.40 g/L at any time in any replicate exposure aquarium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water (laboratory well water) was a mixture of unadulterated on-site well water and de-chlorinated Town of Wareham well water and was characterised as soft water with a typical total hardness of <160 mg/L as CaCO3. The on-site well water was obtained from a 100-meter bedrock well. The Town of Wareham well water was de-chlorinated by the use of ultraviolet (UV) treatment and activated carbon filtration. The two sources of water were individually pumped through 1-µm polypropylene bag filters, de-gassers and then mixed in a 5700-liter polyethylene holding tank. The resulting dilution water was continuously circulated through a de gassing chamber to ensure proper equilibration of dissolved gases with the laboratory atmosphere. The water was pumped from the holding tank through aged PVC piping to a heat exchanger unit that heated the dilution water to test temperature ahead of transfer to the exposure system. During the study, weekly characterisation of the well water established total hardness and alkalinity ranges as CaCO3 of 62 to 74 mg/L and 21 to 23 mg/L, respectively, a pH range of 7.3 to 7.6, and a conductivity range of 370 to 530 µS/cm.

Representative samples of the dilution water source were analysed biannually for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected in any of the water samples analysed at concentrations that are considered toxic, in agreement with ASTM (2007) standard practice. In addition, representative samples of the dilution water source were analysed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 0.72 and 0.82 mg/L for the months of February and March 2020, respectively.

- Total organic carbon: 0.72 and 0.82 mg/L
- Chlorine: None detected
- Alkalinity: 21 to 23 mg/L

- Culture medium different from test medium: No

- Intervals of water quality measurement: Dissolved oxygen concentration, pH, and temperature were measured in all aquaria on day 0. Daily thereafter, these parameters were measured in a single replicate of each concentration and the control; replicates were successively alternated each day (A, then B, then C, etc.). Total hardness, total alkalinity, and conductivity were measured weekly in one replicate of the control, low, and high test concentrations; replicates were successively alternated each week. Test solution temperature was continuously monitored in replicate A of the control using a Control Company minimum/maximum thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light and 8 hours darkness with a 15- to 30 minute transition period,
- Light intensity: light intensity range of 50 to 81 footcandles (540 to 870 lux),

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): embryo hatching success, percentage of embryos that produce live, normal larvae at hatch, larval survival, and larval growth (total length and wet weight)

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
A preliminary range-finding exposure study was conducted with the nominal concentrations of 0.050, 0.016, 0.0049, 0.0015 and 0.00048 mg/L (3.2 dilution factor). A solvent control and control group was also included. The exposure was maintained under flow-through conditions. At least two replicates were used for each exposure concentration and control(s).

The diluter test system equilibrated for approximately one week prior to initiation. The exposure initiated with embryos, continued through larval stage and terminated approximately 14-days post-hatch. The exposure started with 30 embryos per replicate aquaria. Exposure solution samples from one replicate of each concentration and control and from the diluter stock solution were taken during the equilibration period, initiation and termination, respectively.

After completion of hatch, 20 live, normal larvae were indicriminately selected from each incubation cup and transferred into each respective replicate aquarium. All fish were fed three times daily with live brine shrimp nauplii.

At approximately 14-days post-hatch, the larvae from each aquarium were euthanised with a buffered solution of tricaine methanesulfonate (MS-222) counted to determine percentage survival and measured to determine total lengths and wet weights. For wet weight determination, larvae were blotted dry and weighed on an analytical balance. Lengths and weights were measured to the nearest 0.01 mm and 0.1 mg, respectively.
Water quality characteristics (e.g. pH values, dissolved oxygen concentration, temperature) of the exposure solutions were measured daily throughout the exposure.

- Test concentrations: 0.00048, 0.0015, 0.0049, 0.016, and 0.050 mg/L, a control, and a solvent control.
- Results used to determine the conditions for the definitive study: Significant effects on growth observed in highest concentration. Highest concentration was defined as the limit of water solubility of the test substance in test media.
Based on the result from the dose-range finding study, dose group of 0.00034, 0.0010, 0.0031, 0.012 and 0.036 mg/L (0.34, 1.0, 3.1, 12 and 36 µg/L) and a negative control were selected in collaboration with the sponsor. The selected concentrations were intended to include both toxicant-effect and no-effect levels.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 4
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20 per concentration and control per replicate
- Release of alevins from incubation cups to test chamber on day no.: Day 4

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30 per concentration and control per replicate (4 replicates)
- Removal of eggs to check the embryonic development on day no.: At exposure initiation, a subsample of embryos (N = 30) was collected to determine embryonic stage of development. The embryo subsample was preserved in Stockard’s solution (85/6/5/4 mixture of deionized water, glycerin, formaldehyde, and glacial acetic acid, v/v/v/v) for at least 24 hours prior to assessment. Embryonic stage was determined on each individual embryo within the subsample using guidance in the Prehatching Development of the Fathead Minnow Pimephales promelas Rafinesque

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth

Remarks on result:

other: no effects at the highest concentration tested

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth

Details on results:

Survival in the control and solvent control at test termination was 91 and 95%, respectively (pooled control = 93%). Based on the Fisher’s Exact Test with Bonferroni-Holm Adjustment, no significant survival effect was observed in any treatment level compared to the pooled control. No significant effects for neither length nor wet weight compared to the pooled control were observed in any treatment level (Dunnett’s Multiple Comparison Test).
Based on these results, the overall NOEC for this early life-stage exposure was concluded to be 0.036 mg/L (36 µg/L). The LOEC was concluded to be >0.036 mg/L (>36 µg/L).

Reported statistics and error estimates:

Following termination of the early life-stage exposure, data obtained on embryo hatching success, percent live, normal larvae at hatch, larval survival, and larval growth at exposure termination (total length and wet weight) were statistically analysed to establish treatment level effects.
Analyses were performed using the mean organism response in each replicate aquarium rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of the qualifying tests for normality and homogeneity of variance, in which the 99% level of certainty was applied. The following procedures were used:
- Solvent control and control data were first compared using an Equal Variance Two-Sample t-Test (U.S. EPA, 2002). No significant difference was detected between control and solvent control for any of the endpoints; therefore, the pooled control was used to determine treatment effects.
- Statistical analysis of percent hatching success, percent live, normal larvae at hatch, and percent survival was performed. Since the data for these endpoints was non-monotonic, Fisher’s Exact Test with Bonferroni-Holm’s Adjustment was used to determine treatment effects.
- Shapiro Wilks’ Test for normality was conducted to evaluate the distribution of the data. Data for total length and wet weight were normally distributed when treatment data was evaluated.
- Bartlett’s Equality of Variance Test was conducted to evaluate the homogeneity of the data. Data for total length and wet weight met the assumption of homogeneity of variance when treatment data was evaluated.
- Since total length and wet weight data met the assumptions for normal distribution and homogeneity of variance and were non-monotonic, Dunnett’s Multiple Comparison Test, a parametric procedure, was used to establish treatment effects for these endpoints.
CETIS Version 1.9 (Ives, 2019) was used to perform the statistical computations.

Table 1 - Measured test substance concentrations

Nominal concentration (mg/l)	Mean measured concentration (mg/l)	Percent of nominal	% CV	Range from mean measured (%)
Control	N/A	N/A	N/A	N/A
Solvent control	N/A	N/A	N/A	N/A
0.00048	0..00034	71	27	75-140
0.0015	0.0010	68	18	90-130
0.0049	0.0031	64	19	78-120
0.016	0.012	74	13	88-120
0.050	0.036	73	13	83-120
5000 (stock)	4717	Not reported	Not reported	Not reported

Table 2 - Early Life-Stage Exposure of Fathead Minnow (Pimephales promelas) to Vi4D4 - Endpoint summary

			28-days post hatch	28-days post hatch	28-days post hatch
Mean measured concentration (mg/l)	Embryo hatching success (%)	Live, normal larvae at hatch (%)	Larval survival (%)	Total length (mm)	Wet weight (g)
Control	92	94	91	24.02	0.1080
Solvent control	91	95	95	24.08	0.1111
Pooled control	91	94	93	24.05	0.1095
0.00034	97	90	95	24.39	0.1116
0.0010	94	96	89	24.32	0.1143
0.0031	92	91	89	24.51	0.1167
0.012	92	95	94	24.05	0.1072
0.036	91	91	91	23.60	0.1041

Validity criteria fulfilled:

yes

Conclusions:

In an early-life stage toxicity test in Fathead Minnow (Pimephales promelas), conducted according to OECD TG 210 and in compliance with GLP, a NOEC was concluded to be equal to or higher than 0.036 mg/L based on no effects on survival and growth at the highest concentration tested.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-06-04 to 2009-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Twice prior to the start of the definitive exposure, samples from alternating replicates of each concentration and the dilution water control solutions were sampled and analyzed for the concentration of test substance present in each vessel. Results of these pretest analyses were used to judge
whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure.

During the in-life phase of the definitive study, samples were removed from alternate replicates (A/B/C/D) of each treatment level and control on days 0, 5, 12, 19, 21, 26, 28, 33, 40, 47, 54, 61, 64, 68, 70, 76, 80, 82 and 90 for analysis of test substance concentration. Additional samples of the test
solutions were collected at each sampling interval and stored frozen for possible analysis in the event that routine analytical results were variable. All test samples were removed from the approximate midpoint of each aquarium using a pipette.

Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples through the analytical process. 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.

Vehicle:

yes

Details on test solutions:

A 500 μg a.i./mL diluter stock solution was prepared prior to test initiation and as needed during the study by placing, for example, 0.0505 g of the test substance (0.0500 g as active ingredient) in a 100-mL volumetric flask and bringing it to volume with dimethylformamide (DMF, CAS No. 68-12-2). The resultant stock solution was observed to be clear and colorless. A 770 μL/mL solvent stock solution was prepared by diluting 770 mL of DMF to a final volume of 1000 mL with deionized water. The resulting stock solution was observed to be clear and colorless.

Prior to the pretest period, a Harvard Apparatus syringe pump in conjunction with a 100-mL Glenco® gas-tight syringe was calibrated to deliver 0.0660 mL/cycle of the 500 μg a.i./mL stock solution into the diluter system's chemical mixing chamber which also received 1.94 L of dilution water per cycle. The mixing chamber was positioned over a magnetic stir plate and was partially submerged within an ultrasonic water bath which aided in the solubilization of the test substance into the dilution water. The concentration of test substance in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (17 μg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (8.5, 4.3, 2.1 and 1.1 μg a.i./L).

An FMI pump was calibrated to deliver 0.234 mL/cycle of the 770 μL/mL solvent stock solution to 5.30 L 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 all treatment level solutions (34 μL/L). A set of control vessels was also established which contained the same dilution water and was
maintained under the same conditions as the treatment level vessels, but contained no test substance or solvent.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM

- Common name: Rainbow trout

- Source: The "green" rainbow trout eggs and sperm used during this study were obtained from TroutLodge, Inc., a commercial supplier in Sumner, Washington.

- Receipt and handling prior to test: On the day of test initiation, two bags containing the unfertilized gametes were received from TroutLodge, Inc., Sumner, Washington. These bags were received at a temperature of approximately 8 ºC and were warmed in a water bath to 11 ºC gradually over a one-hour period. The unfertilized eggs were approximately 30 hours old upon receipt and were fertilized within 2 hours of arrival.

- Fertilisation of eggs: Fertilization was achieved by adding cold “fertilization buffer” solution to the bag containing the eggs. The bag was rocked gently back and forth a few times and then placed in a water bath at test temperature (11 ºC) for approximately 20 minutes. This procedure was used to rinse away any ovarian fluid sealing the eggs, preventing fertilization. Following the rinsing procedure, the eggs were removed from the water bath and most of the buffer solution was poured off. Sperm and an aliquot of buffer solution were added to the bag containing the eggs. The bag was then rocked gently back and forth and placed in a water bath for 15 minutes to complete the fertilization process. The sperm was then poured off of the eggs and the eggs were then gently rinsed of excess sperm using cold dilution water. The eggs were then placed in a stainless steel bowl containing cold dilution water and allowed to water-harden for approximately 1.5 hours. During the water hardening period, the eggs were maintained at a temperature of 11 ºC.


POST-HATCH FEEDING

Larvae were fed a diet consisting of live brine shrimp nauplii (Artemia salina) three times daily beginning on day 9 post-hatch, when the rainbow trout began to exhibit swim-up behavior. Larvae were not fed during the final 48 hours prior to test termination.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

90 d

Remarks on exposure duration:

60 days post hatch

Hardness:

Total hardness and alkalinity ranges as CaCO3: 48 to 64 mg/L and 18 to 26 mg/L, respectively

Test temperature:

10-14ºC

pH:

6.5-7.8

Dissolved oxygen:

6.6-11 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Solvent control), 1.1, 2.1, 4.3, 8.5 and 17 μg a.i./L

Arithmetic mean measured concentrations: 0 (Control), 0 (Solvent control), 0.92, 2.0, 4.0, 7.8 and 14 μg a.i./L

The results are reported and interpreted with reference to arithmetic mean measured concentrations.

Details on test conditions:

The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967) and a set of 28 exposure vessels. The exposure system was designed to provide five concentrations of the test substance, a dilution water control and a solvent control to four replicate aquaria.

Test vessels were impartially placed in a water bath containing circulating water designed to maintain the test solution at a temperature of 12 ± 2 ºC.
The concentration of test substance in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (17 μg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (8.5, 4.3, 2.1 and 1.1 μg a.i./L).

Dimethylformamide (DMF) was used as a co-solvent. The concentration of DMF in the solvent control vessels was equivalent to the concentration of solvent present in all treatment level solutions (34 μL/L).

Dissolved oxygen concentration, pH and temperature were measured in all test vessels on test day 0 and in alternating replicates daily thereafter. Total hardness and total alkalinity as CaCO3, and specific conductance were measured on day 0 and weekly thereafter in alternating replicates of the low and high concentration and the dilution water control.

Testing was conducted during a 90-day (60-day post-hatch) exposure period, at a temperature range of 10 to 14 ºC, a photoperiod of 16 hours of light at 56 to 80 footcandles (610 to 860 lux) and 8 hours darkness.

On test day 19, mean embryo viability determined in the controls and all treatment levels.

At termination of the early life-stage exposure, data obtained on embryo hatching success, percent normal larvae at hatch, larval survival and larval growth (total length and dry weight) were statistically analyzed to establish treatment level effects. Results of this study are based on arithmetic mean measured concentrations.

The dilution water used during this study was unadulterated water from a 100-meter bedrock well which was pumped into an epoxy-coated concrete reservoir where it was aerated prior to use. During the study, weekly characterization of the well water established total hardness and alkalinity ranges as CaCO3 of 38 to 60 mg/L and 17 to 24 mg/L, respectively, a pH range of 6.3 to 7.3 and a specific conductivity range of 320 to 380 μmhos/cm. Representative samples of the dilution water source were analyzed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected in any of the water samples analyzed at concentrations that are considered toxic, in agreement with ASTM (2005) standard practice. In addition, representative samples of the dilution water source were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water ranged from 0.36 to 0.49 mg/L for the period of June to September 2009.

Reference substance (positive control):

no

Duration:

90 d

Dose descriptor:

NOEC

Effect conc.:

>= 14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching, survival and growth

Duration:

90 d

Dose descriptor:

LOEC

Effect conc.:

> 14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching, survival and growth

Reported statistics and error estimates:

At the termination of the early life-stage exposure, data obtained on embryo hatching success, percent normal larvae at hatch, larval survival and larval growth (total length and dry weight) were statistically analyzed to establish treatment level effects. Analyses were performed using the mean organism response in each replicate aquarium rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of the Bartlett's Test and Shapiro-Wilks' Test, in which the 99% level of certainty was applied.

TOXSTAT® Version 3.5 (West and Gulley, 1996) was used to perform the statistical computations. The lowest arithmetic mean measured concentration that elicited a statistically significant effect on organism performance (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that elicited no statistically significant difference between the exposed organisms and the control (No-Observed-Effect Concentration, NOEC) were determined. Determination of these levels is based on the most sensitive of the performance criteria evaluated (e.g., embryo hatching success, percent of embryos that produce live normal larvae at hatch, organism survival at hatch, larval survival and growth at study termination).

Table 1. Results of analysis of test media

Nominal concentration (μg/L)	Arithmetic mean measured concentration (μg/L) (SD)	N	Mean percentage of nominal
1.1	0.92 (0.16)	14	84
2.1	2.0 (0.35)	14	95
4.3	4.0 (0.67)	14	93
8.5	7.8 (1.3)	14	91
17	14 (2.3)	14	82

Table 2. Test results

Mean measured concentration  (μg/L)	Mean percentage hatch	Mean percentage larval survival to end of test	Mean total length at end of test (mm) (SD)	Mean dry weight at end of test (g) (SD)
0 (Control)	82	92	52.5 (0.65)	0.2394 (0.0132)
0 (Solvent control)	89	90	52.1 (0.42)	0.2382 (0.0171)
0 (Pooled control)	86	91	52.3 (0.30)	0.2388 (0.0008)
0.92	94	92	52.0 (0.96)	0.2399 (0.0174)
2.0	90	92	51.2 (1.02)	0.2317 (0.0164)
4.0	90	90	51.7 (1.05)	0.2398 (0.0157)
7.8	83	90	51.6 (0.96)	0.2358 (0.0152)
14	89	92	51.7 (0.97)	0.2460 (0.0176)

Validity criteria fulfilled:

yes

Conclusions:

A 90-day (60-day post-hatch) NOEC of ≥14 μg/L has been determined for the effects of the test substance on hatching and larval survival and growth of Oncorhynchus mykiss under flow-through exposure conditions.

Description of key information

Long-term toxicity to fish: 28 day NOEC ≥0.036 mg/l, embryo viability, hatching success, larval survival and growth, Pimephales promelas, (guideline OECD 210)

Additional information

A reliable long-term toxicity to fish study is available for the registration substance, 2,4,6,8-tetramethyl-2,4,6,8-tetravinyltetrasiloxane (Vi4-D4, CAS 2554-06-5). A 28 day post hatch No-Observed-Effect Concentration (NOEC) of ≥0.036 µg/l based on geometric mean measured concentrations has been determined for the effects of Vi4-D4 on embryo viability, hatching success, larval survival and growth of fathead minnow (Pimephales promelas), in accordance with OECD TG 210, Fish, Early-Life Stage Toxicity Test and in compliance with GLP (Smithers, 2020). No effects were observed at the maximum achievable concentration of the test substance in test media.

The hydrolysis half-life of the substance, test media preparation methods and exposure system (flow-through) indicate that the test organisms will have been exposed to the parent substance.

The data above are supported by read-across evidence relevant to the impurities. The registration substance has an average purity of >70% Vi4-D4, with <20% 2,4,6,8,10-pentamethyl-2,4,6,8,10-pentavinylcyclopentasiloxane Vi5-D5 (CAS 17704-22-2; Impurity 1) and <10% 2,4,6-trimethyl-2,4,6-trivinylcyclotrisiloxane Vi3-D3 (CAS 3901-77-7; Impurity 2) present as impurities. After due consideration of the properties, the presence of these impurities is not expected to affect the overall hazard profile of the substance.

Read-across studies are in place as supporting studies, to consider the properties of the impurities. Data for Vi5-D5 are read-across from decamethylcyclopentasiloxane D5 (CAS 541-02-6). These siloxanes are expected to have similar properties with regard to aquatic ecotoxicity. Further information is given in a supporting report (PFA, 2017) attached in Section 13 of the IUCLID dossier.

A reliable long-term fish study is available for the siloxane D5 (CAS 541-02-6) and is read-across to Impurity 1 (Springborn Smithers, 2009). This study (90 day, fish early life stage test with Oncorhynchus mykiss) gave a result 90 day NOEC ≥14 µg/l. Effects were not seen within the limit of solubility in test media.

No reliable long-term fish study is available to read-across to Impurity 2. Effects are not anticipated at the limit of solubility and the available data for the main data set are conservative in respect of Impurity 2, which has a much shorter hydrolytic half-life and lower log K_ow. The chemical safety assessment is therefore not sensitive to this data gap.