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Long-term toxicity to fish

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Reference
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 30 April to 1 June 2020 at Smithers, located in Wareham, Massachusetts.
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:
Adopted: 26 July 2013
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the test substance was stored at room temperature in a dark, ventilated cabinet in the original container.

Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Prior to the start of the definitive exposure, samples from one replicate of the treatment level and the control solutions were collected and analyzed for phenyl silsesquioxanes. In addition, a sample of the diluter stock solution was also analyzed. During this pre-test period, the stability of the stock solution concentration was confirmed for at least 11 days. During the exposure stock solutions were prepared as needed throughout the exposure (i.e., every 11 to 12 days). The single stock solution that was prepared after 12 days was not considered to have had a negative impact on the exposure concentration. Results of the pretest analyses were used to judge whether sufficient quantities of phenyl silsesquioxanes 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 the treatment and the controls, 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, 18, 26, 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 analyzed at each sampling interval during the exposure period. All stocks were sampled at least once throughout the duration of the exposure. Additionally, subsamples of the exposure solutions were also collected at each sampling interval and stored frozen as archive samples.

- Sample storage conditions before analysis:
At each interval, samples were analyzed within approximately 8 hours of removal from the exposure system on the day of sampling.
Vehicle:
yes
Remarks:
DMF (solvent control included)
Details on test solutions:
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 (i.e., every 11 to 12 days) thereafter throughout the definitive exposure by adding, for example, 0.2516 g of phenyl silsesquioxanes to a 50-mL volumetric flask and then bringing it to a total volume of 50 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 75 μL/mL solvent stock solution was prepared prior to exposure initiation and as needed throughout the definitive exposure thereafter by diluting 38 mL of DMF to 500 mL with deionized water. The resultant stock solutions were observed to be clear and colorless following mixing by inversions of the flask.
Prior to exposure initiation, a Harvard Apparatus pump, in conjunction with a 10.0-mL Hamilton gas-tight syringe, was calibrated to deliver 0.0194 mL/cycle of the 5.0 mg/mL diluter 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. The continuous stirring (with a Teflon-coated stir bar) aided the solubilization of the test substance in the dilution water. The solution in the mixing chamber was equivalent to that of the nominal limit test concentration (0.050 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 0.28 mL/cycle of the 75 μL/mL solvent stock solution to 2.1 L of dilution water per cycle which was subsequently delivered to the solvent control and treatment vessels. The DMF concentration in the solvent control was 10 μL/L, which was equal to that of the test concentration.
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 phenyl silsesquioxanes concentration was also used to verify proper operation of the diluter system. The exposure system was functioning properly for 13 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.

- Concentrations: Based on results from a solubility trial and preliminary testing at Smithers, and in consultation with Study Sponsor, a single, nominal phenyl silsesquioxanes concentration of 0.050 mg/L was selected for the definitive exposure, which was considered to approximate the functional water solubility of the first and most water soluble constituent comprising the test substance.

- 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 phenyl silsesquioxanes or DMF.



Test organisms (species):
Pimephales promelas
Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Fathead minnow embryos (Smithers Lot No. 20A088) used during this testing were obtained from brood stocks maintained at Smithers. The brood stocks (Smithers Lot Nos. 19A391 and 19A380) used were approximately 21 to 25 weeks old. Mortality of 0.3% 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 24 to 25 °C and the dissolved oxygen ranged from 91.4 to 102% of saturation.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
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-fertilization

POST-HATCH FEEDING
- Start date: day 5 (day 1 post-hatch)
- Type/source of feed: live brine shrimp nauplii (Artemia salina)
- Amount given: 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.
- Frequency of feeding: three times daily
Test type:
flow-through
Water media type:
freshwater
Remarks:
Laboratory well water
Limit test:
yes
Total exposure duration:
32 d
Remarks on exposure duration:
32-day duration (28 days post-hatch)
Hardness:
Weekly characterization of the limit concentration and control solutions established total hardness and alkalinity ranges as CaCO3 of 64 to 76 mg/L and 18 to 22 mg/L, respectively
Test temperature:
24 to 27 °C
pH:
6.8 to 7.6
Dissolved oxygen:
6.26 to 8.25 mg/L (76.1 to 99.8% saturation)
Conductivity:
Weekly characterization of the limit concentration and control solutions established a conductivity range of 520 to 610 μS/cm
Nominal and measured concentrations:
Nominal 0.050 mg/L
Measured: 0.11 mg/L

Based on results from a solubility trial and preliminary testing, and in consultation with Study Sponsor, a single, nominal phenyl silsesquioxanes concentration of 0.050 mg/L was selected for the definitive exposure, which was considered to approximate the functional water solubility of the first and most water soluble constituent comprising the test substance.
Details on test conditions:
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: 12 exposure aquaria. 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. During the 32-day exposure, the aquaria were brushed and siphoned twice weekly to remove excess food and fecal matter. The aquaria were impartially positioned in a water bath containing circulating water designed to maintain the test solution temperatures at 25 ± 1.5 °C. Two Process Technology USA immersion heaters regulated by an Omega CN76000 temperature controller were used to maintain temperatures. Illumination was provided by Sylvania Octron fluorescent lights centrally located above the test aquaria.

- Material, size, headspace, fill volume: The diluter system and exposure aquaria were fabricated of glass and silicone sealant.

- 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 7.3 aquarium volumes per 24-hour period, with a 90% replacement time of approximately 7.5 hours.

- No. of fertilized eggs/embryos per vessel: The embryos were impartially distributed to the embryo incubation cups in the following manner: 12 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 fertilized) 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 35 embryos. To initiate the exposure, the incubation cups were then placed in the respective exposure aquaria (one cup per replicate aquaria).

- 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 0.048 g/L per day under the exposure’s flow-through conditions or 0.35 g/L at any time in any replicate exposure aquarium. These values are within the guideline requirement of ≤0.5 g/L per day and ≤5 g/L of solution at any time.

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 characterized 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 characterization of the well water established total hardness and alkalinity ranges as CaCO3 of 60 to 80 mg/L and 18 to 21 mg/L, respectively, a pH range of 6.7 to 7.3, and a conductivity range of 460 to 580 μS/cm.
Representative samples of the dilution water source were analyzed 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 analyzed at concentrations that are considered toxic, in agreement with ASTM (2007) standard practice. This analysis is presented in Appendix 3. 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 was 0.72, 0.76, and 0.91 mg/L for the months of April, May, and June 2020, respectively.

- 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 and the limit test concentration; 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. Dissolved oxygen concentrations and daily temperature were measured using a Yellow Springs Instrument (YSI) Model 550A dissolved oxygen meter/temperature probe. The pH was measured using a YSI Model pH100A pH meter. Total hardness concentrations presented in this report were measured by the EDTA titrimetric method and total alkalinity concentrations were determined by potentiometric titration to an endpoint of pH 4.5 (APHA et al., 2017). Conductivity was measured using a YSI Model EC300A conductivity meter.

OTHER TEST CONDITIONS
- Adjustment of pH: No

- Photoperiod: A 16-hour light/8-hour darkness photoperiod and with a 15- to 30-minute transition period was provided at the exposure solution’s surface.

- Light intensity: Light intensity was not measured during the study

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).
Following exposure initiation, dead and live embryos were counted daily until the day of hatch (day 3); dead embryos were removed when observed. After hatching began, observations continued until completion of hatch but the embryos and larvae were not handled in order to avoid possible physical damage to the newly-hatched larvae. 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.
At 28 days post-hatch, the exposure was terminated. The surviving larvae in each replicate aquarium were euthanized 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.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: Based on these results of the solubility trial, and consultation with the Study Sponsor, a preliminary exposure was conducted at concentrations of 0.0031, 0.0063, 0.013, 0.025, and 0.050 mg/L and using solvent dosing stock solutions. Based on the physiochemical properties of the test substance and in consultation with the Study Sponsor, the test system was equilibrated for approximately 1 week prior to initiating the exposure. The conditions were similar to those detailed for the definitive exposure. The exposure examined the embryo/larval response to nominal phenyl silsesquioxanes concentrations of 0.0031, 0.0063, 0.013, 0.025, and 0.050 mg/L, a control, and a solvent (DMF) control. Four replicates were maintained for each concentration and control. The exposure was initiated with 30 embryos per replicate. Following hatch, hatched larvae were thinned to 20 larvae per replicate.
- Results used to determine the conditions for the definitive study: No effects observed at highest concentration tested.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 4
- No. of hatched eggs (alevins)/treatment released to the test chamber: 80 organisms per treatment level or control

FERTILIZATION SUCCESS STUDY
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 (US EPA, 1996).
Reference substance (positive control):
no
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Remarks on result:
other: no effects at the highest concentration tested
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.11 mg/L
Nominal / measured:
meas. (arithm. 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:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Remarks on result:
other: no effects at the highest concentration tested
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Remarks on result:
other: no effects at the highest concentration tested
Details on results:
Stage of embryonic development: At exposure initiation, a subsample of 30 embryos was collected and the embryonic stage of development was determined. The mean and median were both 10 (high blastula) and the range of the developmental stages was 9 (late cleavage) to 11 (flat blastula).

Hatching and survival:
All viable embryos in the treatment and control embryo incubation cups had completed hatching by day 4.

At the completion of hatch (day 4), hatching success in the control and solvent control averaged 89 and 84%, respectively (pooled control = 87%). Embryo hatching success in the 0.11 mg/L treatment level averaged 86%. An Unequal Variance Two-Sample t-Test determined no significant reduction in embryo hatching success in the treatment level tested compared to the pooled control (87%).

At the completion of hatch, the percent of live, normal larvae in the control and solvent control averaged 89 and 96%, respectively (pooled control = 93%). Percent of live, normal larvae in the 0.11 mg/L treatment level averaged 94%. An Equal Variance Two-Sample t-Test determined no significant reduction in percent of live, normal larvae in the treatment level tested compared to the pooled control (93%).

At exposure termination (32 days, 28 days post-hatch), larval survival in the control and solvent control both averaged 95% (pooled control = 95%). Larval survival in the 0.11 mg/L treatment level averaged 99%. An Equal Variance Two-Sample t-Test determined no significant reduction in larval survival among organisms exposed to the treatment level tested compared to the pooled control (95%).

Abnormal appearance/behaviour:
At exposure termination, all surviving fish were observed to be normal with some exceptions relating to observations of small fish and small fish with a tail deformity (refer to Table 1 in Any other information on results incl tables).
Due to normal biological variability, a small percentage of fish may be observed to be small and have deformities. The growth variation observed in this study was within expectations for an early life-stage study with this species and did not impact the results or interpretation of this exposure.

Weight:
At exposure termination, mean wet weight of larvae in the control and solvent control averaged 0.0886 and 0.0950 g, respectively (pooled control = 0.0918 g). Wet weight of larvae exposed to the 0.11 mg/L treatment level averaged 0.1003 g. An Equal Variance Two-Sample t-Test determined no significant reduction in wet weight among larvae exposed to the treatment level tested compared to the pooled control (0.0918 g).

Length
At exposure termination, mean total length of larvae exposed to the control and solvent control averaged 22.27 and 21.96 mm, respectively (pooled control = 22.11 mm). The mean total length of larvae exposed to the 0.11 mg/L treatment level was 22.84 mm. An Equal Variance Two-Sample t-Test determined no significant reduction in total length among larvae exposed to the treatment level tested compared to the pooled control (22.11 mm).

(refer to Table 3 in Any other information on results incl tables for Endpoint summary)

- Effect concentrations exceeding solubility of substance in test medium:
No undissolved test substance (e.g., precipitate) was observed in the diluter system (e.g., mixing chamber and chemical cells) or exposure solutions during the pretest period or throughout the exposure period.
Mean measured concentrations for constituent 1, constituent 2, and constituent 3 were 0.077, 0.11, and 0.18 mg/L, respectively. The mean measured value for the sum of the constituents (refer to Table 2 in Any other information on results incl tables) was 220% of nominal and defined the treatment level tested as 0.11 mg/L. Diluter stock solution recoveries ranged from 92 to 110% of nominal concentration. The stock solution results were within expected nominal concentrations for all three constituents and therefore, confirmed that the exposure system was dosed properly. The high recoveries of the exposure solutions were an indication that functional solubility had been exceeded and that there were undissolved particles present in the exposure system. These particles were likely very fine since they were not detected by visual observations. The test substance is a complex mixture of materials which have different functional solubilities. The results from this testing indicate that the recoveries of constituents 2 and 3 were consistently higher than constituent 1 throughout the exposure demonstrating this difference. It should be noted that the test concentration selected for this study (i.e., 0.050 mg/L) was to approximate the functional limit of solubility for constituent 1 while it was known that this nominal dose may be in excess of the other two constituents’ solubility.
Generally speaking, the components being monitored for the test substance appeared to be at their maximum functional solubility for nearly the duration of the exposure with components starting to precipitate out of solution at day 11 in the form of a homogeneous suspension. This functional solubility seems to be above expected water solubility. This high solubility/suspension equilibrium remained relatively constant for the duration of testing, with a relative standard deviation of approximately 22%, with the exception of the last interval. Throughout testing, the third most hydrophobic constituent demonstrated the most variability, suggesting that it was starting to precipitate early on. The relatively high recoveries at test termination were likely the result of a disruption of the suspension equilibrium due to an increase in the undissolved material present. As a result, it is reasonable to conclude that the test organisms were exposed to the functional solubility/suspension equilibrium of the test substance’s constituents over the exposure period. Since no effect on the test organisms was observed at this concentration, the presence of small amounts of fine, suspended particles in the exposure solutions did not have an impact on the evaluation of results of this testing.





Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: See above details on results
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 analyzed to establish treatment level effects. Statistical analyses of the data generally followed the procedures described in the document, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application and the OECD 210 Fish Early-Life Stage Toxicity Test.
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.

No significant response was observed for any endpoints at any test concentration. As a result, ECx conditions for the determination of ECx values were not met due to a significant lack of fit with the available regression models.

Table 1 - Exceptions to normal observations on surviving fish at exposure termination

Mean Measured Concentration (mg/L)

Replicate

Observations

Control

B

One small fish with a tail deformity

C

One small fish

D

One small fish with a tail deformity

Solvent Control

A, D

One small fish

B

Three small fish

C

Two small fish

0.11

A

One small fish

B

Two small fish

Table 2 - Exposure Solution Analyses (Total Constituents) 

Nominal

Concentration

(mg/L)

Measured Concentration (mg/L)

Mean

(SDa)

Percent of Nominala

% CVa

Range of Mean Measured

(%)

 

Day 0

Day 4

Day 11

Day 18

Day 26

Day 32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control

<0.0010b

<0.0010

<0.0010

<0.0010

<0.0010

<0.0010

NAc

(NA)

NA

NA

NA

 

 

 

 

 

 

 

 

 

 

 

Solvent Control

<0.0010

<0.0010

<0.0010

<0.0010

<0.0010

<0.0010

NA

(NA)

NA

NA

NA

 

 

 

 

 

 

 

 

 

 

 

0.050

0.056

0.10

0.12

0.091

0.092

0.19

0.11

(0.044)

220

41

51 - 170

 

 

 

 

 

 

 

 

 

 

 

5000 (stock)

4600

4900

5500

5200

5300

4800

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

QCd#1

0.00235

0.00230

0.00184

0.00295

0.00263

0.00294

 

 

 

 

0.00250

(94.2)

(92.0)

(73.7)

(118)

(105)

(117)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

QC #2

0.0125

0.0143

0.0142

0.0172

0.0151

0.0173

 

 

 

 

0.0150

(83.1)

(95.1)

(94.5)

(115)

(101)

(116)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

QC #3

0.0421

0.0394

0.0380

0.0619

0.0474

0.0852

 

 

 

 

0.0500/0.125e

(84.2)

(78.7)

(76.0)

(124)f

(94.7)

(68.2)f

 

 

 

 

 

aMean measured concentrations, standard deviation (SD), percent of nominal, and coefficients of variation (CV) were calculated using the actual analytical result (unrounded values) and not the rounded values presented in this table.

bConcentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls.

cNA = Not Applicable

dQC = Quality Control sample. The percent recovery for the corresponding QC sample is presented in parentheses.

eNominal fortified concentration for QC #3 was 0.0500 mg/L on days 0, 4, 11, 18, and 26. Nominal fortified concentration for QC #3 was 0.125 mg/L on day 32.

fThe percent recovery of this QC sample was outside of the acceptance criteria (i.e., 70.0 to 120%, Appendix 4). See Section 3.3.2 for additional discussion.

 

Table 3 - Endpoint Summary

Nominal Concentration

(mg/L)

Mean Measured

Concentration

(mg/L)

Replicate

Embryo

Hatching Success

(%)

Live, Normal Larvae

at Hatch

(%)

28 Days Post-Hatch

Larval

Survival

(%)

Total

Length (SDa)

(mm)

Wet

Weight (SD) (g)

 

 

 

 

 

 

 

 

Control

Control

A

94

82

95

22.31 (1.44)

0.0856 (0.0196)

 

 

B

86

97

90

22.66 (2.78)

0.0934 (0.0288)

 

 

C

86

100

95

22.16 (2.98)

0.0910 (0.0342)

 

 

D

91

78

100

21.94 (3.09)

0.0843 (0.0316)

 

 

Mean (SD)

89 (4)

89 (11)

95 (4)

22.27 (0.30)

0.0886 (0.0043)

 

 

 

 

 

 

 

 

Solvent Control

Solvent Control

A

86

97

95

22.27 (2.94)

0.0961 (0.0260)

 

 

B

77

100

100

21.50 (4.46)

0.0904 (0.0417)

 

 

C

89

90

95

21.56 (3.90)

0.0948 (0.0373)

 

 

D

86

97

90

22.48 (3.52)

0.0988 (0.0371)

 

 

Mean (SD)

84 (5)

96 (4)

95 (4)

21.96 (0.50)

0.0950 (0.0035)

 

 

 

 

 

 

 

 

Pooled Control

Pooled Control

Mean (SD)

87 (5)

93 (8)

95 (4)

22.11 (0.42)

0.0918 (0.0050)

0.050

0.11

A

86

90

100

23.22 (2.98)

0.1022 (0.0317)

 

 

B

86

90

100

23.04 (3.25)

0.1081 (0.0353)

 

 

C

86

97

95

22.61 (1.45)

0.0966 (0.0195)

 

 

D

86

100

100

22.48 (1.84)

0.0945 (0.0253)

 

 

Mean (SD)

86 (0)

94 (5)

99 (3)

22.84 (0.35)

0.1003 (0.0061)

 

aSD = Standard Deviation

Validity criteria fulfilled:
yes
Conclusions:
In a 32-day (28 days post-hatch) 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 0.011 mg/L (mean measured concentration) based on no effects on survival and growth at the highest concentration tested.
From the analytical monitoring data it is reasonable to conclude that the test organisms were exposed to the functional solubility/suspension equilibrium of the test substance’s constituents over the exposure period. Since no effect on the test organisms was observed at this concentration, the presence of small amounts of fine, suspended particles in the exposure solutions did not have an impact on the evaluation of results of this testing.

Description of key information

NOEC (32-day (28 days post-hatch)): ≥0.11 mg/l (mean measured), survival and growth, Pimephales promelas.

Key value for chemical safety assessment

Additional information

In a 32-day (28 days post-hatch) early-life stage toxicity test in Fathead Minnow (Pimephales promelas), conducted with the registration substance according to OECD TG 210 and in compliance with GLP, a NOEC was concluded to be equal to or higher than 0.05 mg/l in terms of nominal concentration and 0.11 mg/l mean measured concentration, based on no effects on survival and growth at the highest concentration tested.

The mean measured concentration is higher than the nominal test substance concentration and higher than the limit of water solubility of the test substance. The high recoveries of the exposure solutions are an indication that functional solubility has been exceeded and that there were undissolved particles present in the exposure system. These particles were likely very fine since they were not detected by visual observations. The test substance is a complex mixture of materials which have different functional solubilities. The results from this testing indicate that the recoveries of constituents 2 and 3 were consistently higher than constituent 1 throughout the exposure demonstrating this difference. It should be noted that the test concentration selected for this study (i.e. 0.050 mg/l) was to approximate the functional limit of solubility for constituent 1 although it was known that this nominal dose may be in excess of the other two constituents' solubility.

From the analytical monitoring data it is reasonable to conclude that the test organisms were exposed to the functional solubility/suspension equilibrium of the test substance’s constituents over the exposure period. Since no effect on the test organisms was observed at this concentration, the presence of small amounts of fine, suspended particles in the exposure solutions did not have an impact on the evaluation of results of this testing.

These data are supported by reading across long-term toxicity to fish data from two surrogate substances within the siloxanes category with similar physico-chemical properties, decamethyltetrasiloxane (L4, CAS 141-62-8, EC No. 205-491-7, log Kow 8.2) and dodecamethylcyclohexasiloxane (D6, CAS 540-97-6, EC No. 208-762-8, log Kow 9).

Long-term fish toxicity data are also available for the effects of constituent 1 of phenyl silsesquioxanes, 1,1,1,5,5,5 -hexamethyl-3-phenyl-3-[(trimethylsilyl)oxy]trisiloxane (CAS 2116-84-9, EC No. 218-320-6).

No effects on fish have been reported at the limit of solubility of the substances:

A 60-d (post-hatch) NOEC of ≥0.0079 mg/l (limit of solubility) was determined for the effects of the read-across substance, L4, on hatching, larval survival, abnormal appearance or behaviour (OECD TG 210) in Oncorhynchus mykiss (rainbow trout). The results are expressed as time-weighted mean measured concentrations.

A 45-day NOEC of ≥4.4 μg/l has been determined for the effects of constituent 1 of phenyl silsesquioxanes (1,1,1,5,5,5-hexamethyl-3-phenyl-3-[(trimethylsilyl)oxy]trisiloxane) on mortality and behaviour of Lepomis macrochirus. The results are expressed relative to mean measured concentrations of the substance and were obtained as part of a flow-through test that was undertaken to determine the potential for bioconcentration of the substance from water to fish.

In two further BCF studies, a 49-day NOEC of ≥4.4 μg/l (mean measured) and a 35-day NOEC of ≥5.3 μg/l (meas measured) were determined for the effects of the read-across substances, D6 and L4, respectively, on mortality of Pimephales promelas.

Although an experimental study is available with phenyl silsesquioxanes, these read-across studies are included as evidence that the substance is not toxic at its limit of water solubility and to support the algal data that are also read-across from L4 and D6. The data with the registration substance and the surrogates used for read-across all follow the same pattern of no effects at the limit of solubility of the substances.