Reaction mass of 1,1,1,5,5,5-hexamethyl-3-phenyl-3-((trimethylsilyl)oxy)trisiloxane and 1,1,1,7,7,7-hexamethyl-3,5-diphenyl-3,5-bis[(trimethylsilyl)oxy]tetrasiloxane and 1,1,1,9,9,9-hexamethyl-3,5,7-triphenyl-3,5,7-tris((trimethylsilyl)oxy)pentasiloxane

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-02 to 2008-12-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Prior to test initiation and before division into the replicate exposure chambers, a sample of sediment from each treatment level and control was removed and analyzed. In addition, subsamples of the dosing stock solutions used to dose the sediments were also analyzed for test substance concentration. Results of the pretest analyses were used to judge whether sufficient quantities of D5 had been applied to the sediment.

During the in-life phase of the definitive study, sediment samples were removed and analyzed for D5 concentration on test days 0, 7 and 28. On day 0, samples were removed and analyzed from replicate I of all treatment levels and controls. On day 7, samples were removed and analyzed from replicate J of all treatment levels and controls, while on day 28, samples were removed and analyzed from replicate K of all treatment levels and controls. Overlying water samples were removed from the test vessels by decanting all of the overlying water from each vessel into a graduated cylinder. Following removal of the overlying water, the sediment was then centrifuged at approximately 1200 g for 30 minutes. The pore water generated by centrifugation was then removed by pipet. The sediment samples were then collected from each centrifuge tube with a stainless steel spatula.

In addition, three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure samples throughout the analytical process. These QC samples were prepared in sediment (based on sediment dry weight) at concentrations of D5 similar to the treatment level 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.

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT

- Details of spiking: Six individual dosing stock solutions (49.4, 24.7, 12.4, 6.2, 3.06 and 1.58 mg a.i./mL) were prepared in DMF for application of the test substance to the sediment. All dosing stock solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. For each dose level, a 1.5-kg aliquot of wet sediment (0.8145 kg dry weight based on a percent solids of 54.30%) along with 4.5 L of overlying water (sediment:volume ratio of 3:1) were placed in individual glass jars. Immediately prior to dosing, the contents of each jar were shaken to suspend the sediment. An 8.25-mL volume of each dosing stock solution was then added to each jar. Each jar was shaken again after dosing to distribute the test substance. After a four-hour settling period, the water was decanted from the sediment and the treated sediment was allocated to the individual test vessels.

A solvent control sample was prepared in the same manner as the treated sediment by adding 1.5 kg of wet sediment (0.8145 kg dry weight based on a percent solids of 54.30%) along with 4.5 L of overlying water (sediment:volume ratio of 3:1) and 8.25 mL of DMF in a glass jar and processed in the same manner as the treated sediments. The negative control sediment group was prepared using only untreated sediment and overlying water (no test substance or solvent). The negative control and solvent control vessels were maintained under the same conditions as the treatment vessels.

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM

- Source and culture conditions: The amphipods used during this study were obtained from laboratory cultures maintained at Springborn Smithers. Prior to test initiation, amphipods were maintained in 20-L glass aquaria containing approximately 15 L of culture water under flow-through conditions. The culture water was from the same source as water used as overlying water during the test.

Amphipods (8 days old) used in the exposure were collected from reproducing adult amphipods removed from the main culture tanks 9 days prior to test initiation. The adult amphipods were placed in 9.5-L aquaria (isolation tanks) containing approximately 8 L of water. Juvenile amphipods (< 24 hours old) produced by these isolated adults were then removed from the isolation tanks and pipetted into 1-L beakers containing approximately 0.80 L of laboratory dilution water. The juvenile amphipods were reared under static conditions for 8 days with gentle oil-free aeration. During the holding period, dissolved oxygen ranged from 8.3 to 8.6 mg/L and temperature was maintained at 22 ºC. No mortality was observed in the test population 48 hours prior to test initiation.

During holding and acclimation, the freshwater amphipods were fed 2.5 mL of a combination of yeast, cereal leaves and flaked fish food suspension (YCT) and 2.5 mL of Ankistrodesmus falcatus, a unicellular green algae, every other day. During the 28-day exposure, each replicate test vessel received 1.5 mL of YCT daily. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed. Based on these analyses, food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

Hardness ranged from 44-52 mg/L as CaCO3 and alkalinity 10-20 mg/L as CaCO3

Test temperature:

22 to 24 ºC

pH:

6.7-7.1

Dissolved oxygen:

40 - 100% saturation ( 3.4 - 8.4 mg/L)

Salinity:

Not applicable

Ammonia:

Day 0: 0.44-0.81 mg/L as nitrogen

Day 28: <0.10 mg/L as nitrogen

Nominal and measured concentrations:

Nominal concentrations: 0(Control), 0 (solvent control), 16, 31, 63, 125, 250 and 500 mg a.i./kg

Mean measured concentrations in treatments 18, 28, 62, 130, 230 and 460 mg a.i./kg

The results are interpreted with reference to mean measured concentrations

Details on test conditions:

TEST SYSTEM

- Test container (material, size): The test chambers used in the static-renewal test were 300-mL glass vessels which were chemically cleaned prior to use and rinsed several times using dilution water. Each test vessel had a hole cut on the top edge of the beaker which was covered with 40-mesh Nitex® screen for drainage.

- Sediment volume: Each vessel contained 100 mL (approximately 4-cm layer) of sediment (equivalent to 127 g wet weight per vessel or 69.2 g dry weight per vessel). The total overlying/sediment volume was maintained at approximately 275 mL.

- Overlying water volume: 175 mL. The overlying water was renewed by adding two volume additions (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system.

- Aeration: no


EXPOSURE REGIME
- No. of organisms per container (treatment): 10

- No. of replicates per treatment group: 12 replicates total. Eight replicates (A through H) were used to evaluate the biological response of the test organisms. The remaining four replicates (I through L) were maintained for the purpose of chemical analysis.

- No. of replicates per control / vehicle control: 12

- Feeding regime: During the 28-day exposure, each replicate test vessel received 1.5 mL of YCT daily. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed.


OVERLYING WATER CHARACTERISTCS

The water used during the definitive exposure was characterized as having a total hardness and total alkalinity as calcium carbonate (CaCO3) of 54 mg/L and 20 mg/L, respectively, a pH of 7.1 and a specific conductivity of 230 µmhos/cm. Representative samples of the dilution water source were analyzed for the presence of pesticides, PCBs and metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected in any of the water samples analyzed in agreement with ASTM guidelines (2002). 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 source was 0.69 mg/L for December 2008.


SOURCE OF NATURAL SEDIMENT

The sediment (Springborn Smithers Batch No. 091908) used during this study was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to use and characterization, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterized by Agvise Laboratories, Northwood, North Dakota, as having a percent organic carbon of 4.8%, a particle size distribution of 83% sand, 12% silt, and 5% clay, a pH of 6.0 and a percent moisture at 1/3 bar (water holding capacity) of 28.6%. The percent solids was measured by Springborn Smithers Laboratories to be 54.30%. A sample of the sediment pore water was generated from the sediment batch prior to testing and yielded a measured concentration of 2.4 mg/L as nitrogen (ammonia). This concentration is well below levels of concern for this organism. A representative sample of the sediment source was analyzed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that would be considered to have an adverse impact on the results of the test.


OTHER TEST CONDITIONS

- Lighting: The test area was illuminated with fluorescent bulbs at an intensity range of 840 to 870 lux

- Photoperiod: 16 hours light and 8 hours darkness


EFFECT PARAMETERS MEASURED: All vessels were examined at test initiation and at 24-hour intervals thereafter, until test termination (day 28). Observations of mortality and abnormal behavior were made and the physical characteristics of the test samples were recorded. At test termination (day 28), the total number and growth of surviving amphipods was determined in each test vessel. The remaining sediment was sieved to remove all surviving amphipods. Growth of the surviving amphipods were determined by pooling all surviving amphipods from each replicate in a tared weighing tin and drying at 60 ± 5 ºC for approximately 24 hours. The pooled amphipods were then weighed on a calibrated analytical balance to the nearest 0.01 mg. Growth as dry weight per amphipod for each replicate was calculated using the following equation:

G = TW/NS

Where:
G = Growth as dry weight per amphipod
TW = Total weight of surviving amphipods
NS = Number of surviving amphipods


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

230 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

310 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 210-360

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Reported statistics and error estimates:

The linear interpolation method within TOXSTAT® Version 3.5 was used to determine the LC50 value for survival.

During this study, no concentration tested resulted in ≥ 50% reduction in growth, therefore the EC50 value was empirically estimated to be greater than the highest mean measured sediment concentration that was statistically analyzed.

At the termination of the study, data obtained on amphipod survival and growth (dry weight) were statistically analyzed to identify significant treatment-related effects. The lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant effect (No-Observed-Effect Concentration, NOEC) were determined. Analyses were performed using the mean replicate organism response in each treatment group rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks', Chi-Square Test and Bartlett's Test, in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests.

Table 1. Mean percent survival and mean dry weight during the 28-day static-renewal exposure of freshwater amphipods (Hyalella azteca) to D5 applied to sediment.

 

Mean Measured Sediment Concentration (mg a.i./kg)	Test Day 28
	Mean Percent Survival (SD^a)	Mean Dry Weight Per Larvae in mg (SD)
Control	98 (5)	0.49 (0.07)
Solvent Control	84 (31)	0.54 (0.12)
18	94 (5)	0.51 (0.06)
28	83 (27)	0.36 (0.08)^b
62	98 (7)	0.42 (0.07)
130	93 (7)	0.46 (0.04)
230	64 (37)^c	0.39 (0.08)^d
460	19 (16)^c	0.40 (0.18)^d

 

^aSD = Standard Deviation.

^bSignificantly reduced compared to the negative control, based on Dunnett’s Test. However, due to the lack of statistical significance at the higher treatment levels tested (i.e., 62 and 130 mg a.i./kg), this effect was not considered to be biologically relevant.

^cSignificantly reduced compared to the negative control, based on Steel’s Many-One Rank Test.

^dThis treatment level was excluded from statistical analysis for growth due to the survival effect observed.

Table 2. Concentrations of D5 measured in sediment samples during the 28‑day static-renewal exposure of freshwater amphipods (Hyalella azteca) to D5 applied to sediment.

 

Nominal Sediment Concentration (mg a.i./kg)	Measured Sediment Concentration (mg a.i./kg)				Mean Percent Recovery^d
	Day 0^a	Day 7^b	Day 28^c	Mean^d
Control	< 2.0^e	< 2.0	< 2.0	NA^f	NA
Solvent Control	< 2.0	< 2.0	< 2.0	NA	NA
16	14	22	19	18	110
31	33	23	29	28	91
63	66	63	57	62	99
125	120	150	140	130	110
250	170	220	300	230	92
500	390	500	490	460	92
QC^g#1	3.00	2.34	1.53
2.00	(150)^h	(117)	(76.4)
QC#2	51.4	63.0	49.0
50.0	(103)	(126)^h	(98.0)
QC#3	535	520	550
500	(107)	(104)	(110)

 

^aAnalytical samples were removed from replicate I.

^bAnalytical samples were removed from replicate J.

^cAnalytical samples were removed from replicate K.

^dMean measured and percent recovery values were calculated using the actual analytical results and not the rounded values (two significant figures) presented in this table.

^eConcentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for this analysis was set at 2.0 mg a.i./kg, the lowest validated concentration.

^fNA = Not Applicable.

^gQC = Quality Control sample. Percent of nominal is presented in parentheses below the measured results.

^hPercent recovery for this QC sample is outside of the acceptable range (i.e., 70.0 to 120%,Appendix 2).

 

Validity criteria fulfilled:

yes

Conclusions:

A 28-day LC50 of 310 mg/kg dry weight has been determined for the effects of the test substance on mortality of Hyalella azteca when exposed via sediment. A 28-day NOEC of 130 mg/kg and a LOEC of >130 mg/kg have been determined for effects on growth rate.

Description of key information

NOEC 130 mg/kg dw (135 mg/kg dwt normalised to 5% OC)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

135 mg/kg sediment dw

Additional information

Long-term sediment toxicity studies are not required for Annex IX substances under the REACH Regulation. However, due to the high sediment adsorption potential of the substance and the potential for persistence in the sediment, the compartment has been assessed.

No sediment toxicity data are available for the registration substance, phenyl silsesquioxanes (EC No. 939-487-8).

A category approach is applied to this endpoint and is detailed in the Siloxane Category report (PFA, 2017). The hypothesis for read-across of sediment ecotoxicity evidence within the Siloxanes Category is that no structure-based or property-based pattern is evident from the category dataset of existing studies, although patterns are identifiable associated with extrinsic aspects of test design to which effects may be attributed. The approach will be revisited in the event that reliable new data become available. With this in mind, a single overall interpretation is made across the category. To fulfil the requirements of REACH, a conservative approach is made by reading across on a nearest-neighbour basis the reliable data within the category.

In the context of the RAAF, Scenario 6 is expected to apply to this endpoint. It is considered that effects observed in benthic organisms are associated primarily with extrinsic factors associated with test design and not to structural similarities as such.

 

Over twenty-five sediment toxicity studies for siloxanes are available and have been reviewed in detail. The majority of these studies are of standard duration in standard test species. There is a general trend for studies using natural sediment, which all have pH <~8, to show no effects, or higher NOECs than corresponding studies with artificial sediment. No significant toxicity (NOEC <100 mg/kg) in any organism is found at pH near 7 with natural sediment. The data suggest that it is possible to read across sediment toxicity data between different siloxane structures, especially where natural sediment data are available, given that studies which are not suspected to be confounded by extrinsic factors show relatively minimal effects across the dataset.

READ-ACROSS JUSTIFICATION

No data are available for the effects of the registration substance on sediment organisms. Data are read-across from low functionality siloxanes of a similar molecular weight, decamethylcyclopentasiloxane (D5, CAS 541-02-6, EC No. 208-764-9) and dodecamethylcyclohexasiloxane (D6, 540-97-6, EC No. 208-762-8).

The registered substance (phenyl silsesquioxanes) and read-across substances, D5 and D6, are members of the Reconsile Siloxanes Category. They are methylated siloxanes containing Si atoms linked by oxygen. The registration and read-across substances have similar physicochemical properties (low water solubility, high log K_ow and slow hydrolysis rates), are not readily biodegradable and have high potential for adsorption to sediment.

A result for effects in natural sediment on the invertebrate amphipod Hyalella azteca is read across from D5. The result from that test is a 28 d EC₅₀ value of >130 mg/kg sediment dry weight for growth rate and a NOEC of 130 mg/kg sediment dry weight (135 mg/kg dw normalised to 5% OC) for growth rate.

The following results are read-across from D6 in studies using natural sediment:

A 28-d EC₅₀ value of >420 mg/kg dry weight and a NOEC of ≥420 mg/kg dry weight (≥656 mg/kg dw normalised to 5% OC) have been determined for effects on numbers and biomass of Lumbriculus variegatus. A 28-d EC₅₀ value of >620 mg/kg dry weight and a NOEC of ≥620 mg/kg dry weight (≥969 mg/kg dw normalised to 5% OC) have been determined for effects on emergence and development rate of Chironomus riparius.

The results of all tests are expressed relative to mean measured exposure concentrations in the treated sediment.

The NOEC value normalised to 5% OC of 135 mg/kg dw sediment for effects on Hyalella azteca is used as the key value for the CSA.