Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

GLP compliance:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: Day -4 (pre-test), Day -3 (pre-test) and days 0 (0 and 4 hours), 1, 3, 7, 14, 21, 24, 28, 35, 36, 42 of uptake phase, and on depuration days 1,4,7,14,28,42 and 60.

- Sampling intervals/frequency for test medium samples: Day -4 (pre-test), Day -3 (pre-test) and days 0 (0 and 4 hours), 1, 3, 7, 14, 21, 24, 28, 35, 36, 42 of uptake phase, and on depuration days 1,4,7,14,28,42 and 60.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Two water samples were collected from the solvent control and three samples were collected from each of the two treatment groups. One solvent control samples and two samples from each of the treatment groups were analysed for test substance. The remaining samples were held in reserve as backup samples. All water samples were collected from mid-depth of each test chamber using a volumetric pipette. Water samples were analysed as soon as possible after collection without storage, if possible, otherwise samples were stored frozen until analysis.
A sufficient number of fish were collected at each sampling interval to provide two replicate samples of solvent control fish and four replicate samples of each treatment group fish. Fish were impartially removed from the test chambers and euthanised by severing the spinal cord above the opercular region. The fish were blotted dry and measured for total length and wet weight within approximately 15 minutes of collection, when possible. Each fish was then dissected into edible and nonedible tissue fractions. Dissection was accomplished by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The head, fins and viscera were removed from the body and were considered to be nonedible tissue. The remaining tissue was considered the edible tissue. Tissue samples were transferred to tared vials and weighed. All tissue samples were extracted immediately or stored at approximately -14 °C until extraction.
Selected fish were collected to determine lipid content and tissue solids content. Fish were sampled on Day 0 of uptake, on Day 45 of uptake and on Day 60 of depuration.
Water and fish samples were extracted using hexane. Concentrations of test substance in the hexane extracts were determined using a Hewlett-Packard Model 5890 Gas Chromatograph equipped with a Hewlett-Packard Model 5971 Mass Selective Detector operated in the SIM mode.

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared weekly during the uptake phase of the study. Individual stocks were prepared for each test concentration by dissolving the test substance in dimethylformamide at concentrations of 0.010 and 0.060 mg a.i./ml. The stock solutions were inverted to aid the solubilisation of the test substance.
The two stock solutions and DMF for the solvent control were injected into the diluter mixing chamber where they were mixed with dilution water to achieve the nominal concentrations.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test solutions were clear and colourless in both the mixing chambers and test chambers for all treatments at test initiation and termination.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish

- Source: Osage Catfisheries, Inc. Osage Beach, Missouri 65065

- Age at study initiation (mean and range, SD): Juveniles, hatched April 2, 2002

- Length at study initiation (mean and range): 61 mm (range 46- 74 mm)

- Weight at study initiation (mean and range): 3.15 g (1.04 - 5.82 g)

- Weight at termination (mean and range): 2.26 g (range 1.24 - 3.41 g)

- Feeding during test: Yes

- Food type: Commercially-prepared diet (flake food) supplied by Zeigler Brothers, Inc

- Frequency: once, daily

ACCLIMATION
- Acclimation period: 51 hours

- Acclimation conditions (same as test or not): same

- Health during acclimation (any mortality observed): No mortalities

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

45 d

Total depuration duration:

60 d

Test temperature:

22 ± 1°C

pH:

8 to 8.6

Dissolved oxygen:

Remained ≥6.2 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 106-L stainless steel aquaria filled with approximately 80 L of test solution

- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow diluter with peristaltic pump.

- Renewal rate of test solution (frequency/flow rate): Approximately 6.3 volume additions per 24 hours.

- No. of organisms per vessel: 90

- No. of vessels per concentration (replicates): 1

- No. of vessels per control / vehicle control (replicates): 1

- Biomass loading rate: 0.56 g fish/l/day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered well water, characterised as moderately hard water.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light; 8 hours dark

- Light intensity: 356 lux

Nominal and measured concentrations:

Nominal: 1.0 µg a.i./l; 6.0 µg a.i./l
Mean measured: 0.80 µg a.i./l; 4.4 µg a.i./l

Reference substance (positive control):

no

Lipid content:

5.41 %

Time point:

end of exposure

Remarks on result:

other: 0.80 µg a.i./l

Lipid content:

7.52 %

Time point:

end of exposure

Remarks on result:

other: 4.4 µg a.i./l

Key result

Type:

BCF

Value:

1 011 L/kg

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:0.80 µg a.i./l

Key result

Type:

BCF

Value:

384 L/kg

Basis:

whole body w.w.

Time of plateau:

21 d

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:4.4 µg a.i./l

Key result

Type:

BCF

Value:

2 992 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: Conc.in environment / dose:0.80 µg a.i./l

Key result

Type:

BCF

Value:

1 208 L/kg

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: Conc.in environment / dose:4.4 µg a.i./l

Key result

Elimination:

no

Parameter:

other: DT50 0.80 µg a.i./l

Depuration time (DT):

43 d

Key result

Elimination:

no

Parameter:

other: DT50 4.4 µg a.i./l

Depuration time (DT):

55 d

Details on kinetic parameters:

0.80 µg a.i./l: Uptake rate constant (k1, Day-1) 36.9; Depuration rate constant (k2, Day-1) 0.161; BCFk 2292.
4.4 µg a.i./l: Uptake rate constant (k1, Day-1) 15.1; Depuration rate constant (k2, Day-1) 0.0125; BCFk 1208.

Details on results:

- Mortality of test organisms: All fish in the solvent control and treatment groups appeared normal with no treatment-related signs of toxicity.

Table 1: Bioconcentration factors at different time points and concentrations in water

	Duration of exposure (days)	0hr	4hr	1	3	7	14	21	24	28	35	36	42
Low Concentration level	Concentration in the water (μg a.i./l)	0.815	0.862	0.819	0.947	0.821	0.734	0.828	0.870	0.853	0.772	0.667	0.776	Mean measured test concentration
		0.812	0.837	0.833	0.915	0.788	0.688	0.791	0.781	0.816	0.774	0.604	0.724	0.80 μg a.i./l
Low Concentration level	Concentration in fish (μg a.i./kg)	-	<11.7	<16.7	49.5	123	321	429	-	466	731		542	Mean measured steady-state concentration
		-	<8.23	<18.6	<56.9	154	311	352	-	653	1023		1034	(Days 28, 35,
		-	<7.00	<25.2	56.8	207	392	510	-	836	938		822	42) 809 μg
		-	<11.2	<21.3	68.9	168	449	545	-	699	886		1073	a.i./kg
Low Concentration level	Bioconcentration factor													Steady-State BCF 1011 l/kg
High Concentration level	Concentration in the water (μg a.i./l)	4.59	4.28	4.18	5.19	4.80	4.34	1.97	5.22	5.44	4.55	-	3.78	Mean measured test concentration
		4.69	4.43	3.99	5.15	4.83	4.32	2.07	5.23	5.35	4.55	-	3.80	4.4 μg a.i./l
High Concentration level	Concentration in fish (μg a.i./kg)	-	<19.1	5.97	292	533	1429	1296	-	1441	1142	-	1011	Mean measured steady-state concentration
		-	<21.2	86.8	260	679	735	1177	-	1932	1937	-	2014	(Days 21, 28,
		-	<20.1	89.2	241	639	1333	1578	-	1978	1862	-	1934	35, 42) 1691 μg
		-	<19.5	<79.6	183	607	955	932	-	2022	2165	-	2631	a.i./kg
High Concentration level	Bioconcentration factor	-	134	563	1200	1790	1910	1840	1770	1770	1540	1610	1940	Steady-State BCF 384 l/kg

 Table 2: Depuration

 

	Days	1	4	7	14	28	42	60
Low concentration level	Concentration in the water(µg a.i./l)	<LOQ; <LOQ	<LOQ; <LOQ	<LOQ; <LOQ	<LOQ; <LOQ	<LOQ; <LOQ	<LOQ; <LOQ	<LOQ; <LOQ
		829	1017	814	1063	771	657	242
Low concentration level	Concentration in fish (µg a.i./kg)	797	1014	896	809	721	181	452
		980	977	1016	762	773	598	519
		951	1063	1161	762	750	746	415
High concentration level	Concentration in the water(µg a.i./l)	<0.786	<0.786	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
		<0.786	<0.786	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
		2538	2163	2681	1295	1592	1615	904
		3206	2479	2502	2654	1977	1311	1063
High concentration level	Concentration in fish (µg a.i./kg)	2627	2586	1619	2127	1864	903	1332
		2497	2004	2289	1979	1900	1700	1861

Conclusions:

Steady-state BCF values of 1011 l/kg (0.80 µg a.i./l) and 384 (4.4 µg a.i./l) and kinetic BCF values of 2992 l/kg (0.80 µg a.i./l) and 1208 (4.4 µg a.i./l) were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information

Bioaccumulation: aquatic:

BCFss 1011 l/kg (0.80 µg a.i./l); 384 (4.4 µg a.i./l)

BCFk 2992 l/kg (0.80 µg a.i./l); 1208 (4.4 µg a.i./l)

Lipid normalised (to 5%) values are:

BCFss 934 l/kg (0.80 µg a.i./l); 255 l/kg (4.4 µg a.i./l) and BCFk 2765 l/kg (0.80 µg a.i./l); 803 l/kg (4.4 µg a.i./l).

A BCF value of 2765 is used as a worst case. Depuration rate constants: 0.161 d-1(0.8 µg a.i./l); 0.0125 d-1(4.4 µg a.i./l). 

Key value for chemical safety assessment

BCF (aquatic species):

2 765 L/kg ww

Additional information

Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9) is a multi-constituent substance containing two main constituents; 3,3-diphenylhexamethyltrisiloxane (Constituent 1) and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (Constituent 2).

The constituents of the submission substance have log K_ow values >4.5. The reported values are >9.0 for each constituent, based on a validated QSAR estimation method (See IUCLID Section 4.7). Log K_ow values of 9.1 for Constituent 1 and 13.0 for Constituent 2 were obtained from KOWWIN v1.68 (U.S. EPA, Sept. 2000). At such high log K_ow of 13 for Constituent 2, absorption is expected to be limited due to the very limited solubility. Therefore, Constituent 2 is not expected to be bioaccumulative. Bioaccumulation (BCF) for Constituent 1 is therefore discussed below.

There are no reliable bioaccumulation data available for 3,3-diphenylhexamethyltrisiloxane (Constituent 1), therefore good quality data for the structurally-related substance, 1,1,1,5,5,5-hexamethyl-3-phenyl-3-[(trimethylsilyl) oxy]trisiloxane (PhM3T, CAS 2116-84-9) have been read across.

3,3-Diphenylhexamethyltrisiloxane and PhM3T (CAS 2116-84-9) are within the Siloxanes Category. Substances in this category have similar properties with regard to bioaccumulation.

A review of the data available for substances in this Category indicates that BCF is dependent on log K_ow as well as on chemical structure.

3,3-Diphenylhexamethyltrisiloxane (Constituent 1) and 1,1,1,5,5,5-hexamethyl-3-phenyl-3-[(trimethylsilyl) oxy]trisiloxane (PhM3T, CAS 2116-84-9) are structurally-similar substances, both are siloxanes with phenyl functionality. The target substance 3,3-diphenylhexamethyltrisiloxane is a linear siloxane containing three Si atoms linked by oxygen. The terminal silicon atoms are each substituted with three methyl groups, and the central silicon atom is substituted with two phenyl groups. The source substance 1,1,1,5,5,5-hexamethyl-3-phenyl-3-[(trimethylsilyl) oxy]trisiloxane (PhM3T, CAS 2116-84-9) is a branched structure of four Si atoms, the longest siloxane chain contains three silicon atoms and two oxygen atoms, with a Si-O branch on the central silicon atom in the chain. The terminal silicon atoms are each fully methyl substituted, whilst the central silicon atom has one phenyl group attached.

3,3-Diphenylhexamethyltrisiloxane and 1,1,1,5,5,5-hexamethyl-3-phenyl-3-[(trimethylsilyl) oxy]trisiloxane (PhM3T, CAS 2116-84-9) both have predicted log Kow values of 9. It is therefore considered valid to read-across the results for PhM3T to fill the data gap for Constituent 1 of the submission substance. Additional information is given in a supporting report (PFA 2017at) attached in Section 13.

Steady-state BCF values of 1011 l/kg (0.80 µg a.i./l) and 384 (4.4 µg a.i./l) and kinetic BCF values of 2992 l/kg (0.80 µg a.i./l) and 1208 (4.4 µg a.i./l) were determined for PhM3T in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Lipid normalised (to 5%) values are: BCF_ss= 934 l/kg (0.80 µg a.i./l) and 255 l/kg (4.4 µg a.i./l) and BCF_k= 2765 l/kg (0.80 µg a.i./l) and 803 l/kg (4.4 µg a.i./l).

Fish bioconcentration (BCF) studies are most validly applied to substances with log K_ow values between 1.5 and 6. Practical experience suggests that if the aqueous solubility of the substance is low (i. e. below ~0.01 to 0.1 mg/l) (REACH Guidance R.11; ECHA 2017), fish bioconcentration studies might not provide a reliable BCF value because it is very difficult to maintain exposure concentrations. Dietary bioaccumulation (BMF) tests are practically much easier to conduct for poorly water-soluble substances, because a higher and more constant exposure to the substance can be administered via the diet than via water. In addition, potential bioaccumulation for such substances may be expected to be predominantly from uptake via feed, as substances with low water solubility and high K_oc will usually partition from water to organic matter.

However, there are limitations with laboratory studies such as BCF and BMF studies with highly lipophilic and adsorbing substances. Such studies assess the partitioning from water or food to an organism within a certain timescale. The studies aim to achieve steady-state conditions, although for highly lipophilic and adsorbing substances such steady-state conditions are difficult to achieve. In addition, the nature of BCF and BMF values as ratio values, means that they are dependent on the concentration in the exposure media (water, food), which adds to uncertainty in the values obtained.

For highly lipophilic and adsorbing substances, both routes of uptake are likely to be significant in a BCF study, because the substance can be absorbed by food from the water. 

Dual uptake routes can also occur in a BMF study, with exposure occurring via water due to desorption from food, and potential egestion of substance in the faeces and subsequent desorption to the water phase. Although such concentrations in water are likely to be low, they may result in significant uptake via water for highly lipophilic substances.

Goss et al. (2013) put forward the use of elimination half-life as a metric for the bioaccumulation potential of chemicals. Using the commonly accepted BMF and TMF threshold of 1, the authors derive a threshold value for k_elimination of >0.01 d^-1 (half-life 70 d) as indicative of a substance that does not bioaccumulate.

Depuration rates from BCF and BMF studies, being independent of exposure concentration and route of exposure, are considered to be a more reliable metric to assess bioaccumulation potential than the ratio BCF and BMF values obtained from such studies.

The depuration rate constants of 0.161 d^-1 (0.8 µg a.i./l) and 0.0125 d^-1 (4.4 µg a.i./l) obtained from the BCF study with PhM3T are considered to be valid and to carry most weight for bioaccumulation assessment of 3,3-diphenylhexamethyltrisiloxane. These rates are indicative of a substance which does not bioaccumulate.

Burkhard, L. P. et al., 2012 has described fugacity ratios as a method to compare laboratory and field measured bioaccumulation endpoints. By converting data such as BCF and BSAF (biota-sediment accumulation factor) to dimensionless fugacity ratios, differences in numerical scales and unit are eliminated.

Fugacity is an equilibrium criterion and can be used to assess the relative thermodynamic status (chemical activity or chemical potential) of a system comprised of multiple phases or compartments (Burkhard, L. P. et al., 2012). At thermodynamic equilibrium, the chemical fugacities in the different phases are equal. A fugacity ratio between an organism and a reference phase (e.g. water) that is greater than 1, indicates that the chemical in the organism is at a higher fugacity (or chemical activity) than the reference phase.

The fugacity of a chemical in a specific medium can be calculated from the measured chemical concentration by the following equation:

f = C/Z

Where f is the fugacity (Pa), C is concentration (mol/m³) and Z is the fugacity capacity (mol (m³. Pa)).

The relevant equation for calculating the biota-water fugacity ratio (F_biota-water) is:

F_biota-water= BCF_WD/LW/K_lwx ρ_l/ ρ_B

Where BCF_WD/LW is the ratio of the steady-state lipid-normalised chemical concentration in biota (µg-chemical/kg-lipid) to freely dissolved chemical concentration in water (µg-dissolved chemical/l-water), K_lw is the lipid-water partition coefficient and ρ_l is the density of lipid and ρ_B is the density of biota.

It can be assumed that n-octanol and lipid are equivalent with respect to their capacity to store organic chemicals, i.e. K_lw = K_ow. For some substances with specific interactions with the organic phase, this assumption is not sufficiently accurate. Measurement of K_lw values for siloxane substances is in progress. Initial laboratory work with olive oil as lipid substitute indicates that the assumption that K_lw = K_ow is appropriate (Reference: Dow Corning Corporation, personal communication). However, the calculated fugacity ratios presented here should be used with caution at this stage. 

The table below presents fugacity ratios calculated from the BCF data for PhM3T, using K_ow for K_lw.

Table: Calculated biota-water fugacity ratios for read-across substances PhM3T

Substance	Endpoint	Exposure concentration	BCF Value	Fbiota-water*
PhM3T	BCFss	0.80 µg a. i. /l	1011	2.35E-05
PhM3T	BCFss	4.4 µg a. i. /l	384	6.43E-06
PhM3T	BCFk	0.80 µg a. i. /l	2992	6.97E-05
PhM3T	BCFk	4.4 µg a. i. /l	1208	2.02E-05

*Using log K_ow 9 for PhM3T

 

The fugacity-based BCFs directly reflect the thermodynamic equilibrium status of the chemical between the two media included in the ratio calculations. The fugacity ratios calculated are all below 1, indicating that the chemical in the organism tends to be at a lower fugacity (or chemical activity) than in the water. It should be noted however, that the BCF studies may not have reached true steady-state in the timescale of the laboratory studies. The fugacity ratios indicate that uptake may be less than expected on thermodynamic grounds, suggesting that elimination is faster than might be expected on grounds of lipophilicity alone.

References:

ECHA (2017). Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.11: PBT/vPvB assessment, Version 3.0. June 2017

PFA (2017at). Siloxane Category Report for Environmental Endpoints. PFA.404.114.001