2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane

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

Study period:

22 August 2017 to 05 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms:
uptake phase: Day 7, 14
depuration phase: Day 0, 1, 2, 4, 7, 14, 21, 28, 35, 42, 49, 56
At sampling intervals, fish were removed, euthanised, weighed (wet weight) and measured (total length).

- Sampling intervals/frequency for test medium samples:
pH-value, oxygen saturation: twice per week
temperature: measured continuously, once per hour
TOC, residual chlorine: weekly, from the water supply tank

- Sample storage conditions before analysis: Test item stored at room temperature, protected from light, in tightly closed original container.

Vehicle:

yes

Remarks:

sunflower oil

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

Preparation of food: 500 g commercial fish food was spiked by mixing the test item dissolved in sunflower oil (Heirler Cenovis GmbH, Germany) with a final concentration of 20 µg test item / g food: 10 mg test item were weighed in a 20 mL flask, added with 5 g sunflower oil and treated with ultrasonic for 5 minutes. 5 g sunflower oil were added step-by-step and the suspension was finally heated to 35 °C for approx. 105 min.
500 g fish food was weighed into a 1000 mL graduated flask (corresponding to a bulk volume of approx. 900 mL).
Approx. 100 mL fish food was filled into the next 1000 mL graduated flask and added step-by-step with the suspension whereas the 20 mL flask was washed with further 5 mL oil and added to the food. After continuous shaking the flask on a rotary mixer for 60 min first approx. 200 mL, then 250 mL of the food were added to the spiked food and shaken for 60 min, respectively. Before shaking overnight, the remaining food was added to the spiked food to have the complete amount of 500 g weighed fish food in the flask. The next day the food was filled into 20 mL vials and stored headspace free at at 6 ± 2 °C until using. Control food was treated in an identical manner as the food for the test item but without the addition of the test item. Food containing test item was used no longer than 24 hours after spiking, unless stability was confirmed by concentration control analytics. Spiked control food without test item was stored at room temperature used for up to 14 days. [Palatability of spiked food was evaluated in a non-GLP preliminary test before start of exposure phase.]

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout

- Source: Forellenzucht Harkenbleck, Steinbrink 41, 30966 Hemmingen, Germany. All fish used in the test originated from the same delivery of the supplier

- Age at test start: Juvenile rainbow trout with an age of 15 weeks

- Length at study initiation (mean, SD): 6.74±0.201 (based on ten representative fish sampled from the stock fish at test start)

- Weight at study initiation (mean , SD): 3.20±0.33 g (based on ten representative fish sampled from the stock fish at test start)

- Weight at termination (mean, SD): 11.7±4.5 g (control); 11.0±3.0 g

- Lipid content at test initiation (mean): 5.21%

- Health status: No disease treatments were carried out 2 weeks preceding the start of the test.

- Feeding during test
- Food type: FORELLENFUTTER ZUCHTFUTTER 0.8 – 1.2 mm (supplier: TEICHCENTER-RIEGER, Nüdlingstr. 13, 63607 Wächtersbach-Aufenau, Germany; producer: COPPENS INTERNATIONAL BV, P.O. Box 534, 5700 AM Helmond, the Netherlands). This product is composed of fish products, oils and fats, cereal grains, yeast, byproducts, minerals and vitamins

- Amount: 1.5% of the fish body weight per feeding day

- Frequency: daily

ACCLIMATION
- Acclimation period: Only rainbow trout with at least 12 days of acclimatisation in the laboratory and mortality < 5 % within the last 7 days before start of the exposure were used in the test.

- Acclimation conditions (same as test or not): At least 7 days before testing the fish were held in water of the quality to be used in the test. The stock population was fed with the same type of food used during the test.

- Type and amount of food: same food type as for holding period

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

56 d

Hardness:

Day 0: 66 mg CaCO3/L
Day 70: 62 mg CaCO3/L

Test temperature:

14.0 - 15.8 °C (mean 14.4 °C)

pH:

control group: 7.60 ± 0.25 (range: 7.16 - 7.98)
test item group: 7.58 ± 0.22 (range: 7.18 - 7.89)

Dissolved oxygen:

control group O2 saturation (%) : 96% ± 1.53 (range: 93-100%)
test item group O2 saturation (%) : 96% ± 2.00 (range: 92-100%)

TOC:

1.36±0.23 mg/L (range 1.07 - 1.90)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria (total volume 175 L) covered by glass tops were used.

- Test volume: about 145 L

- Aeration: The test vessels were aerated.

- Renewal rate of test solution (frequency/flow rate): The flow rate was 21 ± 1 L/h. Membrane piston pumps provided the water flow-through. The water volume per test vessel was exchanged approximately 3.5 times per day based on flow rate. The accuracy of flow rates was checked weekly.

- No. of organisms per vessel: ten

- No. of vessels per concentration (replicates): One replicate per test group

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

- Biomass loading rate: Loading did not exceed a range of 0.1 to 1.0 g of fish (wet weight) per litre per day

TEST MEDIUM / WATER PARAMETERS
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 – 250 mg CaCO3/L

pH-value: 6.0 – 8.5

Acidity:
0.1 mmol/L (recent measurement 2018-02-21 and 2018-05-16)

Alkalinity:
1.0 mmol/L (recent measurement 2018-02-21)
0.7 mmol/L (recent measurement 2018-05-14)

Conductivity:
169 µS/cm (recent measurement 2018-02-21)
161 µS/cm (recent measurement 2018-05-14)


OTHER TEST CONDITIONS
- Photoperiod: 16 / 8 h photoperiod daily

- Light intensity: 5.40 - 540 lux

- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): On working days (Mondays to Fridays), food was given in two separate feedings, approximately in the morning and in the afternoon. Otherwise (Saturday s, Sundays, bank holidays), the food was given in one single feeding per day.

- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: The lipid content of 15 % raw fat of the food is provided by the manufacturer.

- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: 16% (after addition of approximately 14 g sunflower oil/500 g food)

RANGE-FINDING / PRELIMINARY STUDY
- Palatability of spiked food was evaluated in a non-GLP preliminary test before start of exposure phase. No differences in feeding behaviour between treated and untreated food was documented.

Nominal and measured concentrations:

Nominal concentration: 20 µg/g food
Geometric mean measured concentration: 14.1 µg/g food

Reference substance (positive control):

no

Lipid content:

5.21 %

Time point:

start of exposure

Lipid content:

4.66 %

Time point:

end of exposure

Remarks on result:

other:

Remarks:

control group

Lipid content:

5.19 %

Time point:

end of exposure

Remarks on result:

other:

Remarks:

exposure group

Lipid content:

7.15 %

Time point:

other: depuration phase, day 28

Remarks on result:

other:

Remarks:

control group

Lipid content:

5.62 %

Time point:

other: depuration phase, day 28

Remarks on result:

other:

Remarks:

exposure group

Lipid content:

7.06 %

Time point:

other: depuration phase, day 56

Remarks on result:

other:

Remarks:

control group

Lipid content:

7.17 %

Time point:

other: depuration phase, day 56

Remarks on result:

other:

Remarks:

exposure group

Key result

Conc. / dose:

14.1 µg/g food

Type:

BMF

Value:

0.162 dimensionless

Basis:

whole body w.w.

Key result

Conc. / dose:

14.1 µg/g food

Type:

BMF

Value:

0.44 dimensionless

Basis:

normalised lipid fraction

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

18.6 d

Remarks on result:

other: growth corrected

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.037

Rate constant:

growth-corrected half-life (d)

Value:

18.6

Rate constant:

growth rate constant (d-1)

Remarks:

control

Value:

0.023

Rate constant:

growth rate constant (d-1)

Remarks:

exposure group

Value:

0.022

Rate constant:

overall depuration rate constant (d-1)

Value:

0.059

Individual and mean concentrations of the Test Item in fish during the update and depuration phase are provided as an attachment to the EPSR.

Calculation of Biomagnification Factors (BMF)

Evaluations are based on a food ingestion rate (I) of 0.015 g food/g fish/day.

Lipid correction factor

 

Lipid in fish at uptake day 14:               5.02 % of weight

Lipid in fish at depuration day 28:          6.75 % of weight

Lipid in fish, mean value:                       5.89 % of weight

Lipid in diet:                                         16 % of weight

                                                           (after addition of approximately 14 g sunflower oil/500 g food)

 

Lipid factor calculation:                         Lipid in fish(mean)        =         5.89%

                                                           Lipid in diet                              16%

 

Lipid factor:                                        0.368

Validity criteria fulfilled:

yes

Conclusions:

A lipid normalised growth corrected BMF value of 0.440 was obtained in a reliable study conducted according to an appropriate test protocol and in compliance with GLP.

Description of key information

BMF 0.162 (kinetic, growth corrected); BMF 0.440 (lipid-normalised, growth-corrected kinetic). A BMF value of 0.440 is selected as the key value for CSA as a worst-case.

Depuration rate constant from BMF study (growth-corrected): 0.0373 d^-1

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.44

Additional information

A growth-corrected kinetic dietary BMF value of 0.162 and a growth-corrected, lipid-normalised kinetic dietary BMF value of 0.440 were determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP. Steady-state conditions were not achieved during the uptake phase of the study, and therefore a steady-state BMF is not reported.

The depuration rate from the BMF studies, being independent of exposure concentration and route of exposure, is considered to be a more reliable metric to assess bioaccumulation potential than the ratio BMF values obtained from such a study. 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.

The depuration rate constant of 0.0373 d^-1(corresponding half-life 18.6 days) obtained in the BMF study is indicative of a substance which does not bioaccumulate.

Annex 8 of the OECD TG 305 summarises some approaches currently available to estimate tentative BCFs from data collected in a BMF study. Since the dietary bioaccumulation study gives a depuration rate constant (k₂), if an uptake rate constant (k₁) can be estimated from available data, then a kinetic BCF could be estimated for aquatic exposure (i.e. kinetic BCF = k₁/k₂). The shortcomings inherent in such an estimation are acknowledged in OECD TG 305, significantly, that the overall rate of depuration is assumed to be independent of the exposure route (i.e. the same elimination processes are occurring), and that there are considerable uncertainties in the available methods for estimation of the uptake rate constant.

A detailed analysis of available methods for estimation of the uptake rate constant is presented by Crookes and Brooke (2011). Methods reported correlate the uptake rate to fish weight, K_ow, or a combination of these (and other) factors. Most models are derived using a limited training set, and substances within the training sets are predominantly neutral organic substances and do not represent a broad range of functionalities.

In accordance with REACH R7c, estimates of k₁ should be derived according to all the models available to give a range of BCFs. Crookes and Brooke (2011) found thirteen methods to be potentially suitable for the estimation of k_1. The outputs of these thirteen models using the substance physico-chemical data and inputs obtained from the BMF study are reported in the table below (details of the methods are not reported here). Only those methods that require inputs which are readily available are reported (i.e. log K_ow, fish weight, dissolved oxygen concentration).

Table 4.3.2 Tentative estimated k₁ and BCF values

Method for estimating k1^(a)	Estimated k1 (l kg^-1day^-1)	Estimated BCF (l kg^-1)
		Not growth corrected	Growth corrected
Sijm et al. (1995)	348	5882	9320
Hayton and Barron (1990)	394	6660	10552
Erickson and McKim (1990a)	552	9339	14797
Barber et al. (1991)	541	9159	14512
Barber (2003) - observed	347	5876	9311
Barber (2003) - calibrated	402	6796	10768
Barber (2001)	566	9575	15171
Streit and Sire (1993)	334	5657	8964
Erickson and McKim (1990b)	428	7234	11462
Hendriks et al. (2001)	526	8906	14111
Tolls and Sijm (1995)	1950	32992	52275
Spacie and Hamelink (1982)	2009	33995	53863
Thomann (1989)a^(b)	18	310	492

 

 

(a)  Estimates based on fish weight of 3.52g at end of uptake phase. Where a log K_owwas required, a value of 9.0 was used.

(b)  This method also requires dissolved oxygen concentration. A DOC of 10.1mg l^-1was calculated from the BMF study data.

Crookes and Brooke tested these methods over the log K_ow range of 3.5 to 8.2, and the training set used did not include any siloxane substances. Poorly soluble substances with a high log K_ow are expected to have very limited uptake rates from aqueous exposure, due to the limited transfer across gill membranes. The log K_ow of the registration substance is 9.0 (calculated by QSAR), and the substance is very poorly soluble (1.3E-06 mg/l at 20-25°C). Given these properties, there is considerable uncertainty in estimation of the uptake rate constant from water using the various methods, and this is reflected in the wide range of tentative BCF values estimated. Bioaccumulation following aqueous exposure to the ultimate degradation product 3,3,3-trifluoropropylmethysilanediol is unlikely, on the basis of a predicted log K_ow value of 1.0.