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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 2nd to 24th May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The WAF and the control were analytically verified via GC MS at least once within every 7 days during the test period in the fresh media at the start of an exposure renewal interval on days 0, 9, 14, 16 and 19 (0 hours) and in the old media at the end of an exposure renewal interval on days 2, 12, 16, 19 and 21 (48 or 72 hours, respectively).
- Sampling method: At the start of an exposure renewal interval (0 hours), samples of the fresh media were taken and analysed after preparation of the WAF.
At the end of an exposure renewal interval (48 or 72 hours), samples of the old media were taken for analysis from separate replicates of the WAF and the control, which were prepared without daphnids and food algae and stored under test conditions until sampling. For the longest exposure interval of 72 hours, samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) at least once within the test period.
At the beginning of one exposure interval (study day 9) 3 separate sample replicates were taken and analysed after preparation of the limit loading. The relative standard deviation was calculated.

- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A WAF at limit loading level of 1 mL/L (0.98 g/L) of the test item in Elendt M4 medium was prepared one day before each water renewal. To reduce the potential of any hydrolysis loss, the pH of each freshly prepared bulk preparation and control were close to neutral. For this purpose, the Elendt M4 medium was adjusted to pH 7 ± 0.2 by addition of 1 M HCl prior to addition of the test item.
A glass flask with a lateral outlet was filled with an appropriate amount of Elendt M4 medium (pH 7) and an appropriate amount of the test item was slowly placed by pipette under the water surface. A slow stirring procedure was applied. Gentle stirring was carried out for 24 hours with a magnetic stirrer at room temperature.  
At the end of the stirring period, the WAF of the test item was removed from the approximate bottom of the aqueous phase and used for the exposure. No defined settling time was applied, with further procedure of the test solutions directly after end of the stirring period, as technically feasible. If necessary, the pH of the WAF was adjusted to pH 7 ± 0.2 again. This procedure was carried out for each exposure interval.

- Controls: Dilution water (Elendt M4) adjusted to pH 7 ± 0.2 without addition of test item

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All concentration levels were visually clear throughout the exposure period. No undissolved particles, aggregations or phase separation was observed during the preparation of the saturated solution. No Tyndall effect was observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: n/a
- Age at study initiation (mean and range, SD): Less than 24 hours old.
- Weight at study initiation (mean and range, SD): The mean values of the dry weight of the parental daphnids were 0.85 mg per daphnid in the WAF and 0.83 mg per daphnid in the control .
- Length at study initiation (length definition, mean, range and SD): The mean values of the body length (excluding the anal spine) of the parental daphnids in the WAF were 4.40 mm per daphnid and 4.60 mm per daphnid in the control group.
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not reported
- Peripheral shell growth removed prior to test initiation: Not reported
- Source: Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany;
Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Feeding during test
Daily feeding per test vessel; Pseudokirchneriella subcapitata (0.789 - 1.14 mL) and Desmodesmus subspicatus (0.317 -0.437 mL) suspension was provided as food corresponding to max. 0.2 mg C per daphnid and day.
There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium Elendt M4, according to OECD 202, Annex 3 (2004), equivalent to OECD 211, Annex 2, modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.

Origin of the food algae Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not specified

Hardness:

The total hardness was between 160 to 180 mg CaCO3/L.

Test temperature:

20 ± 2 °C

pH:

Control fresh media: pH 7.06 - 7.12
Control old media: pH 7.36 - 7.53
Nominal 0.98 g/l fresh media: pH 7.14 - 7.19
Nominal 0.98 g/l old media: pH 7.33 - 7.54
In the cultured medium, pH was 8.2 ± 0.8.

Dissolved oxygen:

The dissolved oxygen concentration ranged from 8.27 to 8.89 mg/L in newly prepared test solutions and from 7.14 to 9.87 mg/L in aged solutions during the exposure period.

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal: 1 mL test item/L, corresponding to 0.98 g test item/L WAF.
Measured: fresh test media: range < LOQ to 66.0 µg/L.
Aged test media: range < LOQ to 44.3 µg/L.
Geometric mean measured concentration: >6.05 µg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (5 (D) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses.
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed 3 times per week after either 48 or 72 hours.
50 g test solution per replicate was weighed out into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.
- No. of organisms per vessel: 1
- No. of vessels per concentration and control: 10 daphnids and 10 replicates were used for the WAF and the control replicates with one daphnid per replicate, which was held individually.
- No. of vessels per vehicle control: One additional replicate without addition of daphnia or food algae was prepared per loading level for analytical purposes.
- Biomass loading rate: 1 daphnid/50 mL of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within every 7 days of exposure, in fresh media at the start of an exposure renewal interval (0 hours) and in old media at the end of an exposure renewal interval (48 or 72 hours), in one replicate of the control and the WAF.
- Total organic carbon: not specified
- Particulate matter: not specified
- Metals: not specified
- Pesticides: not specified
- Chlorine: not specified
- Alkalinity: not specified
- Ca/mg ratio: not specified
- Conductivity: not specified
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: The Elendt M4 medium was adjusted to pH 7 ± 0.2 by addition of 1 M HCl prior to addition of the test item and, if necessary, the pH of the WAF was adjusted to 7 ± 0.2 again at the end of the stirring period.
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED: Survival, reproduction and growth (total body length and dry body weight).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A: limit WAF test.
- Justification for using less concentrations than requested by guideline: No effects at the highest achievable concentration.
- Range finding study: yes, at the concentrations 0.01, 0.10 and 1.00 mL/L.
- Results used to determine the conditions for the definitive study: No effects at the highest concentration tested in the range-finder.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 6.05 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

sum of the three major constituents

Basis for effect:

reproduction

Remarks on result:

other: No effects at the highest concentration achievable

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 0.98 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

>= 0.98 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: The average body length of the parental daphnids in the WAF were 4.40 mm/daphnid and 4.60 mm/daphnid in the control group.
- Other biological observations: No presence of males or any occurrence of ephippia in the control or in the test groups.
- Mortality of control: 93.3 % survival
- Other adverse effects control: None
- Immobilisation of control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? N/a
- Relevant effect levels: N/a

Reported statistics and error estimates:

Normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test) and a two-sample t-test. P values of the normality and the variance homogeneity test were 0.01. The α - value (acceptable probability of incorrectly concluding that there is a difference) was α = 0.01 for the normality test and the variance homogeneity test and α = 0.05 for the multiple test. The coefficients of variation around the mean number of living offspring produced per introduced / survived parent in the control and the test groups were evaluated.
The NOELR and the LOELR given are based on the nominal test item loading and the geometric mean measured test item concentration.

Table 1: The Effects on Reproduction for all parental Daphnids

[g/L]	Mean	SD	CV
0.98	100.3	18.96	18.9
Control	93.3	15.95	17.1

The table shows the mean number of offspring per survived / introduced parental daphnid.

 

Table 2: Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

[g/L]	7 days	14 days	21 days
0.98	0	0	0
Control	0	0	0

The table shows the adult mortality (%) following the nominal test item loading.

Table 3: First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal test item loading [g/L]	No 1:	No 2:	No 3:	No 4:	No 5:	No 6:	No 7:	No 8:	No 9:	No 10:	Mean day:
0.98	9	8	8	10	9	10	10	8	8	8	8.8
Control	10	8	8	8	9	8	8	8	8	8	8.3

The table shows the day of the first appearance of living juveniles at the parental daphnid.

 

Table 4: Total Body Length and Mean Dry Weight of the Parental Daphnids

Nominal test item loading [g/L]	No 1:	No 2:	No 3:	No 4:	No 5:	No 6:	No 7:	No 8:	No 9:	No 10:	MV [mm]	N	Mean dry weight (mg) S	MV
0.98	4.25	3.75	4.25	4.75	4.00	4.75	5.00	4.50	4.25	4.50	4.40	10	8.5	0.85
Control	5.00	4.50	4.50	4.75	4.50	4.00	4.75	4.75	5.00	4.25	4.60	10	8.3	0.83

The table shows the total length of the parent daphnids [mm] where N is the number of the parental daphnids

 

Table 5: Measured Concentrations during the Definitive Test

Sampling date	2017-05-03 day 0 Start of the exposure interval (0 hours)	2017-05-05 day 2 End of the exposure interval (48 hours)	2017-05-12 day 9 Start of the exposure interval (0 hours)	2017-05-15 day 12 End of the exposure interval (72 hours)	2017-05-17 day 14 Start of the exposure interval (0 hours)	2017-05-19 day 16 End of the exposure interval (48 hours)
Start of analysis	2017-05-03	2017-05-05	2017-05-12	2017-05-15	2017-05-17	2017-05-19
Nominal test item loading [g/L]	Meas. test conc. [µg/L]	Meas. test conc. [µg/L]	Meas. test conc. [µg/L]	Meas. test conc. [µg/L]	Meas. test conc. [µg/L]	Meas. test conc. [µg/L]
0.98	66.0^1)	44.3^1)	3.42^2)	< LOQ	< LOQ	37.3^3)
Control	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

The tables show the measured concentrations. The measured test concentration is based on the summation of the 3 major test item constituents.

 Cont’d.  Measured Concentrations during the Definitive Test

Sampling date	2017-05-19 day 16 Start of the exposure interval (0 hours)	2017-05-22 day 19 End of the exposure interval (72 hours)	2017-05-22 day 19 Start of the exposure interval (0 hours)	2017-05-24 day 21 End of the exposure interval (48 hours)
Start of analysis	2017-05-19	2017-05-22	2017-05-22	2017-05-24	Geometric mean
Nominal test item loading [g/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]
0.98	34.4^4)	5.10	< LOQ	< LOQ	6.05
Control	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

Meas. conc.     = measured concentration of the test item, enrichment and dilution factors taken into account

LOQ        = limit of quantification of the analytical method (2 µg/L of the test item)

1)                     = mean value of two dilution factors

2)                     = mean value of three replicates, reanalysed on 2017-05-15, RSD: 16%

3)                     = mean value of two replicates, first one extracted on 2017-05-19, second one extracted on 2017-05-22,
   both diluted and measured on 2017-05-22

4)                     = mean value of three replicates, each with two dilution factors: samples extracted on 2017-05-19,
   measured with dilution factor 10 on 2017-05-19 and with dilution factor 5 on 2017-05-22

5)                     = Results < LOQ taken into account with ½ LOQ (1 µg/L of the test item)

 

 

Validity criteria fulfilled:

yes

Conclusions:

A 21 day NOELR value of ≥980 mg/l has been determined for the effects of phenyl silsesquioxanes on reproduction of Daphnia magna, based on nominal loading rate WAF.
A 21 day NOEC value of ≥0.00605 mg/l has been determined for the effects of phenyl silsesquioxanes on reproduction of Daphnia magna based on geometric mean measured concentrations (sum of the three major constituents).

Executive summary:

A 21-day long term toxicity study to aquatic invertebrates in Daphnia magna was conducted according to OECD TG 211 and in compliance with GLP. Based on the nominal loading concentration of the test item, reaction mass of the monomer, dimer and oligomers of phenyltris(trimethylsiloxy)silane, a NOELR was determined to be ≥ 0.98 g/L and a LOELR was determined to be >0.98 g/L. No significant effects on reproduction, mortality or any other sub-lethal effects up to the achievable limit of solubility under the test conditions were observed.

Description of key information

21 day NOEC ≥0.00605 mg/l, geometric mean measured concentrations, reproduction of Daphnia magna.

Additional information

A chronic Daphnia reproduction test in accordance with OECD TG 211 has been conducted with the registered substance.

No effects at the limit of solubility, using Water Accommodated Fraction (WAF) test methodology, were reported in the test.

A 21 day NOELR value of ≥980 mg/l has been determined for the effects of phenyl silsesquioxanes on reproduction of Daphnia magna, based on nominal loading rate WAF, and a NOEC value of ≥0.00605 mg/l has been determined based on geometric mean measured concentrations (sum of the three major constituents).

These data are supported by reading across long-term toxicity to aquatic invertebrates 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 K_ow 8.2) and dodecamethylcyclohexasiloxane (D6, CAS 540-97-6, EC No. 208-762-8, log K_ow 9). No effects on aquatic invertebrates have been reported at the limit of solubility of the substances:

A 21-day NOEC of ≥4.6 μg/l was obtained for the effects of D6 on the survival, reproduction and growth of Daphnia magna.

A 21-day NOEC of ≥4.9 μg/l was obtained for the effects of L4 based on reproduction of Daphnia magna.

Although an experimental study is available with phenyl silsesquioxanes, these read-across studies are included as evidence 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.