2,4,6,8-tetramethylcyclotetrasiloxane

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:

19th of September 2019 to 15th of January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

yes

Remarks:

Only one concentration used (rather than five)

GLP compliance:

yes (incl. QA statement)

Remarks:

In total, three exceptions are listed, however, these are not expected to have provided any impact on the quality of the study.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (water control) and 12 mg methylsilanetriol/L
- Sampling method: The bulk water control and bulk test solution were sampled at test initiation (day 0) and on days 2, 7, 14 and 19 of the exposure. Spent (pooled replicate) solutions were sampled on days 2, 5, 7 and 14 as well as at exposure termination (day 21). To assess analytical method precision and solution homogeneity, three additional samples were collected on day 0, 7, 14 and 19 from the bulk test solution. The collected samples were analysed for methylsilanetriol by HPLC/MS-MS. Validation of the analytical methodology was conducted concurrently with the study as per standard operating procedure (SOP) MTH-ACL-2 (2014).
- Sample storage conditions before analysis: Not reported.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The definitive test was conducted under static-renewal conditions for 21 days. Ten replicate test vessels, each containing test solution or laboratory dilution water (LDW) were prepared per test level. At test initiation, one daphnid was impartially added to each test vessel within 30 minutes of the addition of the test solutions. The replicate test vessels were placed into a temperature-controlled environmental chamber according to a computer-generated randomization.
A 12 mg/L (target nominal) bulk test solution of methylsilanetriol (MST), equal to the highest (limit) test concentration, was prepared via direct addition (260.5 mg) of the test material (methyltrimethoxysilane (MTM)) to 15 L of lab dilution water (LDW). The amount of methyltrimethoxysilane needed to prepare the 12 mg/L (target nominal) bulk test solution of methysilanetriol was determined as follows:
12 mg MST/L (15 L of solution):
Molecular weight of methyltrimethoxysilane (MTM) = 136.2
Molecular weight of methylsilanetriol (MST) = 94.1
12 𝑚𝑔 𝑀𝑆𝑇 𝐿 x (1 𝑚𝑜𝑙 𝑀𝑆𝑇/94.1 𝑔 𝑀𝑆𝑇) x (1 𝑚𝑜𝑙 𝑀𝑇𝑀/1 𝑚𝑜𝑙 𝑀𝑆𝑇) x (136.2 𝑔/1 𝑚𝑜𝑙 𝑀𝑇𝑀) x 15 𝐿 = 260.5 𝑚𝑔 𝑀𝑇𝑀
Results of preliminary non-GLP methodology work indicated that the test material (methyltrimethoxysilane) would completely hydrolyze (< 24 hours) in aqueous (LDW) solutions to methylsilanetriol and methanol. Based on extensive data completed for methanol, it is considered non-hazardous to the environment and therefore, the projected methanol concentration (12.3 mg/L) present in the test solution did not affect the overall results. Methylsilanetriol and methanol were expected to be completely soluble at the selected test concentration for the study.
The bulk test solution was stirred on a magnetic stir plate utilizing a Teflon lined magnetic stir bar for 24 hours to allow complete hydrolysis of the test material. A bulk solution of LDW was prepared similarly without the addition of the test material and served as the water control. The bulk test solution and bulk water control were utilized for the duration of the study and were stored in the temperature-controlled environmental chamber used for the definitive study. Following preparation, the bulk test solution and water control were apportioned into individual test vessels. Test solutions in the replicate test vessels were renewed three times weekly (M, W, F).
The dispersal of the test material in the surrounding medium (LDW) was considered to represent the most probable route of exposure in the environment.
- Eluate: not specified
- Differential loading: not specified
- Controls: Laboratory dilution water
- Chemical name of vehicle: n/a
- Concentration of vehicle in test medium: n/a
- Evidence of undissolved material: not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: n/a
- Justification for species other than prescribed by test guideline: n/a
- Age at study initiation: Less than 24 hours
- Weight at study initiation: n/a:
- Length at study initiation: Mean length per surviving adult female were 4.81 mm in the water control and 4.85 mm in the 10.0 mg/L test level.
- Stage and instar at study initiation: n/a
- Valve height at study initiation, for shell deposition study: n/a
- Peripheral shell growth removed prior to test initiation: n/a
- Method of breeding: n/a
- Source: In house cultures obtained originally from Aquatic BioSystems, Inc., Fort Collins, Colorado
- Age of parental stock: n/a
- Feeding during test : Daphnids were fed with Raphidocelis subcapitata and YTC (yeast, wheat grass, finfish food suspension) daily from the test initiation (day 0) to day 20, however, not on day 21.
- Food type: Raphidocelis subcapitata and YTC (yeast, wheat grass, finfish food suspension)
- Amount: 0.8 mL Raphidocelis subcapitata and 0.1 mL YTC (yeast, wheat grass, finfish food suspension) daily between day 0-6. Thereafter, 0.9 ml of Raphidocelis subcapitata and 0.1 mL YTC between day 7-20. No food was provided on day 21. Therefore, the final feeding ration resulted in approximately 0.2 mg C/daphnid/day throughout the entire exposure period.
- Frequency: daily

ACCLIMATION
- Acclimation period: n/a
- Acclimation conditions: n/a
- Type and amount of food: n/a
- Feeding frequency: n/a
- Health during acclimation: not specified

QUARANTINE
- Duration: n/a
- Health/mortality: n/a

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: n/a

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

n/a

Hardness:

58-62 mg CaCO3/L

Test temperature:

19–20°C

pH:

7.7-8.9

Dissolved oxygen:

8.0–10.5 mg/L (90–118% air saturation)

Salinity:

not specified

Conductivity:

160-170 µS/cm

Nominal and measured concentrations:

Nominal test concentration: 0 (water control) and 12 mg methylsilanetriol/L
Mean measured test concentrations: < LLQ (water control) and 10.0 mg methylsilanetriol/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 125-mL glass borosilicate jars containing approximately 90 mL of test solution. Each test vessel was loosely covered and uniquely labeled for identification purpose (i.e. replicate, study, test level). Test vessels were replaced with new/clean test vessels on renewal days.
- Type: Open - loosely covered jar
- Material/size/headspace/fill volume: Glass/125 ml/not specified/90 ml
- Aeration: not specified
- Type of flow-through: n/a (semi static)
- Renewal rate of test solution (frequency/flow rate): three times per week (every M, W, F)
- No. of organisms per vessel: 1 daphnia per vessel
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: n/a
- Biomass loading rate: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO2, carbon-filtered, and UV-irradiated.
- Total organic carbon: 1.9 mg/L,
- Particulate matter: < 2 mg/L
- Metals/pesticides/chlorine/alkalinity/Ca/mg ratio: concentrations of contaminants were below levels of concern that may have interfered with the results of this test.
- Conductivity: 160-170 μS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, with gaseous CO2
- Photoperiod: 16 hours light/8 hours dark per day
- Light intensity: 1123-1314 lux.

EFFECT PARAMETERS MEASURED: not specified

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline: no toxicity expected
- Range finding study: No, based on available literature
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: Definitive test concentrations were based on the 72-hr EC50 value of >1000 mg/L, determined from a daphnid acute toxicity test with methylsilanetriol (Annelin, 1976) and the 48-hr EC50 value of >122 mg/L, determined from a daphnid acute toxicity test with methyltrimethoxysilane (Drottar, 2007).

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

MATC

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Details on results:

- Behavioural abnormalities: No
- Observations on body length and weight: The daphnid lengths in the water control were not statistically different to the mean lenght per surviving adult female. No weight is recorded.
- Other biological observations: All biological data were normally distributed and homogenous.
- Mortality of control: 0 %
- Other adverse effects control: None reported
- Immobilisation of control: None reported
- Abnormal responses: None reported
- 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? Yes
- Relevant effect levels: Yes

Reported statistics and error estimates:

Shapiro-Wilk’s test at a Type I error rate of 0.01 was utilised for the testing of normality for reproduction and growth (length). To evaluate the homogeneity of variance, Levene's test at a type I error rate of 0.05 was used. Furthermore, a parametric analysis (ANOVA) was conducted using a Dunnett's test to compare the limit test level with the water control. one-tailed Dunnett’s test, looking for a significant decrease from the water control, was conducted at a Type I error rate of 0.05. Additionally, the standard deviation, coeffcient of variance and mean was calculated for e.g. the individual observations

Table 2 Summary of Biological Observations

Nominal Concentration (mg methylsilanetriol/L)	Mean Measured Concentration (mg methylsilanetriol/L)	Percent Survival	Average Cumulative Offspring Per Adult (mean ± SD)	Length in Millimeters (mean ± SD)
0 (water control)	0 (<LLQ)^a	100	183.1 ± 23.6	4.81 ± 0.13
12	10	100	181.3 ± 37.2	4.85 ± 0.12

^a <LLQ = Less than the lower limit of quantitation = 1.17 mg methylsilanetriol/L

Validity criteria fulfilled:

yes

Conclusions:

A 21 day NOEC value of 10 mg/l (mean measured concentration) (highest concentration tested) has been determined for the effects of methylsilanetriol on reproduction, growth (length) and mortality of Daphnia magna. Additionally,a LOEC for the same endpoints was determined to be >10 mg/L.

Executive summary:

A 21 day chronic toxicity study in Daphnia magna with the test substance methylsilanetriol (pre-hydrolysed from the parent substance methyltrimethoxysilane) was conducted according to OECD TG 211 and in compliance with GLP. A 21 day NOEC value of 10 mg/l (mean measured concentration) (highest concentration tested) was determined for the effects of methysilanetriol on reproduction, growth (length) and mortality. Additionally, a LOEC for the same endpoints was determined to be >10 mg/L.

Description of key information

Long-term toxicity to invertebrates: 21-d NOEC≥10 mg/l (highest concentration tested) (OECD 211), for the effects of the hydrolysis product methylsilanetriol on survival and reproduction of Daphnia magna.

Additional information

There are no reliable long-term invertebrate toxicity data available for 2,4,6,8-tetramethylcyclotetrasiloxane (HD4, CAS 2370-88-9). Due to the rapid hydrolysis of the registered substance, the relevance species for chemical safely assessment is the hydrolysis product methylsilanetriol. Chronic Daphnia toxicity data with methylsilanetriol have been read across.

No adverse effects on Daphnia magna were observed during the test. A 21 day NOEC value of ≥10 mg/l (measured concentration, highest concentration tested) was determined for the effects of methylsilanetriol on survival, growth and reproduction of Daphnia magna.

Refer to Section 6 endpoint summary (Section 7.0 of CSR) for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.