Triethoxyoctylsilane

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:

2017-11-30 to 2018-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

liquid

Analytical monitoring:

yes

Remarks:

measured the parent substance

Details on sampling:

- Concentrations: (nominal) control, solvent control, 0.0313, 0.0625, 0.125, 0.250, 0.500 mg/L
- Sampling method: LC-MS/MS once within every 7 days in fresh media at the start of an exposure interval (0 hours) and in old media at the end of an exposure interval (24 hours).
- Sample storage conditions before analysis: Samples were analysed immediately (as far as technically possible) following sampling, All original samples were stored at room temperature. Prepared samples were stored at room temperature in an autosampler until analysis.

Vehicle:

yes

Remarks:

Solvent used: dimethylformamide, a solvent control was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were freshly prepared for each exposure interval and each concentration level. The stock solutions were prepared by direct addition of the appropriate amount of test item by pipette (the relative density of the test item of 0.877 was taken into account) into DMF. The concentrations of the stock solutions were as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 g/L
- Eluate: not reported
- Differential loading: not reported
- Controls: control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF) was prepared and tested under the same conditions as the test item concentrations and the control.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.05 mL/L in every test concentration and solvent control, 0 ml/L in control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test item solutions were mixed thoroughly by manual agitation, no reported evidence of undissolved material
- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: 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 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.
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): not reported
- Stage and instar at study initiation: not reported
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/a
- Peripheral shell growth removed prior to test initiation: N/a
- Method of breeding: 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
- Source: Institut Dr. Nowak, Mayenbrook 1, 28870 Ottersberg, Germany
- Age of parental stock (mean and range, SD): not reported
- Feeding during test - yes
- Food type: Pseudokirchneriella subcapitata and Desmodesmus subspicatus suspension was provided as food corresponding to 0.2 mg C per daphnid and day.
- Amount: 0.2 mg C per daphnid and day.
- Frequency: Daily feeding per test vessel

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water used for test medium preparation was equivalent to the culture medium.
- Acclimation conditions (same as test or not): N/a
- Type and amount of food: N/a
- Feeding frequency: N/a
- Health during acclimation (any mortality observed): N/a

QUARANTINE (wild caught)
- 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:

no

Total exposure duration:

21 d

Remarks on exposure duration:

none

Post exposure observation period:

none

Hardness:

as CaCO3.
In control group: 216-257.
In solvent control: 224-280
In test concentrations 0.199 and 0.562: 225-260

Test temperature:

In control group: 20.1-20.7
In solvent control: 19.7-20.7
In test concentrations 0.199 and 0.562: 19.7-20.6

pH:

In control group: 7.05-7.47
In solvent control: 7.30-7.44
In test concentrations 0.199 and 0.562: 7.31-7.45

Dissolved oxygen:

In control group: 6.66-9.19
In solvent control: 7.49-9.56
In test concentrations 0.199 and 0.562: 7.95-9.18

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 0.0313, 0.0625, 0.125, 0.250, 0.500 mg/L
Measured: 0.025, 0.043, 0.103, 0.199, 0.562 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: (4.5 (ID) x 9.5 (H) cm) with screw caps were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
- Aeration: Test vessels were not aerated during the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static test so N/a
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed daily. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 10 replicates for all concentrations
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate:N/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Same composition as the culture medium (culture medium Elendt M4)
- Total organic carbon: Not reported
- Particulate matter: not reported
- Metals: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Pesticides: not reported
- Chlorine: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Alkalinity: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Ca/mg ratio: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: once every seven days in fresh media and at the end of an exposure interval (24h)

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to pH 7.0-7.5
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality of parental daphnids (once a day at least), neonates (checked daily), condition of parental daphnids (daily), total body length/mean dry weight of parental daphnids (at the end of the test), first time to brood, intrinsic rate of population increase, number and size of first brood per animal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.0313 - 0.0625 - 0.125 - 0.250 - 0.500 mg/L
- Justification for using less concentrations than requested by guideline: N/a
- Range finding study
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: n/a

Reference substance (positive control):

yes

Remarks:

Test is undertaken once a month at the test facility

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.189 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

(C.I 0.119-0.300)

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.199 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and adult mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and adult mortality

Details on results:

- Behavioural abnormalities: None observed
- Observations on body length and weight: mean length of survived parents for control, solvent control, 0.025, 0.043, 0.103, 0.199, 0.562 mg/L : 4.63, 4.63, 4.70, 4.63, 4.58, 4.50mm and N/a as mortality was 100% in the 0.562 treatment.
- Other biological observations: In the geometric mean measured concentration level of 0.199 mg/L, one parental daphnid was identified as a male during the exposure period. This parental daphnid was treated as accidental mortality and was not taken into account for any further evaluation. No stillborn juveniles or aborted eggs were observed in the control or solvent control during the exposure period of 21 days. In the geometric mean measured concentrations levels of 0.025 mg/L and 0.043 mg/L, a total of 1 and 47 stillborn juveniles were observed, respectively. First appearance of living juveniles mean day for all treatments and controls = day 9 (except for 0.562 mg/L as 100% mortality of parents occurred). No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: 0% mortality in the solvent control and the control
- Other adverse effects control: no adverse effects observed in the control or solvent control
- Immobilisation of control: 0%
- Abnormal responses: described in 'Other biological observations'
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured test item concentration in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 101 to 296% of the nominal concentrations and 16 to 100% in old media at the end of the respective exposure intervals (48 hours). The exact reasons for the determined outliers > 126% are unknown. Possible factors of influence may be the tested concentrations near the functional water solubility of the test item and recoveries near the LOQ of the analytical method. Further, the test item is not stable under test conditions and degradation products may influence recovery rates.
- Effect concentrations exceeding solubility of substance in test medium: no - solvent used

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 1.66 mg/L of potassium dichromate (C.I. <1.00-4.00)
- Other: The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018-03-13 to 2018-03-14.

Reported statistics and error estimates:

Reproduction NOEC/LOEC based on geometric mean concentrations:
Shapiro Wilk's Test on normal distribution: Number of residuals = 55; Shapiro-Wilk´s W = 0.782; p(W) = <0.001; p(W) is smaller than or equal to the selected significance level of 0.010; therefore, treatment data significantly differ from normal distribution.
Levene's Test on Variance Homogeneity: The Levene test indicates variance heterogeneity (p <= 0.010). Variance homogeneity check failed. Also the degree of normal distribution is poor. Therefore, the mediantest (2-by-2 table) with Bonferroni correction is performed.
Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm: A NOEC of 0.1990 mg/L is suggested by the program.

Table 1 – Effects on adult mortality and reproduction for all introduced and for surviving parents (based on the geometric mean measured concentrations of the test item Triethoxyoctylsilane)

Geometric mean measured test item concentration (mg/L)	Adult mortality (%)	Mean number of offspring per introduced parental daphnid - Mean	Mean number of offspring per introduced parental daphnid - SD	Mean number of offspring per introduced parental daphnid - CV	Mean number of offspring per survived parental daphnid – mean	Mean number of offspring per survived parental daphnid – SD	Mean number of offspring per survived parental daphnid – CV
0.562	100	n.a.^1	n.a.^1	n.a.^1	n/a – 100% mortality^1	n/a – 100% mortality^1	n/a – 100% mortality^1
0.199^2	11.1	126	53.7	42.7	142	27.4	19.3
0.103	10	144	51.4	35.8	159	16.1	10.1
0.043	20	154	14.1	9.2	158	12.1	7.6
0.025	0	159	11.3	7.1	159	11.3	7.1
Solvent control	0	131	22.3	17.1	131	22.3	17.1
Control	0	144	21.2	14.7	144	21.2	14.7

¹statistically significant reduction of the reproductive output, compared to pooled controls

²one parental daphnid was identified as male and that replicate was excluded from evaluation

n.a. not applicable, since no juveniles were observed

 

Table 2 – Mortality (%) of the adult Daphnids after 7, 14 and 21 days of exposure (n=10)

Geometric mean measured test item concentration (mg/L)	Adult mortality – 7 days	Adult mortality – 14 days	Adult mortality – 21 days
0.562	100^1	100^1	100^1
0.199^2	11.1	11.1	11.1
0.103	0	10	10
0.043	0	0	20
0.025	0	0	0
Solvent control	0	0	0
Control	0	0	0

¹statistically significant compared

²one parental daphnid was identified as male and that replicate was excluded from evaluation (n=9)

 

Table 3 – Intrinsic Rates of Natural Increase

Geometric mean measured test item concentration (mg/L)	Intrinsic rate of natural increase in replicate no 1	Intrinsic rate of natural increase in replicate no 2	Intrinsic rate of natural increase in replicate no 3	Intrinsic rate of natural increase in replicate no 4	Intrinsic rate of natural increase in replicate no 5	Intrinsic rate of natural increase in replicate no 6	Intrinsic rate of natural increase in replicate no 7	Intrinsic rate of natural increase in replicate no 8	Intrinsic rate of natural increase in replicate no 9	Intrinsic rate of natural increase in replicate no 10	Mean IR MV± SD	CV (%)
0.562	-	-	-	-	-	-	-	-	-	-	-	-
0.199	0.404	0.256	0.405	0.450	-	0.300	0.373	*	0.432	0.300	0.378 ± 0.0563	14.9
0.103	0.396	0.379	0.417	0.406	0.413	0.384	0.361	0.435	0.378	-	0.397 ± 0.0231	5.8
0.043	0.389	0.399	0.386	0.389	-	0.427	0.409	0.393	0.384	-	0.397 ± 0.0145	3.6
0.025	0.407	0.401	0.572	0.390	0.448	0.378	0.397	0.400	0.398	0.372	0.416 ± 0.0585	14.0
Solvent Control	0.379	0.385	0.390	0.410	0.386	0.360	0.406	0.383	0.362	0.341	0.380 ± 0.0209	5.5
Control	0.384	0.41	0.381	0.359	0.363	0.388	0.384	0.390	0.346	0.408	0.381 ± 0.0203	5.3

- Not applicable, due to the mortality of the parental daphnid(s)

* parental daphnid was identified as male and that replicate was excluded from evaluation

Table 4 – First Appearance of Living Juveniles at the Introduced Parental Daphnids in the Individual Groups:

Geometric mean measured test item concentration (mg/L)	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 1	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 2	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 3	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 4	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 5	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 6	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 7	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 8	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 9	Day of first appearance of living juveniles at the survived parental daphnids in replicate no 10	First appearance mean day
0.562	-	-	-	-	-	-	-	-	-	-	-
0.199	9	8	9	8	-	10	8	-*	9	9	9
0.103	9	8	9	9	9	10	9	8	9	8	9
0.043	9	9	9	10	9	9	9	10	8	10	9
0.025	9	9	8	9	8	9	9	9	9	9	9
Solvent Control	9	9	9	9	9	9	9	9	8	9	9
Control	9	9	9	10	9	9	9	9	10	9	9

- no juveniles occurred

* parental daphnid was identified as male and that replicate was excluded from evaluation

 

Table 5 – Total Body Length and Dry Weight of the Survived Parental Daphnids

Geometric mean measured test item concentration (mg/L)	Total length of the survived parent animals (mm). Replicate no 1	Total length of the survived parent animals (mm). Replicate no 2	Total length of the survived parent animals (mm). Replicate no 3	Total length of the survived parent animals (mm). Replicate no 4	Total length of the survived parent animals (mm). Replicate no 5	Total length of the survived parent animals (mm). Replicate no 6	Total length of the survived parent animals (mm). Replicate no 7	Total length of the survived parent animals (mm). Replicate no 8	Total length of the survived parent animals (mm). Replicate no 9	Total length of the survived parent animals (mm). Replicate no 10	MV (mm)	N	Dry weight (mg) – sum	Dry weight (mg) - MV
0.562	-	-	-	-	-	-	-	-	-	-	-	0	-	-
0.199	4.25	4.75	4.5	4.5	-	4.5	4.5	2.5*	4.5	4.5	4.5	8	7.0	0.875
0.103	4.5	4.75	4.75	4.5	4.5	4.75	4.25	4.5	4.75	-	4.58	9	8.6	0.956
0.043	4.5	4.5	4.5	4.75	-	5.0	4.5	4.75	4.5	-	4.63	8	7.4	0.925
0.025	4.75	4.75	4.5	5.0	4.75	4.5	4.75	4.75	5.0	4.25	4.7	10	8.2	0.820
Solvent Control	4.75	4.5	4.5	5.0	5.0	4.5	4.5	4.5	4.5	4.5	4.63	10	8.7	0.870
Control	4.5	4.75	4.5.2	4.75	4.5	4.5	4.75	4.5	5.0	4.5	4.63	10	8.2	0.820

N number of the survived parental daphnid

- not applicable, due to the mortality of the parental daphnid(s)

* parental daphnid was identified as male and excluded from evaluation

Table 6 – Measured Concentrations of the Test Item during the Definitive Test

Sampling day       Nominal test item conc. (mg/L)	Day 0 - Start of the exposure interval (0 hours) - Meas. Conc. (mg/L)	Day 0 - Start of the exposure interval (0 hours) - %	Day 1 – End of the Exposure interval (24 hours) - Meas. Conc. (mg/L)	Day 1 – End of the Exposure interval (24 hours) - %	Day 7 – Start of the exposure interval (0 hours) - Meas. Conc. (mg/L)	Day 7 – End of the exposure interval (0 hours) - Meas. conc. (mg/L)	Day 8 – Start of the exposure interval (24 hours) - Meas. Conc. (mg/L)	Day 8 – End of the exposure interval (24 hours) - Meas. conc. (mg/L)
0.500	1.41	283	0.224	45	n.d.	n.d.	n.d.	n.d.
0.250	0.301	120	0.130	52	0.287	115	0.140	56
0.125	0.152	121	0.0736	59	0.127	101	0.0653	52
0.0625	0.0706	113	0.0101	16	0.0946	151	0.0297	48
0.0313	0.0354	113	0.0312	100	0.0317	101	0.00824	26
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Solvent control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ

 

Sampling day	Day 14 – Start of the exposure interval (0 hours) - Meas. Conc. (mg/L)	Day 7 – End of the exposure interval (0 hours) - %	Day 15 – Start of the exposure interval (24 hours) - Meas. Conc. (mg/L)	Day 15 – End of the exposure interval (24 hours) - %	Geometric mean measured (mg/L)
0.500	n.d.	n.d.	n.d.	n.d.	0.562
0.250	0.340	136	0.116	46	0.199
0.125	0.146	117	0.0882	71	0.103
0.0625	0.107	171	0.0279	45	0.0426
0.0313	0.0928	296	0.00851	27	0.0247
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Solvent control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ

Meas. conc. Measured concentration of the test item, dilution factor taken into account

% percent of the nomnal concentration of the test item

LOQ limit of quantification of the analytical method (30.0 µg test item/L)

n.d. not determined

Validity criteria fulfilled:

yes

Conclusions:

A 21-day reproduction NOEC of ≥0.199 mg/L and EC10 value of >0.189 mg/l (geometric mean measured concentrations) have been determined for the effects of the test substance on reproduction of Daphnia magna. A 21-day adult mortality NOEC of ≥0.199 mg/L (geometric mean measured concentration) has been determined for the effects of the test substance on mortality of Daphnia magna. It is likely that the test organisms were primarily exposed to the parent substance.

Description of key information

Long-term toxicity to invertebrates: 21-day reproduction EC₁₀ >0.189 mg/l and NOEC ≥0.199 mg/l (OECD TG 211), for the test substance triethoxy(octyl)silane (CAS 2943-75-1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

> 0.189 mg/L

Additional information

A long-term toxicity test with Daphnia magna, carried out in accordance with OECD TG 211, has been conducted with the registered substance triethoxy(octyl)silane (CAS 2943-75-1, EC 220-941-2).

The OECD 211 study was performed under the assumption that the WS of the test item would be up to approximately 0.79 mg/l based on a QSAR estimation method due to the lack of a valid OECD 105 study at that point in time.

Prior to the start of the study, 3 preliminary solubility and stability tests were conducted with the substance in test medium (Elendt M4 culture medium) with 1 mg/l as highest test item concentration. Based on the results of these preliminary tests, the definitive study was designed as a daily renewal semi-static study, with test concentrations of 0.0313, 0.0625, 0.125, 0.250 and 0.5 mg/l, in a closed system without headspace, and using an application solvent (dimethylformamide (DMF)).

During the definitive test, significant mortality of parent daphnids (100% mortality) was observed at the highest test concentration of 0.5 mg/l (nominal). No effects on survival or reproduction were observed in the second highest test concentration (0.25 mg/l nominal). The following results were reported by the study author: A 21-day EC₁₀ value of 0.25 mg/l and NOEC value of 0.25 mg/l have been determined for the effects of triethoxy(octyl)silane (CAS 2943-75-1, EC No. 220-941-2) on reproduction of Daphnia magna based on nominal concentrations. The EC₁₀ and NOEC values are 0.189 mg/l and 0.199 mg/l, respectively, based on geometric mean measured concentrations (Noack, 2018).

However, reviewers have subsequently determined that analytically measured test substance concentrations were highly variable (please refer to Table 6 in the results section of the endpoint study record), leading to the conclusion that there is considerable analytical evidence of inhomogeneity in the test medium (i.e., the substance is not fully dissolved). Since the registration substance is a liquid, the sampling methods and treatment would not be able to distinguish between dissolved material and undissolved material present as microdroplets.

Despite the preliminary solubility and stability work carried out prior to the definitive test, the variability in the analytical measurements is considered to be an indicator that the solubility of the test substance had been exceeded. It is noted that the analytically measured concentration in the top dose at day 0 was 1.41 mg/l (approximately 300% of the nominal dose).

In addition, despite the derivation of NOEC and EC₁₀ values, there was a lack of a dose response curve, with daphnids either dying at the highest dose, or surviving with no significant reduction in number of juveniles produced (100% mortality at 0.5 mg/l, no statistically significant effects on survival or reproduction at 0.25 mg/l and lower).

Consequently, the registrants determined that further investigation was required to determine the water solubility of the test substance, and a water solubility test in accordance with OECD TG 105 was performed with triethoxy(octyl)silane. The result was a water solubility of 0.168 mg/l in deionised pure water (IES 2020), i.e., lower than the long-term Daphnia EC₁₀ of 0.189 mg/l (nominal 0.25 mg/l). This value provides further evidence that the chronic Daphnia test was conducted above the limit of water solubility of triethoxy(octyl)silane.

Usually, due to the presence of salts, the solubility of a substance in test media is lower than the solubility in deionised pure water. To resolve the question of whether the Daphnia study was conducted at concentrations exceeding the ‘saturation concentration’, a further study in accordance with OECD TG 105 was performed with triethoxy(octyl)silane in Daphnia culture medium (Elendt M7 medium) (IES 2021).

The following results were reported:

Test medium (M7-medium) without DMF: WS of 385 µg/l

Test medium (M7-medium) with 0.05 mL DMF/L: WS of 494 µg/l.

The results indicate that the measured solubility in Elendt M7 test media with solvent DMF is close to the highest nominal test concentration of 0.5 mg/l used in the Daphnia test but well below the measured test item concentration in the highest dose tested of 1.41 mg/l (at 0 hours). Additionally, the saturation concentrations were not obtained until 48-72 hours. Due to the daily renewal of the test media during the chronic Daphnia test, the test substance would not have had time to reach its saturation concentration in test media. After 24 hours equilibration time, the test substance reached a solubility of 0.319 mg/l without DMF and 0.389 mg/l with DMF. Hence, in ecotoxicological test medium Elendt M7, the highest nominal test concentration of 0.5 mg/l exceeded the solubility of the test substance. In addition, the long-term Daphnia test was conducted in Elendt M4 Medium, as opposed to the M7 Medium used in the solubility test. M7 medium has lower concentrations of some salts, therefore it could be the case that solubility in M4 medium might be even lower than the solubility determined in M7 medium.

The water solubility studies establish that the highest concentration of 0.5 mg/l used in the long-term toxicity to Daphnia test was above the limit of water solubility of triethoxy(octyl)silane in both pure water and Daphnia test media.

During the long-term Daphnia test, the testing laboratory reported that there was no evidence of undissolved material present during the test. However, with a test substance that is a clear liquid and present at low concentrations, it can be almost impossible to observe whether all test substance is dissolved. This is also extremely difficult to determine analytically because all test item concentrations and the control were diluted with acetonitrile directly after sampling. The samples were further diluted with mobile phase prior to analysis by LC-MS. The addition of the solvent used in extraction would dissolve all the test substance so the analysis would not distinguish between any dissolved or undissolved test material that may have been present in the test solution samples.

Triethoxy(octyl)silane is very hydrophobic and is known to coat surfaces. It is therefore feasible that it could form a layer over the exoskeleton of the Daphnia and hence reduce their respiration. In addition, the co-solvent DMF was used to prepare test solutions. ECHA guidance (ECHA 2017) states that “Tertiary solvents are often used in order to prepare stock solutions so that they can be further diluted to provide test solutions. Solvents used at the maximum allowed concentration (100 mg/l) will rarely increase the solubility of the test substance significantly but may lead to emulsion formation which could cause physical effects.”

All these factors support the evidence displayed in the water solubility studies that triethoxy(octyl)silane was tested above its limit of water solubility, resulting in physical effects on the Daphnia that were not caused by true toxicity of the test substance.

It is therefore demonstrated that the highest nominal test concentration of 0.5 mg/l used in the long-term Daphnia test is greater than the solubility of triethoxy(octyl)silane. A re-evaluation of the statistical analysis for the Daphnia study was subsequently conducted using RStudio Software (version 1.1.419), excluding the highest test concentration of 0.5 mg/l nominal. The following observations were made:

Total number of juveniles: None of the treatment groups were significantly different to the control group (all p values were > 0.05).

Adult survival: None of the treatment groups were significantly different to the control group (all p values were > 0.05).

It can be concluded that triethoxy(octyl)silane is not toxic to Daphnia magna at the highest dissolved concentration achieved under the conditions of the study.

The following chronic Daphnia reproduction test results are therefore used for chemical safety assessment:

21-day EC₁₀ and NOEC values of >0.25 mg/l and ≥0.25 mg/l (nominal concentration) respectively, or >0.189 mg/l and ≥0.199 mg/l (geometric mean measured concentration) respectively, were determined for the effects of triethoxy(octyl)silane (CAS 2943-75-1, EC No. 220-941-2) on reproduction of Daphnia magna.

The focus of the chemical safety assessment for this substance is the parent substance, due to its hydrolysis half-life of 30 hours at pH 7. Measures were taken during the Daphnia reproduction test to maximise exposure to the parent, including the use of a solvent for media preparation, keeping the pH neutral and using a semi-static daily renewal system.

In view of the exposure regime, it is likely that the test organisms were exposed predominantly to the parent substance at the beginning of the media renewal period, with increasing amounts of hydrolysis products prior to renewal at 24 hours.

To support the data with the registration substance, data have been read-across from two structurally analogous substances; triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3, EC No. 252-558-1) and trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4, EC No. 242-262-0).

A 21-day NOELR of 32 mg/l (nominal) and a NOEC 0.058 mg/l (arithmetic mean measured concentration) has been determined for the effects of triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3) on reproduction of Daphnia magna under semi-static test conditions. However the result of the study should be treated with caution as the effects were recorded at a nominal concentration 25 times higher than the limit of solubility of the parent substance and it is not possible to discern between effects of the parent and effects of the hydrolysis product. The hydrolysis half-life of this substance is 42 hours at pH 7. The test media preparation included 15 minutes of ultrasonication followed by 96 hours of stirring before introduction of test organisms. It is therefore likely that the test organisms were exposed to high concentrations of the hydrolysis products. The analysis of the parent substance support this theory, with poor measured recoveries of parent test substance. Hence this study is used only as supporting evidence on toxicity of the silanol hydrolysis product.

A 21-day NOEC 32 mg/l (initial mean measured equivalent test substance concentration based on measured TOC, back calculated to concentration of parent substance) has been determined for the effects of trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4) on reproduction of Daphnia magna. The parent substance hydrolyses very rapidly (<1 minute) at pH 7, therefore it is likely that the test organisms were exposed to high concentrations of the hydrolysis products. The results may be expressed in terms of concentration of the hydrolysis product, (2,4,4-trimethylpentyl)silanetriol, by applying a molecular weight correction: (MW of silanol = 192.3 / MW of parent = 247.7) * concentration of parent = 32 mg/l = 25 mg/l.

It is likely that the test organisms in the above studies were predominantly exposed to the hydrolysis products of the substances. These results indicate that the silanol hydrolysis product of the registered substance has low long-term toxicity to aquatic invertebrates and would not cause toxic effects at concentrations that correspond with the solubility limit of the registered substance.

The read-across to the registered substance is considered scientifically justified. Refer to the IUCLID 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.