Octanethioic acid, S-[3-(triethoxysilyl)propyl] ester, reaction products with 2-methyl-1,3-propanediol and 3-(triethoxysilyl)-1-propanethiol

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:

25 September 2020 - 27 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Adopted 2 October 2012

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

Adopted 30 May 2008

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2019 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate), irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken in plastic sample pots from the control and each loading rate WAF test group for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 19 (bulk preparation) and of the expired test preparations on Days 3, 7, 14 and 21 (pooled replicates). On Days 12 and 19, a sample of the freshly prepared 3.2 mg/L loading rate WAF was also taken for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis, if necessary.

Vehicle:

no

Details on test solutions:

Based on the results of the second range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of test concentrations of 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test. Due to the need to test at relatively low concentrations, a single WAF of a nominal loading rate of 3.2 mg/L was prepared from which serial dilutions were made to give the required test concentrations.

The concentration and stability of silicon in the freshly prepared test solutions was verified by chemical analysis on Days 0, 5, 12 and 19 and in the corresponding aged preparations on Days 3, 7, 14 and 21.

The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Test type:

static

Water media type:

freshwater

Remarks:

ISO medium

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim within approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.

Hardness:

244 to 262 mg/L as CaCO3

Test temperature:

19-21 ºC

pH:

7.9 ± 0.3

Dissolved oxygen:

=8.6 mg O2/L

Nominal and measured concentrations:

WAF with nominal loading rates of 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L

Given that the toxicity cannot be attributed to a single component (silicon) or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL plastic beakers which were then covered with a plastic lid to reduce evaporation
- Aeration: Only prior to use
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: With every renewal

OTHER TEST CONDITIONS
- Adjustment of pH: If necessary, the pH was adjusted with NaOH or HCl.
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days
- Light intensity: Not exceeding 1500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The number of Daphnia with eggs or young in the brood pouch was determined daily
- The length of each surviving parent animal was determined at test end

TEST CONCENTRATIONS
- Spacing factor for main test concentrations: Square root of 10 (equals approx. 3.16)
- Range finding study: 2 range-finding tests performed, second with loading rates of 0.0010, 0.010 and 0.10 mg/L

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.028 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

morphology

Remarks:

Body Length.

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

morphology

Remarks:

Body Length.

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

morphology

Remarks:

Body Length.

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 0.32 mg/L

Nominal / measured:

nominal

Basis for effect:

morphology

Remarks:

Body Length.

Details on results:

- Observations on body length and weight: No significant difference in body length in any of the test groups when compared to the control
- Mortality of control: No
- Immobilisation of control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Reported statistics and error estimates:

The 21-Day ELx, NOEL and LOEL values for immobilization based on the nominal loading rates were calculated by Probit analysis using Linear maximum Likelihood Regression or by Fisher’s Exact Binomial Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts.

The ELx, NOEL and LOEL values for body length based on the nominal loading rates were determined by visual inspection of the data.

The ELx, NOEL and LOEL values for reproduction based on the nominal loading rates were calculated by Probit analysis using Linear maximum Likelihood Regression or Williams Multiple Sequential t-test Procedure incorporating Shapiro-Wilk's test on normal distribution, Levene’s test on variance homogeneity and Trend analysis by contrasts.

Verification of Test Concentrations
The test item is a complex mixture from the reaction of 3-octanoylthiopropyltriethoxysilane, 3-mercaptopropyltriethoxysilane and 2-methyl-1,3-propanediol and was known to rapidly hydrolyze. Despite extensive analytical investigations using both GC-MS and LC-MS/MS methods, it was not possible to develop a method of analysis which could accurately quantify the concentration of the hydrolysis product formed. Furthermore, it was considered likely that the hydrolysis product itself was also unstable.
The test item was known to contain 6.01% silicon and, as such, the determination of concentrations of silicon in test media was considered a suitable alternative to compound specific analytical determinations. In an attempt to minimize the background count of silicon due to the use of standard laboratory glassware, all equipment used for the biological phase of the definitive test was a plastic alternative.
Chemical analysis of the control and all loading rate WAF preparations on Days 0, 5, 12 and 19 (fresh media), and Days 3, 7, 14 and 21 (old media) showed no measured concentrations above the LOQ of the analytical method employed, determined to be 0.32 mg Si/L, were obtained. On Days 7, 12, 14, 19 and 21, all measured concentrations were below the LOD, determined to be 0.10 mg Si/L. In an attempt to demonstrate a dose response, on Days 12 and 19 a sample of the freshly prepared 3.2 mg/L loading rate WAF from which dilutions were made to give the required test concentrations was taken for analysis. On both occasions measured concentrations below the LOD were observed.
Due to a technical oversight a sample of the 0.0032 mg/L loading rate WAF was not provided for analysis on Day 19. This was considered to have had no adverse effect on the outcome of the test given that at this concentration, no measurable levels of silicon were detected over the 21-Day period.
Given that the toxicity cannot be attributed to a single component (silicon) or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

Validation Criteria
The following validation criteria were achieved during the test:

	Required	Actual
Control mortality	≤20%	0%
Mean number of live young per surviving adult (control group)	≥60 after 21 days	124
Coefficient of variation for control group∗	≤25%	7%
No ephippia produced	0	0
Dissolved oxygen	>3 mg O2/L	≥8.6 mg O2/L
pH (control group)	6 to 9 variation ≤1.5	7.0 to 8.5 1.5

 

Lethal Effects on the Parental Generation (P₁)

Mortality (immobilization) occurred in the 0.10 and 0.32 mg/L loading rate WAF test groups only (10% and 20%, respectively). Statistical analysis of the mortality data by Fisher’s Exact Binomial Test with Bonferroni Correction incorporating Qualitative Trend Analysis by Contrasts showed that the observed mortalities in the 0.10 and 0.32 mg/L loading rate WAF test groups were not significantly different (P≥0.05) when compared to the control group. However, when compared to the pattern of immobilization observed in the second range-finding test where 40% immobilization occurred at 1.0 mg/L loading rate WAF, it was considered that the effects seen at 0.10 and 0.32 mg/L loading rate WAF were the start of a dose response. As such, the immobilization seen was attributed to exposure of the Daphnia to the test item rather than being considered to have been an incidental response.

Nominal Loading Rate (mg/L)	Parental (P1) Generation Mortalities (Initial Population = 10)	Parental (P1) Generation Mortalities (Initial Population = 10)	Parental (P1) Generation Mortalities (Initial Population = 10)	Total Number of Live Young
	Accidental	Inadvertent	Total
Control	0	0	0	1240
0.0032	0	0	0	1257
0.010	0	0	0	1287
0.032	0	0	0	1200
0.10	0	0	1	1135
0.32	0	0	2	956

 

Sub-lethal Effects on the Parental Generation (P₁)

There was no significant effect on the size and color of the daphnids in the control, 0.0032, 0.010 and 0.032 mg/L loading rate WAF test groups.
A single adult in the 0.10 mg/L loading rate WAF test group was observed to be pale on Days 13 and 14 but appeared normal between Days 15 and 21. A single adult in the 0.32 mg/L loading rate WAF test group was observed to be pale on Days 10, 11, 13 but appeared normal between Days 14 and 21.

Inspection of the body length data showed that was no significant difference in body length in any of the test groups when compared to the control.

 

Effects on Reproduction

After 21 days there were no statistically significant differences (P≥0.05) between the control and the 0.0032, 0.010, 0.032 and 0.10 mg/L loading rate WAF test groups in terms of the number of live young produced per adult. However, a significant difference (P<0.05) was determined between the control and the 0.32 mg/L loading rate WAF test group in terms of number of live young produced per adult after 21 days exposure to the test item (approximately a 23% reduction in number of live young produced at 0.32 mg/L loading rate WAF compared to the control group).

 

Effects on the Filial Generation (F₁)

Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by the 0.0032, 0.010, 0.032 and 0.10 mg/L loading rate WAF test groups to be in the same general conditions as the young produced in the control group over the duration of the test. Five broods produced on Day 19 and one brood on Day 20 in the 0.32 mg/L loading rate WAF test group were observed to be small and/or pale in comparison to those produced in the control group.
Young were first produced in the control test group on Day 8 of the test.
On Day 20, three out of the eight control replicates which produced young were observed to contain dead neonates. These amounted to 46 out of the 236 in total produced by the control group on this day (19%). It was not evident from the data as to the cause of the deaths but the 14% loss was considered to have had no adverse effect on the outcome of the test.
There were no unhatched eggs or dead young in any of the treatment groups surviving to maturation.

 

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

 

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF.
At both the start and end of the mixing period and following a 1-hour standing period the 3.2 mg/L loading rate was observed to be a clear colorless water column with test item adhered to the plastic slide suspended under the water surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
At the start and throughout the test all control solutions were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated following OECD/EC guidelines and gave a 21-day NOELR of 0.1 mg/L loading rate WAF for reproduction and 0.032 mg/L loading rate WAF for immobilisation.

Description of key information

The toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated following OECD/EC guidelines and gave a 21-day (semi-static) NOELR of 0.1 mg/L loading rate WAF for reproduction and 0.032 mg/L loading rate WAF for immobilisation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.032 mg/L