3-(triethoxysilyl)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:

2020-11-18 to 2021-11-03.

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analyzed via GC-MS.

- Sampling method: The test item concentrations of 3-(Triethoxysilyl)propanethiol were analytically verified via GC-MS/MS in the fresh media at the start of the exposure-renewal intervals on days 0, 7, 14 (0 hours) and in the old media at the end of the exposure intervals on days 1, 8, 15 (24 hours) in all concentration levels (with surviving daphnids) and in the control. Three samples were taken and extracted immediately by direct sampling in the extraction vials with added extraction medium at each sampling point. Two replicates were analysed. In case the confirmation of the results was necessary, the third replicate was also analysed.

- Sample storage conditions before analysis: Immedate extraction by direct sampling into extraction vials.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of the non-GLP preliminary range finding test, the definitive reproduction test was carried out with five concentrations of the test item of 25.0 - 44.5 - 79.1 - 141 - 250 µg/L (separation factor: 1.78). The definitive test was performed under semi-static conditions with a daily renewal of the test solutions.
A stock solution of 10.0 mg/L of the test item was prepared with the culture medium (adjusted to pH 7 ± 0.1 by addition of 1 M HCl) for each exposure-renewal interval. The dispersion was stirred with a magnetic stirrer at approximately 1100 rpm for 1 hour at room temperature.
Five concentrations of the test item were prepared by dilution of the stock solution with the dilution water (dilution water is the same media as the culture medium) and tested at follows:
25.0 - 44.5 - 79.1 - 141 - 250 µg/L (separation factor: 1.78) which corresponds to the time-weighted mean measured test item concentrations of: 10.7 – 19.0 – 27.2 – 81.0 – 158 µg/L

50 g test solution per replicate were weighed out into each test vessel. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

- Controls: 10 replicates in dilution water without test item, adjusted to pH 7 ± 0.1 by addition of 1 M HCl, incubated under the same conditions as the test groups.

- Test concentration separation factor: 1.78

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Age at study initiation (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, 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 the following period 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: not reported
- Source: continuous laboratory cultures. Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany. Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany.
- Age of parental stock (mean and range, SD): not reported
- Feeding during test
- Food type: Pseudokirchneriella subcapitata (0.327 – 0.418 mL) and Desmodesmus subspicatus (0.420 – 0.619 mL) suspension was provided as food corresponding to max. 0.2 mg C per Daphnia and day.
The density of the algae suspension varied, but it was the same for all test groups on each feeding day.
- Amount: max. 0.2 mg C per Daphnia and day
- Frequency: Daily

ACCLIMATION
- Acclimation period: At least 2 hours in the dilution water adjusted to pH 7 ± 0.1 by addition of 1 M HCl
- Acclimation conditions (same as test or not): Yes
- Feeding frequency: n/a
- Health during acclimation (any mortality observed): none reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, 16 hours illumination; light intensity of max. 1500 lx
Culture medium Elendt M4, according to OECD 211, is used.
Feeding of the culture stocks 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. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

240275 CaCO3

Test temperature:

20.4-21.3 °C

pH:

6.89-7.67

Dissolved oxygen:

7.78-9.23 mg/l

Salinity:

n/a

Nominal and measured concentrations:

Nominal: Control, 25.0, 44.5, 79.1, 141, 250 µg/L
Measured (time weighted mean): Control, 10.7, 19.0, 27.2, 81.0, 158 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity filled with 50 ml test solution, loosely covered with watch glasses
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: not reported

Additionally, for the days with specific analysis, replicates without daphnids and algae were prepared, sampled at start and after 24 h hours and analyzed to check the stability of the test item under exposure conditions.

Application 50 g test solution per replicate were weighed out into each test vessel. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Renewal of the test solutions The test solutions were renewed every day. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same composition as the culture medium: Culture Medium is Elendt M4 according to OECD 211, Annex 2.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water quality parameters (i.e. dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in fresh media at the start (0 hours) and in old media at the end (24 hours), in one replicate of the control and the highest concentration of the test item with a living parent animal. The pH was measured daily from fresh and old medium of at least one replicate per test group.
The water quality parameters in fresh media at the start of an exposure-renewal interval were measured in an additional replicate without daphnids of the highest test item concentration and the control. At the end of an exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest test item concentration and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test period with a thermo-hygrograph.

OTHER TEST CONDITIONS
- Adjustment of pH: Dilution water adjusted to pH 7 ± 0.1 by addition of 1 M HCl
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality of parental daphnids The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the offspring was counted. Dead daphnids were removed as soon as they were detected.

Neonates First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of observation were recorded.

Additional observations / Condition of parental daphnids Abnormalities (e.g. abnormal swimming behavior, number of males and winter eggs) were observed and recorded on each day of observation.

Total body length / Mean dry weight of parental daphnids At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all test concentrations and the control were determined. As stated in the OECD protocol, this may be useful additional information but is not used for determination of a NOEC.

Additional endpoints The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions with a renewal of the test solutions every day over a period of 21 days. A control and three test concentrations of the test item of 0.006, 0.030 and 0.150 mg/L were tested in the range finding test. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). The pH value of the stock solution was measured daily and was in the range from 6.92 to 7.07. Five daphnids, one daphnia per replicate, were exposed to each concentration level and the control. The biological results are presented in Table 1 in "Any other information on materials and methods incl. tables".
The test item concentrations of the control and the concentration levels 0.006 and 0.150 mg/L were analytically verified via GC-MS in the fresh media at the start of the exposure and in the old media at the end of an exposure-renewal interval. For extraction, 10 ml of the media was sampled without further treatment into 50 ml headspace vials. The samples were immediately transferred to the analytical facility and extracted by adding sodium chloride and cyclohexane and manual agitation for 2 minutes.
- Results used to determine the conditions for the definitive study: No effects on mortality or reproduction at the 0.006 and 0.03 mg/l concentrations. Effects on mortality at the 0.15 mg/l concentration.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and adult mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.158 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.148 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.158 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and adult mortality

Details on results:

- Behavioural abnormalities: none reported
- Observations on body length and weight: The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 10.7 to 158 µg/L were in the range of 4.00 to 4.40 mm per daphnid and 4.55 mm per daphnid in the control group.
The mean dry weight of the survived parental daphnids was in the range of 0.20 to 0.70 mg per daphnid in the concentration levels 10.7 to 158 µg/L and 0.74 mg per daphnid in the control.
- Other biological observations: The first appearance of living juveniles was observed between days 6 and 9 at the introduced parental daphnids of the control and the concentration levels 10.7 to 158 µg/L.
No ephippia were observed in the control or in the test groups during the test. 
- Mortality of control: No mortality
- Other adverse effects control: None reported
- 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

Reported statistics and error estimates:

NOEC Determination
Shapiro-Wilk´s Test on Normal Distribution
Levene´s Test on Variance Homogeneity (with Residuals)
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).

Normal-distribution and variance-homogeneity requirements are fulfilled.
The user-selected multiple test is performed.

Dunnett`s Multiple t-test Procedure

Calculation of the EC10 and the EC50 for the Reduction of the Reproductive Output:
Estimated parameters of the 3-param. normal CDF
Shapiro Wilk`s test on normal distribution of residuals
Analysis of Variance and Test for Lack of Fit for the 3-param. normal CDF
Point estimates from the 3-param. normal CDF
Since the slope of the relationship was found to be not significant, no EC and confidence limits are provided.
As the slope of the curve is not significantly different from zero no EC calculation is possible. The EC10 and EC50 value was empirically derived from the data.
Adult Mortality
Cochran-Armitage test procedure
Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response)
Step-down Cochran-Armitage Test Procedure
Probit analysis using linear max. likelihood regression

Table 2: Effects on Reproduction for all Introduced Parents (based on the time-weighted mean measured concentrations of the test item)

Time-weighted mean measured test item concentrations [µg/L]	Mean number of offspring per introduced parental daphnid
	Mean	SD
158	71.4	27.5
81.0	102	25.7
27.2	92.3	17.9
19.0	90.1	23.9
10.7	102	18.1
Control	120	24.7

Table 3: Effects on Reproduction for all Survived Parents (based on the time-weighted mean measured concentrations of the test item)

Time-weighted mean measured test item concentrations [µg/L]	Mean number of offspring per survived parental daphnid
	Mean	SD	CV
158	73.5	8.39	11.4
81.0	106	23.3	22.0
27.2	92.3	17.9	19.4
19.0	90.1	23.9	26.5
10.7	102	18.1	17.8
Control	120	24.7	20.6

Table 4: Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

Time-weighted mean measured test item concentrations [µg/L]	Adult mortality [%]
	7 days	14 days	21 days
158	0	10	50 ^1)
81.0	0	0	10
27.2	0	0	0
19.0	0	0	0
10.7	0	0	0
Control	0	0	0

¹⁾ = statistically significant (Step down Cochran-Armitage Test Procedure, α = 0.05)

Table 5: Measured Concentrations of the Test Item during the Second Definitive Test (Days 0, 1, 7 and 8)

Sampling date	Day 0 Fresh medium 0 hours		Day 1 Old medium with algae 24 hours		Day 7 Fresh medium 0 hours		Day 8 Old medium with algae 24 hours		Day 14 Fresh medium 0 hours		Day 15 Old medium with algae 24 hours		Time weighted mean measured Test Item concentration
Nominal concentration of the test item [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	[µg/L]
250	202 (200/205)	81	120 (114/126)	48	212 (202/222)	85	85.6 (82.1/89.1)	34	205 (204/206)	82	101 (103/100)	41	158
141	111 (111/110)	78	65.7 (64.1/67.2)	47	112 (104/121)	80	49.9 (51.5/48.2)	35	120 (120/120)	85	49.8 (47.8/51.7)	35	81.0
79.1	60.6 (59.7/61.5)	77	5.47 ^1) (5.46/5.22/5.73)	7	60.8 (57.5/64.0)	77	8.11 (8.42/7.81)	10	68.7 (68.7/68.7)	87	12.2 (12.4/12.0)	15	27.2
44.5	34.8 (33.0/36.7)	78	10.5 (10.8/10.2)	24	34.8 (32.0/37.5)	78	6.08 (5.98/6.18)	14	35.9 (34.3/37.5)	81	10.0 (10.1/9.96)	22	19.0
25.0	19.2 (19.7/18.8)	77	7.37 (6.63/8.12)	29	16.4 (14.6/18.1)	65	2.80 (2.56/3.04)	11	20.3 (19.4/21.2)	81	6.33 (6.83/5.83)	25	10.7
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc.      = measured concentration of the test item, mean value of 2 / 3 replicates, enrichment and dilution factors taken into account, single values in brackets

%                     = percent of the nominal concentration of the test item

LOQ               = limit of quantification (1.0 µg/L of the test item)

1)                     = mean value of three replicates

Table 6: Measured Concentrations of the Test Item in Old medium without Algae during the Second Definitive Test (Days 1, 8 and 15)

Sampling date	Day 1 Old medium without algae 24 hours		Day 8 Old medium without algae 24 hours		Day 15 Old medium without algae 24 hours
Nominal concentration of the test item [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
250	148 (145/151)	59	112 (106/117)	45	143 (141/146)	57
141	88.7 (84.7/92.6)	63	79.5 (79.7/79.3)	56	80.0 (79.0/80.9)	57
79.1	42.6 (42.9/42.2)	54	32.3 (32.7/32.7)	41	37.2 (37.1/37.30)	47
44.5	23.8 (22.8/24.7)	53	17.6 (17.2/18.12)	40	21.2 (21.0/21.5)	48
25.0	9.83 (9.82/9.84)	39	10.2 (9.67/10.6)	41	10.4 (9.91/11.0)	42
Control	< LOQ		< LOQ		< LOQ

Meas. conc.       = measured concentration of the test item, mean value of two replicates, enrichment and dilution factors taken into account, single values in brackets

%                      = percent of the nominal concentration of the test item

LOQ                 = limit of quantification (1.0 µg/L of the test item)

It can be seen from Tables 5 and 6 above, when comparing the measured concentrations in old test media when test organisms were present with the replicates for analysis without the test organisms and algae, that higher recoveries of test substance are measured in the media without test organisms.  These recoveries are consistent with the expected hydrolysis rate. This suggests that the substance is potentially interacting with biological matter, perhaps adsorbing to it, and this may account for the variability observed in analytical recoveries in test media when test organisms and algae are present. This also indicates that losses of parent material in test solution may not just be due to hydrolysis.

Validity criteria fulfilled:

yes

Conclusions:

A 21-day NOEC value of 0.081 mg/l has been determined for the effects of the test substance on survival and reproduction of Daphnia magna, based on time weighted average measured concentrations of the parent substance.
The LOEC for reproduction and adult survival was determined to be 0.158 mg/l (the highest concentration tested).
The EC10 / 50 values for the reproductive output could not be determined. The slope of the curve was not significantly different from zero. The EC10 and EC50 value were empirically derived from the observed data.
The test substance hydrolyses in water at a moderately rapid rate and the focus of the study was to assess the effects of the parent substance. Based on the semi-static exposure regime with daily renewal of test media, neutral pH of test media and measured analytical recoveries of parent substance, it is likely that the test organisms were primarily exposed to the parent substance at the beginning of the test media renewal period, with increasing amounts of hydrolysis products towards the end of the media renewal period.

Description of key information

Long-term toxicity to invertebrates: 21-day NOEC 0.081 mg/l, effects of the test substance on survival and reproduction of Daphnia magna, based on time weighted average measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.081 mg/L

Additional information

A 21-day NOEC value of 0.081 mg/l has been determined for the effects of the test substance (3-(triethoxysilyl)propanethiol (CAS 14814-09-6, EC No. 238-883-1)) on survival and reproduction of Daphnia magna, based on time weighted average measured concentrations of the parent substance (Noack, 2022). The study was conducted in accordance with OECD TG 211 and in compliance with GLP.

The LOEC for reproduction and adult survival was determined to be 0.158 mg/l (the highest concentration tested).

The EC₁₀ and EC₅₀ values for the reproductive output could not be determined. The slope of the curve was not significantly different from zero. The EC₁₀ and EC₅₀ values were empirically derived from the observed data to be <0.0107 mg/l and >0.158 mg/l respectively, for effects on reproduction. However, because there was no dose response curve and reproductive output did not follow a trend in effects, and only the highest dose was statistically different to the control, the EC₁₀ value is not considered to be robust. The NOEC value has therefore been selected as the key value.

The 21-day LC₅₀ for adult mortality was determined to be 0.148 mg/l and the LC₂₀ was 0.0997 mg/l.

The test substance hydrolyses in water at a moderately rapid rate and the focus of the study was to assess the effects of the parent substance. Based on the semi-static exposure regime with daily renewal of test media, neutral pH of test media and measured analytical recoveries of parent substance, it is likely that the test organisms were primarily exposed to the parent substance at the beginning of the test media renewal period, with increasing amounts of hydrolysis products towards the end of the media renewal period.