1,3-bis(trimethylsilyl)urea

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 - 29 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

Gas chromatography (GC)

Details on sampling:

- Concentrations: Fresh media (0 and 24 h) and aged media (24 and 48 h) of the control, reference and all test groups
- Sampling method: The fresh media samples (at 0 and 24 h) were taken from the bulk test preparation and aged media samples (at 24 and 48 h) were taken from pooled replicates.
- Sample storage conditions before analysis: All samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preliminary solubility work indicated that the test item is practically insoluble in water using traditional methods. Therefore, the test item qualifies as a difficult substance according to the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). A modification of the standard method for the preparation of aqueous media was performed and a saturated solution was prepared by stirring an excess (100 mg/L, i.e. 1100 mg in 11 L) of test item in test water for a period of 24 h (propeller at 1500 rpm) followed by the removal of any remaining undissolved test item by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 1 L used to pre-condition the filter was discarded), to give a saturated solution of the test item (100% v/v).
- Range-finding test: The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 80 mg/L could be obtained using a saturated solution method of preparation.
- Controls: Reconstituted water (Elendt M4 medium)
- Evidence of undissolved material: Yes, considering the filtration step.
- Other relevant information: At the start and throughout the test all control, test and reference solutions were observed to be clear colorless solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at study initiation: < 24 h
- Stage and instar at study initiation: First instar
- Method of breeding/culturing: Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C and a light cycle of 16 h light and 8 h darkness with 20 min dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension). Culture conditions ensured that reproduction occured by parthenogenesis. Gravid adults were isolated the day before test initiation, such that the young daphnids produced overnight were < 24 h old. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Source: In-house
- Feeding during test : No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L = 250 mg/L CaCO3

Test temperature:

20 - 21 °C (fresh media at 0 and 24 h)
20 °C (aged media at 24 and 48 h)

pH:

7.6 - 7.8 (fresh media at 0 and 24 h)
7.6 - 7.8 (aged media at 24 and 48 h)

Dissolved oxygen:

9.0 - 9.4 mg O2/L (fresh media at 0 and 24 h)
9.0 - 9.1 mg O2/L (aged media at 24 and 48 h)

Nominal and measured concentrations:

Control, 100% v/v (nominal)
Not determined, 73 mg/L trimethylsilanol (fresh media at 0 h)
Not determined, 19.9 mg/L trimethylsilanol (aged media at 24 h)
Not determined, 79.8 mg/L trimethylsilanol (fresh media at 24 h)
Not determined, 43.7 mg/L trimethylsilanol (aged media at 48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels containing approximately 100 mL test preparation
- Type: Covered to reduce evaporation
- Aeration: No
- Renewal rate of test solution: 24 h , daphnids were transferred by wide bore pipette
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water (Elendt M4 medium)
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h darkness with 20 min dawn and dusk transition periods
- Light intensity: 200 - 1200 lux

EFFECT PARAMETERS MEASURED:
- Immobilization or adverse reactions: at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test a limit test was conducted as definitive test at a concentration of 100% v/v saturated solution (corresponding to approximately 80 mg/L, based on the preliminary media preparation trial) to confirm that at highest attainable test concentration no significant immobilization was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 70 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: reference item trimethylsilanol (hydrolysis product)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Immobilisation of control: 5%

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the positive control was conducted between 23 Jan to 07 Feb 2020 and the results from the positive control were within the normal range for the tested reference item potassium dichromate (see 'Other').
- Dose-response test: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L potassium dichromate
- EC50 (24 h) = 0.90 mg/L (C.I. = 0.80 - 1.0 mg/L)
- EC50 (48 h) = 0.79 mg/L (C.I. = 0.71 - 0.87 mg/L)
- NOEC (24 h/ 48 h) = 0.56 mg/L
- LOEC (24 h/ 48 h) = 1.0 mg/L
- Other: Analysis of immobilization data was carried out using the Weibull analysis (linear maximum-likelihood regression) at 24 and 48 h using the ToxRat Professional computer software package.
- Other: The results of the inter laboratory tests and Technical Corrigendum to ISO 6341 give an EC50 (24 h) of 0.6 to 2.1 mg/L for potassium dichromate.

ANALYTICAL RESULTS

Test concentrations and stability

Analysis of the fresh test item preparations at 0 and 24 h resulted in measured test item concentrations of 73 and 80 mg/L, respectively. Analysis of the aged test item preparations at 24 and 48 h resulted in measured test item concentrations of 20 and 44 mg/L, respectively (Table 1).

Analysis of the fresh reference item (TMS) preparations at 0 and 24 h resulted in 94 and 103 mg/L, respectively. Analysis of the aged test preparations at 24 and 48 h resulted in 42 and 58 mg/L, respectively (Table 1).

The recovery rates in the reference item ranged from 94 to 103% in fresh media and from 42 to 58% in aged media (Table 1).

Table 1. Analytical results for test samples.

Time [h]	Nominal concentration of test item Cnom [% v/v saturated solution]	Measured concentration of trimethylsilanol in test sample C [mg/L]	Percentage of nominal concentration [%]
0	Control	Not detected	-
	100 (test item)	73.0	-
	100 (TMS)	94.1	94
24 (aged)	Control	Not detected	-
	100 (test item)	19.9	-
	100 (TMS)	42.2	42
24 (fresh)	Control	Not detected	-
	100 (test item)	79.8	-
	100 (TMS)	103	103
48 (aged)	Control	Not detected	-
	100 (test item)	43.7	-
	100 (TMS)	58.3	58

              

Given that not all concentrations were within 80% of the nominal concentration, it was considered justifiable to base the results on the geometric mean measured test item concentrations in order to give a worst-case analysis of the data.

The geometric mean measured concentrations for each period of media renewal (0 - 24 and 24 - 48 h) were calculated and the arithmetic mean of the two values determined. The geometric mean measured test concentration of the test item and reference item were 49 and 70 mg/L, respectively.

Nitrate, Nitrite and Ammonia Analysis

Analysis of test samples at 0 and 24 h showed that there were no significant changes in the levels of nitrate, nitrite or ammonia during the course of the definitive test (Table 2).

Table 2. Nitrate, Nitrite and Ammonia Analysis From the Definitive Test

Geometric Mean Measured Test concentration [mg/L]	Time point [h]	Ammonia as NH3 [mg/L]	Nitrite NO2 [mg/L]	Nitrate NO3 [mg/L]
Control	0	0.0005	0.0	0.18
Test item 49		0.014	0.002	0.14
Reference item 70		0.00086	0.002	0.17
Control	24	0.0011	0.004	0.12
Test item 49		0.00034	0.003	0.15
Reference item 70		0.00024	0.002	0.16

                      

BIOLOGICAL RESULTS

No sub-lethal effects of exposure were observed throughout the test.

Test item.

5% immobilization was observed in the control and at 49 mg/L of the test item at 48 h (Table 3).

For the test item, the experimental findings resulted in an EC50 (48 h) > 49 mg/L and a NOEC (48h) of 49 mg/L based on the geometric mean measured test concentration (Table 4).

Reference item.

No immobilization was observed at 70 mg/L of the reference item (Table 3).

For the reference item, the experimental findings resulted in an EC50 (48 h) > 70 mg/L and a NOEC (48 h) of 70 mg/L based on the geometric mean measured test concentration (Table 4).

Table 3. Cumulative immobilization data and observations

Geom. Mean meas. Conc. [mg/L]	24 h
	Cumulative Immobilized Daphnia (initial population: 5 per replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Test item 49	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Ref. item 70	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Geom. Mean meas. Conc. [mg/L]	48 h
	Cumulative Immobilized Daphnia (initial population: 5 per replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	1	0	0	0	1	5	4 N	5 N	5 N	5 N
Test item 49	1	0	0	0	1	5	4 N	5 N	5 N	5 N
Ref. item 70	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R = replicate; N = normal

Table 4. Effect concentrations for the immobilization of Daphnia magna

Geometric mean measured test concentration	Time	EC50
[mg/L]	[h]	[mg/L]
Test item 49	24	> 49
	48	> 49
Reference item 70	24	> 70
	48	> 70

 

VALIDATION CRITERIA 

The experimental study fulfilled the validity criteria defined by the OECD testing guideline 202 (Table 5).

Table 5. Validity criteria for OECD 202 (2004)

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, not more than 10% of the daphnids should have been immobilized.	In the control, the observed immobilization was 5%.	YES
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	The dissolved oxygen concentration at the end of the test was 9.0 mg O2/L.	YES

 

Description of key information

No effects up to the observed limit of water solubility in the test medium

EC50 (48 h) > 49 mg/L (geom. mean measured, OECD 202, D. magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 49 mg/L

Additional information

One experimental study is available for the short-term toxicity of 1,3-bis(trimethylsilyl)urea (CAS No. 18297-63-7) to aquatic invertebrates, which was conducted according to OECD testing guideline 202 (2004) and GLP.

The test item is poorly soluble and hydrolyses rapidly (DT50 < 10 min, OECD 111) to trimethylsilanol (TMS), hexamethyldisiloxane and urea. TMS has low volatility and relatively high solubility and was considered to be a stable degradant representative of the test item.

Preliminary solubility work indicated that a testing solution could not be obtained using traditional methods of preparation. Therefore, a saturated solution of the test item (100% v/v) was prepared by stirring an excess of test item in test water (1100 mg in 11 L) using a propeller stirrer at approximately 1500 rpm for 24 h followed by filtration (0.2 µm Sartorius Sartopore filter, first 1 L discarded) to remove any undissolved test item. In this way, a dissolved test item concentration (i.e. measured as TMS) of approximately 80 mg/L was obtained, indicating the limit of water solubility of this test item under test conditions.

The concentration and stability of the test item in the test preparations was verified by chemical analysis of TMS concentrations in the fresh bulk preparations (at 0 and 24 h) and from the pooled replicates of the aged media (at 24 and 48 h) using gas chromatography. Ammonia, nitrates and nitrites were also measured in the test water (fresh and aged media) by spectrophotometry to assess if there could be an increase in urea throughout the test.

In a semi-static test with a 24 h renewal rate, Daphnia magna were exposed for 48 h to an aqueous saturated solution (100% v/v) of the test item or TMS, the latter of which was used as reference item to ensure that if toxicity was observed it could be related to this constituent and not any other degradant. The 100% v/v saturated solution of the reference item TMS was prepared in an identical manner to that described for the test item (100 mg/L stirred for 24 h followed by filtration). Immobilization and sub-lethal effects were recorded after 24 and 48 h.

Analytical findings showed that there were no significant changes in the levels of nitrate, nitrite or ammonia over the course of the test.

Analysis of the fresh test item preparations (100% v/v) at 0 and 24 h resulted in measured test item concentrations of 73 and 80 mg/L TMS, respectively. Analysis of the aged test item preparations at 24 and 48 h resulted in measured test item concentrations of 20 and 44 mg/L TMS, respectively.

Analysis of the fresh reference item (TMS) preparations (100% v/v) at 0 and 24 h resulted in 94 and 103 mg/L, respectively. Analysis of the aged test preparations at 24 and 48 h resulted in 42 and 58 mg/L, respectively. The recovery rates in the reference item ranged from 94 to 103% in fresh media and from 42 to 58% in aged media.

Given that not all concentrations were within 80% of the nominal concentration, it was considered justifiable to base the results on the geometric mean measured test item concentrations. The geometric mean measured concentrations for each period of media renewal (0 - 24 and 24 - 48 h) were calculated and the arithmetic mean of the two values was determined. The geometric mean measured test concentration of the test item and reference item were 49 and 70 mg/L, respectively.

After 48 h, no sub-lethal effects were observed.

For the test item (incl. all possible hydrolysis products which occurred within the duration of the test), the experimental findings resulted in an EC50 (48 h) > 49 mg/L and a NOEC (48 h) of ≥ 49 mg/L based on the geometric mean measured test concentration. For the reference item, the experimental findings resulted in an EC50 (48 h) > 70 mg/L and a NOEC (48 h) of ≥ 70 mg/L based on the geometric mean measured test concentration.

The study fulfilled the validity criteria defined by the testing guideline.