1,3-bis(trimethylsilyl)urea

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May - 11 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Deviations:

yes

Remarks:

Minor deviations (reduced volume of test media, increased loading rate, plastic tanks) did not affect the integrity or validity of the study (see "Any other information on results incl. tables" for details).

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

Gas chromatography

Details on sampling:

- Concentrations: Control, 100% v/v saturated test item solution, 100% v/v saturated reference item solution
- Sampling method: Duplicate samples were taken from freshly prepared bulk control, test item and reference item solutions (at 0, 24, 48 and 72 h) and from aged test item solutions (at 24, 48, 72 and 96 h)
- Sample storage conditions before analysis: Samples were analyzed on the day of sampling. Duplicate samples from fresh and aged media were stored frozen fur 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 was practically insoluble in water and qualified as being a 'difficult substance' as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019) for which traditional methods of media preparation (i.e. ultrasonication & high shear mixing) do not work.
- Preparation of test solution: A nominal amount of 2200 mg test item was dispersed in 22 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 h. After 24 h the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 L used to pre-condition the filter was discarded) to give a 100% v/v saturated solution. Similarly, a 100% v/v saturated solution of the reference item trimethylsilanol was prepared.
- Controls: Test water only (dechlorinated tap water), maintained under identical conditions.
- Evidence of undissolved material: The control, test item and reference item preparations were observed to be clear colorless solutions throughout the test.
- Verification of test item concentration and stability: Chemical analysis of fresh media at 0, 24, 48 and 72 h and of old media at 24, 48, 72 and 96 h was conducted to verify concentrations and stability.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: In-house culture, originally obtained from Brow Well Fisheries Limited, Hebden, Yorkshire, UK (29 Sep 2020)
- Mean length at study end: 6.7 cm (SD = 0.8)
- Mean body weight at study end: 2.73 g (SD = 0.96)
- Maintenance of the brood fish: Fish were maintained in a plastic tank with a single pass water renewal system.
- Breeding conditions: 16 h light and 8 h dark cycle with 20 min dawn and dusk transition periods; 13 - 14 °C; Dissolved oxygen ≥ 9.8 mg O2/L

ACCLIMATION
- Acclimation period: 30 Oct - 06 Nov 2020
- Acclimation conditions: Same as test
- Type of food during acclimation: Commercial trout pellets
- Health during acclimation: No mortality 7 d prior to the start of the test

FEEDING DURING TEST
- Frequency: Feeding was discontinued approximately 25 h prior to the start of the definitive test

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

140 mg/L CaCO3

Test temperature:

13 - 14 °C

pH:

Control: 7.6 - 7.8 (fresh media) and 7.7 - 8.1 (aged media)
Test item solutions: 7.7 - 8.2 (fresh media) and 7.8 - 8.0 (aged media)
Reference item solutions: 7.7 - 8.0 (fresh media) and 8.0 (aged media)

Dissolved oxygen:

Control: 10.2 - 10.8 mg O2/L (fresh media) and 8.3 - 9.7 mg O2/L (aged media)
Test item solutions: 9.9 - 10.3 mg O2/L (fresh media) and 9.3 - 9.8 mg O2/L (aged media)
Reference item solutions: 9.8 - 10.2 mg O2/L (fresh media) and 9.6 - 9.9 mg O2/L (aged media)

Nominal and measured concentrations:

Nominal: Control, 100% v/v
Measured concentration of freshly prepared test item solutions (at 0, 24, 48 and 72 h): 82.1, 86.1, 89.3, and 96.0 mg/L
Measured concentration of aged test item solutions (at 24, 48, 72 and 96 h): 82.5, 80.7, 79.9 and 75.0 mg/L
Measured concentration of freshly prepared reference item solutions (at 0, 24, 48 and 72 h): 99.5, 97.1, 92.7, and 100 mg/L
Measured concentration of aged reference item solutions (at 24, 48, 72 and 96 h): 84.0, 95.6, 86.9 and 94.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Covered 25 L glass exposure vessels filled with 20 L test media (with the exception of the reference item tank where only 12 L was added at 48 h in error).
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution: A semi-static test was performed with daily renewal of test preparations to prevent the build-up of nitrogenous waste products.
- No. of organisms per vessel: 7 fish per test vessel
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.96 g body weight/L at the end of the test, which is greater than the 0.8 g body weight/L allowed by the study plan. Also, an error had occured during the filtration procedure during which approximately 10L of the reference test media was lost and only 12 L were available to dose the test system at 48 h, instead of the required 20 L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water was dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination unit) and partly softened (Elga Nimbus 1248D Duplex Water Softener).
- Hardness: approximately 140 mg/L CaCO3
- Culture medium different from test medium: The test water was the same as that used to maintain the stock fish.
- Intervals of water quality measurement: Water temperature (using a Testo 106 digital thermometer), pH and dissolved oxygen concentrations (using a Hach Flexi handled meter) were recorded daily throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark cycle with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED
- Mortality: recorded at 1, 3, 6, 24, 29, 48, 52, 72, 76 and 96 h
- Sub-lethal effects: recorded at 1, 3, 6, 24, 29, 48, 52, 72, 76 and 96 h

TEST CONCENTRATIONS
- Limit test: 100% v/v saturated solution
- Range finding study : No
- Other relevant information: In accordance with REACh, the test was conducted according to the recommended threshold approach in which the lowest EC50 value from either the algal growth inhibition study or acute toxicity to aquatic invertebrates study is set as the threshold concentration for a 'limit test'. If no mortalities are observed at this concentration level, this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 values obtained from both the algal study (study number SN22HF) and aquatic invertebrates study (study number MV79QL) were based on 100% v/v saturated solutions. Therefore, a limit test at the single nominal concentration of 100% v/v saturated solution was conducted.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: reference item trimethylsilanol (hydrolysis product)

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: There were no sub-lethal effects of exposure.
- Mortality of control: One of the seven control fish was found dead at the 48-hour observation. This was considered to be due to an injury sustained during the 24-hour water change and un-related to the conditions of the test.
- Other adverse effects control: One control fish was found to have sustained fin damage and was swimming erratically at 29 h. This observation is considered to result from an error during the water change at 24 h and is considered to be un-related to the exposure.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The control, test item and reference item preparations were observed to be clear colorless solutions throughout the test.

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

ANALYTICAL RESULTS

 

Test item. Analysis of the freshly prepared test item solutions at 0, 24, 48 and 72 h resulted in measured test item concentrations of 82 to 96 mg/L. Analysis of the corresponding aged preparations at 24, 48, 72 and 96 h resulted in measured concentrations ranging from 75 to 83 mg/L (Table 1).

 

Reference item. Analysis of the freshly prepared reference item solutions at 0, 24, 48 and 72 h resulted in measured reference item concentrations of 93 to 100 mg/L. Analysis of the corresponding aged preparations at 24, 48, 72 and 96 h resulted in measured concentrations in the range of 84 to 96 mg/L (Table 1).

 

Table 1. Measured test concentrations.

Time point	Nominal concentration of test item	Measured concentration of trimethylsilanol	Percentage of nominal concentration
	Cnom	C
[h]	[% v/v saturated solution]	[mg/L]	[%]
0	Control	ND	-
	100	82.1	-
	100*	99.5	100
24** (old)	Control	ND	-
	100	82.5	-
	100*	84.0	84
24*** (fresh)	Control	ND	-
	100	86.1	-
	100*	97.1	97
48*** (old)	Control	ND	-
	100	80.7	-
	100*	95.6	96
48*** (fresh)	Control	ND	-
	100	89.3	-
	100*	92.7	93
72*** (old)	Control	ND	-
	100	79.9	-
	100*	86.9	87
72** (fresh)	Control	ND	-
	100	96.0	-
	100*	100	100
96*** (old)	Control	ND	-
	100	75.0	-
	100*	94.1	94

* Sample prepared using the trimethylsilanol analytical standard.

** 24 h (old) samples were analyzed alongside the 72 h (fresh) samples.

*** 24 h (fresh), 48 h (old), 48 h (fresh), 72 h (old) analyzed alongside the 96 h samples.

ND = not detected

- = not applicable

 

Nitrate, Nitrite and Ammonia.Analysis of the test water samples at 0 and 24 h showed that there were no changes in the levels of nitrate or nitrite during the course of the definitive study. However, there was an increase in ammonia which may be attributable to the presence of urea, which is a known degradant of the test item (Table 2).

 

Table 2. Measured nitrate, nitrite and ammonia concentrations.

Geometric mean measured test concentrations	Time point	Ammonia as NH3	Nitrite NO2	Nitrate NO3
[mg/L]	[h]	[mg/L]	[mg/L]	[mg/L]
Control	0	0.000025	0.002	1.6
Test item 84		0.0026	0.0	2.1
Reference item 94		0.00011	0.002	2.1
Control	24	0.0037	0.001	1.9
Test item 84		0.018	0.001	2.0
Reference item 94		0.0058	0.003	2.1

 

Since not all of the measured concentrations were within 80 to 120% of the nominal concentration, results were based on the geometric mean measured test concentrations of the test item and reference item, respectively, in order to give a “worst-case” analysis of the data.

The geometric mean measured concentration was determined for 24 h intervals (i.e. 0 to 24, 24 to 48, 48 to 72 and 72 to 96 h) and the arithmetic average was calculated from these values. The geometric mean measured test concentration of the test item and reference item are 84 and 94 mg/L, respectively (Table 3).

 

Table 3. Geometric mean measured test concentrations.

	Geometric mean measured test concentration
Test item	84 mg/L
Reference item	94 mg/L

 

 

BIOLOGICAL RESULTS

No mortalities in the 7 fish exposed to either 84 mg/L test item or 94 mg/L reference item was recorded after the exposure period of 96 h, resulting in an LC50 (96 h) of > 84 mg/L and an LC50 (96 h) > 94 mg/L for the test item and reference item, respectively (Table 5).

 

Table 4. Cumulative Mortality Data

Geometric mean measured test concentration	Cumulative mortality (initial population = 7)										Mortality
[mg/L]	Sampling time [h]										[%]
	1	3	6	24	29	48	52	72	76	96
Control	0	0	0	0	0	1	0	0	0	0	14
Test item 84	0	0	0	0	0	0	0	0	0	0	0
Reference item 94	0	0	0	0	0	0	0	0	0	0	0

 

 

Table 5. Effect values

	Test item [mg/L]	Reference item [mg/L]
LC50 (96 h)	> 84	> 94
NOEC (96 h)	≥ 84	≥ 94

 

 

DEVIATIONS FROM STUDY PLAN

During the preparation of the aqueous test media, an error had occurred during the filtration procedure through which approximately 10 L of the reference test media was lost, leaving only 12 L available for the test system instead of the required 20 L at 48 h. There was no impact on the study as the reference fish did not show any signs of stress or any other reactions and the reduction in test media did not affect the concentration received over the 24 h period.

 

Furthermore, the fish loading rate was 0.96 g body weight/L at the end of the test, which is greater than the 0.8 g body weight/L allowed by the study plan. Also, when the reference item tank only received 12 L of test media the loading rate would have been much greater than 0.8 body weight/L (approximately 1.6 g body weight/L). However, no signs of stress were noted in the fish throughout the exposure period and so significant depletion of the test item was noted. Therefore, the higher loading rates are considered not to have affected the purpose or validity of this study.

 

Lastly, the fish were maintained in plastic tanks prior use on the study instead of in glass or fiberglass tanks, as documented in the Study Plan. However, this was not considered to have had an impact on the test since the fish passed the validity criteria prior to test start and were found to be suitably healthy for use on the test.

 

VALIDATION CRITERIA

The study fulfilled the validation criteria laid down by the guideline and is therefore considered to be adequate and reliable (Table 6).

 

Table 6. Validity criteria for OECD testing guideline 203 (2019)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	One control fish died due to an injury sustained during the water change at 24 h.	Yes
The dissolved oxygen concentration must have been at least 60% of the air saturation value throughout the test	The oxygen concentration at the end of the test was≥60% of ASV (6.4 mg O2/L at 12 °C) in the control and test vessels.	Yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80% of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	Results were based on the geometric mean measured test concentration given that not all measured concentrations were within 80 to 120%.	Yes

Description of key information

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

LC50 (96 h) > 84 mg/L (geom. mean measured, OECD 203, O. mykiss)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 84 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 fish, which was conducted according to OECD testing guideline 203 (2019) 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 a nominal amount of 2200 mg test item in 22 L of test water with the aid of propeller stirring 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 91 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 preparation was verified by chemical analysis of TMS concentrations in fresh test media at 0, 24, 48 and 72 h and aged test media at 24, 48, 72 and 96 h by gas chromatography. Ammonia, nitrates and nitrites were also measured in fresh and aged test water by spectrophotometry to assess if there could be an increase in urea throughout the test.

In a semi-static limit test, rainbow trout Onchorhynchus mykiss was exposed to an aqueous saturated solution of 100% v/v 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 saturated solution of the reference item TMS was prepared in the same way as that of the test item. Mortality and sub-lethal effects were recorded at 1, 3, 6, 24, 29, 48, 52, 72, 76 and 96 h.

Analytical results showed that the test item and reference item were not stable under the experimental conditions. Furthermore, an increase of ammonia was recorded, which may be attributable to the presence of urea, which is a known degradant of the test item.

Analysis of the 100% v/v test item preparations resulted in measured test concentrations of 82 to 96 mg/L in fresh media (at 0, 24, 48 and 72 h) and of measured test item concentrations of 75 to 83 mg/L in aged media (at 24, 48, 72 and 96 h). Analysis of the 100% v/v reference item preparations resulted in measured reference item concentrations of 93 to 100 mg/L in fresh media (at 0, 24, 48 and 72 h) and of measured reference item concentrations ranging from 84 to 96 mg/L in aged media (at 24, 48, 72 and 96 h).

Therefore, results were based on the geometric mean measured test item and reference item concentrations, which were 84 and 94 mg/L for the test item and reference item, respectively.

After the 96 h exposure period, no mortality was observed in any test preparation, resulting in an LC50 (96 h) of > 84 mg/L for the test item (incl. all possible hydrolysis products which occurred within the duration of the test) and an LC50 (96 h) of > 94 mg/L for the reference item.

The study fulfilled the validity criteria and is thus considered reliable and valid.