1,3-bis(trimethylsilyl)urea

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May - 04 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Gas chromatography (GC)

Details on sampling:

- Concentrations: Control, 100% v/v saturated solution of test item (with and without algal cells at 0, 24, 48 and 72 h) and 100% v/v saturated solution of reference item (TMS) (with and without algal cells at 0, 24, 48 and 72 h)
- Sampling method: Samples were taken from the bulk test preparation at 0 and from the pooled replicates at 72 h. Additional samples of each concentration containing algal cells were prepared and incubated alongside the test to provide samples for analysis at 24 and 48 h. An additional sample of each concentration without algal cells was incubated alongside the test to provide samples for uninoculated analysis at 24, 48 and 72 h.
- Sample storage conditions before analysis: All samples were analyzed on the day of sampling. Duplicate samples were taken at each occasion 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 it was not possible to prepare a test solution using traditional methods (e.g. ultrasonication and high shear mixing). Therefore, a saturated solution was prepared by dispersing 1100 mg test item in 11 L of culture medium 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. A 100% v/v saturated solution of the control reference item trimethylsilanol was prepared the same way.
- Controls: Test solution without test item/reference item
- Evidence of undissolved material: At the start of the test all control, test and reference item cultures were observed to be clear colorless solutions. After 72 h all cultures wer green dispersions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater unicellular alga
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 3 - 4 d
- Method of cultivation: Algae were held in flasks, which were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 8.6E05 cells per mL.
- Culturing media and conditions: Culure medium same as test medium

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

0 h: 8.0 (control), 8.0 (test item), 7.9 (reference item)
72 h (pooled): 7.9 (control), 8.4 (test item), 8.4 (reference item)

Nominal and measured concentrations:

Nominal: Control, 100% v/v saturated solution test item and 100% v/v saturated solution reference item TMS
Measured concentrations of test item with algal cells at 0, 24, 48 and 72 h: 60 mg/L, 56 mg/L, 50 mg/L and 20 mg/L
Measured concentrations of test item without algal cells at 0, 24, 48 and 72 h: 60 mg/L, 55 mg/L, 46 mg/L and 40 mg/L
Measured concentrations of TMS with algal cells at 0, 24, 48 and 72 h: 90 mg/L, 86 mg/L, 77 mg/L and 34 mg/L
Measured concentrations of TMS without algal cells at 0, 24, 48 and 72 h: 90 mg/L, 82 mg/L, 66 mg/L and 51 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical glass flasks filled with 100 mL test solution, plugged with polyurethane foam bungs
- Initial cell density: 5.0E03 cells/mL
- Control end cell density: 2.8E05 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Inoculation: Inoculation of 2000 mL of test medium with 11.6 mL of algal suspension (8.6E05 cells/mL) gave an initial cell density of 5.0E03 cells/mL and had no significant dilution effect on the final test concentration.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionized water
- pH: Adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: ph was measured at test start and end (0 and 72 h); Temperature and appearance of test media were recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux, warm white light (380 to 730 nm)
- Other: Constant shaking at approximately 150 rpm for 72 h

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter (Coulter Multisizer Particle Counter) at 24, 48 and 72 h
- Other: Visual inspection of algal cells by microscopy

TEST CONCENTRATIONS
- Range finding study : Yes
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution of test item and reference item trimethylsilanol (TMS)
- Results used to determine the conditions for the definitive study: Yes. The results showed no effect on growth at test concentrationd of 0.1, 1.0, 10 and 100% saturated solution of test item or at 100% v/v saturated solution of the reference item.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (20 Jan 2020 and 06 Mar 2020)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 50 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: reference item trimethylsilanol (hydrolysis product)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: reference item trimethylsilanol (hydrolysis product)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes, the cell concentration of the control cultures increased by a factor of 56 after 72 h.
- Observation of abnormalities: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes, a positive control was conducted between 20 Jan and 06 Mar 2020
- ErC50 (72 h): 1.2 mg/L, 95% confidence limits 1.1 to 1.3 mg/L
- NOErC (72 h): 0.25 mg/L
- EyC50 (72 h): 0.5 mg/L, 95% confidence limits 0.45 to 0.58 mg/L
- NOEyC (72 h): 0.125 mg/L

Reported statistics and error estimates:

As no inhibition was noted in growth or yield in either the test or reference item exposures the NOEC values were determined by inspection of the data.

ANALYTICAL RESULTS

Test item

Analysis of the test item preparation at 0 h showed a measured test item concentration of 60 mg/L. Analysis of the test preparations at 24, 48 and 72 h showed measured test concentrations of 56, 50 and 20 mg/L, respectively. Analysis of an additional solution at 24, 48 and 72 h, which had been prepared without algae showed measured test concentrations of 55, 46 and 40 mg/L, respectively (Table 1).

The decline in measured test item concentrations observed between 0 and 72 h indicates that the test item was not stable under the testing conditions. The decline in test item concentrations was more marked in the test preparations containing algal cells, indicating that the test item was also adsorbing to the algal cells.

 

Reference item trimethylsilanol

Analysis of the reference item at 0 h showed a measured test concentration of 90 mg/L. Analysis of the test preparations after 24, 48 and 72 h showed measured test concentrations of 86, 77 and 34 mg/L, respectively. Analysis of an additional solution after 24, 48 and 72 h, which had been prepared without algae showed measured test concentrations of 82, 66 and 51 mg/L, respectively. This decline in measured test concentrations over time indicates that the reference item was unstable under the testing conditions. The decline was more marked in the reference item preparations containing algal cells, indicating that the reference item was also adsorbing to the algal cells.

 

Table 1. Test sample concentrations

Time	Nominal concentration of test item in test sample	Determined concentration of trimethylsilanol	Percentage of nominal
[h]	[% v/v saturated solution]	[mg/L]	[%]
0	Control	ND	-
	100	60.4	-
	100*	90.3	90
24	100	55.7	-
	100 (no algae)	54.7	-
	100*	85.8	-
	100 (no algae)*	82.2	-
48	100	50.1	-
	100 (no algae)	45.6	-
	100*	77.1	77
	100 (no algae)*	66.4	66
72	Control	ND	-
	100	19.9	-
	100 (no algae)	40.2	-
	100*	34.4	34
	100 (no algae)*	51.1	51

* samples were prepared using the trimethylsilanol analytical standard

ND = not detected

- = not applicable

 

Geometric mean measured test concentrations

Given the decline in measured test concentrations and following the recommendations of the test guideline, it was considered justifiable to base the results on the geometric mean measured test concentrations. The geometric mean measured concentration was calculated based on the 0, 24, 48 and 72 h no algae measured concentrations as it can be considered that whilst the test item was adsorbing to the algal cells, they were still exposed to the levels present. The geometric mean measured test concentration of the test item and reference item were therefore determined to be 50 and 71 mg/L, respectively (Table 2).

 

Table 2. Geometric mean measured concentration

Nominal test concentration	Geometric mean measured test concentration	Expressed as a % of the 0 h measured test concentration
[% v/v saturated solution]	[mg/L]	[%]
Test item: 100	50	83
Reference item: 100	71	79

 

 

Nitrate and ammonia concentrations

No significant changes in the levels of nitrate or ammonia were noted (Table 3).

 

Table 3. Nitrate and Ammonia analysis in the definitive test.

Geometric mean measured test concentrations	Time point	Ammonia as NH3	Nitrate NO3	Time Point	Ammonia as NH3	Nitrate NO3
[mg/L]	[h]	[mg/L]	[mg/L]	[h]	[mg/L]	[mg/L]
Control	0	0.0	4.4	72	0.0011	3.6
Test item 50	0	0.024	4.3	72	0.028	3.9
Reference item 71	0	0.0	4.3	72	0.0042	3.6

 

BIOLOGICAL RESULTS

No effects of the test item or reference item were observed on the growth rate of the tested algae (Table 4 and Table 5). As no inhibition was noted in growth rate in either the test or reference item exposures, the NOEC values were determined by inspection of the data.

 

Table 4. Cell densities in the definitive test.

Geometric mean measured test concentrations		Cell densities*
[mg/L]		[cells/mL]
		24 h	48 h	72 h
Control	R1	1.53E+04	5.40E+04	2.29E+05
	R2	1.32E+04	6.62E+04	3.23E+05
	R3	1.55E+04	9.18E+04	3.49E+05
	R4	1.32E+04	5.68E+04	2.48E+05
	R5	1.21E+04	5.70E+04	2.08E+05
	R6	1.31E+04	7.55E+04	3.24E+05
	Mean	1.37E+04	6.69E+04	2.80E+05
Test item 50	R1	1.63E+04	7.98E+04	3.75E+05
	R2	2.13E+04	1.22E+05	4.79E+05
	R3	2.00E+04	1.11E+05	4.16E+05
	R4	1.90E+04	1.04E+05	4.62E+05
	R5	2.14E+04	1.14E+05	5.59E+05
	R6	1.78E+04	1.30E+05	6.33E+05
	Mean	1.93E+04	1.10E+05	4.87E+05
Reference item 71	R1	1.73E+04	9.28E+04	3.68E+05
	R2	1.57E+04	9.14E+04	4.04E+05
	R3	1.68E+04	1.14E+05	4.43E+05
	R4	1.58E+04	7.73E+04	3.74E+05
	R5	2.07E+04	9.11E+04	4.26E+05
	R6	2.09E+04	1.08E+05	5.34E+05
	Mean	1.79E+04	9.57E+04	4.25E+05

* mean number of cells per mL, calculated from the mean of the cell counts from 3 counts for each of the replicate flask.

R = replicate

 

Table 5. Inhibition of growth rate and yield

Geometric mean measured test concentrations [mg/L]		Growth rate [cells/mL/h]		Yield [cells/mL]
		0 to 72 h	% inhibition	0 to 72 h	% inhibition*
Control	R1	0.053	-	2.24E+05	-
	R2	0.058		3.18E+05
	R3	0.059		3.44E+05
	R4	0.054		2.43E+05
	R5	0.052		2.03E+05
	R6	0.058		3.19E+05
	Mean	0.056		2.75E+05
	SD	0.003		5.89E+04
Test item 50	R1	0.060	[7]	3.70E+05
	R2	0.063	[13]	4.74E+05
	R3	0.061	[9]	4.11E+05
	R4	0.063	[13]	4.57E+05
	R5	0.066	[18]	5.54E+05
	R6	0.067	[20]	6.28E+05
	Mean	0.063	[13]	4.82E+05	[75]
	SD	0.003		9.48E+04
Reference item 71	R1	0.060	[7]	3.63E+05
	R2	0.061	[9]	3.99E+05
	R3	0.062	[11]	4.38E+05
	R4	0.060	[7]	3.69E+05
	R5	0.062	[11]	4.21E+05
	R6	0.065	[16]	5.29E+05
	Mean	0.062	[10]	4.20E+05	[53]
	SD	0.002		6.07E+04

* In accordance with the OECD test guideline only the mean value for yield for each test concentration was calculated

R = replicate

- = not applicable

SD = Standard Deviation

[] = Increase in growth compared to controls

 

Effect values

The resulting effect values for the test item are an ErC50 (72 h) > 50 mg/L and a NOErC (72 h) of 50 mg/L, based on the geometric mean measured concentration of the test item (Table 6).

 The resulting effect values for the reference item are an ErC50 (72 h) > 71 mg/L as well as a NOErC (72 h) of 71 mg/L, based on the geometric mean measured concentration of the reference item (Table 6).

 

Table 6. Effect values for inhibition of growth rate and yield

Effect value	Test item	Reference item
ErC10 (72 h) / EyC10 (72 h)	> 50 mg/L	> 71 mg/L
ErC50 (72 h) / EyC50 (72 h)	> 50 mg/L	> 71 mg/L
NOEC (72 h)	≥ 50 mg/L	≥ 71 mg/L

 

 

VALIDATION CRITERIA

 

The study fulfilled the validity criteria laid down by the guideline and is thus considered valid and reliable (Table 7).

 

Table 7. Validity criteria for OECD testing guideline 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The cell concentrations of the control cultures increased by a factor of 56 after 72 h. 0 h: 5.00E+03 cells/mL 72 h: 2.80E+03 cells/mL	Yes

 

Description of key information

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

ErC50 (72 h) > 50 mg/L (geom. mean measured, OECD 201, P. subcapitata)

ErC10 (72 h) > 50 mg/L (geom. mean measured, OECD 201, P. subcapitata)

NOErC (72 h) = 50 mg/L (geom. mean measured, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

50 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

One experimental study is available for the toxicity of 1,3-bis(trimethylsilyl)urea (CAS No. 18297-63-7) to aquatic algae, which was conducted according to OECD testing guideline 201 (2006) and GLP.

The test item is poorly soluble and hydrolyses rapidly (DT50 < 10 min, OECD 111) to trimethylsilanol (TMS), hexamethyldisiloxane and urea. TMS has a 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 fiter, 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 60 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 of the control, test and reference groups was verified by chemical analysis of TMS concentrations in the fresh bulk preparations (at 0 h) and from the pooled replicates of the aged media (at 72 h) using gas chromatography. Additional samples of each test concentration (test and reference) containing algal cells were prepared and incubated alongside the test to provide samples for analysis at 24 and 48 h. Additional samples of each test concentration (test and reference) without algal cells was incubated alongside the test to provide samples for uninoculated analyses at 24, 48 and 72 h.

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 static limit test, Pseudokirchneriella subcapitata were exposed for 72 h to a 100% v/v saturated aqueous solution of test item and 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). Cell concentrations were recorded daily with a Coulter Counter.

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

 

Analysis of the fresh test item preparations (100% v/v) at 0 h resulted in a measured test item concentration of 60 mg/L TMS. Analysis of the aged test item preparations at 24, 48 and 72 h resulted in measured test item concentrations of 56, 50 and 20 mg/L TMS, respectively. The analysis of the additional solution without algae at 24, 48 and 72 h resulted in measured test item concentrations of 55, 46 and 40 mg/L, respectively.

 

Analysis of the fresh reference item (TMS) preparations (100% v/v) at 0 h resulted in 90 mg/L.Analysis of the aged test preparations at 24, 48 and 72 h resulted in measured test concentrations of 86, 77 and 34 mg/L, respectively. Analysis of the additional solutions without algal cells at 24, 48 and 72 h resulted in measured concentrations of 82, 66 and 51 mg/L, respectively.

The analytical results indicate that the test item is unstable under the testing conditions of the present study. Furthermore, the decline in test concentrations was more marked in preparations containing algal cells, indicating that adsorption to algal cells possibly also occurred.

 

Given the decline of the measured test concentrations, the results were based on the geometric mean measured test concentrations based on the 0, 24, 48 and 72-hour measured concentrations without algae, as it can be considered that whilst the test item was adsorbing to the algal cells, they were still exposed to the levels present.

The geometric mean measured test concentration of the test item and reference item were 50 and 71 mg/L, respectively.

After 72 h, no toxic effects toward aquatic algae were recorded at the saturation level. The study resulted in an ErC50 (72 h) of > 50 mg/L and a NOErC (72 h) of≥50 mg/L for the test item itself (incl. all possible hydrolysis products which occurred within the duration of the test) and in an ErC50 (72 h) of > 71 mg/L and a NOErC (72 h) of≥71 mg/L for the reference item TMS itself.

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