1,1’,1’’-((methylsilanetriyl)tris(oxy))tris(butan-2-amine)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-08 to 2020-06-11

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:

Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentrations in the test solutions, all three test vessels were analysed at the start and at the end of the study. In case of control sample, all six test vessels were analysed at the start and at the end of the study.
- Sample storage conditions before analysis: no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test was prepared by mechanical dispersion without using of any solubilising agent.
For preparation of test item solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.0303 g test item in 303 mL OECD medium. The test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution.
- Controls: negative (blank) control, positive control
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany.
- Age of inoculum: 3 days (exponentially growth)
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for three days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

not specified

Test temperature:

22.7 – 23.2 °C

pH:

7.81 – 9.18

Dissolved oxygen:

not specified

Salinity:

not relevant

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 0 (blank control), 0.26, 0.64, 1.6, 4.0 and 10 mg/L
measured: 0.18, 0.52, 1.4, 3.7 and 9.3 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with permeable stoppers
- Material, size, fill volume: glass, ~250 mL nominal volume,100 mL fill volume
- Aeration: agitated by using an orbital shaker
- Initial cells density: 10^4 cells/mL in each test flask
- Control end cells density: 10^4 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (OECD medium, according to OECD 201) prepared with deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: 7112 lux, wavelength range: 400 – 700 nm

DETERMINATION OF CELL CONCENTRATION
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: performed
- Test concentrations: 0 (control), 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: No morphological abnormalities were observed in the control and in the concentrations of 0.18, 0.52 mg/L (measured) during the test.
Swollen cells, thin cells or pinches on cells were observed in the concentrations of 1.4, 3.7 and 9.3 mg/L (measured).
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: none
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

The results of the statistical evaluations showed that the 0 - 72 h average specific growth rate and yield were statistically significantly different from the untreated control at concentrations of 1.4, 3.7 and 9.3 mg/L (measured) and not significantly different at concentrations of 0.18, 0.52 mg/L (measured).
The cell density in the control has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to 74.67× 104 cells at the end of the test (72 h).

Results with reference substance (positive control):

72h ErC50: 0.52 mg/L (95 % confidence limits: 0.46 – 0.58 mg/L)
72h EyC50: 0.31 mg/L (95 % confidence limits: 0.28 – 0.35 mg/L)

Reported statistics and error estimates:

For determination of the ECx values Probit analysis by SPSS software was performed.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (Dunnett T3) by SPSS software.

Table 1: Algal cell number (× 10⁴cell/mL) during the test period of 72 hours and Yield

Concentration			Number of Cells (× 10^4/mL)				Yield
nominal [mg/L]	measured [mg/L]		0 h	24 h	48 h	72 h
Control	Not detected		1	2	17	77	76
			1	3	14	72	71
			1	3	16	71	70
			1	2	18	83	82
			1	2	16	67	66
			1	2	19	78	77
		Mean	1.00	2.33	16.67	74.67	73.7
		SD	0.00	0.52	1.75	5.75	5.75
0.26	0.18		1	2	18	74	73
			1	2	20	78	77
			1	2	14	69	68
		Mean	1.00	2.00	17.33	73.67	72.7
		SD	0.00	0.00	3.06	4.51	4.51
0.64	0.52		1	2	17	67	66
			1	3	17	74	73
			1	3	17	71	70
		Mean	1.00	2.67	17.00	70.67	69.7
		SD	0.00	0.58	0.00	3.51	3.51
1.6	1.4		1	2	11	13	12
			1	2	10	13	12
			1	2	12	15	14
		Mean	1.00	2.00	11.00	13.67	12.7
		SD	0.00	0.00	1.00	1.15	1.15
4.0	3.7		1	2	3	4	3
			1	2	2	2	1
			1	2	4	3	2
		Mean	1.00	2.00	3.00	3.00	2.0
		SD	0.00	0.00	1.00	1.00	1.00
10	9.3		1	1	1	1	0
			1	1	1	1	0
			1	1	1	1	0
		Mean	1.00	1.00	1.00	1.00	0.0
		SD	0.00	0.00	0.00	0.00	0.00

Table 2: The specific Growth Rates during the test period of 72 hours

Concentration			Growth Rates (µ)
nominal [mg/L]	measured [mg/L]		0 – 24 h	0 – 48 h	0 – 72 h
Control	Not detected		0.0289	0.0590	0.0603
			0.0458	0.0550	0.0594
			0.0458	0.0578	0.0592
			0.0289	0.0602	0.0614
			0.0289	0.0578	0.0584
			0.0289	0.0613	0.0605
		Mean	0.0345	0.0585	0.0599
		SD	0.0087	0.0022	0.0011
0.26	0.18		0.0289	0.0602	0.0598
			0.0289	0.0624	0.0605
			0.0289	0.0550	0.0588
		Mean	0.0289	0.0592	0.0597
		SD	0.0000	0.0038	0.0009
0.64	0.52		0.0289	0.0590	0.0584
			0.0458	0.0590	0.0598
			0.0458	0.0590	0.0592
		Mean	0.0401	0.0590	0.0591
		SD	0.0098	0.0000	0.0007
1.6	1.4		0.0289	0.0500	0.0356
			0.0289	0.0480	0.0356
			0.0289	0.0518	0.0376
		Mean	0.0289	0.0499	0.0363
		SD	0.0000	0.0019	0.0011
4.0	3.7		0.0289	0.0229	0.0193
			0.0289	0.0144	0.0096
			0.0289	0.0289	0.0153
		Mean	0.0289	0.0221	0.0147
		SD	0.0000	0.0073	0.0048
10	9.3		0.0000	0.0000	0.0000
			0.0000	0.0000	0.0000
			0.0000	0.0000	0.0000
		Mean	0.0000	0.0000	0.0000
		SD	0.0000	0.0000	0.0000

Table 3: Growth Rates (µ) and percentage inhibition of µ during the test period

Concentration		Growth Rates (µ)and % Inhibition ofµ
nominal [mg/L]	measured [mg/L]	0–24 h		0–48 h		0–72 h
		µ	%	µ	%	µ	%
Control	Not detected	0.0345	–	0.0585	–	0.0599	–
0.26	0.18	0.0289	16.3	0.0592	-1.2	0.0597	0.3
0.64	0.52	0.0401	-16.3	0.0590	-0.9	0.0591	1.2
1.6	1.4	0.0289	16.3	0.0499	14.7	0.0363	39.4
4.0	3.7	0.0289	16.3	0.0221	62.3	0.0147	75.4
10	9.3	0.0000	100.0	0.0000	100.0	0.0000	100.0

Table 4: Morphological changes of algal cells observed after 24, 48 and 72 hours of exposure

Concentration		Morphological changes
nominal [mg/L]	measured [mg/L]	24 h	48 h	72 h
Control	Not detected	- normal cells	- normal cells	- normal cells
0.26	0.18	- normal cells	- normal cells	- normal cells
0.64	0.52	- normal cells	- normal cells	- normal cells
1.6	1.4	- normal cells	- normal cells - swollen cells - thin cells	- swollen cells pinches on cells
4.0	3.7	- normal cells - swollen cells - thin cells	- swollen cells	- swollen cells
10	9.3	- swollen cells	- swollen cells pinches on cells	- swollen cells

Table 5: Concentration of Test Item measured in the Test Solutions

Nominal concentration mg/L	Mean of the measured concentrations
	Start(June 08, 2020)		End(June 11, 2020)
	Concentration (mg/L)	% of the nominal	Concentration (mg/L)	% of the nominal
Control	not detected	-	not detected	-
0.26	0.230	89	0.140	54
0.64	0.597	93	0.453	71
1.6	1.477	92	1.381	86
4	3.702	93	3.654	91
10	9.274	93	9.353	94

Validity criteria fulfilled:

yes

Conclusions:

In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.

Executive summary:

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 0.26, 0.64, 1.6, 4.0 and 10 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.18, 0.52, 1.4, 3.7 and 9.3 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.

Description of key information

In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.92 mg/L

EC10 or NOEC for freshwater algae:

0.74 mg/L

Additional information

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 0.26, 0.64, 1.6, 4.0 and 10 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.18, 0.52, 1.4, 3.7 and 9.3 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.