1,1,1"((methylsilanetriyl)tris)(oxy))tris(2-methylpropan-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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L and 8 mg/L
- Sample storage conditions before analysis: Test samples were analysed immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method:The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L.
were prepared using M7 medium in a 250 mL measuring cylinder.
- Controls: M7 Medium (Control),
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s)
including control(s)): Not applicable

Test organisms (species):

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

Details on test organisms:

Test organisms (species)
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capr
icornutum)
Details on test organisms
TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Bio
logical Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained
in
Laboratory.
- Method of cultivation: OECD medium
ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae
was OECD medium.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23-24°C

pH:

7.81-8.44

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 0.5, 1, 2, 4 and 8 mg/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control
GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(3000-4000Lux)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cellcounts were measured using microscope.
- Chlorophyll measurement: No data

- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item) Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 0, 0.5, 1, 2, 4 and 8 mg/L (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of expo
sure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control
medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the
study period. This is to provide constant shaking to the algal cells to keep them in suspension and to
ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Details on results
The microscopic observations were also noted in each of the experimental flasks. All the cells appea
red healthy, sickle shaped and green throughout the test duration in the control.

Results with reference substance (positive control):

Results with reference substance valid?
- EC50: 0.868 mg/l

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined

Assessment of Dose Range concentrations

 

Sr. No	Concentrations mg/L	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	198	0.0003	25°C
2	0.25	198	0.0298	25°C
3	0.5	198	0.0357	25°C
4	1	198	0.0463	25°C
5	2	198	0.0801	25°C
6	4	198	0.1518	25°C
7	6	198	0.2172	25°C
8	8	198	0.3149	25°C
9	10	198	0.3509	25°C
10	12	198	0.4458	25°C

 

 

		0 Hours	0 Hours	72 Hours	72 Hours
SR. No	concentrations (mg/L)	Absorbance (mean)	Analytical concentrations	Absorbance (mean)	Analytical concentrations
1	control	0.00	0.00	0.00	0.01
2	0.5	0.02	0.52	0.02	0.52
3	1	0.04	1.35	0.04	1.04
4	2	0.08	2.1	0.07	2.0
5	4	0.12	3.49	0.13	3.55
6	8	0.24	8.53	0.214	8.30

 

pH AND TEMPERATURE

 

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 Hours	72 Hours	0 Hours	72 Hours
control	R1	7.81	8.44	23	24
control	R2	7.82	8.21	23	24
control	R3	7.96	8.31	23	24
0.5	R1	8.25	9.68	23	24
0.5	R2	8.16	9.61	23	24
0.5	R3	7.94	9.68	23	24
1	R1	8.07	8.78	23	24
1	R2	7.86	9.77	23	24
1	R3	7.97	9.68	23	24
2	R1	7.85	9.74	23	24
2	R2	7.96	9.72	23	24
2	R3	8.01	9.64	23	24
4	R1	7.75	9.68	23	24
4	R2	7.82	9.66	23	24
4	R3	7.87	9.89	23	24
8	R1	8.00	9.78	23	24
8	R2	8.23	9.68	23	24
8	R3	8.41	9.23	23	24

The pH was measured at the beginning of the test and after 72 hr of exposure. The pH of the control medium did not increase by more than 1.5 units during the test.

		DAY 0				DAY 1				DAY 2				DAY 3
Experimental Sets	TEST CONC.s (mg/L)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Daily Growth rate 0-1 days	Daily Growth rate 1-2 days	Daily Growth rate 2-3 days	Average Growth rate 0-3 days	Standard deviation	Mean	Coefficient of Variation	Inhibition	% Inhibition
Solvent 1	0.0	10000	10000	10000	9.21	30000	30000	30000	10.31	90000	90000	90000	11.41	280000	200000	240000	12.39	1.10	1.10	0.98	1.06	0.010	1.07	0.010
Solvent 2	0.0	10000	10000	10000	9.21	30000	30000	30000	10.31	80000	90000	85000	11.35	250000	260000	255000	12.45	1.10	1.04	1.10	1.08
Solvent 3	0.0	10000	10000	10000	9.21	30000	30000	30000	10.31	90000	90000	90000	11.41	230000	270000	250000	12.43	1.10	1.10	1.02	1.07
Conc. 1REP 1	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	80000	80000	80000	11.29	190000	190000	190000	12.15	1.10	0.98	0.86	0.98	0.019	0.97	0.020	0.10	9.76
Conc. 1REP 2	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	90000	70000	80000	11.29	190000	180000	185000	12.13	1.10	0.98	0.84	0.97
Conc. 1REP 3	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	80000	80000	80000	11.29	170000	170000	170000	12.04	1.10	0.98	0.75	0.94
Conc. 2REP 1	1	10000	10000	10000	9.21	30000	20000	25000	10.13	70000	70000	70000	11.16	150000	150000	150000	11.92	0.92	1.03	0.76	0.90	0.000	0.90	0.000	0.16	15.69
Conc. 2REP 2	1	10000	10000	10000	9.21	30000	20000	25000	10.13	70000	70000	70000	11.16	150000	150000	150000	11.92	0.92	1.03	0.76	0.90
Conc. 2REP 3	1	10000	10000	10000	9.21	20000	20000	20000	9.90	70000	70000	70000	11.16	140000	160000	150000	11.92	0.69	1.25	0.76	0.90
Conc. 3REP 1	2	10000	10000	10000	9.21	30000	20000	25000	10.13	70000	60000	65000	11.08	100000	100000	100000	11.51	0.92	0.96	0.43	0.77	0.018	0.75	0.023	0.30	29.94
Conc. 3REP 2	2	10000	10000	10000	9.21	20000	20000	20000	9.90	60000	60000	60000	11.00	90000	100000	95000	11.46	0.69	1.10	0.46	0.75
Conc. 3REP 3	2	10000	10000	10000	9.21	20000	20000	20000	9.90	50000	50000	50000	10.82	80000	100000	90000	11.41	0.69	0.92	0.59	0.73
Conc. 4REP 1	4	10000	10000	10000	9.21	20000	20000	20000	9.90	50000	60000	55000	10.92	80000	60000	70000	11.16	0.69	1.01	0.24	0.65	0.026	0.68	0.038	0.37	36.64
Conc. 4REP 2	4	10000	10000	10000	9.21	20000	20000	20000	9.90	60000	50000	55000	10.92	80000	80000	80000	11.29	0.69	1.01	0.37	0.69
Conc. 4REP 3	4	10000	10000	10000	9.21	20000	20000	20000	9.90	50000	50000	50000	10.82	80000	80000	80000	11.29	0.69	0.92	0.47	0.69
Conc. 5REP 1	8	10000	10000	10000	9.21	10000	20000	15000	9.62	50000	50000	50000	10.82	60000	60000	60000	11.00	0.41	1.20	0.18	0.60	0.000	0.60	0.000	0.44	44.21
Conc. 5REP 2	8	10000	10000	10000	9.21	20000	10000	15000	9.62	30000	30000	30000	10.31	60000	60000	60000	11.00	0.41	0.69	0.69	0.60
Conc. 5REP 3	8	10000	10000	10000	9.21	10000	10000	10000	9.21	30000	30000	30000	10.31	60000	60000	60000	11.00	0.00	1.10	0.69	0.60

									Criteria No. 1)≥0.92/day		Criteria No. 3) ≤ 7%
Daily Growth rate 0-1 days	Daily Growth rate 1-2 days	Daily Growth rate 2-3 days	Average Growth rate 0-3 days	Standard deviation	Mean	Coefficient of Variation	% CV
12.39	1.10	1.10	0.98	1.06	0.010	1.07	0.0096	0.96
12.45	1.10	1.04	1.10	1.08
12.43	1.10	1.10	1.02	1.07
			Mean	1.09861	1.07956	1.03370
			SD	0.00000	0.03300	0.05981		Culture increased by Fator	24.83
			CV	0.0000	0.0306	0.0579
			% of CV	0.0	3.1	5.8
Criteria No. 2 ) ≤ 35%			Mean of % CV of Average specific / section by section growth rate			2.95

Validity criteria fulfilled:

yes

Remarks:

The average increase in cell count in control group is >16 fold. * The coefficent of varion be tween replictes was <7 % * coefficent of variation in the in control f % CV of Average specific <35%.

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis).

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. T The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.81 in all the replicates at day 0 and followed by between 8.44 on day 3. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. T The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.81 in all the replicates at day 0 and followed by between 8.44 on day 3. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

9.66 mg/L

Additional information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. T The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.81 in all the replicates at day 0 and followed by between 8.44 on day 3. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control

vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

The effect of test chemical on green algae was estimated using ECOSAR model for the exposure period of 72 hours and median effective concentration was found to be 2.047 mg/l. Based on the values it can be classified into aquatic chronic category 2 as per CLP classification criteria.

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.