A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime

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:

February 15 - March 3, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to EU Method C.3, with GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test substance was added drop by drop to the nutrient medium and was dissolved by stirring for about one hour, followed by filtration of the solution (cellulose acetate filter, 0.2 µm, Sartorius). The solution was filtered because in a previous experiment without filtering, "particles" were formed during the incubation period, interfering with the counting of the algae.
90 mL of the filtrate were combined with 10 mL of the algal inoculum to give the highest test concentration. Aliquots of the filtrate were further diluted with nutrient medium to obtain lower concentrations.
The preparations were made freshly before the start of the exposure.
The test was performed without adjustment of the pH of the test substance cultures.


- Controls:
One negative control culture (reference substance: nutrient medium) was set up concurrently.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae (Selenastrum capricornutum)
- Strain: Selenastrum capricornutum ATCC (American Type Culture Collection) 22662
- Source (laboratory, culture collection): obtained directly from ATCC, 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in 250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature of 23 ± 2°C under permanent light with an intensity of at least 6000 lux. In about weekly intervals 1 mL of the stock culture is diluted 100-fold with nutrient medium for precultivation and incubation is continued.
Precultures were prepared by incubating a new stock culture for three days. At the start of the experiment the cell density was determined and adjusted to about 105 algae/mL by diluting with nutrient medium.

ACCLIMATION
- Culturing media and conditions (same as test or not):
Concentrated nutrient medium:
Stock solution 1 100 mL
Stock solution 2 10 mL
Stock solution 3 10 mL
Stock solution 4 10 mL
sterile deionised water ad 1000 mL

Storage: Refrigerator.

Nutrient medium:
As nutrient medium for the stock cultures, for precultivation and for the test media a tenfold dilution of the "concentrated nutrient medium" with sterile deionised water was used.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

All the culture and the blank were incubated at 22 ± 1 ºC

pH:

The pH was between 7.4 and 7.6 at the start of the incubation in the test cultures and it was 7.2 in the control cultures.
After 72 hours of incubation the pH was 8.3 in the test cultures and it was 8.4 in the control cultures.
The pH in the control cultures changed by 1.2 units during the 72 hours of incubation.

Nominal and measured concentrations:

Nominal concentrations: 100.0, 50.0, 25.0, 12.5, and 6.3 mg test substance/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (erlenmeyer)
- Initial cells density: 10 exp.5 algae/mL
- Control end cells density: At incubation time of 72 hours aliquots were taken from each flask (i.e. from each replicate) and the cell densities of the algae were determined by a Coulter Counter
- No. of organisms per vessel: 10 exp4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
Nutrient medium: Sterile deionised water and sterile concentrated nutrient medium 9+1 (v/v).

Concentrated nutrient medium:
Stock solution 1 100 mL
Stock solution 2 10 mL
Stock solution 3 10 mL
Stock solution 4 10 mL
sterile deionised water ad 1000 mL


OTHER TEST CONDITIONS
- Photoperiod: permanent light
- Light intensity and quality: at least 6000 lux (wavelength 400 - 700 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell counts by a Coulter Counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

- Range finding study
- Test concentrations: As no data were available on algal toxicity, the highest test substance concentration in this study was 100 mg/L, the same as to be used for a limit test. The next four concentrations were each about one half of the previous one.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

cell number

Remarks on result:

other: Based on the area under the growth curve

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: Based on the average growth rates

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: Based on the area under the growth curve

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on the average growth rates

Details on results:

- Exponential growth in the control (for algal test): The increase in cell density in the control cultures during the 72 hours incubation period was about 82-fold, corresponding to about 6.4 generations and showing suitable growth conditions for the algae.

Reported statistics and error estimates:

Based on the areas under the growth curves as well as on the average growth rates two "lowest observed effective concentrations" (LOECs) are calculated by comparison of the data of the three replicates of each test substance culture with the negative control (analysis of variance, followed by the Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived from these results (highest concentration with no statistically significant difference to the control).

Mean areas under the growth curves, average specific growth rates and percent inhibition.

Negative values indicate enhanced growth compared to the control cultures.

 

concentration of test substance (mg/L)	mean area under growth curve	average specific growth rate (µ x 10^-3) 0-72 h	percent inhibition based on
			area	growth rates
0 (control)	14411	60.4	0.0	0.0
6.3	13800	60.0	4.2	0.7
12.5	15635	61.8	-8.5	-2.3
25.0	17031	63.0	-18.2	-4.2
50.0	15422	60.6	-7.0	-0.4
100.0	16654	61.6	-15.6	-1.9

µ: Specificgrowth rate

 

 Cell densities and mean areas under the growth curves.

 

Concentration of test substance  (mg/L)	cell density			Mean cell density (cells x 10^3/ml	Mean area under growth curve
	(cells x 10^3/mL
	replicate 1	replicate 2	replicate 3
Incubation time: 24 h					0-24 h
0 (control)	46	58	45	50	480
6.3	47	46	49	47	450
12.5	52	51	43	49	464
25.0	52	46	53	50	485
50.0	55	48	35	46	430
100.0	63	47	50	53	521
Incubation time: 48 h					24-48 h
0 (control)	139	218	146	168	2373
6.3	177	170	182	177	2448
12.5	201	208	146	185	2564
25.0	241	156	229	209	2868
50.0	259	212	118	196	2665
100.0	320	183	207	237	3241
Incubation time: 72 h					48-72 h
0 (control)	619	1204	623	815	11558
6.3	730	797	729	752	10902
12.5	1042	1027	588	886	12608
25.0	982	704	1168	951	13678
50.0	1093	1027	433	851	12327
100.0	1106	719	749	858	12892

Validity criteria fulfilled:

yes

Conclusions:

Two (identical) NOEC (0-72h)-values and two (identical) EC50(0-72h)-values were derived:
The NOEC (0-72h) based on the area under the growth curves was 100 mg/mL and the NOEC (0-72h) based on the average growth rates was 100 mg/mL.
The EbC50 (0-72 h) based on the areas under the growth curves was > 100 mg/mL and the ErC50 (0-72 h), based on the average growth rates, was > 100 mg/mL
The concentrations are nominal test substance concentrations.

Executive summary:

An Alga Growth Inhibition test was performed to determine the possible effects of the test substance on the growth of an unicellular green algae, Selenastrum capricomutum (According to EU Method C). Five concentrations (100.0, 50.0, 25.0, 12.5, 6.3 mg of test substance per liter of nutrient medium) were tested. A negative control (nutrient medium only) was included in the test. There were 3 replicates for each test and control culture.

In each vessel the gel density of the algae was determined 24, 48 and 78 hours after the onset of incubation. The pH was measured at the beginning and at the end of the incubation period. During the 72 hours of incubation, the cell density in the control cultures was increased by a factor of about 82 and the pH changed by 1.2 units (from 7.2 to 8.3). No chemical analysis of the actual test substance concentration was performed, neither at the start nor at the end of the exposure.

Two identical NOEC (0-72h) values and two identical EC50(0-72h) values were derived:

The NOEC (0-72h) based on the area under the growth curves was 100 mg/mL and the NOEC (0-72h) based on the average growth rates was 100 mg/mL.

The EbC50(0-72 h) based on the areas under the growth curves was > 100 mg/ml and the ErC50(0-72 h), based on the average growth rates, was > 100 mg/mL .

 

Description of key information

An Alga Growth Inhibition test was performed to determine the possible effects of the test substance on the growth of an unicellular green algae, Selenastrum capricomutum (According to EU Method C). The NOEC (0-72h) based on the area under the growth curves was 100 mg/mL and the NOEC (0-72h) based on the average growth rates was 100 mg/mL.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Five concentrations (100.0, 50.0, 25.0, 12.5, 6.3 mg of test substance per liter of nutrient medium) were tested. A negative control (nutrient medium only) was included in the test. There were 3 replicates for each test and control culture.

In each vessel the gel density of the algae was determined 24, 48 and 78 hours after the onset of incubation. The pH was measured at the beginning and at the end of the incubation period. During the 72 hours of incubation, the cell density in the control cultures was increased by a factor of about 82 and the pH changed by 1.2 units (from 7.2 to 8.3). No chemical analysis of the actual test substance concentration was performed, neither at the start nor at the end of the exposure.

Two identical NOEC (0-72h) values and two identical EC50(0-72h) values were derived:

The NOEC (0-72h) based on the area under the growth curves was 100 mg/mL and the NOEC (0-72h) based on the average growth rates was 100 mg/mL.

The EbC50(0-72 h) based on the areas under the growth curves was > 100 mg/mland theErC50(0-72 h), based on the average growth rates, was > 100 mg/mL.