A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)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.
- Controls: Yes (nutrient medium)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Selenastrum capricornutum ATCC (American Type Culture Collection) 22662
- Source (laboratory, culture collection): 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 10E+05 algae/mL by diluting with nutrient medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 1 ºC

pH:

7.2-8.8

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 6.3, 12.5, 25.0, 50.0 and 100.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks (Erlenmeyer).
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 250 mL (volume was 100 mL)
- Initial cells density: 10E+05 algae/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: permanent light
- Light intensity and quality: at least 6000 lux (wavelength 400-700 nm)
- Other: shaking

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At incubation times of 0 and 72 hours aliquots were taken from each flask and the cell densities of the algae were determined by a Coulter Counter, model ZF, Coulter Electronics, UK (pore size 100 µm).To achieve total cell counts of less than 50000, appropriate amounts of the cell suspensions were added to 20 mL of isotonic solution (Isoton 11,Coulter Electronics).
- Other: The pH was determined in all test cultures, in the control and in the blank at the start of the exposure and 72 hours thereafter. The temperature of the incubation chamber was protocolled at the begin of incubation and each time when cell counts were performed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: 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.
- Test concentrations: 6.3, 12.5, 25.0, 50.0 and 100.0 mg/L.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance equivalents (variety of degradation products)

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:

other: test substance equivalents (variety of degradation products)

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:

other: test substance equivalents (variety of degradation products)

Basis for effect:

cell number

Remarks on result:

other: Based on the area under the growth curve

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance equivalents (variety of degradation products)

Basis for effect:

growth rate

Remarks on result:

other: Based on the average growth rates

Details on results:

Compared to the negative controls, algal growth was only slightly inhibited at the highest tested concentration (by 24.4 % based on the area under the growth curves or by 7.5 % based on the average growth rates). At all other test substance concentrations algal growth was slightly enhanced (by 1.6 to 10.2 %, depending on the method of calculation).

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 per cent 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	per cent inhibition based on
			area	growth rates
0 (control)	14428	60.4	0.0	0.0
6.3	15904	61.7	-10.2	-2.1
12.5	15314	61.4	-6.1	-1.6
25.0	15837	61.9	-9.8	-2.4
50.0	15842	61.9	-9.8	-1.9
100.0	10905	55.9	24.4	7.5

 

µ: Specific growth 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	478
6.3	49	62	47	52	508
12.5	52	50	50	51	487
25.0	49	56	47	51	487
50.0	56	51	52	53	513
100.0	36	39	47	41	371
Incubation time: 48 h					24-48 h
0 (control)	139	219	146	168	2373
6.3	177	264	145	195	2729
12.5	202	213	170	195	2706
25.0	204	223	171	200	2763
50.0	250	200	166	206	2859
100.0	135	198	134	155	2113
Incubation time: 72 h					48-72 h
0 (control)	620	1205	625	817	11577
6.3	813	1192	636	880	12667
12.5	861	915	730	835	12121
25.0	800	1051	757	869	12588
50.0	997	897	667	854	12470
100.0	483	698	519	867	8422

Validity criteria fulfilled:

yes

Remarks:

(The 72h incubation period cell density increase in the control cultures was about 82-fold (> 16-fold), showing suitable growth conditions for the algae)

Conclusions:

The EbC50 (72 h) based on the areas under the growth curves and the ErC50 (72 h), based on the average growth rates, for test item Wasox-MMAC2 were determined to be > 100 mg/L.

Executive summary:

An Alga Growth Inhibition test was performed to determine the possible effects of the test substance Wasox-MMAC2 on the growth of an unicellular green algae, Selenastrum capricomutum (According to EU Method C.3). 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 cell 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 1.4 units (from 7.2 to 8.8). No chemical analysis of the actual test substance concentration was performed, neither at the start nor at the end of the exposure.

The NOEC (72h) based on the area under the growth curves and the NOEC (72h) based on the average growth rates, were 100 mg/L. The EbC50 (72 h) based on the areas under the growth curves and the ErC50 (72 h), based on the average growth rates, were > 100 mg/L.

Description of key information

Key study: EU method C.3 and GLP. The EbC50 (72 h) based on the areas under the growth curves and the ErC50 (72 h), based on the average growth rates, for Wasox-MMAC2 were determined to be > 100 mg/L under test conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

Key study: An Alga Growth Inhibition test was performed to determine the possible effects of the test substance Wasox-MMAC2 on the growth of an unicellular green algae, Selenastrum capricomutum (According to EU Method C.3 and GLP). The EbC50 (72 h) based on the areas under the growth curves and the ErC50 (72 h), based on the average growth rates, were determined to be > 100 mg/L under test conditions.