N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

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:

September 4, 2017 - September 7, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 mg/mL.
- Sampling method: Test solutions and control sample were determined at the beginning and at the end of the test. The samples were properly diluted and analysed by an HPLC-UV method.
- Sample storage conditions before analysis: Saturated solution and about 30 fold diluted saturated solution were stored at test conditions. At the end of the storage period formulations were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (ISO medium) using ultrasonic bath (aprox. 30 minutes). As a Limit test was carried out, further dilution of stock solution was not performed.
- Controls: The dilution water (ISO medium) was used without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum).
- Strain: 61.81 SAG (identical strains. CCAP 278/4; UTEX 1648; ATCC 22662).
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): 3 days.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. Stock cultures were small algal colonies that are inoculated onto agar regularly. These were transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days.

ACCLIMATION
- Acclimation period: no.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.7 - 22.8

pH:

7.86 - 8.76

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100 mg/L.
Measured: 6.62, 13.10, 25.65, 50.85 and 100.44 mg/L. As the analytically mesured concentration deviated not more than 20 per cent from the nominal during the test, the biological results are based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks.
- Type: The flasks were covered with air-premeable stoppers.
- Material, size, headspace, fill volume: glass, size 250 mL, fill volume 100 mL test solution.
- Initial cells density: 1 E04 cells/mL.
- Control end cells density: 67.67 E04 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water.
- Culture medium different from test medium: No.

OTHER TEST CONDITIONS
- Sterile test conditions: no.
- Adjustment of pH: no.
- Photoperiod: continuous ilumination.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8335 lux (equivalent to ~113 µE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Range finding study: A concentration range-finding test was conducted to determined the approximate toxicity of the test item so that appropiate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Because inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

For the evaluation of the quality of algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07-10 March 2017.
- The 72h ErC 50: 0.88 ,g/L, (95% confidence limits: 0.81 - 0.96 mg/L).
- The 72h EyC 50: 0.54 ,g/L, (95% confidence limits: 0.50 - 0.59 mg/L).
- The 72h EbC 50: 0.64 ,g/L, (95% confidence limits: 0.59 - 0.70 mg/L).
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence intervals were calculated using Probit analysis by TOXSTAT software
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (alpha = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Table 3:Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

NominalConcentration [mg/L]	Growth rate (m) and % inhibition ofm
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0498	0.0	0.0590	0.0	0.0585	0.0
6.25	0.0578*	-16.1	0.0582	1.4	0.0575	1.7
12.5	0.0458	8.0	0.0569	3.6	0.0580	0.8
25.0	0.0458	8.0	0.0573	2.9	0.0578	1.2
50.0	0.0458	8.0	0.0479*	18.8	0.0524*	10.5
100.0	0.0458	8.0	0.0371*	37.1	0.0328*	43.9

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 4:Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

NominalConcentration [mg/L]	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	28.0	0.0	248.0	0.0	1240.0	0.0
6.25	36.0*	-28.6	256.0	-3.2	1184.0	4.5
12.5	24.0	14.3	220.0*	11.3	1164.0	6.1
25.0	24.0	14.3	224.0	9.7	1160.0	6.5
50.0	24.0	14.3	156.0*	37.1	776.0*	37.4
100.0	24.0	14.3	108.0*	56.5	284.0*	77.1

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 5:Yield (Y) and Percentage Inhibition of Y during the Test Period

NominalConcentration [mg/L]	Yield (Y) and % inhibition of Y
	0–72 h
	Y	%
Control	66.7	0.0
6.25	62.0	7.0
12.5	64.3	3.5
25.0	63.3	5.0
50.0	42.7*	36.0
100.0	9.7*	85.5

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 6: Influence ofN,N’-(ethoxymethylsilylene)bis[N-methylbenzamide]on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
		Calculation based on the nominal concentrations
EC50	> 100		57.25	62.40
95 % conf. limits	not determined		49.50 – 66.22	53.16 – 73.24
NOEC	25.0		25.0	25.0
LOEC	50.0		50.0	50.0

 

Table 7:pH-Values in the Test Media at the Start and End of the Test

NominalConcentration [mg/L]	pH-values
	Start	End
Control	7.98	8.05
		7.98
		8.01
		8.15
		8.20
		8.31
6.25	7.95	8.76
		8.59
		8.63
12.5	7.94	8.12
		8.10
		8.13
25.0	7.91	8.11
		8.27
		8.35
50.0	7.86	8.35
		8.31
		8.28
100.0	7.89	8.24
		8.20
		8.13

Table 8:Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.8	22.8	22.8	22.7
min / max temperature (ºC) of the climate chamber	max	23.1	23.1	23.0	22.9
	min	22.6	22.2	22.2	21.9

Table 9:Cell Number (x 10⁴cell/mL) determined in the Main Experiment

 

NominalConcentration [mg/L]		Number of cells
		0 h	24 h	48 h	72 h
Control		1	3	16	63
		1	4	18	75
		1	4	16	69
		1	3	17	66
		1	3	17	63
		1	3	18	70
	Mean	1.00	3.33	17.00	67.67
	SD	0.0	0.5	0.9	4.6
6.25		1	4	16	63
		1	4	16	66
		1	4	17	60
	Mean	1.00	4.00	16.33	63.00
	SD	0.0	0.0	0.6	3.0
12.5		1	3	15	68
		1	3	16	60
		1	3	15	68
	Mean	1.00	3.00	15.33	65.33
	SD	0.0	0.0	0.6	4.6
25.0		1	3	17	63
		1	3	15	65
		1	3	15	65
	Mean	1.00	3.00	15.67	64.33
	SD	0.0	0.0	1.2	1.2
50.0		1	3	10	45
		1	3	9	38
		1	3	11	48
	Mean	1.00	3.00	10.00	43.67
	SD	0.0	0.0	1.0	5.1
100.0		1	3	6	12
		1	3	7	10
		1	3	5	10
	Mean	1.00	3.00	6.00	10.67
	SD	0.0	0.0	1.0	1.2

 

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met (see "Overall remarks"), therefore the study can be considered as valid.

Conclusions:

The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal), based on growth rate, and 57.25 and 62.40 mg/l based on yield and biomass respectively. The NOEC was determined to be 25 mg/L and the LOEC 50 mg/mL, both based on growth rate, yield and biomass.

Executive summary:

A freshwater algae growth inhibition test was performed on the test item, according to OECD 201 and EU Method C.3, following GLP. Based on the results obtained from a preliminary range-finding test, where significant inhibition was observed at the highest examined concentration level, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. Control and test concentrations were analytically determined (validated method) at the beginning and at the end of the experiment and did not deviate more than 20%. The following concentrations were tested: 6.25, 12.5, 25, 50 and 100% saturated solutions. The corresponding measured geometric mean test item concentration were: 6.62, 13.10, 25.65, 50.85 and 100.44 mg/L. Three replicates at each concentration and six replicates for the untreated controls were performed. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met. The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal), based on growth rate, and 57.25 and 62.40 mg/L based on yield and biomass respectively. The NOEC was determined to be 25 mg/L and the LOEC 50 mg/mL, both based on growth rate, yield and biomass.

Description of key information

Key study: OECD 201 and EU method C.3. GLP study. The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal), based on growth rate, and 57.25 and 62.40 mg/l based on yield and biomass respectively. The NOEC was determined to be 25 mg/L and the LOEC 50 mg/mL, both based on growth rate, yield and biomass.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

Key study: A freshwater algae growth inhibition test was performed on the test item, according to OECD 201 and EU Method C.3, following GLP. Based on the results obtained from a preliminary range-finding test, where significant inhibition was observed at the highest examined concentration level, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. Control and test concentrations were analytically determined (validated method) at the beginning and at the end of the experiment and did not deviate more than 20%. The following concentrations were tested: 6.25, 12.5, 25, 50 and 100% saturated solutions. The corresponding measured geometric mean test item concentration were: 6.62, 13.10, 25.65, 50.85 and 100.44 mg/L. Three replicates at each concentration and six replicates for the untreated controls were performed. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met. The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal), based on growth rate, and 57.25 and 62.40 mg/l based on yield and biomass respectively. The NOEC was determined to be 25 mg/L and the LOEC 50 mg/mL, both based on growth rate, yield and biomass.