2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

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:

The study was conducted between 02 February 2018 and 15 March 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 15-0735
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range finding test:
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive test:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding test:
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Definitive Test:
A nominal amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.6 - 7.7.

Nominal and measured concentrations:

Range-finding test - nominal concentration:
0.10, 1.0, 10 and 100 mg/L

Definitive test - nominal concentrations:
1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.08 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 4.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

- No. of vessels per concentration (replicates):
2

- No. of vessels per control (replicates):
2

GROWTH MEDIUM
- Standard medium used: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

- Photoperiod:
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination provided by warm white lighting (380 to 730 nm and constantly shaken at approximately 150 rpm for 72 hours.

- Light intensity and quality:
intensity approximately 7000 lux

- Salinity (for marine algae):
Not applicable

- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 18, 43 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.

- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 and 72 hours showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test:
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94% to 104% of nominal and so the results are based on nominal test concentrations only.

Inhibition of Growth Rate
ErC10 (0 to 72 hour): 6.5 mg/L
ErC20 (0 to 72 hour): 11 mg/L
ErC50 (0 to 72 hour): 30 mg/L; 95% confidence limits 24 to 38 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 4.0 mg/L
EyC20 (0 to 72 hour): 4.8 mg/L
EyC50 (0 to 72 hour): 7.3 mg/L; 95% confidence limits 6.2 to 8.6 mg/L
Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant decrease in yield (P≥0.05), between the control, 1.0 and 3.2 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 3.2 mg/L. Correspondingly the LOEC based on yield was 10 mg/L.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Cell Densities and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.00E+03	1.62E+06	-	-
	R2	8.35E+03	1.95E+06
	Mean	6.68E+03	1.79E+06
0.10	R1	6.65E+03	1.83E+06	1	4
	R2	6.65E+03	1.60E+06
	Mean	6.65E+03	1.72E+06
1.0	R1	5.00E+03	1.72E+06	[1]	4
	R2	6.65E+03	1.72E+06
	Mean	5.83E+03	1.72E+06
10	R1	1.00E+04	2.73E+05	36	81
	R2	8.35E+03	4.20E+05
	Mean	9.18E+03	3.47E+05
100	R1	5.00E+03	3.35E+03	106	100
	R2	6.65E+03	5.00E+03
	Mean	5.83E+03	4.18E+03

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3  fields of view for each of the replicate flasks

R   = Replicate

-    = Not applicable

[ ]    =Increase in growth compared to controls

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	18 Hours	43 Hours	72 Hours	72 Hours
Control	R1	7.7	2.80E+04	1.93E+05	1.37E+06	8.5
	R2		2.96E+04	2.00E+05	1.48E+06
	R3		2.60E+04	1.90E+05	1.65E+06
	R4		2.59E+04	2.08E+05	1.60E+06
	R5		2.56E+04	1.97E+05	1.45E+06
	R6		2.64E+04	1.94E+05	1.47E+06
	Mean		2.69E+04	1.97E+05	1.50E+06
1.0	R1	7.7	2.77E+04	2.10E+05	1.65E+06	8.4
	R2		2.71E+04	1.95E+05	1.67E+06
	R3		2.54E+04	1.91E+05	2.01E+06
	Mean		2.67E+04	1.99E+05	1.78E+06
3.2	R1	7.6	2.52E+04	1.78E+05	1.68E+06	8.4
	R2		2.27E+04	1.69E+05	1.50E+06
	R3		2.29E+04	1.46E+05	1.38E+06
	Mean		2.36E+04	1.64E+05	1.52E+06
10	R1	7.6	1.91E+04	7.77E+04	4.69E+05	8.2
	R2		1.99E+04	7.25E+04	3.87E+05
	R3		2.14E+04	7.52E+04	5.21E+05
	Mean		2.01E+04	7.51E+04	4.59E+05
32	R1	7.7	1.49E+04	3.02E+04	1.04E+05	7.9
	R2		1.26E+04	2.95E+04	8.02E+04
	R3		1.31E+04	2.57E+04	7.05E+04
	Mean		1.35E+04	2.85E+04	8.48E+04
100	R1	7.7	8.15E+03	1.08E+04	1.30E+04	7.8
	R2		8.04E+03	8.83E+03	1.21E+04
	R3		8.80E+03	8.92E+03	1.06E+04
	Mean		8.33E+03	9.51E+03	1.19E+04

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R   = Replicate

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.096	0.077	0.068
	R2	0.099	0.076	0.069
	R3	0.092	0.080	0.075
	R4	0.091	0.083	0.070
	R5	0.091	0.082	0.069
	R6	0.093	0.080	0.070
	Mean	0.094	0.080	0.070

R   = Replicate

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.078	-	1.36E+06	-
	R2	0.079		1.48E+06
	R3	0.081		1.64E+06
	R4	0.080		1.59E+06
	R5	0.079		1.45E+06
	R6	0.079		1.47E+06
	Mean	0.079		1.50E+06
	SD	0.001		1.02E+05
1.0	R1	0.081	[3]	1.64E+06
	R2	0.081	[3]	1.67E+06
	R3	0.083	[5]	2.01E+06
	Mean	0.082	[4]	1.77E+06	[18]
	SD	0.001		2.04E+05
3.2	R1	0.081	[3]	1.68E+06
	R2	0.079	0	1.49E+06
	R3	0.078	1	1.38E+06
	Mean	0.079	[1]	1.52E+06	[1]
	SD	0.002		1.52E+05
10	R1	0.063	20	4.64E+05
	R2	0.060	24	3.82E+05
	R3	0.065	18	5.16E+05
	Mean	0.063	21	4.54E+05	70
	SD	0.003		6.79E+04
32	R1	0.042	47	9.88E+04
	R2	0.039	51	7.52E+04
	R3	0.037	53	6.55E+04
	Mean	0.039	50	7.98E+04	95
	SD	0.003		1.71E+04
100	R1	0.013	84	7.99E+03
	R2	0.012	85	7.09E+03
	R3	0.010	87	5.59E+03
	Mean	0.012	85	6.89E+03	100
	SD	0.002		1.21E+03

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R   = Replicate

-    = Not applicable

SD   =Standard Deviation

[ ]    =Increase in growth compared to controls

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 301 after 72 hours.  This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours            :       5.00 x 10³ cells per mL

Mean cell density of control at 72 hours              :       1.50 x 10⁶ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 15% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L. A few misshapen/enlarged cells were observed at 10 mg/L, some cell breakdown was observed at 32 mg/L and no intact cells were observed at 100 mg/L.

Water Quality Criteria

Temperature was maintained at 24 ±1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.5 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were light green dispersions; the 32 mg/L test cultures were extremely pale green dispersions whilst the 100 mg/L test cultures were clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and NOEC were 30 mg/L and 10 mg/l based on growth rate.

Executive summary:

A study was performed to assess the effect of FRET 15-0735 on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The results of the range-finding test showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94% to 104% of nominal and so the results are based on nominal test concentrations only.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	30	24	-	38	3.2	10
Yield	7.3	6.2	-	8.6	3.2	10

Description of key information

A study was performed to assess the effect of FRET 15-0735 on the growth of the green alga Pseudokirchneriella subcapitata.The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The results of the range-finding test showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94% to 104% of nominal and so the results are based on nominal test concentrations only.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	30	24	-	38	3.2	10
Yield	7.3	6.2	-	8.6	3.2	10

Key value for chemical safety assessment

EC50 for freshwater algae:

3.2 mg/L