N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

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:

14 December 2017 - 19 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32, 100 mg/L
- Sampling method: 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 each occasion and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: Frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water-accomodated fractions: Nominal amounts of test item (20, 32, 20, 64 and 200 mg) were each separately added to the surface of 20, 10, 2.0, 2.0 and 2.0 liters of culture medium to give loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.7 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.
- Eluate: N/A
- Differential loading: N/A
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At both the start and end of the mixing period, and following a 1-Hour standing period, all loading rate WAFs were observed to have formed clear colorless media columns with solidified test item floating at the media surface. At the start of stirring all test item immediately became free of the glass slide. Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: N/A
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: The master cultures were maintained in the laboratory under constant agitation by orbital shaker (100 to 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 (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

ACCLIMATION
- Acclimation period: N/A
- 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

Remarks on exposure duration:

Guideline-recommended

Post exposure observation period:

N/A

Hardness:

N/A

Test temperature:

24 ±1 °C

pH:

7.3 - 7.4

Dissolved oxygen:

N/A

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured:
Chemical analysis of 10 and 100 mg/L loading rate WAF test preparations, 0 hours: 0.080 and 0.72 mg/L respectively.
A decline in measured test concentrations observed at 72 hours to less than the LOQ, determined to be 0.071 mg/L, and 0.14 mg/L for the 10 and 100 mg/L loading rate WAF test preparations respectively indicated the test item was unstable under the conditions of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Volumetric flask
- Type (delete if not applicable): Closed - plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: Glass, 250 mL conical flasks; containing 100 mL test solution; constant shaking maintained throughout test.
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 5.00 x 10^3 cells/mL
- Control end cells density: 0 h: 5.00 x 10^3 cells/mL; 72 h: 1.99 x 10^6 cells/mL
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: The pH of the control and each test concentration was determined at test initiation and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: The culture medium pH was adjusted to 7.5 with 0.1N NaOH or HCl
- Photoperiod: Constant illumination
- Light intensity and quality: Intensity approximately 7000 lux, warm white lighting (380 to 730 nm)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Samples were taken at 19, 44 and 72 hours
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- Chlorophyll measurement: N/A
- Other: The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Yes: Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (9.5 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAFs.
- Test concentrations: 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Growth was observed to be reduced at both 10 and 100 mg/L loading rate WAF, prompting the definitive test concentration range.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

mg/L loading rate WAF

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an ErL50 value as no loading rate tested resulted in 50% inhibition of growth rate.

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

mg/L loading rate WAF

Effect conc.:

49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: None
- Colour differences: 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, 3.2, 10 and 32 mg/L loading rate WAF test cultures were observed to be green dispersions. Replicate 1 of the 100 mg/L loading rate WAF was observed to be a pale green dispersion, replicate 2 was a very pale green dispersion and replicate 3 was an extremely pale green dispersion.
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Other: The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.
- Any stimulation of growth found in any treatment: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. It is evident from this work that increasing the stirring period significantly increased the amount of dissolved test item in the WAF between the 24-Hour (0.049 mg/L measured concentration) and 96-Hour (0.13 mg/L measured concentration) preparation periods and so preparation of the WAF was a 95-Hour stirring period followed by a 1-Hour standing period. Therefore, any risk of differences between emasured and nominal values was minimised.
- Effect concentrations exceeding solubility of substance in test medium: Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.071 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
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
- Other:
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) 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.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all loading rates 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 between the control, 1.0, 3.2, 10 and 32 mg/L loading rate WAFs (P≥0.05), however the 100 mg/L loading rate was significantly different (P<0.05) and, therefore the NOEL based on growth rate was 32 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 100 mg/L loading rate WAF.
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ELx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Table 1. Cell densities and percentage inhibition of growth from the range-finding test

Nominal Loading Rate (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	1.11E+03	6.59E+05	-	-
	R2	6.81E+03	6.69E+05
	Mean	3.96E+03	6.64E+05
10	R1	1.59E+04	6.22E+05	27	4
	R2	1.43E+04	6.73E+05
	Mean	1.51E+04	6.47E+05
100	R1	1.08E+04	2.86E+04	90	99
	R2	1.32E+04	9.83E+03
	Mean	1.20E+04	1.92E+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

- = not applicable

Table 2. Cell densities and pH values in the definitive test

Nominal Loading Rate (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0h	19 h	44 h	72 h	72 h
Control	R1	7.3	3.62E+04	2.10E+05	1.89E+06	7.8
	R2		3.83E+04	2.35E+05	1.91E+06
	R3		3.95E+04	2.32E+05	2.15E+06
	R4		3.94E+04	2.32E+05	2.09E+06
	R5		3.96E+04	2.38E+05	1.87E+06
	R6		3.77E+04	2.22E+05	2.04E+06
	Mean		3.84E+04	2.28E+05	1.99E+06
1.0	R1	7.3	3.57E+04	2.34E+05	1.91E+06	7.9
	R2		3.45E+04	2.13E+05	2.02E+06
	R3		3.43E+04	2.07E+05	1.98E+06
	Mean		3.48E+04	2.18E+05	1.97E+06
3.2	R1	7.4	3.99E+04	2.09E+05	2.04E+06	7.9
	R2		3.59E+04	2.05E+05	1.45E+06
	R3		3.40E+04	2.06E+05	2.00E+06
	Mean		3.66E+04	2.07E+05	1.83E+06
10	R1	7.3	3.49E+04	2.04E+05	1.81E+06	7.9
	R2		4.03E+04	2.20E+05	1.63E+06
	R3		2.93E+04	2.00E+05	1.29E+06
	Mean		3.49E+04	2.08E+05	1.58E+06
32	R1	7.4	2.99E+04	1.80E+05	1.10E+06	7.9
	R2		3.26E+04	1.96E+05	1.74E+06
	R3		3.10E+04	2.12E+05	1.71E+06
	Mean		3.12E+04	1.96E+05	1.52E+06
100	R1	7.3	1.74E+04	1.19E+05	6.34E+05	7.8
	R2		7.04E+03	4.61E+04	2.49E+05
	R3		7.57E+03	3.75E+04	1.51E+05
	Mean		1.07E+04	6.75E+04	3.45E+05

*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

h - hours

Table 3. Daily specific growth rate (cells/mL/hour)

Treatment			Daily Specific Growth Rate (cells/mL/hour)
			Day 0 to 1			Day 1 to 2	Day 2 to 3
Control		R1	0.104			0.070	0.078
		R2	0.107			0.073	0.075
		R3	0.109			0.071	0.080
		R4	0.109			0.071	0.078
		R5	0.109			0.072	0.074
		R6	0.106			0.071	0.079
		Mean	0.107			0.071	0.077

Table 4. Inhibition of growth rate and yield in the definitive test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.082	-	1.88E+06	-
	R2	0.083		1.91E+06
	R3	0.084		2.15E+06
	R4	0.084		2.08E+06
	R5	0.082		1.87E+06
	R6	0.084		2.04E+06
	Mean	0.083		1.99E+06
	SD	0.001		1.18E+05
1.0	R1	0.083	0	1.91E+06
	R2	0.083	0	2.01E+06
	R3	0.083	0	1.98E+06
	Mean	0.083	0	1.96E+06	1
	SD	0.000		5.39E+04
3.2	R1	0.083	0	2.04E+06
	R2	0.079	5	1.44E+06
	R3	0.083	0	1.99E+06
	Mean	0.082	2	1.82E+06	8
	SD	0.002		3.30E+05
10	R1	0.082	1	1.81E+06
	R2	0.080	4	1.63E+06
	R3	0.077	7	1.28E+06
	Mean	0.080	4	1.57E+06	21
	SD	0.003		2.68E+05
32	R1	0.075	10	1.09E+06
	R2	0.081	2	1.73E+06
	R3	0.081	2	1.70E+06
	Mean	0.079	5	1.51E+06	24
	SD	0.003		3.61E+05
100	R1	0.067	19	6.29E+05
	R2	0.054	35	2.44E+05
	R3	0.047	43	1.46E+05
	Mean	0.056	32	3.40E+05	83
	SD	0.010		2.55E+05

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

R = replicate

SD = standard deviation

- = Not applicable

Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 0.080 and 0.72 mg/L respectively were obtained. A decline in measured test concentrations was observed at 72 hours to less than the LOQ, determined to be 0.071 mg/L, and 0.14 mg/L for the 10 and 100 mg/L loading rate WAF test preparations respectively indicating that the test item was unstable under the conditions of the test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test, the exposure to the test 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

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

The test item was prepared as a water accomodated fraction given its low solubility and complexity. Following a range-finding test (10 and 100 mg/L), a definitive test was conducted at 1.0, 3.2, 10, 32 and 100 mg/L nominal loading rates alongside controls.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.071 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

No cell abnormalities were recorded in any control or cell cultures; and the control, and 1.0 - 32 mg/L cultures were observe to be green dispersions by the end of the test.

All validity criteria were fulfilled (the control cell concentration increased by a factor greater 16 during the test; the mean of the coefficients of variation of the section by section specific growth rates in the control cultures (Days 0 to 1, 1 to 2 and 2 to 3) did not exceed 35%; the coefficient of variation of average specific growth rate in replicate control cultures did not exceed 7%). The positive reference control was also valid.

Exposure of the algae to the test substance gave the following results:

Inhibition growth rate

ErL10 (0 to 72 hour) : 45 mg/L loading rate WAF

ErL20 (0 to 72 hour) : 70 mg/L loading rate WAF

ErL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Inhibition yield

EyL10 (0 to 72 hour) : 13 mg/L loading rate WAF

EyL20 (0 to 72 hour) : 21 mg/L loading rate WAF

EyL50 (0 to 72 hour) : 49 mg/L loading rate WAF; 95% confidence limits 42 to 57 mg/L loading rate WAF

Description of key information

ErL50 (0 to 72 hour, growth rate inhibition) : >100 mg/L loading rate WAF; EyL50 (0 to 72 hour, yield inhibition) : 49 mg/L loading rate WAF OECD 201; Vryenhoef (2018).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

A single key study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

The test item was prepared as a water accomodated fraction given its low solubility and complexity. Following a range-finding test (10 and 100 mg/L), a definitive test was conducted at 1.0, 3.2, 10, 32 and 100 mg/L nominal loading rates alongside controls.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.071 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

No cell abnormalities were recorded in any control or cell cultures; and the control, and 1.0 - 32 mg/L cultures were observe to be green dispersions by the end of the test.

All validity criteria were fulfilled (the control cell concentration increased by a factor greater 16 during the test; the mean of the coefficients of variation of the section by section specific growth rates in the control cultures (Days 0 to 1, 1 to 2 and 2 to 3) did not exceed 35%; the coefficient of variation of average specific growth rate in replicate control cultures did not exceed 7%). The positive reference control was also valid.

Exposure of the algae to the test substance gave the following results:

Inhibition growth rate

ErL10 (0 to 72 hour) : 45 mg/L loading rate WAF

ErL20 (0 to 72 hour) : 70 mg/L loading rate WAF

ErL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Inhibition yield

EyL10 (0 to 72 hour) : 13 mg/L loading rate WAF

EyL20 (0 to 72 hour) : 21 mg/L loading rate WAF

EyL50 (0 to 72 hour) : 49 mg/L loading rate WAF; 95% confidence limits 42 to 57 mg/L loading rate WAF