N,N'-methylenedistearamide

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:

22 Aug 2018 to 22 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

as Method C.3 of Commission Regulation (EC) No 761/2009.(2006)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100% v/v saturated solution 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. A duplicate set of samples was taken at each occasion and stored frozen for further analysis if necessary.
- Preparation of the test samples: The test samples were thawed in a water bath. The test samples (200 mL) plus 60 g of sodium chloride were extracted three times with 50 mL of chloroform. The combined organic extracts were dried using anhydrous sodium sulfate and evaporated to dryness using rotary evaporation. The residues were dissolved in 5 mL of tetrahydrofuran and then an aliquot was taken for instrumental analysis.

Vehicle:

no

Details on test solutions:

- Method: Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions. A nominal amount of test item (275 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. An aliquot (1 liter) of this saturated solution was inoculated with algal suspension (20.8 mL) to give the required test concentration of 100% v/v saturated solution. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source: Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23 - 25 °C

pH:

- At t=0h: 7.6 - 7.8
- At t=72h: 8.4 - 8.5

Nominal and measured concentrations:

- Nominal concentrations: 0 (control) and 100 % (v/v) saturated solution.
- Mean measured concentratoins:

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks, constantly shaken at approximately 150 rpm for 72 hours
- Type: closed with polyurethane foam bungs to reduce evaporation.
- Fill volume: 100 mL
- Aeration: No
- Incubator: INFORS Multitron(R) Version 2 incubator
- Initial cells density: 5.00E+03 cells/mL
- Control end cells density: 8.98E+03 cells/mL
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (AAP growth medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water (*Elga Optima 15+ or Elga Purelab Option R-15 BP)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: Approximately 7000 lux provided by warm white lighting (380 to 730 nm)

WATER QUALITY PARAMETERS
- pH: The pH of the control and 100% v/v saturated solution test group was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.
- Temperature: The temperature within the incubator was recorded daily.
- Test solution appearance: The appearance of the test media was recorded daily.

EFFECT PARAMETERS MEASURED: cell density, appearance
Samples were taken at 23, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

RANGE FINDING TEST
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 72 hours. The results showed no effect on growth at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate; the positive control was conducted between 06 November 2017 and 09 November 2017.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: % (v/v) saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects observed. The ErC10 was determined as greater than the maximum water solubility of test item (<0.00026 mg/L).

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % (v/v) saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effects observed. The ErC50 was determined as greater than the maximum water solubility of test item (<0.00026 mg/L).

Details on results:

- Test solution appearance: 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 and test cultures were observed to be green dispersions.
- Growth rate and yield: Growth rate and yield of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.
- Appearance: There were no abnormalities detected in any of the control or test cultures at 72 hours.

Results with reference substance (positive control):

The 72-h ErC50 was determined to be 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L. The 72-h EyC50 was determined to be 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L. The NOECs and LOECs for growth rate and yield were 0.25 mg/L and 0.50 mg/L, respectively. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100% v/v saturated solution test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table: Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	23 Hour	47 Hour	72 Hour	72 Hours
Control	R1	7.6	1.77E+04	1.22E+05	9.19E+05	8.5
	R2		1.76E+04	1.07E+05	9.14E+05
	R3		2.05E+04	1.04E+05	9.01E+05
	R4		1.78E+04	1.06E+05	8.79E+05
	R5		1.59E+04	1.06E+05	9.36E+05
	R6		1.53E+04	9.17E+04	8.38E+05
	Mean		1.75E+04	1.06E+05	8.98E+05
100	R1	7.8	1.90E+04	1.34E+05	1.08E+06	8.4
	R2		1.69E+04	9.87E+04	8.15E+05
	R3		1.68E+04	1.21E+05	8.88E+05
	R4		1.49E+04	8.86E+04	8.29E+05
	R5		1.58E+04	1.07E+05	9.42E+05
	R6		1.46E+04	8.59E+04	7.14E+05
	Mean		1.63E+04	1.06E+05	8.78E+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

 

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

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.055	0.080	0.081
	R2	0.055	0.075	0.086
	R3	0.061	0.068	0.086
	R4	0.055	0.074	0.085
	R5	0.050	0.079	0.087
	R6	0.048	0.075	0.088
	Mean	0.054	0.075	0.086

R = Replicate

 

Table: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.072	-	9.14E+05	-
	R2	0.072		9.09E+05
	R3	0.072		8.96E+05
	R4	0.072		8.74E+05
	R5	0.073		9.31E+05
	R6	0.071		8.33E+05
	Mean	0.072		8.93E+05
	SD	0.001		3.49E+04
100	R1	0.075	[4]	1.08E+06
	R2	0.071	1	8.10E+05
	R3	0.072	0	8.83E+05
	R4	0.071	1	8.24E+05
	R5	0.073	[1]	9.37E+05
	R6	0.069	4	7.09E+05
	Mean	0.072	0	8.73E+05	2
	SD	0.002		1.25E+05

* 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

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'.

Conclusions:

Based on these findings, the 72-h EC50 values for growth rate and yield/biomass are determined to be >100% v/v of a saturated aqueous solution. The 72-h ErC10 was also determined to be >100% v/v of a saturated aqueous solution for both endpoints.

Executive summary:

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (Envigo, 2018). In this study, exponentially growing freshwater alga (Pseudokirchneriella subcapitata) were exposed in a limit test (based on a preliminary range-finding study) to saturated aqueous solutions of the test substance of 0, (control) and 100% (v/v) for 72 hours under static conditions. The test was performed in six replicates per test concentration. The initial concentrations and the maintenance of the exposure concentrations during the test were analytically determined. Cell density was measured after 24, 48 and 72 hours exposure and the growth rate, yield and biomass per time point were determined. Throughout the test, no effects on the growth rate and yield were observed. Based on these findings, the 72-h EC50 values for growth rate and yield/biomass are determined to be >100% v/v of a saturated aqueous solution. The 72-h ErC10 was also determined to be >100% v/v of a saturated aqueous solution for both endpoints.

Description of key information

72-h ErC50: >100% (v/v) of a saturated aqueous solution (Pseudokirchneriella subcapitata)

72-h ErC10: >100% (v/v) of a saturated aqueous solution (Pseudokirchneriella subcapitata)