N,N-Dimethyl-9-dodecenamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2021 - 31 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions (from a replicate of each treatment with algae dedicated exclusively to chemical analyses) at the start and the end of the test.

Vehicle:

no

Details on test solutions:

Preparation of stock and test solutions
The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water was added. Then 100.1 mg test item were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1 L of stock solution and closed. Mixing was initiated with the vortex in the centre extending to maximally around 10% of the vessel depth from the top to the bottom of the vessel. After approx. 22 hours of gentle stirring in the dark at room temperature, the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port and samples were taken from the stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary and fixed amount of inoculum (5 E+03 cells/mL per vessel) to obtain the required test concentrations in the test vessels (based on the concentration measured in the stock solution (around 75 mg/L)) that were immediately sealed after filling with a minimum headspace.
- Test concentration separation factor: Approx 2.11
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of the test, the test solution in test vessels was observed to be clear and colourless (Tyndall effect, checked via laser beam, was negative).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions, according to the test guidelines.
- Stock culture: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
- Acclimation period: 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1×104 cells/mL.
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature in the incubator was situated between 21.9 and 23.6°C throughout the test (average value: 23.4°C)

pH:

See Table 2 below

Nominal and measured concentrations:

Nominal: 0.5, 1.1, 2.2, 4.7 and 10.0 mg/L
Geometric mean measured concentration: 0.28, 0.86, 2.18, 5.08 and 10.70 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass flasks
- Type: closed with ground glass stopper
- Material, size, headspace, fill volume: completely filled with test solution with minimum headspace
- Aeration: No
- Initial cells density: An initial cell density of 5 E+03 cells/mL using the exponentially growing pre-culture.
- Control end cells density: 150.6 E+04 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration for counting. Moreover, additional replicates of each treatment with algae were prepared for chemical analyses (destructive samples) in order to determine maintenance of actual concentrations.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 were added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L, according to the study plan.


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: The mean light intensity was of 5521 lux (range: 5140-5767 lux),

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 2.11
- Range finding study
- Test concentrations: 1.3, 3.2, 10.0. 32.0, 100.0 mg test item/L
- Results used to determine the conditions for the definitive study: See table below

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.67 mg/L

95% CI:

>= 0.51 - <= 0.82

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

95% CI:

>= 0.22 - <= 0.47

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.01 mg/L

95% CI:

>= 0.83 - <= 1.17

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.48 mg/L

95% CI:

>= 0.34 - <= 0.59

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

95% CI:

>= 1.97 - <= 2.44

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.85 mg/L

95% CI:

>= 0.72 - <= 0.97

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- 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):

On January 18, 2022 (KA22-001; most recent test), the 72h-EC50 was 0.791 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-ErC50: 0.65 mg/L to 1.73 mg/L).

Reported statistics and error estimates:

See Appendix VII attached

Range-finding test results

Algal cell densities during the range-finding test (expressed as density of algal cells/mL×104).

	Replicate	Analytically confirmed concentration (geometric mean) (mg/L)
		Control	1.0	3.2	10.0	32.0	100.0
t=24 h	1	2.4	4.8	2.8	1.2	0.8	1.2
	2	4.8	4.4	2.8	0.8	0.8	0.4
	3	5.2	3.2	1.2	0.8	0.4	0.4
	Mean	4.1	4.1	2.3	0.9	0.7	0.7
	Std. Dev.	1.51	0.83	0.92	0.23	0.23	0.46
	% Inh. GR	-	-2.0	30.0	70.6	88.4	93.0
	% Red. Y	-	0.0	51.4	88.1	95.4	95.4
t=48 h	1	32.8	29.6	1.6	0.8	0.4	0.4
	2	27.2	22.0	3.6	0.8	0.4	0.8
	3	26.8	22.0	2.0	0.4	0.4	0.4
	Mean	28.9	24.5	2.4	0.7	0.4	0.5
	Std. Dev.	3.35	4.39	1.06	0.23	0.00	0.23
	% Inh. GR	-	4.2	62.8	94.1	105.5	99.8
	% Red. Y	-	15.5	93.3	99.4	100.4	99.9
t=72 h	1	121.6	88.0	4.0	0.8	0.4	0.1
	2	112.0	98.0	3.6	1.2	0.8	0.4
	3	84.0	62.0	6.8	0.4	0.4	0.8
	Mean	105.9	82.7	4.8	0.8	0.5	0.4
	Std. Dev.	19.54	18.58	1.74	0.40	0.23	0.35
	% Inh. GR	-	4.7	58.4	93.0	99.9	108.5
	% Red. Y	-	22.0	95.9	99.7	100.0	100.1

At test start 5000 algal cells/mL were incubated; 3 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

% Inh. GR: %Inhibition of growth rate relative to the control determined by ToxRat.

% Red. Y: %Reduction in yield relative to the control determined by ToxRat.

Table 2. pH-values during the final test.

	Analytically confirmed concentration (geometric mean) (mg/L)
	Control	0.28	0.86	2.18	5.08	10.70
Start t=0 h	8.22	8.22	8.25	8.28	8.30	8.23
End t=72 h	10.34	10.25	9.68	9.79	8.40	8.40

Table 3. Algal cell densities during the final test (expressed as density of algal cells/mL×10⁴)

	Replicate	Analytically confirmed concentration (geometric mean) (mg/L)
		Control	0.28	0.86	2.18	5.08	10.70
t=24 h	1	8.8	7.2	5.2	2.0	0.8	0.8
	2	8.4	9.6	3.6	2.4	1.2	0.4
	3	10.4	8.8	6.4	2.0	2.4	0.4
	4	7.6
	5	8.0
	6	6.4
	Mean	8.3	8.5	5.1	2.1	1.5	0.5
	Std. Dev.	1.33	1.22	1.40	0.23	0.83	0.23
t=48 h	1	56.0	53.2	29.6	2.0	1.2	0.0
	2	40.4	56.0	22.4	2.4	1.6	0.4
	3	41.2	52.8	20.4	2.0	0.0	0.4
	4	66.4
	5	42.0
	6	42.0
	Mean	48.0	54.0	24.1	2.1	0.9	0.3
	Std. Dev.	10.76	1.74	4.84	0.23	0.83	0.23
t=72 h	1	162.0	110.0	70.8	6.4	2.0	0.8
	2	132.4	164.4	74.0	9.2	2.0	0.8
	3	150.4	152.8	87.6	5.2	1.2	0.4
	4	168.0
	5	124.8
	6	166.0
	Mean	150.6	142.4	77.5	6.9	1.7	0.7
	Std. Dev.	18.26	28.65	8.92	2.05	0.46	0.23

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

Table 4. Yield of algal cells during the final test.

	Replicate	Analytically confirmed concentration (geometric mean) (mg/L)
		Control	0.28	0.86	2.18	5.08	10.70
t=24 h	1	8.3	6.7	4.7	1.5	0.3	0.3
	2	7.9	9.1	3.1	1.9	0.7	-0.1
	3	9.9	8.3	5.9	1.5	1.9	-0.1
	4	7.1
	5	7.5
	6	5.9
	Mean	7.8	8.0	4.6	1.6	1.0	0.0
	Std. Dev.	1.33	1.22	1.40	0.23	0.83	0.23
	% Red.	-	-3.4	41.2	79.0	87.6	99.6
t=48 h	1	55.5	52.7	29.1	1.5	0.7	-0.4
	2	39.9	55.5	21.9	1.9	1.1	-0.1
	3	40.7	52.3	19.9	1.5	-0.4	-0.1
	4	65.9
	5	41.5
	6	41.5
	Mean	47.5	53.5	23.6	1.6	0.5	-0.2
	Std. Dev.	10.76	1.74	4.84	0.23	0.78	0.17
	% Red.	-	-12.6	50.2	96.6	99.0	100.4
t=72 h	1	161.5	109.5	70.3	5.9	1.5	0.3
	2	131.9	163.9	73.5	8.7	1.5	0.3
	3	149.9	152.3	87.1	4.7	0.7	-0.1
	4	167.5
	5	124.3
	6	165.5
	Mean	150.1	141.9	77.0	6.4	1.2	0.2
	Std. Dev.	18.26	28.65	8.92	2.05	0.46	0.23
	% Red.	-	5.5	48.7	95.7	99.2	99.9

Values were extracted from the computer program ToxRat

% Red.: %Reduction in yield relative to the control determined by ToxRat

Table 5. Mean specific growth rate in P. subcapitata during the final test.

	Replicate	Analytically confirmed concentration (geometric mean) (mg/L)
		Control	0.28	0.86	2.18	5.08	10.70
t=0 h - t=24 h	1	2.868	2.667	2.342	1.386	0.470	0.470
	2	2.821	2.955	1.974	1.569	0.875	-0.223
	3	3.035	2.868	2.549	1.386	1.569	-0.223
	4	2.721
	5	2.773
	6	2.549
	Mean	2.795	2.830	2.288	1.447	0.971	0.008
	Std. Dev.	0.1611	0.1475	0.2914	0.1053	0.5555	0.4002
	% Inh.	-	-1.3	18.1	48.2	65.2	99.7
t=0 h - t=48 h	1	2.359	2.334	2.040	0.693	0.438	-0.805
	2	2.196	2.359	1.901	0.784	0.582	-0.112
	3	2.206	2.330	1.854	0.693	-0.805	-0.112
	4	2.444
	5	2.215
	6	2.215
	Mean	2.273	2.341	1.932	0.724	0.072	-0.343
	Std. Dev.	0.1038	0.0160	0.0968	0.0526	0.7623	0.4002
	% Inh.	-	-3.0	15.0	68.2	96.9	115.1
t=0 h - t=72 h	1	1.927	1.798	1.651	0.850	0.462	0.157
	2	1.860	1.932	1.666	0.971	0.462	0.157
	3	1.902	1.907	1.722	0.781	0.292	-0.074
	4	1.939
	5	1.840
	6	1.935
	Mean	1.900	1.879	1.680	0.867	0.405	0.080
	Std. Dev.	0.0417	0.0713	0.0375	0.0963	0.0983	0.1334
	% Inh.	-	1.1	11.6	54.4	78.7	95.8

Values were extracted from the computer program ToxRat

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat

Analytical results

The results of analysis of the samples taken during the final test are described in the Table 7 of the attached Analytical Report (Appendix V). Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine actual concentrations of the test item.

Chemical analysis revealed that test item levels were relatively stable, with losses of test item < 20% (except at 1.1 mg/L where a 45% loss was found). Furthermore, geometric means of measured concentrations were within ± 20% of the nominal concentrations except at the two lowest test concentrations (see Table 7 of Appendix V for details). Therefore, the evaluation of the effects on P. subcapitata was based on the geometric means of the analytically confirmed concentrations.

Validity criteria of the study:

Cell density in controls: 301-fold increase within 72 hours.

Coefficient of variation:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 12% in 72 hours.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.2%.

Thus, the validity criteria were respected in this study.

Validity criteria fulfilled:

yes

Remarks:

See Any other information on results

Executive summary:

A study was performed to assess the test item N,N-dimethyldodec-9-enamide for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD TG 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Methods.

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 0.5, 1.1, 2.2, 4.7 and 10.0 mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

Results.

The test item levels were found to be stable, with losses of test item < 20% (except at 1.1 mg/L with 45% loss). Therefore, the evaluation of the effects on P. subcapitatawas based on the geometric means of the analytically confirmed concentrations: 0.28, 0.86, 2.18, 5.08 and 10.70 mg/L.

 

The ErCx, EyCx, NOErC and NOEyC values after 72 hours were as follows:

 

Parameter	Growth rate in mg/L (and 95% conf. limits)	Yield in mg/L (and 95% conf. limits)
72-hour EC10	0.67 (0.51 – 0.82)	0.36 (0.22 – 0.47)
72-hour EC20	1.01 (0.83 – 1.17)	0.48 (0.34 – 0.59)
72-hour EC50	2.20 (1.97 – 2.44)	0.85 (0.72 – 0.97)
72-hour LOEC	0.86	0.86
72-hour NOEC	0.28	0.28

Statistical analyses were performed by the computer program ToxRat (8).

Conclusion.

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 2.20 mg/L and 0.85 mg/L, respectively. The 72-hour EC10 for growth rate was 0.67 mg/L and for yield was 0.36 mg/L. The 72-hour NOEC for both parameters was 0.28 mg/L.

Description of key information

The toxic effect of the test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test performed according to OECD TG 201. Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 2.20 mg/L and 0.85 mg/L, respectively. The 72-hour EC10 for growth rate was 0.67 mg/L and for yield was 0.36 mg/L. The 72-hour NOEC for both parameters was 0.28 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.2 mg/L

EC10 or NOEC for freshwater algae:

0.67 mg/L

Additional information

A study was performed to assess the test item N,N-dimethyldodec-9-enamide for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD TG 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 0.5, 1.1, 2.2, 4.7 and 10.0 mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

The test item levels were found to be stable, with losses of test item < 20% (except at 1.1 mg/L with 45% loss). Therefore, the evaluation of the effects on P. subcapitatawas based on the geometric means of the analytically confirmed concentrations: 0.28, 0.86, 2.18, 5.08 and 10.70 mg/L.

 

The ErCx, EyCx, NOErC and NOEyC values after 72 hours were as follows:

 

Parameter	Growth rate in mg/L (and 95% conf. limits)	Yield in mg/L (and 95% conf. limits)
72-hour EC10	0.67 (0.51 – 0.82)	0.36 (0.22 – 0.47)
72-hour EC20	1.01 (0.83 – 1.17)	0.48 (0.34 – 0.59)
72-hour EC50	2.20 (1.97 – 2.44)	0.85 (0.72 – 0.97)
72-hour LOEC	0.86	0.86
72-hour NOEC	0.28	0.28

Statistical analyses were performed by the computer program ToxRat (8).