Nonanamide, N,N-dimethyl-

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:

2016-12-12 to 2016-12-15

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

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Vehicle:

no

Details on test solutions:

Stock solution
100 mg test item/L were freshly prepared with dilution water. The dispersion was agitated until the stock solution was clear.

Test concentrations
5 concentrations were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg test item/L. The concentrations are based on the results of a preliminary range finding test (non-GLP).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.46 8.12
31.6 7.51 8.32
10.0 7.58 9.49
3.16 7.68 9.64
1.00 7.79 9.51
Control 7.97 9.42

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2330531 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6




GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 4440 - 8880 lux, mean value: 5035 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 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:

EC10

Effect conc.:

16.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 14.6 - 18.2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

20.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 18.5 - 21.6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 28.2 - 29.3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 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

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 9.29 - 11.7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 10.9 - 14.3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: CI: 13.7 - 19.2

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.763 ± 0.514
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.

The following statistical tests were conducted:

Monotonicity was tested by trend analysis by contrasts (significance level 0.05).

Shapiro-Wilk’s test on normal distribution was done with a significance level to 0.01.

Levene’s test on variance homogeneity was done with a significance level to 0.01.

William’s test on significant differences was done for growth rate with a significance level to 0.05.

Multiple sequentially-rejective welsh-t-test after Bonferroni-holm was done for yield with a significance level to 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•ToxRat, TOXRAT SOLUTIONS GMBH
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test itemconcentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6305	< LOQcell density	< LOQcell density	< LOQcell density
	2	6305	< LOQcell density	< LOQcell density	< LOQcell density
	3	6305	< LOQcell density	< LOQcell density	< LOQcell density
	Mean	6305	n.a.	n.a.	n.a.
31.6	1	6305	12250	24293	71745
	2	6305	10522	18618	65761
	3	6305	14525	25821	78618
	Mean	6305	12432	22911	72041
10.0	1	6305	35222	324638	2124603
	2	6305	45092	320804	2158769
	3	6305	38064	385325	2090324
	Mean	6305	39459	343589	2124565
3.16	1	6305	46901	430531	2238829
	2	6305	60808	549538	2364444
	3	6305	42662	447625	2396467
	Mean	6305	50124	475898	2333247
1.00	1	6305	48037	493439	2498966
	2	6305	54200	449542	2435257
	3	6305	42174	458698	2183295
	Mean	6305	48137	467226	2372506
Control	1	6305	38960	395541	2381527
	2	6305	42774	342589	2266850
	3	6305	46431	451989	2452340
	4	6305	45551	399251	2296731
	5	6305	50862	413741	2331010
	6	6305	51730	484982	2292559
	Mean	6305	46051	414682	2336836

< LOQ_{cell density}= below the limit of quantification (2736 cells/mL)                          n.a. = data not determinable

 Evaluation after 72 hours

             Statistically significant differences of growth rates and yield compared to
                  control values are marked (+), not significant differences are marked (-).

 

Nominal test itemconcentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		n.a.	100		n.a.	100
	2		n.a.	100		n.a.	100
	3		n.a.	100		n.a.	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
31.6	1		0.811	59		65440	97
	2		0.782	60		59456	97
	3		0.841	57		72313	97
	Mean	(+)	0.811	59	(+)	65736	97
10.0	1		1.94	2		2118298	9
	2		1.95	1		2152464	8
	3		1.94	2		2084019	11
	Mean	(+)	1.94	2	(+)	2118260	9
3.16	1		1.96	1		2232524	4
	2		1.98	0		2358139	-1
	3		1.98	0		2390162	-3
	Mean	(-)	1.97	0	(-)	2326942	0
1.00	1		1.99	-1		2492661	-7
	2		1.99	-1		2428952	-4
	3		1.95	1		2176990	7
	Mean	(-)	1.98	0	(-)	2366201	-2
Control	1		1.98			2375222
	2		1.96			2260545
	3		1.99			2446035
	4		1.97			2290426
	5		1.97			2324705
	6		1.97			2286254
	Mean		1.97			2330531

 

negative inhibition = increase of growth

n.a. = not available

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.82	2.32	1.80	1.98	0.294	14.9	12.5
	2	1.92	2.08	1.89	1.96	0.104	5.3
	3	2.00	2.28	1.69	1.99	0.292	14.7
	4	1.98	2.17	1.75	1.97	0.211	10.7
	5	2.09	2.10	1.73	1.97	0.210	10.6
	6	2.11	2.24	1.55	1.97	0.363	18.5
				Mean	1.97
				SD ±	0.01
				CV [%]	0.49

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, N,N-Dimethylnonanamide was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item concentrations): The NOEC (0-72 h) for inhibition of growth and inhibition of yield was 3.16 mg/L. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 28.8 (28.2 – 29.3) mg/L and 15.6 (13.7 – 19.2) mg/L, respectively. All effect values are based on the nominal test item concentrations of N,N-Dimethylnonanamide.

Executive summary:

The toxicity of N,N-Dimethylnonanamide to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009 Method C.3 from2016-12-12 to 2016-12-15 at the test facility. The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 100 mg/L was freshly prepared with dilution water. Five concentrations were tested in a geometrical series with a dilution factor of √10:1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of thetest item N,N-Dimethylnonanamide in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure.
The measured concentrations of N,N-Dimethylnonanamidein the fresh media (0 h) were in the range of 98 to 109% of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 88 to 105% of the nominal values of all concentrations.

Therefore, all effect values given are based on the nominal test item concentrations of N,N-Dimethylnonanamide.

 

 

NOEC, LOEC, EC-values and 95% Confidence Intervals ofN,N-Dimethylnonanamide(0 - 72 hours)

                  based on nominal test item concentrations [mg/L]

	Inhibition of Growth Rate
NOEC	3.16
LOEC	10.0
ErC10	16.3 (14.6 – 18.2)
ErC20	20.1 (18.5 – 21.6)
ErC50	28.8 (28.2 – 29.3)
	Inhibition of Yield
NOEC	3.16
LOEC	10.0
EyC10	10.2 (9.29 – 11.7)
EyC20	11.9 (10.9 – 14.3)
EyC50	15.6 (13.7 – 19.2)

 

Description of key information

In this study, N,N-Dimethylnonanamide was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item concentrations): The NOEC (0-72 h) for inhibition of growth and inhibition of yield was 3.16 mg/L. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 28.8 (28.2 – 29.3) mg/L and 15.6 (13.7 – 19.2) mg/L, respectively. All effect values are based on the nominal test item concentrations of N,N-Dimethylnonanamide.

Key value for chemical safety assessment

EC50 for freshwater algae:

28.8 mg/L

EC10 or NOEC for freshwater algae:

16.3 mg/L