N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan - 28 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

EC Number:

263-218-7

EC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Cas Number:

61792-31-2

Molecular formula:

C17H36N2O2

IUPAC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: Single samples (2 mL) were taken after 0, 24 and 72 h from all test item concentrations and control; At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling
- Sample storage conditions before analysis: Samples were stored in the freezer (≤ -15°C) until the day of analysis. On the day of analysis, the test samples were thawed at room temperature. Afterwards, the samples were diluted in a 1:1 (v:v) ratio with methanol and analyzed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solution of 10 mg/L was prepared and 3 to 3¼ hours of magnetic stirring was applied to completely dissolve the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium.
- Method of cultivation: The pre-culture was maintained under the same conditions as used in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23 °C

pH:

8.2 - 8.5 (Control )
8.0 - 8.5 (Test item concentrations)

Nominal and measured concentrations:

0.10, 0.32, 1.0, 3.2 and 10 mg/L (nominal)
0.047, 0.14, 0.44, 2.0 and 11 mg/L (measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Initial cells density: 1 x 10E^04 cells/mL.
- Control end cells density:
- No. of vessels per concentration (replicates): 3 for each concentration
- No. of vessels per control (replicates): 6 replicates for the control

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water and with the following composition
NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO4 1.6 mg/L; FeCl3.6H2O 64 μg/L; Na2EDTA.2H2O 100 μg/L; H3BO3 185 μg/L; MnCl2.4H2O 415 μg/L; ZnCl2 3 μg/L; CoCl2.6H2O 1.5 μg/L; CuCl2.2H2O 0.01 μg/L; Na2MoO4.2H2O 7 μg/L; NaHCO3 50 mg/L;
- pH: 8.1 ± 0.2
- Culture medium different from test medium: yes
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Pre-cultre and Test medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water (see above).
- Intervals of water quality measurement: pH was measured at test start and test end; Temperature was measured continously; At the end of the final test microscopic observations were performed at all test concentrations to observe for any abnormal appearance of the algae compared to the control.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 97 to 103 μExm-2xs-1
- Other Incubation conditions: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber at 24-hour intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1
Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Percentage Inhibition of Growth Rate0.1 mg/L: 1%, 1.0 mg/L: 39%, 10 mg/L: 88% and 100 mg/L: >100%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits

Remarks:

0.24 - 0.34 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits

Remarks:

0.080 - 0.17

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.047 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: since the inhibition of growth rate was 1.4% at the lowest test item concentration of 0.047 mg/L the statistical significance might be a artefact. Therefore the EC10 was taken into account.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No, at the end of the test the algae cells at all test item concentrations appeared normal and healthy when compared to the control.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Multiple Sequentially rejective Welsh-t-test after Bonferroni-Holm correction, α=0.05, one-sided, smaller). Calculation of ErCx-values was based on logit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Analytical Results of the definitve test

The measured concentrations at the start of the test were 0.12, 0.37, 1.1, 3.4 and 12 mg/L at nominally 0.10, 0.32, 1.0, 3.2 and 10 mg/L, respectively. Only the highest concentration remained stable during the test period and was 115% of nominal at the end of the test. All lower concentrations decreased to 0.68-14% of nominal. No test item was detected in the control samples. Based on these results, the average exposure concentrations were calculated and used to express effect parameters.

Table 1 :Nominal, Measured and TWA Concentrations

Test item	Measured concentration (mg/L)
Nominal concentration (mg/L)	t=0h	t=24h	t=72 h	TWA (mg/L)
0.10	0.122 (122)	0.116 (116)	0.00099 (0.99)	0.047 (47)
0.32	0.372 (116)	0.366 (114)	0.0022 (0.68)	0.14 (44)
1.0	1.10 (110)	1.08 (108)	0.012 (1.2)	0.44 (44)
1.0*	1.13 (113)	1.15 (115)	0.0097 (0.97)	0.45 (45)
3.2	3.43 (107)	3.55 (111)	0.452 (14.1)	2.0 (63)
10	12.1 (121)	11.1 (111)	11.5 (115)	11 (114)

* incubated without algae

( ) % relative to nominal

Biological Results

Due to a low inhibition of 1.4% at 0.047 mg/L the EC10 was used as toxicological endpoint since effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant. According to the Guidance on Application of CLP criteria (Version 5.0, July 2017) the preference is given to EC10 to be used for classification and risk assessment. Following this strategy the EC10 and EC50 were used as toxicological endpoints.

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period

N-[3-(dimethyloxidoamino)propyl]-dodecanamide TWA concentration [mg/L]	Mean	Std. Dev.	n	%Inhibition
Control	1.756	0.0168	6
0.047	1.731	0.0146	3	1.4*
0.14	1.568	0.0052	3	11*
0.44	0.423	0.0503	3	76*
2.0	0.314	0.1114	3	82*
11	0.321	0.1278	3	82*

* Effect was statistically significant.

 

Table 3: Effect Parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	<0.047	0.13	0.18	0.29
	lower 95%-cl		0.080	0.12	0.24
	upper 95%-cl		0.17	0.22	0.34
Yield	Value	<0.047	0.073	0.094	0.15
	lower 95%-cl		0.059	0.077	0.12
	upper 95%-cl		0.090	0.12	0.20

cl – confidence limit

Table 4: Individual Cell Densities (x10E^04 cells/mL)

Time	Replicate	N-[3-(dimethyloxidoamino)propyl]-dodecanamide TWA conc. (mg/L)
		Control	0.047	0.14	0.44	2.0	11
0 h	1	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
n:		6	3	3	3	3	3
Mean:		1.0	1.0	1.0	1.0	1.0	1.0
Std.Dev.:		0.0	0.0	0.0	0.0	0.0	0.0
CV:		0.0	0.0	0.0	0.0	0.0	0.0
24 h	1	6.500	5.000	4.750	2.500	4.750	2.500
	2	5.250	4.250	4.250	1.500	4.000	1.500
	3	5.750	4.750	3.000	3.250	3.000	4.000
	4	4.250
	5	5.500
	6	4.500
n:		6	3	3	3	3	3
Mean:		5.3	4.7	4.0	2.4	3.9	2.7
Std.Dev.:		0.8	0.4	0.9	0.9	0.9	1.3
CV:		15.6	8.2	22.5	36.3	22.4	47.2
48 h	1	24.000	35.170	23.500	3.750	3.750	5.000
	2	27.000	31.000	23.750	5.000	3.250	4.250
	3	28.500	26.500	24.250	4.250	4.500	4.500
	4	25.500
	5	22.500
	6	27.750
n:		6	3	3	3	3	3
Mean:		25.9	30.9	23.8	4.3	3.8	4.6
Std.Dev.:		2.3	4.3	0.4	0.6	0.6	0.4
CV:		8.9	14.0	1.6	14.5	16.4	8.3
72 h	1	206.250	171.000	109.500	3.000	2.250	1.750
	2	189.750	184.500	112.500	4.000	2.000	2.750
	3	207.000	184.500	109.500	3.750	3.750	3.750
	4	189.000
	5	189.000
	6	183.750
n:		6	3	3	3	3	3
Mean:		194.1	180.0	110.5	3.6	2.7	2.8
Std.Dev.:		9.9	7.8	1.7	0.5	0.9	1.0
CV:		5.1	4.3	1.6	14.5	35.5	36.4

Table 5: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	194	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	0.96%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	16%	Yes

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

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