N-[3-(dimethylamino)propyl]stearamide

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:

2013-02-04 to 2013-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]stearamide

EC Number:

231-609-1

EC Name:

N-[3-(dimethylamino)propyl]stearamide

Cas Number:

7651-02-7

Molecular formula:

C23H48N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]octadecanamide

Test material form:

solid: pellets

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10.0 - 31.6 - 100 - 316 - 1000 µg/L
- Sampling method: All test item concentrations and the control were analytically verified via LC-MS/MS at the beginning and end of the test. Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with demineralised water. The pH-value of the demineralised water was adjusted to pH 2 ± 0.1 by adding 1 M HCl. The test item dispersion was stirred with a magnetic stirrer for 18.5 hours. The test concentrations were prepared from this stock solution by dilution with natural river water without further adjustment of the pH-value.
- Eluate: Water of the river Innerste was used. The river water was supplemented with 50 % of the OECD medium to enable a sufficient growth of the algae (total application volume 6.5 mL/L).

River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2012-09-17
Weather on Day of Sampling Sunny, slightly clouded; approx. 18 °C
Colour Yellowish to brownish, clear
pH 7.56
Conductivity [µS/cm] 898
Dissolved Oxygen [mg O2/L] 8.83
NPOC [mg C/L] 3.13
Ammonium-N [mg N/L] 0.05
Nitrate-N [mg N/L] 1.70
Total Nitrogen [mg N/L] 2.70
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] < 0.20
Suspended Matter [mg/L] 16.4
Total Hardness [mg CO3/L] 283
Storage conditions 15 ± 2°C

The natural river water was stored at 15 ± 2 °C and at - 18  2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity.

Composition of Dilution Water

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2

- Differential loading: 10.0 - 31.6 - 100 - 316 - 1000 µg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Test temperature:

min.: 21.0 max.: 22.5, mean value: 21.8 °C

pH:

Nominal concentration pH-value
Test item
[µg/L] Start; 0 hours End; 72 hours
1000 8.05 8.30
316 8.06 8.43
100 8.06 8.50
31.6 8.06 8.53
10.0 8.05 8.53
Control 8.04 8.54

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

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 2 - 5 x 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 687640 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  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  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: 60 - 120 µE x m-2 x s-1, mean value: 73.2



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.



TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 100 100
0.100 14 39
0.0100 -5 -17

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

140 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 321-427 µg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

71 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 63-79 µg/L

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
- 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):

- Results with reference substance valid? Yes

Actual Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. For growth rate the data were transformed (y = 10y) to achieve normally distributed data.

Any other information on results incl. tables

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 hours		24 hours	48 hours	72 hours
1000		1		4332		2844		3344
		2		4332				3992
		3		4332				3180
		Mean		4332		n.a.	n.a.	3505
316		1		4332		2608		5464
		2		4332		3995		6391
		3		4332		2567		8477
		Mean		4332		3057	n.a.	6777
100		1		4332		10879	24544	173608
		2		4332		9294	27998	237499
		3		4332		5671	18431	144155
		Mean		4332		8615	23658	185087
31.6		1		4332		16349	108802	780958
		2		4332		17761	128716	808005
		3		4332		14861	126798	840933
		Mean		4332		16324	121439	809965
10.0		1		4332		14078	113174	753345
		2		4332		11933	103456	738060
		3		4332		13084	101455	658775
		Mean		4332		13032	106028	716727
Control		1		4332		14968	108022	695099
		2		4332		16518	106827	658837
		3		4332		18220	111507	747306
		4		4332		14575	98065	637514
		5		4332		13531	100965	742180
		6		4332		13257	91322	644905
		Mean		4332		15178	102785	687640

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 item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1000	1		-0.086	100		-988	100
	2		-0.027	100		-340	100
	3		-0.103	100		-1152	100
	Mean	(+)	-0.0722	100	(+)	-827	100
316	1		0.077	95.4		1132	99.8
	2		0.130	92.3		2059	99.7
	3		0.224	86.8		4145	99.4
	Mean	(+)	0.144	91.5	(+)	2445	99.6
100	1		1.23	27.1		169276	75.2
	2		1.34	21.0		233167	65.9
	3		1.17	30.8		139823	79.5
	Mean	(+)	1.24	26.3	(+)	180755	73.6
31.6	1		1.73	-2.55		776626	-13.7
	2		1.74	-3.23		803673	-17.6
	3		1.76	-4.01		836601	-22.4
	Mean	(+)^#	1.74	-3.26	(+)^#	805633	-17.9
10.0	1		1.72	-1.84		749013	-9.62
	2		1.71	-1.44		733728	-7.38
	3		1.68	0.81		654443	4.22
	Mean	(-)	1.70	-0.82	(-)	712395	-4.26
Control	1		1.69			690767
	2		1.68			654505
	3		1.72			742974
	4		1.66			633182
	5		1.71			737848
	6		1.67			640573
	Mean		1.69			683308

negative inhibitions = increase of growth

^#) significance caused by increase of growth

Section-by-Section and Average Specific Growth Rates of 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	1.24	1.98	1.86	1.69	0.396	23.4	22.7
	2	1.34	1.87	1.82	1.68	0.292	17.5
	3	1.44	1.81	1.90	1.72	0.247	14.4
	4	1.21	1.91	1.87	1.66	0.391	23.5
	5	1.14	2.01	2.00	1.72	0.499	29.1
	6	1.12	1.93	1.96	1.67	0.476	28.5
				Mean	1.69
				SD ±	0.02
				CV [%]	1.38

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study N-[3-(dimethylamino)propyl]stearamide was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 µg/L. The LOEC-values for both inhibition of growth rate and yield after 72 hours were 100 µg/L. The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 140 (130 – 154) µg/L. The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 74.3 (68.1 – 82.0) µg/L.
All effect levels are given based on nominal concentrations of N-[3-(dimethylamino)propyl]stearamide.

Executive summary:

The toxicity of N-[3-(dimethylamino)propyl]stearamide (batch number: PA02309460) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-02-04 to 2013-02-13, with the definitive exposure phase from to The aim of the study was to assess the effects on growth rate and yield in natural river water over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4332 cells/mL. Based on a preliminary test, 5 concentration levels were tested in a geometrical series with a dilution factor of (nominal): 10.0 - 31.6 - 100 - 316 - 1000 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

N-[3-(dimethylamino)propyl]stearamide is a tertiary straight chain amine. The test item had low water solubility and sorbed to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item was difficult to test in artificial water (e.g. sorption to the test organism and walls of the test vessel). Natural river water contained particulate as well as dissolved organic carbon to which the test item could sorb partially preventing that the test item settled onto surfaces. The sorbed fraction of the test item was difficult to extract from the test system which normally led to low analytical recoveries. Nevertheless the test item was present in the test system and therefore available for exposure (dissolved in water and sorbed). Sorption of the test item to the glass ware of the test system was monitored as appropriate. Due to the properties of the test item nominal concentrations were used instead of measured ones.

All test concentrations of N-[3-(dimethylamino)propyl]stearamide and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 h) and the end of the exposure (72 h).
The measured concentrations of N-[3-(dimethylamino)propyl]stearamide at the start of the exposure were in the range of 95 % to 117 % of the nominal values. At the end of the exposure, they were in the range of  LOQ_M to 2 %. The adsorption samples were analytically verified by LC-MS/MS at the end of the exposure. Adsorption to the glass walls were in the range % for samples with algae and at 13 % for the sample without algae.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of N-[3-(dimethylamino)propyl]stearamide
(0-72 hours)

                   based on nominal test item concentrations [µg/L]

                               

	Growth Rate Inhibition
NOEC	31.6
LOEC	100
ErC10	70.8 (62.7 – 78.7)
ErC20	89.8 (82.9 – 96.7)
ErC50	140 (130 – 154)
	Yield Inhibition
NOEC	31.6
LOEC	100
EyC10	44.9 (40.8 – 49.0)
EyC20	50.9 (46.7 – 55.3)
EyC50	74.3 (68.1 – 82.0)