N-[3-(dimethylamino)propyl]stearamide

Long-term toxicity to fish

Administrative data

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-04 to 2012-12-21

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 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

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: 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L (dilution factor √10)
- Sampling method: Samples of test media including control were taken from alternating test replicates in 3 sampling intervals from freshly prepared and corresponding 24 h old test solutions.
- 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 2 M HCl. The test item dispersion was stirred with a magnetic stirrer for about 24 hours. The test media were prepared from this stock solution by dilution with natural river water without further adjustment of the pH-value. The test media were mixed with an ultraturrax (20 sec, 18000 rpm).
- Eluate: Dilution water
- Differential loading:0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L (dilution factor √10)
- Controls: 30 eggs in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbuundesamt, Schichauweg 58, 12307 Berlin).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning rectangular dishes (26 cm x 14 cm x 6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. After approx. 3 h the glass dishes were gently removed. About 300 eggs were taken and immediately distributed to the test solution and dilution water (for control).
- Subsequent handling of eggs: After 4 hours eggs were checked for fertilization. Under a stereo microscope every embryo was checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were discarded. Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Test conditions

Hardness:

Total hardness 326 mg CaO3/L (control), 199 mg CaCO3 (10 mg/L)

Test temperature:

Period of measurements 2012-12-05 to 2012-12-14
Minimum temperature [°C] 21.9
Maximum temperature [°C] 26.1
Mean temperature
± Standard deviation [°C] 25.2 ± 0.8

Water Temperature in the Test Media

Study day Test media Water temperature [°C]
Nominal test item concentration [mg/L]
Control 0.100 0.316 1.00 3.16 10.0
0 new 25.0 24.9 24.8 24.9 25.0 25.0
1 old 25.0 25.0 24.9 24.9 24.5 24.8
new 25.1 24.9 24.8 24.8 24.9 24.8
2 old 24.9 25.0 25.1 25.1 25.2 24.9
new 24.9 25.2 25.2 25.0 25.2 -
3 old 25.2 24.5 24.4 24.6 24.5 -
new 24.8 24.8 25.0 24.7 24.9 -
4 old 24.8 25.2 25.0 25.0 24.8 -
new 25.0 25.1 25.1 25.1 25.1 -
5 old 25.4 25.2 25.1 25.3 25.7 -
new 24.9 24.9 24.9 24.7 24.8 -
6 old 25.2 24.9 25.2 25.1 25.3 -
new 25.4 25.2 25.1 25.0 25.2 -
7 old 25.0 24.9 25.0 24.8 25.1 -
new 25.4 25.2 25.1 25.1 25.3 -
8 old 25.3 25.2 25.3 25.3 25.4 -
new 25.4 25.1 24.9 25.1 - -
9 old 25.0 24.9 24.9 24.7 - -
Mean 25.1 25.0 25.0 25.0 25.1 24.9
SD ± 0.21 0.19 0.20 0.21 0.32 0.10
Min. 24.8 24.5 24.4 24.6 24.5 24.8
Max. 25.4 25.2 25.3 25.3 25.7 25.0

pH:

pH Values in the Test Media

Study day Test media pH value
Nominal test item concentration [mg/L]
Control 0.100 0.316 1.00 3.16 10.0
0 new 8.24 8.27 8.27 8.16 7.88 7.20
1 old 8.08 8.29 8.36 8.38 8.35 8.20
new 7.76 7.88 7.97 7.94 7.71 7.23
2 old 8.30 8.43 8.44 8.46 8.41 8.11
new 8.15 8.18 8.20 8.15 8.01 -
3 old 7.81 8.09 8.13 8.26 8.30 -
new 7.82 7.95 7.79 7.78 7.58 -
4 old 8.44 8.27 8.39 8.42 8.41 -
new 8.03 8.02 7.95 7.93 7.90 -
5 old 7.99 8.22 8.27 8.23 8.26 -
new 8.27 8.30 8.32 8.32 8.25 -
6 old 7.88 8.27 8.40 8.44 8.42 -
new 8.22 8.26 8.29 8.24 8.01 -
7 old 8.09 8.22 8.26 8.30 8.26 -
new 7.83 7.84 7.91 7.90 7.80 -
8 old 7.96 8.19 8.32 8.40 8.27 -
new 7.90 8.02 8.10 8.16 - -
9 old 8.12 8.27 8.44 8.48 - -
Mean 8.05 8.17 8.21 8.22 8.11 7.69
SD ± 0.20 0.16 0.20 0.21 0.27 0.54
Min. 7.76 7.84 7.79 7.78 7.58 7.20
Max. 8.44 8.43 8.44 8.48 8.42 8.20

Dissolved oxygen:

Dissolved Oxygen in Percent Air Saturation Value

Study day Test media Dissolved Oxygen [%]
Nominal test item concentration [mg/L]
Control 0.100 0.316 1.00 3.16 10.0
0 new 100 100 100 100 100 100
1 old 100 99 99 98 98 100
new 100 100 100 100 100 100
2 old 91 97 96 99 93 75
new 88 89 93 91 93 -
3 old 83 95 82 94 90 -
new 100 100 100 100 97 -
4 old 90 95 89 92 91 -
new 96 100 99 99 100 -
5 old 100 100 99 100 98 -
new 94 97 95 99 100 -
6 old 98 100 99 99 96 -
new 98 100 100 100 100 -
7 old 100 99 100 100 100 -
new 100 100 99 100 96 -
8 old 100 99 100 100 98 -
new 99 100 97 99 - -
9 old 100 99 99 99 - -
Mean 97 98 97 98 97 94
SD ± 5 3 5 3 3 13
Min. 83 89 82 91 90 75
Max. 100 100 100 100 100 100

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dishes (inner diameter 13.5 cm, water height about
5 cm) were used. The volume of the test media in the dishes was about 500 mL.
- Aeration: Gentle aeration was provided. Semi-static conditions with daily renewal of the test media was performed.
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5


OTHER TEST CONDITIONS
- pH: 6 – 8.5 within a range of 0.5 °C
- Photoperiod: 16 h photoperiod daily
- Light intensity: 0.1 - 10 µmol photons • m-2 • s-1on water surface (diffuse light)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biological parameters Observations were made daily.

Hatched eggs The number of hatched eggs was determined daily until 5 days post-hatch.

Post hatch period Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the control group have hatched (day 4 of the study).

Mortality Criteria for mortality vary according to life stage:

For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, was checked daily. Dead eggs were discarded.

For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.

For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/or lack of reaction to mechanical stimulus.

Further effects
Abnormal appearance and behaviour were also recorded.The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour will also be recorded by visually inspecting each replicate.

Measurement of fish size Fish size was determined at the end of the exposure (post-hatch day 5) of the control and of two test groups where no significant mortality (0.316 mg/L) and mortality close to 50 % (1.00 mg/L) were observed, respectively.
The fish were euthanized in a Benzocaine solution and the total length of each fish was measured to the nearest 0.001 mm with a microscope camera and corresponding software (ImageFocus, EUROMEX BV).


Measurement of
dry body weight At the end of exposure (post-hatch day 5) the mean fish dry weight from pooled survivors per replicate were determined from the control and of two test groups where no significant mortality (0.316 mg/L) and mortality close to 50 % (1.00 mg/L) was observed. The fish were dried for at least 24 h at 60 °C. Dry biomass weight was measured to the nearest 0.001 mg



Chemical parameters

Water quality and Temperature, pH value and oxygen saturation were measured
light intensity daily in freshly prepared and old test media in one replicate per
measurements test concentration and the control, respectively. Temperature was recorded continuously in the dilution water by a datalogger. Total hardness was measured at the beginning of exposure from one replicate of the control and the highest test concentration, respectively. The light intensity on the surface of the test vessels was measured at start of the exposure and on study day 5.



VEHICLE CONTROL PERFORMED: no


Overall Survival and Mortality in the Preliminary Test
(30 eggs / concentration)

Nominal
test item concentration [mg/L] Survival [n/30] Post hatch survival Overall survival Overall mortality
Study day 3 Study day 6 Study day 7 Study day 8 [%] [%] [%] 10.0 6 1 --- --- n.a. 3 97
1.00 29 23 21 21 91 70 30
0.100 29 28 28 26 93 87 13
Control 28 27 26 25 93 83 17

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: abnormal behaviour, most sensitive endpoint

Effect concentrations 2

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Posthatch survival, Overall survival, Length, Weight

Details on results:

- Mortality/survival at embryo and larval stages:
The post hatch success in all control replicates met the guideline criteria. The post hatch success was calculated from the study day with the maximum hatch rate per test group (post hatch day 1: control, 0.100 – 1.00 mg/L; post hatch day 0: 3.16 mg/L) and the surviving larvae on post hatch day 5. The post hatch survival at the end of the study was 100 % in the control group and ranged from 0 to 100 % post hatch success in the tested concentration levels, respectively.
One way analysis of variance and DUNNETT’S test were carried out for post hatch survival at the end of the study. Statistically significant differences were found for the test concentrations of 1.00 – 10.0 mg/L when compared with the control.


- Overall mortality/survival:
verall survival at the end of the study was 97 % in the control group and ranged from 0 to 97 % in the tested concentration levels, respectively. One way analysis of variance and DUNNETT’S test were carried out for the results of overall survival on post hatch day 5 (end of the study). Statistically significant differences were found for the test concentrations of 1.00 – 10.0 mg/L when compared with the control.


- Days to hatch and numbers hatched:
Egg hatch began on study day 3 in the control and all tested concentrations and continued until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 93 %.
Statistical procedures (one way analysis of variance) of the test concentrations of 0.100 – 3.16 mg/L were applied for study days 3, 4 and 5 (post hatch days -1, 0 and 1). Statistically significant differences were found on post hatch days 0 and 1 for the concentration level of 3.16 mg/L. Determination of statistical differences of hatch in the highest test concentration (10.0 mg/L) was not applicable, since 100 % mortality occurred on study day 2.


- Data for length and weight of surviving fish:
The fry growth, expressed as length and dry weight, was determined on study day 9 (post hatch day 5).
One way analysis of variance and DUNNETT’S test were carried out on post hatch day 5 (end of the study) for the data of the control and the test concentrations of 0.316 and 1.00 mg/L. For the weight data a statistically significant difference was found for the test concentration of 1.00 mg/L. For the length data of the concentration of 1.00 mg/L a statistically significant difference was found.


- Type of and number with morphological abnormalities:
On study day 4 one fish larva in the test concentration of 3.16 mg/L showed scoliosis. This larva was removed on death on study day 5. From study day 6 to 9 quiescence marked by abnormally low activity or inactivity, as well as arresting unusually long on the ground was observed for further concentration levels. One way analysis of variance and DUNNETT’S test were carried out on post hatch day 5 for the observed effects of the concentration level of 0.316 and 1.00 mg/L on study 9. These effects were determined to be statistically significant different compared to the control.
Other tested concentrations and the control showed no morphological and behavioural effects.

Reported statistics and error estimates:

One way analysis of variance (ANOVA) and DUNNETT’S test was used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first. For the parameters hatch, post hatch survival and overall fry survival (mortality), growth (length and dry weight), the following statistical tests were conducted:
Hatching data of study days 3 to 5 (post hatch days -1 to 1) were analysed with ANOVA. Post hatch survival and overall survival data (mortality), dry weight and length data, of study day 9 (post hatch day 5) were analysed with ANOVA. Normality test failed for post hatch survival and overall survival data. No transformations were carried out with these data because the data sets were not estimated to follow a normal distribution. Behavioral effects (quiescence and arresting on the ground) observed on study day 9 were analysed with ANOVA and DUNNETT’S test. The statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The -value (acceptable probability of incorrectly concluding that there is a difference) was 0.05.

Any other information on results incl. tables

Egg Hatch / Hatching Time

Nominal test item concentration [mg/L]	Replicate	Egg hatch [%]
		Post hatch day -2 (Study day 2)	Post hatch day -1 (Study day 3)	Post hatch day 0 (Study day 4)	Post hatch day 1  (Study day 5)	Post hatch day 2   (Study day 6)
10.0	1	0	n.a.	n.a.	n.a.	n.a.
	2	0	n.a.	n.a.	n.a.	n.a.
	3	0	n.a.	n.a.	n.a.	n.a.
	Mean	0	n.a.	n.a.	n.a.	n.a.
3.16	1	0	30	30	30	n.a.
	2	0	40	60	60	n.a.
	3	0	40	40	40	n.a.
	Mean	0	37	+       43	+       43	n.a.
1.00	1	0	100	100	100	100
	2	0	40	80	90	90
	3	0	70	80	80	80
	Mean	0	70	87	90	90
0.316	1	0	90	90	100	100
	2	0	80	90	90	90
	3	0	70	90	90	90
	Mean	0	80	90	93	93
0.100	1	0	90	90	100	100
	2	0	90	100	100	100
	3	0	90	90	100	100
	Mean	0	90	93	100	100
Control	1	0	40	90	90	90
	2	0	100	100	100	100
	3	0	90	90	100	100
	Mean	0	77	93	97	97

Overall Survival and Mortality on Study Day 9 (Post Hatch Day 5)

Nominal test item concentration [mg/L]	Replicate	Vital larvae on post hatch day 5 (Study day 9) n/10	Overall survival [%]	Mortality [%]
10.0	1	0	0	100
	2	0	0	100
	3	0	0	100
	Mean	0	+        0	100
3.16	1	0	0	100
	2	0	0	100
	3	0	0	100
	Mean	0	+        0	100
1.00	1	9	90	10
	2	2	20	80
	3	4	40	60
	Mean	5	+       50	50
0.316	1	10	100	0
	2	9	90	10
	3	9	90	10
	Mean	9	93	7
0.100	1	9	90	10
	2	10	100	0
	3	10	100	0
	Mean	10	97	3
Control	1	9	90	10
	2	10	100	0
	3	10	100	0
	Mean	10	97	3

Fry Growth: Length and Dry Weight on Study Day 9 (Post Hatch Day 5)

Nominal test item concentration [mg/L]	Replicate	Number of fish larvae	Post hatch day 5 (Study termination)
			Mean length per fish larvae [mm]	Pooled dry weight [mg]	Mean dry weight per fish larva [mg]
1.00	1	9	3.808	0.364	0.0404
	2	2	3.722	0.254*	0.0423*
	3	4	3.873
	Mean		+      3.801	0.309	+        0.0414
0.316	1	10	3.928	0.466	0.0466
	2	9	3.965	0.406	0.0451
	3	9	3.947	0.404	0.0449
	Mean		3.947	0.425	0.0455
Control	1	9	4.071	0.412	0.0458
	2	10	4.030	0.470	0.0470
	3	10	4.033	0.442	0.0442
	Mean		4.045	0.441	0.0457

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

N-[3-(dimethylamino)propyl]stearamide caused significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions at the dosage levels of 0.316 to 10.0 mg/L (nominal test item concentration). The LC50 on study day 9 (post hatch day 5) was determined to be 0.999 (0.488 – 2.18) mg/L. The NOEC for the parameter hatch was 1.00 mg/L. The NOEC for the parameters post hatch and overall survival (mortality), growth (expressed as length and dry weight) was 0.316 mg/L.
Due to the properties of the test item, all effect levels were based on the nominal concentrations of N-[3-(dimethylamino)propyl]stearamide.

Executive summary:

The effects of the test item N-[3-(dimethylamino)propyl]stearamide to the embryo and sac-fry stages of the zebrafish (Danio rerio) were determined according to OECD Guideline 212 from 2012-12‑04 to 2012-12-21, with the definitive exposure phase from 2012-12-05 to 2012-12-14 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with natural river water and daily renewal of the test media was performed with the nominal test item concentrations of 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L (dilution factor √10).

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio rerio were exposed per test concentration and control (3 replicates with 10 eggs each), respectively.

On day four 93 % of the control larvae have hatched. Therefore, study day 4 was defined as post hatch day 0 (PHD 0).

Different toxic endpoints were determined: egg hatch, time to hatch, post hatch survival, overall fry survival and mortality, fry growth (expressed as length and weight), morphological and behavioural effects. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results.

N-[3-(dimethylamino)propyl]stearamide is a tertiary straight chain amine. The test item had a 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 (please refer to part 4.2.3.1). Due to the properties of the test item nominal concentrations were used instead of measured ones.

All concentrations of N-[3-(dimethylamino)propyl]stearamide and the control were analytically verified by LC-MS/MS at the start and the end of three exposure intervals. The measured concentrations of N-[3-(dimethylamino)propyl]stearamide were in the range of 103 to 134 % in freshly prepared solutions and 37 to 60 % in 24 h aged solutions for the first exposure interval, for the fifth exposure interval they were in the range of 60 to 73 % in freshly prepared solutions and LOQ_Mto 3 % in 24 h aged solutions and for the seventh exposure interval the measured concentration were in the range of 75 to 84 % in freshly prepared solutions and 1 to 4 % in 24 h aged solutions.

Adsorption to glass walls of the test vessels was determined exemplarily at the concentration levels of 3.16 and 0.316 µg/L. Adsorption to the glass walls was determined in 48 h aged solutions after the first exposure interval and was in the range of 3 to 16 % of the nominal test item concentration. After the fifth exposure interval adsorption to the glass walls was in the range of 1 to 3 % in 48 h aged solutions and after the seventh exposure interval adsorption to the glass walls was in the range of 2 to 110 % in 48 h aged solutions. Adsorption values are expressed in per cent calculated from the nominal concentrations.

NOEC, LOEC: Hatch, Fry Survival, Growth, Behaviour

                based on nominal test item concentrations [mg/L]

Parameter	NOEC	LOEC
Hatch	1.00	3.16
Post hatch survival	0.316	1.00
Overall survival	0.316	1.00
Length	0.316	1.00
Weight	0.316	1.00
Abnormal behaviour (Quiescence, Remaining unusually long at the bottom)	0.100	0.316

LC₅₀-Value with 95 % Confidence Interval on Study Day 9 (Post Hatch Day 5)

                based on nominal test item concentrations [mg/L]

LC50	0.999
95 % confidence interval	0.488 – 2.18