N-[3-(dimethylamino)propyl]stearamide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-05 to 2013-02-08

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 202 (Daphnia sp. Acute Immobilisation 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: 62.5 - 125 - 250 - 500 - 1000 µg/L (dilution factor 2).
- Sampling method: All concentration levels and the control were analytically verified by LC-MS/MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h).
Samples at the start of the exposure interval (0 h) were taken immediately after preparation of the test concentrations.
Samples at the end of the exposure interval (24 h) were taken directly from the test vessels containing daphnids.
Additionally, the freshly prepared stock solutions of 100 mg/L in demineralised water (adjusted with 1 M HCl to pH 2 ± 0.2) were analysed at the start of the exposure intervals (0 and 24 h). Sorption to the walls of the test vessel was checked as specified below. For this purpose, additional replicates without daphnids were prepared and incubated under test conditions for quantification of sorption to the glassware. Due to the test item properties, the nominal concentrations were used for calculation of the effect values.
The sorption of the test item on glass was quantified. Additional replicates of the three highest concentration levels without daphnids were prepared. After sampling for the test media analysis (aqueous phase), the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure, the test vessels were carefully rinsed with demineralised water to remove the remaining test media.
- 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:The stock solutions (100 mg/L test item were weighed out) were prepared with demineralised adjusted with 1 M HCl to a pH of 2 ± 0.2 one day prior to the start of the exposure phase (0 h) and the water renewal (24 h). The stock solution was stirred with approximately 1100 rpm for 24 h at room temperature.
- Eluate: Natural river waterof the river Innerste
- Differential loading: 62.5 - 125 - 250 - 500 - 1000 µg/L (dilution factor 2).
- Controls: Natural river water without test item tested under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:
Dilution water Natural river wter of the river Innerste
River Innerste
Location Ruther Strasse, 31157 Sarstedt, Germany
latitude:52.24°, longitude: 9.84°
Sampling date 2012-08-02
Weather on day of sampling Sunny, approx. 25 °C
Colour Slightly yellowish , clear
pH 8.11
Conductivity [µS/cm] 997
Dissolved oxygen [mg O2/L] 8.82
DOC [mg C/L] 2.72
TOC [mg C/L] 3.01
Ammonium-N [mg NH4-N/L] 0.11
Nitrate-N [mg NO3-N/L] 2.3
Total nitrogen [mg N/L] 4.6
o-phosphate-p [mg P/L] < 0.20
Total phosphate [mg P/L] < 0.20
Suspended matter [mg/L] 11.4
Total hardness [mg CO3/L] 299
Storage conditions 15 ± 2°C

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 269 (mg CaCO3/L)
Dilution water after 24 h: 279 (mg CaCO3/L)

Test temperature:

18.0 °C (Water)

pH:

Water Quality Parameters at the Start of the 1st Exposure Interval (0 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[µg/L] pH-Value
1000 8.16
500 8.22
250 8.24
125 8.24
62.5 8.23
Control 8.27

Water Quality Parameters at the End of the 1st Exposure Interval (24 h)
(measured in all replicates)
Nominal concentration
of the test item
[µg/L] pH-Values
Replicates
1 2 3 4
1000 8.29 8.28 8.28 8.28
500 8.31 8.30 8.30 8.30
250 8.32 8.31 8.32 8.32
125 8.31 8.32 8.33 8.32
62.5 8.34 8.34 8.35 8.29
Control 8.30 8.33 8.34 8.33

Water Quality Parameters at the Start of the 2nd Exposure Interval (24 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[µg/L] pH-Value
1000 8.25
500 8.30
250 8.34
125 8.35
62.5 8.33
Control 8.31

Water Quality Parameters at the End of the 2nd Exposure Interval (48 h)
(measured in all replicates)
Nominal concentration
of the test item
[µg/L] pH-Values
Replicates
1 2 3 4
1000 8.21 8.24 8.24 8.23
500 8.24 8.24 8.25 8.21
250 8.28 8.30 8.27 8.27
125 8.30 8.32 8.30 8.31
62.5 8.30 8.31 8.32 8.30
Control 8.30 8.31 8.31 8.31

Dissolved oxygen:

Water Quality Parameters at the Start of the 1st Exposure Interval (0 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[µg/L] Dissolved O2-Concentration
1000 9.16
500 9.16
250 9.16
125 9.11
62.5 9.04
Control 9.37

Water Quality Parameters at the End of the 1st Exposure Interval (24 h)
(measured in all replicates)
Nominal concentration
of the test item
[µg/L] Dissolved O2-Concentration
Replicates
1 2 3 4
1000 7.96 7.94 7.77 8.01
500 7.94 8.14 8.08 8.11
250 8.35 8.36 8.36 8.37
125 8.41 8.46 8.48 8.46
62.5 8.47 8.46 8.45 8.50
Control 8.34 8.39 8.38 8.39

Water Quality Parameters at the Start of the 2nd Exposure Interval (24 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[µg/L] Dissolved O2-Concentration
1000 9.26
500 9.29
250 9.14
125 9.07
62.5 9.19
Control 9.05

Water Quality Parameters at the End of the 2nd Exposure Interval (48 h)
(measured in all replicates)
Nominal concentration
of the test item
[µg/L] Dissolved O2-Concentration
Replicates
1 2 3 4
1000 7.63 7.96 8.00 7.90
500 7.94 7.71 7.92 6.92
250 7.74 8.21 7.62 7.58
125 7.99 8.28 8.06 8.27
62.5 8.13 8.22 8.22 8.11
Control 8.18 8.00 8.05 7.92

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): semi-static test (with a renewal of the test solutions after 24 h)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start:947 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start, at water renewal after 24 h and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was determined in all groups after 24 h and 48 h.

TEST CONCENTRATIONS
- Range finding study:
Immobilization Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
10.0 100 100 100 100 100 100
1.00 30 40 35 100 100 100
0.100 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

381 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 321-427 µg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

249 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 62.5 - 345 µg/L

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.23 (CI 1.14 - 1.37) mg/L

Reported statistics and error estimates:

The EC10- and EC50-values after 24 h, the EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were estimated directly from the observation data, without mathematical calculation.
The EC10- and EC50-values after 48 h were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The concentration effect relationship after 48 h is shown graphically. The EC50-value for the reference item and its confidence limits were calculated accordingly.

Any other information on results incl. tables

Immobilization Rates after 24 and 48 h of Exposure in the Definitive Test

                 (n = 20, divided into 4 replicates with 5 daphnids each)

N-[3-(dimethylamino)propyl]stearamide	IMMOBILISATION [%]
Nominal test item concentration [µg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
1000	20	0	20	0	10	100	100	100	100	100
500	0	0	0	0	0	80	80	80	100	85
250	0	0	0	0	0	20	0	0	20	10
125	0	0	0	0	0	20	0	0	20	10
62.5	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item N-[3-(dimethylamino)propyl]stearamide the 48 h- EC50 for Daphnia magna was estimated to be 381 µg/L (95 % confidence limits: 321 – 427 µg/L).
The NOEC after 48 h was 250 µg/L. The LOEC after 48 h was 500 µg/L.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item N-[3-(dimethylamino)propyl]stearamide (batch number: PA02309460) were determined according to OECD 202 (2004) from 2013-02-05 to 2012-02-08, with the definitive exposure phase from 2013-02-06 to 2013-02-08, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under semi-static conditions over a period of 48 h with 5 concentrations of the test item N-[3-(dimethylamino)propyl]stearamide in the range of 62.5 to 1000 µg/L, prepared with natural river water in a geometric series with a separation factor of 2. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the test item N-[3-(dimethylamino)propyl]stearamide were analytically verified by LC-MS/MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control. Additionally, the freshly prepared stock solutions of 100 mg/L, prepared in demineralised water (adjusted with 1 M HCl to pH 2 ± 0.2), were analysed at the start of the exposure phase (0 h) and at the water renewal (24 h). N-[3-(dimethylamino)propyl]stearamide is a tertiary straight chain amine. The test item has a low water solubility and sorbs 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 is difficult to test in artificial water (e.g. sorption to the test organism and walls of the test vessel). Natural river water contains particulate as well as dissolved organic carbon to which the test item can sorb partially preventing that the test item settles onto surfaces. The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries. Nevertheless the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed). Due to the properties of the test item the nominal concentrations were used for assessment of the endpoints instead of measured ones.

The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 95 to 120 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 9 to 68 % of the nominal values. The recovery rates of the stock solutions of 100 mg/L were 109 % (0 h) and 105 % (24 h) of the nominal value.

Adsorption to the glass walls of the test vessels was determined exemplarily at the three highest concentration levels of 250, 500 and 1000 µg/L in separate replicates without daphnids. Adsorption to the glass walls in the range of 10 to 18 % of the nominal test item concentrations was determined at the concentration levels of 250 to 1000 µg/L.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀-Values, NOEC and LOEC

                   (based on the nominal concentrations of N-[3-(dimethylamino)propyl]stearamide)

Endpoints	Test duration [h]	N-[3-(dimethylamino)propyl]stearamide
		Nominal test item concentrations [µg/L]	95 % confidence limits [µg/L]
EC10	24	1000	Not applicable
	48	249	62.5 - 345
EC50	24	> 1000	Not applicable
	48	381	321 – 427
EC100	24	> 1000	Not applicable
	48	1000	Not applicable
NOEC	48	250	Not applicable
LOEC	48	500	Not applicable