Nonanamide, N,N-dimethyl-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-02-01 to 2017-02-24, with the definitive exposure phase from 2017 02-22 to 2017-02-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 in fresh media at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours). Due to 100% mortality after 24 hours in all replicates of the three highest concentration levels 20.7 to 100 mg/L of the test item, the respective concentration levels were analyzed at 0 and 24 hours only.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytica monitoring
At the start of the exposure and at renewal (0 and 24 hours), samples were taken from the freshly prepared concentration levels and the control and analyzed.
At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

Range (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (10 mg/L of the test item was weighed out) was freshly prepared with dilution water (see Table 2) prior to the start of the exposure and at renewal (0 and 24 hours). The stock solution was mixed thoroughly by manual agitation.

Test concentrations
6 test item concentrations in a geometric series with a separation factor of 2.2, prepared by dilution of the stock solution 10 mg/L with dilution water were tested as follows: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100 mg/L
The test concentrations mentioned above were selected based on the results of a non-GLP preliminary range finding test and the first definitive test under GLP conditions.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

160 mg CaCO3/L start of exposure
163 mg CaCO3/L after 24 hours

Test temperature:

20.8°C start of exposure
20.7°C after 24 hours

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal (0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Nominal
test item
concentration
[mg/L] 0 hours 24 hours
pH-value Dissolved
O2 concentration [mg/L] pH-value Dissolved
O2 concentration [mg/L]
100 8.45 9.50 Not determined,
due to 100% mortality after 24 hours
45.5 8.40 8.46
20.7 8.44 8.63
9.39 8.40 8.64 8.04 9.47
4.27 8.39 8.71 7.96 9.42
1.94 8.37 9.61 7.81 9.10
Control 8.14 10.7 7.99 10.6
— = not determined, due to 100% mortality after 24 hours


Water Quality Parameters in old Media at Renewal (24 hours) (measured in all replicates containing daphnids)
Nominal
test item
concentration
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 7.71 7.77 7.74 7.74 8.01 7.86 8.02 7.73
45.5 7.68 7.74 7.73 7.74 8.19 8.13 8.26 7.93
20.7 7.69 7.72 7.72 7.72 8.46 8.25 8.42 8.18
9.39 7.63 7.66 7.68 7.71 8.65 8.47 8.53 8.56
4.27 7.51 7.51 7.60 7.62 8.59 8.64 8.66 8.53
1.94 7.38 7.47 7.54 7.54 8.69 8.59 8.66 8.65
Control 7.50 7.46 7.49 7.45 8.33 8.24 8.54 8.48

 
Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in all replicates containing daphnids)
Nominal
test item
concentration
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
100 Not determined, due to 100% mortality after 24 hours
45.5
20.7
9.39 7.44 7.48 7.47 7.50 8.64 8.35 8.50 8.56
4.27 7.41 7.42 7.46 7.45 8.30 8.36 8.62 8.66
1.94 7.36 7.39 7.42 7.39 8.75 8.58 8.76 8.78
Control 7.39 7.36 7.37 7.37 8.63 8.70 8.79 8.75

Dissolved oxygen:

see pH

Conductivity:

420 µS/cm after 0 hours
443 µS/cm after 24 hours

Details on test conditions:

Test method
The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours, since the measured concentrations were expected not be stable during an exposure period of 48 hours.

Test duration 48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same as culture medium.

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were used per concentration level and control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).


Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

9.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 7.01 - 17.0

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 1.94 - 7.37

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

11.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 9.55 - 17.8

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 7.34 - 13.0

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The tested concentration levels were visually clear throughout the exposure period.
Neither immobility nor any adverse effects were observed at the concentration level 1.94 mg/L and in the control.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 02-16 to 2017-02-17.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.00 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.80 - 2.28 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EC100-values (after 24 and 48 hours) were empirically derived from the observation data (lowest concentration level resulting in 100% immobilization).
All effect concentrations (EC10 / 50 / 100) given are based on the nominal test item concentrations, since the measured test item concentrations were stable within ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours are shown graphically.

EC-values and statistical statistical analyses
The EC10- and the EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression with the software GraphPad Prism5. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

 Biological Data of the Definitive Test

 

The percentage of immobility, determined in all concentration levels and in the control after 24 and 48 hours under semi-static conditions, is given inTable5. The absolute numbers of immobile daphnids is presented inTable6. Other adverse effects did not appear.

 

Immobilization Rates after 24 and 48 hours ofExposure during the Definitive Test

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

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Mean	1	2	3	4	Mean
100	100	100	100	100	100	100% mortality after 24 hours
45.5	100	100	100	100	100
20.7	100	100	100	100	100
9.39	0	20	0	0	5	60	80	20	20	45
4.27	20	0	0	0	5	60	20	0	0	20
1.94	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobileDaphnids after 24 and 48 h ofExposure in the Definitive Test

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

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
45.5	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
20.7	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
9.39	0 / 5	1 / 5	0 / 5	0 / 5	1 / 20	3 / 5	4 / 5	1 / 5	1 / 5	9 / 20
4.27	1 / 5	0 / 5	0 / 5	0 / 5	1 / 20	3 / 5	1 / 5	0 / 5	0 / 5	4 / 20
1.94	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

The concentration-effect relationships after 24 and 48 hours of exposure are graphically illustrated inFigures1and2. Since all measured test item concentrations were within±20% of the nominal concentrations, the effect concentrations presented inTable1were based on the nominal concentrations of the test item.

 

 

Additional Observations during the Definitive Test

 

The tested concentration levels were visually clear throughout the exposure period.

Neither immobility nor any adverse effects were observed at the concentration level 1.94 mg/L and in the control.

 Measured Exposure Concentrations during the Definitive Test

 

The exposure concentrations of the test itemN,N-Dimethylnonanamidewereanalytically verified via LC‑MS/MS in fresh media of all concentration levels and the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours aged media at renewal and at the end of the test (24 and 48 hours). Details of the analytical method are presented in section14.

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 103 to 111% of the nominal values and 99 to 112% in aged media at renewal and at the end of the test (24 and 48 hours).

The measured test item concentrations were all within±20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

 

 Measured Concentrations of the Test ItemN,N-Dimethylnonanamideduring the Definitive Test

Sampling date	2017-02-22 Start of the  exposure interval 0 hours		2017-02-23 End of the  exposure interval 24 hours		2017-02-23 Start of the  exposure interval 24 hours		2017-02-24 End of the  exposure interval 48 hours
Start of analysis	2017-02-22		2017-02-23		2017-02-23		2017-02-24
Nominal test item concentration [mg/L]	N,N-Dimethylnonanamide
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	103	103	103	103	Not analyzed, due to 100% mortality after 24 hours
45.5	47.3	104	46.1	101
20.7	21.9	106	21.8	105
9.39	10.2	108	10.1	107	10.4	111	9.61	102
4.27	4.47	105	4.71	110	4.62	108	4.24	99
1.94	2.15	111	2.16	111	2.12	109	2.17	112
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc.= measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percent of the nominal concentration of the test item

LOQ             = limit of quantification of theanalytical method(0.05 mg/L of the test item)

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item, the 48 hours-EC50 for Daphnia magna was 9.63 mg/L (95% confidence limits: 7.34 – 13.0 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item N,N‑Dimethylnonanamide were determined at the test facility according to OECD 202 (2004) from 2017-02-01 to 2017-02-24, with the definitive exposure phase from 2017‑02-22 to 2017-02-24.

The study was conducted under semi-static conditions over a period of 48 hours with six nominal concentrations of the test item N,N-Dimethylnonanamide in the range of 1.94 to 100 mg/L, prepared in a geometric series with a separation factor of 2.2.

A stock solution with a nominal concentration of 100 mg/L of the test item was freshly prepared with dilution water before the start of the exposure period and at renewal (0 and 24 hours) and was used as the highest concentration level and as the stock solution for the preparation of all further tested concentration levels.

The tested concentration levels were visually clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The exposure concentrations of the test item N,N-Dimethylnonanamide were analytically verified via LC‑MS/MS in fresh media of all concentration levels and the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours aged media at renewal and at the end of the test (24 and 48 hours). 

The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 103 to 111% of the nominal values and 99 to 112% in aged media at renewal and at the end of the test (24 and 48 hours).

The measured test item concentrations were all within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all effect concentrations are based on the nominal concentrations of the test item N,N-Dimethylnonanamide.

The validity criteria of the test guidelines were fulfilled.

 

EC₁₀-, EC₅₀- (with 95% Confidence Limits) and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

N,N-Dimethylnonanamide
Effect concentrations	Test duration [hours]	Nominal test item concentrations [mg/L]
EC10 (with 95% confidence limits)	24	9.89	(Cl: 7.01 – 17.0)
	48	3.88	(Cl: 1.94 – 7.37)
EC50 (with 95% confidence limits)	24	11.5	(Cl: 9.55 – 17.8)
	48	9.63	(Cl: 7.34 – 13.0)
EC100	24	20.7
	48	20.7

Description of key information

Based on the nominal concentrations of the test item, the 48 hours-EC50 for Daphnia magna was 9.63 mg/L (95% confidence limits: 7.34 – 13.0 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

9.63 mg/L