N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

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:

2016

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
The saturated solution and the control were analytically verified via LC-MS/MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the saturated solution. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels.

Quality criteria
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentration.

Vehicle:

no

Details on test solutions:

Test concentration
The effects of the saturated solution of the test item were tested in a limit test. The limit concentration is based on the results of a non-GLP preliminary range finding test.

Preparation of the saturated solution
A dispersion with a nominal concentration of 100 mg/L of the test item was prepared with dilution water (ISO test water as specified below) three days prior to the start of each exposure interval (at -72 and -48 hours).
100.3 mg test item (at -72 hours) and 100.2 mg test item (at -48 hours) were weighed out and transferred with 1000 mL of the dilution water into a brown glass flask, respectively. The dispersions were stirred with a magnetic stirrer for 72 hours with approximately 1100 rpm at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). This procedure was repeated for the renewal of the test media after 24 hours.

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:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Dilution water
dated: pH-values Dissolved O2 concentration [mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
0 hours: 2016-03-16 7.62 9.10 19.9 669 270
24 hours: 2016-03-17 7.75 8.73 20.3 680 270

Test temperature:

see above

pH:

Water Quality Parameters at the Start of the Exposure Intervals (0 and 24 hours)
(measured in one additional replicate of the saturated solution and the control)
Geometric mean
measured test item
concentration
[µg/L] 0 hours 24 hours
pH-values Dissolved
O2 concentration [mg/L] pH-values Dissolved
O2 concentration [mg/L]
13.1* 7.62 7.02 7.70 7.13
Control 7.62 9.10 7.75 8.73
* = saturated solution

Water Quality Parameters at the End of the Exposure Interval (24 hours)
(measured in all replicates of the saturated solution and the control)
Geometric mean
measured test item
concentration
[µg/L] pH-values Dissolved O2 concentration [mg/L]]
Replicates Replicates
1 2 3 4 1 2 3 4
13.1* 7.40 7.44 7.49 7.51 8.10 8.47 8.52 8.56
Control 7.37 7.41 7.47 7.50 8.70 8.57 8.65 8.71
* = saturated solution

Water Quality Parameters at the End of the Exposure Interval (48 hours)
(measured in all replicates of the saturated solution and the control)
Geometric mean
measured test item
concentration
[µg/L] pH-values Dissolved O2 concentration [mg/L]]
Replicates Replicates
1 2 3 4 1 2 3 4
13.1* 7.58 7.58 7.59 7.60 8.61 8.61 8.66 8.56
Control 7.59 7.58 7.56 7.56 8.55 8.55 8.55 8.56
* = saturated solution

Dissolved oxygen:

see above

Conductivity:

see above

Details on test conditions:

Test method
The study was performed with a semi-static test design (with a renewal of the test solutions after 24 hours), since the measured concentration of the test item in the preliminary test decreased markedly within 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 in each test replicate (see ‘Application’)

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’).

Dilution water
ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

 
Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for the saturated solution and the control.

Age of the daphnids at the start of the exposure
2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase in the dilution water of 2 hours. No first brood progeny was used for the test.

Acclimatisation
At least 2 hours in dilution water

Application
20 g test solution per replicate were weighed out into the test vessels. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

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

Illumination (target)
Diffuse light, light intensity of max. 20 µEm-2  s-1 (max. 1340 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:

other: EC 10/50/100

Effect conc.:

> 13.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC 10/50/100

Effect conc.:

> 13.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The saturated solution was visually clear throughout the exposure period.

Results with reference substance (positive control):

The percentage of immobility for the reference item was determined after 24 hours. The EC50-value with 95% confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.95 mg/L (Cl: 1.14 - 3.30 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the required concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

Methods of evaluation
The effects of the saturated solution concentration were deduced directly from the observed immobilisation rates. All effect levels (EC10 / 50 / 100) given were based on the geometric mean measured concentration of the test item.

EC-values and statistica analyses for reference item
An EC50-value was calculated only for the reference item by sigmoidal dose-response regression. 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.

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

The percentage of immobility, determined in all test and control groups after 24 and 48 h under semi-static conditions, is given in the Table.

 

Immobilisation Ratesafter 24 and 48 h of Exposure in the Definitive Test

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

Geometric mean measured concentration of the test item [µg/L]	IMMOBILISATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
13.1*	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

*    = saturated solution

 

The saturated solution was visually clear throughout the exposure period.

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in the saturated solution and the control. Details of the analytical method are presented in section 12.

The measured concentrations were in the range of 6.19 to 22.1 µg/L at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours).

 

 Measured Concentrations of the Test Item during the Definitive Test

Sampling date:	2016-03-16 0 hours Start of the exposure interval	2016-03-17 24 hours End of the exposure interval		2016-03-17 24 hours Start of the exposure interval	2016-03-18 48 hours End of the exposure interval
Start of analysis:	2016-03-16	2016-03-17		2016-03-17	2016-03-18
Nominal test item concentration   [mg/L]	Test item						Geometric mean measured concentration [µg/L]
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
100*	10.5	6.10	58	20.6	22.1	107	13.1
Control	< LOQ	< LOQ		< LOQ	< LOQ

*                       = saturated solution

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

%                     = percent of the initially measured concentration of the test item

LOQ                = limit of quantification of the analytical method (3.00 µg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

The saturated solution of the test item, which corresponds to a geometric mean measured concentration of 13.1 µg/L, induced no effects on Daphnia magna. Thus, the test item is not toxic at the level of the water solubility.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of a saturated solutionof the test item were determined according to OECD 202 (2004) from 2016-03-13 to 2016-03-18, with the definitive exposure phase from 2016-03-16 to 2016-03-18 at the test facility.

The limit test was conducted under semi-static conditions over a period of 48 hours. The test item is a yellowish, highly viscous liquid. A saturated solution with a nominal concentration of 100 mg/L of the test item was prepared. The saturated solution was visually clear during both exposure intervals.

Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the saturated solution and the control.

The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in the saturated solution and the control.

The measured concentrations were in the range of 6.19 to 22.1 µg/L at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours).

The effect levels given are based on the geometric mean measured concentration of the test item.

The validity criteria of the test guideline were fulfilled.

 

EC_{10 / 50 / 100}-Values (based on the geometric mean measured concentration of the test item)

Effect values	Test duration [h]	Geometric mean measured test item concentration [µg/L]
EC10 / 50 / 100	24	> 13.1
	48	> 13.1

Description of key information

The saturated solution of the test item, which corresponds to a geometric mean measured concentration of 13.1 µg/L, induced no effects on Daphnia magna. Thus, the test item is not toxic at the level of the water solubility.