2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

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 2016-11-28 to 2016-12-01, with the definitive exposure phase from 2016-11-29 to 2016 12-01.

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 substance:
All tested loading levels and the control were analytically verified via HPLC-DAD at the start (0 h) and at the end of the exposure period (48 h). 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 (0 h), sampling of the fresh media was carried out after preparation of the test substance concentrations. At the end of the exposure (48 h), samples of the old media were taken from additional replicates, which were prepared with test media, but without daphnids and incubated under test conditions until sampling.

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

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution:
The saturated solution (100 mg/L test substance was weighed out) was prepared with dilution water one day before the start of the exposure (at -24 h).The test substance was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 h at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 10 min to allow adsorption and saturation of the filter material with dissolved test substance. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water. During filtration, the filter was always kept covered.
The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was observed. Thus, no undissolved test substance was present.

Test loadings:
5 loading levels of the test substance in a geometric series with a separation factor of 2, prepared by dilution of the saturated solution with dilution water, were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, which corresponds to nominal loading level of the test substance of 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. The test substance loadings were selected based on the results of a non-GLP preliminary range finding test.

Control:
Dilution water without test substance 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 selection of 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 h illumination; light intensity of max. 1500 lux

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 > 10E+6 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:

see any other information on material and methods incl. tables

Test temperature:

see any other information on material and methods incl. tables

pH:

see any other information on material and methods incl. tables

Dissolved oxygen:

see any other information on material and methods incl. tables

Conductivity:

see any other information on material and methods incl. tables

Details on test conditions:

Test method:
The study was performed with a static test design.

Test duration:
48 h

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’)

Dilution water:
Same as culture medium

Number of daphnids and replicates:
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading level and the control.

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

Acclimatization:
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application:
20 g test solution per replicate was 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 by a pipette.

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

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

Photoperiod (target):
16/8 h light/dark cycle

Feeding:
The daphnids were not fed during the study.

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

22.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 15.5 - 24.8

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

18.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 16.3 - 20.5

Results with reference substance (positive control):

The percentage of immobility for the reference substance potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 h.

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 1.01 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 0.767 - 1.24 mg/L

Reported statistics and error estimates:

Methods of evaluation:
The EL100-values (after 24 and 48 h) were empirically derived from the observed immobilization rates. The effect levels (EL10 / 50 / 100) given are based on the nominal loadings of the test substace, since the main component of the test substance could not be detected and presence of the test substance is indicated by the signal of the impurities. The loading-effect relationships of the test substance after 24 and 48 h are shown graphically.

EC- / EL-values and statistical analyses:
The EL10- and the EL50-values after 24 and 48 h for the test substance were calculated 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. The loading-effect relationships are shown graphically. The EC50-value for the reference substance and its 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

Immobilization rates after 24 and 48 h of exposure in the definitive test:

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

Disperse Orange 30   Nominal loading level of the test substance [mg/L]	IMMOBILIZATION [%]
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100*	100	100	100	100	100	100% mortality after 24 h
50.0	100	80	100	100	95	100	100	100	100	100
25.0	40	60	100	60	65	80	80	100	100	90
12.5	0	0	0	0	0	0	0	20	0	5
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

*    = saturated solution

 Absolute numbers of immobile Daphnids after 24 and 48 h of exposure in the definitive test

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

Disperse Orange 30   Nominal loading level of the test substance [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
50.0	5 / 5	4 / 5	5 / 5	5 / 5	19 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
25.0	2 / 5	3 / 5	5 / 5	3 / 5	13 / 20	4 / 5	4 / 5	5 / 5	5 / 5	18 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	1 / 5	0 / 5	1 / 20
6.25	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

*    = saturated solution

 

 Additional Observations during the Definitive Test

 

All tested concentration levels were visually clear and showed a loading related orange colour throughout the exposure period.

The highest loading level was orange and the lowest concentration was light orange.

 

 

 Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test substance Disperse Orange 30 were analytically verified via HPLC-DAD at the start (0 h) and at the end of the

exposure (48 h) in all concentration levels and in the control. The measured concentrations of the main component of the test substance

were below the lowest calibration level in all loading levels at the start of the exposure (0 h) and also at the end of the exposure

(48 h). Instead of the main component a signal with a different retention time was observed. As a result of adequate investigations,

this signal was considered to originate from impurities of the test substance. Thus, the test substance was present in the test solutions.

 

 

 Water Quality Parameters

 

The measured water quality parameters(i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature)

were within the acceptable limits.

 Water Quality Parameters at the Start of the Exposure (0 h)

(measured in one additional replicate (without daphnids) per loading level and control)

Nominal loading level of the test substance [mg/L]		pH-value		Dissolved O2concentration [mg/L]
100		7.94		5.37
50.0		8.02		8.07
25.0		8.06		8.76
12.5		8.07		9.14
6.25		7.95		9.30
Control		7.71		9.42

 

 

Water Quality Parameters at the End of the Exposure (48 h)

(measured in all replicates containing daphnids)

Nominal loading level of the test substance [mg/L]		pH-values					Dissolved O2concentration [mg/L]
		Replicates					Replicates
		1		2		3		4		1		2		3		4
100		7.56		7.58		7.55		7.56		6.75		6.36		6.92		6.73
50.0		7.60		7.61		7.63		7.58		6.95		6.76		7.18		6.37
25.0		7.63		7.63		7.60		7.60		7.36		6.61		7.23		6.06
12.5		7.61		7.62		7.61		7.61		7.44		7.58		7.61		7.92
6.25		7.49		7.63		7.63		7.62		7.83		7.69		7.98		8.02
Control		7.27		7.31		7.30		7.30		8.09		8.01		7.99		8.10

 

 Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 h)

Dilution water dated:	pH-value	Dissolved O2concentration [mg/L]	Temperature   [°C]	Conductivity   [µS/cm]	Total hardness   [mg CaCO3/L]
2016-11-29	7.71	9.42	20.0	442	171

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the test conditions, the 48 h EL50 of the test substance for Daphnia magna was 18.6 mg/L (95% confidence limits: 16.3 - 20.5 mg/L).

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance in Daphnia magna according to OECD Guideline 202, in compliance with GLP. The study was conducted under static conditions over a period of 48 h with a saturated solution at a nominal loading of 100 mg/L of the test substance and further four dilution levels in a geometric series with a separation factor of 2 (6.25 to 50% of the saturated solution, corresponding to nominal loading levels of 6.25 to 50.0 mg/L of the test substance). All tested levels were visually clear and showed a loading related orange colour throughout the exposure period. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading level and the control. The concentrations of the test substance were analytically verified via HPLC-DAD at the start (0 h) and at the end of the exposure (48 h) in all concentration levels and in the control. The measured concentrations of the main component of the test substance were below the LOQ in all loading levels. Instead of the main component a signal with a different retention time was observed. As a result of adequate investigations, this signal was considered to originate from impurities of the test substance. For this reason, the effect levels given are based on the nominal loading levels of the test substance. The validity criteria of the test guideline were fulfilled. At 48 h, all daphnids at the loading levels 50 and 100 mg/L were found to be immobile. At 25 mg/L, 18 out of 20 daphnids were immobile. Under the test conditions, the 48 h nominal EL50 of the test substance was determined to be 18.6 mg/L (Klix, 2017).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

18.6 mg/L

Additional information

A study was conducted to determine the short-term toxicity of the test substance in Daphnia magna according to OECD Guideline 202, in compliance with GLP. The study was conducted under static conditions over a period of 48 h with a saturated solution at a nominal loading of 100 mg/L of the test substance and further four dilution levels in a geometric series with a separation factor of 2 (6.25 to 50% of the saturated solution, corresponding to nominal loading levels of 6.25 to 50.0 mg/L of the test substance). All tested levels were visually clear and showed a loading related orange colour throughout the exposure period. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each loading level and the control. The concentrations of the test substance were analytically verified via HPLC-DAD at the start (0 h) and at the end of the exposure (48 h) in all concentration levels and in the control. The measured concentrations of the main component of the test substance were below the LOQ in all loading levels. Instead of the main component a signal with a different retention time was observed. As a result of adequate investigations, this signal was considered to originate from impurities of the test substance. For this reason, the effect levels given are based on the nominal loading levels of the test substance.The validity criteria of the test guideline were fulfilled. At 48 h, all daphnids at the loading levels 50 and 100 mg/L were found to be immobile. At 25 mg/L, 18 out of 20 daphnids were immobile. Under the test conditions, the 48 h nominal EL50 of the test substance was determined to be 18.6 mg/L (Klix, 2017).