4,4'-ethylenedianiline

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

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:

4,4'-ethylenedianiline

EC Number:

210-716-7

EC Name:

4,4'-ethylenedianiline

Cas Number:

621-95-4

Molecular formula:

C14H16N2

IUPAC Name:

4-[2-(4-aminophenyl)ethyl]aniline

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): 4,4’-Ethylenedianiline (DABY)
- Molecular formula: C14H16N2
- Molecular weight: 212.30 g/mol
- Purity of main component: 98.7 % (w/w)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via
LC-MS/MS at the start (0 h) and at the end of the exposure (48 h).
At the start of the exposure (0 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure (48 h), samples were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Recoveries of the test item should be within ± 20 % of the nominal or initially measured concentrations.
The effect levels can be given based on the nominal concentrations, if this quality criteria is fulfilled.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution (750 µg/L of the test item were weighed out) was freshly prepared with dilution water before the start of the exposure.
The stock solution will be stirred with approximately 1100 rpm for 24 hours at room temperature.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding:
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. 20 µE x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: 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
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

258 mgCaCO3/L

Test temperature:

18 - 22°C, constant within ± 1°C

pH:

pH at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)

NNominal
test item
concentrations
[µg/L] pH-value

750 7.78
341 7.86
155 7.87
70.4 7.94
32.0 7.82
Control 7.87


pH at the End of the Exposure (48 h)
(measured in all replicates)

Nominal
test item
concentrations
[µg/L] pH-values
Replicates
1 2 3 4
750 7.58 7.59 7.57 7.55
341 7.53 7.58 7.56 7.59
155 7.59 7.57 7.58 7.59
70.4 7.65 7.62 7.62 7.59
32.0 7.56 7.64 7.63 7.66
Control 7.52 7.50 7.53 7.46

Dissolved oxygen:

O2-concentration at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)

NNominal
test item
concentrations
[µg/L]
Dissolved
O2-concentration
[mg/L]
750 8.75
341 8.93
155 9.03
70.4 9.03
32.0 9.03
Control 9.16



O2-concentration at the End of the Exposure (48 h)
(measured in all replicates)

Nominal
test item
concentrations
[µg/L] Dissolved O2 -concentration [mg/L]
Replicates
1 2 3 4
750 8.30 8.23 8.53 8.16
341 8.68 8.42 8.49 8.55
155 8.68 8.65 8.69 8.68
70.4 8.62 8.64 8.79 8.79
32.0 8.70 8.69 8.59 8.74
Control 8.57 8.60 8.43 8.64

Nominal and measured concentrations:

32.0 - 70.4 - 155 - 341 - 750 µg/L (factor 2.2)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 h):
pH-value DissolvedO2-concentration[mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
7.87 9.16 19.8 655 258

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0h) and test end (48h)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 h interval

TEST CONCENTRATIONS
- Test concentrations: 32.0 - 70.4 -155 - 341 - 750 µg/L (spacing factor 2.2 )

- Range finding study:
The most recent range finding test was conducted with three concentrations of the test item of 0.100, 1.00 and 10.0 mg/L. In the preliminary range finding test, two replicates per concentration level each with 10 daphnids were tested. The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure. The biological results are presented and the analytical results showed stable recoveries during the range finding test are presented in the tables below.

Immobilisation 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] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
10.0 60 60 60 90 80 85
1.00 100 70 85 100 100 100
0.100 0 0 0 20 40 35
Control 0 0 0 0 0 0

MV = mean value

Measured Concentrations of the Test Item 4,4’-Ethylenedianiline (DABY) during the non GLP Range Finding Test

Sampling date 2014-10-14 2014-10-16
0 h 48 h
Start of the exposure End of the exposure

Nominal test item concentration
[mg/L] 4,4’-Ethylenedianiline (DABY)
Meas. conc. Meas. conc
[mg/L] % [mg/L] %
10.0 10.1 101 9.91 99
1.00 1.02 102 0.992 99
0.100 0.111 111 0.106 106
Control < SYSQL < SYSQL

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

155 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 104 - 217

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

155 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 151 - 163

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

341 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 272 - 436

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

167 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 162 - 172

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 750 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

341 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: not applicable

Effect concentrations 7

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

155 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 8

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

341 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

See below

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 2.05 mg/L (CI 1.90 - 2.18 mg/L)

The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed 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.

Any other information on results incl. tables

Biological Data

The percentage immobility, determined in all test and control groups after 24 and 48 h under static conditions, is given in the table below.

 

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

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

4,4’-Ethylenedianiline(DABY)	IMMOBILISATION [%]
Nominal test item concentration [µg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
750	80	60	60	80	70	100	100	100	100	100
341	40	40	60	60	50	100	100	100	100	100
155	40	0	0	0	10	40	0	0	0	10
70.4	0	0	0	0	0	0	0	0	0	0
32.0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

MV = mean value

 

All test concentrations were visually clear throughout the exposure period.

Measured Exposure Concentrations during the Definitive Test

The concentrations of the test item4,4’-Ethylenedianiline (DABY) were analytically verified via LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (48 h) in all concentration levels and the control.

The measured concentrations of the test item at the start of the exposure (0 h) were in the range of 100 to 111 % of the nominal values. At the end of the exposure (48 h), the measured concentrations of the test item were in the range of 96 to 102 % of the nominal values.The analytical results are presented in the table below.

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 and Percent of the Nominal Concentrations of the Test Item
4,4’-Ethylenedianiline (DABY)during the Definitive Test

Sampling date	2014-12-09 Start of the exposure, 0 h		2014-12-11 End of the exposure, 48 h
Date of analysis	2014-12-09		2014-12-11*
Nominal test item concentration [µg/L]	4,4’-Ethylenedianiline (DABY)
	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
750	773	103	743	99
341	342	100	328	96
155	164	106	150	97
70.4	75.8	108	71.8	102
32.0	35.5	111	32.7	102
Control	< LOQ		< LOQ

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

%                     = percent of the nominal concentration of the test item

LOQ                = limit of quantification (5.00 µg test item/L)

*                       = reinjected on 2014-12-11

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item 4,4’-Ethylenedianiline (DABY), the 48 h-EC50 for Daphnia magna was 167 µg/L (95 % confidence limits: 162 – 172 µg/L).
The NOEC after 48 h was 155.0 µg/L. The LOEC after 48 h was 341 µg/L.

Executive summary:

In the acute immobilisation test with Daphnia magna(STRAUS), the effects of the test item 4,4’-Ethylenedianiline(DABY) were determined according to OECD 202 (2004) from 2014-12-08 to 2014-12-11, with the definitive exposure phase from 2014-12-09 to 2014-12-11.

The study was conducted under static conditions over a period of 48 h with5concentrations of the test item 4,4’-Ethylenedianiline(DABY) in the range o f32.0 - 750 µg/L, prepared with dilution water in a geometric series with a separation factor of 2.2.

The test item 4,4’-Ethylenedianiline(DABY)is a beige powder with a water solubility of 48 mg/L (20 °C).The tested solutions were visually clear throughout the exposure period.Twenty daphnids were exposed to each concentration leveland the control.

The concentrations of the test item 4,4’-Ethylenedianiline (DABY) were analytically verified via LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (48 h) in all concentration levels and the control.

The measured concentrations of the test item at the start of the exposure (0 h) were in the range of 100 to 111 % of the nominal values. At the end of the exposure (48 h), the measured concentrations of the test item were in the range of 96 to 102 % of the nominal values.The analytical results arepresented.

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. The effect levels given are based on the nominal concentrations of the test item4,4’-Ethylenedianiline (DABY).

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (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 the test item4,4’-Ethylenedianiline (DABY))

4,4’-Ethylenedianiline(DABY)
Effect levels	Test duration [h]	Nominal test item concentrations [µg/L]
EC10 (with 95 % confidence limits)	24	155	(Cl: 104 – 217)
	48	155	(Cl: 151 – 163)
EC50 (with 95 % confidence limits)	24	341	(Cl: 272 – 436)
	48	167	(Cl: 162 – 172)
EC100	24	> 750
	48	341
NOEC	48	155
LOEC	48	341