4,4'-benzylidenedianiline

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:

From 16th to 18th February 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13th 2004 version

Deviations:

yes

Remarks:

According to the study report, test vessels were aerated during the test.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-benzylidenedianiline

EC Number:

210-038-1

EC Name:

4,4'-benzylidenedianiline

Cas Number:

603-40-7

Molecular formula:

C19H18N2

IUPAC Name:

4-[(4-aminophenyl)(phenyl)methyl]aniline

Test material form:

solid

Details on test material:

Identification: DATIPH
Physical form: Solid
Solubility in water: < 1 g/L
Colour: yellow brown
CAS no.: 603-40-7
Chemical name: 4,4’-Benzylidenedianiline
Molecular formula: C19H18N2
Test substance storage: At room temperature protected from light
Stability: Stable under storage conditions
Expiry date: 31 October 2010

Specific details on test material used for the study:

Identification: DATIPH
Chemical name: 4,4’-Benzylidenedianiline
Physical form: solid
Colour: yellow brown
Solubility in water: <1 g/l (virtually insoluble)
CAS no.: 603-40-7
EINECS no.: 210-038-1
Molecular formula: C19H18N2

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The water accommodated fractions of DATIPH were prepared by adding the respective amounts of an acetonic stock solution to the empty test vessels. After complete evaporation of the solvent, the Daphnia medium was applied. The test solutions were moderately stirred overnight followed by filtration (glass fibre filter). The resulting water accommodated fractions were used in the test.
About 2 h before the test, the vessels were filled with the formulated media to allow equilibration of pH, oxygen and temperature.

Test solutions

Vehicle:

yes

Remarks:

The vehicle is the formulated Daphnia medium (see water media type)

Details on test solutions:

Based on the data of a non-GLP range finding test using water accommodated fractions of DATIPH a definitive test with nominal concentration of DATIPH of 6.4, 16, 40, 100, and 250 mg/l was performed.
A spacing factor greater than the recommended maximum factor of 2.2 was selected because of the unusually flat concentration-response curve (presumably due to the limited water solubility).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism: Daphnia magna (Straus, 1820)
Breeding: Parental and young Daphnia will be held in 40 I glass aquaria at 20 ± 2 °C.
Illumination: 16 h per day
Medium: Continuously aerated Elendt M4 medium (Tables 1 and 2); prepared with deionised water (conductivity <1.5 ¿/S/cm).
Feed: 15 g sera micropan (sera GmbH, D-52525 Heinsberg) homogenized in 1 I deionised water
Frequency of feeding: Once a day, except weekends.
Control of sensitivity: Periodically conducted acute reference test with potassium dichromate.

Study design

Test type:

static

Water media type:

other: Elendt M4 medium

Remarks:

Separate stock solutions (I) of trace elements are first prepared in deionised water. From these different stock solutions (I) a second single stock solution (II) is prepared, which contains all trace elements (combined solution, see tables given below).

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Daphnia were acclimatised to the medium for about 2 h before introduction into the test media.

Post exposure observation period:

Not relevant.

Test conditions

Hardness:

As given by the composition of the prepared Elendt M4 medium.

Test temperature:

20 ±2 °C.

pH:

Between 7.1 and 7.9.

Dissolved oxygen:

Between 5.9 and 6.8 mg/L.

Salinity:

As given by the composition of the prepared Elendt M4 medium.

Conductivity:

< 1.5 µS/cm.

Nominal and measured concentrations:

Nominal concentrations (mg/L) Measured concentrations (mg/L)
6.4 2.22
6.4 2.22
16 3.07
16 3.03
40 5.00
40 5.00
100 5.50
100 5.53
250 11.8
250 11.9

Details on test conditions:

Test vessel: 100 ml flasks, all-glass, with 50 ml of test medium
Test medium: Continuously aerated Elendt M4 medium (Tables 1 and 2); prepared with deionised water (conductivity <1.5/uS/cm)
Number of Daphnia: 40 individuals per test concentration, 20 per vessel
Size: <2 mm
Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes
Temperature: 20 ±2 °C (temperature-controlled room)
Test type: Static exposure conditions
Test duration: 48 hours

Daphnia, which were selected in size, were acclimatised to the medium for about 2 h before introduction into the test media. The pH was adjusted before the test, if necessary.
For each test series the following number of test flasks was set up:
• Test suspension (Tn); containing test medium, test substance, two replicate samples with 20 Daphnia each;
• Blank control (Bn); containing test medium, three replicate samples with 20 Daphnia each.

Reference substance (positive control):

yes

Remarks:

Periodically conducted acute reference test with potassium dichromate.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 11.7 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 6.76 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits for EC50 (mg/l): 5.75 - 8.32

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

ca. 5.26 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 11.7 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

ca. 2.27 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 12.4 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Measured concentrations in tested substance

Basis for effect:

mobility

Details on results:

EFFECTS ON MOBILITY:
After 24 h of incubation the following percentage of immobilization of the total of 40 individuals was observed at the respective nominal concentrations:
250 mg/l (45%). No significant effects (≤ 10% of a total of 40 individuals) were observed at 100, 16 and 6.4 mg/l and in the blank control. The 15% immobilization observed at 40 mg/l were considered to be an outlier.
After 48 h of incubation the following percentage of immobilization of the total of 40 individuals was observed at the respective nominal concentrations: 250 mg/l (82.5%), 100 mg/l (45%), 40 mg/l (25%), 16 mg/l (15%). No significant effects (≤ 10% of a total of 40 individuals) were observed at 6.4 mg/l and in the blank control. Immobilization at 48 h was increased as compared to the effects after 24 h.

DETERMINATION OF EC50, EC0 AND EC100:
Based on the immobilization data the median effect concentration (EC50) of DATIPH to Daphnia magna for 24 h was estimated to be >11.7 mg/l with respect to the time- weighted average of the measured concentrations. The 48 h EC50 was calculated to be 6.76 mg/l (95% confidence limits: 5.75 - 8.32 mg/l).
The experimentally determined no-effect concentration (NOEC, EC0) was 5.50 and 2.27 mg/l with respect to the time-weighted average of the measured concentrations after an exposure of 24 and 48 h.
100% immobility (EC10o) was not observed
The calculated or estimated data for EC0 and EC100 were:
EC0 for 24 h: 5.26 mg/l
EC100 for 24 h: >11.7 mg/l
EC0 for 48 h: 2.27mg/l
EC100 for 48 h: >12.4 mg/l
The coefficients (r^2) of the regression curves for the 24 and 48 h observations were 0.970 and 0.920, respectively.

Results with reference substance (positive control):

Not reported.

Reported statistics and error estimates:

95 % confidence limits for EC50 (mg/l): 5.75 - 8.32.

Any other information on results incl. tables

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours of exposure to the test material

Nominal concentration (mg/l)	Code	Number of individuals per test vessel	ImmobileDaphnia(No./vessel) 24 h	ImmobileDaphnia(No./vessel) 48 h	% immobileDaphniaafter 24 h of exposure	% immobileDaphniaafter 48 h of exposure
Control	A	20	0	1	0	5
0	B	20	0	0	0	0
	C	20	0	0	0	0
6.4	A	20	1	1	5	5
	B	20	1	1	5	5
16	A	20	0	4	0	20
	B	20	0	2	0	10
40	A	20	1	4	5	20
	B	20	5	6	25	30
100	A	20	1	8	5	40
	B	20	0	10	0	50
250	A	20	8	17	40	85
	B	20	10	16	50	80

Table 2: pH and oxygen values concentrations at the start and at the end of the test

Nominal concentration (mg/l)	Code	pH-values 0h	pH-values 48h	Oxygen concentration (mg/l),0h	Oxygen concentration (mg/l), 48 h
0 (control)	A B C	7.3	7.8	6.3	6.8
6.4	A B	7.3	7.8	6.0	6.4
16	A B	7.2	7.9	6.0	6.3
40	A B	7.1	7.8	6.1	6.0
100	A B	7.1	7.9	6.1	6.3
250	A B	7.1	7.9	5.9	6.0

Table 3: Analytically determined concentrations in the definitive test

Nominal concentration (mg/L)	Code	Test concentrations determined at different points in time after start of exposure (mg/L)
		Time (hours)			TWA
		0	24	48	24 hours	48 hours
6.4	A B	2.22 2.22	2.16 2.13	2.31 2.34	2.16 2.13	2.26 2.27
16	A B	3.07 3.03	3.00 3.01	3.19 3.23	3.00 3.01	3.13 3.16
40	A B	5.00 5.00	4.96 4.94	5.31 5.20	4.96 4.94	5.19 5.11
100	A B	5.50 5.53	5.55 5.45	5.93 5.81	5.55 5.45	5.80 5.69
2.5	A B	11.8 11.9	11.7 11.8	12.7 12.7	11.7 11.8	12.3 12.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Not more than 10% of the Daphnia in the control have been immobilized or trapped at the surface of the water. The dissolved oxygen concentration at the end of the test was >3 mg O2/l.

Conclusions:

Based on the immobilization data, the median effect concentration (EC50) of DATIPH to Daphnia magna for 24 h was estimated to be >11.7 mg/l with respect to the time-weighted average of the measured concentrations. The 48 h EC50 was calculated to be 6.76 mg/l (95% confidence limits: 5.75 - 8.32 mg/l).

Executive summary:

The median effective concentration (EC₅₀) of DATIPH (4,4'-Benzylidenedianiline) to Daphnia magna was investigated under static exposure conditions over a period of 48 h.

40 individual Daphnia divided in 2 test vessels were exposed to each concentration of the test substance.

The nominal concentrations of DATIPH, as selected by a preceding non-GLP range finding test, were 6.4, 16, 40, 100 and 250 mg/l, respectively.

Due to the limited water solubility of the test material, only the water accommodated fractions of DATIPH were tested.

After 24 h of incubation the following percentage of immobilization of the total of 40 individuals was observed at the respective nominal concentrations:

250 mg/l (45%). No significant effects (≤ 10% of a total of 40 individuals) were observed at 100, 16 and 6.4 mg/l and in the blank control. The 15% immobilization observed at 40 mg/l were considered to be an outlier.

After 48 h of incubation the following percentage of immobilization of the total of 40 individuals was observed at the respective nominal concentrations:

250 mg/l (82.5%), 100 mg/l (45%), 40 mg/l (25%), 16 mg/l (15%). No significant effects (<10% of a total of 40 individuals) were observed at 6.4 mg/l and in the blank control. Immobilization at 48 h was increased as compared to the effects after 24 h.

Based on the immobilization data the median effect concentration (EC₅₀) of DATIPH to Daphnia magna for 24 h was estimated to be >11.7 mg/l with respect to the time-weighted average of the measured concentrations. The 48 h EC₅₀was calculated to be 6.76 mg/l (95% confidence limits: 5.75 - 8.32 mg/l).

The experimentally determined no-effect concentration (NOEC, EC₀) was 5.50 and 2.27 mg/l with respect to the time-weighted average of the measured concentrations after an exposure of 24 and 48 h.