4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 21 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

EC Number:

235-985-8

EC Name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

Cas Number:

13080-86-9

Molecular formula:

C27H26N2O2

IUPAC Name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test item concentrations
- Sampling method: 2 mL samples were taken after 0, 24 and 72 h. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the highest test item concentration but without algae and samples for analysis were taken at the start, after 24 h of exposure and at the end of the test period.
- Sample storage conditions before analysis: The samples were analyzed on the day of sampling

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
- Controls: Yes
- Differnetial loading: No
- Eluate: No
- Evidence of undissolved material: No; All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): pre-culture was used (3 days old)
- Method of cultivation: pre-culture was maintained under the test conditions as used in the test

ACCLIMATION
- Culturing media and conditions: for stock cultures M1 medium was used; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (corresponding to 24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

8.0 - 8.4 (Control)
8.0 - 8.3 (Test item concentrations)

Nominal and measured concentrations:

control, 1.0, 10 and 100% of a SS prepared at a concentration of 100 mg/L corresponding to 0.62 µg/L (time weighted average, over a test period of 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass, 100 mL, headspace: 50 mL, fill volume: 50 mL
- Type: open (capped vessels)
- Material, size, headspace, fill volume:
- Initial cells density: 1 x 10E+04 cells/mL
- Control end cells density: 371 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 6 (for the highest test item concentration 100 mg/L) and 3 (for the other test item concentrations 1 and 10 mg/L).
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test for the control and the highest test concentration; Temperature was recorded continously in a seperate vessel;

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuously
- Light intensity and quality: 81 to 85 µE/m2/s with TLD-lamps

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Spectrophotometric measurement were used at 680 nm with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Other: At the end of the test, microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range-finding test: A range-finding test in limit-test design was performed. Since no biological effects were observed no main test was required.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.62 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

filtered solution

Basis for effect:

other: growth rate and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.62 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

filtered solution

Basis for effect:

other: growth rate and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.62 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

filtered solution

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72 h ErC50) was 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72 h ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (STUDENT-t test for Homogeneous Variances, α=0.05, one-sided, smaller). No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Individual Cell Densities (x10E+04 cells/mL)

Time	Replicate	4,4’-[isopropylidenebis (4,1-phenyleneoxy)]dianiline; % SS prep. at 100 mg/L
		Control	1.0	10	100
0 h	1	1	1	1	1
	2	1	1	1	1
	3	1	1	1	1
	4	1	--	--	1
	5	1	--	--	1
	6	1	--	--	1
n:		1	1	1	1
Mean:		0	0	0	0
Std.Dev.:		6	3	3	6
CV:		0	0	0	0
24 h	1	11.3	n.d.	n.d.	8.7
	2	10.2	n.d.	n.d.	9.5
	3	9	n.d.	n.d.	9.2
	4	9.3	--	--	11.3
	5	10.2	--	--	9.5
	6	11	--	--	10.8
n:		10.2	n.a.	n.a.	9.8
Mean:		0.91	n.a.	n.a.	1.02
Std.Dev.:		6	n.a.	n.a.	6
CV:		8.91	n.a.	n.a.	10.35
48 h	1	85.7	n.d.	n.d.	48
	2	62.3	n.d.	n.d.	55.7
	3	63.9	n.d.	n.d.	53.3
	4	61.4	--	--	71.2
	5	68.5	--	--	55
	6	69	--	--	70
n:		68.5	n.a.	n.a.	58.9
Mean:		9	n.a.	n.a.	9.49
Std.Dev.:		6	n.a.	n.a.	6
CV:		13.14	n.a.	n.a.	16.11
72 h	1	421.5	320.6	365	274.3
	2	344.5	313.1	333.7	325.4
	3	345	355.3	334.6	302.7
	4	356.6	--	--	370.1
	5	396.7	--	--	326.4
	6	359.5	--	--	362.4
n:		370.6	329.7	344.4	326.9
Mean:		31.38	22.53	17.85	35.98
Std.Dev.:		6	3	3	6
CV:		8.47	6.83	5.18	11.01

n.d.: not determined; n.a.: not applicable

Table 2: Growth Rate and Percentage Inhibition For The Total Test Period

Test item^1 % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.971	0.0275	6
1.0	1.932	0.0224	3	2.0^2
10	1.947	0.0170	3	1.2^2
100	1.928	0.0373	6	2.2*^#

¹4,4’-[isopropylidenebis(4,1-phenyleneoxy)]dianiline;²was statistically not compared to control;
* effect was statistically significant;^#effect was biologically not relevant

Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	317	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	16%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata.	1.4%	yes

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no significant inhibition of growth rate was recorded at any of the concentrations of 4,4’-[isopropylidenebis(4,1-phenyleneoxy)]dianiline tested.
The EC50 for growth rate inhibition (72 h ErC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.62 µg/L being considered the maximum soluble concentration of the test item in test medium. Also the ErC10 was determined to be > 0.62 µg/L. The 72 h NOEC for growth rate inhibition was >= 0.62 µg/L, based on biological relevance (since the measured effect was below 10% of inhibition).