Benzenamine, 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(oxy)]bis-

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in compliance with EU Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. L383A, 1992) Part C, Method 3 "Algal Inhibition Test". The reported deviation from the protocol is not thought to have affected the validity or integrity of the study since the validity criteria for this type of study were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

On Day 3 of the definitive test, teh maximum temperature recorded for incubator was 24.6 °C, which exceeded the range stated in the protocol (21 to 24 °C).

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline

EC Number:

700-102-1

Cas Number:

105112-76-3

Molecular formula:

C24H20N2O2

IUPAC Name:

3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline

Details on test material:

- Name of test material (as cited in study report): 3,3-(4,4-Biphenylenedioxy)dianiline
- Physical state: Off-white powder
- Analytical purity: >99 %
- Lot/batch No.: 20070501
- Expiration date of the lot/batch: 30 April 2008
- Storage condition of test material: Room temperature
- Other: Date received 28 August 2007

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

No data

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (20 mg) was dissolved in dimethylformamide (DMF; 10 ml) to provide a primary stock solution at 2 mg/ml. This primary solution was serially diluted, with DMF, to produce two intermediate solutions at 20 and 200 μg/ml. An aliquot (200 μl) of the solvent stock at 20 μg/ml was added to sterile algal culture medium (total volume, 2 l) in a volumetric flask and the contents of the flask were shaken vigorously before use.
- Eluate:
- Differential loading:
- Controls: As an intermediate vehicle was used to facilitate the preparation of the test medium, an additional control group containing dimethylformamide and sterile algal culture medium (100 μl/l) was included in the study. The solvent control vessel was filled with dilution medium containing the same concentration of auxiliary substance as present in the test concentration. The solvent selected for use in this study was based on information provided by the Sponsor.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test medium was colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 22 January 2008.
- Age of inoculum (at test initiation):
- Method of cultivation:


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

no data

Test temperature:

22.8 to 23.1 °C

pH:

7.55 to 9.95

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal concentration was 2 microgram/L. Geometric mean of the measured concentration was 0.7 microgram/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): loosely plugged with a foam
- Material, size, headspace, fill volume: Conical glass flasks, 250 mL volume, filled with 100 mL of the inoculated test medium
- Aeration:
- Initial cells density:10000 cells/mL
- Control end cells density: 1248406 cells/mL (control) and 1119683 (solvent control)
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): six
Plus two additional controls, which contained the test substance but no algal cells


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water, which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm)
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: Continuous illumination
- Light intensity and quality: approximately 7202 to 7396 lux provided by six 30 W "cool white" 1 metre fluorescent tubes
- Salinity (for marine algae):


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution medium at each test concentration. The presence of any abnormal cells was also noted during screening of each flask.



TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: The range finding test employed nominal test concentrations of 0.1, 1.0 and 10 μg/l.
- Results used to determine the conditions for the definitive study: After 72
hours, no inhibition had occurred at 10 μg/l. Based on this result, the definitive (limit) test was conducted at a nominal 2 μg/l; this concentration intentionally slightly exceeded the limit of the solubility of the test substance (stated value, 1.7 μg/l) to ensure that the algal cells were exposed to the maximum attainable concentration.

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

cell number

Details on results:

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate. After 72 hours, no significant inhibition of algal growth had been observed.

The following values were derived from the data:
Area under the growth curve: EbC50 (72 h) : >0.70 μg/l
Average specific growth rate: ErC50 (0 - 72 h) : >0.70 μg/l

No observed effect concentration (NOEC) : 0.70 μg/l

The mean coefficient of variation (CoV) for daily growth rates in control and solvent cultures ranged between 1.4 and 7.4% during the definitive test and the CoV for the average specific growth rates of the control and solvent control cultures was 2.7 and 1.5%, respectively, during the 72-hour exposure period.

No microscopic abnormalities of the cells were detected.

Any other information on results incl. tables

Table 1: Measured concentrations of the test substance

Nominal exposure concentration* (microgram/L)	Measured concentration of test substance* (microgram/L)					Overall geometric mean
	0 hours	%N	72 hours	%N	%t0
Control	nd, nd	-	nd, nd	-	-	-
Solvent control	nd, nd	-	nd, nd	-	-	-
2	2.09, 1.85	99	nd, nd**	-	-	0.70**
2 A	-	-	nd	-	-	-

nd: none detected ( 0.25 microgram/L)

%N: measured concentration expressed as percentage of the nominal concentration (calculated using unrounded values but expressed to 3 significant figures)

%t0: measured concentration after 72 hours expressed as percentage of the starting concentration

A: culture medium incubated under test conditions without algal cells

*: 3,3-(4,4-biphenylenedioxy)dianiline

**: for calculation of the test results, the limit of detection (0.25 microgram/L) has been used as the mean measured concentration present at 72 hours

Table 2: Cell densities

Exposure groups (microgram/L)		Replicate number	Cell densities (cells/mL)
Nominal	Measured		24 hours	48 hours	72 hours
Control		R1	57583	273633	1309717
		R2	62117	274167	1283983
		R3	65550	277367	1103550
	nd	R4	60483	281667	1497550
		R5	58950	258367	1033683
		R6	58783	266933	1261950
		Mean	60578	272022	1248406
Solvent control		R1	60317	267633	1211650
		R2	59083	255867	1035517
		R3	60083	277367	1163317
	nd	R4	60483	279000	1168150
		R5	57050	263967	1013783
		R6	57817	258800	1125683
		Mean	59139	267106	1119683
2*		R1	55150	260400	1145117
		R2	58950	253800	1060350
		R3	57083	254300	1211283
	0.70**	R4	58717	264367	1045283
		R5	54417	237300	1008783
		R6	57717	265700	1029517
		Mean	57006	255978	1083389

*: 3,3-(4,4-biphenylenedioxy)dianiline

**: geometric mean measured concentration

Table3: Inhibition of growth

Parameter	Concentration	Sample size	Mean	% Inhibition	p
Area under curve to 72 hours	Control	6	22.4	-8.2	0.068
	Solvent control	6	20.7	0.0	-
	2 microgram/L	6	19.9	3.6	0.396
Growth rate to 72 hours	Control	6	0.066	-2.2	0.080
	Solvent control	6	0.065	0.0	-
	2 microgram/L	6	0.065	0.7	0.56
Rate0_24	Control	6	0.075	-1.3	0.279
	Solvent control	6	0.074	0.0	-
	2 microgram/L	6	0.072	2.1	0.096
Rate24_48	Control	6	0.062	0.3	0.810
	Solvent control	6	0.062	0.0	-
	2 microgram/L	6	0.062	0.4	0.780
Rate48_72	Control	6	0.063	-6.0	0.102
	Solvent control	6	0.059	0.0	-
	2 microgram/L	6	0.060	-0.7	0.844

p values are for the comparison with solvent control using the t-test

* p 0.05, ** p 0.01, *** p 0.001

Table 4) Environmental parameters

a) Temperature and pH

Exposure groups (microgram/L)		Temperature °C		pH
Nominal	Measured	0 hours	72 hours	0 hours	72 hours
Control	nd	22.7	23.0	7.52	9.30
Solvent control	nd	22.7	22.8	7.53	10.01
2*	0.70**	22.8	23.1	7.55	9.95

nd: none detected (0.25 microgram/L)
*: 3,3-(4,4-biphenylenedioxy)dianiline

**: geometric mean measured concentration

b) Light intensity

Incubator position	Light intensity (lux)
	Day 0	Day 1	Day 2	Day 3	Mean
Top, left	7230	6980	7370	7320	7225
Bottom, left	6720	6780	6570	6860	6733
Centre	7930	7640	7640	7840	7763
Top, right	7480	7360	7640	7610	7523
Bottom, right	7260	7250	7670	7350	7383
Mean	7234	7202	7378	7396
Range (%)	-8.3, +8.3	-5.9, +6.1	-11.0, +4.0	-7.2, +6.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

3,3-(4,4-biphenylenedioxy)dianiline was not acutely toxic to the alga Pseudokirchneriella subcapitata when tested at a starting concentration that approximately the limit of aqueous solubility of the test substance (1.7 μg/l).

After 72 hours of exposure to 3,3-(4,4-biphenylenedioxy)dianiline, neither the EbC50 nor the ErC50 could be determined but both must be >0.70 μg/l (mean measured concentration).

The “no observed effect concentration” (NOEC) was 0.70 μg/l (mean measured
concentration).

Executive summary:

The effect of 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions (Wilby et al. 2008c). The study was conducted in accordance with EU Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 3 “Algal Inhibition Test” and OECD Guideline for Testing of Chemicals No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, adopted 23 March 2006.

Six algal cultures, with an initial cell density of 1 x 104/mL, were exposed to 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline at a nominal concentration of 2 μg/L, which was slightly in excess of the limit of solubility of the test substance (stated value, 1.7 μg/L) to ensure that the algal cells were exposed to the maximum attainable concentration. To aid dissolution, the test substance was dissolved in an auxiliary solvent before its addition to the culture medium. To minimise losses by adsorption, test glassware was pre-exposed to the test substance before use in the definitive test. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 23.2 to 24.6°C for 72 hours. At the start of the test, the mean measured concentration of 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline in samples of test cultures was 1.97 μg/L. After 72 hours, the concentration in expired medium had decreased to below the limit of detection of the analytical method (0.25 μg/L), indicating that 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline was not stable under the conditions of the test. Based on a geometric mean, an overall mean measured concentration of 0.70 μg/L was calculated and this value has been used in test calculations. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate and the following values were derived from the data:

Area under the growth curve EbC50 (72 h): >0.70 μg/L

Average specific growth rate ErC50 (0-72 h): >0.70 μg/L

NOEC: ≥0.70 μg/L

The NOEC must be greater 0.7 μg/L. 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline was not acutely toxic to the alga Pseudokirchneriella subcapitata when tested at a starting concentration (1.97 μg/L) exceeding the aqueous solubility of the test substance (1.7 μg/L).