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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

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 accordance with EU Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 1 “Acute Toxicity For Fish” and the OECD Guideline for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test” (1992). No deviations from the protocol were reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

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: Sample received 28 August 2007

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: On each occasion, 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 (2 ml) of the solvent stock at 20 μg/ml was added to diluent water (3 l) in a volumetric flask (5 l). The contents of the flask were shaken vigorously before being made up to volume (5 l) and poured into a test vessel (glass aquarium) containing diluent water (10 l). The volumetric was re-filled with water and this was poured into the test vessel to give a final volume of 20 litres.
- 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 dilution water (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 medium. 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
- 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 clear and colourless.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain:
- Source: The fish were supplied by a commercial fish farm in the UK and were reared at the farm from eggs that hatched in September 2007.
- Age at study initiation (mean and range, SD): at least four months
- Length at study initiation (length definition, mean, range and SD): 4.71 cm
- Weight at study initiation (mean and range, SD): 1.85 g
- Method of breeding:
- Feeding during test
- Food type:
- Amount: No food was given during the 24-hour period immediately before exposure o9r during the exposure period itself.
- Frequency:


ACCLIMATION
- Acclimation period: The stock of fish was obtained from the supplier on 21 November 2007 and they were held in an aerated supply of diluent water under flow-through conditions until use.
- Acclimation conditions (same as test or not): During the 14-day period immediately before the definitive test, temperatures remained within the range 14.9 to 15.1°C, pH values within the range 7.68 to 7.74, dissolved oxygen concentrations within the range 91 to 95% air saturation value (ASV) and total hardness within the range 170 to 180 mg/l as CaCO3.
- Type and amount of food: commercial fish food (TROUW (UK) Ltd; Nutra Fry 02); 1 % of the total wet-weight of fish in the holding tank.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No medication was given during the holding period, and mortalities were recorded as <7% during the previous 14 days before the definitive test, with less than 2.6% occurring during the preceding 7 days.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

180 mg/L as CaCO3

Test temperature:

13.8 to 15.2 °C

pH:

7.22 to 7.95

Dissolved oxygen:

86 to 99 % air saturation value

Salinity:

no data

Nominal and measured concentrations:

The nominal concentration during the limit test was 2.0 μg/L.

During the definitive test, the measured concentrations of 3,3(4,4-biphenylenedioxy)dianiline ranged from 1.52 μg/l to 1.79 μg/l in samples of freshly prepared medium; between 76 and 90% of the nominal value. After 24 hours, in samples of expired media, the achieved concentrations had decreased, with measured levels ranging from above the limit of detection of the analytical method (0.25 μg/l) to 0.52 μg/l; between ca. 13 and 27% of the nominal value and between 16 and 30% of the starting value. Based on a geometric mean, fish were exposed to an overall mean measured concentration of 0.77 μg/l and this value has been used in test calculations. The results indicate that the test substance was not stable under the conditions of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open glass aquarium
- Material, size, headspace, fill volume: glass, containing 20 litres of medium to a depth of 18.5 cm
- Aeration: supplementary aeration was provided via a narrow bore glass tube
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): daily batch renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: initial static loading of 0.93 g bodyweight/litre


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water, dechlorinated and softened by passage through a water purification system. It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply.
- Total organic carbon: 0.54 mg/L
- Particulate matter: 5 mg/L
- Metals: approximately 0.08 mg/L
- Pesticides: organochlorine pesticides < 0.0072 ug/L; polychlorinated biphenyls < 0.02 ug/L; organophosphorous pesticides < 0.0058 ug/L
- Chlorine: 0.01 mg/L
- Alkalinity:
- Ca/mg ratio: 13.84
- Conductivity: 300 uS/cm
- Culture medium different from test medium:
- Intervals of water quality measurement:


OTHER TEST CONDITIONS
- Adjustment of pH: 7.22 to 7.95
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity:


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to physical stimulation of the caudal peduncle.
In addition to observations on mortality at approximately 2, 4, 24, 48, 72 and 96 hours, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish.

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 concentrations of 0.1, 1.0 and 10 μg/l. After 96 hours, no mortality or significant sub-lethal effects on the fish were observed. 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 fish were exposed to the maximum attainable concentration.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.77 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mortality (fish)

Details on results:

No mortality or adverse treatment-related effects on the fish were exhibited during the study.
Based on these findings the following values have been estimated:
96-hour LC50 value : >0.77 μg/l
“No observed effect concentration” : 0.77 μg/l

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1: Measured concentrations of the test substances (microgram/L)

Nominal exposure concentration (ug/L)**	Measured concentration of test substance (ug/L)										Overall geometric mean
	0 hours (fresh)
	ug/L	%N	ug/L	%N	%ti	ug/L	%N	ug/L	%N	%ti
Control	nd	-	nd	-		nd	-	nd	-		nd
	nd	-	nd	-		nd	-	nd	-
Solvent control	nd	-	nd	-		nd	-	nd	-		nd
	nd	-	nd	-		nd	-	nd	-
2	1.72	86	0.51	26	30	1.52	76		13	16	0.77
	1.79	90	0.54	27		1.55	78

nd : none detected (0.25 microgram/L)

LOQ: limit of quantification (0.5 microgram/L)
%ti: mean measured concentration after 24 or 96 hours expressed as a percentage of the mean starting concentration (0 and 72 hours)

%N: mean measured concentration expressed as a percentage of the nominal concentration

*: measured levels were above the limit of detection but below the limit of quantification, so a concentration of 0.25 microgram/L has been used in test calculations.

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

Table 2: Environmental parameters

Exposure group		Temperature °C		pH		Dissolved oxygen % ASV		Total hardness mg/L as CaCO3
Nominal	Measured	Min	Max	Min	Max	Min	max	0 hours
Control	nd	13.7	15.1	7.14	7.75	84	99	162
Solvent control	nd	13.7	15.0	7.2	7.89	86	99	-
2 ug/L*	0.77**	13.8	15.2	7.22	7.95	86	99	168

ASV: air saturation value

nd: none detected ( 0.25 microgram/L)

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

**: geometric mean measured concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, 3,3-(4,4-biphenylenedioxy)dianiline was not found to be acutely toxic to rainbow trout at a mean measured concentration of 0.77 μg/l. Consequently, the 96-hour LC50 value for 3,3-(4,4-biphenylenedioxy)dianiline with rainbow trout could not be calculated but must be >0.77 μg/l, and the “no observed effect concentration” was 0.77 μg/l.

Executive summary:

A study was performed to assess the acute toxicity 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline to rainbow trout (Oncorhynchus mykiss) under semi-static conditions (Wilby et al. 2008a). 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 1 “Acute Toxicity For Fish” and the OECD Guideline for Testing of Chemicals No. 203 “Fish, Acute Toxicity Test” (1992).

A group of ten juvenile fish was exposed to 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline dispersed in water 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 fish were exposed to the maximum attainable concentration. To aid dissolution, the test substance was dissolved in an auxiliary solvent before its addition to the diluent water (dechlorinated tap water, hardness 180 mg/L as CaCO3). To minimise losses by adsorption, test vessels were pre-exposed to the test substance before use in the definitive test. Measured concentrations of 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline ranged between 1.52 and 1.79 μg/L in samples of freshly prepared media and decreased to between 0.250 and 0.538 μg/L in samples of expired (24-hour-old) media, giving an overall geometric mean measured level of 0.77 μg/L. These results indicate that the test substance was not stable under the conditions of the test. Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of exposure. No mortality or treatment-related effects on the fish were exhibited during the study. Based on these findings, the following values have been determined:

96-hour LC50 value: >0.77 μg/L;

No observed effect concentration (NOEC): ≥0.77 μg/L.

The NOEC must be greater than 0.77 μg/L. Under the conditions of the study, 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline was not found to be acutely toxic to Oncorhynchus mykiss when tested at a starting concentration (1.79 μg/L) that exceeded the limit of aqueous solubility of the test substance (1.7 μg/L).