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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in compliance with EC Directive 88/302 Method C.11, "Biodegradatin - Activated Sludge Respiration Inhibition Test". 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:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition 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: Date of receipt was 28 August 2007

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

DILUTION WATER
The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.

SYNTHETIC SEWAGE
Synthetic sewage feed for activated sludge was prepared by dissolving the following in one litre of RO water:
peptone - 16.0 g
meat extract - 11.0 g
urea - 3.0 g
sodium chloride - 0.7 g
calcium chloride dihydrate - 0.4 g
magnesium sulphate heptahydrate - 0.2 g
di-potassium hydrogen phosphate - 2.8 g

(3,5-DICHLOROPHENOL)
A concentrated solution of 3,5-DCP (500 mg/l) was prepared, for the test, by dissolving 0.5 g in 10 ml of 1N sodium hydroxide and diluting to approximately 30 ml with RO water.
Sulphuric acid (1N) was added to the point of incipient precipitation and the solution made up to a final volume of one litre with RO water. The pH of this solution was then measured.
Nominal concentrations of 3, 10 and 32 mg/l were prepared by dilution of this concentrated solution.

TEST METHODS
The results of a preliminary formulation trial showed that the test substance formed a dispersion in RO water. Therefore, at test initiation appropriate weights were established in one-litre test beakers, RO water (284 ml) was added and the mixtures then treated with ultrasound for fifteen minutes in order to form dispersions. The pH of each preparation was then determined; adjustment of the pH was unnecessary.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works, which treats predominantly domestic waste. In the laboratory, the samples were maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.

On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed.

The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated.

Synthetic sewage (50 ml/l) was added to the stock of activated sludge and this was aerated overnight.

On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/l by the addition of tap water. The pH and temperature of the sludge was also measured.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

no data

Test temperature:

Initial between 19.9 and 20.2 °C; final between 19.6 and 19.9 °C

pH:

Initial between 7.8 and 7.9; final between 8.3 and 8.4

Dissolved oxygen:

see table

Salinity:

no data

Nominal and measured concentrations:

10, 100 and 1000 mg/L

Details on test conditions:

Addition of synthetic sewage and microbial inoculum were made at fifteen-minute intervals as detailed in the schedule below:

The prepared mixtures were covered loosely with aluminium foil and aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory supply of oil-free compressed air (ca. one litre/minute). Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity, 270 ml). The rate of oxygen consumption was measured, over a period of approximately ten minutes or until the dissolved oxygen concentration fell below 2 mgO2/l, using a Yellow Springs Instruments (YSI) dissolved oxygen meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (3,5-DCP), 99.9 %

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Dissolved oxygen concentrations and measurement times are given in Table 1. Temperature, pH and measurements of respiration rate in the test are given in Table 2.
Measurements of the pH of the aqueous stock solution of the reference substance and of the activated sludge (4 g/l) before the start of the test are given below:

3,5-DCP stock solution: pH 7.9; activated slude: pH 8.0 (adjusted from pH 8.2)

The specific respiration rate of the control culture established at the end of the test (16.2 mgO2/g/h) was 102% of the rate of that established at the start (15.8 mgO2/g/h). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition.

3,3-(4,4-biphenylenedioxy)dianiline was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The respiration rate in one mixture containing the test substance at 1000 mg/l was decreased by 1%. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 1000 mg/l, the highest level tested.

Results with reference substance (positive control):

Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 11.3 mg/l (95% confidence limits, 8.4 - 15.0 mg/l).

Any other information on results incl. tables

Table 1: Dissolved oxygen concentrations and measurement times

Test mixture	Initial DO concentration in the culture (mg/L)	Initial measure DO concentration (mg/L)	Final measured DO concentration (mg/L)	Measurement time (minutes)
Control (1)	7.6	6.5	3.0	8.3
3,3-(4,4-biphenylenedioxy)dianiline (mg/L)
10	7.8	6.5	3.0	8.0
100	8.0	6.5	3.0	8.1
1000	7.5	6.5	3.0	7.5
1000	8.0	6.5	3.0	7.5
1000	8.0	6.5	3.0	8.3
3,5-DCP (mg/L)
3	7.9	6.5	3.4	8.9
10	8.0	7.7	5.0	10.8
32	8.8	8.6	7.7	10.0
Control (2)	8.2	6.5	3.0	8.1

DO: Dissolved Oxygen

Table 2: Temperature, pH and measurements of respiration rate

Test mixture	Temperature °C		pH		Specific respiration rate (mgO2/g/h)	% inhibition
	Initial	Final	Initial	Final
Control (1)	20.1	19.9	7.8	8.4	15.8	-
3,3-(4,4-biphenylenedioxy)dianiline (mg/L)
10	20.2	19.9	7.8	8.4	16.4	0
100	20.1	19.9	7.8	8.3	16.2	0
1000	20.1	19.9	7.9	8.4	17.5	0
1000	20.0	19.8	7.9	8.3	17.5	0
1000	19.9	19.8	7.9	8.3	15.8	1
3,5-DCP (mg/L)
3	20.1	19.8	7.9	8.4	13.1	18
10	20.0	19.7	7.9	8.4	9.4	41
32	20.0	19.6	7.9	8.4	3.4	79
Control (2)	20.1	19.8	7.9	8.3	16.2	-

The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may be observed in the respiration rate calculation if rounded values are used to calculate the data. This minor discrepancy is not considered to be significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The three-hour EC50 for 3,5-DCP (11.3 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/L), and that relating to the respiration rates in the control (variation not greater than 15 %) was also s

Conclusions:

3,3-(4,4-biphenylenedioxy)dianiline was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 1000 mg/l, the highest level tested.

Executive summary:

The effect of 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline on the respiration rate of activated sludge was assessed by the methods detailed in EU Directive 88/302, Method C.11, ‘Biodegradation - Activated Sludge Respiration Inhibition Test’ and OECD Test Guideline 209, ‘Activated Sludge, Respiration Inhibition test’ (Dickinson 2008).

Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 10, 100 and 1000 mg/L for three hours. Single mixtures were prepared at 10 and 100 mg/L, and the highest level was prepared in triplicate. Their rates of oxygen consumption were determined and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of the culture series. The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed at 3, 10 and 32 mg/L, as a positive control. The specific respiration rate of the control culture established at the end of the test series (16.2 mg O2/g/h) was 102% of the rate of that established at the start (15.8 mg O2/g/h). The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 11.3 mg/L (95% confidence limits, 8.4-15.0 mg/L). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition. 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline was considered to have had no biologically significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 1000 mg/L, the highest level tested.