[1,1'-Biphenyl]-2,2'-diol, 3,3'-bis(1,1-dimethylethyl)-5,5'-dimethoxy-

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 January 2009 and 22 January 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols/ Current OECD Guidelines and to GLP standards, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 19/08/08 Date of signature: 04/03/09

Test material

Test material information

Constituent 1

Reference substance name:

L17_Precursor

IUPAC Name:

L17_Precursor

Details on test material:

Sponsor's identification : L17_Precursor
Description : pale yellow powder
Batch number : DALA046312
Date received : 11 November 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
For the purpose of the definitive test, the test material was dispersed directly in water. The following test concentrations were assigned to the test: 10, 32, 100, 320, and 1000 mg/l.
Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 hours. All test vessels were shielded from the light during mixing. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320 and 1000 mg/l.
The control group was maintained under identical conditions but not exposed to the test material.

- Eluate:
Not applicable

- Differential loading:
Not applicable.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10 and 32 mg/l contained dark brown dispersions with few yellow particles of test material dispersed throughout. The test concentrations of 100, 320 and 1000 mg/l contained dark brown dispersions with particles of test material dispersed throughout.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
A mixed population of activated sewage sludge micro-organisms was obtained on 22 January 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

- Pretreatment:
Not applicable

- Initial biomass concentration:
The suspended solids concentration was equal to 3.8 g/l prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.

pH:

The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
The pH was maintained at 7.9 ± 0.1 throughout the study.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal Concentration of 10, 32, 100, 320, 1000 mg/l

Details on test conditions:

TEST SYSTEM
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described in Section 3.4.1. Finally a second control was prepared.

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.


TEST MEDIUM / WATER PARAMETERS
The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

OTHER TEST CONDITIONS
- Adjustment of pH:
pH not adjusted

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.

% inhibition = [ 1 – 2 RS / RC1 + RC2 ] x 100

where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line.

The EC15, EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the EC50 values for the reference material only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).

The No Observed Effect Concentration (NOEC) was taken as being the EC15 value.

The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
10 - 100 - 100, x 10, 32 - 320, x 10

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study
Not applicable

- Results used to determine the conditions for the definitive study:
For the purpose of the definitive test, the test material was dispersed directly in water. The following test concentrations were assigned to the test: 10, 32, 100, 320, and 1000 mg/l.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367)

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

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:

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 3%.

The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 mg O2/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentration of 10 mg/l contained a clear colourless water column with a few yellow particles of test material dispersed throughout. The test concentration of 32 mg/l contained a slightly cloudy water column with particles of test material dispersed throughout and on the surface. The test concentrations of 100, 320 and 1000 mg/l contained cloudy water colums with particles of test material dispersed throughout and on the surface.

Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10 and 32 mg/l contained dark brown dispersions with few yellow particles of test material dispersed throughout. The test concentrations of 100, 320 and 1000 mg/l contained dark brown dispersions with particles of test material dispersed throughout.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes

Please see Table 1 for results.

Reported statistics and error estimates:

None.

Any other information on results incl. tables

Table 1         Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	6.6	10	1.9	0.47	-
	R2	6.6	8	2.6	0.50	-
Test Material	10	6.2	9	1.8	0.49	[1]
	32	6.5	10	1.7	0.48	1
	100	6.4	9	2.0	0.49	[1]
	320	6.0	9	1.6	0.49	[1]
	1000	6.7	9	2.6	0.46	5
3,5-dichlorophenol	3.2	7.0	10	3.2	0.38	22
	10	7.8	10	5.8	0.20	59
	32	8.4	10	7.7	0.07	86

[Increase in respiration rate as compared to controls]

R₁– R₂= Replicates 1 to 2

Table 2              pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.8	7.9
	R2	7.8	7.9
Test Material	10	7.8	7.9
	32	7.8	7.9
	100	7.8	8.0
	320	7.8	8.0
	1000	7.8	7.9
3,5-dichlorophenol	3.2	7.8	8.0
	10	7.8	8.1
	32	7.8	8.1

 

R₁– R₂= Replicates 1 to 2

Table3              Observations on the Test Preparations Throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Material	10	Few yellow particles of test material visible in a colourless water column[*]	Dark brown dispersion, with a few yellow particles of test material visible dispersed throughout	Dark brown dispersion, with a few yellow particles of test material visible dispersed throughout
	32	Particles of test material visible dispersed throughout and on the surface of a slightly cloudy water column*	Dark brown dispersion, with a few yellow particles of test material visible dispersed throughout	Dark brown dispersion, with a few yellow particles of test material visible dispersed throughout
	100	Particles of test material visible dispersed throughout and on the surface of a cloudy water column *	Dark brown dispersion, with yellow particles of test material visible dispersed throughout	Dark brown dispersion, with yellow particles of test material visible dispersed throughout
	320	Particles of test material visible dispersed throughout and on the surface of a cloudy water column *	Dark brown dispersion, with yellow particles of test material visible dispersed throughout	Dark brown dispersion, with yellow particles of test material visible dispersed throughout
	1000	Particles of test material visible dispersed throughout and on the surface of a cloudy water column *	Dark brown dispersion, with yellow particles of test material visible dispersed throughout	Dark brown dispersion, with yellow particles of test material visible dispersed throughout
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

R₁– R₂= Replicates 1 to 2

[*]Observations prior to addition of activated sewage sludge and synthetic sewage

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Council Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC₅₀value of 8.2 mg/l, 95% confidence limits
6.5 - 10 mg/l.