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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 June 2009 and 17 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect 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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

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 beige coloured powder
Batch number : DALA046312
Date received : 11 November 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

EXAMPLE:
- Concentrations:
A volume of test sample had 30% (w/v) sodium chloride added and was then extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in dichloromethane to give a final theoretical concentration of 10 mg/l

- Sampling method:
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) for quantitative analysis.

- Sample storage conditions before analysis:
Samples were used immedialtely upon sampling. Duplicate samples were taken and stored at approximately -20°C for further analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

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

- Method:
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
An amount of test material (2100 mg) was added to the surface of 21 litres of dechlorinated tap water to give the 100 mg/l loading rate. After the addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Visual inspection of the WAF showed test material to still be present in the water column. Therefore the WAF was further filtered through Postlip filter paper. Microscopic observations were performed on the WAF after filtering and showed the WAF to be a clear, colourless solution with no undissolved test material or micro-dispersions to be present.
This method of preparation was conducted in duplicate to give replicates R1 and R2.


- Eluate:
Not applicable

- Differential loading:
Not applicable

- Controls:
Maintained under identical conditions but not exposed to to 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 on the test media were carried out during the mixing and testing of the WAFs.
At the start of each mixing period the 100 mg/l loading rate was observed to be a clear, colourless water column with test material floating at the surface and a small amount dispersed throughout. At end of each mixing period, and after the 1-Hour settlement period the 100 mg/l loading rate was observed a cloudy homogenous dispersion with some undissolved test material at the surface. Microscopic examination of the WAF showed visible test material and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Visual inspection of the WAF showed test material to still be present in the water column. The WAF was further filtered through Postlip filter paper. Microscopic observations were performed on the WAF after filtering and showed the WAF to be a clear, colourless solution with no undissolved test material or micro-dispersions to be present.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

EXAMPLE:
TEST ORGANISM
- Common name:
Rainbow Trout

- Strain:
Not stated

- Source:
Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK

- Age at study initiation (mean and range, SD):
Juvenile.

- Length at study initiation (length definition, mean, range and SD):
The fish had a mean standard length of 5.3 cm (sd = 0.1)

- Weight at study initiation (mean and range, SD):
A mean weight of 2.35 g (sd = 0.22)

- Method of breeding:
Not stated

- Feeding during test
Not fed during study.

ACCLIMATION
- Acclimation period:
Fish were acclimatised to test conditions from 17 September 2008 to 29 September 2008.

- Acclimation conditions (same as test or not):
Same as test conditions.

- Type and amount of food:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Feeding frequency:
Not stated

- Health during acclimation (any mortality observed):
There was less than 1% mortality in the 7 days prior to the start of the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Test conditions

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The water temperature was recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer.. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations.

Temperature was maintained at approximately 14 deg C throughout the test

Please see Appendix 5 for more details.

pH:

The pH was recorded daily throughout the test using a WTW pH/Oxi 340I pH.

pH range of 7.8 +/- 0.2.

Please see Appendix 5 for more details.

Dissolved oxygen:

The dissolved oxygen concentration was measured using a dissolved oxygen meter.

mg O2/l range of ~ 9.7 +/- 0.3

Please see Appendix 5 for more details.

Salinity:

Not applicable

Nominal and measured concentrations:

In the range-finding test fish were exposed to a nominal loading rate of 100 mg/l. The test material was prepared as a WAF. A single loading rate was used as results from the Acute Toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 1571/0096) indicated that toxicity was not expected at this concentration.

Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no mortalities or sub-lethal effects of exposure were observed.

Details on test conditions:

TEST SYSTEM
- Test vessel:
In the definitive test 20 litre glass exposure vessels were used for each test concentration.
The test vessels were then covered to reduce evaporation

- Aeration:
The test vessels were aerated via narrow bore glass tubes

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable

- Renewal rate of test solution (frequency/flow rate):
Daily renewal of the test preparations to ensure test concentrations of soluble components of the test material were maintained over the test and to prevent the build up of nitrogenous waste products.

- No. of organisms per vessel:
At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.

-No. of vessels per concentration (replicates):
Range finding study: 1
Definitive study: 2

- No. of vessels per control (replicates):
1

- No. of vessels per vehicle control (replicates):
Not applicable

- Biomass loading rate:
Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was 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.

-Total Organic Carbon Analysis:
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of Total Organic Carbon (TOC), as an indicator of soluble organic materials, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for TOC analysis.
The results are summarised as follows:

Nominal
Loading Rate
(mg/l) Time (Hours)
24 96
mg C/l mg C/l Corrected for Control mg C/l mg C/l Corrected for Control
Control 100 1.47 1.47 1.56 1.56

It is evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

- Particulate matter:
Not measured
- Metals:
Al <12.692 µg/l
Pb <1.138 µg/l
Fe <13.481 µg/l
Cd <0.111 µg/l
Cr <0.551 µg/l
Cu <0.408 mg/l
Ni <1.788 µg/l
Hg <0.012 µg/l
Mn <1.546 µg/l
- Pesticides:
0 µg/l
- Chlorine: Not Stated
- Alkalinity:
Not Stated
- Ca/mg ratio:
Not stated
- Conductivity:
421.385 µS/cm at 20°C
- Culture medium different from test medium:
No
- Intervals of water quality measurement:
Not Stated


OTHER TEST CONDITIONS
The test vessels were then sealed and maintained at approximately 14ºC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline:
Not applicable
- Range finding study
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test fish were exposed to a nominal loading rate of 100 mg/l. The test material was prepared as a WAF. A single loading rate was used as results from the Acute Toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 1571/0096) indicated that toxicity was not expected at this concentration.
An amount of test material (2100 mg) was added to the surface of 21 litres of dechlorinated tap water to give the 100 mg/l loading rate. After the addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Visual inspection of the WAF showed test material to still be present in the water column. Therefore the WAF was further filtered through Postlip filter paper. Microscopic observations were performed on the WAF after filtering and showed the WAF to be a clear, colourless solution.
In the range-finding test 3 fish were added to each test and control vessel containing 10 litres and maintained at approximately 14C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The control group was maintained under identical conditions but not exposed to the test material.
Data from the control group was shared with similar concurrent studies.
Each vessel was covered to reduce evaporation. After 3, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. The 6-Hour observations were not completed due to time restraints. Given that no mortlaities or sub-lethal effects were observed, the omission of this information has no impact on the study.


- Results used to determine the conditions for the definitive study:Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: * LL = Lethal Loading rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
None recorded.

- Observations on body length and weight:
None recorded

- Other biological observations:
The results of the definitive test showed the highest loading rate resulting in 0% mortality to be greater than or equal to 100 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.


- Mortality of control:
None recorded

- Other adverse effects control:
None recorded

- Abnormal responses:
There were no sub-lethal effects of exposure observed in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of each mixing period the 100 mg/l loading rate was observed to be a clear, colourless water column with test material floating at the surface and a small amount dispersed throughout. At end of each mixing period, and after the 1-Hour settlement period the 100 mg/l loading rate was observed a cloudy homogenous dispersion with some undissolved test material at the surface. Microscopic examination of the WAF showed visible test material and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Visual inspection of the WAF showed test material to still be present in the water column. The WAF was further filtered through Postlip filter paper. Microscopic observations were performed on the WAF after filtering and showed the WAF to be a clear, colourless solution with no undissolved test material or micro-dispersions to be present.

- Effect concentrations exceeding solubility of substance in test medium:
None recorded

Results with reference substance (positive control):

- Results with reference substance valid?
Not applicable

- Mortality:
Not applicable

- LC50:
Not applicable

- Other:
Not applicable

Reported statistics and error estimates:

None recorded

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table1              Cumulative Mortality Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population = 3)
	3 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0
100	0	0	0	0	0

 

Table2              Cumulative Mortality Data in the DefinitiveTest

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population =7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

Table 3              Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	34.5	34.5	35.0	35.0	35.0	35.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

SECOND MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	35.5	35.5	36.0	36.0	35.5	35.5
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

THIRD MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	35.5	35.5	35.0	35.0	35.0	35.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

FOURTH MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	35.0	35.0	35.5	35.5	36.0	36.0
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

R₁- R₂= Replicates 1 and 2

*= Start of mixing period

+= End of mixing period

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL50* value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.
Given that no toxicity was observed at the maximum test concentration employed of 100 mg/l loading rate WAF in accordance with Council Directive 67/548/EEC the test material will not be classified as being harmful to aquatic organisms.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LL₅₀*based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Analysis of the test preparations at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) showed measured concentrations to range from 0.328 to 0.374 mg/l with the exception of the 100 mg/l loading rate WAF replicate R₂at 24 hours which showed a measured concentration of 0.167 mg/l. Analysis of the corresponding frozen duplicate sample showed a measured concentration of 0.139 mg/l. A reason for this low result could not be determined from the data given that the corresponding
0-Hour result showed a measured concentration of 0.338 mg/l and no decline in measured concentration was observed from the remaining samples. This was considered not to affect the integrity of the test given the consistency of the remaining results and that no effects were observed throughout the test.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

Given that no toxicity was observed at the maximum test concentration employed of 100 mg/l loading rate WAF in accordance with Council Directive 67/548/EEC the test material will not be classified as being harmful to aquatic organisms.

*LL = Lethal Loading rate