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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 9 February 2009 and 13 March 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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, OECD 2000 and Singer et al 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 yellow 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:
Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sampling method:
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.

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

Test solutions

Vehicle:

no

Details on test solutions:

EXAMPLE:
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 (1000 mg) was added to the surface of 10 litres of reconstituted water to give the 100 mg/l loading rate. After the addition of the test material, the reconstituted 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 approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test material present.

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1069) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
Microscopic examination after filtering showed there to be no micro-dispersions of test material present.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

EXAMPLE:
TEST ORGANISM
- Common name:
Water Flea

- Source:
Derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

- Feeding during test:
Received no food during exposure

ACCLIMATION
- Acclimation period:
Not stated


- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 20°C to 22°C throughout the test

The temperature of the control and 100 mg/l loading rate WAF test vessels were measured to be slightly above the 20 ± 1°C range given in the protocol at 0 hours. This was considered not to affect the outcome of the test as no adverse effects of exposure were observed throughout the test and the temperature range was within the Test Guideline limits.

The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

See Appendix 4 for results.

pH:

The reconstituted water had a pH of 7.9 - 8.0 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.

See Appendix 4 for results.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.

mg O2/l range of ~ 9.0 +/- 0.3

See Appendix 4 for results.

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10, and 100 mg/l.
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 immobilisation or adverse reactions to exposure were observed.

Details on test conditions:

EXAMPLE
TEST SYSTEM
- Test vessel:
In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used.
- Aeration:
None.

- Type of flow-through (e.g. peristaltic or proportional diluter):
None.

- Renewal rate of test solution (frequency/flow rate):
The test preparations were not renewed during the exposure period.

- No. of organisms per vessel:
At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations.

- No. of vessels per concentration (replicates):
Four replicate test vessels were prepared

- No. of vessels per control (replicates):
Four replicate control vessels were prepared

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

- Biomass loading rate:
Not recorded.

- Source/preparation of dilution water:

Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

- 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

- Adjustment of pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Photoperiod:
A photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity:
Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
x 10

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

- Range finding study
Due to the low aqueous solubility and complex nature of the test material for the purposes of the test the test material was prepared as a Water Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10, and 100 mg/l.

Amounts of test material (2.5, 25 and 250 mg) were each separately added to the surface of 2.5 litres of reconstituted water to give the 1.0, 10 and 100 mg/l loading rates respectively. After the addition of the test material, the reconstituted 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 mixtures allowed to stand for 1 hour. Microscopic observations made on the 100 mg/l loading rate 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 WAFs 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 approximate 75-100 ml discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test material present.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 deg C to 22ºC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.

The temperature of the control and 100 mg/l loading rate WAF test vessel was measured to be slightly above the 20 ± 1°C range given in the protocol at 0 hours. This was considered not to affect the outcome of the test as no adverse effects of exposure were observed throughout the test and the temperature range was within the Test Guideline limits.

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

- Results used to determine the conditions for the definitive study:
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 immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

immobilisation

Details on results:

- Behavioural abnormalities:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.
There was no significant immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours.

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon no significant immobilisation at this loading rate.

- Other biological observations: None recorded

- Immobilisation of control:
There was no significant immobilisation in the control.

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded

- Effect concentrations exceeding solubility of substance in test medium:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 100 mg/l loading rate was observed to have formed a clear colourless water column with test material at the water surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/l loading rate was observed to have formed a cloudy water column with test material at the water surface and at the bottom of the mixing vessel. Microscopic examination of the WAF showed there to be a significant amount of dispersed test material present in the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed there to be no micro-dispersions of test material present.
During the test the 100 mg/l loading rate was observed to be a clear, colourless solution.

Results with reference substance (positive control):

EXAMPLE
- Results with reference substance valid?
Yes

- Mortality:
No mortalities recorded.

- EC50/LC50:

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Project No: 0039/1069) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:


Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
3 > 3.2 -
24 0.82 0.71 - 0.94
48 0.71 0.61 - 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

- Other:
None.

Reported statistics and error estimates:

Not recorded

Any other information on results incl. tables

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	0	0

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate (mg/l)		Cumulative ImmobilisedDaphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	0	0	0	0	1	5
	R2	0			0
	R3	0			1
	R4	0			0

R₁– R₄= Replicates 1 to 4

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	1	1	2	10	2	2	4	20
1.0	0	0	0	0	7	8	15	75	9	9	18	90
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Table 4              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Water Column (cm)	26	26	26	26
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

*= 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 invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 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 Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

 Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 20°C to 22ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

 The 48-Hour EL*₅₀ for the test material Daphnia magna 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.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 2.3 to 2.9 mg/l. A decline in measured concentrations was observed at 48 hours in the range of 1.0 to 1.1 mg/l. As the preliminary stability analyses conducted indicated that the test material was stable over the test duration this decline was considered to be due to adsorption of the test material to the biological matter present. A study to determine the adsorption co-efficient of the test material (Harlan Laboratories Ltd Project Number 1571/0100) indicated that the test material had a log₁₀ k_oc value of 5.3 which would suggest that it would be easily adsorbed.

However, given that toxicity cannot be attributed to a single component or a 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.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 – 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

*EL = Effective Loading Rate