4,4-bis(methoxymethyl)biphenyl

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 2 February 2009 and 5 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 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

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 19/08/08. Date of signature: 14/06/09.

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-bis(methoxymethyl)-1,1'-biphenyl

EC Number:

700-008-0

Cas Number:

3753-18-2

Molecular formula:

C16 H18 O2

IUPAC Name:

4,4'-bis(methoxymethyl)-1,1'-biphenyl

Constituent 2

Reference substance name:

4,4-bis(methoxymethyl)-biphenyl

IUPAC Name:

4,4-bis(methoxymethyl)-biphenyl

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finding (nominal): 0.0, 0.022, 0.22, 2.2, 22 mg/l
Definitive (nominal): 0.0, 0.22, 0.40, 0.70, 1.2, 2.2, 4.0, 7.0, 12.0 and 22.0 mg/l

- Sampling method: Water samples were taken from the control (replicates R1 – R2 pooled) and each test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis. The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.
The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, Shardlow, UK.

- Sample storage conditions before analysis: 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:
Pre-study media preparation trial
Information provided by the Sponsor indicated that the test material was insoluble in water. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 2.0 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide.
Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation
An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
Centrifugation at 10000 g for 30 minutes
Centrifugation at 40000 g for 30 minutes
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 500 ml discarded in order to pre-condition the filter)

Solvent spike preparation
An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 µl) of the 200 mg/10 ml solvent stock solution was dispersed, in 2 litres of reconstituted water, in triplicate with the aid of magnetic stirring for periods of approximately either 10 minutes, 24 hours or 48 hours to give the required test concentration of 2.0 mg/l prior to taking samples for chemical analysis after the following pre-treatments:
Untreated
Centrifugation at 10000 g for 30 minutes
Centrifugation at 40000 g for 30 minutes
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 500 ml discarded in order to pre-condition the filter)

Further saturated solution preparation
The results obtained from the initial saturated solution preparations showed an increase in the dissolved test material concentration obtained when the preparation period was extended from 24 to 48 hours. It was therefore considered appropriate to prepare further saturated solutions stirred for periods of 72 and 96 hours in order to ensure that the maximum dissolved test material concentration was obtained.
An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of either 72 or 96 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximate 500 ml discarded in order to pre-condition the filter)


- Eluate:
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

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.

- Differential loading: no data

- 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): methanol

- Concentration of vehicle in test medium (stock solution and final test solution): not stated in report

- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: not applicable
- Source: in-house laboratory cultures.
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: Parthenogenesis
- Feeding during test
not fed during exposure period


ACCLIMATION - not applicable

QUARANTINE (wild caught)
- not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

h

Post exposure observation period:

None

Test conditions

Hardness:

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

Test temperature:

Temperature was maintained at approximately 20°C throughout the test.

pH:

7.9 at 0 hours. 7.9 - 8.0 at 48 hours.

Dissolved oxygen:

8.9 mg O2/l at 0 hours
8.8 - 8.9 O2/l at 48 hours

Salinity:

No data

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 83% to 113% of nominal value and so the results are based on nominal test concentrations only.

The test material was to be prepared as a saturated solution with any undissolved test material removed by filtration to give a nominal test concentration of 22 mg/l. Aliquots (10, 18, 32, 54.5, 100, 180, 320 and 545 ml) of the 22 mg/l test concentration were each separately dispersed in a final volume of 1 litre of reconstituted water to give the remainder of the test series of 0.22, 0.40, 0.70, 1.2, 2.2, 4.0, 7.0 and 12 mg/l respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type: closed
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 200 ml of test preparation
- Aeration: Test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 2 (duplicates)
- No. of vessels per control (replicates): 2 (duplicates)
- No. of vessels per vehicle control (replicates): not applicable, as no vehicle
- Biomass loading rate: not stated in report


TEST MEDIUM / WATER PARAMETERS
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

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.


OTHER TEST CONDITIONS
- Adjustment of pH: not conducted during study
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: not stated in report


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.

An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.0, 0.22, 0.40, 0.70, 1.2, 2.2, 4.0, 7.0, 12.0 and 22.0 mg/l

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

- Range finding study
- Test concentrations: 22, 2.2, 0.22 and 0.022 mg/l.

- Results used to determine the conditions for the definitive study: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.022, 0.22, 2.2, and 22 mg/l.
An amount of test material (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 22 mg/l. A series of dilutions were performed in reconstituted water from the 22 mg/l test concentration to give the remainder of the test series of 2.2, 0.22 and 0.022 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room 20°C to 22°C 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. A single temperature was measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: 95% confidence limits of 1.8 – 2.5 mg/l.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Details on results:

- Behavioural abnormalities: Please see data in the following section "Remarks on Results including tables and figures"
- Observations on body length and weight: immobilisation
- Other biological observations: none
- Mortality of control: immobilization only observed. Mortality not assessed.
- Other adverse effects control: none stated in report
- Abnormal responses:not described in report
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: The test preparations were observed to be clear colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

- Results with reference substance valid? - Yes . Please see data in the following section "Remarks on Results including tables and figures"
- Mortality: not applicable.
- EC50/LC50: The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.21).
- Other:

Any other information on results incl. tables

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. 

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)
24	4.3	3.6 – 5.2
48	2.1	1.8 – 2.5

The No Observed Effect Concentrations after 24 and 48 hours exposure were 1.2 and 0.70 mg/l respectively. 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   4.8 (SE = 0.8) and 6.5 (SE = 1.2) respectively.

Positive control

Cumulative immobilisation data from the exposure ofDaphnia magnato the reference material (Harlan Laboratories Ltd Project No: 0039/1069) during the positive control are given in Table 3.

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).

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.022	0	0
0.22	0	0
2.2	3	3
22	10	10

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.22	0	0	0	0	0	0	0	0
0.40	0	0	0	0	0	0	0	0
0.70	0	0	0	0	0	0	0	0
1.2	0	0	0	0	0	2	2	10
2.2	1	0	1	5	4	5	9	45
4.0	6	5	11	55	10	10	20	100
7.0	8	7	15	75	10	10	20	100
12	10	10	20	100	10	10	20	100
22	10	10	20	100	10	10	20	100

 

R₁– R₂= Replicates 1 and 2

Table3              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

 

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 EC50 value of 2.1 mg/l with 95% confidence limits of 1.8 – 2.5 mg/l. The No Observed Effect Concentration at 48 hours was 0.70 mg/l.

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.Information provided by the Sponsor indicated that the test material was insoluble in water. Pre-study solubility work conducted indicted that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 22 mg/l was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 0.22, 0.40, 0.70, 1.2, 2.2, 4.0, 7.0, 12 and 22 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. The test material solutions were prepared by stirring an excess (50 mg/l) of test material in reconstituted water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 100 ml discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a measured concentration of 21 mg/l. This saturated solution was then further diluted as necessary, to provide the remaining test groups. The number of immobilised Daphnia 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 understatic test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was 2.1 mg/l with 95% confidence limits of 1.8 – 2.5 mg/l. The No Observed Effect Concentration was 0.70 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 83% to 113% of nominal value and so the results are based on nominal test concentrations only.

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.