4,4-bis(methoxymethyl)biphenyl

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 4 February 2009 and 19 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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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: 19/08/2008. Date of signature: 01/05/2009.

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: final theoretical concentrations of approximately 11 to 14 mg/l.

- Sampling method: 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.

A Strata X solid phase extraction (SPE) cartridge (packed with glass wool) was sequentially pre-conditioned with methanol and water*. ( Prepared by ELGA Purelab Option R-15BP water purification). A volume of test sample was eluted through the cartridge and the cartridge dried. The test material was eluted from the cartridge with methanol and made to volume to give final theoretical concentrations of approximately 11 to 14 mg/l.

Standard solutions of test material were prepared in methanol at a nominal concentration of 10 mg/l.

- Sample storage conditions before analysis: Preliminary test samples were prepared, analysed initially and then after storage in sealed glass vessels at ambient temperature in light and dark conditions for approximately 72 hours (equivalent to the test exposure period). In addition a test sample was tested for stability without prior mixing (sonication) of the test sample bottle to assess for losses due to adsorption and/or insolubility.

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 (approximately 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximately 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 (approximately 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximately 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 (approximately 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)


- Eluate: same as culture media
- Differential loading: no
- 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): none stated in the report

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not stated in report
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable to algae study

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not stated in report.

Test temperature:

24 ± 1°C.
The temperature within the incubator was recorded daily.

pH:

7.4 - 7.5 at 0 hours.
7.7 - 8.1 at 72 hours.
pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not stated in report

Salinity:

Not stated in report.

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.0022, 0.022, 0.22, 2.2 and 22 mg/l for a period of 72 hours.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.38, 2.75, 5.5, 11 and 22 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: closed - plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable, as static test condition.
- Renewal rate of test solution (frequency/flow rate): not applicable as static test condition
- Initial cells density: 4 x 10^3 cells per ml
- Control end cells density: 1.2 x 10^5 cells/ml
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): three flasks each containing 100 ml were used for each treatment group.
- No. of vessels per control (replicates): Six flasks each containing 100 ml of solution were used for the control
- No. of vessels per vehicle control (replicates): not applicable


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable


TEST MEDIUM / WATER PARAMETERS
An amount of test material (550 mg) was dispersed in 11 litres of culture medium 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 Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution with a 0-Hour measured concentration of 18 mg/l. A series of dilutions was made from this saturated solution to give further stock solutions of 1.1, 2.1, 4.2 and 9.2 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with 11.2 ml algal suspension to give the test concentrations of 1.1, 2.1, 4.2, 9.2 and 18 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.


NaNO3: 25.5 mg/l
MgCl2.6H2O: 12.164 mg/l
CaCl2.2H2O: 4.41 mg/l
MgSO4.7H2O: 14.7 mg/l
K2HPO4: 1.044 mg/l
NaHCO3: 15.0 mg/l
H3BO3: 0.1855 mg/l
MnCl2.4H2O: 0.415 mg/l
ZnCl2: 0.00327 mg/l
FeCl3.6H2O: 0.159 mg/l
CoCl2.6H2O: 0.00143 mg/l
Na2MoO4.2H2O: 0.00726 mg/l
CuCl2.2H2O: 0.000012 mg/l
Na2EDTA.2H2O: 0.30 mg/l
Na2SeO3.5H2O: 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
* Elga Optima 15+ or Elga Purelab Option R-15 BP


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Salinity (for marine algae): not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: no
- Other: not applicable


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: nominal test concentrations of 0.0022, 0.022, 0.22, 2.2 and 22 mg/l for a period of 72 hours
- Results used to determine the conditions for the definitive study:
Cell Densities and Percentage Inhibition of Growth from the Range-finding Test
Nominal Concentration
(mg/l) Cell Densities (cells per ml) Inhibition Values (%)
0 Hours 72 Hours Growth Rate Yield/Biomass Integral
Control R1 4.68E+03 3.18E+05
R2 4.36E+03 3.31E+05 - -
Mean 4.52E+03 3.25E+05
0.0022 R1 4.88E+03 3.27E+05
R2 4.92E+03 3.29E+05 2 [1]
Mean 4.90E+03 3.28E+05
0.022 R1 4.53E+03 4.13E+05
R2 4.93E+03 3.99E+05 [5] [25]
Mean 4.73E+03 4.06E+05
0.22 R1 4.25E+03 4.56E+05
R2 4.76E+03 5.07E+05 [10] [49]
Mean 4.50E+03 4.81E+05
2.2 R1 4.46E+03 4.04E+05
R2 4.09E+03 4.67E+05 [8] [35]
Mean 4.28E+03 4.36E+05
22 R1 4.26E+03 1.01E+04
R2 4.34E+03 6.46E+03 85 99
Mean 4.30E+03 8.26E+03



The results showed no effect on growth at the nominal test concentrations of 0.0022, 0.022, 0.22 and 2.2 mg/l. However, growth was observed to be reduced at 22 mg/l.
Based on this information nominal test concentrations of 1.38, 2.75, 5.5, 11 and 22 mg/l were selected for the definitive test. Chemical analysis of the test preparations at 0 hour showed measured test concentrations of 1.1, 2.1, 4.2, 9.2 and 18 mg/l were obtained. The slight differences observed between the measured concentrations obtained in the pre-study media preparation trial and definitive test were considered to be due to the different diluent types used.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 3.1 - 3.7 mg/L (95% CL). See comments in "Remarks on results including tables and figures" section.

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 3.3-3.9 mg/L (95% CL). See comments in "Remarks on results including tables and figures" section.

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: See comments in "Remarks on results including tables and figures" section.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium:No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
ErC50 (0 – 72 h) : 0.52 mg/l, 95% confidence limits 0.43 – 0.62 mg/l
EyC50 (0 – 72 h) : 0.29 mg/l, 95% confidence limits 0.25 – 0.33 mg/l
EbC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.26 – 0.34 mg/l

- Other:
No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.125 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral: 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.1 to 18 mg/l. Analysis of the test preparations at 72 hours showed no significant change in the measured test concentrations and so it was considered justifiable to base the results on the 0-Hour measured test concentrations only.

Pre-study media preparation trial

The results obtained from the pre-study media preparation trial conducted indicated that significantly higher dissolved test material concentrations were obtained following the saturated solution method of preparation compared to a solvent spike method. In addition an increase in measured concentrations was observed when the preparation period of the saturated solution was extended from 24 hours to 48 hours. A further media preparation trial conducted using a saturated solution method of preparation stirred for periods of 72 and 96 hours showed no increase in measured concentrations compared to those obtained following a 48 hour preparation period.

Based on this information the test material was prepared as a saturated solution, stirred for a period of 48 hours prior to the removal of any undissolved test material by filtration through a 0.2μm Gelman Acrocap filter (first approximate 100 ml discarded).

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.

The results showed no effect on growth at the nominal test concentrations of 0.0022, 0.022, 0.22 and 2.2 mg/l. However, growth was observed to be reduced at 22 mg/l.

Based on this information nominal test concentrations of 1.38, 2.75, 5.5, 11 and 22 mg/l were selected for the definitive test. Chemical analysis of the test preparations at 0 hour showed measured test concentrations of 1.1, 2.1, 4.2, 9.2 and 18 mg/l were obtained. The slight differences observed between the measured concentrations obtained in the pre-study media preparation trial and definitive test were considered to be due to the different diluent types used.

DefinitiveTest

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

 Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 31 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours        :   3.88 x 10³cells per ml
Mean cell density of control at 72 hours      :   1.20 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 30% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass (b) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72‑Hour exposure period.

Accordingly the following results were determined from the data based on the 0-Hour measured test concentrations:

Inhibition of growth rate

E_rC₁₀(0 - 72 h)           : 3.3 mg/l
E_rC₂₀(0 - 72 h)           : 3.6 mg/l
E_rC₅₀(0 - 72 h)           : 5.5 mg/l*

where E_rC_x is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant decreases in growth rate between the control, 1.1 and 2.1 mg/l test concentrations (P ≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 2.1 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 4.2 mg/l.

Inhibition of yield

E_yC₁₀(0 - 72 h)          : 3.0 mg/l
E_yC₂₀(0 - 72 h)          : 3.1 mg/l
E_yC₅₀(0 - 72 h)          : 3.6 mg/l; 95% confidence limits 3.3 – 3.9 mg/l

where E_yC_x is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 5.3.2.1. There were no statistically significant decreases in yield between the control, 1.1 and 2.1 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 2.1 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 4.2 mg/l.

 Inhibition of biomass integral

E_bC₁₀(0 - 72 h)          : 2.8 mg/l
E_bC₂₀(0 - 72 h)          : 2.9 mg/l
E_bC₅₀(0 - 72 h)          : 3.4 mg/l; 95% confidence limits 3.1 – 3.7 mg/l

where E_bC_x is the test concentration that reduced biomass integral (area under the growth curve) by x%.

Statistical analysis of the biomass integral data was carried out as in Section 5.3.2.1. There were no statistically significant decreases in biomass integral between the control, 1.1 and 2.1 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 2.1 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 4.2 mg/

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	4.68E+03	3.18E+05
	R2	4.36E+03	3.31E+05	-	-
	Mean	4.52E+03	3.25E+05
0.0022	R1	4.88E+03	3.27E+05
	R2	4.92E+03	3.29E+05	2	[1]
	Mean	4.90E+03	3.28E+05
0.022	R1	4.53E+03	4.13E+05
	R2	4.93E+03	3.99E+05	[5]	[25]
	Mean	4.73E+03	4.06E+05
0.22	R1	4.25E+03	4.56E+05
	R2	4.76E+03	5.07E+05	[10]	[49]
	Mean	4.50E+03	4.81E+05
2.2	R1	4.46E+03	4.04E+05
	R2	4.09E+03	4.67E+05	[8]	[35]
	Mean	4.28E+03	4.36E+05
22	R1	4.26E+03	1.01E+04
	R2	4.34E+03	6.46E+03	85	99
	Mean	4.30E+03	8.26E+03

 

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

 

Table2              Cell Densities and pH Values in the DefinitiveTest

0-Hour Measured Test Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	4.06E+03	1.42E+04	5.24E+04	1.31E+05	8.1
	R2	7.5	3.50E+03	1.67E+04	5.01E+04	1.30E+05	8.0
	R3	7.4	3.82E+03	1.09E+04	3.57E+04	1.23E+05	7.8
	R4	7.4	4.08E+03	2.06E+04	3.76E+04	1.17E+05	7.8
	R5	7.4	3.78E+03	1.85E+04	3.39E+04	1.10E+05	7.8
	R6	7.4	4.05E+03	1.96E+04	3.88E+04	1.10E+05	7.7
	Mean		3.88E+03	1.67E+04	4.14E+04	1.20E+05
1.1	R1	7.3	4.10E+03	1.26E+04	5.50E+04	3.94E+05	7.8
	R2	7.3	4.09E+03	1.29E+04	6.46E+04	3.78E+05	7.8
	R3	7.3	4.01E+03	1.22E+04	3.80E+04	2.37E+05	7.8
	Mean		4.06E+03	1.26E+04	5.26E+04	3.36E+05
2.1	R1	7.3	4.07E+03	1.11E+04	4.64E+04	1.96E+05	7.8
	R2	7.3	4.10E+03	1.32E+04	4.28E+04	2.28E+05	7.8
	R3	7.3	4.16E+03	9.90E+03	5.77E+04	2.80E+05	7.8
	Mean		4.11E+03	1.14E+04	4.90E+04	2.35E+05
4.2	R1	7.3	4.03E+03	5.06E+03	1.19E+04	3.29E+04	7.7
	R2	7.3	4.06E+03	6.36E+03	1.41E+04	4.18E+04	7.6
	R3	7.3	4.09E+03	6.48E+03	1.43E+04	4.33E+04	7.6
	Mean		4.06E+03	5.97E+03	1.34E+04	3.93E+04
9.2	R1	7.3	4.11E+03	4.17E+03	1.01E+04	1.88E+04	7.6
	R2	7.3	4.03E+03	4.53E+03	8.58E+03	1.16E+04	7.5
	R3	7.3	4.14E+03	6.05E+03	8.98E+03	1.58E+04	7.5
	Mean		4.10E+03	4.91E+03	9.21E+03	1.54E+04
18	R1	7.3	3.90E+03	4.70E+03	8.93E+03	1.31E+04	7.5
	R2	7.3	3.38E+03	4.07E+03	1.05E+04	1.73E+04	7.5
	R3	7.3	3.94E+03	4.90E+03	7.59E+03	1.21E+04	7.4
	Mean		3.74E+03	4.55E+03	9.01E+03	1.41E+04

 

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

			Daily Specific Growth Rate (cells/ml/hour)
			Day 0 - 1		Day 1 - 2		Day 2 - 3
Control		R1		0.053		0.054		0.038
		R2		0.060		0.046		0.040
		R3		0.042		0.050		0.052
		R4		0.068		0.025		0.047
		R5		0.064		0.025		0.049
		R6		0.066		0.028		0.043
		Mean		0.059		0.038		0.045

 

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth Rate, Yield and BiomassIntegral in the Definitive Test

0-Hour Measured Test Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)		Biomass Integral (cells/ml/hour)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R1	0.048		1.27E+05		2.93E+06
	R2	0.048		1.26E+05		2.92E+06
	R3	0.048		1.20E+05		2.36E+06
	R4	0.047	-	1.13E+05	-	2.55E+06	-
	R5	0.046		1.06E+05		2.34E+06
	R6	0.046		1.06E+05		2.48E+06
	Mean	0.047		1.16E+05		2.60E+06
	SD	0.001		9.32E+03		2.65E+05
1.1	R1	0.064	[36]	3.90E+05		6.12E+06	[136]
	R2	0.063	[34]	3.74E+05		6.16E+06	[137]
	R3	0.057	[21]	2.33E+05		3.80E+06	[46]
	Mean	0.061	[30]	3.32E+05	[186]	5.36E+06	[106]
	SD	0.004		8.68E+04		1.35E+06
2.1	R1	0.054	[15]	1.92E+05		3.50E+06	[35]
	R2	0.056	[19]	2.24E+05		3.84E+06	[48]
	R3	0.059	[26]	2.75E+05		4.74E+06	[82]
	Mean	0.056	[20]	2.30E+05	[98]	4.02E+06	[55]
	SD	0.003		4.21E+04		6.41E+05
4.2	R1	0.029	38	2.88E+04		5.60E+05	78
	R2	0.033	30	3.78E+04		7.53E+05	71
	R3	0.033	30	3.92E+04		7.78E+05	70
	Mean	0.032	33	3.53E+04	70	6.97E+05	73
	SD	0.002		5.63E+03		1.19E+05
9.2	R1	0.021	55	1.47E+04		3.27E+05	87
	R2	0.015	68	7.58E+03		2.14E+05	92
	R3	0.019	60	1.16E+04		3.10E+05	88
	Mean	0.018	61	1.13E+04	90	2.84E+05	89
	SD	0.003		3.56E+03		6.12E+04
18	R1	0.016	66	9.15E+03		2.44E+05	91
	R2	0.020	57	1.39E+04		3.18E+05	88
	R3	0.015	68	8.14E+03		2.05E+05	92
	Mean	0.017	64	1.04E+04	91	2.55E+05	90
	SD	0.003		3.09E+03		5.75E+04

  *In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

1.                          REFERENCES

Dunnett, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc50, 1096-1121.

Environment Directorate, Organisation for Economic Co-operation and Development (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Monograph No. 26 (1996) Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances.

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experiments. J Pharmacol Exp Ther96, 99-113.

SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc,,,.

Sokal, R R and Rohlf, F J (1981) Biometry.: W H Freeman and Company.

Xlfit, ID Business Solutions Ltd.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test material gave the following results based on the 0-Hour measured test concentrations:
Response Variable EC50 (mg/l) 95% Confidence Limits (mg/l) NOEC (mg/) Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate 5.5 * 2.1 4.2
Yield 3.6 3.3 - 3.9 2.1 4.2
Biomass 3.4 3.1 - 3.7 2.1 4.2
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.1 to 18 mg/l. Analysis of the test preparations at 72 hours showed no significant change in the measured test concentrations and so it was considered justifiable to base the results on the 0-Hour measured test concentrations only.

Executive summary:

Introduction

A study was perford to assess the effect of the test material on the growth of the green algae Desmodesmus subspicatus. Thethod followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 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, Desmodesmus subspicatus was exposed to solutions of the test material at 0-Hour measured concentrations of 1.1, 2.1, 4.2, 9.2 and 18 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (50 mg/l) of test material in culture medium 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 Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a measured concentration of 18 mg/l. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 

Results. 

Exposure of Desmodesmus subspicatus to the test material gave the following results based on the 0-Hour measured test concentrations:

Response Variable	EC50(mg/l)	95% Confidence Limits (mg/l)			No Observed Effect Concentration (NOEC) (mg/)	Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate	5.5		*		2.1	4.2
Yield	3.6	3.3	-	3.9	2.1	4.2
Biomass	3.4	3.1	-	3.7	2.1	4.2

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.1 to 18 mg/l. Analysis of the test preparations at 72 hours showed no significant change in the measured test concentrations and so it was considered justifiable to base the results on the 0-Hour measured test concentrations only.

*It was not possible to calculate 95% confidence limits for the E_rC₅₀ value as the data generated did not fit the models available for the calculation of confidence limits.