3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

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 28 May 2009 and 18 June 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

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

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 ECETOC 1996 and within OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by centrifugation at 40000 g for 30 minutes to give a saturated solution of the test material.

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:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

EC Number:

700-341-1

Cas Number:

105194-34-1

Molecular formula:

C24H15ClN2O2

IUPAC Name:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Constituent 2

Reference substance name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

IUPAC Name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

Details on test material:

Sponsor's identification : PDE-28
Description : red powder
Purity : 97%
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
0.019mg/l

- Sampling method:
Saturated Solution Preparation
An amount of test material (550 mg) was dispersed, in duplicate, in 11 litres of reconstituted water to give initial test material dispersions of 50 mg/l. These were stirred using a propeller stirrer at approximately 1500 rpm at approximately 21oC for periods of 24 and 48 hours.
Samples were taken for analysis following removal of any undissolved test material by centrifugation at 10000 or 40000 g for 30 minutes or following filtration through 0.2 μm Sartorius Sartopore filters with the first approximate 1 or 2 litres being discarded.

Solvent Spike Preparation:
An amount of test material (50 mg) was dissolved in 50 ml of tetrahydrofuran to give a 50 mg/50 ml solvent stock solution. An aliquot (1000 μl) of this solvent stock solution was dispersed in 10 litres of reconstituted water with the aid of magnetic stirring for approximately 10 minutes to give a nominal test concentration of 0.10 mg/l. This test concentration was then stirred by magnetic stirrer at approximately 21oC with samples taken for analysis initially and after stirring for periods of 24 or 48 hours.

Verification of test concentrations:
Samples were taken from the control (replicates R1 - R6 pooled) and the 0.019 mg/l test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
Samples were analysed untreated, following centrifugation at 10000 or 40000 g for 30 minutes or following filtration through 0.2 μm Gelman Acrocap filters with the first approximate 100 or 500 ml being discarded.


- Sample storage conditions before analysis:
A preliminary test sample was 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).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Due to the low aqueous solubility and high purity of the test material the test concentration used in the definitive test was a saturated solution prepared from an initial test material dispersion at a concentration of 50 mg/l.
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 for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by centrifugation at 40000 g to give a saturated solution with a 0-Hour mean measured concentration of 0.019 mg/l. An aliquot (2 litres) of this saturated solution was inoculated with 63 ml algal suspension to give the test concentration of 0.019 mg/l.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project Number: 0039/1088) used potassium dichromate as the reference material. D The positive control was conducted between 26 May 2009 and 29 May 2009.

A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 – 72 h): 0.79 mg/l*
EyC50 (0 – 72 h): 0.30 mg/l, 95% confidence limits 0.27 – 0.34 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- 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 recorded.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
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.

- Any deformed or abnormal cells observed:
None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

The temperature within the incubator was recorded daily. The temperature was maintained at 24 ± 1ºC throughout the test.

pH:

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.

The pH values of the control cultures were observed to increase from pH 7.2 at 0 hours to pH 7.6 – 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

A nominal concentration of 0.013 mg/l was used for the limit test.
Measured test concentrations were in the range from 0.018 to 0.019 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml conical flask
- Aeration: ). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1C.

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

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.27 x 105 cells per ml. Inoculation of 2 litres of test medium with 63 ml of this algal suspension gave an initial nominal cell density of 4 x 103 cells per ml and had no significant dilution effect on the final test concentration.

- Control end cells density:
Not stated

- No. of organisms per vessel:
not applicable

- No. of vessels per concentration (replicates):
6

- No. of vessels per control (replicates):
6 replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM

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 0.10 mg/l (by visual inspection) was obtained using a preliminary solution in tetrahydrofuran.

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 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 Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
 Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Under continuous illumination

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution were used for the control and 0.019 mg/l treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.27 x 105 cells per ml. Inoculation of 2 litres of test medium with 63 ml of this algal suspension gave an initial nominal cell density of 4 x 103 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 


- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a nominal concentration of 0.013 mg/l to confirm that at the highest attainable concentration no effect on algal growth was observed

- Range finding study:

The results obtained from the pre-study media preparation trial conducted indicated that a dissolved test material concentration of approximately 0.010 mg/l could be obtained using a saturated solution method of preparation.
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.0010 and 0.010 mg/l for a period of 72 hours.
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 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by centrifugation at 40000 g for 30 minutes to give a saturated solution with a nominal concentration of 0.010 mg/l. A dilution was made from this saturated solution to give a further stock solution of 0.0010 mg/l. An aliquot (250 ml) of each of the stock solutions was separately inoculated with algal suspension (4.7 ml) to give the required test concentrations of 0.0010 and 0.010 mg/l.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

POSITIVE CONTROL:
A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 – 72 h) : 0.79 mg/l*
EyC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.27 – 0.34 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.50 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.


VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
- The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
- The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

For all tables (please see the any other information on results section). Figure 1 is shown in the attachment.

Observations on test material solubility
Pre-Study Media Preparation Trial
The results obtained from the pre-study media preparation trial conducted (see Appendix 3) indicated that significantly higher dissolved test material concentrations were obtained following a saturated solution method of preparation rather than a solvent spike method of preparation. The results obtained from the filtered test samples indicated that the test material was adsorbing to the filter matrices so filtration was considered inappropriate to remove any undissolved test material. There were no significant increases in the measured dissolved test material concentrations obtained when the preparation period of the saturated solution was extended beyond 24 hours.
Based on this information the test material was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/l, stirred for a period of 24 hours prior to removal of any undissolved test material by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of approximately 0.010 mg/l.


- Any stimulation of growth found in any treatment:
None

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

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 test concentrations of 0.0010 and 0.010 mg/l.
Based on this information a single test concentration of six replicates, of 0.010 mg/l was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration of 0.010 mg/l no effect on growth was observed.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 119 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 : 4.66 x 103 cells per ml
Mean cell density of control at 72 hours : 5.55 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Inhibition of growth rate

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a 0-Hour mean measured test concentration of 0.019 mg/l over the 72 Hour exposure period.

The test concentration of 0.019 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.

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

ErC10 (0 - 72 h) : > 0.019 mg/l
ErC20 (0 - 72 h) : > 0.019 mg/l
ErC50 (0 - 72 h) : > 0.019 mg/l

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

Statistical analysis of the growth rate data was carried out for the control and 0.019 mg/l test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P0.05), between the control and 0.019 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.019 mg/l.


Inhibition of yield


EyC10 (0 - 72 h) : > 0.019 mg/l
EyC20 (0 - 72 h) : > 0.019 mg/l
EyC50 (0 - 72 h) : > 0.019 mg/l
where EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences (P0.05), between the control and 0.019 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.019 mg/l.

Observations on cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility:
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control (Safepharm Laboratories Project No: 0039/0994) are given in Table 6 and Figure 2. Daily specific growth rates for the control cultures are given in Table 7 whilst growth rate, yield and biomass integral values are given in Table 8. Percentage inhibition values are plotted against test concentration in Figure 3, Figure 4 and Figure 5.
Accordingly the following results were determined from the data:
ErC50 (0 - 72 h) : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
EyC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
EbC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 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 range for this reference material.

Reported statistics and error estimates:

 Statistical analysis
A Student’s t-test incorporating's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 0.019 mg/l test concentration 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

Verification of test concentrations:

Analysis of the test preparations at 0 hours (see Appendix 3) showed measured test concentrations to be to range from 0.018 to 0.019 mg/l. A decline in measured concentrations was observed at 72 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.0019 mg/l to 0.0042 mg/l. Given that 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 algal cells present. The preliminary recovery analyses conducted in the presence of algal cells indicated that adsorption may occur.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀ values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.00094 mg/l) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be:

0-Hour Mean Measured Test Concentration (mg/l)	Geometric Mean Measured Test Concentration (mg/l)
0.019 R1-R3	0.0042
0.019 R4-R6	0.0087

The following results were determined from the data based on the geometric mean measured test concentrations:

Growth rate

E_rC₁₀(0 - 72 h)           : > 0.0065 mg/l
E_rC₂₀(0 - 72 h)           : > 0.0065 mg/l
E_rC₅₀(0 - 72 h)           : > 0.0065 mg/l
Yield

E_yC₁₀(0 - 72 h)          : > 0.0065 mg/l
E_yC₂₀(0 - 72 h)          : > 0.0065 mg/l
E_yC₅₀(0 - 72 h)          : > 0.0065 mg/l

The use of the geometric mean measured test concentrations in the calculation of the EC₅₀and NOEC values had no significant effect on the outcome of the study as no toxicity was observed at saturation.

Care should be taken in the interpretation of the results of the study based on geometric mean measured test concentrations as, whilst there was a significant decline in measured test concentrations over the test period, the test material was shown to be stable in aqueous medium and hence the decline was considered to be due to adsorption to algal cells. As such it may be considered that the algal cells were exposed to nominal concentrations of test material throughout the test period.

R₁-R₆= Replicates 1 to 6

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
Control	R1	3.98E+03	3.18E+05
	R2	3.66E+03	3.33E+05	-	-
	Mean	3.82E+03	3.25E+05
0.0010	R1	4.64E+03	5.55E+05
	R2	3.29E+03	5.48E+05	[11]	[70]
	Mean	3.96E+03	5.51E+05
0.010	R1	3.61E+03	4.28E+05
	R2	2.17E+03	4.09E+05	[11]	[29]
	Mean	2.89E+03	4.18E+05

*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

Table 2              Cell Densities and pH Values in the DefinitiveTest

0-Hour Mean 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	7.2	4.62E+03	2.35E+04	7.60E+04	5.34E+05	7.6
	R2	7.2	4.81E+03	2.11E+04	7.24E+04	5.35E+05	7.7
	R3	7.2	4.09E+03	2.44E+04	7.64E+04	5.75E+05	7.6
	R4	7.2	4.83E+03	2.35E+04	7.70E+04	5.94E+05	7.6
	R5	7.2	5.10E+03	2.57E+04	8.27E+04	5.51E+05	7.6
	R6	7.2	4.54E+03	1.81E+04	7.10E+04	5.42E+05	7.6
	Mean		4.66E+03	2.27E+04	7.59E+04	5.55E+05
0.019	R1	7.1	3.66E+03	2.48E+04	7.77E+04	5.81E+05	7.5
	R2	7.1	4.34E+03	2.79E+04	8.00E+04	5.21E+05	7.5
	R3	7.1	5.46E+03	2.86E+04	8.66E+04	5.38E+05	7.5
	R4	7.1	5.07E+03	2.68E+04	7.61E+04	5.49E+05	7.4
	R5	7.1	4.26E+03	2.62E+04	7.88E+04	6.44E+05	7.4
	R6	7.1	4.37E+03	2.53E+04	7.71E+04	6.36E+05	7.5
	Mean		4.53E+03	2.66E+04	7.94E+04	5.78E+05

 

*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	0.074	0.049	0.081
	R2	0.069	0.051	0.083
	R3	0.075	0.048	0.084
	R4	0.074	0.050	0.085
	R5	0.077	0.049	0.079
	R6	0.063	0.057	0.085
	Mean	0.072	0.051	0.083

 

R₁- R₆= Replicates 1 to 6

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

0-Hour Mean Measured Test Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.068		5.29E+05
	R2	0.068		5.30E+05
	R3	0.069		5.71E+05
	R4	0.069	-	5.89E+05	-
	R5	0.068		5.46E+05
	R6	0.068		5.38E+05
	Mean	0.068		5.50E+05
	SD	0.001		2.43E+04
0.019	R1	0.069	[1]	5.77E+05
	R2	0.068	0	5.17E+05
	R3	0.068	0	5.32E+05
	R4	0.068	0	5.44E+05
	R5	0.071	[4]	6.40E+05
	R6	0.070	[3]	6.32E+05
	Mean	0.069	[1]	5.74E+05	[4]
	SD	0.001		5.21E+04

[ ]

 

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

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and based on the 0-Hour mean measured test concentrations gave EC50 values of greater than 0.019 mg/l. Correspondingly the No Observed Effect Concentration was 0.019 mg/l.
Based on the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.0065 mg/l. Correspondingly the No Observed Effect Concentration was 0.0065 mg/l.

Executive summary:

Introduction:

A study was perford to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method 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:

 Pre-study solubility work conducted indicated 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 0.010 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 a solution of the test material at a 0-Hour mean measured test concentration of 0.019 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/l) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by centrifugation at 40000g for 30 minutes to produce a saturated solution of the test material with a 0-Hour mean measured concentration of 0.019 mg/l.

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 based on the 0-Hour mean measured test concentrations gave EC₅₀ values of greater than 0.019 mg/l and correspondingly the No Observed Effect Concentration was 0.019 mg/l.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.018 to 0.019 mg/l. A decline in measured concentrations was observed at 72 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.0019 mg/l to 0.0042 mg/l. Given that 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 algal cells present. The preliminary recovery analyses conducted in the presence of algal cells indicated that adsorption may occur.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geotricanasured test concentrations in order to give a "worste case" analysis of the data.

The EC₅₀ values based on the geometric measured test concentrations were greater than 0.0065 mg/l and correspondingly the No Observed Effect Concentration was 0.0065 mg/l.

This study showed that there were no toxic effects at saturation.