1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-, (6E)-, hydrogenated

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22 September 2011 and 22 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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 items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item 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 item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Farnasene

IUPAC Name:

Farnasene

Constituent 2

Reference substance name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

EC Number:

242-582-0

EC Name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

18794-84-8

IUPAC Name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Test material form:

other: liquid

Details on test material:

Sponsor's identification: Farnesene
Description : clear colourless liquid

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES of Farnesene (surrogate)
- Melting point: 253K
- Boiling point: 250 - 260 C
- Vapour pressure:
- Water solubility (under test conditions):<0.0001 g/L at 20 C
- log Pow: >6.5
-Density: 0.83
Molecular weight = 204
Molecular Formula C15H24

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Pre-study media preparation trial

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. An estimate of the water solubility for the test item using EPIWIN (Version 3.20) indicated that the solubility was approximately 0.0090 mg/l.
Based on this information the test item 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 item under test conditions.

Range-finding test

The results obtained from the pre-study media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
An amount of test item (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 item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (5.6 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
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 item.
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.
Samples of each test concentration were taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Method:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Experimental Preparation:

An amount of test item (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 item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 ml) of each of the stock solutions was separately inoculated with 6.8 ml of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Verification of test concentrations:

Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20°C prior to 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.
The method of analysis, recovery and test solution analyses are described in the attached Appendix 5.

Observations on test item 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 pale green dispersions.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test Species:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.
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 10E+03 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 ± 1ºC until the algal cell density was approximately 10E+04 - 10E+05 cells/ml.



- 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 (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.


- Any deformed or abnormal cells observed:

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

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

pH:

The pH values of the control and each test preparation are given in Table 2.
The pH value of the control cultures (see Table 2) was observed to increase from pH 8.1 at 0 hours to pH 8.3 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 applicable

Salinity:

freshwater used

Nominal and measured concentrations:

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
The method of chemical analysis developed for use during the range-finding test was considered to be insufficiently sensitive to detect the low dissolved test item concentrations present. Therefore the results obtained were considered to be unreliable and hence have not been presented within this report.

Details on test conditions:

TEST SYSTEM

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 three flasks each containing 100 ml were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 6.57E+05 cells per ml. Inoculation of 900 ml of test medium with 6.8 ml of this algal suspension gave an initial nominal cell density of 5.00E+03 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.


- Control end cells density: algal cell density was approximately 1.18E+06 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 5.00E+03 cells/ml.

- No. of vessels per concentration (replicates):
Three replicate flasks per concentration.

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


OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable


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.


- Chlorophyll measurement:
Not recorded


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 specific 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:

LOEC

Effect conc.:

100 other:

Nominal / measured:

nominal

Conc. based on:

other: % v/v saturated solution

Basis for effect:

other: growth rate and yield

Remarks on result:

other: Not applicable

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 other:

Nominal / measured:

nominal

Conc. based on:

other: % v/v saturated solution

Basis for effect:

other: growth rate and yield

Remarks on result:

other: Not applicable

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other:

Nominal / measured:

nominal

Conc. based on:

other: % v/v saturated solution

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other:

Nominal / measured:

nominal

Conc. based on:

other: % v/v saturated solution

Basis for effect:

cell number

Remarks:

Yield

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Details on results:

- Exponential growth in the control (for algal test): yes

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.


Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding test are given in Table1.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 233 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 : 5.05E+03 cells per ml
Mean cell density of control at 72 hours : 1.18E+06 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% 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 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72 Hour exposure period.

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 rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 please see attached


Accordingly the following results were determined from the data:

Inhibition of growth rate

ErC10 (0 - 72 h) : >100% v/v saturated solution
ErC20 (0 - 72 h) : >100% v/v saturated solution
ErC50 (0 - 72 h) : >100% v/v saturated solution

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 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 differences between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P>0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield

EyC10 (0 - 72 h) : 22% v/v saturated solution
EyC20 (0 - 72 h) : 46% v/v saturated solution
EyC50 (0 - 72 h) : >100% v/v saturated solution

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

Statistical analysis of the yield data was carried out as for the growth rate. There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P>0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100% v/v saturated solution.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 41104038) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.

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

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 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 item.

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 and yield 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

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.12E+03	1.16E+06
	R2	6.55E+03	8.98E+05	-	-
	Mean	6.34E+03	1.03E+06
0.10	R1	4.89E+03	8.84E+05
	R2	5.17E+03	1.12E+06	[4]	2
	Mean	5.03E+03	1.00E+06
1.0	R1	5.09E+03	8.28E+05
	R2	5.67E+03	1.11E+06	[1]	6
	Mean	5.38E+03	9.70E+05
10	R1	5.81E+03	5.80E+05
	R2	5.53E+03	7.15E+05	7	37
	Mean	5.67E+03	6.48E+05
100	R1	4.39E+03	1.07E+05
	R2	5.11E+03	7.92E+04	42	91
	Mean	4.75E+03	9.32E+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.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	5.04E+03	2.79E+04	1.67E+05	7.61E+05	8.3
	R2		5.06E+03	3.82E+04	2.19E+05	1.33E+06
	R3		5.04E+03	3.36E+04	2.05E+05	1.16E+06
	R4		5.01E+03	3.23E+04	1.89E+05	1.18E+06
	R5		5.06E+03	3.82E+04	2.49E+05	1.51E+06
	R6		5.09E+03	3.34E+04	2.39E+05	1.12E+06
	Mean		5.05E+03	3.39E+04	2.11E+05	1.18E+06
1.0	R1	8.0	5.10E+03	3.37E+04	1.94E+05	9.73E+05	8.2
	R2		5.05E+03	3.56E+04	1.94E+05	1.30E+06
	R3		4.90E+03	3.30E+04	1.46E+05	9.74E+05
	Mean		5.02E+03	3.41E+04	1.78E+05	1.08E+06
3.2	R1	7.9	5.17E+03	2.80E+04	1.51E+05	8.73E+05	8.1
	R2		5.07E+03	3.62E+04	2.22E+05	1.07E+06
	R3		5.03E+03	2.59E+04	1.63E+05	8.15E+05
	Mean		5.09E+03	3.00E+04	1.79E+05	9.20E+05
10	R1	7.8	5.00E+03	2.37E+04	1.73E+05	8.79E+05	8.1
	R2		5.03E+03	3.54E+04	1.66E+05	1.20E+06
	R3		5.06E+03	3.34E+04	2.04E+05	1.25E+06
	Mean		5.03E+03	3.09E+04	1.81E+05	1.11E+06
32	R1	7.8	5.05E+03	2.53E+04	1.24E+05	8.75E+05	8.1
	R2		4.85E+03	3.23E+04	2.08E+05	1.24E+06
	R3		5.14E+03	3.40E+04	1.91E+05	1.06E+06
	Mean		5.01E+03	3.06E+04	1.74E+05	1.06E+06
100	R1	7.7	4.35E+03	2.12E+04	1.37E+05	7.54E+05	8.1
	R2		5.14E+03	3.06E+04	1.33E+05	5.68E+05
	R3		5.09E+03	2.60E+04	1.37E+05	8.48E+05
	Mean		4.86E+03	2.59E+04	1.36E+05	7.23E+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	0.072	0.075	0.063
	R2	0.085	0.073	0.075
	R3	0.079	0.075	0.072
	R4	0.078	0.074	0.076
	R5	0.085	0.078	0.075
	R6	0.079	0.082	0.064
	Mean	0.080	0.076	0.071

R₁- R₆= Replicates 1 to 6

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

Nominal Concentration (% v/v Saturated Solution)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.070		7.56E+05
	R2	0.078		1.33E+06
	R3	0.076		1.15E+06
	R4	0.076	-	1.17E+06	-
	R5	0.079		1.50E+06
	R6	0.075		1.11E+06
	Mean	0.076		1.17E+06
	SD	0.003		2.50E+05
1.0	R1	0.073	4	9.68E+05
	R2	0.077	[1]	1.30E+06
	R3	0.073	4	9.69E+05
	Mean	0.074	2	1.08E+06	8
	SD	0.002		1.89E+05
3.2	R1	0.072	5	8.68E+05
	R2	0.075	1	1.07E+06
	R3	0.071	7	8.10E+05
	Mean	0.073	4	9.15E+05	22**
	SD	0.002		1.35E+05
10	R1	0.072	5	8.74E+05
	R2	0.076	0	1.19E+06
	R3	0.077	[1]	1.24E+06
	Mean	0.075	1	1.10E+06	6
	SD	0.003		2.00E+05
32	R1	0.072	5	8.70E+05
	R2	0.077	[1]	1.24E+06
	R3	0.074	3	1.05E+06
	Mean	0.074	2	1.05E+06	10
	SD	0.003		1.84E+05
100	R1	0.070	8	7.49E+05
	R2	0.066	13	5.62E+05
	R3	0.071	7	8.43E+05
	Mean	0.069	9	7.18E+05	39
	SD	0.003		1.43E+05

[ ]

*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]

** Value shown to not be statistically significantly different due to variability observed in the control cultures

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

ErC50 (0-72 h) >100% v/v saturated solution
EyC50 (0-72 h) >100% v/v saturated solution

No Observed Effect Concentration based on growth rate: 32% v/v saturated solution
No Observed Effect Concentration based on yield: 32% v/v saturated solution
Lowest Observed effect Concentration based on growth rate: 100% v/v saturated solution
Lowest Observed effect Concentration based on yield: 100% v/v saturated solution

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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 761/2009.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. An estimate of the water solubility for the test item using EPIWIN (Version 3.20) indicated that the solubility was approximately 0.0090 mg/l. A pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.

Following a preliminary range-finding test,Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (50 mg/l) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50 (% v/v Saturated Solution)	No Observed Effect Concentration (NOEC) (% v/v Saturated Solution)	Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution)
Growth Rate	>100	32	100
Yield	>100	32	100

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.00013 mg/l. This does not infer that no test item was in solution, just that which was, was at a concentration of less than 0.00013 mg/l.