6,10-Dodecadienal, 3,7,11-trimethyl-, (3S,6E)-

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 23 July 2015 and 16 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Test material

Test material information

Constituent 1

Reference substance name:

(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal

EC Number:

810-298-1

Cas Number:

194934-66-2

Molecular formula:

C15H26O

IUPAC Name:

(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Two further samples of each test concentration were prepared and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 liters 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 (4.4 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 liters 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 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 7.5 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.

Test organisms

Test organisms (species):

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

Details on test organisms:

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 constant illumination at 21 ± 1 °C.

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 1E+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 ± 1 °C until the algal cell density was approximately 1E+04 - 1E+05 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

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

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.2 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.

Nominal and measured concentrations:

Range-Finding Test: Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitive Test: Nominal test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Procedure
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized 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 preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 1.4 mg/L could be obtained using a saturated solution method of preparation.

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.

A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 liters 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 (4.4 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 ± 1 ºC 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.

A sample of each test concentration was 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 frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
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
A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 liters 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 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 7.5 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.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours (see Appendix 5).


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 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 characterized by a cell density of 5.99 x 105 cells per mL. Inoculation of 900 mL of test medium with 7.5 mL of this algal suspension gave an initial nominal cell density of 5 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.


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

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Data Analysis
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1n Nn – 1n N1) / tn – t1

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = ((µc - µt) / µc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture


Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


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 computerized interpolation using the Xlfit software package (IDBS). values 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).

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0% v/v saturated solution. Enlarged cells were observed in the 3.2% v/v saturated solution test cultures, cell debris was observed in the 10 and 32% v/v saturated solution test cultures and no intact cells were observed to be present in the test cultures at 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
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.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.0090 mg/L) to 0.89 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the LOQ in all test samples indicating that the test item was unstable over the test duration.


Definitive Test
Growth Data
From the data given, 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.

Accordingly the following results were determined from the data:


Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.14 mg/L
ErC20 (0 - 72 h): 0.15 mg/L
ErC50 (0 - 72 h): 0.18 mg/L; 95% confidence limits 0.17 – 0.18 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 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, 0.0045, 0.0070 and 0.034 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 0.034 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.17 mg/L.


Inhibition of Yield
EyC10 (0 - 72 h): 0.023 mg/L
EyC20 (0 - 72 h): 0.034 mg/L
EyC50 (0 - 72 h): 0.066 mg/L; 95% confidence limits 0.053 – 0.082 mg/L

Where:

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

Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control, 0.0045, 0.0070 and 0.034 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 yield was 0.034 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.17 mg/L.

Results with reference substance (positive control):

A positive control (Study Number 41501180) 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): 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/L
EyC50 (0 – 72 h): 0.49 mg/L; 95% confidence limits 0.42 – 0.58 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:

Statistical Analysis
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).

Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 - R3 pooled:

GM = √(C0 x C1)

Where:
GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

The geometric mean measured concentration was determined for each 24-Hour interval (0-24, 24-48 and 48-72-Hour) and the arithmetic average calculated of these values.

Any other information on results incl. tables

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0090 mg/L, to 1.1 mg/L. A decline in measured test concentrations was observed following each 24-Hour interval in the range of less than the LOQ to 0.85 mg/L at 24 hours, less than the LOQ to 0.40 mg/L at 48 hours and less than the LOQ to 0.16 mg/L at 72 hours. These results indicate that the test item was unstable over the test duration.

 

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀values. 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. 0.0045 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
1.0	0.0045	NA
3.2	0.0070	21
10	0.034	31
32	0.17	78
100	0.60	53

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 122 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: 3.91 x 10³cells per mL

Mean cell density of control at 72 hours: 4.79 x10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% 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 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10% v/v saturated solution test cultures were observed to be green dispersions. The 32% v/v saturated solution test cultures were observed to be pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be clear colorless solutions.

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

Nominal Concentration (% v/v Saturated Solution)		Cell Densities* (cells permL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.98E+03	1.04E+06	-	-
	R2	3.98E+03	1.07E+06
	Mean	4.48E+03	1.06E+06
0.10	R1	4.32E+03	1.11E+06	[1]	[5]
	R2	4.10E+03	1.11E+06
	Mean	4.21E+03	1.11E+06
1.0	R1	4.02E+03	1.23E+06	[7]	[19]
	R2	3.57E+03	1.27E+06
	Mean	3.79E+03	1.25E+06
10	R1	4.45E+03	4.16E+05	17	62
	R2	4.26E+03	4.00E+05
	Mean	4.35E+03	4.08E+05
100	R1	3.50E+03	1.22E+04	79	99
	R2	3.76E+03	1.02E+04
	Mean	3.63E+03	1.12E+04

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

R₁and R₂= Replicates 1 and 2

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities* (cells permL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.7	4.08E+03	2.03E+04	1.17E+05	3.81E+05	8.2
	R2		4.13E+03	1.96E+04	1.20E+05	4.32E+05
	R3		3.74E+03	2.03E+04	1.32E+05	5.07E+05
	R4		4.06E+03	2.09E+04	1.52E+05	6.56E+05
	R5		3.73E+03	2.02E+04	1.23E+05	4.76E+05
	R6		3.71E+03	1.94E+04	1.31E+05	4.20E+05
	Mean		3.91E+03	2.01E+04	1.29E+05	4.79E+05
1.0	R1	7.8	4.11E+03	1.58E+04	8.93E+04	5.39E+05	8.3
	R2		4.16E+03	1.74E+04	1.11E+05	5.39E+05
	R3		4.08E+03	1.37E+04	1.22E+05	4.24E+05
	Mean		4.12E+03	1.56E+04	1.08E+05	5.01E+05
3.2	R1	7.8	4.10E+03	2.28E+04	1.31E+05	4.69E+05	8.3
	R2		3.90E+03	1.93E+04	1.22E+05	6.03E+05
	R3		4.11E+03	1.78E+04	1.02E+05	4.30E+05
	Mean		4.04E+03	2.00E+04	1.18E+05	5.01E+05
10	R1	7.8	3.62E+03	1.63E+04	9.12E+04	3.86E+05	8.2
	R2		4.40E+03	1.60E+04	8.48E+04	3.80E+05
	R3		4.14E+03	1.72E+04	1.04E+05	3.78E+05
	Mean		4.05E+03	1.65E+04	9.34E+04	3.81E+05
32	R1	7.8	4.22E+03	1.11E+04	3.16E+04	6.70E+04	8.2
	R2		3.97E+03	1.22E+04	3.75E+04	6.51E+04
	R3		4.05E+03	1.31E+04	3.84E+04	6.88E+04
	Mean		4.08E+03	1.21E+04	3.58E+04	6.70E+04
100	R1	7.8	3.65E+03	3.89E+03	4.13E+03	5.07E+03	8.2
	R2		3.73E+03	3.17E+03	3.58E+03	5.22E+03
	R3		4.35E+03	2.40E+03	2.80E+03	3.94E+03
	Mean		3.91E+03	3.15E+03	3.50E+03	4.74E+03

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

R₁- R₆= Replicates 1 to 6

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	0.058	0.073	0.049
	R2	0.057	0.075	0.053
	R3	0.058	0.078	0.056
	R4	0.060	0.083	0.061
	R5	0.058	0.075	0.056
	R6	0.056	0.079	0.049
	Mean	0.058	0.077	0.054

R₁- R₆= Replicates 1 to 6

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	0.060		3.77E+05
	R2	0.062		4.28E+05
	R3	0.064		5.03E+05
	R4	0.068	-	6.52E+05	-
	R5	0.063		4.72E+05
	R6	0.062		4.16E+05
	Mean	0.063		4.75E+05
	SD	0.003		9.72E+04
1.0	R1	0.065	[3]	5.35E+05
	R2	0.065	[3]	5.35E+05
	R3	0.062	2	4.20E+05
	Mean	0.064	[1]	4.96E+05	[5]
	SD	0.002		6.66E+04
3.2	R1	0.063	0	4.65E+05
	R2	0.067	[6]	5.99E+05
	R3	0.062	2	4.26E+05
	Mean	0.064	[1]	4.97E+05	[5]
	SD	0.003		9.05E+04
10	R1	0.060	5	3.82E+05
	R2	0.060	5	3.75E+05
	R3	0.060	5	3.73E+05
	Mean	0.060	5	3.77E+05	21
	SD	0.000		4.77E+03
32	R1	0.036	43	6.28E+04
	R2	0.036	43	6.11E+04
	R3	0.036	43	6.48E+04
	Mean	0.036	43	6.29E+04	87
	SD	0.000		1.83E+03
100	R1	0.000	100	1.42E+03
	R2	0.001	98	1.49E+03
	R3	-0.003	105	-4.16E+02
	Mean	-0.001	101	8.32E+02	100
	SD	0.002		1.08E+03

*    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 the control]

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 based on the geometric mean measured test concentrations gave the following results:
The 72 h EC50 (growth rate) was 0.18 mg/L with 95 % confidence limits of 0.17 - 0.18 mg/L.
The 72 h EC50 (yield) was 0.066 mg/L with 95 % confidence limits of 0.053 - 0.082 mg/L.
The NOEC was 0.034 mg/L an the LOEC was 0.17 mg/L.