Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 02 April 2018 and 03 May 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 14-0383
Physical state/Appearance: Colorless liquid
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

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 immediate quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

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.

Preparation
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm 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 Acrcaop filter (approximately 500 mL discarded in order to pre condition the filter)

Results
Stirring Period and Treatment Concentration Found
(mg/L)
24 Hours Centrifuged 10000 g 9.25
24 Hours Centrifuged 40000 g 8.28
24 Hours Filtered ~ 100 mL discarded 8.85
24 Hours Filtered ~ 500 mL discarded 9.52
48 Hours Centrifuged 10000 g 9.22
48 Hours Centrifuged 40000 g 8.30
48 Hours Filtered ~ 100 mL discarded 9.20
48 Hours Filtered ~ 500 mL discarded 11.0

Discussion
It was evident from the results obtained that there was no significant increase in the dissolved test item concentration obtained when the preparation period was extended beyond 24 hours. Furthermore, there were no significant differences between the pre-treatment types.

Therefore, for the purposes of testing the test item was prepared as a saturated solution at an initial loading rate of 50 mg/L, stirred via propeller stirrer for 24 hours prior to the removal of any undissolved test item by filtration through a 0.2µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a nominal concentration of 9.5 mg/L.

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of 9.5 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 (550 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 Gelman Acrocap filter (first approximate 500 mL 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 (900 mL) of each of the stock solutions was separately inoculated with algal suspension (2.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

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.
An initial experiment was terminated after 24 hours exposure due to lack of growth in the control cultures.

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 kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 10.2 at 72 hours.

Nominal and measured concentrations:

Range-finding test: 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.
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.

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. The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 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

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive tests, additional sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use.

Range-Finding Test
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers 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 sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 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 immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.

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.
An initial experiment was terminated after 24 hours exposure due to lack of growth in the control cultures.

Experimental Preparation
A nominal amount of test item (550 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 Gelman Acrocap filter (first approximate 500 mL 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 (1700 mL) of each of the stock solutions was separately inoculated with 7.9 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 prepared concentration 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 and 72 hours.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with solution were used for the control and three flasks each completely filled 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 1.08 x 10^6 cells per mL. Inoculation of 1700 mL of test medium with 7.9 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 22, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
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.

Water Quality Criteria
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 temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

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 immediate quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
A further sample of each test concentration containing no algal cells was incubated alongside the test to provide samples for uninoculated analysis at 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 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 0.025 to 12 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.015 mg/L, to 10 mg/L indicating possible adsorption of the test item to the algal cells present.

Definitive Test
Growth Data
Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 0.27 mg/L
ErC20 (0 to 72 hour): 0.44 mg/L
ErC50 (0 to 72 hour): 0.99 mg/L; 95% confidence limits 0.76 to 1.3 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 and 0.16 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.16 mg/L. Correspondingly the LOEC based on growth rate was 0.35 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.11 mg/L*
EyC20 (0 to 72 hour): 0.17 mg/L
EyC50 (0 to 72 hour): 0.38 mg/L; 95% confidence limits 0.29 to 0.49 mg/L
Where EyCx is the test concentration that reduced yield by x%.
Statistically significant differences (P<0.05) were observed between the control and all test concentrations. As such it was not possible to determine either a NOEC or LOEC value based on inhibition of yield.
* Value determined from the equation for the fitted line as at the lowest test concentration employed, 24% inhibition of yield occurred.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 137 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.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.84 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 to 72 hours) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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 0.16 mg/L test cultures. Some slightly enlarged cells were observed in the 0.35 mg/L test cultures, cell debris was observed in the 1.6 and 4.0 mg/L test cultures whilst no cells were observed in the 13 mg/L test cultures.

Water Quality Criteria
Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 10.2 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

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 and 0.16 mg/L test cultures were observed to be green dispersions. The 0.35 mg/L test cultures were light green dispersions, the 1.6 and 4.0 mg/L test cultures were extremely pale green dispersions whilst the 13 mg/L test cultures were slightly hazy dispersions.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) 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 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration 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.

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 18 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.13 and 9.4 mg/L (52% to 84% of the 0-Hour measured test concentrations). Analysis of additional samples prepared at the start of the test which contained no algal cells showed measured test concentrations in the range of 0.10 to 8.2 mg/L were obtained at 72 hours indicating that the losses observed were not due to adsorption, rather instability and/or losses through volatilization despite having taken all precautionary measures against such losses.

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. . 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 Percentage of the 0-Hour Measured Test Concentration
1.0	0.16	87
3.2	0.35	80
10	1.6	88
32	4.0	93
100	13	72

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	6.95E+03	6.20E+05	-	-
	R2	7.36E+03	5.02E+05
	Mean	7.16E+03	5.61E+05
0.10	R1	7.63E+03	5.97E+05	[2]	[7]
	R2	6.45E+03	6.07E+05
	Mean	7.04E+03	6.02E+05
1.0	R1	6.37E+03	4.99E+05	0	10
	R2	6.16E+03	5.11E+05
	Mean	6.26E+03	5.05E+05
10	R1	6.22E+03	2.24E+04	75	98
	R2	5.51E+03	1.16E+04
	Mean	5.87E+03	1.70E+04
100	R1	7.89E+03	7.86E+03	103	100
	R2	7.04E+03	5.10E+03
	Mean	7.47E+03	6.48E+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

R   = Replicate

-    = Not applicable

[ ] = Increase in growth compared to controls

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	22 Hour	47 Hour	72 Hour	72 Hours
Control	R1	7.7	1.65E+04	1.02E+05	7.45E+05	10.2
	R2		1.36E+04	5.03E+04	4.99E+05
	R3		1.85E+04	9.84E+04	6.71E+05
	R4		1.60E+04	1.01E+05	7.16E+05
	R5		1.59E+04	8.82E+04	7.35E+05
	R6		1.65E+04	8.10E+04	7.36E+05
	Mean		1.62E+04	8.69E+04	6.84E+05
0.16	R1	7.8	1.16E+04	5.96E+04	5.41E+05	10.1
	R2		1.42E+04	5.64E+04	4.89E+05
	R3		1.19E+04	5.22E+04	5.42E+05
	Mean		1.26E+04	5.61E+04	5.24E+05
0.35	R1	7.8	1.11E+04	2.90E+04	4.16E+05	9.9
	R2		1.13E+04	3.15E+04	4.06E+05
	R3		1.12E+04	2.33E+04	3.82E+05
	Mean		1.12E+04	2.79E+04	4.01E+05
1.6	R1	7.8	9.36E+03	9.30E+03	2.34E+04	8.3
	R2		8.71E+03	8.36E+03	1.44E+04
	R3		9.97E+03	1.01E+04	1.47E+04
	Mean		9.35E+03	9.24E+03	1.75E+04
4.0	R1	7.8	9.94E+03	9.15E+03	1.36E+04	8.1
	R2		8.57E+03	9.15E+03	1.01E+04
	R3		8.33E+03	9.15E+03	1.10E+04
	Mean		8.95E+03	9.15E+03	1.15E+04
13	R1	8.1	6.37E+03	5.25E+03	6.42E+03	8.2
	R2		7.01E+03	4.61E+03	6.13E+03
	R3		5.54E+03	4.49E+03	8.48E+03
	Mean		6.31E+03	4.78E+03	7.01E+03

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from3 counts for each of the replicate flasks

R      = Replicate

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

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.054	0.073	0.079
	R2	0.046	0.052	0.092
	R3	0.059	0.067	0.077
	R4	0.053	0.074	0.078
	R5	0.053	0.068	0.085
	R6	0.054	0.064	0.088
	Mean	0.053	0.066	0.083

R   =Replicate

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.069	-	7.40E+05	-
	R2	0.064		4.94E+05
	R3	0.068		6.66E+05
	R4	0.069		7.11E+05
	R5	0.069		7.30E+05
	R6	0.069		7.31E+05
	Mean	0.068		6.79E+05
	SD	0.002		9.42E+04
0.16	R1	0.065	4	5.36E+05
	R2	0.064	6	4.84E+05
	R3	0.065	4	5.37E+05
	Mean	0.065	5	5.19E+05	24
	SD	0.001		3.03E+04
0.35	R1	0.061	10	4.11E+05
	R2	0.061	10	4.01E+05
	R3	0.060	12	3.77E+05
	Mean	0.061	11	3.96E+05	42
	SD	0.001		1.71E+04
1.6	R1	0.021	69	1.84E+04
	R2	0.015	78	9.40E+03
	R3	0.015	78	9.67E+03
	Mean	0.017	75	1.25E+04	98
	SD	0.003		5.11E+03
4.0	R1	0.014	79	8.55E+03
	R2	0.010	85	5.06E+03
	R3	0.011	84	5.97E+03
	Mean	0.012	83	6.53E+03	99
	SD	0.002		1.81E+03
13	R1	0.003	96	1.42E+03
	R2	0.003	96	1.13E+03
	R3	0.007	90	3.48E+03
	Mean	0.004	94	2.01E+03	100
	SD	0.002		1.28E+03

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

R   = Replicate

SD   =Standard Deviation

-    = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC of FRET 14-0383 were 0.99, 0.27 and 0.16 mg/L.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 9.5 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 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 through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL 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. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 18 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.13 and 9.4 mg/L (52% to 84% of the 0-Hour measured test concentrations). Analysis of additional samples prepared at the start of the test which contained no algal cells showed measured test concentrations in the range of 0.10 to 8.2 mg/L were obtained at 72 hours indicating that the losses observed were not due to adsorption, rather instability and/or losses through volatilization despite having taken all precautionary measures against such losses. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.16, 0.35, 1.6, 4.0 and 13 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	0.99	0.76	-	1.3	0.16	0.35
Yield	0.38	0.29	-	0.49	Not determined due to nature of data	Not determined due to nature of data

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 9.5 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 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 through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL 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. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 18 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.13 and 9.4 mg/L (52% to 84% of the 0-Hour measured test concentrations). Analysis of additional samples prepared at the start of the test which contained no algal cells showed measured test concentrations in the range of 0.10 to 8.2 mg/L were obtained at 72 hours indicating that the losses observed were not due to adsorption, rather instability and/or losses through volatilization despite having taken all precautionary measures against such losses. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.16, 0.35, 1.6, 4.0 and 13 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations: 

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	0.99	0.76	-	1.3	0.16	0.35
Yield	0.38	0.29	-	0.49	Not determined due to nature of data	Not determined due to nature of data

Key value for chemical safety assessment

EC50 for freshwater algae:

0.99 mg/L

EC10 or NOEC for freshwater algae:

0.16 mg/L