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EC number: 229-059-2
CAS number: 6408-50-0
The following data show that the cell concentration of the control
cultures increased by a factor of 201 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 103 cells per mL
Mean cell density of control at 72 hours : 1.00 x 106 cells per mL
The mean coefficient of variation for section by section specific growth
rate for the control cultures was 16% and hence satisfied the validation
criterion given in the OECD Guideline which states the mean must not
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%.
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
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
7.8 at 0 hours to pH 7.9 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.
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 100% v/v saturated solution test cultures were observed to be green
A study was performed to assess the effect of the test item on the
growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.
Information provided by the Sponsor indicated the test item was
insoluble in water. Preliminary 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 and high shear
A preliminary media preparation trial indicated that a saturated
solution method of preparation was appropriate for this test item.
Following a preliminary range-finding test, Pseudokirchneriella
subcapitata was exposed to a solution of the test item at a nominal
concentration of 100% v/v saturated solution (six replicate flasks) for
72 hours, under constant illumination and shaking at a temperature of 24
± 1 °C. The test item solution was 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 liters discarded in order to
pre-condition the filter) to produce a 100% v/v saturated solution of
the test item.
Samples of the algal populations were removed daily and cell
concentrations determined for each control and treatment group, using a
Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 and 72 hours showed measured test
concentrations of less than the limit of quantification (LOQ) of the
analytical method employed were obtained which was determined to be
0.057 mg/L. This does not infer that no test item was in solution, just
that any dissolved test item present was at a concentration of less than
Exposure of Pseudokirchneriella subcapitata to the test item gave EC50
values of greater than 100% v/v saturated solution. The No Observed
Effect Concentration was determined to be 100% v/v saturated soltuion.
This study showed that there were no toxic effects at saturation.
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