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EC number: 947-656-2 | CAS number: -
The limits of quantification of test substance in this study were 0.02 mg/L for the 0 h time point samples and 0.2 mg/L for the 72 h time point samples. All analytical values were quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 90-97% of nominal and at the end were 83-90% of nominal. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 30% and less than 20% at levels greater than the LOQ. On the basis of the analytical data the nominalconcentrations were used for the calculation and reporting of results. Please find attached method validation report "Analytical Method Validation in Algal media" in the below attached background material section.
Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.
The growth rate (0 to 72 h) was calculated for each replicate culture. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).
The mean growth rates, are given in below table together with the rates expressed as percentages of the control.
Nominal concentration of test substance
Mean measured concentration of test substance
Mean growth rate/day
Percentage ofcontrol (%)
Culture medium control
* Significant differences (p <0.05) from the control
All biological measurements are quoted to 3 decimal places and percentages to the nearest integer.
The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:
95% Confidence Limits
The response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).
The yield mean values are shown in below table, together with the yields expressed as percentages of the control.
Mean yield (0-72 h) 95% CI
Mean yield values are quoted to 3 significant figures and percentages to the nearest integer.
The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:
Additional biological data
The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.625 and 1.25 mg/L test concentrations appeared normal. Algal cells from the nominal 2.5 mg/L concentration appeared deformed and enlarged. Algal cells from the nominal 5.0 and 10 mg/L concentrations appeared deformed.
The validity criteria specified in the OECD 201 guideline are;
1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test, cell particle density increase (measured as a surrogate for biomass) was 85.6 over the 72 h for the control.
2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 8%.
3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and in this test, was calculated to be 1.1%.
Based on the study results, it was concluded that the study has fulfilled validity criteria.
A study was conducted to determine the acute toxicity study of the test substance, C10-12 and C18-unsatd. DEA, to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.892 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.527 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.54, 1.2, 2.3, 4.6 and 8.8 mg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50, ErC10 values (for growth rate) and EyC50, EyC10 values (Cell particle density) were found to be 2.9, 1.4 and 1.8, 0.9 mg/L respectively. The NOEC and the LOEC values were found to be 1.25 mg/L and 2.5 mg/L, respectively, for growth rate and 0.625 mg/L and 1.25 mg/L, respectively, for biomass. All validity criteria were considered to be met. Under the study conditions, the 72 h ErC50, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively (Scymaris, 2018).
Based on the study results, the 72 h ErC50, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively.
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