Alga

Four days before the start of each test, an aliquot of the permanent culture was brought into pre-culture medium and incubated for 96 hours. The resulting culture is growing exponentially.

Before usage, the pre – culture was checked for the absence of cell aggregates and the cell number of culture was determined via photometric measurement.

Performance of the Study

For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.41 mL) to achieve a cell concentration of approximately 2-5 * 103 cells/mL. In this mixture, the pH-value was measured. Samples for the analytical determination were taken.

For the control, nutrient medium was used instead of test item solution. The test vessels were filled with 45 ± 1 ml with the respective test solution and incubated for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was calculated based on the determination of the light absorption at 440 nm. After the test, the pH value in treatments and control was measured again.

At the end of the test, the treatments were examined microscopically in order to assess the appearance of the alga and detect abnormalities (e.g. caused by the exposure to the test item).

Experimental Conditions

Date: 06. – 09. January 2019
Treatments tested: 1.0 / 1.0 / 100 mg/L nominal concentration
Number of replicates six replicates for the control
three replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 21.3 – 22.8 °C
Lighting: 5300 Lux
Control: deionized water with nutrient medium and alga
Treatment: test solution and alga
Date: 06. – 09. January 2019
Treatments tested: 1.0 / 1.0 / 100 mg/L nominal concentration
Number of replicates six replicates for the control
three replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 21.3 – 22.8 °C
Lighting: 5300 Lux
Control: deionized water with nutrient medium and alga
Treatment: test solution and alga

CALCULATIONS

The data were evaluated after the test with Excel®. Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

Cell Numbers

From the absorption values, cell numbers were calculated using the following equation:

y(t) = A + B * x(t)

with:

A 0
B 1078
y (t) nominal number of cells at time t
x (t) absorption (40 mm cuvette, 440 nm) at time t

The equation was calculated through microscopic measurement of the cell concentration and the absorption of seven different algal concentrations. The data were evaluated using linear fit.

Growth Rate

The specific growth rate μ was calculated using the following equation:

with:

μ  growth rate;
tn   time of the last measurement in days
N0 nominal number of cells at time t0
Nn  nominal number of cells at time tn 

Yield

The yield was calculated using the following equation:

Yield = N72 -N0

N0 number of cells at time t = 0 h
N72 number of cells at time t = 72 h

Inhibition

All inhibition values were calculated by comparing the value for the endpoint of the treatment with the respective mean value of all controls (100%). Inhibition values are given in percent.