Aim, materials and methods – Study report of toxicity – GREENQUAT® BT

Test System

Specification
Unicellular freshwater green alga.

Genus                               Desmodesmus

Species                             subspicatus

SAG Strain Number        86.81

Taxonomic position         Chlorophyta – Chlorophyceae

Origin and Culture

The culture of Desmodesmus subspicatus was obtained in February 2020. The algae are kept as stock culture on solid agar at 7°C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

CONDUCT OF THE STUDY

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

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

Microscopical Observations

In the following table, the appearance of the alga at the end of the test is stated:

Growth Rate and Yield

From the cell numbers, the Growth Rate μ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table: