A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

When a chemical company wants to put a new chemical or another material to the market, they need to deliver data from a series of ecotoxicity tests. The algal test is one of the three mandatory tests that needs to be performed. The algal test suffers from the fact that it's quite challenging to perform.

For this reason, we have developed the LEVITATT testing platform. The LEVITATT uses LED light to avoid problems with excessive heat generation. Furthermore, it ensures a uniform light field.

It has a compact size, meaning that you don't need much space to perform the algae test, but at the same time, it allows for using enough sample volume to perform chemical analysis and characterization before, during, and after testing. This is especially important when you test nanomaterials. This video will demonstrate the use of LEVITATT for algal toxicity testing of nanomaterials and other difficult substances.

The demonstration consists of the following four steps. First, describe the components of the LEVITATT. The second step is to prepare the solution for algae growth media.

And the third step is to give an overview of the procedure of how to perform an algae toxicity test. The final step is to measure the increase in algal biomass by fluorometer. The LEVITATT can be placed in the temperature-controlled cabinet of room.

This allows for stable temperature throughout the exposure. The setup is placed on an orbital shaker to allow for rotation of the samples during the test. The test is conducted in 20-milliliter scintillation vials containing four milliliters exposure medium.

A screw cap with a drilled hole allows for CO2 exchange with air during the exposure. The LED source is sandwiched between two plastic sheets and located in the center of each casing. The placement creates a homogeneous light field and minimizes shading between the samples.

Each casing is mounted with clamps and can hold up to five samples. The compact size minimizes the space required to perform the experiment. To prepare the stock solutions, weigh out the appropriate amount of each compound.

The stock solution consists of various salts vital for the growth of the algae. When the appropriate amount of salts have been weighed out, they are diluted with ultra-pure water. The stock solutions are sterilized either by filtrating or autoclaving.

After sterilization, the stock solutions can be stored in the dark at four degrees Celsius. Prepare a series of exposure concentrations. The factor between the exposure concentrations should not exceed 3.2.

The appropriate volume of exponentially growing algae is added to each test concentration. The final cell density should be approximately 10, 000 algal cells per milliliter. It is key that the pre culture is exponentially growing to fulfill the validity criteria of the test.

The measuring vials are mixed to allow for a homogeneous distribution of algae and compound before sampling. The samples are poured into beakers, and four milliliters is pipetted into 20-milliliter scintillation vials. The scintillation vials are now capped.

Pipette 0.4 milliliter from each concentration. Add 1.6 milliliter acetone to disrupt the cell wall and release the chlorophyll to the liquid phase. Each glass vial is now close tightly to avoid evaporation of acetone during storage.

The capped scintillation vials are now randomly distributed in the LEVITATT and incubated for 72 hours. Measure the light intensity and the temperature at the start and end of incubation to ensure compliance with desired guidelines. Sub-sampling is done at 24 hours'interval by pipetting 0.4 milliliters from each scintillation vials into glass vials.

Add 1.6 million acetone to disrupt the cell wall and release the chlorophyll to the liquid phase. After acetone is added, screw the lids on tightly and leave the samples in the dark until analysis. Use a fluorometer to quantify the biomass in the sample acquired throughout the testing period.

The settings on the fluorometer should be 420 nanometers as excitation wavelength and an emission wavelength of 671 nanometers. Each individual sample is measured three times, and the relative values are noted. The relative values from the fluorometer is used to calculate the growth rate which will be used for further data treatment.

Use a statistical software to fit a nonlinear regression curve to the calculated growth rate data. An example of a nonlinear regression curve could be a log-logistic of viable function. The associated 95%confidence interval is shown alongside the model fit.

Regularly effective concentration values such as 10, 20, and 50%inefficient are used to report the result of an ecotoxicity test. In conclusion, the LEVITATT provides a compact platform for algal toxicity testing of regular chemicals compliant with international standardized guidelines. Furthermore, the setup provides a robust platform for testing of difficult substances that interfere with the passage of light towards the algal, such as nanomaterials or colored substances.