Introduction

In recent years, many studies have been carried out to investigate the effect of manufactured nano-objects (MNOs) on aquatic organisms and their ecosystem. Development and more common use of MNOs in consumer products lead to an increased exposure, and hence a higher possibility of impact on human health and the environment, in case the MNO cause adverse effects. Nanoparticles are used for example in various household products, industrial processes, and in products spanning applications from construction to health and fitness, and MNOs can end up in the environment, for example, bound to wastewater sludge, ultimately entering into the aquatic environment.

Various aquatic organisms (such as fish, daphnia, artemia, algae) are currently used to predict the potential harmful effects of chemicals, including MNOs, on the aquatic environment. Unicellular protozoa of the genus Tetrahymena sp. are freshwater organisms with widespread distribution in aquatic environments and are at the bottom of the aquatic food chain. Tetrahymena sp. (Protozoa, Ciliata, Oligohymenophorea) are non-pathogenic, free-living eukaryotes and ubiquitously distributed in nature and constituting an important connection between the highly productive and nutrient retaining microbial loop and the metazoans of the classical food chain. This unicellular eukaryote which is bigger than many mammalian cells (approximately 30 µm to 50 µm), can be found in temperate freshwater environments and exhibits nuclear dimorphism (two types of cell nuclei). They have a larger, non-germline macronucleus and a small, germline micronucleus. Tetrahymena sp. has a fast generation time, shows a high level of complexity and it is a typical eukaryotic cell resembling cells in multicellular organisms including humans. In addition, although it is unicellular, it possesses many core processes conserved across a wide diversity of eukaryotes (including humans) that are not found in other single-celled model systems (e.g. the yeasts Saccharomyces cerevisiae).

The protozoan Tetrahymena sp. is an established experimental model in biological studies and it has been extensively used for more than six decades as a toxicological model organism to test the toxicity of different substances using several endpoints.^[¹²^] During the last several years, considerable effort has been devoted to computational modelling of the toxicity of chemicals to Tetrahymena pyriformis for medium and large sized data sets using computational modelling.^[²⁷^] It means that data from standardized tests is highly needed. In recent years, viability of cells of Tetrahymena sp. has been suggested also as a routine test of MNOs toxicity.^[¹^]-[²⁴^] There are several advantages to using Tetrahymena sp. as a biological model for a toxicological test model system in freshwater aquatic toxicology and in bioassimilation experiments:

— abundant information is available about using Tetrahymenasp. in cellular biology, ecology and ecotoxicology and its role in the microbial food web;
— cells of Tetrahymena sp. can easily be cultured at high densities;
— Tetrahymena sp. possesses features of both single eukaryotic cells and whole organisms;
— Tetrahymena sp. plays an important role as grazers of microbes in aquatic environments and balancing bacterio-plankton production;
— Tetrahymena sp. has acceptable sensitivity to exposure to different xenobiotics;
— some species of Tetrahymena possess a genetically fully sequenced macronucleus, thus facilitating the study of changes in gene expression patterns under pollution stress (toxicogenomics);
— Tetrahymena sp. is an invertebrate, lacks the characteristic of vertebrates but can still be used to replace the use of animals in toxicity testing at initial stages of testing;
— Tetrahymena sp. eats anything that fits into their mouth; it has a highly developed system for the internalization of nanoscale and microscale particles which makes them an ideal model system in nanotoxicity and material cellular internalization (bioassimilation) research.

To ensure the sustainable development of nanotechnology, there is a need for hazard identification and risk assessment of MNOs. This document provides a standard protocol intended to generate reliable toxicity and bioassimilation data by using Tetrahymena sp. for evaluation of MNOs in any experimental suspension of MNOs of interest or in samples from freshwater ecosystems.

Tetrahymena is positioned as a primary consumer in the freshwater food chain, so it is considered as a potential vehicle of environmental contaminants. Tetrahymena phagocytic activity is a cost-effective, suitable and rapid assessment tool towards cell internalization (uptake and possible assimilation) of pollutants including particles.^[⁴^] It can act as a very early and sensitive indicator for the toxic effect of various xenobiotic compounds as well as an indication of internalization / bioassimilation of xenobiotics. The effect of MNOs on Tetrahymena can be induced by the ingested (phagocytosed) MNOs, but also by the contact with MNOs (without internalization) or by the metal ions released from metal-containing MNOs in the suspension. The effect of ingested (phagocytosed) material is measured via cell viability (endpoint of effect) measurements. Phagocytic activity is particle internalisation by cells which, in this case, can be measured by the number and appearance of food vacuoles. Detection of MNOs in living cells exposed to a suspension indicates that the suspension contains MNOs that can be internalized by living cells. This can be taken as a characteristic of biological significance of a suspension containing NMOs. “Biological significance” in this case means that material can be internalized (phagocytosed) by cells. In case of exposure to MNOs, the number and appearance of food vacuoles can also be used as a measure that particles of a defined size (which fit into their mouth) are present in a suspension. This can be used as a biological indication of exposure and in parallel the effects of ingested material can be studied. Tetrahymena sp. possesses features of both single eukaryotic cells and whole organisms. Several studies have highlighted their potential as models in in vitro toxicological assessment of chemical pollutants using various endpoints. Tetrahymena based pilot ring test has been initiated by the German Federal Environmental Agency for ecological risk assessment^[¹¹^] and further elaborated by OECD for activated sludge.^[²⁶^] Although the OECD’s working party on manufactured nanomaterials has recently reviewed the relevance of its various test guidelines on traditional experimental models for the testing of MNOs (see Reference [31]), Reference [31] did not review any methods that utilize the Tetrahymena sp. phagocytic activity, as mentioned earlier, is a cost-effective physiological endpoint, which can act as a very early and sensitive indicator for the toxic effect of various xenobiotic compounds as well as an indication of internalization or bioassimilation of xenobiotics. In case of MNO exposure, this endpoint can also serve as a measure of exposure to MNOs in any suspension of MNOs where their cellular internalization is of interest.

PD ISO/TS 4988:2022

Nanotechnologies. Toxicity assessment and bioassimilation of manufactured nano-objects in suspension using the unicellular organism Tetrahymena sp.

Current

Published:

31 Aug 2022