Exploring the applicability of the RTgill-W1 and ZFL cell line assays in aquatic hazard assessment of mineral and organic nanomaterials.

Many fish have been used annually to evaluate chemical hazards to aquatic environments; thus, cell-based methods, such as the RTgill-W1 and ZFL cell line assays, have been proposed as alternatives to provide information on acute toxicity to fish, mostly in screening approaches and as part of a weight of evidence (WoE) approach. These methods have been developed using soluble chemicals, which can pose challenges for testing novel types of chemicals, such as nanomaterials. Cellulose nanofiber (CNF) kraft-bleached pulp, chitosan nanoparticles (CS-NP), and silica nanoparticles (SiO-NP) were evaluated regarding their toxicity to fish cells using the RTgill-W1 and ZFL cell line assays (in vitro). Experiments were performed using the original protocols of each assay and modified versions to enhance the bioavailability of nanoparticles (NPs) during in vitro testing. NPs characterization in the exposure media (mean diameter, polydispersity index) was also performed. Both cell lines were exposed to CNF (39-1250 mg/L), CS-NP (4.88 mg/L to 156.25 mg/L), and SiO-NP (7.81 mg/L to 250 mg/L) for 24 h under static culture (original protocols) and orbital shaking at 150 rpm (modified protocols). Fish cell acute toxicity was determined based on cell viability readouts. There was no effect on cell viability for CNF in both cell lines, while SiO-NP and CS-NP presented toxicity. The toxic effect levels of SiO-NP and CS-NP were higher in the static culture than in the orbital shaking. For SiO-NP, ZFL cells showed higher sensitivity to SiO-NP toxicity than RTgill-W1; however, the in vitro data did not correlate with the available acute fish toxicity data (in vivo), and if these data were used for classification purposes under the United Nations' Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), this could lead to overprotective measurements. The NP characterization data demonstrated that all tested NPs exhibit higher aggregation tendencies, which helps explain the observed biological effects. Overall, these results underscore the need for improved in vitro methods to assess the toxicity of NPs to fish cells as well as provide insights into the toxicity of NPs to fish cells.