GNOME – Investigation of the mode of action when co-exposing the nematode Caenorhabditis elegans to Cd and TiO2 nanoparticles

The use of industrial manufactured nano-scale particles is rising continuously
in consumer products and industrial areas. A study of Keller and Lazareva
(2013) estimated that in 2010 8 to 28 % of 260000 to 309000 metric tons produced
nanoparticles world wide were released into soils. Consequently, the
environmental fate of nano-materials in aquatic and soil systems has been the
focus of many recent studies (Schaumann et al., 2015, Petersen and Henry,
2012), but information on ecotoxicity of nanoparticles is still limited (Handy
et al., 2012b, Guzman et al., 2006, Nowack et al., 2012). Among currently
applied nano-materials, nano-scale titanium dioxide (n-TiO2) is probably the
most environmentally relevant nanoparticles with regard to potential exposure
in sediments in Europe of 1.9 mg kg-1yr-1 (Sun et al., 2013). A major
gap in our knowledge on the toxicity of n-TiO2 is the interaction with other
contaminants that are potentially present in environmental matrices. If n-
TiO2 accumulates in the ppm range in European sediments as the projections
suggest, it will likely interact with other contaminants in the environmental
matrices, potentially increasing toxicity for different organisms. Due to the
photo-catalytic activity of tested n-TiO2 (P 25), exposure to solar radiation
may add an additional risk component. Thus, particularly during low water
level and mudflats sediments of industrial rivers, where organisms are often exposed
to multiple contaminants and periodically to sun light could be at risk.
This project quantifies the effect that co-occurrence of n-TiO2 and cadmium,
with and without exposure to sun light, have on nematodes as frequent members
of the sediment meiofauna, and investigates the mixture toxicity model
and the mode of action. According to ISO 10872 Caenorhabditis elegans is
used in the nematode test in medium with the toxic endpoints of growth and
reproduction. Nematodes were exposed to n-TiO2 (P 25, with a primary particle
size of 21 nm) and Cd in single and co-exposure for 72 h. Additionally
two test conditions were tested. The incubation under dark conditions and
treatment with simulated solar radiation for 30 min.

Single substance tests in the dark resulted in an EC50 value (for reproduction)
of 0.6 mgL-1 for Cd and > 200 mgL-1 for n-TiO2. Results from the nematode
test showed that solar light significantly increased toxicity of n-TiO2 by
a factor of 2 and confirmed previous results by et al. (2014). Supplementing
the n-TiO2 suspension (40 mgL-1) with 50 μgL-1 Cd inhibited reproduction
and amplified this effect by a factor of 2 compared to n-TiO2 alone under solar
light. Further, exposure to solar light led to synergistic inhibition effect (80
% reproduction inhibition) in the mixture. Assuming that the photo-catalytic
activity of n-TiO2 damages cell membranes under solar light. Therewith Cd
uptake to the cells is facilitated and promoted adverse effects of cadmium. A
cell-impermeant stain (NucGreen Dead 488) was used to test for membrane
integrity under solar radiation and co-exposure. However, no membrane instability
caused by n-TiO2 could be detected with NucGreen. An alternative
hypothesis focused on interference with the cellular calcium metabolism:
as Cd is known to induce intracellular Ca signalling as part of protective cell
processes, the effect of the mixture on intracellular calcium release was investigated.
The red fluorescent dye rhodamine was loaded into the intestinal cells
to measure the free intracellular calcium level during single and co-exposure
and under solar radiation and dark conditions. Cd increased the free calcium
level in the cells (in single and co-exposure) in opposite to n-TiO2 alone when
incubated in the dark. Under solar radiation, co-exposure of n-TiO2/Cd decreased
intracellular calcium signalling compared to the control. This could be
explained by ROS production through solar light exposure of n-TiO2 followed
by lipid peroxidation and loss of membrane integrity. This could provide the
possibility of Cd influx into the cells, resulting in a blockage of calcium release
from the calcium stores. This model would explain for the first time the sensitization
of Caenorhabditis elegans towards Cd in the presence of n-TiO2 and
solar light with its major consequences for the nematodes’ life cycle.