Project MFW-1: Marine organismal nanotoxicology: Studying Nanomaterial Interactions at the
Molecular, Cellular, Organ, and Systemic Levels
Gary N. Cherr, Elise Fairbairn, Bryan Cole, Carol Vines
Abstract: Engineered NMs are likely to exhibit differing toxicological effects on individual organisms,
populations, and ecosystems in different marine habitats. In this project we are utilizing ecologically
relevant model marine organisms that include algae and invertebrates, as well as both embryonic and
adult life stages. The differences in fate and transport of diverse NMs, as well as differences in their
interactions with structurally and physiologically diverse organisms must be accounted for when
predicting NM impacts to marine populations and ecosystems. The individual components within this
project include determining: 1) the physiological effects of NMs on adult marine organisms’ cells and
tissues, 2) the effects of NMs on developing marine embryos, and 3) effects of NMs on both the cellular
and population-level endpoints in marine phytoplanktion (in collaboration with Theme 5, Project 24). We
are utilizing a broad range of organisms that include single cell marine phytoplankton (diatoms and green
algae), developing embryos from marine invertebrates (sea urchin, abalone, mussel), and adult marine
bivalve molluscs in order to address the hypothesized diverse responses we expect across phyla, as well
as in different habitats (water column and benthic environments). The mechanistic effects of NM
exposure being investigated include endpoints and high content screening (HCS) approaches have been
directly adapted from high throughput screening (HTS) results from in vitro studies with mammalian cells
in culture by Theme 2. These include: generation of reactive oxygen species (ROS), mitochondrial
function, membrane integrity, apoptosis, phagocytosis, inflammation, etc. These basic parameters of
cytotoxicity and sublethal responses are being linked directly to other projects within Theme 5 at the
larger-scale organismal/population levels. Our overall goal is to understand differences and similarities in
the responses of very different organisms and life stages from distinct habitats to diverse NMs. These
aims are described below.
Research on this project is being conducted in close collaboration with CEIN researchers in Themes 1, 2,
and 3. Relating to Aim 1 (physiological effects of NMs on cells and tissues), collaborative studies with
Theme 3 on the behavior of metal oxide NMs in seawater have been published (Keller et al., 2010; Env.
Sci. Tech.) and new approaches for stabilizing CNTs in seawater and physiologically relevant media are
being developed by Theme 1 in collaboration with the current project. In addition, the trophic transfer of
NMs through a simplified system has been published in a collaborative study with Theme 4 (Werlin et al.,
2011; Nat. Nano.); our data contribution was on oxidative damage as a result of the consumption of NMexposed bacteria. Our current focus is on marine mussel (bivalve mollusc) blood cells or hemocytes, both
in vitro and in vivo, for the assessment of sublethal cellular endpoints of toxicity and function. Since
hemocytes are the immune cells of marine invertebrates, their functional capabilities are critical for
organism and population level health. In vitro exposure to ZnO NMs inhibits hemocyte phagocytosis of
fluorescent yeast particles. In vivo exposure of mussels to ZnO NMs (collaboration with Theme 5, Project
24) did not reduce hemocyte phagocytosis and may relate to adaptive mechanisms for sequestration of
free Zn++ at the organismal level. For hemocytes, ZnO NMs alter plasma membrane membrane integrity
and induce reactive oxygen species, and these responses are related to the decrease in phagocytosis. The
impacts of selected metal oxide NMs (TiO2, CeO2, and ZnO ) are being prepared for publication.
Recently we have studied other NMs in order to compare responses of hemocytes to different NMs. The
effects of SWCNTs on hemocyte function as well as toxicity have been investigated. These SWCNTs
were introduced as nanotori due to the solvent dispersion methodology. We are now initiating exposures
of cells to SWCNTs using a dispersion method with natural organic matter. In vitro exposure of mussel
hemocytes to SWCNTs caused increased production of reactive oxygen species (ROS) and in some cases
can impair the hemocytes’ ability to phagocytose foreign particles. These effects occur at different
concentrations, depending on the SWCNTs used:



HiPCO SWCNTs interfered with phagocytosis at concentrations as low as 0.4 ppm whereas there
was no effect of exposure to SG65 or P2 CNTs even above 10 ppm;
HiPCO and SG65 SWCNTs caused increased cellular production of ROS at concentrations as
low as 1 and 6.5 ppm, respectively. This effect was apparent after just 3 hours when exposed to
SG65 and at 20 hours exposure time with HiPCO CNTs.;
The P2 SWCNTs had no effect on mussel hemocytes, although greater concentrations (probably
not environmentally relevant) still need to be tested.
While distinct differences in subcellular and celluar responses of mussel hemocytes, to different SWCNT
exposures was apparent, the basis for these differences is not clear at present. These differences may be
related to SWCNT size and form, purity, or the catalyst used. The results of these initial studies with
SWCNT nanotori need to be repeated with CNTs dispersed with natural organic matter rather than
solvents.
Based on new findings from Theme 2 regarding inflammasome activation in mammalian cells in culture
by CNT exposure, we have adapted the inflammasome assay (Magic Red detection of cathepsin enzyme
activity) to mussel hemocytes using scanning laser confocal microscopy. We have modified the assay for
a cooler, higher osmotic medium using a positive control and will now assess the effects of CNTs on
inflammasome activation in these invertebrate blood cells.
In Aim 2, we are investigating the responses of developing embryos to NMs as these early life stages are
typically more sensitive than the adults, and impacts on development direcly relate to population-level
changes. Since all externally developing embryos exhibit distinct phases of development (early cleavage
stages with little gene transcription and later differentiation/gene activation stages), toxicological impacts
of NMs on development can be very significant to normal embryo development and thus recruitment of
new individuals to the population. To date we have linked the physical behavior of metal oxide NMs in
seawater with embryo toxicity (Fairbairn et al., 2011, J. Haz. Mat.). Several key findings were that ZnO
NMs were toxic to developing sea urchin embryos at the low ppb range and the Fe-doped ZnO NMs were
not significantly less toxic than non-doped particles; this differs from what has been observed in
mammalian cells and underlines the importance of comparative toxicological studies. Related to this
study we have found that ZnO NMs induce apoptosis in developing embryos at specific stages of
development, and that this may be related to observed abnormal development in later stage embryos.
These experiments are being repeated and will be prepared for publication. Another metal oxide NM,
CuO, has also been investigated as recently CuO NMs have been found to specifically inhibit hatching in
zebrafish embryos (Theme 2, Project 8). When zebrafish embryos were exposed to 0.5-200 ppm CuO
NM, there is a significant and dramatic decrease in hatching success with no corresponding increase in
either mortality or gross developmental abnormalities. We expanded on these results by investigating the
effects of CuO NM on marine invertebrate embryos, including sea urchins, abalone, and mussels. The
hatching enzyme in sea urchins shares homology with the zebrafish hatching enzyme so we hypothesized
that we would observe a similar inhibition of hatching in sea urchins. While little is known regarding the
hatching enzymes of mussels and abalone, the embryos of all three species are known to be very sensitive
to Cu2+, with EC50s in the low ppb range. Embryos were exposed to CuO NM from the early cleavage
stage until hatching, or from 9 hours post fertilization until hatching, and were then assessed for mortality,
developmental abnormalities, and hatch success. There was no specific effect of the CuO NM on
hatching, even though developmental abnormalities were observed at the low ppb range. Unlike results
with tropical freshwater zebrafish embryos where inhibition of hatching was independent of
developmental abnormalities, we did not observe an effect on hatching success in temperate marine
embryos but did observe developmental abnormalities at very low NM levels.. Once again, these results
highlight the importance of assessing NM effects on different biological systems under different
environmental conditions.
In Aim 3, we are focusing on the toxicological responses of marine phytoplankton to different NMs.
Phytoplankton are important organisms as they form the base of marine food webs and may also be a
route of exposure for higher trophic organisms. In fact phytoplankton are the primary source of food for
larval marine invertebrates such as sea urchins. We have been establishing for the first time high content
assays with phytoplankton for assessing impacts of NM exposure. These endpoints include oxidative
stress, membrane integrity, and mitochondrial function. Results from these fluorescence-based assays
with phytoplankton (four species) are being linked to ecosystem-level responses including phytoplankton
growth and photosynthesis (collaboration with Theme 5, Project 24). In two species of marine
phytoplankton, Dunaliella tertiolecta and Isochrysis galbana, we have performed high-content screening
for multiple cytotoxicological endpoints of exposure to metal oxide nanomaterials (ZnO, AgO, CuO). In
most cases, each species responded differently to a particular nanomaterial, though all materials tested
were found to have an effect at some concentration. This work is being combined with information on the
effects of the same nanomaterials on the growth rates and primary production being conducted by the
Theme 5, Project 24, and is being prepared for publication. To date we have found:
 ZnO nanoparticles were found to significantly impact mitochondrial membrane potential in
Dunaliella at concentrations as low as 1 ppm, but did not cause damage to membranes over a 48
hr exposure period. Conversely, there was no effect of ZnO on Isochrysis mitochondria, but
membrane damage was observed at 10 ppm.
 CuO nanoparticles significantly impacted mitochondria and increased production of ROS in both
study species at all concentrations tested (1-10 ppm). Further work will examine the effects at
lower concentrations.
 Ag nanoparticles significantly impacted Dunaliella mitochondria, but were not directly cytotoxic,
nor did they cause production of ROS. In Isochrysis Ag NMs were found to be directly cytotoxic
at concentrations above 5 ppm, and significantly increased ROS production and impacted
mitochondrial function at lower, sublethal doses.
Differences in responses of phytoplankton may relate to physiological differences between species.
These include a formidable silica frustule protecting diatoms but the presence of a typical cell wall in
green algae. The approaches we have developed for phytoplankton (as well as for mussel hemocytes) will
be applied to organisms that are part of larger mesocosm experiments in theme 5, Project 24. We are also
expanding our CNT investigations using marine embryos and phytoplankton, and SWCNTs dispersed in
natural organic matter.
Our results in marine organisms highlight the importance of toxicity assessments from both in vitro and in
vivo models, and from organisms from a variety of ecosystems and developmental life stages. For
example, CuO NMs had very different results with freshwater zebrafish embryos compared to marine
invertebrate embryos. Furthermore, our results with ZnO NM indicated that Fe-doped ZnO NMs were
not less toxic to developing sea urchin embryos compared to pure ZnO NMs, in contrast to the results
observed in cell culture. However, the basic inflammasome response appears conserved between
mammalian and invertebrate systems and we hypothesize that both could be initiated by CNTs. It is
important that stakeholders understand that effects of NMs in one biological system/environmental
medium may not be similar to responses observed in other organisims from different environments. This
concept is essential to understanding/predicting the global toxicological impacts of engineered NMs.