Project ID Assignment: Project TER-1: Nanotoxicology in terrestrial microcosms
Yuan Ge, John Priester, Allison Horst, Josh Schimel, Roger Nisbet, Jorge Gardea Torresdey,
Patricia A. Holden
Abstract: In soils, ENMs could negatively impact microbial and plant populations, microbial
communities, and ecosystem processes in nutrient cycling. Impacts could occur through either
proximate or distal processes. However, little is known regarding the fates and effects of ENMs
in soil, including agricultural systems. This project aims to quantify the impacts of ENMs to
model terrestrial ecological systems as a function of ENM type and concentration. The
hypotheses are: 1) effects on ecosystem-level processes will be observable through the effects of
bioavailable ENMs (specifically metal oxide nanoparticles) on sensitive or “key” microbial taxa,
2) ENMs can affect soil physical properties that influence water and nutrient availability, thereby
indirectly impacting soil microbes, plants, and their interactions, and 3) the effects of ENMs in
planted, e.g. agricultural, systems, will be through effects on plant-microbe interactions that
exceed the effects on either alone. Our hypotheses are being tested in three related domains,
involving terrestrial environments of differing complexity: grassland soil microcosms to evaluate
the effects of ENMs on 1) microbial community composition and 2) soil – water relations (which
may in turn influence community composition), and agricultural microcosms to evaluate 3) plant
uptake and damage, plus plant-microbe interactions and interactive effects with soil microbial
communities.
We previously reported the time-course effects of nano-TiO2 and -ZnO nanoparticles at varying
concentrations (0, 0.5, 1.0 and 2.0 mg g-1 soil for TiO2, 0.05, 0.1 and 0.5 mg g-1 soil for ZnO) on
a California grassland soil (Sedgwick Reserve, Santa Barbara County) in unplanted microcosms.
The now-published (Ge et al., ES&T, 2011) results include that nano-TiO2 and -ZnO reduced
microbial biomass (as measured by total extractable DNA and substrate induced respiration or
SIR) and altered soil bacterial community diversity and composition (as measured by PCRTRFLP profiles, genotypic richness and Shannon index). We initiated a pyrosequencing
approach in the Fall of 2010 to explore the taxa-specific response of soil bacteria to nano-TiO2
and -ZnO. The voluminous data have been analyzed, which reveal functionally-important,
sensitive taxa; a manuscript has been submitted. The insights into ENM-sensitive taxa--of
significance to N and C cycling--will be extremely valuable to the theme “Molecular, cellular
and organism high-throughput screening for hazard assessment (Theme 2)” which can recruit the
environmentally-relevant, sensitive bacterial taxa for HTS. This project will next assess
treatment effects to ammonia oxidation, where amoA gene copies will be quantified by qPCR to
examine the abundance of the bacterial functional group associated with ammonium oxidization,
TRFLP analysis and associated multivariate statistics will be used to characterize the effects of
nano-TiO2 and -ZnO on ammonia oxidizer community structure, and net and gross N
mineralization and nitrification analyses will indicate ecosystem-level consequences. The linkage
between nitrifier community and ecosystem process (N cycle) will allow for testing the
hypothesis that effects on ecosystem-level processes will be observable through the effects of
nanoparticles on sensitive or “key” microbial taxa.
The observed effects of SRMs on soil microbial community composition and function, as
described above, may be due to proximate or distal effects of ENMs, i.e. on organisms in direct
contact with ENMs or of ENMs affecting soil properties that, in turn, alter organism function.
Because of their high specific surface area, ENMs, even in relatively low amounts, may alter soil
water relations or nutrient availability, which could impose distal effects on biota. For example,
increased water retention—due to the presence of high specific surface area ENMs in soil-- could
increase water content under very dry conditions, thus promoting comparatively greater nutrient
and water availability as soils dry. Alternatively, comparatively lowered water activity due to
higher surface interactions between water and ENMs could impose water stress to soil
organisms. However, little is known regarding the fundamental effects of ENMs on altered soil
water relations. Working from theoretical and empirical frameworks in soil physics and
hydrology, we designed and conducted an experiment to equilibrate nano-TiO2–amended soils
across a range of water potentials, both higher and lower than the native water potential of the asexcavated (grassland, as above) study soil. The change in water content of the soils was
monitored over time, and equilibration was reached. Following equilibration, the soils were
evaluated for final soil water content, nutrient concentrations, and microbial activity; DNA was
extracted to characterize bacterial community shifts. Data are under analysis and manuscripts are
in preparation, but synthesis thus far indicates very modest effects of ENMs on soil water
holding characteristics, but measureable effects on microbial communities according to soil
dryness and ENM addition. This work will contribute to our understanding of possible “distal”
effects that ENMs can have in soil, i.e. of changing soil water holding characteristics relevant to
plant growth and microbial processes.
The third component of this project involves planted microcosms to evaluate plant-microbe
interactions, plant uptake and damage, soil microbial communities, and interactions between
microbes and plants in effecting ENM influences. This project builds upon the considerable
progress made in TER-4 in which Jorge Gardea-Torresdey’s research has shown ENM uptake
and damage in hydroponically-grown and soil-cultivated plants, with magnitudes varying with
plant species and ENM type. During this period, we designed and conducted a highly
collaborative project whereby soybean plants were grown in nano-CeO2 and –ZnO amended
organic farm soil. Plants were grown to maturity (60 days, for dwarf plants), and analyses were
performed to assess food crop consequences, metal compartmentalization and transformation,
and plant damage, microbial community alterations, and N-fixing symbioses impacts. Most
datasets are now complete (e.g. ICP for total metals in all plant tissues and soils, all plant growth
data, plant ultrastructure, N2 fixation potential, plant genetic damage, leaf ROS and chlorophyll,
plant photosystem II, and plant macro/micronutrients); some measurements are in progress (e.g.
microbial community analysis, and plant oxidative damage indicators). Key findings include that
N-fixation is interfered with by CeO2 in a dose-dependent fashion. The result is particularly
interesting, given that N2-fixing symbiotic units (nodules, on soybean root) were similar in
number and mass across all treatments, but their potential for N2 fixation (as measured by the
acetylene reduction assay) appeared significantly affected by CeO2 at high concentrations. The
implications of this finding are that ENMs could affect self-fertilization by soybean, thereby
requiring increased synthetic N administration which, practically, would have profound impacts
economically and environmentally. We also observed copious translocation of Zn into aboveground biomass, suggesting that the food supply is vulnerable. Combined with the unplanted soil
microcosm work described above, the results of this experiment will provide much needed
insight into the effects of ENMs on terrestrial agricultural ecosystems, with an emphasis on food
supply and crops that promote soil fertility.