Evidence for this can be found in Figure 3.2, which reveals that, contrary to the second hypothesis, these ENMs in fact increase ion release from contaminated soils. However, the identity and amounts of ions released was dependent on soil type, with the total change in ion release for a given soil being roughly proportional to its CEC . Fig. 3.2B shows that all three ENMs increase release of Mg2+ in grass soil, which may be the cause of the pH increases seen in grass soil since Mg2+ is a basic cation. While no consistent corresponding changes in ion concentration were seen in farm soil, the decreases in pH may have been due to the release of H+ stored in the soil. The farm soil used here had relatively low amounts of basic Mg2+ and K+ and low cation exchange capacity and so has the lowest buffering capacity of the soils in this study. Potting soil had the highest concentrations of basic ions and CEC and correspondingly showed no changes in pH due to the presence of ENMs. Cu2 consistently increased Na and S levels in the soil because both of these elements are major components of the soluble composite matrix the Cu2 ENMs are embedded in and are released as the composite dissolves in water. Similarly, it was found that these ENMs either had no effect or slightly increased the amount of water extractable or bio-available P . This is likely due to the same mechanism described above, namely the replacement of PO4 3- ions on soil surfaces by ENMs. Additionally, since these ENMs already possess or rapidly develop negative surface charges in soil solution they will not attract negatively charged phosphate ions as readily and thus would not inhibit their mobility or bio-availability.Given the range of sizes, morphologies,garden plastic pots and chemical properties they encompass, ENMs may be taken up into plant root tissues and transported through the vascular system in several ways.
It has been known for some time that metal ions can be transported both apoplastically1 and symplastically in plants, but it has only been fairly recently that more definite mechanisms of ENM transport between different plant tissues have been put forth and tested . One of the key barriers in plant roots that play a role in uptake is the Casparian strip , a hydrophilic thickening of the primary cell wall and middle lamella of root cells in the endodermis that is composed primarily of lignin. The CS blocks apoplastic flow into the cortex and vascular bundle and so water and ions must either penetrate the cellular membranes of these endodermal cells or flow symplastically through plasmodesmata in order to reach the xylem and phloem and so be transported throughout the plant.Another possible pathway for ENMs to penetrate into the vascular bundle is to enter through the root tip, which does not have a CS. This has implications for the uptake of ENMs by plant roots, for if ENMs are bound in aggregates or complexes, or if their primary particle size is above a certain threshold, they will not be able to pass through endodermal cell pores or plasmodesmata .Reports of ENM uptake in both soil and hydroponic systems vary and likely depend on the interaction of several factors, including ENM composition, crystal structure, primary particle size, and coating, as well as plant type, soil composition, and solution chemistry. Under most conditions, ENMs in any solution rapidly aggregate to tens, hundreds, or even thousands times their primary particle size.However, there are three possible mechanisms by which ENMs could reach sizes small enough to pass through the barriers listed above. Plant roots are known to release protons to free Ca2+ and Mg2+ ions from clays4 and so may locally decrease the pH of the soil solution. This could potentially cause the release of primary particles from ENM aggregates by a similar mechanism. Alternately, free ENM primary particles likely exist in equilibrium with ENMs in aggregates and so there may be a small fraction of particles that will be bio-available at all times.
A third possible mechanism is that ENMs will be coated and dispersed by natural organic matter such as humic or fulvic acids or inorganic substances like phosphate and so be more bio-available. However, Schwabe, et al. found that coating CeO2 ENMs with fulvic acid decreased their uptake into pumpkin shoots grown hydroponically, which may be due to increased aggregate size of NOM-coated particles . Research on plant uptake of ENMs dates back less than a decade,so as of yet these mechanisms are largely hypothetical. However, Sabo-Attwood, et al. found Au ENMs above a size limit were excluded from vascular tissue in tobacco plants, which lends support to the hypothesis that particles must be small enough to pass through cell pores or plasmodesmata to enter the cortex. Similarly, Larue, et al. 3 found that TiO2 ENMs above 140 nm did not accumulate in root tissues, those above 36 nm did not enter the cortex, but that ENMs 14 nm in diameter were able to pass through the CS, enter the vascular tissue, and be translocated throughout the plant. Judy, et al. found that uptake can be species-dependent, as citrate and tannate coated Au ENMs were taken up in tobacco but not wheat. One aspect of plant/ENM interactions that has as yet received little attention is the influence of abiotic environmental conditions on plant uptake and translocation of ENMs. These include factors such as water and nutrient availability, temperature, soil salinity and pH, and light intensity. Plant performance depends heavily on environmental conditions, as physiological processes adapt to conditions that may be more or less favorable to growth. This has been shown for several non-nano pollutants. For example, high light intensities resulted in higher concentrations of As and Cd22 in sunflower and duckweed due to increased transpiration. Additionally, it was found in pea seedlings that nutrient stress increased the expression of transporter proteins that, in turn, increase cellular uptake of metals such as Cd.In these experiments I investigated the uptake and translocation of three metal oxide ENMs, CeO2, TiO2, and Cu2, in soil-grown Clarkia unguiculata , radish , and wheat in different soils, illumination, and/or nutrient levels. In the first part of this study, C. unguiculata were grown in potting soil under different light and nutrient levels and exposed to a range of ENM concentrations in order to discern how uptake trends depend on soil ENM content.
In the second part of this study C. unguiculata was again used as a model organism, but was grown in two natural soils under different light levels to gain insight into how soil properties influence ENM uptake. Finally, two crop plants were grown in natural soils to see how the trends seen in C. unguiculata vary with plant species. Radishes and wheat were selected as model crop plants representing the two major groups of angiosperms, Dicots and Monocots. Additionally, they have edible parts arising from different tissue types, which may influence ENM accumulation; namely, the radish hypocotyl is derived from the stem while wheat grains arise from reproductive tissue. C. unguiculata is an annual wildflower often used in ecological and genetic studies, and was selected here for its ease of growth, distinct tissues, and moderate lifespan that would allow for subchronic effects to be detected. I used C. unguiculata individuals from wild populations with greater genetic variability than crop plants typically used in nanotoxicological studies,raspberry plant pot which may mean results seen in this model organism are conservative with respect to detecting the effects of ENM exposure on plant uptake and performance. Here, I hypothesized that ENM uptake and distribution would vary between plant and soil type due to differences in plant physiology and ENM behavior, but that ENMs would in general be found in highest concentrations in the roots as the point of uptake, followed by leaves as the endpoint of transpiration, then stems as an intermediary between the two. Second, I predicted that plants grown in high light would uptake and accumulate higher concentrations of ENMs in leaves due to higher rates of transpiration. Third, I hypothesized that P would be positively correlated with ENM concentration in tissues due to sorption of phosphate from the soil. Natural metal oxides such as clays are known to strongly and preferentially sorb phosphate over other organic and inorganic ligands,and research has shown that metal oxide ENMs can also sorb phosphorous and thereby potentially affect its bio-availability in soils and other environmental media.Metals from ENMs were taken up into all tissues in all treatments, although the amounts depended on ENM type, soil ENM concentrations, growth condition , high light and limited nutrient , low light and excess nutrient , and low light and limited nutrient, and tissue type. Mean tissue metal concentrations of C. unguiculata grown in potting soil can be seen in Figure 4.1 and results from multiple regressions can be seen in Figures 4.2-4.4 and Table 4.1. In general, Ce and Ti were found in highest concentration in roots while Cu was primarily found in leaves , although relatively high concentrations of Ti were also seen in stems . Background concentrations of Ti and Cu were found in all three tissues, while background Ce was only found in roots. Among individuals in the Control group , it is likely that Ce was not found in stems or leaves because it was not present in the soil at concentrations as high as Ti , nor is it an essential micro-nutrient as is Cu.
Of the three ENMs to which plants were exposed, those exposed to CeO2 and TiO2 followed the pattern of distribution described in my first hypothesis, with concentrations being consistently highest in the roots followed by leaves then stems . In Cu2-exposed plants, however, Cu concentrations were roughly an order of magnitude higher in leaves than in roots . Plants from all groups showed statistically significant positive correlations between exposure concentration and metal concentration in roots and, with a few exceptions, tended to have the highest metal concentrations at the highest exposure level in all tissues. The most notable exceptions to this trend are the variable Ce and Ti content of leaves from plants grown under high light, excess nutrient and high light, limited nutrient conditions . This reflects the high inter-leaf metal content variability for Ce and Ti and may be due to a randomized or patchy accumulation of these nanoparticles between leaves. There were no significant associations between leaf metal content and leaf node number, which is indicative of order of production . Since C. unguiculata leaves are produced in a temporal sequence along the height of the plant and are also larger lower on the plant, this indicates that ENM uptake into leaves was independent of both stage of growth and leaf size.Particle charge likely plays a large role in determining distribution as well. Figures 2.3 C-D shows that all three ENMs used here had a weak negative charge in potting soil pore solution, although this was likely due to the high ionic strength and organic content of this soil shielding the particle surfaces and not a result of a direct alteration of the ENM crystal surface. Wang, et al. and Zhu, et al. found that under hydroponic conditions, well-dispersed particles coated with positively charged polymers are more readily taken up into plant roots compared to those coated with negatively charged polymers , which had higher accumulation in leaves. The results seen here provide confirmation of the importance of surface charge in ENM uptake and distribution in plants under more environmentally relevant conditions, i.e., in soil and with polydisperse ENMs. In addition to its surface charge, the tendency of Cu2 to dissolve at low pH,such as is found in the soil used in this study , likely also contributes to its uptake behavior. Rhizosphere pH tends to be more acidic than the surrounding soil due to the release of protons by roots to stimulate and counterbalance the uptake of ions from the soil;one effect of this acidity may be to dissolve a portion of the Cu2. Dissolved Cu would, in turn, encounter less size exclusion than ENMs and be retained less in the roots and stems in addition to being actively transported to the leaves.