Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K⁺) as a nutrient, and ammonia/ammonium (NH₃/NH₄⁺) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.     

Using physiology and behaviour to understand the responses of fish early life stages to toxicants

The use of early life stages of fishes (embryos and larvae) in toxicity testing has been in existence for a long time, generally utilizing endpoints such as morphological defects and mortality. Behavioural endpoints, however, may represent a more insightful evaluation of the ecological effects of toxicants. Indeed, recent years have seen a considerable increase in the use of behavioural measurements in early life stages reflecting a substantial rise in zebrafish Danio rerio early life‐stage toxicity testing and the development of automated behavioural monitoring systems. Current behavioural endpoints identified for early life stages in response to toxicant exposure include spontaneous activity, predator avoidance, capture of live food, shoaling ability and interaction with other individuals. Less frequently used endpoints include measurement of anxiogenic behaviours and cognitive ability, both of which are suggested here as future indicators of toxicant disruption. For many simple behavioural endpoints, there is still a need to link behavioural effects with ecological relevance; currently, only a limited number of studies have addressed this issue. Understanding the physiological mechanisms that underlie toxicant effects on behaviour so early in life has received far less attention, perhaps because physiological measurements can be difficult to carry out on individuals of this size. The most commonly established physiological links with behavioural disruption in early life stages are similar to those seen in juveniles and adults including sensory deprivation (olfaction, lateral line and vision), altered neurogenesis and neurotransmitter concentrations. This review highlights the importance of understanding the integrated behavioural and physiological response of early life stages to toxicants and identifies knowledge gaps which present exciting areas for future research.     

Profiling of plants at petroleum contaminated site for phytoremediation

The paucity of information in the literature on the characteristics of plants that could be used for phytoremediation of petroleum hydrocarbons (PHC)-contaminated sites was the principal reason for this study. The aim of the study was to identify indigenous plants growing in PHC-impacted soil in Umuahia in eastern-Nigeria that have the ability to phytoremediate soils contaminated with hydrocarbons under tropical monsoon climate conditions. A total of 28 native plant species from different families growing in and around hydrocarbon-impacted soil in the vicinity of vandalized pipelines carrying petroleum products were collected and studied for their ability to grow in a hydrocarbon-impacted soil and remove the PHC from the impacted soil. Some of the plants demonstrated the ability to grow in soil with high levels of the total petroleum hydrocarbons (TPH), which shows that they may be tolerant to hydrocarbons in soil and could potentially phytoremediate a hydrocarbon-contaminated soil. Chromolaena odorata, Aspilia africana, Chloris barbata, Pasparlum vaginatum, Bryophyllum pinnatum, Paspalum scrobiculatum, Cosmos bipinnatus, Eragrostis atrovirens, Cyperus rotundus, and Uvaria chamae showed tendencies to phytoremediate contaminated soil. By using bioaccumulation coefficient (BAC) as a measure of phytoremediation, results showed that C. odorata, A. africana, and U. chamae demonstrated the highest potentials to phytodegrade hydrocarbons in soil.     

Evaluation of Mycorrhizal Influence on the Development and Phytoremediation Potential of Canavalia Gladiata in Pb-Contaminated Soils

Soil contamination by heavy metals is a serious problem to humans due to its high level of toxicity. The heavy metal lead (Pb) is commonly used in industries and if the disposal of residues that contain this element is not done properly may result in tragic consequences to the organisms. In this experiment we assessed the potential of a forrage leguminous, Canavalia gladiata, to phytoremediate lead-contaminated soil under mycorrhizal influence. The experimental design was composed of 4 Pb doses (0, 250, 500, and 1000 mg kg^?1 of soil) and the plants were inoculated or uninoculated with arbuscular mycorrhizal fungi (AMF). We observed that the nodulation was severely affected by the presence of Pb independently of the mycorrhizal status; most of the elements analyzed were affected independently of the mycorrhizal status with exception of P. The mycorrhizal colonization was able to restrict the entrance of Pb in plants under high concentrations of Pb but promoted it's accumulation in both organs under intermediate concentrations of this element. Besides the mycorrhization did not promote plant growth under Pb stress, the use of this plant may be considered to be used for phytostabilization purposes.     

Respiratory response of cell-based sensors to toxicants measured by using pseudo-random signals

Cell-based sensors using such as Pseudomonas putida and Bacillus subtilis were applied to examine the toxicity of chemicals. Both toxicant and substrate solutions were introduced into the sensor system according to M-series and anti-symmetric M-series pseudo-random binary signals, Xn (n=15, minimal pulse width 4 min, period 60 min) and Yn (n=30, minimal pulse width 4 min, period 120 min). Toxicants such as Ag+, formaldehyde, azide, and hypo-chlorite were used to demonstrate the proposed method. The individual responses of substrate and toxicant were obtained at a time by calculating the cross-correlation function between the input and the output signals. Effects of toxicant on the response of substrate and the response of toxicant itself can be observed at a time, so that the method appears to be useful in studying the behavior of microorganisms in the presence of toxicants.     

The Role of Soil Microbial Tests in Ecological Risk Assessment: Differentiating between Exposure and Effects

The use of soil microorganisms in ecological risk assessment is hampered by an unclear dose-response relationship for most contaminants. Establishing dose-response curves for soil microbial communities requires that one have a clear estimate of exposure at the site of toxic action and a response free of confounding environmental factors. It is not clear what methods can estimate toxicant dose at the site of toxic action or determine microbial response to a toxicant. Pollution-induced community tolerance (PICT) is one possible estimate of microbial toxicant exposure. The PICT hypothesis is that the tolerance of a microbial community is proportional to the in situ dose. This method automatically corrects for differences due to differences in soil physical-chemical variables between samples. Various components of the soil nitrogen cycle can act as microbial bioindicators of toxicant impacts. Estimating denitrifica-tion activity presents a number of advantages over other components of the nitrogen cycle. Denitrifying bacteria come from a diversity of habitats, can be autotrophic or heterotrophic, and denitrification is a well-defined enzymatic system, which allows the use of molecular tools. Determining denitrification may be a good estimate of effects of toxicants on microbial communities. However, given the state of our ignorance regarding soil microbial community structure and function, redundant estimates of exposure and effect are necessary to adequately characterize the response of microbial communities to toxicants.     

Formation of fluorine technogenic anomalies in above-ground ecosystems of Siberia: Biological sorption,monitoring, possibility of lowering the negative impact

This paper studies how aluminum smelter emissions into the atmosphere affect the accumulation of fluorine by soil and plants, as well as change the chemical composition in leaves of woody plants. The ratio between total and extractable fluorine is ascertained to vary widely depending on the plant species. Meanwhile, gas resistant plants are characterized by an increased ash content in leaves (at the expense of K, P, Ca, etc.) and growth in the ratio between total and water-soluble fluorine. Plant resistance to fluorine that enters tissues depends on the capability of an organism to transform the toxicant into insoluble forms, which are not involved in physiological processes, i.e., on the presence of tissue elements with high precipitating capability. The gas-resistant species are ascertained to be characterized by high capability of limiting the intake of fluorine through roots, as well as its migration through a plant (especially to the organs that determine the further development of an organism).     

Landscape-level toxicant exposure mediates infection impacts on wildlife populations

Anthropogenic landscape modification such as urbanization can expose wildlife to toxicants, with profound behavioural and health effects. Toxicant exposure can alter the local transmission of wildlife diseases by reducing survival or altering immune defence. However, predicting the impacts of pathogens on wildlife across their ranges is complicated by heterogeneity in toxicant exposure across the landscape, especially if toxicants alter wildlife movement from toxicant-contaminated to uncontaminated habitats. We developed a mechanistic model to explore how toxicant effects on host health and movement propensity influence range-wide pathogen transmission, and zoonotic exposure risk, as an increasing fraction of the landscape is toxicant-contaminated. When toxicant-contaminated habitat is scarce on the landscape, costs to movement and survival from toxicant exposure can trap infected animals in contaminated habitat and reduce landscape-level transmission. Increasing the proportion of contaminated habitat causes host population declines from combined effects of toxicants and infection. The onset of host declines precedes an increase in the density of infected hosts in contaminated habitat and thus may serve as an early warning of increasing potential for zoonotic spillover in urbanizing landscapes. These results highlight how sublethal effects of toxicants can determine pathogen impacts on wildlife populations that may not manifest until landscape contamination is widespread.     

Plant compensatory growth: a conquering strategy in plant–herbivore interactions?

We present a theoretical analysis that considers the phenotypic trait of compensatory growth ability in a context of population dynamics. Our model depicts a system of three interactors: herbivores and two different plant types referred to as ordinary and compensating. The compensating plant type has the ability to increase its intrinsic rate of biomass increase as a response to damage. This compensatory growth ability is maintained at the expense of a reduced growth rate in the absence of damage, where the ordinary plant type has the higher growth rate. Analysis of this system suggests that, even though a compensatory capacity of this kind will not imply an increase in equilibrium plant density, it will give a competitive advantage in relation to other plants, in the presence of a sufficiently efficient herbivore. Invasion of compensating plants into a population of non-compensating plants is facilitated by a high compensatory growth ability and a high intrinsic rate of plant biomass increase. Conversely, an ordinary plant can invade and outcompete a compensating plant when the herbivore is characterised by a relatively low attack rate, and/or when plant intrinsic growth rate is decreased.     

Betula pendula: A Promising Candidate for Phytoremediation of TCE in Northern Climates

Betula pendula (Silver birch) trees growing on two contaminated sites were evaluated to assess their capacity to phytoscreen and phytoremediate chlorinated aliphatic compounds and heavy metals. Both locations are industrially-contaminated properties in central Sweden. The first was the site of a trichloroethylene (TCE) spill in the 1980s while the second was polluted with heavy metals by burning industrial wastes. In both cases, sap and sapwood from Silver birch trees were collected and analyzed for either chlorinated aliphatic compounds or heavy metals. These results were compared to analyses of the surface soil, vadose zone pore air and groundwater. Silver birch demonstrated the potential to phytoscreen and possibly phytoremediate TCE and related compounds, but it did not demonstrate the ability to effectively phytoextract heavy metals when compared with hyperaccumulator plants. The capacity of Silver birch to phytoremediate TCE appears comparable to tree species that have been employed in field-scale TCE phytoremediation efforts, such as Populus spp. and Eucalyptus sideroxylon rosea.     

Attack frequency and the tolerance to herbivory of Neotropical savanna trees

Tolerance is the ability of a plant to regrow or reproduce following damage. While experimental studies typically measure tolerance in response to the intensity of herbivory (i.e., the amount of leaf tissue removed in one attack), the impact of how many times plants are attacked during a growing season (i.e., the frequency of damage) is virtually unexplored. Using experimental defoliations that mimicked patterns of attack by leaf-cutter ants (Atta spp.), we examined how the frequency of herbivory influenced plant tolerance traits in six tree species in Brazil’s Cerrado. For 2 years we quantified how monthly and quarterly damage influenced individual survivorship, relative growth rate, plant architecture, flowering, and foliar chemistry. We found that the content of leaf nitrogen (N) increased among clipped individuals of most species, suggesting that Atta influences the allocation of resources in damaged plants. Furthermore, our clipping treatments affected tree architecture in ways thought to promote tolerance. However, none of our focal species exhibited a compensatory increase in growth (increment in trunk diameter) in response to herbivory as relative growth rates were significantly lower in clipped than in unclipped individuals. In addition, the probability of survival was much lower for clipped plants, and lower for plants clipped monthly than those clipped quarterly. For plants that did survive, simulated herbivory dramatically reduced the probability of flowering. Our results were similar across a phylogenetically distinct suite of species, suggesting a potential extendability of these findings to other plant species in this system.     

Fish emulsion as a food base for rhizobacteria promoting growth of radish (Raphanus sativus L. var. sativus) in a sandy soil

Commercial fish emulsion was evaluated as a plant growth medium and as a nutrient base to enhance radish (Raphanus sativus L. var. sativus) growth by bacterial and actinomycete isolates. Six bacterial isolates including three actinomycetes were selected from a screening of 54 bacteria (including 23 actinomycetes) based on their ability to produce plant growth regulators (PGRs) and to colonize radish roots. These isolates were tested in the presence and absence of autoclaved or non-autoclaved fish emulsion or inorganic fertilizers. The nutrient contents and types and levels of PGRs in tissues of treated plants were assayed to determine the basis of growth promotion. Fish emulsion was found to support plant growth in a sandy soil as effectively as an applied inorganic fertilizer. The plant growth promotion by bacterial and actinomycete isolates was most pronounced in the presence of autoclaved or non-autoclaved fish emulsion than in the presence of the inorganic fertilizers. The bacterial and actinomycete isolates were capable of producing auxins, gibberellins and cytokinins and appeared to use fish emulsion as a source of nutrients and precursors for PGRs. PGR levels in planta following combined treatments of the bacterial and actinomycete isolates and fish emulsion were found to be significantly enhanced over other treatments. The effect of fish emulsion appears to be more related to its role as a nutrient base for the bacterial and actinomycete isolates rather than to the increased activity of the general microflora of treated soil. This is the first report of fish emulsion as a nutrient base for plant growth promoting rhizobacteria. These results also indicate that the successful treatment can be effective and economical for horticultural production in sandy soils such as those found in the United Arab Emirates where fish emulsion is already in use as a substitute or supplement for inorganic fertilizer.     

Measurement of chronic toxicity using the opossum shrimpMysidopsis bahia

The chronic toxicity of silver and endosulfan to the opossum shrimpMysidopsis bahia was determined using continuous-flow bioassays. The 28-day bioassays measured survival, fecundity, and growth (length and weight measurements). Maximum acceptable toxicant concentrations (MATC) were estimated from measured toxicant concentrations. MATC values were similar using either brood size or growth as a criterion for sublethal effects. As an alternative to the determination of fecundity impairment, measurement of growth reduction in response to exposure to toxicants may provide a useful tool in the assessment of chronic toxicity inMysidopsis life-cycle bioassays.     

The evolution of growth rate, resource allocation and competitive ability in seeder and resprouter tree seedlings

Many woody plant species in fire disturbed communities survive disturbance events by resprouting. The resprouting life history is predicted to be costly to plants as resources are diverted into storage for post-fire regrowth rather than allocated to current growth, and resprouting species typically grow more slowly than seeder species (species that do not resprout after disturbance events). Differences in allocation to current growth are also predicted to make resprouter species poorer competitors compared to seeder species. We tested the predictions that the evolution of a resprouter life history is associated with slow growth, increased allocation to storage, and low competitive ability in woody plant seedlings. We grew eight phylogenetically independent pairs of seeder and resprouter species in competition and no competition treatments in a field experiment near Sydney, Australia. The presence of competitors reduced plant growth rates across taxa and fire response life histories. However, relative to seeder species, resprouter species were not slower growing, they did not allocate more resources to storage, and they did not have lower competitive abilities. We propose that differences in resource allocation to storage are not responsible for differences in growth rate and competitive ability. Rather, growth rate and competitive ability in seedlings are associated with key aspects of plant life history such as life-span and body size at maturity. These traits that are sometimes, but not always, related to fire response life histories.     

Arbuscular mycorrhizal fungi reduce the construction of extrafloral nectaries in <Emphasis Type="Italic">Vicia faba</Emphasis>

Arbuscular mycorrhizal fungi (AMF) can alter the physiology and morphology of their host plant, and therefore may have indirect effects on insect herbivores and pollinators. We conducted this study to test the hypothesis that AMF can also affect insects involved in protection-for-food mutualisms. We examined the constitutive and inducible production of food rewards [extrafloral (EF) nectaries] in Vicia faba plants by manipulating the presence/absence of AMF and by simulating various levels of herbivory. Plants inoculated with AMF produced significantly fewer EF nectaries than uninoculated plants, even after accounting for differences in plant growth. In contrast to earlier studies, EF nectaries were not inducible: damaged plants produced significantly fewer EF nectaries than undamaged plants. Moreover, the effects of mycorrhizal and damage status on EF nectary production were additive. The reduction in EF nectaries in mycorrhizal plants potentially represents a mechanism for indirect effects of AMF on the protective insects that exploit EF nectaries as a food source (e.g., ants). Reduced reward size should result in reduced protection by ants, and could therefore be a previously unappreciated cost of the mycorrhizal symbiosis to host plants. However, the overall effect of AMF will depend upon the extent to which the reduction of EF nectaries affects the number and activity of ants and the extent to which AMF alter other aspects of host plant physiology. Our results emphasize the complexity of multitrophic interactions, particularly those that span belowground and aboveground ecology.     

Nep1-like proteins from plant pathogens: recruitment and diversification of the NPP1 domain across taxa

An emerging group of proteins found in many plant pathogens are related to their ability to cause plant cell death. These proteins may be identified by the presence of a common NPP1 (necrosis-inducing Phytophthora protein) domain, and have collectively been named NLPs (Nep1-like proteins). The NLPs are distinguished by their wide distribution across taxa and their broad spectrum of activity against dicotyledonous plants. The function of NLPs is not known but there is strong evidence that they may act as positive virulence factors, accelerating disease and pathogen growth in plant hosts. Interest in NLPs is gaining momentum as more members of this protein family are discovered in more species of plant pathogens.     

Continuous culture algal bioassays for organic pollutants in aquatic ecosystems

Short-term responses of phytoplankton to organic pollutants are highly transitory. Time-course studies of non-steady state cells in continuous culture showed varying growth or photosynthetic responses such as enchancement, inhibition, adaptation (or development of resistance) or rebound, depending on the direction of changes in the intracellular toxicant concentration and the duration of exposure. However, steady-state cells in a two-stage chemostat system exhibited an increased tolerance to toxicants and subtle physiological effects such as photosynthetic enhancement which was accompanied by a considerable leakage of photosynthesates. It is important to understand such steady-state responses for the prediction and assessment of ecological impact by organic pollution on phytoplankton, since the time scale of changes in the toxicant/biomass ratio in most natural waters is long enough to approximate an equilibrium state.     

A Hypothetical Application of the Pollution-Induced Community Tolerance Concept in Megafaunal Communities Found at Contaminated Sites

The purpose of our paper is to suggest a novel, hypothetical and yet untested use of the pollution-induced community tolerance concept (PICT). Historically, PICT has been applied to determine whether toxicants are deleterious in microfaunal aquatic and terrestrial communities. We hypothesize that it may be possible to apply PICT to megafaunal organisms (e.g., vertebrates). In doing so, researchers could (1) identify which toxicant, in a complex mixture of toxicants, is harmful since only those contaminants that exert selection pressure are biologically relevant and will result in changes in community structure and (2) determine the transfer of toxicants across trophic levels found within that community. We suggest that community tolerance could be measured in megafaunal communities by measuring biomarkers of exposure and effect in either a number of individuals that make up different populations of animals comprising a community, or in the community as a whole. In this article we discuss the theoretical suitability of our megafaunal PICT approach to the assessment of contaminated sites and some of the potential pitfalls associated with its use. Our intention is that this paper will generate debate and commentary surrounding PIGT and its potential uses in the future. Whether this potential approach is feasible remains to be determined.     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.