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Earthmaster and the University of Waterloo have successfully developed plant growth promoting rhizobacteria (PGPR) - Enhanced Phytoremediation Systems (PEPSystems™) which have been deployed across Canada for the treatment of soil contaminated with petroleum hydrocarbons (PHCs), including CCME fractions F2 and F3. A challenge with phytoremediation is to predict the length of time to remediate a site so that site owners will be inclined to use the technology. In previous field trials of PEPSystems, it was determined that PHC was mostly degraded by microbes in the rhizosphere, following first-order exponential decay kinetics. Using new PEPSystems data collected from multiple commercial remediation sites across Western Canada, the kinetic equations of PHC decay were tested to determine if remediation time was accurately predicted. In general, when compared to the predicted time to remediation endpoint, data from recent commercial field applications showed that 35% and 20% less time was needed to reach remediation endpoints for fractions F2 and F3, respectively. As a result, the predictive kinetic equation for fraction F2 degradation was updated to reflect current remediation outcomes. Insufficient data were available to update the F3 equation. Being able to more accurately predict remediation timelines will enhance the value and utilization of PEPSystems.

Soil salinization negatively impacts plant growth and soil structure, which leads to environmental stress and agricultural/economic losses. Improved plant growth during salt-induced ionic and osmotic plant stress is the key to successful phytoremediation of salt-impacted sites. Using plant growth-promoting rhizobacteria (PGPR) in PGPR-Enhanced Phytoremediation Systems (PEPS), positive effects of PGPR on plant biomass and health have been observed in greenhouse and field experiments. Revegetation is arguably the most important aspect of salt phytoremediation and substantial biomass increases occur in PGPR-treated plants in both sodic and saline soils. PGPR protect against inhibition of photosynthesis and plant membrane damage, which suggests that they confer tolerance to plants under salt stress. Using PEPS, decreases in soil salinity are observed due to uptake of sodium and chloride from the soil into foliar plant tissue. Although rates of uptake do not change due to PGPR inoculation, higher plant biomass due to PGPR enhancement of plant performance leads to greater salt uptake on a per area basis relative to that of untreated plants. Significant improvements in plant growth and commensurate sodium chloride uptake, and the results of mass balance studies used to assess the direct impact of ion uptake on actual observed changes in soil salinity, provide evidence that phytoremediation of salt-impacted soil is feasible within acceptable time frames using PEPS.

Phytoremediation is the use of plants to extract, immobilize, contain and/or degrade contaminants from soil, water or air. It can be an effective strategy for on site and/or in situ removal of various contaminants from soils, including petroleum hydrocarbons (PHC), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), solvents (e.g., trichloroethylene [TCE]), munitions waste (e.g., 2,4,6-trinitrotoluene [TNT]), metal(loid)s, salt (NaCl) and radioisotopes. Commercial phytoremediation technologies appear to be underutilized globally. The primary objective of this opinion piece is to discuss how to take phytoremediation from a proven technology to an accepted practice. An overview of phytoremediation of soil is provided, with the focus on field applications, to provide a frame of reference for the subsequent discussion on better utilization of phytoremediation. We consider reasons why phytoremediation is underutilized, despite clear evidence that, under many conditions, it can be applied quite successfully in the field. We offer suggestions on how to gain greater acceptance for phytoremediation by industry and government. A new paradigm of phytomanagement, with a specific focus on using phytoremediation as a "gentle remediation option" (GRO) within a broader, long-term management strategy, is also discussed.

Bioavailability of metals in soil is a major factor influencing estimates of risk associated with exposure of ecological receptors. Metal concentrations in soil are often compared to ecological screening benchmarks, which are based on total concentrations in soil. Often, the total concentration is not correlated with toxicity. No standardised method exists for determining the bioavailability of metals in soil to ecological receptors. Several surrogate measures of bioavailability were compared to the results of a battery of toxicity tests using copper (Cu), lead (Pb), and zinc (Zn)-contaminated soils collected from a former industrial area. A calcium chloride (CaCl(2)) extraction, cyclodextrin (HPCD) extraction, simulated earthworm gut (SEG) test, and earthworm bioaccumulation test were performed using the soils. Extractable metals using the CaCl(2) solution were not correlated with any biological responses of earthworms (Eisenia andrei), collembola (Folsomia candida), northern wheatgrass (Elymus lanceolatus), or alfalfa (Medicago sativa L.). Concentrations of metals in the HPCD extracts were highly variable and were not adequate for revealing adverse effects. E. andrei tissue concentrations were variable but were predictive of adverse effects to invertebrates. The results of the SEG test correlated with most of the biological endpoints. Bioavailable Cu was correlated with adverse effects to invertebrates and plants using the SEG test. Overall, coefficients of determination associated with the relationships between the biological responses and each measure of bioavailability indicated that those for the SEG test were greater than those for the other surrogate measures of bioavailability. Further validation is required before this test is routinely used to estimate metal bioavailability and toxicity.

Plant growth-promoting rhizobacteria (PGPR) can have positive effects on vigour and productivity, especially under stress conditions. In asparagus ( Asparagus officinalis L.) field culture, seeds are planted in high-density nurseries, and 1-year-old crowns are transplanted to production fields. Performance can be negatively affected by water stress, transplant shock, and disease pressure on wounded roots. PGPR inoculation has the potential to alleviate some of the stresses incurred in the production system. In this study, the effects of PGPR ( Pseudomonas spp.) treatment were determined on 3-week-old greenhouse-grown seedlings and germinating seeds of 2 asparagus cultivars. The pots were irrigated to a predetermined level that resulted in optimum growth or the plants were subjected to drought or flooding stress for 8 weeks. The cultivars responded differently to PGPR: single inoculations of seedlings enhanced growth of ‘Guelph Millennium’ under optimum conditions and ‘Jersey Giant’ seedlings under drought stress. Seed inoculations with PGPR resulted in a positive response only for ‘Guelph Millennium’, for which both single or multiple inoculations enhanced plant growth under drought stress.

Over the past few decades there has been avid interest in developing in situ strategies for remediation of environmental contaminants. Major foci have been on persistent organic chemicals and metals. Phytoremediation, a strategy that uses plants to degrade, stabilize, and/or remove soil contaminants, has been extensively investigated. Rhizoremediation, a specific type of phytoremediation that involves both plants and their associated rhizosphere microbes, can occur naturally, or can be actuated by deliberately introducing specific microbes. These microbes can be contaminant degraders and/or can promote plant growth under stress conditions. Because initial phytoremediation research showed great promise as a cost-effective remedial strategy, considerable effort has been devoted to making the transition from the laboratory to commercialization. Despite our understanding of the mechanisms of remediation, and the success of studies in the laboratory and greenhouse, efforts to translate phytoremediation research to the field have proven challenging. Although there have been many encouraging results in the past decade, there have also been numerous inconclusive and unsuccessful attempts at phytoremediation in the field. There is a need to critically assess why remediation in the field is not satisfactory, before negative perceptions undermine the progress that has been made with this promising remedial strategy. Two general themes have emerged in the literature: (1) Plant stress factors not present in laboratory and greenhouse studies can result in significant challenges for field applications. (2) Current methods of assessing phytoremediation may not be adequate to show that contaminant concentrations are decreasing, although in many cases active remediation may be occurring. If phytoremediation is to become an effective and viable remedial strategy, there is a need to mitigate plant stress in contaminated soils. There is also a need to establish reliable monitoring methods and evaluation criteria for remediation in the field. This review will focus on the challenges and the potential of phytoremediation, particularly rhizoremediation, of organic contaminants from soils.

Plectonema boryanum UTEX 485 cells were grown at 29 degrees C and 150 mumol m-2 s-1 photosynthetically active radiation (PAR) and exposed to PAR combined with ultraviolet-A radiation (UV-A) at 15 degrees C. This induced a time-dependent inhibition of photosystem II (PSII) photochemistry measured as a decrease of the chlorophyll a fluorescence ratio, Fv/Fm, to 50% after 2 h of UV-A treatment compared to nontreated control cells. Exposure of the same cells to PAR combined with UV-A + ultraviolet-B radiation (UV-B) caused only a 30% inhibition of PSII photochemistry relative to nontreated cells. In contrast, UV-A and UV-A + UV-B irradiation of cells cultured at 15 degrees C and 150 mumol m-2 s-1 had minimal effects on the Fv/Fm values. However, cells grown at 15 degrees C and lower PAR irradiance (6 mumol m-2 s-1) exhibited similar inhibition patterns of PSII photochemistry as control cells. The decreased sensitivity of PSII photochemistry of P. boryanum grown at 15 degrees C and 150 mumol m-2 s-1 to subsequent exposure to UV radiation relative to either control cells or cells grown at low temperature but low irradiance was correlated with the following: (1) a reduced efficiency of energy transfer to PSII reaction centers; (2) higher levels of a carotenoid tentatively identified as myxoxanthophyll; (3) the accumulation of scytonemin and mycosporine amino acids; and (4) the accumulation of ATP-dependent caseinolytic proteases. Thus, acclimation of P. boryanum at low temperature and moderate irradiance appears to confer significant resistance to UV-induced photoinhibition of PSII. The role of excitation pressure in the induction of this resistance to UV radiation is discussed.

Polycyclic aromatic hydrocarbons (PAHs) are known to exhibit photoinduced toxicity to organisms, especially in the presence of ultraviolet irradiation. Oxidation is the most common photochemical reaction of anthracene (ANT) and ANT derivatives. Quinones, hydroxy-quinones, diols, and aldehydes are the resultant products. Different ANT photoproducts have different impact on biological organisms. The structure of these molecules contains information concerning their toxicity. From two main bioenergetic pathways in aerobic organisms (respiration and photosynthesis), we have previously reported the inhibition of photosynthesis by ANT derivatives. We showed that inhibition of photosynthesis is a reliable assay of xenobiotic stress. To study the correlation between chemical structures and toxicity, precise structure of anthracene (ANT), anthraquinone (ATQ) and eleven hydroxyanthraquinones (hATQs) were constructed. A numerical comparison of molecular shape codes was used for shape comparison and similarity analysis. The data sets for growth inhibition showed good correlations to the shape similarity. Both the one ring and whole molecule similarities of ANT, ATQ and hATQs showed a good correlation with the empirical whole organism toxicity data. The IC50 values for FV/FM and FQ/FM were plotted against hyperbolic dependencies of one-ring and whole molecule similarity to determine their predictive capacity. The two data sets for FQ/ FM and FV/FM have reasonable regression coefficients, and both were found to be excellent bioindicators of effects. In this study correlation between the shape of chemicals and the biological activity provides necessary information related to toxicity of hazard chemicals based on chemical and biological properties of their reactions.

Multiple techniques that affect different aspects of contaminant removal can improve remediation of persistent hydrocarbons from soils. We have developed a multi-process phytoremediation system (MPPS) that is composed of land-farming (aeration and light exposure), contaminant degrading bacteria, plant-growth-promoting rhizobacteria (PGPR), and growth of the contaminant-tolerant plant, Tall Fescue (Festuca arundinacea). In this study, the MPPS was applied to a contaminated soil acquired from the Imperial Oil land farm site in Sarnia, Ontario, Canada. This soil was contaminated with oil refinery sludge to a level of approximately 5% (w/w) total petroleum hydrocarbons (TPHs). Over an initial 4-month period, the average efficiency of removal of persistent TPHs by the MPPS was twice that of land-farming alone, 50% more than bioremediation alone, and 45% more than phytoremediation alone. Importantly, the MPPS removed oil fractions 2, 3 and 4 with equal efficiency. Therefore, the highly hydrophobic, recalcitrant TPH fractions were remediated from the soil with the MPPS. After a second 4-month period, the MPPS removed 90% of all fractions of TPHs from the soil. Phytoremediation alone was able to remove only about 50% of TPHs in the same time period. The key elements for successful phytoremediation were the use of a plant species that can proliferate in the presence of high levels of contaminants, and strains of PGPR that increase plant tolerance and accelerate plant growth in heavily contaminated soils.

To improve phytoremediation processes, multiple techniques that comprise different aspects of contaminant removal from soils have been combined. Using creosote as a test contaminant, a multi-process phytoremediation system composed of physical (volatilization), photochemical (photooxidation) and microbial remediation, and phytoremediation (plant-assisted remediation) processes was developed. The techniques applied to realize these processes were land-farming (aeration and light exposure), introduction of contaminant degrading bacteria, plant growth promoting rhizobacteria (PGPR), and plant growth of contaminant-tolerant tall fescue (Festuca arundinacea). Over a 4-month period, the average efficiency of removal of 16 priority PAHs by the multi-process remediation system was twice that of land-farming, 50% more than bioremediation alone, and 45% more than phytoremediation by itself. Importantly, the multi-process system was capable of removing most of the highly hydrophobic, soil-bound PAHs from soil. The key elements for successful phytoremediation were the use of plant species that have the ability to proliferate in the presence of high levels of contaminants and strains of PGPR that increase plant tolerance to contaminants and accelerate plant growth in heavily contaminated soils. The synergistic use of these approaches resulted in rapid and massive biomass accumulation of plant tissue in contaminated soil, putatively providing more active metabolic processes, leading to more rapid and more complete removal of PAHs.

Research on toxicant-responsive genes is providing new and important bioindicators for environmental biologists. Identifying genes whose expression is modulated by toxicant exposure provides important clues into the mechanisms underlying toxicity. In addition, toxicant-responsive genes can be developed as molecular end points that are likely to be sensitive tools for environmental assessment. Differential display polymerase chain reaction (ddPCR) is a useful approach for screening and analyzing the expression of genes. A ddPCR protocol was optimized to investigate gene expression in the cladoceran Daphnia magna. The modified protocol requires submicrogram quantities of total RNA (from

Metals and ultraviolet (UV) radiation are two environmental stressors that can cause damage to plants. These two types of stressors often impact simultaneously on plants and both are known to promote reactive oxygen species (ROS) production. However, little information is available on the potential parallel stress responses elicited by metals and UV radiation. Using the aquatic plant Lemna gibba, we found that copper and simulated solar radiation (SSR, a light source containing photosynthetically active radiation (PAR) and UV radiation) induced similar responses in the plants. Both copper and SSR caused ROS formation. The ROS levels were higher when copper was combined with SSR than when applied with PAR. Higher concentrations of copper plus PAR caused toxicity as monitored by diminished growth and chlorophyll content. This toxicity was more pronounced when copper was combined with SSR. Because the generation of ROS was also higher when copper was combined with SSR, we attributed this enhanced toxicity to elevated levels of ROS. In comparison to PAR-grown plants, SSR treated plants exhibited elevated levels of superoxide dismutase (SOD) and glutathione reductase (GR). These enzyme levels were further elevated under both PAR and SSR when copper was added at concentrations that generated ROS. Interestingly, copper treatment in the absence of SSR (i.e. copper plus PAR) induced synthesis of the same flavonoids as those observed in SSR without copper. Finally, addition of either dimethyl thiourea or GSH (two common ROS scavengers) lowered in vivo ROS production, alleviated toxicity and diminished induction of GR as well as accumulation of UV absorbing compounds. Thus, the potential of ROS being a common signal for acclimation to stress by both copper and UV can be considered.

Selenastrum capricornutum was grown in two lake waters of differing dissolved organic carbon content (1.8 vs. 9.1 mg DOCl(-1)) to determine the responses of population dynamics and photosynthesis to Cu, and to assess the modifying effects of varying ultraviolet radiation (UVR) exposure. In the absence of UVR, the mean EC(50) for Cu effect on population growth rate was 2.3-2.6 microg l(-1) in the low DOC water and 17.4-26.2 microg l(-1) in the high DOC water. The variable chlorophyll a fluorescence ratio, F(v)/F(m), decreased approximately in parallel with the diminished growth rates. Exposure of the higher DOC lake water to full spectrum artificial radiation caused an increase of Cu(2+) concentration, compared to samples held in darkness or in photosynthetically active radiation (PAR) only. Full spectrum exposures also resulted in a lower (although not significantly so) EC(50) for Cu effect on growth rate, consistent with response to the moderately elevated Cu(2+) concentration. Cu(2+) concentration was unaffected by radiation exposure in the low DOC water, and EC(50)s for growth were also unaffected except in the most severe UVR treatment, which was40% inhibited even in the absence of added Cu. Using F(v)/F(m) as an end-point, there was no evidence of interactions between UVR and Cu under the relatively low PAR exposures used here. Algal growth and photosynthesis was extremely sensitive to Cu in these soft lake waters, with EC(50)s close to current water quality standards in the low DOC water.

To assess photosynthesis as a bioindicator of toxicity from polycyclic aromatic hydrocarbons (PAHs), the response of chlorophyll-a fluorescence to creosote exposure was compared with effects on population-level plant growth. Large, outdoor, freshwater microcosms containing Eurasian watermilfoil (Myriophyllum spicatum) received either a single application or multiple applications of liquid creosote at nominal concentrations from 0.109 to 32.7 mg L(-1). For several weeks following treatment, photosynthetic electron transport was measured using pulse amplitude-modulated chlorophyll-a fluorescence. The maximum efficiency of photosystem II electron transport (Fv/Fm) and the quantum yield of photochemistry (deltaF/F'm) were diminished in plants shortly after the addition of creosote. In microcosms that received a single treatment of creosote only, the 50% effective concentrations (EC50s), expressed as the aqueous concentration of 15 of the most abundant PAHs, were 0.28 mg L(-1) for Fv/Fm and 0.30 mg L(-1) for deltaF/F'm. Chlorophyll-a fluorescence was diminished to a greater extent in microcosms that received multiple treatments of creosote, with EC50s of 0.13 mg L(-1) for Fv/Fm and 0.10 mg L(-1) for deltaF/F'm. Plant biomass accumulation was inhibited in a concentration-dependent manner in all microcosms treated with creosote, but this inhibition occurred to a greater degree in microcosms treated with multiple creosote applications. The response of chlorophyll-a fluorescence, measured only 8 d after creosote treatment, was similar to plant growth over the entire growing season, indicating that this technique can be used to estimate potential effects of contaminants before detrimental impacts on populations.

Pulse amplitude modulated (PAM) chlorophyll-a fluorescence is a simple, rapid technique for measuring photosynthetic efficiency in plants and algae that could be a useful biomarker of toxicity in the aquatic environment. PAM Chlorophyll fluorescence was used to detect inhibition of photosynthesis in natural assemblages of Lake Erie phytoplankton incubated with both intact and photomodified polycyclic aromatic hydrocarbons. The maximum efficiency of electron transport in photosystem II (Fv/Fm) and the effective yield of photochemistry (or steady state photosystem II activity) (μF/Fm′) were measured from phytoplankton exposed for 30 min in sunlight to polycyclic aromatic hydrocarbons at concentrations from 40 to 2,000 μgL−1. Anthracene, fluoranthene, and phenanthrenequinone were most toxic to phytoplankton, with EC50s for ΔF/Fm′ inhibition of 314, 118 and 90 μgL−1, respectively. Anthraquinone, 1,2-dihydroxyanthraquinone, and phenanthrene were less toxic, with EC50s ranging from 684 to > 2,000 μgL−1. Recovery of photosynthetic function in darkness occurred to varying degrees, and was related to the regions of the photosynthetic apparatus on which the chemicals are thought to act. Inhibition of chlorophyll fluorescence parameters demonstrated a reciprocity-like response between concentration and duration of exposure, implying chronic exposures to lower concentrations of PAHs, such as those found in the Great Lakes, could cause inhibition of photosynthesis in phytoplankton assemblages.

Sunlight can greatly enhance the toxicity of polycyclic aromatic hydrocarbons (PAHs). Photosensitization reactions (e.g., generation of singlet-state oxygen) and photomodification reactions (e.g., photooxidation of PAHs to more toxic species) are both pathways of photoinduced toxicity of PAHs. Previously, a quantitative structure-activity relationship (QSAR) was developed for PAHs showing that a photosensitization factor (PSF) and photomodification factor (PMF) can be additively combined to describe photoinduced toxicity. That QSAR model was developed for the photoinduced toxicity of 16 PAHs to the higher plant Lemna gibba. The objective of this study was to apply the QSAR model developed for L. gibba to another organism. The organism chosen was the luminescent marine bacteria Vibrio fischeri. Toxicity data used for the QSAR model were inhibition of luminescence and inhibition of growth of V. fischeri. Both short-term (15 min) and long-term (18 h) assays of toxicity were used. Light did not impact on PAH toxicity in the short-term assay, and thus the QSAR model did not correlate well with these data. Conversely, light greatly enhanced toxicity when the long-term assay was employed. The PMFs for the PAHs from the L. gibba QSAR showed a moderate correlation to bacterial toxicity in the long-term assay, whereas the PSFs showed only a weak correlation to toxicity. As was the case for L. gibba, summing the PMF and the PSF resulted in a strong correlation to toxicity that had predictive value. Thus, a QSAR model derived for plants accurately described the toxicity of PAHs to a bacterial species. This indicates that the bipartite mechanism of PAH-photoinduced toxicity may be applicable to other organisms.

The impact of natural solar ultraviolet radiation (UVR), particularly UVB (297–320 nm), on phytoplankton primary production in Lake Erie was investigated during the spring and summer of 1997. Radiocarbon incorporation and size-selective filtration was used to trace total production and its distribution among particulate and dissolved pools. On average, 1-h exposures produced half the UVB-dependent inhibition of total production realized in 8-h exposures, indicating rapid kinetics of photoinhibition. Cumulative UVB-dependent photoinhibition averaged 36% in 8-h simulated surface exposures. The efficiency of photoinhibition was greater for N-deficient than N-replete communities, but was not related to phytoplankton light history, P limitation, or the dominant genera. The proportion of recently fixed carbon occurring in the dissolved pool after 8-h exposures was significantly greater in higher-UVB treatments, whereas the share in picoplankton (

Growing concern over environmental contamination has stimulated rigorous efforts to establish reliable biological monitoring assays. Methodology was developed for measuring photoinduced short- and long-term toxicity of an important group of contaminants, polycyclic aromatic hydrocarbons (PAHs), using the luminescent bacterium Vibrio fischeri. The toxicity of most PAHs can be greatly enhanced on exposure of a living organism and/or the chemicals to ultraviolet (UV) radiation. There are two major mechanisms involved in photoinduced toxicity of PAHs: photosensitization and photomodification. In the former, production of singlet oxygen leads to cellular damage. In the latter, photooxidation of PAHs results in new compounds (usually oxygenated PAHs) that are often more toxic than their parent PAHs. Microbial toxicity assays were developed to measure short- and long-term photoinduced toxicity of PAHs. The bioassays were based on inhibition of luminescence and growth of V. fischeri. The short-term assay should detect toxicity of chemicals that are taken up rapidly and/or whose photosensitization activity is immediate. The long-term assay should identify chemicals where the rate of assimilation is slow and/or time is required for photoinduced effects to be realized. The assays were tested with 12 different PAHs. The short-term assay did not reveal photoinduced toxicity for any of the test chemicals. However, photoinduced toxicity was apparent in the long-term assay, indicating that short-term assays may be opaque to this key mechanism of PAH toxicity.

There is surprisingly little data on the photooxidation of polycyclic aromatic hydrocarbons (PAHs) under environmentally relevant lighting conditions. The aqueous photooxidation reactions of naphthalene (the simplest and most water soluble PAH) were investigated using natural sunlight as a light source.Six of the major reaction products were identified, including 1-naphthol, coumarin, and two hydroxyquinones. The reactionproducts were consistent with initial [2,2] or [2,4] photocycloaddition reactions, with subsequent oxidations and/or rearrangements. The oxidation reactions in aqueous phase favoredproducts different from those observed in atmospheric oxidation reactions. However, similar photoproducts were observed withtitanium catalysts or in the presence of hydrogen peroxide. Theproducts from aqueous photooxidation were also similar to the products resulting from naphthalene metabolism. The observedphotooxidation products were generated by mechanisms that areexpected to occur with other PAHs as well, and thus naphthaleneoxidation provides a model for possible photoreactions of largerPAHs.

Plant growth-promoting bacteria (PGPB) strains that contain the enzyme 1-amino-cyclopropane-1-carboxylate (ACC) deaminase can lower stress ethylene levels and improve plant growth. In this study, ACC deaminase-producing bacteria were isolated from a ) salt-impacted ( 50 dS/m) farm field, and their ability to promote plant growth of barley 1): and oats in saline soil was investigated in pouch assays (1% NaCI), greenhouse trials (9.4 dS/m), and field trials (6-24 dS/m). A mix of previously isolated PGPB strains UW3 (Pseudomonas sp.) and UW4 (P. sp.) was also tested for comparison. Rhizobacterial isolate CMH3 (P. corrugata) and UW3+UW4 partially alleviated plant salt stress in growth pouch assays. In greenhouse trials, CMH3 enhanced root biomass of barley and oats by 200% and 50%, respectively. UW3+UW4, CMH3 and isolate CMH2 also enhanced barley and oat shoot growth by 100%-150%. In field tests, shoot biomass of oats tripled when treated with UW3+UW4 and doubled with CHM3 compared with that of untreated plants. PGPB treatment did not affect salt uptake on a per mass basis; higher plant biomass led to greater salt uptake, resulting in decreased soil salinity. This study demonstrates a method for improving plant growth in marginal saline soils. Associated implications for salt

Chlorophyll-a fluorescence induction is a rapid technique for measuring photosynthetic electron transport in plants. To assess chlorophyll-a fluorescence as a bioindicator of effects of polycyclic aromatic hydrocarbon mixtures, chlorophyll-afluorescence parameters and plant growth responses to exposure to the wood preservative creosote were examined in the aquatic plants Lemna gibba and Myriophyllum spicatum. Exposure to creosote inhibited growth of L. gibba (EC50 = 7.2 mg/L total polycyclic aromatic hydrocarbons) and M. spicatum (EC50 = 2.6 mg/L) despite differences in physiology. Creosote also diminished maximum PSII efficiency (Fv/Fm) (EC50 = 36 and 13 mg/L for L. gibba and M. spicatum) and the effective yield of photosystem II photochemistry (ΔF/F′m) (EC50 = 13 and 15 mg/L for L. gibba and M. spicatum). The similarity between growth and chlorophyll-a fluorescence EC50s and slopes of the response curves suggests a close mechanistic link between these end points. The predictive power of chlorophyll-a fluorescence as a bioindicator of whole-organism effects applied to complex contaminant mixtures is discussed.

Summary. In mixture experiments, one may be interested in estimating not only main effects but also some interactions. Main effects and significant interactions in a mixture may be estimated through appropriate mixture experiments, such as simplex-centroid designs. However, for mixtures with a large number of factors, the run size for these designs becomes impractically large. A subset of a full simplex-centroid design may be used, but the problem remains regarding which factor-level settings should be selected. In this paper, we propose a solution that considers design points with either one or p individual nonzero factor-level settings. These fractional simplex designs provide a means of screening for interactions and of investigating the behavior of many-component mixtures as a whole while greatly reducing the run size compared with full simplex-centroid designs. The means of construction of the design arrays is described, and designs for ≤31 factors are presented. Some of the proposed methodology is illustrated using generated data.

Photoinhibition was examined in natural assemblages of phytoplankton from Lake Erie exposed to ambient solar radiation. The impacts on photosynthesis of photosynthetically active radiation (400-700 nm) (PAR), ultraviolet-A radiation (320-400 nm) (UVA), and ultraviolet-B radiation (295-320 nm) (UVB) were assessed at three sites on the lake using pulse amplitude modulated chlorophyll fluorescence. Short exposures (-2) resulted in the rapid loss of up to 60% of photosystem II efficiency (in the dark-adapted state) (Fv/Fm) and quantum yield (in the light-adapted state) (ΔF/F'm). Exposure to UVA (46-105 mmol·m-2) generally diminished Fv/Fm and, to a lesser extent, ΔF/F'm. Short exposures to PAR (733-1588 mmol·m-2) had no significant effects on electron transport. Recovery from UVA- or UVB-induced photoinhibition was complete for Fv/Fm and 90% complete for ΔF/F'm after 2 h in low light. The results indicate that exposures of phytoplankton to surface radiation need only be short in duration to cause substantial UV inhibition of photosynthesis. However, depending on the kinetics of mixing of the water column, recovery of photosynthesis is possible if there is sufficient time for repair of UV damage. Future elevated levels of solar UVB due to ozone depletion could significantly inhibit primary production in mesotrophic lakes such as Lake Erie.

Recently, there has been a trend toward less turbid water and greater light penetration in parts of western Lake Erie. This could lead to greater phototoxicity from sediment-bound polycyclic aromatic hydrocarbons. To test photosynthesis as a bioindicator of contaminant impacts on algae, water samples containing natural assemblages of phytoplankton were collected from the western and central basins of Lake Erie. These samples were incubated with 0.2 to 2 mg L−1 anthracene or its photomodified product 1,2-dihydroxyanthraquinone for 60 min in darkness or in 50% sunlight, to mimic exposure of phytoplankton in the photic zone of a mixed water column. Photosynthetic efficiency was determined from filtered phytoplankton immediately after exposure using a pulse-amplitude modulated chlorophyll fluorometer. Phytoplankton incubated with chemicals in the dark demonstrated chlorophyll fluorescence values similar to those of controls. However, exposure to anthracene or 1,2-dihydroxyanthraquinone in sunlight diminished photosystem II photosynthetic efficiency and photosynthetic quantum yield in a concentration-dependent manner. Anthracene inhibited photosynthesis at lower concentrations than 1,2-dihydroxyanthraquinone, which is consistent with the different modes of action and toxic strengths of these two contaminants. These results demonstrate that phytoplankton in Lake Erie can be subject to phototoxicity from intact and photomodified polycyclic aromatic hydrocarbons after very short exposures. Further, chlorophyll fluorescence was found to be an effective bioindicator in the field for this form of chemical stress.

Solar radiation spectra were measured in mesotrophic Lake Erie in 1997 to determine the principal factors influencing ultraviolet radiation (UVR) attenuation. Water clarity ranged widely, with the diffuse vertical attenuation coefficient for photosynthetically active radiation (KdIPAR) varying from 0.26 to 17.2 m-1, and UVR attenuation varied positively and significantly with KdIPAR. Particle concentration (total suspended solids, TSS) was the single most useful water quality characteristic for predicting broadband UVB (305-320 nm) and UVA (320-400 nm) attenuation. Dissolved organic carbon concentration (DOC) and fluorescence were much less important than TSS in predicting UVR attenuation over the typical range of water clarity (KdIPAR < 2 m-1). The predominant role of TSS in UVR attenuation could not be explained by phytoplankton or other absorbing organic matter in the particulate phase, suggesting instead an important role for scattering. Attenuation spectra through the UV region were not monotonic at the higher particle concentrations and could not be modelled solely as a function of DOC as previously reported for other lakes. Attenuation at 310 and 320 nm confirmed the conclusion from spectrally integrated measurements that particles are the dominant agents of variable UVR attenuation in Lake Erie.

The influence of ultraviolet (UV) irradiation on creosote toxicity was investigated with the rainbow trout ( Oncorhynchus mykiss) gill cell line, RTgill-W1, and two indicator dyes, alamar Bluey and 5-carboxyfluorescein diacetate acetoxymethyl ester. These monitor redox potential and membrane integrity, respectively. After solubilization and chemical analysis, creosote was presented to cells in the dark to measure cytotoxicity or concurrently with UV irradiation to evaluate photocytotoxicity. Additionally, creosote was photomodified b y2ho f UVirradiation before presentation to cells in the dark or together with UV. Cytotoxicity was detected only at high nominal creosote concentrations, but photocytoxicity occurred at creosote concentrations 35-fold lower. All the aromatic hydrocarbons in creosote appeared to contribute to cytotoxicity, but photocytotoxicity was due only to the fluoranthene, pyrene, anthracene, and benzo( a)anthracene in the mixture. Photomodified creosote was much more cytotoxic than intact creosote and this difference was most pronounced in the alamar Blue assay. Likely, this was due to photomodification products that impaired the mitochondrial electron transport chain. Photomodified creosote was slightly less photocytotoxic than intact creosote. Overall these results indicate that UV irradiation potentially enhances the toxicity of creosote to fish in several different but significant ways.

The toxicity of polycyclic aromatic hydrocarbons (PHAs) is known to be enhanced by light via photosensitization reactions (production of active oxygen) and photomodification of the chemicals (e.g., oxidation) to more toxic compounds. Anthracene (ANT) toxicity in particular has been found to increase dramatically following photomodification. The objective of this study was to identify the photooxidation products of ANT and assess the toxicity of selected photoproducts. High performance liquid chromatography (HPLC) analysis of anthracene photooxidation revealed a complex array of oxidation products; prevalent among these were anthraquinone (ATQ) and hydroxy-anthraquinones (hATQs). Eleven of these compounds were tested for toxicity using growth inhibition of the duckweed Lemna gibba L. G-3. All but one of the compounds tested were found to be toxic, and when UV radiation was present in the light source toxicity was generally enhanced. The chemicals were also irradiated under SSR prior to toxicity testing. In about half the cases, the ATQ compounds were rapidly photooxidized and the resultant photoproducts were more toxic than the parent compounds. Interestingly, 2-hydroxyanthraquinone, which was not subject to photooxidation, was the most toxic of the compounds tested. As a light stable compound it presents the risk of a persistent environmental hazard.

— The population and photosynthetic responses of a microscopic green alga (Selenastrum capricornutum) to realistic levels of UV radiation (UVA and UVB) were assessed in natural lake waters of different dissolved organic carbon (DOC) concentration. Specific growth rates and photosynthetic competence (as reflected by Fv/Fm [measure of maximal quantum efficiency of photosystem II] and t1/2 [estimate of electrons transported to the plastoquinone pool] measured by in vivo variable chlorophyll a fluorescence) were compared between two exposure levels of UVR and two concentrations of DOC (2.5 mg C L−1 7.7 mg C L−1). Exposure periods of 6–9 days (five to nine generations) were used. Exposure to UVA primarily affected the efficiency of photosystem II, as evidenced by significant decreases of Fv/Fm but not growth rates or t1/2 Exposure to UVB, in the presence of UVA, did not cause significant additional decreases of Fv/Fm but did diminish growth rates. In the low DOC water, t1/2 was also diminished, suggesting different proximate sites of action from those for UVA. The high DOC water decreased the effective exposure to both UVA and UVB and diminished the negative impact of UV radiation on the cells, but the apparent protection was not explicable solely by the shading action of the DOC. Control values for Fv/Fm, growth rates and t1/2 were all lower in the high DOC water, suggesting a negative side effect to the apparent protective action of the DOC against UVB.

The influence of a commercial humic acid (Aldrich; AHA) on the development of anthracene photoinduced toxicity to the duckweed Lemna gibba was examined using both room- and low-temperature (77°K) chlorophyll fluorescence assays. Plants were exposed to 2 mg liter -1 anthracene both with and without 6.2 mg liter -1 of AHA and grown under either visible light or simulated solar radiation that mimics the natural abundance of UV radiation. Exposure periods ranged from 1 to 48 h to examine temporal changes in chlorophyll degradation and chlorophyll a fluorescence induction in response to these light and HA treatments. The onset of anthracene photoinduced toxicity followed a definite sequence; chlorophyll a fluorescence induction parameters (F v /F m , and t 1 /2) responded earliest to anthracene exposure, with observable chlorophyll degradation requiring up to 24 to 48 h. Of these, t 1 /2 was the most sensitive, with significant inhibition apparent within I h of exposure. Throughout the entire 48-h exposure, 6.2 mg liter 1 AHA ameliorated the photoinduced toxicity of anthracene, in terms of both chlorophyll degradation and F v / F m inhibition. In contrast, AHA delayed the complete inhibition of t 1 /2 by only I to 24 h rather than permanently protecting the plants from anthracene damage to PS2. This suggests that AHA may slow, but not prevent, the entrance of either intact anthracene or its photooxidized byproducts under these exposure conditions.

The quantitative shape-activity relationship (QShAR) methodology, based on accurate three-dimensional electron den- sities and detailed shape analysis methods, has been applied to a Lemna gibba photoinduced toxicity data set of 16 polycyclic aromatic hydrocarbon (PAH) molecules. In the first phase of our studies, a shape fragment QShAR database of PAHs was developed. The results provide a very good match to toxicity based on a combination of the local shape features of single rings in comparison to the central ring of anthracene and a more global shape feature involving larger molecular fragments. The local shape feature appears as a descriptor of the susceptibility of PAHs to photomodification and the global shape feature is probably related to photosensitization activity. Keywords—Quantitative shape-activity relationships Polycyclic aromatic hydrocarbons Toxicity Electron density Molecular topology

We have shown in previous work that Brassica napus synthesizes epidermal flavonoids in response to UVB radiation (290–320 nm) and that these compounds are effective at screening the leaf mesophyll from UVB (Wilson and Greenberg, Photochem. Photobiol. 57, 556–563, 1993). This route of acclimation is common to many plant species. However, flavonoids are a highly diverse group of compounds that vary greatly from species to species, and little is known about the specific flavonoids synthesized in response to UVB. To address this, we extracted flavonoids from the leaves of B. napus plants exposed to photosynthetically active radiation (PAR: 400–700 nm), PAR + UVA (320–400 nm) and PAR + UVA + UVB. The compounds were resolved by HPLC and their structures were elucidated. Twelve distinct flavonoid glucosides with quercetin and kaempferol backbones were found. In some cases, a hydroxycinnamic acid moiety was linked via an ester bridge to a glucose. Of the 12 compounds identified, the leaf concentrations of 6 increased in response to UVB: kaempferol-3-O-sophoroside (K2), kaempferol-3-O-sophoroside-7-O-glucoside (Q3), quercetin-3-O-sophoroside (Q2), quercetin-3-O-sophoroside-7-O-glucoside (Q3), K3-coumaroyl ester and Q3-caffeoyl ester. Of these six compounds, K2, K3, Q2 and Q3 accumulated to high enough concentrations to contribute to UVB screening. Interestingly, the extractable amounts of the other six compounds identified were lower in the plants exposed to UVA or UVA + UVB compared to plants exposed only to PAR. Thus, in B. napus UV exposure seems to cause a shift in the population of flavonoid glycosides, with four of the UVB-induced flavonoids being generated in high concentrations.

Although UV-B is a minor component of sunlight, it has a disproportionately damaging effect on higher plants. Ultraviolet-sensitive targets include DNA, proteins and membranes, and these must be protected for normal growth and development. DNA repair and secondary metabolite accumulation during exposure to UV-B have been characterized in considerable detail, but little is known about the recovery of photosynthesis, induction of free-radical scavenging and morphogenic changes. A future challenge is to elucidate how UV-B-exposed plants balance damage, repair, acclimation and adaptation responses in a photobiologically dynamic environment.

Photoinduced toxicity of polycyclic aromatic hydrocarbons (PAHs) occurs via photosensitization reactions (e.g., generation of singlet-state oxygen) and by photomodification (photooxidation and/or photolysis) of the chemicals to more toxic species. The quantitative structure-activity relationship (QSAR) described in the companion paper predicted, in theory, that photosensitization and photomodification additively contribute to toxicity. To substantiate this QSAR modeling exercise it was necessary to show that toxicity can be described by empirically derived parameters. The toxicity of 16 PAHs to the duckweed Lemna gibba was measured as inhibition of leaf production in simulated solar radiation (a light source with a spectrum similar to that of sunlight). A predictive model for toxicity was generated based on the theoretical model developed in the companion paper. The photophysical descriptors required of each PAH for modeling were efficiency of photon absorbance, relative uptake, quantum yield for triplet-state formation, and the rate of photomodification. The photomodification rates of the PAHs showed a moderate correlation to toxicity, whereas a derived photosensitization factor (PSF; based on absorbance, triplet-state quantum yield, and uptake) for each PAH showed only a weak, complex correlation to toxicity. However, summing the rate of photomodification and the PSF resulted in a strong correlation to toxicity that hadmore » predictive value. When the PSF and a derived photomodification factor (PMF; based on the photomodification rate and toxicity of the photomodified PAHs) were summed, an excellent explanatory model of toxicity was produced, substantiating the additive contributions of the two factors.« less

Light (particularly ultraviolet B) results in photomodification of polycyclic aromatic hydrocarbons (PAHs) to products with increased polarity and water solubility and enhanced toxicity relative to the parent compounds. The uptake and depuration kinetics of three representative PAHs, anthracene (ANT), phenanthrene (PHE), and benzo[a]pyrene (BAP), and their photomodified products were determined for Lemna gibba. The {sup 14}C-labeled PAHs were delivered to the plants in their aqueous growth medium either via a dimethylsulfoxide (DMSO) carrier or adsorbed directly to sand placed in the medium. Assimilation was carried out under simulated solar radiation (SSR) and in darkness. The potential sites of PAH action within the plants were defined by identifying the subcellular location of both intact and photomodified PAHs following assimilation. Lemna gibba had a high capacity for intact ANT, PHE, and BAP in the dark regardless of the two routes of delivery. Depuration was also rapid. Net assimilation of all three PAHs in the dark was always higher when the chemicals were delivered with DMSO than from sand, although first-order kinetics were apparent with both delivery systems. The relative levels of assimilation were PHE > ANT > BAP. Polycyclic aromatic hydrocarbons were rapidly assimilated under SSR, albeit net assimilation for both themore » intact and photomodified forms was generally lower under SSR compared with darkness. This was also reflected in the bioconcentration factors, which were highest in darkness for each PAH and dropped significantly under SSR and after photomodification. Both intact and photooxidized PAHs accumulated preferentially in the thylakoids and microsomes of L. gibba, suggesting these to be the subcellular compartments most at risk from PAH damage.« less

Polycyclic aromatic hydrocarbon (PAH) toxicity is enhanced by light, especially ultraviolet (UV) radiation. To examine a potential mechanism(s) of photoinduced toxicity of PAHs to plants, the effects of anthracene and its photoproducts on photosynthesis were investigated using the aquatic higher plant Lemna gibba L. G-3 (duckweed). Photosynthetic activity was monitored both in vivo and in vitro by measuring chlorophyll a (Chl a) fluorescence, carbon fixation, and electron transport. In simulated solar radiation (a light source with a visible light: UV-A: UV-B ratio similar to sunlight), inhibition of photosynthesis was more rapid with photomodified anthracene than with intact anthracene, and intact anthracene appeared to only inhibit photosynthesis following its photomodification. The primary site of action of photomodified anthracene was found to be electron transport at or near photosystem I (PSI). This was followed by inhibition of photosystem II (PSII), probably due to excitation pressure on PSII once the downstream electron transport through PSI was blocked. Accordingly, higher chemical concentrations and/or longer exposures were required to inhibit PSII than PSI. Net photosynthesis (carbon fixation) was also inhibited, implying that the inhibition of electron transport in PSI by photomodified anthracene can lead to diminished primary productivity. A linkage between inhibition of photosynthesis and inhibition of plant growth was established in terms of the initial site of action (PSI) and primary productivity (carbon fixation), which suggested that Chl a fluorescence can be used as a bioindicator of PAH impacts on plants.

Phenanthrene (PHE) undergoes a significant increase in toxicity after exposure to simulated or natural sunlight in aqueous media, coincident with the appearance of PHE photoproducts. To investigate whether the primary photoproduct of PHE, 9,10- phenanthrenequinone (PHEQ), contributes to the increased hazards of solutions containing photomodified PHE, toxicity assays were conducted using the marine bacteria Photobacterium phosphoreumand the aquatic plant Lemna gibba (duckweed). Photo- bacterium phosphoreumwas exposed to PHE, PHEQ, a photomodified PHE mixture containing known amounts of PHE and PHEQ (pmPHE), and a mixture mimicking the amounts of PHE and PHEQ in the pmPHE mixture. The bacteria were found to be equally sensitive to PHE in simulated solar radiation (SSR, a light source with a visible light : UVA : UVB ratio similar to that of sunlight) or darkness, with an EC50 of 0.53 mg/L. In both darkness or SSR, solutions containing PHEQ (with or without PHE) all exhibited an EC50 of 0.06 to 0.10 mg/L based on PHEQ concentrations, indicating that PHEQ was the primary active component of the pmPHE mixture. Lemna gibba was tested in SSR and visible light with PHE, PHEQ, and the pmPHE mixture. The calculated EC50 for PHE was 3.5 mg/L in SSR and 10.8 mg/L in visible light, showing that the presence of UV radiation in the SSR source increased the phytotoxicity of PHE. Strikingly, PHEQ was much more toxic to L. gibba than PHE in a light-independent manner (an EC50 of 0.53 and 0.57 mg/L PHEQ in dark and SSR, respectively). Thus, for both P. phosphoreum and L. gibba the major photooxidation product of PHE in SSR, PHEQ, is the more toxic of the two chemicals.

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to a gill cell line from rainbow trout for future use in screening polycyclic aromatic hydrocarbons for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without foetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the fluoranthene to remain in solution and blocked photocytotoxicity if present during UV irradiation. DMSO had little effect on fluoranthene distribution in cell cultures but caused cells to be slightly more sensitive to the phototoxicity of fluoranthene. The indicator dyes alamar BlueTM and 5-carboxyfluorescein diacetate acetoxymethyl ester were used to quantify cytotoxicity in two different ways-singly in two separate assays, and mixed together in a novel single assay, which saved time and material. With UV irradiation for 2 hr at a photon fluence rate of either 1.4 mu mol UV-B/m(2)/sec (UV-A:UV-B, 1.5) or 1.1 mu mol UV-B/m(2)/sec (UV-A:UV-B, 9.7), both dyes indicated increasing loss of viability with increasing doses of fluoranthene. EC, values ranged from 18 to 44 ng/ml (89-217 nM), with the alamar Blue assay being slightly more sensitive. (C) 1997 Elsevier Science Ltd.

Simulated solar radiation and natural sunlight can enhance polycyclic aromatic hydrocarbon (PAH) toxicity, previously reported in terms of inhibited production of Lemna gibba leaves and diminished growth of Brassica napus seedling roots. This work has been extended to examine the photoinduced impact of PAHs on the foliar regions of terrestrial plants. To carry out these experiments two crop species, B. napus (canola) and Cucumbis sativus (cucumber), were chosen to test the photoinduced toxicity of six PAHs (anthracene, phenanthrene, benzo(a)anthracene, benzo(a)pyrene, flouranthene, and pyrene). Aqueous solutions containing PAHs were sprayed on the foliage of the plants. It was found that all the PAHs tested had negative impacts on the foliage of the plants and the concentrations that induced toxicity were consistent with those observed for inhibition of growth of L. gibba. The impacts were observed as diminished biomass accumulation, induction of chlorosis, and inhibition of photosynthesis. It may be concluded from this work that PAHs in rain and surface waters could be harmful to photosynthetic tissues of terrestrial plants.

— Although the effects of ultraviolet B (UVB, 290–320 nm) radiation have been studied in plants extensively, little is known about the potential impacts on maturation of chloroplasts. To address this problem, the effects of supplementary UVB on chloroplast development were examined in the aquatic higher plant Spirodela oligorrhiza. Dark-grown Spirodela-containing proplastids were exposed to photosynthetically active radiation (PAR) and ultraviolet A (UVA, 320–400 nm), plus supplementary UVB equivalent to 1% of PAR on a photon basis. The biosynthesis and assembly of chlorophyll (Chi) into reaction centers was followed for 4 days in situ by low temperature (77 K) Chi fluorescence. Impacts on chloroplast development were detected after only 1 h incubation in light with supplementary UVB. Fluorescence emission signals from Chi associated with the photosystem (PS) II antenna, PSII reaction centers and PSI reaction centers were detected at the same time with or without UVB, but the magnitude of PS fluorescence was diminished up to 60% in plants incubated in UVB. The Chi content was also lower in UVB-treated plants, but to a lesser degree than anticipated by low temperature fluorescence, suggesting lack of organization and/or association of Chi with PS. Electron transport, measured with room temperature fluorescence induction, was not consistently different in plants exposed to UVB. These results suggest that with UVB, fewer and/or smaller PS form during chloroplast development, but there is not a large inhibition of Chi synthesis or PSII activity.

It has recently been demonstrated that light dramatically enhances the toxicity of polycyclic aromatic hydrocarbons (PAHs) to the duckweed Lemna gibba L. G-3 (L. Ren, X.-D. Huang, B.J. McConkey, D.G. Dixon, and B.M. Greenberg, 1994, Ecotoxicol. Environ. Saf. 28, 160-171). To extend this research to terrestrial plants, Brassica napus L. (oil seed rape) seeds were germinated in the presence of three PAHs; anthracene (ANT), benzo[a]pyrene (BAP), and fluoranthene. The chemicals were applied both in intact form and following photomodification in UV-B radiation; toxicity was assessed in simulated solar radiation (SSR), a light source with a visible light:UV-A:UV-B ratio similar to that of sunlight. Germination efficiency, root and shoot growth, and chlorophyll content, measured after 6 days of exposure, were used as toxicity endpoints. Intact and photomodified PAHs had little impact on shoot fresh weight or chlorophyll content, but markedly inhibited root fresh weight, with the photomodified PAHs having greater impacts than the intact PAHs. The decline in root fresh weight was not attributable to a decline in germination frequency or delayed germination. However, the seedlings produced shorter roots in the presence of either intact or photomodified PAHs. To explore the role of actinic radiation on PAH toxicity, seedlings were incubated in SSR, visible light and darkness with either intact or photomodified PAHs. Inhibition of root growth was only achieved by the intact chemicals if actinic radiation was present. However, with photomodified ANT or photomodified BAP, root fresh weight accumulation was inhibited in SSR, visible light and darkness. Thus, intact PAHs are hazardous to terrestrial plants in the presence of light, but once the compounds are photomodified, actinic radiation is no longer an absolute requirement for phytotoxic activity.

Plants can balance the relative levels of excitation energy reaching the two photosystems of photosynthesis via state transitions. This process was investigated in vivo using the aquatic higher plant Spirodela oligorrhiza . State transitions were followed by 77 K chlorophyll a (Chl a ) fluorescence and phosphorylation of the Chl a b light harvesting complex (LHCII). A response spectrum for the state transition indicated that light absorbed predominantly by Chl b led to state 2 and light absorbed predominantly by Chl a resulted in state 1. The kinetics of LHCII phosphorylation ( t 1 2 = 4 min ) during a state 1 to state 2 transition were similar to the rise in fluorescence at 77 K from Photosystem I (PS I) relative to Photosystem II (PS II) ( t 1 2 = 3 min ). As well, for the transition from state 2 to state 1, the kinetics of LHCII dephosphorylation ( t 1 2 = 13 min ) and the rate of loss of fluorescence from PS I relative to PS II ( t 1 2 = 10 min ) were comparable. The phosphatase inhibitor, NaF, suppressed both LHCII dephosphorylation and the decrease in the PS I/PS II fluorescence emission ratio, thus showing prevention of a transition to state 1 under PS I illumination. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which indirectly inactivates the LHCII kinase, triggered a transition to state 1 in PS II light. Upon exposure to PS II light, phosphorylated LHCII was observed to appear in granal thylakoid fractions prior to its appearance in stromal thylakoid fractions, indicating vectoral movement between the two membrane compartments. Determinations of the absolute yield of PS II and PS I fluorescence showed a complementary decrease and increase in PS II and PS I emission, respectively, in state 2 compared to state 1. Our results strongly support the hypothesis that LHCII phosphorylation and antenna migration between the photosystems are key components of the state transition mechanism in vivo.

The soil-contact exposure pathway can be the main driver of ecological risk assessments. There is currently no standard method to measure bioavailability of metals in soil to ecological receptors, yet the influence of metal bioavailability on toxicity has been known for decades and is a major factor influencing risk to ecological receptors. Bioavailability is to a large degree governed by varying soil characteristics within and among sites, yet ecological screening benchmarks are often derived on a total-concentration basis. We compared a calcium chloride (CaCl2) extraction, cyclodextrin extraction, simulated earthworm gut (SEG) test, earthworm kinetic bioaccumulation test, and metal residues in plant tissues with a battery of invertebrate and toxicity tests using mining soils consisting of high organic-matter content cocontaminated with copper (Cu) and zinc (Zn). Earthworm (Eisenia andrei) tissue concentrations of Cu and Zn were regulated and were not predictive of invertebrate toxicity. All chemical measures of bioavailability correlated with several biological responses; however, CaCl2-extractable Cu and SEG-extractable Cu and Zn best predicted effects to E. andrei. Total Cu concentrations in soil best correlated with effects to plants. Overall, a chemical measure was the best predictor of toxicity to each organism compared with biological measures, although the exact measure was dependent on organism and end point. Chemical-extraction techniques provide relatively quick, inexpensive indicators of essential metal bioavailability compared with biological measures; however, no single measure was indicative of all effects to all organisms.

The toxicity of polycyclic aromatic hydrocarbons (PAHs) can be enhanced by both biotic and abiotic processes. This is exemplified by light, which, by virtue of the extensive π-orbital systems of PAHs, can be a major factor in PAH toxicity. Light activation of PAHs is known to occur via photosensitization reactions (generation of singlet oxygen and superoxide) and potentially by photomodification of the chemicals (photooxidation and/or photolysis) to more toxic species. To examine the modes of PAH action in the light and determine if the photomodified compounds are hazardous, we investigated the photoinduced toxicity of anthracene, phenanthrene and benzo[a]pyrene to the aquatic higher plant Lemna gibba (a duckweed). Toxicity end points were inhibition of growth and extent of chlorosis. Light did indeed activate the phytotoxicity of PAHs, with UV radiation more effective than visible light. Dose-response curves based on chemical concentration and light intensity revealed the order of phytotoxic strength to be anthracene > phenanthrene > benzo[a]pyrene. To explore whether photomodified PAHs were contributing to toxicity, the chemicals were irradiated before toxicity testing. The rates of photomodification of the three PAHs were rapid (half-lives in hours), and the relative velocities were coincident with the order of toxic strength. Furthermore, the photomodified PAHs were more hazardous to Lemna than the intact compounds. Because interpretations of the potential impacts of PAHs in the environment are based mostly on measurements of the structurally intact chemicals, the severity of PAH hazards is possibly underestimated.

As depletion of the stratospheric ozone layer continues, the biosphere will most likely be exposed to higher levels of ultraviolet-B (UV-B) irradiation (290–320nm). For plants, damage from UV-B can occur at several molecular targets with the photosynthetic apparatus being especially vulnerable. We are interested both in the mechanisms of UV-B-induced damage and identifying adaptation processes that can confer protection from UV-B. Toward this end, Brassica napus (oil seed rape) plants grown under visible light plus a low level of UV-B radiation (adapted plants) were compared to plants grown under visible light alone (control plants). Relative to the control plants, the adapted plants showed little evidence of damage at the levels of morphology or photosynthesis, indicating that B. napus has some tolerance of UV-B and that the plants may have protection mechanisms. Consistent with this, a strong UV-B adaptation process was observed in the plants-accumulation of flavonoids in the epidermis. These pigments seemed to screen a molecular target in the mesophyll. Namely, the D1 photosystem II reaction center protein, which is rapidly degraded in UV-B, was partially protected from degradation in UV-B in the adapted plants. Moreover, the extent that the half-life of the D1 protein increased in the adapted plants was on par with the elevation in total flavonoid concentrations. These experiments demonstrate that degradation of the D1 protein can be used as an in vivo assay of penetration of UV-B photons to the mesophyll.

Plants accumulate flavonoids in response to a myriad of environmental challenges, especially when exposed to ultraviolet (UV) radiation or situations causing oxidative stress. However, the origin and nature of the signal triggering their accumulation remain obscure. In this study, a group of flavonoids belonging to the flavone class was identified in Lemna gibba (duckweed). These flavones accumulated upon exposure to UV radiation, low temperature, copper and the photosynthetic electron transport (PET) inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and 1,2-dihydroxyanthraquinone (DHATQ). All of these stressors were also shown to promote PET chain (PETC) reduction; however, in the co-presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or a light regime that oxidized the PETC, flavonoid accumulation ceased. Chloroplast-derived reactive oxygen species (ROS) were not associated with all of the stress conditions that promoted both PETC reduction and flavonoid synthesis, indicating that ROS were not a strict requisite for flavonoid accumulation. Transcripts for the flavonoid biosynthetic genes, chalcone synthase (CHS) and chalcone isomerase, were similarly responsive to the PETC redox state, as were a panel of transcripts revealed by differential display PCR. Collectively, these results provide evidence that PETC redox status is one of the factors affecting flavonoid biosynthesis.

In a petroleum impacted land-farm soil in Sarnia, Ontario, compound-specific natural abundance radiocarbon analysis identified biodegradation by the soil microbial community as a major pathway for hydrocarbon removal in a novel remediation system. During remediation of contaminated soils by a plant growth promoting rhizobacteria enhanced phytoremediation system (PEPS), the measured Delta(14)C of phospholipid fatty acid (PLFA) biomarkers ranged from -793 per thousand to -897 per thousand, directly demonstrating microbial uptake and utilization of petroleum hydrocarbons (Delta(14)C(PHC) = -1000 per thousand). Isotopic mass balance indicated that more than 80% of microbial PLFA carbon was derived from petroleum hydrocarbons (PHC) and a maximum of 20% was obtained from metabolism of more modern carbon sources. These PLFA from the contaminated soils were the most (14)C-depleted biomarkers ever measured for an in situ environmental system, and this study demonstrated that the microbial community in this soil was subsisting primarily on petroleum hydrocarbons. In contrast, the microbial community in a nearby uncontaminated control soil maintained a more modern Delta(14)C signature than total organic carbon (Delta(14)C(PLFA) = +36 per thousand to -147 per thousand, Delta(14)C(TOC) = -148 per thousand), indicating preferential consumption of the most modern plant-derived fraction of soil organic carbon. Measurements of delta(13)C and Delta(14)C of soil CO(2) additionally demonstrated that mineralization of PHC contributed to soil CO(2) at the contaminated site. The CO(2) in the uncontaminated control soil exhibited substantially more modern Delta(14)C values, and lower soil CO(2) concentrations than the contaminated soils, suggesting increased rates of soil respiration in the contaminated soils. In combination, these results demonstrated that biodegradation in the soil microbial community was a primary pathway of petroleum hydrocarbon removal in the PEPS system. This study highlights the power of natural abundance radiocarbon for determining microbial carbon sources and identifying biodegradation pathways in complex remediation systems.

Although higher plants represent a significant portion of the total biomass in some aquatic environments, their use in ecosystem evaluation has lagged behind that of other organisms. This is partly due to a lack of convenient aquatic higher plant systems that can be employed for ecotoxicological assessment. However, the aquatic C-3 monocot Lemna gibba has many attributes that makes it useful for ecosystem health assessment. In this report, using examples from the literature and our research, some of the applications Lemna has for environmental research are discussed. Toxicant impacts on Lemna can be readily assessed in terms of growth; the plants multiply quickly and changes in biomass (which doubles approximately every 2 days) can be accurately measured by counting leaves. The plants are small, allowing for simultaneous multiple replication. The small size also makes the lighting conditions easy to control; sunlight can be accurately simulated and specific spectral regions can be enhanced or deleted. Lemna is amenable to in vitro chlorophyll and photosynthesis assays, which make excellent companion endpoints for growth. The plants assimilate chemicals directly from the growth medium, facilitating controlled toxicant application. Furthermore, Lemna has a high bioconcentration capacity, indicating a potential for use in bioremediation technologies.