TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo

Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids.     

Genetic diversity of culturable bacteria in oil-contaminated rhizosphere of Galega orientalis

A collection of 50 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-toluate also degraded m-toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.     

Effects of mercury on visible/near-infrared reflectance spectra of mustard spinach plants (Brassica rapa P.)

Mustard spinach plants were grown in mercury-spiked and contaminated soils collected in the field under controlled laboratory conditions over a full growth cycle to test if vegetation grown in these soils has discernible characteristics in visible/near-infrared (VNIR) spectra. Foliar Hg concentrations (0.174-3.993ppm) of the Mustard spinach plants were positively correlated with Hg concentration of soils and varied throughout the growing season. Equations relating foliar Hg concentration to spectral reflectance, its first derivative, and selected vegetation indices were generated using stepwise multiple linear regression. Significant correlations are found for limited wavelengths for specific treatments and dates. Ratio Vegetation Index (RVI) and Red Edge Position (REP) values of plants in Hg-spiked and field-contaminated soils are significantly lower relative to control plants during the early and middle portions of the growth cycle which may be related to lower chlorophyll abundance or functioning in Hg-contaminated plants.     

Evaluation of copper availability to plants in copper-contaminated vineyard soils

The repeated use of copper (Cu) fungicides to control vine downy mildew has led to long-term accumulation of Cu in vineyard soils which now raises the issue of the potential bioavailability of Cu for various living organisms including plant species. The bioavailable Cu can be defined as the portion of soil Cu that can be taken up by roots, for a given plant species. In order to evaluate the bioavailability of Cu to plants, a pot experiment was conducted in glasshouse conditions with a crop species (maize) and 12 soils sampled in the upper horizon of 10 vineyard plots (total Cu ranging from 38 to 251 mg kg-1) and two woodland plots (control soils that had not received any Cu application; total Cu amounting to 20-26 mg kg-1). These soils were selected for their diverse physical (large range of particle size distribution) and chemical (from acid to calcareous soils) properties. After 35 days of growth, plant shoots were harvested for analysis. The roots were separated from soil particles for further analysis. The concentrations of Cu in the roots and aerial parts of the maize were then compared with the amounts of Cu extracted from the soil by a range of conventional extractants. Observed Cu concentrations in maize roots which have grown in contaminated vineyard soils were very high (between 90 and 600 mg kg-1), whereas Cu concentrations in the aerial parts varied only slightly and remained low (< 18 mg kg-1). Root Cu concentrations observed for maize increased with increasing total Cu content in the soil and with decreasing soil CEC. Cu accumulation in maize roots may be as high in calcareous soils as in acid soils, suggesting that soil pH had little influence. In the case of the vineyard soils studied, the lack of correlation found for maize between Cu concentrations in roots and in the aerial parts, suggests that an analysis of the aerial parts would not be a good indicator of plant Cu uptake, as it provides no insight into the real amount of Cu transferred from the soil to the plant. For maize, our results show that extraction with organic complexing agents (EDTA, DTPA) and extraction with ammonium acetate seem to provide a reasonably good estimate of root Cu concentration.     

Phytoextraction of lead from firing range soil by Vetiver grass

Phytoextraction techniques utilizing a sterile strain of Vetiver grass (Vetiveria zizanoides) along with soil amendments were evaluated for removing lead and other elements such as Zn, Cu, and Fe from the soil of a 50-year old active firing range at the Savannah River Site (SRS). Lead-contaminated soil (300-4500 ppm/kg) was collected, dried, placed in pots, fertilized, and used as a medium for growing transplanted Vetiver grass plants in a greenhouse. The uptake of metals by the plants was evaluated in response to various fertilization and pre-harvest treatment schemes. Baseline metal concentrations in the soil of all pots were measured prior to planting and when the plants were harvested. Plants grew better when fertilized with Osmocote fertilizer in comparison to plants fertilized with 10-10-10 (NPK) fertilizer. Application of a chelating agent, EDTA, one week prior to harvest significantly increased the amount of lead that was phytoextracted. Lead concentrations of up to 1390-1450 ppm/kg in tissue samples were detected. Maximum Pb levels were observed in root tissues. The addition of non-lethal doses of a slow-release herbicide in combination with EDTA did not appear to further enhance phytoextraction or the translocation of Pb into shoots. The study indicated that the use of Vetiver grass coupled with the use of chelating soil amendments has considerable potential for use as a remedial strategy for lead-contaminated soils such as those associated with firing ranges.     

Effects of chronic ozone exposure on growth, root respiration and nutrient uptake of rice plants

To clarify the response of growth and root functions to low concentrations of ozone (O(3)), rice plants (Oryza sativa L.) were exposed to O(3) at 0.0 (control), 0.05 and 0.10 ppm for 8 weeks from vegetative to early heading stages. Exposure to 0.05 ppm O(3) tended to slightly stimulate the dry weight of whole plants up to 5 weeks and then slightly decrease the dry weight of whole plants. However, these effects were statistically significant only at 6 weeks. Exposure to 0.10 ppm O(3) reduced the dry weight of whole plants by 50% at 5 and 6 weeks, and thereafter the reduction of the dry weight of whole plants was gradually alleviated. Those changes in dry weight can be accounted for by a decrease or increase in the relative growth rate (RGR). The changes in the RGR caused by 0.05 and 0.10 ppm O(3) could be mainly attributed to the effect of O(3) on the net assimilation rate. Root/shoot ratio was lowered by both 0.05 and 0.10 ppm O(3) throughout the exposure period. The root/shoot ratio which had severely decreased at 0.10 ppm O(3) in the first half period of exposure (1-4 weeks) became close to the control in the latter part of exposure (5-8 weeks). Time-course changes in NH(4)-N root uptake rate were similar to those in the root/shoot ratio especially for 0.10 ppm O(3). On the other hand, root respiration increased from the middle to later periods. Since it is to be supposed that plants grown under stressed conditions change the ratio of plant organ weight to achieve balance between the proportion of shoots to roots in the plant and their activity for maintaining plant growth, these changes in root/shoot ratio and nitrogen uptake rate under long-term exposure can be considered to be an adaptive response to maintain rice growth under O(3) stress.     

Organism-induced accumulation of iron, zinc and arsenic in wetland soils

Four year old spruce (Picea abies (L.) Karst.) seedlings were planted in sand pots and supplied with nutrient solution. Three groups were formed, differing only in manganese nutrition (0.5 ppm, 2.5 ppm, 12.5 ppm, respectively). After three months, five individuals of each group were transferred to a dew chamber. For the next seven weeks the trees were sprayed in the evenings, the relative humidity overnight was kept high and the droplets were collected directly from the needles in the mornings. The trees were sprayed with HNO3 (pH 3.4) during the first three weeks to reduce the natural buffering capacity of the needles. After this time, the trees were sprayed with KCl (1 mM) solution, and NaHSO3 was added to the chamber resulting in SO2 concentrations usually between 50 and 150 microg m(-3). Needles and water samples were analysed. Foliar Ca seemed to be only a short-time buffer even under optimal Ca supply. A highly significant influence of managanese supply on manganese in needles and droplets was observed, as well as on sulphate, H+ and calcium concentrations in the droplets. The SO2 flux to trees treated with 12.5 ppm Mn was about twice as high as to trees treated with 0.5 ppm Mn. The conclusion is that this is due to a synergism between manganese leaching and catalysis of the SO2 oxidation by the leached Mn2+ ions. The results suggest a positive feedback between (moderate) acidification of soils and SO2 and NH3 inputs to terrestrial ecosystems.     

Comparative studies of the fate of atrazine‐14C and pentachlorophenol‐14C in various laboratory and outdoor soil‐plant systems

Abstract

Mass balance and fate of atrazine‐ ¹⁴C and pentachlorophenol‐ ¹⁴C (PCP‐ ¹⁴C) were studied in short‐term tests in a closed aerated laboratory soil‐plant system, using two concentrations in soil and two plant species, as well as under outdoor conditions for one vegetation period. In the laboratory, for both pesticides bioaccu‐mulation factors of radiocarbon taken up by the roots into plants were low. They were higher for lower (1 ppm) than for higher soil concentrations (6 ppm for atra‐zine, 4 ppm for pentachlorophenol) and varied with the plant species. Mineralization to ¹⁴CO₂ in soil was negatively related to soil concentration only for PCP‐ ¹⁴C. Conversion rates in soil including the formation of soil‐bound residues were higher for the lower concentrations of both pesticides than for the higher ones; conversion rates in plants were species‐dependent. In 14 terms of CO₂ formation and of conversion rates, PCP was less persistent in soil than was atrazine. For both pesticides, laboratory data on conversion and mineralization gave a rough prediction of their persistence in soil under long‐term outdoor conditions, whereas bio‐accumulation factors in plants under long‐term outdoor conditions could not be predicted by short‐term laboratory experiments.     

Speciation and uptake of arsenic accumulated by corn seedlings using XAS and DRC-ICP-MS

ICP-MS was used to investigate the uptake of As(III) and As(V) from hydroponics growth media by corn seedlings. It was found that arsenic uptake by the plant roots for the arsenic(V) and arsenic(III) treatments were 95 and 112 ppm, respectively. However, in the shoots of the arsenic (V) treatments had 18 ppm whereas arsenic(III) treatments had 12 ppm. XANES studies showed that As for both treatments arsenic was present as a mixture of an As(III) sulfur complex and an As(V) oxygen complex. The XANES data was corroborated by the EXAFS studies showing the presence of both oxygen and sulfur ligands coordinated to the arsenic. Iron concentrations were found to increase by 4 fold in the As(V) contaminated growth media and 7 fold in the As(III) treatment compared to the control iron concentration of 500 ppm. Whereas, the total iron concentration in the shoots was found to decrease by approximately the same amount for both treatments from 360 ppm in the control to approximately 125 ppm in both arsenic treatments. Phosphorus concentrations were found to decrease in both the roots and shoots compared to the control plants. The total sulfur in the roots was found to increase in the arsenic(III) and arsenic(V) treatments to 560 ppm and 800 ppm, respectively, compared to the control plants 358 ppm. In addition, the total sulfur in shoots of the plants was found to remain relatively constant at approximately 1080 ppm. The potassium concentrations in the plants were found to increase in the roots and decrease in the shoots.     

Growth responses of crop and weed species to heavy metals in pot and field experiments

Greenhouse and field studies were performed to examine the growth responses and possible phytoremediation capacity towards heavy metals of several Brassicaceae (Brassica alba, Brassica carinata, Brassica napus and Brassica nigra) and Poaceae (durum wheat and barley). Soils used featured total concentrations of Cr, Cu, Pb and Zn largely exceeding the maximum levels permitted by the Italian laws. Different organic amendments were tested such as a compost and the plant growth-promoting rhizobacterium Bacillus licheniformis. In the greenhouse experiment, plant length, leaf area index and shoots dry matter were evaluated periodically for the Brassicaceae examined. Whereas plant length, grains production, weight of 1,000 seeds, ear fertility and tiller density were determined under field conditions at the end of the crop cycle for wheat and barley. In general, the species tested appeared to be tolerant to high heavy metal concentrations in soil, and slightly significant differences were found for all parameters considered. A marked growth increase was shown to occur for Brassicaceae cultivated on compost- and bacillus-amended contaminated soils, with respect to non-amended contaminated soils. With some exception, higher growth parameters were measured for wheat and barley plants cropped from contaminated soils in comparison to non-contaminated soils. Further, bacillus amendment enhanced the length of wheat and barley plants in both non-contaminated and contaminated soils, while different effects were observed for the other parameters evaluated.     

Effect of copper on growth of an aquatic macrophyte,Elodea canadensis

Elodea canadensis has been proposed as a potential biomonitor due to its wide distribution and apparent ability to accumulate pollutants in aquatic ecosystems. We investigated the effects of copper sulfate on growth in E. canadensis to determine its effectiveness as a biomonitor of copper pollution in aquatic systems and whether growth is a suitable index of sub-lethal stress. Copper sulfate significantly slowed or stopped growth at all concentrations (low: 1 ppm, medium: 5 ppm, high: 10 ppm of copper sulfate) used. Final plant drymass was significantly lower in medium and high copper treatments compared with controls. E. canadensis appears to be very sensitive to copper levels, and may be useful as a biomonitor of copper levels in aquatic systems. However, its utility as a bioaccumulator may be limited, because we observed senescence of most leaves in all copper-treated plants following 4 weeks of treatment.     

Trace elements in soil and biota in confined disposal facilities for dredged material

We studied the relation of trace element concentrations in soil to those in house mice (Mus musculus), common reed (Phragmites australis) and ladybugs (Coccinella septempunctata at five disposal facilities for dredged material. The sites had a wide range of soil trace element concentrations, acid soils and a depauperate fauna. They were very poor wildlife habitat because they were dominated by the common reed. Bioassay earthworms exposed to surface soils from three of the five sites died, whereas those exposed to four of five soils collected a meter deep survived, presumably because the deeper, unoxidized soil, was not as acid. Concentrations of Ni and Cr in the biota from each of the sites did not seem to be related to the concentrations of the same elements in soil. Although Pb, Zn and Cu concentrations in biota were correlated with those in soil, the range of concentrations in the biota was quite small compared to that in soil. The concentrations of Pb detected in mice were about as high as the concentrations previously reported in control mice from other studies. Mice from the most contaminated site (530 ppm Pb in soil) contained only slightly more Pb (8 ppm dry wt) than did mice (2-6 ppm dry wt) from sites containing much less Pb (22-92 ppm in soil). Despite the acid soil conditions, very little Cd was incorporated into food chains. Rather, Cd was leaching from the surface soil. We concluded that even the relatively high concentrations of trace elements in the acid dredged material studied did not cause high concentrations of trace elements in the biota.     

Mobility of antimony in soil and its availability to plants

In a historical mining area residual material has been filled on land and these locations are used today as agricultural soils or house gardens. The antimony concentrations in these soils are up to 500 mg/kg. Antimony transfer into 19 vegetable and crop species was investigated. In grain and other storage organs up to 0.09 mg Sb/kg were found, whereas maximum antimony concentrations in shoots and leaves were determined to be 0.34 mg Sb/kg and 2.2 mg Sb/kg, respectively. Despite the high antimony contamination of the soils, concentrations in the investigated plants in general corresponded to concentrations only reported for uncontaminated soils. NH4NO3 extraction of some of the soils indicated that the mobile fraction of antimony present was only 0.06-0.59%. In contrast, in leaves of spinach grown under controlled conditions in soils with a high mobile antimony content an accumulation of the element could be observed: a maximum value of 399 mg Sb/kg was detected, and a correlation between the mobile fraction in the soils and antimony in leaves was found.     

Hybrid-Type Electrostatic Precipitator

Three experiments were designed to determine the effects of SO₂ concentration on two Southeastern oat varieties—Caroiee and Coker 227. The plants were grown under uniform conditions prior to and after the experimental growth period. During the 7 to 14 day experimental growth period the plants were subjected to four growth temperatures (18/14, 22/18, 26/22 and 30/26° C day/night temperatures) and exposed 2 or 4 times to specific concentrations of SO₂ (from 0 to 4 ppm) for 1.5 or 3.0 hr periods. In the first two experiments, the exposure temperatures were varied (18° to 30° C). In the third design, the exposure temperatures remained constant and two exposure relative humidities (55 and 80%) were used. Injury and growth reductions were more severe in Coker 227 than in Caroiee. Root dry weight (RDW) was affected more than top dry weight (TDW). Plants were more sensitive at the higher growth temperatures. Exposure temperatures had little to variable effects. Plants were more sensitive at 80% than at 55% relative humidity. Biomass was reduced at 0.4 ppm SO₂ after four 3 hr exposures and at 0.75 ppm after two 1.5 hr exposures, but growth was also increased as often. These concentrations approached the accepted threshold for adverse effects to sensitive vegetation. Foliar injury was highly correlated with growth reductions.     

Cd accumulation and phytostabilization potential of dominant plants surrounding mining tailings

The objectives of the present study were to compare nine dominant plant species growing in mine tailings and nonmining areas in terms of biomass and Cd concentrations and to search for Cd accumulation and tolerance. Also, more detailed experiments were conducted on Athyrium wardii using a pot experiment to assure its Cd-accumulation ability and tolerance as a potential phytostabilizer of Cd-polluted soils. Nine dominant plant species growing on Pb/Zn mine tailings and their corresponding nonmining ecotypes were investigated for their potential to phytostabilize Cd. The performance of A. wardii exposed to high levels of Cd was investigated under controlled conditions. A field study revealed that the Cd concentrations in the roots of these plants ranged from 0.21 to 251.07?mg?kg^?1, and the highest concentrations were found in A. wardii, which reached a concentration of 69.78, 251.07, and 126.35?mg?kg^?1 during the early growth stage (May), vigorous growth stage (August), and late growth stage (October), respectively. The Cd concentrations of roots among the nine mining ecotypes were positively correlated with available content of Cd in the rhizosphere soils, whereas a negative correlation was observed in the nonmining ecotypes. A pot experiment showed that the mining ecotype of A. wardii had a higher biomass production and Cd retention capacity in roots than that of the nonmining ecotype. Due to the relatively high tolerance to Cd and the capacity of roots to retain this metal, A. wardii may be useful for the phytostabilization of soils contaminated by Cd.     

Uptake of metals by food plants grown on soils 10 years after biosolids application

Potentially hazardous trace elements such as Cd, Cu, Cr, Ni and Zn are expected to accumulate in biosolids–amended soil and remain in the soil for a long period of time. In this research, uptake of metals by food plants including cabbage, carrot, lettuce and tomato grown on soils 10 years after biosolids application was studied. All the five metals were significantly accumulated in the biosolids-amended soils. The accumulation of metal in soil did not result in significant increase in concentrations of Cu, Cr and Ni in the edible plant tissues. However, the Cd and Zn concentrations of the edible tissues of plants harvested from the biosolids receiving soils were significantly enhanced in comparison with those of the unaffected soils. The plant uptake under Greenfield sandy loam soil was generally higher than those under the Domino clayey loam soil. The metal concentration of edible plant tissue exhibited increasing trends with respect to the concentrations of the ambulated metals. The extents of the increases were plant species dependent. The indigenous soil metals were absorbed by the plants in much higher rates than those of the biosolids–receiving soils. It appeared that the plant uptake of the indigenous soil-borne metal and the added biosolids-borne metals are independent of one another and mathematically are additive.     

Comparing growth development of <Emphasis Type="Italic">Myriophyllum</Emphasis> spp. in laboratory and field experiments for ecotoxicological testing

BACKGROUND, GOALS AND SCOPE: Risk assessment of herbicides and the evaluation of contaminated sediments based on algae and the macrophyte Lemna sp. alone may underestimate the potential hazard of certain compounds. Therefore, various test systems with Myriophyllum spp. have been developed recently to assess the phytotoxicity in surface waters and natural sediments. In the present study, experiments investigating the growth development of Myriophyllum spp. were performed in the laboratory under defined conditions and in mesocosms under environmentally realistic exposure conditions to evaluate the suitability of these species as potential standard test organisms in ecotoxicological testing. This study provides data on the endpoints biomass, plant length and root development. MATERIALS AND METHODS: Six independent experiments were performed to investigate the plant development of Myriophyllum spp. under control conditions. The main difference in the experiments was the complexity of the test systems ranging from simple laboratory experiments to complex outdoor mesocosm studies. At the start of each experiment, uniform cuttings of Myriophyllum spp. were placed in vessels with or without sediments to reduce variability between replicates. The endpoints considered in this investigation were biomass (fresh weight of the whole plant), length of the main shoot, length of the side shoots, total length of the plant (calculated from the length of the main and side shoots) and root formation. Root to shoot ratios were calculated as a further measure for plant development. Relative growth rates (RGR) based on plant length (RG(L)R) and on biomass (RG(B)R) were calculated. RESULTS: Despite the various experimental conditions, comparable growth was obtained in all test systems and the variability of endpoints, such as total length and biomass of plants, was low. It was observed that the RGR of M. spicatum in the simple laboratory test system with sediment were comparable to growth data obtained for M. verticillatum and M. spicatum grown in indoor and outdoor mesocosms, thus indicating that Myriophyllum growth tends to increase by the addition of sediment. High variability was determined for the endpoints length of the side shoots, total root length and biomass of roots. DISCUSSION: One challenge for a test design to investigate phytotoxicity on aquatic plants is to obtain good growth of the plants. From the results, it can be concluded that the experimental conditions in the various test systems were suitable to study the plant development of Myriophyllum spp. because obtained growth rates were comparable between laboratory and field investigations. Another challenge for developing a plant biotest system is the definition of sensitive endpoints. Low variability is preferred to detect minor effects of chemicals or polluted sediments on plant development. In our studies, the variability of the endpoints biomass and total length of plant was low and, therefore, they have much potential as endpoints for assessing toxicity. CONCLUSIONS: The methodologies presented in this study have applications within the risk assessment for aquatic plants and have the advantage of assessing effects taking into account the relevant exposure pathways via water and/or sediment for compounds under investigation. RECOMMENDATIONS AND PERSPECTIVES: Setting safe quality criteria for surface water and sediments is one of the challenges authorities are facing today. Myriophyllum spp. is recommended as suitable test species to investigate phytotoxicity in surface water and sediments. These results, thus, might serve as a basis for the compilation of a new harmonised guideline for ecotoxicological testing with aquatic macrophytes.     

Biosolids-amended soils: Part I. Effect of biosolids application on soil quality and ecotoxicity.

Investigations of potential risk from biosolids generally indicate that land application does not threaten human or ecosystem health, but questions continue to arise concerning the environmental effects of this practice. This research project was initiated to evaluate ecotoxicity resulting from the amendment of soils with biosolids from municipal wastewater treatment plants. Toxicity was evaluated using standard tests, including earthworm mortality, growth, and reproduction; seedling germination and root elongation; microbial respiration; and nematode mortality and reproduction. Nineteen municipal wastewater treatment plants were identified to participate in an initial screening of toxicity, and five were chosen for a more detailed evaluation. In addition, two soils with historically high applications of high-metal biosolids were evaluated. Contaminants examined were zinc, copper, nickel, chromium, arsenic, cadmium, lead, and coplanar polychlorinated biphenyls (PCBs). Single applications had no effect on soil metal concentrations. Coplanar PCBs were not detectable in any of the soils or biosolids. All target organisms were sensitive to reference toxicants. Limited toxicity was observed in a small number of the amended soils, but no patterns emerged. Approximately one-half of the negative effects of biosolids on bioindicators could be attributed to routine properties, such as slight depression of pH and/or elevated salinity. None of the accumulated metal concentrations were excessive, and most would not be considered elevated. These observations suggest that current regulations for application of biosolids to soils are providing adequate ecosystem protection.     

Eutrophication of Lake Tasaul,Romania—proposals for rehabilitation

Background, aim, and scope  The reclamation of nonferrous metal-polluted soil by phytoremediation requires an overall and permanent plant cover. To select the most suitable plant species, it is necessary to study metal effects on plants over the time, thereby checking that metals remain stored in root systems and not transferred to aerial parts. In this purpose, the seasonal and annual variations of metal bioaccumulation, transfer, and phytotoxicity in Trifolium repens and Lolium perenne grown in a Cd-, Pb-, and Zn-contaminated soil were also studied. Materials and methods  The experimental site was located near a closed smelter. In spring 2004, two areas were sown with T. repens and L. perenne, respectively. Thereafter, the samplings of plant roots and shoots and surrounding soils were realized in autumn 2004 and spring and autumn 2005. The soil agronomic characteristics, the Cd, Pb, and Zn concentrations in the surrounded soils and plant organs, as well as the oxidative alterations (superoxide dismutase [SOD], malondialdehyde [MDA], and 8-hydroxy-2′-deoxyguanosine [8-OHdG]) in plant organs were carried out. Results  Whatever the sampling period, metal concentrations in soils and plants were higher than background values. Contrary to the soils, the fluctuations of metal concentrations were observed in plant organs over the time. Bioaccumulation and transfer factors confirmed that metals were preferentially accumulated in the roots as follows: Cd>Zn>Pb, and their transfer to shoots was limited. Foliar metal deposition was also observed. The results showed that there were seasonal and annual variations of metal accumulation in the two studied plant species. These variations differed according to the organs and followed nearly the same pattern for the two species. Oxidative alterations were observed in plant organs with regard to SOD antioxidant activities, MDA, and 8-OHdG concentrations. These alterations vary according to the temporal variations of metal concentrations. Discussion  Metal concentrations in surrounded soils and plant organs showed the effective contamination by industrial dust emissions. Metals absorbed by plants were mainly stored in the roots. With regard to this storage, the plants seemed to limit the metal transfer to their aerial parts over the time, thereby indicating their availability for metal phytostabilization. Aerial deposition was another source of plant exposure to nonferrous metals. Despite the occurrence of metal-induced oxidative alterations in plant organs, both plant species seemed to tolerate a high metal concentration in soils. Conclusions  Taken together, these results indicated that T. repens and L. perenne were able to form a plant cover on highly Cd-, Pb-, and Zn-polluted soils, to limit the metal transfer to their aerial parts and were relatively metal-tolerant. All these characteristics made them suitable for phytostabilization on metal-contaminated soils. These findings also highlighted the necessity to take into account seasonal and annual variations for a future phytomanagement. Recommendations and perspectives  In this work, the behavior of plant species grown in metal-polluted soil has been studied during 2 years. Obviously, this time is too short to ensure that metals remain accumulated in the root system and few are transferred in aerial parts over the time. It is why regular monitoring should be achieved during more than a decade after the settlement of the plant cover. This work will be completed by the study of the T. repens and L. perenne effects on mobility of metals in order to evaluate the quantities of pollutants which could be absorbed by the biota and transferred to groundwater. Bioaccessibility tests could be also realized on polluted soils in order to evaluate the phytostabilization impacts on the exposition risks for humans.     

The influence of ozone exposure dynamics on the growth and yield of kidney bean

A field experiment was conducted in open-top chambers to assess the importance of peak exposure concentration and exposure frequency on the responses of kidney bean plants to O3. There were five treatments in the study: charcoal-filtered air, constant exposure to 0.05 ppm O3 (131 microg m(-3)) daily. fluctuating exposure to 0.08 ppm O3 on three alternate days, cluster exposure to 0.08 ppm O3 on three consecutive days, and peak exposure to 0.12 ppm O3 on two consecutive days. Exposures lasted 4 h and produced an average weekly exposure-period concentration of approximately 0.05 ppm in the O3-addition treatments and 0.025 ppm in the charcoal-filtered treatment. Exposures began on June 23 and terminated on September 8. Plants were harvested weekly and assessed for the number, area, and dry mass of leaves; dry mass of stems; dry mass of roots; the number of pods; and the incidence of foliar O3 injury. Yield was assessed at the end of the study. There were no consistent differences between the plants receiving charcoal-filtered air and those receiving O3 exposure. Significant differences were detected among the treatments for several of the growth variables assessed at the interim harvests, but in the final two harvests these differences had mostly disappeared. There were no significant effects of the O3-addition treatments on yield when compared to the plants receiving charcoal-filtered air. This indicates that there were no cumulative impacts on plants exposed to 0.12 ppm O3 for 4 h on two consecutive days followed by filtered air compared to plants receiving charcoal-filtered air. The seasonal 7-h average concentrations of O3 in the peak and filtered air treatments were approximately 0.040 and 0.025 ppm, respectively.