Heavy metal accumulation by Nicotiana glauca Graham in a solid waste disposal site

Nicotiana glauca Graham, is the only perennial shrub growing in a solid waste contaminated site in the Negev desert of Israel. The concentration of heavy metals (Cu, Fe, Mn, Zn, Ni, Cd and Pb) in the upper soil layer was significantly higher (p<0.01) than in non-contaminated desert soil. In root and shoot of N. glauca, growing in the site, the concentration of Cu, Zn and Fe was significantly higher (p<0.05) than in plants of a non-contaminated site. In a controlled experiment, the concentrations of Zn and Cu in root of plants grown, in a mixture of contaminated and non-contaminated soil (1:1) was 9.5 and 4.7 higher than that of plants grown in non-contaminated soil, respectively. While Zn was accumulated in shoot of plants grown in contaminated soil (531 mgkg(-1)) in significantly higher concentration than in plants grown in non-contaminated soil (56 mgkg(-1)), no significant differences were found in Cu accumulation. Growth of N. glauca was inhibited on contaminated soil, but no other obvious stress symptoms were apparent. Therefore, long term experiments under controlled conditions are planned to study the mechanism of heavy metal tolerance and accumulation in N. glauca.     

Effects of sewage sludge amendment on heavy metal accumulation and consequent responses of Beta vulgaris plants

Use of sewage sludge, a biological residue produced from sewage treatment processes in agriculture is an alternative disposal technique of waste. To study the usefulness of sewage sludge amendment for palak (Beta vulgaris var. Allgreen H-1), a leafy vegetable and consequent heavy metal contamination, a pot experiment was conducted by mixing sewage sludge at 20% and 40% (w/w) amendment ratios to the agricultural soil. Soil pH decreased whereas electrical conductance, organic carbon, total N, available P and exchangeable Na, K and Ca increased in soil amended with sewage sludge in comparison to unamended soil. Sewage sludge amendment led to significant increase in Pb, Cr, Cd, Cu, Zn and Ni concentrations of soil. Cd concentration in soil was found above the Indian permissible limit in soil at both the amendment ratios.

The increased concentration of heavy metals in soil due to sewage sludge amendment led to increases in heavy metal uptake and shoot and root concentrations of Ni, Cd, Cu, Cr, Pb and Zn in plants as compared to those grown on unamended soil. Accumulation was more in roots than shoots for most of the heavy metals. Concentrations of Cd, Ni and Zn were more than the permissible limits of Indian standard in the edible portion of palak grown on different sewage sludge amendments ratios. Sewage sludge amendment in soil decreased root length, leaf area and root biomass of palak at both the amendment ratios, whereas shoot biomass and yield decreased significantly at 40% sludge amendment. Rate of photosynthesis, stomatal conductance and chlorophyll content decreased whereas lipid peroxidation, peroxidase activity and protein and proline contents, increased in plants grown in sewage sludge-amended soil as compared to those grown in unamended soil.

The study clearly shows that increase in heavy metal concentration in foliage of plants grown in sewage sludge-amended soil caused unfavorable changes in physiological and biochemical characteristics of plants leading to reductions in morphological characteristics, biomass accumulation and yield. The study concludes that sewage sludge amendment in soil for growing palak may not be a good option due to risk of contamination of Cd, Ni and Zn and also due to lowering of yield at higher mixing ratio.     

The effects of biochars from rice residue on the formation of iron plaque and the accumulation of Cd, Zn, Pb, As in rice (Oryza sativa L.) seedlings

A historically multi-metal contaminated soil was amended with biochars produced from different parts of rice plants (straw, husk and bran) to investigate how biochar can influence the mobility of Cd, Zn, Pb and As in rice seedlings (Oryza sativa L.). Rice shoot concentrations of Cd, Zn and Pb decreased by up to 98%, 83% and 72%, respectively, due to biochar amendment, though that of As increased by up to 327%. Biochar amendments significantly decreased pore water concentrations (C_pw) of Cd and Zn and increased that of As. For Pb it depended on the amendment. Porewater pH, dissolved organic carbon, dissolved phosphorus, silicon in pore water and iron plaque formation on root surfaces all increased significantly after the amendments. The proportions of Cd and Pb in iron plaque increased by factors 1.8-5.7 and 1.4-2.8, respectively; no increase was observed for As and Zn. Straw-char application significantly and noticeably decreased the plant transfer coefficients of Cd and Pb. This study, the first to investigate changes in metal mobility and iron plaque formation in rice plants due to amending a historically contaminated soil with biochar, indicates that biochar has a potential to decrease Cd, Zn and Pb accumulations in rice shoot but increase that of As. The main cause is likely biochar decreasing the C_pw of Cd and Zn, increasing the C_pw of As, and increasing the iron plaque blocking capacity for Cd and Pb.     

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.     

Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape

Two lead (Pb)-resistant endophytic bacteria were isolated from rape roots grown in heavy metal-contaminated soils and characterized. A pot experiment was conducted for investigating the capability of the two isolates to promote the growth and Pb uptake of rape from Pb-amended soil. The two isolates were identified as Pseudomonas fluorescens G10 and Microbacterium sp. G16 based on the 16S rDNA gene sequence analysis. Strains G10 and G16 exhibited different multiple heavy metal and antibiotic resistance characteristics and increased water-soluble Pb in solution and in Pb-added soil. Root elongation assays demonstrated increases in root elongation of inoculated rape seedlings compared to the control plants. Strain G16 produced indole acetic acid, siderophores and 1-aminocyclopropane-1-carboxylate deaminase. Increases in biomass production and total Pb uptake in the bacteria-inoculated plants were obtained compared to the control. The two strains could colonize the root interior and rhizosphere soil of rape after root inoculation.     

Influence of early stages of arbuscular mycorrhiza on uptake of zinc and phosphorus by red clover from a low-phosphorus soil amended with zinc and phosphorus

In a pot experiment, red clover (Trifolium pratense) was grown in sterilized Zn-amended low available P soil (0, 50 or 400 mg Zn kg(-1)) with or without 100 mg kg(-1) added P and with or without inoculation with the arbuscular mycorrhizal (AM) fungus G. mosseae. When the plants were harvested after 40 days, AM colonization of the roots was still at an early stage, with only 14-38% of total root length colonized on average. AM colonization was highest in low-P soil, and was lowest in soil amended with 400 mg Zn kg(-1). Shoot yields were highest in AM plants with added P, but root yields were unaffected by AM inoculation. Shoot and root yields were higher with 100 mg added P kg(-1) soil, but lower with 400 mg Zn kg(-1) than 50 mg Zn kg(-1) or controls unamended with Zn. Shoot and root P concentrations were seldom higher in AM plants, but shoot P offtakes were higher in AM plants with added P. Concentrations of Zn and Cu were much higher in the roots than in the shoots. Shoot and root Zn and shoot Cu were lower, but root Cu was higher, in AM plants. Soil residual pH after plant growth was higher in AM treatments, and residual total Zn was also higher, indicating lower Zn uptake by AM plants. Soil solution pH was higher in AM treatments, and soil solution Zn was lower in the presence of mycorrhiza. The results are discussed in terms of AM protection of the plants against excessive shoot Zn uptake.     

Promising role of plant hormones in translocation of lead in Sesbania drummondii shoots

Lead (Pb) accumulation in Sesbania drummondii shoots was enhanced by 654 and 415% in the presence of 100 microM IAA and 100 microM NAA, respectively, compared to control plants (Pb alone). However, when IAA or NAA was added along with EDTA, Pb accumulation further increased in shoots by 1349% and 1252%, respectively. Scanning electron microscopic observations revealed that Pb particles in both leaf and stem of Pb+EDTA+IAA and Pb+EDTA+NAA treated plants were concentrated in the region of vascular bundles. In root tissue, Pb particles were present between epidermis to stele region. Plant growth in both treatments (Pb+100 microM IAA and Pb+100 microM NAA) was comparable to control plants; however, it was significantly inhibited in the treatments containing Pb+EDTA and EDTA at concentrations of 10 microM of IAA or NAA. Moreover, the photosynthetic efficiency and strength of the treated plants were not affected in the presence of IAA or NAA and EDTA.     

Effect of gamma-irradiated sludge on the growth and yield of rice (Oryza sativa L. var. GR-3)

The effects of gamma-irradiated sludge on the growth and yield of rice (Oryza sativa L. var. GR-3) in pot cultures have been studied. Compared to plants grown only in soil, shoot length, root length, fresh weight, dry weight, total proteins, total soluble sugars, starch and chlorophyll content of plants grown in soil supplemented with unirradiated or gamma-irradiated sludge were found to be significantly increased. Irradiation of sludge significantly stimulated the linear growth of shoot and root systems as well as fresh and dry weights of plants, compared to those grown in soil containing unirradiated sludge. There was also an improvement in the grain yield (weight of seed) when plants were grown in soil supplemented with irradiated sludge. The results obtained suggest that the gamma-irradiated sewage sludge can be beneficially recycled for agricultural uses.     

Ca(2+) -dependent plant response to Pb(2+) is regulated by LCT1

Tobacco plants transformed with TaLCT1 were cultured on Knop's medium with modified calcium concentrations (0.01-3 mM) in the presence of Pb(2+), and in soil contaminated by lead. A 4-5 microM Pb(2+) administered in the presence of 1 mM Ca(2+) inhibited the root growth of transgenic plants to much lesser degree than of control plants, whereas in the presence of 3mM Ca(2+) no differences were found between the studied lines. The reduction of Pb(2+) toxicity in the presence of 1 mM Ca(2+) was not accompanied by a change in the lead tissue concentration. However, when Ca(2+) level in the medium was lowered to 0.01 mM, several fold higher root/shoot Pb ratio in transgenic plants was observed, twofold increase in the total amount of metal accumulated, and lower concentration of Pb in the xylem sap. Results suggest the involvement of TaLCT1 in the regulation of Ca-dependent Pb-detoxification, and under conditions of low calcium in lead uptake and distribution.     

Allocation and source attribution of lead and cadmium in maize (Zea mays L.) impacted by smelting emissions

Plants grown in contaminated areas may accumulate trace metals to a toxic level via their roots and/or leaves. In the present study, we investigated the distribution and sources of Pb and Cd in maize plants (Zea mays L.) grown in a typical zinc smelting impacted area of southwestern China. Results showed that the smelting activities caused significantly elevated concentrations of Pb and Cd in the surrounding soils and maize plants. Pb isotope data revealed that the foliar uptake of atmospheric Pb was the dominant pathway for Pb to the leaf and grain tissues of maize, while Pb in the stalk and root tissues was mainly derived from root uptake. The ratio of Pb to Cd concentrations in the plants indicated that Cd had a different behavior from Pb, with most Cd in the maize plants coming from the soil via root uptake.     

Bioaccumulation and physiological effects of excess lead in a roadside pioneer species Sonchus oleraceus L

Seedlings of Sonchus oleraceus L. were transplanted to soil supplied with lead acetate at dosages of 0, 800, 1600 and 3200 mg kg(-1) DW. Measures of chlorophyll content, peroxidase (POD) activity, shoot length, biomass and Pb content in the plant tissues were obtained from the experimental plants. With increasing amounts of Pb in the soil, the chlorophyll content, shoot length and biomass decreased, while POD activity and Pb content in the plant tissues increased. At 3200 mg kg(-1) Pb treatment, Pb content in the plant leaf, stem and root were 65.67, 149.82 and 1113.24 mg kg(-1), respectively. Only at 3200 mg kg(-1) Pb treatment did chlorophyll content, shoot length and biomass significantly increase by 18, 15 and 44%, respectively, while POD decreased by 39% over the control. The potential of applying this species in phytoremediation of Pb contaminated roadside soils and thus restoration of the roadside vegetation are discussed.     

Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry

Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PC_n, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg⁻¹ dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PC_n, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg ⁻¹ EDTA, vetiver accumulated 4460 and 480 mg Pb kg⁻¹ dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC₁) (m/z 308.2), along with chelated complexes like Pb-EDTA (m/z 498.8) and PC₁-Pb-EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass.     

Assessment of the phytoextraction potential of high biomass crop plants

A hydroponic screening method was used to identify high biomass crop plants with the ability to accumulate metals. Highest values of shoot accumulation were found in maize cv. Ranchero, rapeseed cv. Karat, and cardoon cv. Peralta for Pb (18 753 mg kg(-1)), Zn (10 916 mg kg(-1)), and Cd (242 mg kg(-1)), respectively. Subsequently, we tested the potential of these three cultivars for the phytoextraction of a metal spiked compost, finding out that, in cardoon and maize plants, increasing Zn and Cd concentrations led to lower values of root and shoot DW. By contrast, rapeseed shoot growth was not significantly affected by Cd concentration. Finally, a metal polluted soil was used to check these cultivars' phytoextraction capacity. Although the soil was phytotoxic enough to prevent the growth of cardoon and rapeseed plants, maize plants phytoextracted 3.7 mg Zn pot(-1). We concluded that the phytoextraction performance of cultivars varies depending on the screening method used.     

Heavy metal accumulation in wheat plant grown in soil amended with industrial sludge

The concentrations of different forms of Zn, Cu, Mn, Ni, Cd, Cr, Pb and Fe metals were determined for the roadside sludge collected from pickling-rolling and electroplating industrial area. In sludge the relative abundance of total heavy metals were Fe>Mn>Cr>Ni>Cu>Pb>Zn>Cd and DTPA-extractable metals were in the order--Fe>Ni>Mn>Cr>Cu>Zn>Pb>Cd. Pot-culture experiment was conducted in soils amended with sludge (0%, 10%, 20%, 30%), pretreated with lime (0%, 0.5% and 1%). The soils were alkaline in nature (pH>8.3) with organic carbon contents were 0.34% and 0.72%. The most abundant total and bio-available metal was Fe. Two wheat seedlings were grown in each pot containing 3kg sludge-amended or control soil and the experiment was conducted till harvesting. Application of sludge increased both total and bio-available forms of metals in the soils, while lime application decreased the bioavailability of heavy metals in sludge-amended soils. The content of organic carbon showed positive correlation with all metals except Zn, Cr and Pb. CEC also showed a strong positive correlation (R2>0.7) with Fe, Mn, Cu, Ni and Cd. Though wheat plants are not accumulators, the translocation efficiency was appreciably high. The translocation factor from shoot to grain was found smaller than that of root to shoot of wheat plants. This makes an implication that the heavy metal accumulation was proportionally lesser in grain than in shoot. In, 10% sludge with 0.5% lime-amended soils; each of these toxic heavy metals was found to be within permissible range (USEPA). Hence, on the basis of present study, the best possible treatment may be recommended.     

Changes in soil solution Zn and pH and uptake of Zn by arbuscular mycorrhizal red clover in Zn-contaminated soil

Red clover plants inoculated with Glomus mosseae were grown in a sterile pasture soil containing 50 mg Zn kg(-1) in 'Plexiglas' (acrylic) containers with nylon net partitions (30 microm mesh) designed to separate the soil into a central root zone and two outer zones for hyphal growth with no root penetration. Two porous plastic soil moisture samplers were installed in each pot, one in the root compartment and the other in one of the hyphal compartments. The soil in the outer compartments was amended with one of the four application rates of Zn (as ZnSO4) ranging from 0 to 1000 mg kg(-1). Non-mycorrhizal controls were included, and there were five replicates of each treatment in a randomised block in a glasshouse. Uninoculated plants received supplementary P to avoid yield limitation due to low soil P status. Plants grew in the central compartment for nine weeks. Soil moisture samples were collected 4, 24 and 62 days after sowing to monitor changes in the Zn concentration and pH of the soil solution. At harvest, the mean mycorrhizal infection rate of inoculated plants ranged from 29% to 34% of total root length and was little affected by Zn application. Root and shoot yields were not affected by mycorrhizal infection. Plant Zn concentration and uptake were lower in mycorrhizal plants than non-mycorrhizal controls, and this effect was more pronounced with increasing Zn application rate to the soil. Soil solution Zn concentrations were lower and pH values were higher in mycorrhizal treatments than non-mycorrhizal controls and the mycorrhiza effect was more pronounced at higher Zn application rates. The protective effect of mycorrhiza against plant Zn uptake may have been associated with changes in Zn solubility mediated by changes in the soil solution pH, or by immobilisation of Zn in the extraradical mycelium.     

Effect of La3+ on the activities of antioxidant enzymes in wheat seedlings under lead stress in solution culture

In order to improve the plant ability to resist lead stress, effect of 0.05 mg/l La(NO₃)₃ on the activities of catalase (CAT), superoxide dismutase (SOD), the level of malondialdehyde (MDA) in wheat seedlings under lead stress was studied. The effect of La³⁺ on plant growth, chlorophyll content in wheat seedlings after adding 0, 50, 100 mg/l Pb(NO₃)₃ to the nutrient solution for 12 days was observed. The plants were grown in nutrient solution in a strictly controlled climate growth room. Effects of La³⁺ (with La treatment) compared with check groups was evidently observed. The activities of SOD and CAT in root were enhanced 0.45–1.69 times and 33.20–77.77% respectively and MDA content was reduced 11.05–27.49% in root after treatments from the second day till the end of the experiment. The activities of SOD and CAT was found to be increased slightly (P<0.05) and MDA content decreased in shoot and root of wheat seedlings by La³⁺ under lead stress within five days after treatments compared with Pb1 and Pb2 groups. It was assumed that antioxidant enzymes was found to be increased by La(NO₃)₃, the antioxidant potential of the wheat seedlings to resist lead stress enhanced. It is suggested that La³⁺ could be used to resist lead stress at the beginning under stress while the stress was not so serious.     

The arbuscular mycorrhizal fungus Glomus mosseae induces growth and metal accumulation changes in Cannabis sativa L

The effect of arbuscular mycorrhiza on heavy metal uptake and translocation was investigated in Cannabis sativa. Hemp was grown in the presence and absence of 100 microg g-1 Cd and Ni and 300 microg g-1 Cr(VI), and inoculated or not with the arbuscular mycorrhizal fungus Glomus mosseae. In our experimental condition, hemp growth was reduced in inoculated plants and the reduction was related to the degree of mycorrhization. The percentage of mycorrhizal colonisation was 42% and 9% in plants grown in non-contaminated and contaminated soil, suggesting a significant negative effect of high metal concentrations on plant infection by G. mosseae. Soil pH, metal bioavailability and plant metal uptake were not influenced by mycorrhization. The organ metal concentrations were not statistically different between inoculated and non-inoculated plants, apart from Ni which concentration was significantly higher in stem and leaf of inoculated plants grown in contaminated soil. The distribution of absorbed metals inside plant was related to the soil heavy metal concentrations: in plant grown in non-contaminated soil the greater part of absorbed Cr and Ni was found in shoots and no significant difference was determined between inoculated and non-inoculated plants. On the contrary, plants grown in artificially contaminated soil accumulated most metal in root organ. In this soil, mycorrhization significantly enhanced the translocation of all the three metals from root to shoot. The possibility to increase metal accumulation in shoot is very interesting for phytoextraction purpose, since most high producing biomass plants, such as non-mycorrhized hemp, retain most heavy metals in roots, limiting their application.     

The role of citric acid on the phytoremediation of heavy metal contaminated soil

Adsorption and hydroponics experiments were conducted to study the role of citric acid on the phytoremediation of heavy metal contaminated soil. The results show that addition of citric acid decreased the adsorption of both lead and cadmium, such an effect was bigger for cadmium than for lead. The decrease in the adsorption of Pb and Cd was mainly due to a decrease of pH in the presence of citric acid. The presence of citric acid could alleviate the toxicity of Pb and Cd to radish, and stimulate their transportation from root to shoot. The studies of heavy metal forms using sequential extraction demonstrated that lead was mainly existed as FHAC (a lower bioavailable form) in the root, while F(HCl) was the dominant form in the leaf. The addition of citric acid to the soil changed the concentration and relative abundance of all the forms. The detoxifying effect of citric acid to Pb in shoots might result from the transformation of higher toxic forms into lower toxic forms. Cadmium was mainly present as F(NaCl), therefore, it had higher toxicity than lead. The addition of citric acid increased the abundance of F(H2O) + F(NaCl), indicating that citric acid treatment could transform cadmium into more transportable forms.     

Effects of ozone on three open-pollinated families of Pinus taeda L. grown in two substrates

Seedlings from three open-pollinated loblolly pine (Pinus taeda L.) families grown in a mixture of commercial peat moss and grade 3 vermiculite (1:3 by volume) or a mixture of mineral soil and peat (1:1 by volume) were exposed to 0, 160 or 320 ppb ozone (O3) for 6h/day, 4 days/week for 8 weeks beginning 12 weeks after transplanting. Before exposures began, seedlings grown in the vermiculite-peat substrate were taller but smaller in diameter than those grown in the mineral soil-peat substrate. After 8 weeks of exposure, seedlings grown in the mineral soil-peat substrate were significantly larger in diameter and total biomass than those grown in the vermiculite-peat substrate. Primary needle and secondary needle injury increased with increasing O3 concentrations. Suppression of diameter growth, shoot weight and root weight was linear as O3 concentration increased. The effect of O3 on height or diameter growth or shoot biomass was not influenced by substrate type; but the suppression of root biomass due to O3 was dependent on substrate, with greater suppression in biomass occurring in the vermiculite-peat substrate. Foliar injury due to O3 was slightly greater in family 8-103, but growth suppression due to O3 was not significantly different among the families. Based on root biomass, response of seedlings to O3 was substrate-dependent.