Accumulation Of Heavy Metals By Azolla spp. 

Anju Arora, National Centre for Conservation and Utilisation of Blue Green Algae, Indian Agricultural Research Institute, N.Delhi, India.110012, Tel. 91-11-5788431  Fax 91-11-5766420
Anjuli Pabby, National Centre for Conservation and Utilisation of Blue Green Algae, Indian Agricultural Research Institute, N.Delhi, India.110012, Tel. 91-11-578843, Fax 91-11-5766420, 5751719
P.K. Singh, National Centre for Conservation and Utilisation of Blue Green Algae, Indian Agricultural Research Institute, N.Delhi, India.110012, Tel. 91-11-5788431, Fax 91-11-5766420, 5751719

Toxic wastes from various industrial and municipal operations are disposed into soil and water, the two most important compartments of environment where these accumulate and enter food chain leading to health hazards. Conventional physicochemical means for treating polluted waters are expensive.  On the other hand, bioremediation of wastewaters using various microorganisms and aquatic plants offers eco-friendly and economical ways for in situ treatment of wastewaters.  The efficiency of bio-treatment process using aquatic plants for stripping metals from waste waters depends on the abundance of plant, its ability to grow in water bodies and propensity to accumulate high levels of metals. Free floating aquatic fern Azolla, which occurs in symbiotic association with N₂ fixing blue green alga Anabaena azollae, is particularly useful because of its high rate of multiplication, worldwide distribution, ability to grow in varied conditions from dilute to polluted water bodies, remarkable capacity to accumulate heavy metals in its biomass.  Azolla processes may prove superior to other traditional processes especially when environmental and ecological constraints exist and when concentrations of toxic metals in waste waters are extremely low (1-100 ppm) so that at such concentration no chemical means are effective. Azolla processes may be operated in two forms active and passive. The active process can be applied in situ to treat diluted effluents and for concentration or recycling of metals.  This study presents ability of three species of Azolla namely A.microphylla, A.pinnata and A.filiculoides to grow in presence of different concentrations of toxic heavy metals like Pb and Cd and accumulate them in their biomass.  Cd severely inhibits growth allowing production of only 40-60% of control biomass at 1 ppm concentration and no growth beyond 1 ppm level. At 5 and 10ppm concentration however, fronds of Azolla spp. are able to survive and accumulate high amount of metal ions. A.pinnata shows  highest capacity for concentrating Cd with its concentration reaching 2760 ppm in the biomass of A.pinnata after 7 days incubation (275 fold ).  Cd accumulates more when it is present in high concentration  in the medium. But this high amount of uptaken Cd severely affects growth.  On the contrary, Pb is less inhibitory to growth of all the three species and even at 80 ppm of lead in the medium the Azolla spp. produce 75-91 percent of control biomass.  A filiculoides and A.pinnata are more tolerant than A.microphlla.  At 40 ppm of Pb, uptake appears to be saturated and concentration of metal in biomass is more at lesser amounts of Pb dissolved in the medium. Thus, Azolla species can be employed to design suitable outdoor processes for removal and concentration of heavy metals from metal contaminated effluent.

Phytoremediation’s Possibility of Food-Chain Contamination

Sandra Benson, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-6410, Email: denali60@hotmail.com
David Coyle, US Forest Service, Savannah River Site, Aiken, SC 29802, Tel: 803-725-1758
Lee Newman, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-4795and The Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, Email: newman@srel.edu

Phytoremediation is a process where plants absorb pollutants from the surrounding soil or groundwater via their root systems. In the case of organic compounds, plants can take up and degrade the contaminant within the plant tissues.  Trichloroethylene (TCE) research has shown that plants will reduce TCE to CO_2, water, and organic metabolites, such as trichloroacetic acid (TCA) and dichloroacetic acid (DCA). This research looks at the two different types of insects, Ostrinia nubilalis (European corn borers) and Malacosoma disstria (forest tent caterpillars) and examines the affects of feeding on TCE exposed plants.  Malacosoma disstria, which naturally feed on poplars, are defoliants and thus might be affected by the TCE metabolites produced in the leaves.  Ostrinia nubilalis, though they do not naturally infest poplars, are wood boring insects and consume the cambium of plants, which is where the parent pollutant is being transferred through the plant.  Larval size, pupae weight, duration of the pupal stage and emergence to adults, and adult biomass are examined.  Analysis is also done to determine the presence of any TCE or metabolites in the insects.

Dewatering, Remediation, and Evaluation of Dredged Sediments

Paul Biery, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-6410, Email: solo49@hotmail.com
Lee Newman, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-4795 and  The Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, Email: newman@srel.edu

In order to keep U.S. waters navigable large dredging operations may be required.  Dredging operations remove large quantities of sediments from many industrial and urbanized harbors and waterways.  The presence of contaminated sediments not only contributes to environmental degradation but it also inhibits the efficiency of the US Army Corps of Engineers to dredge, transport, and relocate sediments.  Currently, contaminated sediments are stored in confined placement facilities (CPFs), capped, treated, or simply not dredged. 

Our research project focuses on the use of plants (primarily grasses and trees) to accelerate the removal of water from the sediments and to promote the degradation/extraction of contaminants.  Research plants will be evaluated based on their ability to dewater and degrade/extract contaminants.  Monitoring will provide real-time data relevant to the progress of dewatering based on moisture content and dissolved oxygen in the sediments.  Ultimately, the success of the project will be based on the final concentrations of the contaminants (compared to regulated concentrations) and residual toxicity (based on seedlings, soil microorganisms, earthworm toxicity test).  Remediation of contaminated sediments will allow it to become suitable for beneficial use such as industrial fill, construction, or even reintroduction to open water.

Soil and Plant Analysis in a Metal Impacted Estuarine System in South Carolina

Jaclin A. DuRant, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-6410, Email: jaclindurant@yahoo.com
Lee Newman, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-4795 and The Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, Email: newman@srel.edu

Industrial pollution is a major concern in US rivers, streams, and estuaries. Though recent advances in technology and regulatory mechanisms have helped lessen the amount of pollution discharged, large amounts of industrial waste still effect aquatic environments. This research focuses on determining the concentration of several different metals in soil and plant tissue samples taken from sample sites along the Sampit river near Georgetown, SC. Two major industries are located on and discharge into the Sampit. The metals contained within the effluent from these industries will be the focus of analysis.

Sediment cores as well as root, stem, and leaf tissue samples from the dominant marsh plant, Spartina alterniflora, are taken from eight sample sites located upstream, throughout, and downstream from the major industrial section of the Sampit, located in the vicinity of Georgetown harbor. The concentrations of metals found in the sediment and plant tissues should help to assess the possibility of an ecological impact on this estuarine ecosystem due to industrial pollution.

Using Sediment Slurry Batch Reactors to Evaluate Mercury Methylation with Regard to Sulfate Concentration

Sarah M. Harmon, University of South Carolina, Department of Environmental Health Sciences, School of Public Health, Columbia, SC   29208, Tel: 803-725-0441, Fax: 803-725-7673, Email: sarah.harmon@srs.gov
G. Tom Chandler, University of South Carolina, Department of Environmental Health Sciences, School of Public Health, Columbia, SC   29208, Tel: 803-777-4795, Fax: 803-777-3391
Lee Newman, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-4795 and  The Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, Email: newman@srel.edu

Mercury is a common environmental contaminant which becomes much more toxic in its methylated form because of methylmercury's ability to easily cross cell membranes, accumulate in biological tissues, then biomagnify through the food chain.  From the standpoint of human health risk, it is the accumulation of methylmercury in the edible tissue of fish which has caused recent health advisories and much public apprehension.  Inorganic mercury in the environment is converted to methylmercury through bacterial activity and, specifically, through the activity of anaerobic sulfate-reducing bacteria (SRB). It has been shown that wetlands are a major source of mercury methylation in natural systems, presumably due to anaerobic nature of a wetland environment and subsequent elevation of anaerobic bacterial populations.   This research addresses the potential risk of mercury methylation in a constructed treatment wetland which has been amended with sulfate to enhance the wetland's capability to sequester metals from the water column.  It is generally accepted that the presence of sulfate in sediment will enhance the activity of SRB and thus stimulate methylmercury production. It has also been shown that the accumulation of sulfide (the product of sulfate reduction) will inhibit mercury methylation due to the formation of solid HgS.  Therefore, there is believed to be a range of sulfate concentrations which is optimal for the stimulation of methylmercury production.  This study attempts to identify this optimal range for a particular wetland system and explore the ways in which bacterial population dynamics affect mercury methylation.  A more thorough understanding of these concepts may help future treatment wetland designers to optimize the benefits of bacterial activity while avoiding this dangerous optimal sulfate concentration range.  Anaerobic batch reactors were used to simulate natural wetland sediment conditions. Test vessels containing sediment slurries with varying sulfate concentrations were incubated in an anaerobic chamber and periodically spiked with aqueous mercury.  Aliquots were regularly removed for measurement of sulfate, sulfide, total mercury, and methylmercury concentrations, in addition to SRB population estimates.  Data from this research allows an evaluation of  mercury methylation rate with respect to sulfate reduction rate and SRB population growth, as well as a correlation between initial sulfate concentration and final methylmercury production.  Through this research, a more detailed understanding of the geochemistry of sulfate and mercury in one particular wetland treatment system has been developed.

Estimating Efficiencies of Tritium Phytoremediation at the Savannah River Site

Daniel R. Hitchcock, Center for Forested Wetlands Research, USDA Forest Service - Savannah River, P. O. Box 700, New Ellenton, SC  29809-0700, Tel:  803-725-2968, Fax:  803-725-0311, Email:  dhitchcock@fs.fed.us
Karin T. Rebel, Department of Earth and Atmospheric Sciences, Cornell University, Ithaca, NY  14853, Tel:  607-255-2286, Email:  ktr5@cornell.edu
Chris Barton, Center for Forested Wetlands Research, USDA Forest Service - Savannah River, P. O. Box 700, New Ellenton, SC  29809-0700, Tel:  803-725-8157, Email: barton@srel.edu
John Seaman, UGA Savannah River Ecology Laboratory, Savannah River Site, Tel:  803-725-0977, Email:  seaman@srel.edu
Susan J. Riha, Department of Earth and Atmospheric Sciences, Cornell University, Ithaca, NY  14853, Tel:  607-255-1729, Email: sjr4@cornell.edu
John I. Blake, USDA Forest Service - Savannah River, P. O. Box 700, New Ellenton, SC  29809-0700, Tel:  803-725-8721, Email: j.blake@srs.gov

Current activities at the Savannah River Site (SRS), formerly a Cold War nuclear material production and storage site, include environmental remediation and restoration.  Specifically, the U.S. Department of Energy, the Westinghouse Savannah River Corporation, and the USDA Forest Service are working in collaboration to design, operate, maintain, and monitor phytoremediation irrigation systems for the remediation of low-level radioactive and mixed waste at SRS.  Current Forest Service projects at SRS include the phytoremediation of groundwater containing the hydrogen isotope tritium.  A sprinkler irrigation system has been constructed on an approximately 22-acre plot of pines and hardwoods for the distribution of tritiated groundwater seep discharge that is collected by a pond.  The irrigation system has been in operation since April 2001. Daily irrigation scheduling is based on continuous soil water deficit calculations using observed rainfall data and calculated evapotranspiration.  Instrument clusters on certain plots consist of time-domain reflectometry (TDR) tubes for measuring soil water content, tensiometers for measuring soil water tension, and piezometer for measuring the depth to the water table.  Clusters also contain lysimeters and vapor tubes for collecting water samples for tritium analyses.  Tritium mass balances are being calculated and used to estimate remediation efficiency.  Future work involves developing relationships between daily irrigation rates, calculated treatment efficiencies, and soil and vegetation characteristics within irrigation plots.          

Microbiota Involved in Vegetation of Sulphidic Mine Tailings: Greenhouse Experiments

Ioana G. Petrisor, University of Southern California, Department of Civil and Environmental Engineering, 3620 S. Vermont Ave., KAP 210 – MC 2531, Los Angeles, CA 90089-2531, Tel: 213-740-0594, Fax: 213-744-1426, Email: petrisor@usc.edu
Mugur Stefanescu, Institute of Biology of Romanian Academy, Spl. Independentei 296, CP 56-53, sector 6, 79651 Bucharest, Romania, Tel: 401-2239072, Fax: 401-2219071, Email: mstef@ibiol.ro
Smaranda Dobrota, Institute of Biology of Romanian Academy, Spl. Independentei 296, CP 56-53, sector 6, 79651 Bucharest, Romania, Tel: 401-2239072, Fax: 401-2219071, Email: sdobr@ibiol.ro
Anca Voicu, Institute of Biology of Romanian Academy, Spl. Independentei 296, CP 56-53, sector 6, 79651 Bucharest, Romania, Tel: 401-2239072, Fax: 401-2219071, Email: avoic@ibiol.ro
Chris M. Teaf, Institute for International Cooperative Environmental Research, Florida State University, 226 Morgan Building, 2035 East Paul Dirac Drive, Tallahassee, FL 32310-3700, Tel: 850-644-5524, Fax: 850-574-6704, Email: cteaf@mailer.fsu.edu

Intensive mining and ore processing activities from the Black Sea Coastal area have resulted in the production of millions of tons of mine tailings (wastes) directly disposed on land. A large sulphidic tailing dump, located next to flotation stations for metalliferous ore processing at Baia locality - on the Black Sea Coast, is considered a potential source for environmental pollution with heavy metals. This paper presents greenhouse experiments for establishing vegetation on sulphidic tailings from Baia, discussing the interactions between plants and microbiota at the rhizosphere level. These interactions are involved in fertilization of the arid growing support. The paper emphasizes two aspects referring to the dynamics of some microorganisms monitored over 12 months, and the enzymatic studies performed periodically in order to establish global dehydrogenase activity. It was demonstrated the presence, at plant rhizosphere level, of microbial groups involved in carbon, nitrogen, phosphorous and sulfur cycles, inducing good substrate (tailings) fertilization, high enzymatic activity and increased plant growth.

Phytoremediators: Possible Implications in Restoration Ecology and Molecular Medicine

J.Rajiv, Centre for Environmental Management of Degraded Ecosystems, University of Delhi, Delhi-110 007, India, Email: janardhanan_r@hotmail.com
K.K.Aggarwal, School of Biotechnology, GGS Indraprastha University, Kashmere Gate, Delhi-110 006, India    
C.R.Babu, Centre for Environmental Management of Degraded Ecosystems, University of Delhi, Delhi-110 007, India

The problem of ecosystem damage is international and probably no country in the world is unaffected. One of the biggest challenges today in achieving sustainability is to reverse the trend of ecosystem damage through restoration and rehabilitation of ecosystems. Phytoremediation is a low input cost effective approach, which preserves the topsoil and reduces the amount of the hazardous materials generated during the cleanup.  Legumes have been extensively used as biological inputs in revegetation/reclamation technologies as they are not only a major source of fixed nitrogen in land based systems, but also known to tolerate moderate concentrations of heavy metals. Root exudates - secretions from the plant roots, continuously produced and secreted into the environment are known to have important role in the biological processes and the unexplored chemical diversity of the root exudates is an obvious area to search for novel biologically active compounds including antimicrobials. Recently, we have isolated a chemical compound possessing the properties of a siderophore from the root exudates of a Legume plant, Tephrosia purpurea. Potentiality of this compound to serve as a bioremediator in the soils contaminated with toxic metals has been suggested (Aggarwal et al., 1999).

The same purified compound when assessed for its anti-microbial activities was found to inhibit the growth of M tuberculosis H₃₇ RA under in vitro conditions (Rajiv et al., 2001). Therefore a definite search based on phytoremediators may not only lead to the development of soil amendments for site specific restoration technologies but also may lead to the development of new avenues in molecular medicine.

REFERENCES
Aggarwal K. K., Rajiv, J., and Babu, C.R. (1999): A Rock-Iron solubilizing compound from root exudates of Tephrosia purpurea. J Chem. Ecol. 25: 2327-2336.

Rajiv, J., Dam, T., Kumar, S., Bose, M., Aggarwal, K. K. and Babu, C.R.(2001) Inhibition of the in-vitro growth of Mycobacterium tuberculosis by a phytosiderophore.

J. Med. Microbiol. 50: 916-918.

Cultural Approaches to Reduce Nitrate Contamination of Groundwater

Raj K. Shreatha, Soil Science Division, Nepal Agri. Research Council, Khumaltar, Lalitpur, NEPAL, Tel: 977-1-521149, Fax: 977-1-220807, Email: rajkumarshrestha@hotmail.com
Jagdish K. Ladha, Soil & Water Sciences Division, Int. Rice Research Inst., DAPO Box 7777, Manila, The Philippines, Tel: (63-2) 845-0563, 845-0569, Fax: (63-2) 845-0606

Drinking nitrate contaminated water can cause Methaemoglobinaemia (blue-baby syndrome), which impairs the oxygen carrying capacity of human blood resulting bluish-tinged or oxygen starved baby. The risk of drinking nitrate-contaminated water is likely to be in rural areas in developing countries where nitrate fertilizer applications are increasing without monitoring water quality. Nitrate levels in groundwater have been increasing over recent decades in most countries as a result of excess use of fertilizers to feed increasing population. The input-intensive cropping system has resulted in a problem of a large leakage of N into the environment, thereby polluting the water. Excessive use of N fertilizer in high-value crops grown in DS is economically motivated. Application of 600 kg nitrogen ha^-1 by farmers in dry season crops has contaminated sixty percent of tube well containing near or above World Health Organization’s (WHO) NO3-N limit for drinking water of 10 ppm. Residual soil mineral N (upper 100 cm) in farmers' field reached up to 694 kg ha^-1. Growing catch crops like indigo (Indigofera tinctoria L.)  and corn (Zea mays L.)  in dry-to-wet transition period reduced residual nitrate level up to 68%. In fallow plots, nitrate was moved down to lower soil profile demonstrating NO3 leaching in absence of crop. On an average, catch crops were able to reduce nitrate leaching by 40%. As catch crop alone was not able to reduce nitrate leaching substantially, a strategy of increasing N use efficiency of the cropping system by applying N-fertilizer at proper time and amount and judicious management on frequency of irrigation in sandy soils was recommended in addition to including N-catch crops.

Sarah Strycharz, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-6410, Email: sarahstry@yahoo.com
Lee Newman, University of South Carolina, School of Public Health, 800 Sumter Street, Columbia, SC 29208, Tel: 803-777-4795 and The Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, Email: newman@srel.edu

Phytoremediation of trichloroethylene (TCE) from contaminated groundwater has been performed using fast-growing tree species that maintain a high water demand. Tree species with these characteristics make excellent candidates for phytoremediation applications due to their ability to uptake large amounts of groundwater, and therefore contaminant. Several metabolites of TCE have been identified in the tissue of poplars including trichloroethanol, di- and trichloroacetic acids. The presence of these metabolites indicates that TCE degradation is taking place through natural metabolism of exogenous compounds in the plant system. However, it is important to expand the range of plants that can be utilized in varying areas of the country for phytoremediation. In this study, we will be examining native tree and plant species of the Southeast and studying their interaction with TCE. By screening native tree species for the ability to take up and degrade TCE we hope to identify phytoremediation candidates suitable for the Southeast. This study is a greenhouse based project that simulates the effects of groundwater TCE on the plant system.

Utilization of Apoplasmic Space in Tobacco by Genetic Manipulation for Accumulation of Cadmium

Toshihiro Yoshihara, Fumiyuki Goto, and Taro Masuda, Bio-Science Dep., Central Res Inst Electric Power Ind., 1646 Abiko, Chiba, Japan, 270-1194, Tel: +81 471 82 1181, Fax: +81 471 83 3347

A concept, called phytoremediation, is one of the solution to reduce the harmful effects of heavy metals without additional energy and large costs. Many natural plants are identified as a “hyper-accumulator” to isolate heavy metals from soil; however, the ability is not enough and there is a need to breed more powerful one. At this point, genetically manipulation techniques are helpful. Two strategies can be recognized to accumulate a metal in living plant cells; firstly, to directly increase the intake flow of the metal; and secondly, to increase the storage capacity of the metal. Our group recently demonstrated the effect of the second strategy on fortifying the iron content in crops (eg. tobacco, lettuce and rice) by over-expressing a gene of the iron storage protein, “ferritin”. However, these strategies may not work well to accumulate non-essential metals, because no specific uptake system for non-essential metals is recognized. Actually, recent reports showed that over-expression of a metal-binding molecule like metallothionein in cell could not enhance the metal intake into cell, while it extinguished the harmful effect of metals. So, we tried to create the third way to accumulate non-essential metals in plant. In our system, it is based on a hypothesis that a metal-binding molecule existed in the apoplasmic space may trap metals independent of the specific uptake system. Alpha-domain of human metallothionein is used for the metal-binding molecule. It is expressed under the regulation of CaMV 35S promoter, and transported to the apoplasmic space of tobacco by the role of transit peptide of tobacco invertase-inhibitor. When the T1 tobaccos were grown in the cadmium containing media, it accumulated about 55% more cadmium than the non-transformant. This is the first experiment to exhibit the possibility that the third way contribute to accumulate non-essential metals in plant.

Aromatic Plant Production on Polluted Soils

Valtcho Zheljazkov, Department of Plant and Animal Sciences, Nova Scotia Agricultural College, PO Box 550, Truro, NS, B2N 5E3 Canada, Tel: 902-893-7859, Fax: 902-897-9762 Email:vjeliazkov@nsac.ns.ca

A two year field experiment was conducted in the vicinities of Pb-Zn smelter near Plovdiv Bulgaria, at the presence of both soil and aerosol metal pollution to evaluate productivity and phytoremediation potential of coriander, sage, dill, and chamomile.  Crops were grown in the vicinities of the smelter at distances of 0.8, 3.0, 6.0 and 9.0 km (the latter was unpolluted and regarded as a control). 

The level of pollution at 0.8 km from the smelter reduced yields of fresh herbage and essential oil from all tested crops compared to the control and the yields at 3 km from the smelter.   Despite the yield reduction of 12-20 %  (relative to the control), coriander, sage, dill and chamomile can be successfully grown and remain profitable crops on heavy metal polluted sites.  Heavy metal concentration in the plant tissue reflected the level of soil and aerosol pollution in the area.  The highest removal of metals with the yields was as follows: Cd up to 180 g/ha; Pb up to 660 g/ha; Cu up to 180 g/ha, Mn up to 350 g/ha, Zn up to 205 g/ha.

The accumulation of heavy metals in the aboveground herbage of the four crops is tabulated. Overall, there was less variation in Zn content and uptake between plant species than with other elements.

Essential oils from the four crops were free of metals.  The concentration of Cd, Pb, Cu, Mn, and Zn in the essential oils was below 0.06, 0.62, 0.25 and 0.3 mg/L respectively, i.e. below the detection limit of AAS.

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