Phytoremediation of Toxic Metals Metal accumulating plants Bioavailability, stability of metals in soil Mechanisms of metal hyperaccumulation in plants

Phytoremediation of Toxic Metals

• Metal accumulating plants• Bioavailability, stability of metals in soil• Mechanisms of metal hyperaccumulation in

plants• Mechanisms of metal resistance:

Phytochelatins and metallothioneins• Molecular mechanisms of ion transport in

plant cells

Metals are of nutritional values at low concentrations in plant tissues. However, unique plants known as hyperaccumulators have the natural ability to accumulate and detoxify metals such as Ni, Zn, Cu, and Mn at very high concentrations in their shoots (0.1-5% foliar dry biomass)

Accumulator of nickel (Ni) and Zinc (Zn), for example, have been reported to contain as much as 5% of these metals on a dry-weight basis.

5% = 50,000 mg/kg

• If the soil has metal concentration of 5,000 mg/kg, growth of these plant results in 10-fold bioaccumulation factor.

• If the plant produces a significant amount of biomass while accumulating high concentration of metal, an important quantity can be removed from the soil

Reference for phytoaccumulators

Baker and Brook. 1989. Terrestrial higher plants which accumulate metallic elements: A review of their distribution, ecology, and phytochemistry. Biorecovery 1:81-126.

Problems

According to Baker et al. (1994), using even the best metal accumulator identified in a recent field trial, Alpine pennygrass, would take 13 to 14 years of continuous cultivation to clean the site.

Reasons:• These plants are relatively small• have slow rates of biomass production• lack any established cultivation, pest

management and harvesting practices

Wenzel et al., 1999

To overcome these limitations:

High biomass metal-hyperaccumulator crops need to be developed by modifying traditional crop plants.

• screening existing genotypes• cultivars• mutant lines for metal accumulation in shoots,

or by transferring genetic material to high biomass crop plant from wild hyperaccumulators species via somatic hybridization or genetic engineering.

New development

Certain soil-applied chelating agents greatly increase translocation of heavy metals, including Pb, from soil into the shoots of high biomass crop plants such as corn and pea and Indian mustard.

E.g. EDTA is effective in facilitating the phytoextraction of Cd, Cu, Ni, Pb, and Zn from contaminated soils.

Application of 10 mmoles/kg of EDTA to soil containing 1200 mg/kg Pb resulted in the accumulation of 1.6% Pb, on a dry weight basis, in the shoots of Indian mustard.

Raskin and Enzley, 2000

Wenzel et al., 1999






Fate of metal-loaded plant materials:

1. Can be collected and removed from the site using established agricultural practices.

2. The biomass can be then be recycled to reclaim the metals that may have an economic importance.

3. Alternatively, postharvest biomass treatment, including composting, compaction thermal treatments, can be employed to reduce the volume and/or the weight of biomass for disposal as a hazardous waste if necessary.

Three main factors influence and determine the ability of phytoextraction to effectively remediate a metal-contaminated site

1. Selection of a site conducive to phytoextraction.

2. Metal solubility and availability for uptake.

3. The ability of the plant to accumulate metals in the harvestable plant tissue.

Concentration and regulatory limits

• Concentration: <1 mg/kg -- 100,000 mg/kg

• Metal contamination is specific for each contaminant.

• Regulatory limits for metal concentrations in soil vary considerably by state and even by site.

• Limits may be negotiated depending on site-specific factors and specific land use restrictions.

• Limits may be set up based on human health impacts from direct soil contact or on ecological risk or secondary exposure pathways,



Phytostabilization

Soil amendments are applied to contaminated soil to reduce the bioavailability of the contaminants. Also termed as inplace inactivation or phytorestoration.


Soil amendments should be

1. Be inexpensive2. be easy to handle and apply3. Be safe to the workers handling the

amendment4. Be compatible with and nontoxic to the plants

selected for revegetation5. Be readily available or easy to produce6. No cause additional environmental impact to

the site

Suggested mechanisms of phytostabilization include:

PrecipitationHumificationSorptionRedox transformation


Phosphate + Pb Pb-pyromorphites

• Extremely insoluble even under very acidic conditions (e.g. the conditions in the stomach of a fasting human)

• Changes in Pb leachability, solubility, or bioavailability

• The changes are rapid, stable and long-lasting

Problems:1. Application rate:

5000 mg P/kg vs. 15-30 mg P/kg for agricultural crops

2. Excess soil P may cause environmental concern

The role of plants

• Physically stabilizing the soil with dense root systems to prevent erosion

• By protecting soil surface from human contact and rain impact with a dense canopy

• Minimize water percolation through the soil, further reducing contaminant leaching

• Provide surfaces for sorption or precipitation of metal contaminants

• Chemically altering the form of the contaminants that inactivate the contaminant. E.G. Inducing the formation of insoluble metal compounds inside plant tissues or on root surfaces.

Water Balance for the root zone of a phytastabilization effort using trees Raskin and Enzley, 2000

Hydrolic Control--refers to the “solar pump” that is established when trees are deeply and densely rooted and growing at a site. The system must act as a “sponge and pump”.

Storage = precipitation-evaporation-percolation-runoff

..

Monthly water balance terms for average climatological conditions at Burlington, IA (unit: Inches of water per month)


=EvapoTranspiration

Selection of plants:

• Poor translocators of metal contaminants to aboveground plant tissues that could be consumed by humans or animals.

• Be tolerant of the soil metal levels as well as the other initial site conditions.

• Must quickly to establish ground cover, have dense rooting systems and canopies and have relatively high transpiration rates to effectively dewater the soil

• Must be easy to establish and care for, and have a relatively long life or be able to self-propagate.

Determining hazard reduction

1. Chemical test. For example: U.S. regulatory test TCLP and SPLP.

• TCLP: The Toxicity characteristic leaching procedure (U.S. EPA, 1990). Designed to measure the leachability of contaminants under landfill condition.

• SPLP: The simulated precipitation leaching procedure (U.S. EPA, 1995). Designed to measure the leachability of contaminants under acid rain conditions.

2. Characterize a contaminant’s potential bioavailability. For example, Physiologically based extraction test (PBET). It was designed to simulate the conditions in the stomach and intestines of a young fasting child (pH, digestive enzymes, organic acids, temperature, and residence time)

• Results from PBET appear to correlate well with swine and rat dosing studies for Pb, but not as good for As.

• Currently, animal dosing studies are more widely accepted for estimating Pb and As availability to children, although chemical extraction tests have been accepted as a measure of bioavailability in a few specific cases.

3. To measure hazard reduction include bioassays that target specific organisms, such as plant assays, earthworm assays, animal dosing studies, or human dosing studies.

• Most bioassays are organism-specific, their results may have limited applicability to other organisms.

• Animal tests are expensive and time-consuming. Physiologically relevant tests, such as the PBET, may provide an easy and inexpensive alternative to animal dosing studies.



Bioavailability and Risk Assessment

Bioavailability: The portion of contaminant that can enter the human circulatory system following ingestion.

IEUBK: Integrated exposure and uptake biokinetic model

• was developed by U.S. EPA (1994) to estimate the percentage of children who are at risk in areas contaminated with Pb.

• incorporates potential Pb exposure from multiple sources, including air, food, water, soil, and dust.

• considers age, nutritional status, and exposed population.

Model Output:

• assumption: 30% of soil Pb is bioavailable to a child,

• Thus, soils cannot contain more than 400 mg Pb/kg before more than 5% of the children exhibit blood Pb level above 10 g Pb/dL blood.

• By changing soil Pb bioavailability from 30% to 10% for a soil containing 2000 mg Pb/kg, the percentage of children predicted to be at risk falls from over 50% to 11%.



Phytofiltration

The use of plant roots to absorb, concentrate, and precipitate heavy metals from water. e.g. the roots of sunflowers have been used to treat water containing lead, uranium, strontium, cesium, cobalt, and zinc to concentrations below the accepted water standards.

Hydroponically cultivated roots of several terrestrial plants were discovered to be effective in absorbing, concentrating, or precipitating toxic metals from polluted effluents. This was termed rhizofiltration.

Recently, hydroponically grown seedlings of some terrestrial plants could be also used for metal removal from solution.

The efficiency of rhizofiltration compares favorably with that of currently employed water treatment technologies.

The precise mechanisms are largely unknown, suggested mechanisms include:

• Extracellular precipitation• Cell wall precipitation and adsorption• Intracellular uptake followed by cytoplasmic

compartmentalization or vacuolar deposition.

The introcellular uptake of toxic metals may employ the same mechanisms that are responsible for the uptake of essential ions such as K+, Ca2+, Mg2+, NO3-, and SO4

2-.

Solute transport across membranes may be both a passive process along the concentration gradient or a process linked to energy-consuming mechanism.


Rhizofiltration

• Biofilter formed by biologically active, high-surface-area plant roots.

• Can potential be very effective.

• Examples:• Cynodon dactylis has been shown to

accumulate > 10,000 mg As kg-1 dry matter in root from contaminated soil

• among 50 compounds tested, horseradish roots have been found to be effective in removing 99% of 27 compounds.

An idea plant for rhizofiltration:

• Exhibit characteristics that provide the maximum toxic metal removal from a contaminated stream

• Easy handling• Low maintenance cost,• A minimum of secondary waste requiring disposal• Desirable if can produce hydroponically

significant amounts of root biomass or surface area.

• Accumulate significant amounts of the contaminant.

• Tolerate high levels of a toxic metal• have high root:shoot ratio and grow safely in

controlled environments

Rhizofiltration plant growth unit

It is important to provide the plant with adequate nutrition without the addition of nutrients to the treated water.

Rhizofiltration units may be organized according to differing engineering designs to accommodate specific site conditions



Lead concentrations in roots ranged from 5.6 to 16.9% on a dry-weight basis.

Among the fast growing crop plants, root capacity to accumulate lead declined in the order:

Sunflower > Indian mustard > Tobacco > Rye > Spinach > Corn


The roots of Indian mustard were more effective in the removal of Cd2+, Cr6+, Cu2+, Ni2+, and Zn2+.

Sunflower plants tested in batch experiments in a growth chamber significantly reduced water concentrations of Cd2+, Cr6+, Cu2+, Mn2+, Ni2+, and Pb2+ within the first hour of treatment.

However, the plants were much less efficient in removing anionic species such as AsO2

- and SeO4

2-.

Sunflower plants were superior to Indian mustard for removing radio nuclides from water

The most rapid removal was demonstrated: Uranium concentration decreased 10-fold in 1 h. After 48 h, an equilibrium was reached at 10 g/L.

Sunflower root concentrated uranium from solution by up to 10,000-fold


Blastofiltration

The technology of using plant seedling to remove toxic metals from water.

Many plant species, including Brassica juncea L. Czern (Indian mastard)Brassica napus L. Brassica rapa L. Medicago sativa L. (alfalfa) Oryza sativa L. (rice)

can germinate and grow for up to 10 days in aerated water in the absence of light and nutrients.

Example:

5-day-old seedlings of B. juncea (i.e. Indian Mustard) were able to concentrate divalent cationic metals Pb, Sr, Cd, and Ni by a factor of 500-2000 over the concentration in solution




Phytovolatilization

• Removal of soil contaminants by plant-assisted volatilization into the atmosphere.

• The volatile compounds could be volatile elemental form or volatile methyl or dimethyl compounds of some metals, metalloids, and halides.

Example: merA

HgSR+ + NADPH Hgo + RSH + NADP+

• merA: bacterial mercuric ion reductase genes• have been manipulated into transgenic

Arabidopsis plants to establish plant-assisted reduction of thiolsalts to Hgo and its subsequent volatilization.

• Transgenic Arabidopsis was highly resistant to Hg, can tolerate up to 100 M Hg as HgCl2. The resistance was associated with enhanced reduction and volatilization of Hg.

In hydroponic experiments, 15 crop species were tested for their potential to volatilize Se (Table 18-7)

Rice (Oryza sativa L.) broccoli (Brassica oleracea botrytis L.) cabbage (Brasssica oleracea L.)

volatilized Se at the fastest rates (1500 to 2500 g Se kg-1 plant dry mass d-1)

Wenzel et al., 1999

Documents

Phytoremediation of Toxic Metals Metal accumulating plants Bioavailability, stability of metals in soil Mechanisms of metal hyperaccumulation in plants