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
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
Raskin and Enzley, 2000
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 sitespecific 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,
Raskin and Enzley, 2000
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 inexpensive
2. be easy to handle and apply
3. Be safe to the workers handling the
amendment
4. Be compatible with and nontoxic to the plants
selected for revegetation
5. Be readily available or easy to produce
6. No cause additional environmental impact to
the site
Suggested mechanisms of
phytostabilization include:
Precipitation
Humification
Sorption
Redox 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-percolationrunoff
..
=EvapoTranspiration
Monthly water balance terms for average climatological
conditions at Burlington, IA (unit: Inches of water per month)
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 selfpropagate.
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 mg 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%.
Raskin and Enzley, 2000
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 SO42-.
Solute transport across membranes may be both a passive
process along the concentration gradient or a process
linked to energy-consuming mechanism.
Raskin and
Enzley, 2000
Rhizofiltration
• Biofilter formed by biologically active, highsurface-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
Raskin and Enzley, 2000
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
Raskin and Enzley, 2000
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
SeO42-.
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 mg/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 5002000 over the concentration in solution
Raskin and Enzley, 2000
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)