Download Metal fate and sensitivity in the aquatic tropical vegetable Ipomoea aquatica

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Metal fate and sensitivity in the
aquatic tropical vegetable
Ipomoea aquatica
Agneta Göthberg
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©Agneta Göthberg, Stockholm 2008
ISBN 978-91-7155-653-0, pp 1 – 39
Printed in Sweden by Universitetsservice, US-AB, Stockholm 2008
Distributor: Department of Applied Environmental Science, Stockholm University
Front cover: Ipomoea aquatica, photo by Karin Karlsson
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Abstract
The aquatic plant Ipomoea aquatica is a widely consumed vegetable in
Southeast Asia. It often grows in waters serving as recipients for domestic and
other types of wastewater. These waters contain not only nutrients, but also a
wide variety of pollutants, including heavy metals, posing a risk to the
population. In addition, fertilizers are generally used in the commercial
cultivation of I. aquatica for the food market.
The aim of this thesis was to estimate the accumulation and toxic effects of
mercury (Hg), methyl-Hg, cadmium (Cd) and lead (Pb) in I. aquatica and how
these are affected by ambient nutrient concentrations. The potential for these
metals to affect plant growth and the hazards they may pose to human health
have also been evaluated.
In plants from cultivations in the Bangkok area, Thailand, the
concentrations of Cd and Pb in the shoots were well beneath recommended
maximum values for human consumption, but at some sites the Hg
concentrations were high. It was demonstrated that I. aquatica has the capacity
to accumulate Cd and Pb in the shoots at concentrations on the order of a
hundred times higher than found in field-cultivated I. aquatica from Bangkok,
before exhibiting toxic symptoms. The Hg concentrations in field-cultivated I.
aquatica, however, occasionally reached levels that are toxic for the plant. At
most sites up to11% of total-Hg was in the form of methyl-Hg, the most toxic
Hg species, though at one site methyl-Hg made up 50-100% of the total-Hg. To
study if methyl-Hg is formed or degraded in I. aquatica, plants were exposed to
inorganic Hg through the roots. Methyl-Hg was formed in the shoots of I.
aquatica, especially in the young, metabolically active parts, but there was no
sign of Hg demethylation activity. A major proportion of the absorbed metals
was retained by the roots, which had a higher tolerance than the shoots for high
internal metal concentrations.
The nutrient level did influence accumulation and effects of Hg, Cd and Pb
in I. aquatica. Lower external nutrient levels resulted in increased metal
accumulation in the shoots and metal-induced toxic effects in the plant at lower
external metal levels. A generous supply of sulphur or nitrogen induced the
formation of glutathion and other thiol-rich peptides in I. aquatica, compounds
that have a metal detoxifying effect in plants.
To conclude, the levels of Cd and Pb in I. aquatica from the Bangkok area
do not pose any apparent threat to human health or risk for reduced plant
growth. The levels of Hg however, were high at some sites and could be a
health threat, for children and foetuses in particular, and especially considering
the presence of methyl-Hg. The use of fertilizers in cultivations of I. aquatica is
favourable as it reduces the risk for increased metal concentrations in the plants
and reduced crop yields.
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List of papers
This thesis is based on four papers, three of them published. The papers will be
referred to by their Roman numerals.
I. Göthberg A, Greger M and Bengtsson B-E. 2002. Accumulation of heavy
metals in Water Spinach (Ipomoea aquatica) cultivated in the Bangkok region,
Thailand. Environ Toxicol Chem 21 (9): 1934 - 1939.
II. Göthberg A, Greger M, Holm K and Bengtsson B-E. 2004. Influence of
nutrient levels on uptake and effects of mercury, cadmium, and lead in Water
Spinach. J Environ Qual 33: 1247 - 1255.
III. Göthberg A and Greger M. 2006. Formation of methyl mercury in an
aquatic macrophyte. Chemosphere 65: 2096-2105.
IV. Göthberg A, Landberg T, Greger M. 2008. Formation of thiol-rich peptides
in water spinach at exposure to metals and nutrients. Submitted to Aquatic
Toxicology
My contributions to the papers were as follows: I was responsible for writing all
papers with help from the coauthors. I planned the experiments for papers II-III
with the help and advice of the coauthors, I initiated and planned the
experiments for paper IV. I performed all the laboratory work and the analyses
of total-Hg (Paper II, III) and thiol-rich peptides (Paper IV).
Reprints of the papers I, II and III were made with the permission of the
publishers involved.
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Contents
Introduction....................................................................
Background....................................................................
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Hg, Cd and Pb effects on human health..................................
Cultivation of Water Spinach (Ipomoea aquatica)..............
Metal uptake in aquatic plants..............................................
Availability in the aquatic environment ..........................
Accumulation in plants........................................................
Uptake in roots..........................................................
Uptake in plant cells..................................................
Translocation to the shoot.........................................
Distribution in the plant.............................................
Uptake in leaves........................................................
Effects of metal stress in plant...........................................
Toxic effects of Hg, Cd and Pb.................................
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Aims.................................................................................
Comments on materials and methods.............................
Results and discussion…………………………...…………
Accumulation of Hg, Cd and Pb in Ipomoea aquatica........
Concentrations of total-Hg,Cd and Pb..............................
Influence of nutrient levels ........................................
Concentrations of methyl-Hg.............................................
Influence of nutrient levels........................................
Concentrations in different tissues.....................................
Distribution........................................................................
Influence of nutrient levels........................................
Toxic effects............................................................................
Toxic effects at high and low nutrient levels.............
Highest recommended human intake of metals related to
concentrations in I. aquatica............................................ 30
Conclusions..................................................................... 31
Suggestions for future research......................................... 31
Acknowledgements........................................................................ 32
References……………………………………………………. 33
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Introduction
Metals occur naturally in the environment at low levels and are present in
soil, sediment, water, air and biota. They also enter the environment as a result
of human activities. Because of antropogenic emissions to the atmosphere, their
global distribution, and precipitation, the levels of metals are increased
worldwide. On a global scale coal combustion is the main source of
atmospheric mercury (Hg) and combustion of leaded gasoline continues to be
the major source of atmospheric lead (Pb). Non-ferrous metal production is the
largest source of atmospheric cadmium (Cd) (Pacyna 2005). Atmospheric heavy
metal emissions from Asia account for about half the total global emissions of
Hg, Cd and Pb (Pacyna 2005). Antropogenic emissions directly to water courses
from point sources and diffuse sources additionally increase the levels locally
and regionally. Increased metal concentrations constitute a serious problem
since they may cause biological disruptions even at relatively low levels.
Plants take up and accumulate metals and metals may therefore be a
problem in the cultivation of food crops. The semi-aquatic plant water spinach
(Ipomoea aquatica) is found in freshwater courses throughout Southeast Asia,
either wild or cultivated, and is a widely consumed vegetable in the region.
Many of the waters where I. aquatiaca grows serve as recipients for domestic
and other types of wastewater and fertilizers are frequently used where I.
aquatiaca is cultivated for the food market. The question is therefore if metal
concentrations in this crop may pose a risk to human health or plant growth and
if fertilization makes it worse.
Background
Hg, Cd and Pb effects on human health
The accumulation of heavy metals in plants consumed by people may be
deleterious for human health. Mercury, Cd and Pb do not have any essential
function what we know so far, but are detrimental, even in small quantities, and
accumulate because of a slow biological turnover rate. Mercury and Pb
adversely affect the central nervous system, especially in small children and
foetuses, who are especially susceptible, and impact the neurologic,
psychomotoric and intellectual development of the child (Richardson and
Gangolli 1995). Lead exposure may also cause anaemia (Goyer 1996). The
most toxic effect from Hg is caused by organic methyl Hg as it is lipophilic and
efficient in crossing cell membranes (Richardson and Gangolli 1995). In the
1950´s in Japan, methyl-Hg in fish and shellfish caused the so-called
“Minamata disease”, a severe intoxication of the central nervous system (Ui
1969). Cadmium accumulates in the liver and, above all, in the kidneys where
injuries first appear at a more advanced age, depending on the amount that has
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been accumulated (Richardson and Gangolli 1995). Renal tubular dysfunction is
the main effect of long-term exposure to Cd. This may eventually lead to
abnormalities of calcium metabolism and osteomalacia, as in the so called “ItaiItai disease” observed in Japan in1946 (Hallenbeck 1986). There are threshold
values for highest tolerable weekly intake for humans of total-Hg, methyl-Hg,
Cd and Pb set up by the World Health Organization (1993, 1999).
Cultivation of water spinach (Ipomoea aquatica Forsk.).
Ipomoea aquatica, in English called water spinach, is a semi-aquatic
tropical macrophyte. Its precise natural distribution is unknown due to extensive
cultivation, though it is found throughout the tropical and subtropical regions of
the world. It is a herbaceous, fast growing, perennial vine. The stems are hollow
and the leaves are entire, 7-14 cm in length, with long petioles (Figure 1).
Figure 1. Water spinach (Ipomoea aquatica).
From IFAS, Center for Aquatic Plants, University of Florida, USA.
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Ipomoea aquatica grows prostrate or floating (Jacquat 1990) with shoots
that are in contact with both water and air. The roots are readily produced from
the nodes into the water or may penetrate into wet soil or sediment. It flourishes
in nutrient-enriched waters, is frost-sensitive, with an optimum growth
temperature between 24 oC to 30 oC and zero growth below 10 oC (Edie and Ho
1969). In South China, India and Southeast Asia, this plant is traditionally much
appreciated as a leaf vegetable, having nutritional benefits similar to spinach. It
is high in Vitamins A and C, has good quality protein and is rich in minerals,
especially iron (Chughtai 1995). Untreated urban wastewater from major cities
is discharged to periurban wetlands that serve as sites for natural treatment and
extensive aquatic food production. I. aquatica is one of the most important
vegetables produced in these periurban wetlands (Edie and Ho 1969, Jacquat
1990). It is cultivated in moist soil, small and big watercourses, e.g. flooded
soil, ditches, ponds, canals and rivers and it is harvested once a week under
normal tropical conditions (Karlsson 2001). I. aquatica utilizes the nutrients in,
and brings about a certain purification of, the wastewater (Edie and Ho 1969,
Abe et al. 1992). In these waters I. aquatica is often also exposed to a wide
variety of pollutants from human activities, including heavy metals such as Hg,
Cd and Pb (Kwei Lin, pers. comm.). Moreover, fertilizers are frequently used
where Ipomoea aquatica is cultivated commercially for the food market.
Because of I. aquatica´s importance as a commonly consumed vegetable
and the fact that it is cultivated in polluted areas that seem unsuitable for food
production, information about the possible accumulation of metals was needed
(B-E Bengtsson pers. comm.). It also was necessary to consider the influence of
nutrients on the uptake of metals and metal induced effects on biomass
production.
Metal uptake in aquatic plants
Availability in the aquatic environment
In natural aquatic environments, metals may be in an available or not
available form for plants to take up. It is generally assumed that free metal ions
are the form primarily available to biota (Nelson and Donkin 1985, Campbell
1995). In the water column most metals exist as cations that are complexed to
varying degrees by inorganic and organic ligands. There are a number of forms
including (Borg 1995):
Free metal ions
Inorganic complexes (CO32-, OH-, Cl-)
Organic complexes with fulvic and humic acids.
Associated with colloidal and particulate materials such as clay
minerals and biogenic material as living algae and detritus.
Once bound with organic or inorganic colloid and particulate material, metals
may, more or less rapidly, settle to the sediment.
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In the soils and sediments metals are bound to colloids, which roughly
consist of inorganic clay minerals and organic substances. These colloids are
negatively charged because of hydroxyl groups and electron pairs of oxygen in
the structure of clay minerals and carboxyl and phenolic groups of organic
substances (Mengel and Kirkby 1982). The positive metal ions are attracted to
these charges. Low pH increases metal availability since the hydrogen ion has a
higher affinity for negative charges on the colloids than the metal ions which
are therefore released (Vesely and Majer 1994). In sediments with low redox
potential, metals bind to sulfides and are thus immobilized (Förstner 1981).
Plants may lower the pH in the rhizosphere by excreting protons which replace
metal ions on the colloids and thus release the metal and make it available to the
plant (Fig 2).
a)
b)
O2
root
Men+
O2 + MeS + 2H2O
Men+ + SO42- + 4H+
Figure 2. Influence by plant-induced a) decrease in soil- or sediment-pH b) increase of
redox potential on metal uptake in plant root .
Plants also manage to increase the redox potential in the rhizosphere by
excreting oxygen and thus oxidise sulfides and release metals (Sand-Jensen et
al. 1982, Chen and Barko 1988) (Figure 2). Several root exudates, such as malic
acid, citric acid, piscidic acid, and phenolics, form chelates with metal ions in
the rhizosphere and thus make them available for the plant (Morel et al. 1986,
Mensch et al. 1988).
In aquatic environments inorganic Hg may be converted to methyl-Hg.
This increases its availability and its toxic effects in living organisms, as
methyl-Hg is lipophilic and easily crosses cell membranes. Microbial
methylation of inorganic Hg is regarded as the main mechanism for methylation
of Hg in aquatic environments (Furutami and Rudd 1980, St Louis et al. 1994)
and has been observed in flooded soil and in fresh and marine water areas
(Lucotte et al. 1999, Roulet et al. 2001, Ullrich et al. 2001). Methyl-Hg is
biomagnified along food chains and has a very slow biological metabolism and
excretion rate (Förstner and Wittmann 1981), which in the end may pose a risk
for human and animal health.
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Accumulation in plants
Higher aquatic plants may grow submersed or emergent, i. e. completely or
partially below the water surface and are able to take up metals from the
sediment through the root, from the water through the root and the shoot and
from the air through the shoot. In addition to the uptake, there is also a release
of metals back into the water and sediment from plant tissue and to the air of
metals in gaseous form from the leaves. Thus, the net metal accumulation
depends on both uptake into the tissue and release to the surrounding medium
(Greger 1999).
Uptake in roots
The roots may penetrate into sediment, mud or wet soil, or simply remain
in the free water. Metal ions are first taken up by diffusion or mass flow into the
apparent free space (AFS) of the root cell walls (Marschner 1995). Older parts
of the root have hydrophobic incrustations (suberin), called the Casparian strip,
in the radial cell walls (stage I) of the endodermis, which act as a barrier to the
passive movement of water and solutes into the vascular cylinder (Marschner
1995) (Figure 3, 4a).
Figure 3. Ion transport across the root, showing the cellular and apoplastic pathways.
From Taiz and Zeiger 1998.
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a)
xylem
endodermis
cortex
b)
endodermis
xylem
cortex
phloem
exodermis
Figure 4. Fluorescence microscopic pictures of a transverse section of a I. aquatica root. a)
Four cm from root tip, stage I. Stained with berberine. b) Nine cm from root tip,
stage II. Stained with Fluoral yellow 088. Photos by the author.
The Casparian strip can also be formed in the exodermis. Gradually the
entire cell wall is suberinized (stage II) (Figure 4b). There is a certain
symplastic transport of solutes across the Casparian band into the vascular
cylinder, but there are different opinions as to whether this is the case for heavy
metals. For this reason it may be that it primarily is metal ions that have been
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taken up by the tip of the root that enter the xylem and are transported to the
shoot. The distance from the root tip to the Casparian strip (stage II) is larger in
emersed plants, such as I. aquatica, than in terrestrial plants, which likely
promotes metal uptake into the root (data not shown).
Metals that have been taken up in plant roots are bound to negative charges
of the cell wall structure, and for instance Beauford et al. (1977), Wierzbicka
(1998) and Wang (2004) have shown that the major amount of Hg and Pb in
roots is bound in the cell walls. Some of the metal is transported further within
the cell walls and some is transported into the cytoplasm of the root cells
(Greger 1999) (Figure 5).
Figure 5. Schematic drawing of the uptake of metals (Me) into the root tissue. Metal ions can
be: a) trapped by negative charges in the cell walls b) transported apoplastically
c) transported into the cell. PC=phytochelatin. From Greger 1999.
Uptake in plant cells
Metal ions are probably taken up into cells by membrane transport proteins
designed for acquisition of nutrient metals, but not entirely specific for intended
nutrient metal (Cohen et al. 1998, Arazi et al. 1999, Fortin and Campbell 2001).
In the cytoplasm, metals bind to inorganic anions, structural compounds and
various macromolecules such as organic acids and sulfur-rich polypeptides, like
phytochelatins (PC´s) (Grill et al. 1985, Prasad 1999). Synthesis of PC´s is
induced by metals and they are considered to be indicators of metal stress
(Gupta et al. 1995, 1998, Maserti et al. 1998, Prasad 1999). Phytochelatins may
function as shuttles for transport over the tonoplast into the vacuole, where the
metal is released and may be bound to an organic acid (Mathys 1977, Steffens
1990, Harmens et al. 1994) (Figure 5). These mechanisms, evolved for storage
of micro nutrient ions, also counteract toxic concentrations of free heavy metal
cations in plasmatic compartments and is a way to prevent heavy metals from
interfering with sensitive metabolic reactions and from development of
oxidative stress (Woolhouse 1983, Ernst et al. 1992). Competition for
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membrane transport sites and for intracellular metabolic binding sites can
substantially influence the uptake of both nutrient and toxic metals and resultant
effects on e. g. growth.
Translocation to the shoot
Some of the metal that is taken up into the root cells is transported further
into the xylem vessels (Figure 3) and translocated to the shoot. How the metals
are transported into the xylem is still unknown. In the xylem vessels the metals
are probably translocated to some extent in complexed form but also by
interactions with nondiffusible anions of the cell wall of the xylem vessels,
which leads to a separation of cation transport from the water flow, as for Cd
(Wolterbeek 1987). The xylem is thought to act as an ion exchange column that
has the potential to impede the movement of the metal (Bell and Biddulph
1963). In the shoot the metal may be bound in the cell walls or transported into
the cells.
Distribution in the plant
The distribution of metals between the different compartments of the cell
and of the plant body depends on the metal and the plant genotype. Different
plant species accumulate metals to various degrees in different parts (Guillizoni
1991, Berrow and Burridge 1991). During their transport through the plant
metals are bound largely in the cell walls, which explains why most of the metal
taken up often is found in the roots and smaller amounts are translocated to the
shoot (Siedlecka 1995, Greger 1999). Submersed plant species are known to
translocate metals both from root to shoot and from shoot to root depending on
the metal being accumulated (Eriksson and Mortimer 1975, Greger 1999). In
submersed plants about 40 % of the Cd that had been taken up in the shoot was
translocated to the roots and about 20 % of the Cd that had been taken up in the
roots was translocated to the shoot (Greger 1999). On the contrary to submersed
plants, terrestrial plants often have a one-way translocation of metals, from root
to shoot (Greger 1999).
Uptake in leaves
Heavy metals are absorbed by the leaves from air or water to different
degrees, depending on the metal species involved (Lagerwerff 1972, Little
1973). In gaseous form, for instance Hg0, metals can be taken up from the air
through the stomata (Browne and Fang 1978, Gaggi et al.1991). In ionic form
metals can be taken up through the cuticle. For example, Cd, Zn and Cu showed
greater leaf penetration than Pb (Little and Martin 1972), which is, to a big part,
adsorbed to the epicuticular lipids at the surface (Paper I; Figure 3). Permeation
of solutes through the cuticle takes place because of negative charges
(presumabely mainly from polygalacturonic acids) within the cuticle, which are
increasing in density from the outside to the inside (i. e. the cutinized layer and
the cell wall interface) (Marschner 1995). Accordingly, permeation of cations
along this gradient is enhanced and anions are repulsed (Tyree et al. 1990).
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Submersed plants do not need a non-permeable cuticle to decrease transpiration
since there is no need for that in the water and thus leaves of submersed plants
are most permeable to metal ions (Greger 1999).
Effects of metal stress in plants
Metal induced effects in plants are to a great deal caused by disruptions on
a cellular level. The knowledge of the biochemical basis of metal toxicity
symptoms in plants is sketchy. It is known that heavy metals, to a varying
degree, have affinity for sulfur (Pais and Jones 1997, Dietz et al. 1999). As
almost all proteins contain sulfur, metals may disturb almost any function in
which proteins are involved. The result is, e. g., inhibition of enzymatic
reactions and disturbance of metabolism (van Assche and Clijsters 1990,
Marschner 1995). Toxic metals may replace essential metals, such as Mg in
chlorophyll and they may bind to plasma membranes, with resultant increased
rigidity and leakage of the membranes. Heavy metals also stimulate formation
of reactive oxygen species and free radicals and the onset of oxidative stress,
which, among other things, cause peroxidation of lipids with resultant
rigidification of membranes (Halliwell and Gutteridge 1984, Elstner 1990).
Plants also have a range of potential mechanisms that might be involved
in detoxification of metals. These all appear primarily to avoid the build-up of
toxic metal concentrations at sensitive sites and thus prevent oxidative stress
(Hall 2002). The strategies for avoiding metal build-up are diverse and include
binding to the cell wall and root exudates, reduced influx across the plasma
membrane, active efflux into the apoplast, chelation in the cytosol by various
ligands, and compartmentalization in the vacuole. Chelation in the cytosol is
performed by organic and inorganic anions, amino acids, organic acids and
sulphur-rich polypeptides, such as phytochelatins (Grill et al. 1985, Prasad
1999). In order to counteract oxidative stress, plants have evolved a complex
antioxidant defence system, including a number of enzymic and non-enzymic
antioxidants. Examples of antioxidant enzymes are superoxide dismutases,
catalases, ascorbate peroxidases and glutathione peroxidases.
Examples of non-enzymic antioxidants are glutathione, ascorbate, tocopherol
and carotenoids. Under non-stress or moderate stress conditions this system is
sufficient to ensure redox homeostatis and thus prevent oxidative damage
(Foyer et al. 1994).
Toxic effects of Hg, Cd and Pb
The capacity for metal uptake and accumulation in plants varies,
depending on metal and plant species (Mortimer 1985, Guillizoni 1991, Berrow
and Burridge 1991). Plants that are grown in metal polluted environments often
do not show visible symptoms of intoxication even if they contain elevated
concentrations of toxic metals (Clemens 2001). Heavy metals may cause a
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change in metabolic pathways, which can be measured in reduced biomass
production.
Symptoms of Hg toxicity are stunting of seedling growth and root
development (Godbold 1991, Pais and Jones 1997, Mishra and Choudhuri
1999), decreased chlorophyll content and inhibition of photosynthesis (Bernier
et al. 1992, Pais and Jones 1997). The detrimental effect of Hg is strengthened
when inorganic Hg is transformed into organic methyl-Hg (Mortimer 1985,
Richardson and Gangolli 1995). Methyl-Hg is lipophilic and efficient in
crossing cell membranes and the Hg atom still can bind to sulphur of proteins
and thus inactivate metabolic enzymes and structural proteins. (Mortimer 1985,
Richardson and Gangolli 1995).
Cadmium interferes with mineral assimilation (Khan and Khan 1983),
decreases the diameter of xylem vessels and reduces the water transport to the
shoot (Lamoreaux and Chaney 1977, Barcelo and Poschenrieder 1990). The
latter causes a decreased stomatal aperture followed by a reduced CO2 uptake,
which in combination with a Cd-induced decrease in chlorophyll content lead to
a decreased net photosynthesis (Lamoureaux and Chaney 1977, Baszynski et al.
1980, Schlegel et al. 1987). Visible cadmium toxicity symptoms are leaf
chlorosis and necrosis followed by leaf abscission (Pais and Jones 1997).
Lead reacts with important functional groups in macromolecules and the
activity of several enzymes is influensed, some of which are important in
photosynthesis and nitrogen metabolism. Symptoms are reduced growth,
productivity and photosynthesis (Singh et al. 1997).
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Aims
The overall aim of this thesis was to estimate potential risks for human health
and reduced plant growth due to accumulation and toxicity of total-mercury
(total-Hg), methyl-Hg, cadmium (Cd) and lead (Pb) in the tropical vegetable
water spinach, Ipomoea aquatica.
The approach taken was to:
1. find out the concentrations and distribution of total-Hg, methyl-Hg, Cd
and Pb in I. aquatica cultivated for the local food market in Bangkok,
Thailand, and compare them with guidelines for the maximum
recommended intake by humans, in order to estimate a potential health
risk.
2. find out the capacity to accumulate these metals before metal-induced
toxic effects appear in I. aquatica, in order to estimate a potential risk for
affected plant growth.
3. estimate the influence of nutrient availability on accumulation and toxic
effects of these metals in I. aquatica, since the waters where I. aquatica is
cultivated often contain high nutrient levels or are fertilized.
4. estimate the formation of thiol-rich peptides in I. aquatica at varied
nutrient strength and at metal treatment, which could be a sign of stress
and a help to cope with heavy metal stress.
5. Find out if methyl-Hg is formed in the tissues of I. aquatica, since this Hg
species is more toxic than the inorganic one.
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Comments on materials and methods
In order to estimate the concentrations of total-Hg, methyl-Hg, Cd and Pb
in Ipomoea aquatica that is sold on the food market, nine sites of cultivation
were sampled in the greater Bangkok region, Thailand. In the food market, the
upper 50 cm of the shoot of I. aquatica is used. Therefore, that part of the plants
was sampled. The samples were cut into three parts of equal length (16-17 cm),
which all consisted of stems and leaves, and were analysed for metals. A
complementary sampling was done at three of the sites. These samples were
separated into leaves and stems and analysed for total-Hg only (Paper I).
Climate chamber tests were performed with plants of I. aquatica that
originally had been collected in Bangkok, Thailand (Paper II, III) or bought as
fresh plants in an Asian food store in Stockholm, Sweden (Paper IV) and had
been cultivated in a green house before start of tests. In all tests nutrients and
metals were supplied in the traditional mode, i. e. initially at the start of tests.
The tests were performed at 27 oC, 14/10 h light dark cycle and a photon
flux density of 100 to 110 µmol m-2 s-1 (Paper II) or 28/26 oC day/night, 16/8 h
light dark cycle and 200 µmol m-2 s-1 (Paper III, IV). The light period and light
intensity were increased in Paper III and IV in order to improve growth
conditions.
Plants were exposed or not exposed to Hg-, Cd- or Pb-spiked nutrient
solution (Paper II), Hg-spiked nutrient solution (Paper III, IV), Hg-, methyl-Hgor Cd-spiked nutrient solution (Paper IV) or Hg-spiked soil that was flooded
with nutrient solution (Paper III). Each plant was exposed with the lower part of
the stem and the roots beneath the surface of the nutrient solution in a dark
plastic container and the shoot upright above the surface (Paper II, IV). In some
tests special experimental chambers were used in order to prevent that volatile
Hg, originating from the nutrient solution, reached the shoot. These chambers
were equipped with a transparent shoot container on top of the dark root
container in a way that prevented air from the root container to reach the shoot
container (Paper III).
In order to determine concentrations and distribution of metals in different
parts of plants, leaves, stems and roots were analysed for total-Hg, Cd and Pb
(Paper II), total-Hg and methyl-Hg (Paper III) or total-Hg (Paper IV). Leaves
and roots were analysed for thiol-rich peptides in order to study any relation to
nutrient and metal availability (Paper IV).
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Results and discussion
Accumulation of Hg, Cd and Pb in Ipomoea aquatica
In the current thesis it is shown that Hg, Cd and Pb were accumulated in
the shoots of I. aquatica that was cultivated in the greater Bangkok region.
There were wide ranges between lowest and highest concentrations of total-Hg,
methyl-Hg, Cd and Pb in I. aquatica from Bangkok (Table 1) (Paper I).
Table 1. Concentration ranges of metals in shoots of
I. aquatica from Bangkok.
µg/kg DW
Total-Hg
Methyl-Hg
12 – 2 590
0.8 – 220
Cd
< 10 – 120
Pb
40 – 530
Concentrations of total-Hg, Cd and Pb
Concentrations of total-Hg, Cd and Pb in the shoots of aquatic vascular
plants have been reported from polluted and not polluted, tropical and temperate
areas (Table 2). Compared with metal concentrations in aquatic macrophytes
reported in other investigations, Cd and Pb concentrations in I. aquatica from
the Bangkok region varied from about 100 times lower up to about the same
range. Mercury concentrations in I. aquatica from Bangkok were in the same
range as Hg concentrations in aquatic macrophytes reported in other
investigations. The Hg concentrations at one of three sites that were sampled
twice were very much increased at the second sampling occasion (Paper I). This
indicates a possible discontinous Hg pollution. Episodically increased metal
concentrations in the environment and the fast growth of I. aquatica may
contribute to big variations with time of metal concentrations in I. aquatica.
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Table 2. Metal concentrations in freshwater aquatic plants (µg/kg dry weight) found in the literature. (ref)=reference site (poll)=polluted site
TROPICAL and SUBTROPICAL
Species
Hg
Cd
Pb
Area
Reference
Floating
Eichhornia crassipes
710
230
2 600
Harbeespoort Dam, S Africa
Greichus et al. 1977
Eichhornia crassipes
16 000
River San Juan, Cuba
(ref) Gonzales et al. 1989
Eichhornia crassipes
51 000
River Sagua Grande, Cuba (poll) Gonzales et al. 1989
Eichhornia crassipes
(shoot)
1 100
1 600
Okayama, Japan
Muramoto and Oki 1983
Eichhornia crassipes
(leaves) 60
Bot garden, Havanna, Cuba
Gonzales et al. 1994
Eichhornia crassipes
(stem)
13
Bot garden, Havanna, Cuba
Gonzales et al. 1994
Eichhornia crassipes
(leaves)
4 200
9 400
Columbia, Mississippi, US
Chigbo et al. 1982
Eichhornia crassipes
(stem)
2 200
7 400
Columbia, Mississippi, US
Chigbo et al. 1982
Pistia stratiotes
310
930
22 600
Lower Volta River, Ghana
Biney 1991
Floating/Emersed
Ipomoea aquatica
(leaves) 40
Caustic Soda Factory, Thai (ref) Suckcharoen 1978
Ipomoea aquatica
(leaves) 950
Caustic Soda Factory, Thai (poll) Suckcharoen 1978
Ipomoea aquatica
(stem)
40
Caustic Soda Factory, Thai (ref) Suckcharoen 1978
Ipomoea aquatica
(stem) 430
Caustic Soda Factory, Thai (poll) Suckcharoen 1978
Ipomoea aquatica
(shoot)
<80 - 800
1 800 - 3 800
Hanoi, Vietnam
Jörgensen 2005
Ipomoea aquatica
(shoot)
300 - 1000
<100 - 1 800
Phnom Penh, Cambodia
Marcussen et al. 2005
Submersed
Ceratophyllum sp.
<50
2 700
River Nile, Egypt
(ref) Fayed, Abd El-Shafty 1985
Ceratophyllum sp.
300
22 200
River Nile, Egypt
(poll) Fayed, Abd El-Shafty 1985
Ceratophyllum sp.
370
990
17 400
Lower Volta River, Ghana
Biney 1991
Hydrilla verticillata
20 000
Lake Moondarra, Queensland, Au Finlayson et al. 1984
Lagarosiphon ilicifolius
(shoot)
1 200
15 100
Lake Kariba, Zimbabwe
Berg et al. 1995
Najas tenuifolia
240
930
Flood plain, northern Australia
Mc Bride, Noller 1995
Potamogeton octandrus
250
<200
9 400
Lower Volta River, Ghana
Biney 1991
Potamogeton octandrus
(shoot)
1 200
15 500
Lake Kariba, Zimbabwe
Berg et al. 1995
Potamogeton schweinfurtii (shoot)
1 300
12 600
Lake Kariba, Zimbabwe
Berg et al. 1995
Vallisneria aethiopica
130
1 330
23 200
Lower Volta River, Ghana
Biney 1991
Vallisneria aethiopica
(shoot)
2 000
19 100
Lake Kariba, Zimbabwe
Berg et al. 1995
21
Table 2. (continued)
TEMPERATE
Species
Hg
“Aquatic macrophytes”
(shoot)
35- 50
“Aquatic macrophytes”
(shoot) 250
Emersed
Equisetum arvense
(shoot)
Eriocaulon septangulare (leaves)
30
Heteranthera dubia
Pontederia cordata
(shoot)
Floating/Emersed
Nuphar lutea
(leaves)
20
Nuphar lutea
(stem)
10
Nuphar variegatum
6- 36
Nymphea odorata
(shoot)
Submersed
Elodea canadensis
58-230
Elodea canadensis
(shoot)
Elodea canadensis
(shoot)
Heteranthera dubia
(shoot)
Myriophyllum
(shoot)
40
Myriophyllum
(shoot) 1 600
Myriophyllum spicatum
(shoot)
Myriophyllum spicatum
(shoot)
Myriophyllum spicatum
63-240
Myriophyllum spicatum
(shoot)
Myriophyllum verticulatum (shoot)
Potamogeton crispus
80- 85
Potamogeton perfoliatus
40
Potamogeton sp
(shoot)
Vallisneria americana
(shoot)
Cd
100-2 400
580
100-6 000
200
100 - 400
Pb
Area
Reference
Ottawa River, Ontario, Can (ref) Mortimer 1985
Ottawa River, Ontario, Can (poll) Mortimer 1985
1 900 - 3 400
2 600
10 000- 11 000
Nepisiguit River, N Brunsw, Can
Bentshoe Lake, Ontario, Can
Flowing waters, Austria
Flowing waters, Austria
Ray, White 1979
Coquery, Welbourn 1995
Ledl et al. 1981
Mudrock, Capabianco 1979
2 000 - 32 000
River Pappilanjoki, Finland
River Pappilanjoki, Finland
St Lawrence River, Can
Flowing waters, Austria
Lodenius 1991
Lodenius 1991
Thompson et al. 1999
Mudrock, Capabianco 1979
St Lawrence River, Can
St Lawrence River, Can
Flowing waters, Austria
St Lawrence River, Can
St Lawrence River, Can
(ref)
St Lawrence River, Can
(poll)
The Stockholm area, Swed (ref)
The Stockholm area, Swed (poll)
St Lawrence River, Can
St Lawrence River, Can
Flowing waters, Austr
St Lawrence River, Can
River Pappilanjoki, Finland
St Lawrence River, Can
St Lawrence River, Can
Thompson et al. 1999
Désy et al. 2002
Mudrock, Capabianco 1979
Désy et al. 2002
Mortimer 1985
Mortimer 1985
Greger, Kautsky 1990, 1993
Greger, Kautsky 1990, 1993
Thompson et al. 1999
Désy et al. 2002
Mudrock, Capabianco 1979
Thompson et al. 1999
Lodenius 1991
Désy et al. 2002
Désy et al. 2002
790
2 100-3 400
560
25 000 – 33 000
700
2 500
10 000
60 000
500
500-1 400
380
880
8 000 – 31 000
22
Influence of nutrient levels
This study showed in controlled laboratory tests that the lower the nutrient
levels in the medium were, the higher were the Hg, Cd and Pb concentrations in
leaves and stems, and the Cd and Pb concentrations in roots (Paper II). This is
probably due to a higher concentration of plant available metal species in the
medium and less competition with nutrient cations at uptake sites at a low than
at a high nutrient level. According to an equilibrium model (Puigdomenech
1983), the Cd2+ concentration was higher at a low nutrient level (Figure 5).
25 % nutrient solution
100 % nutrient solution
Figure 5. Speciation of Cd in 25 and 100 % Hoagland nutrient medium according to an
equilibrium model (Puigdomenech 1983).
According to the same reference Hg(OH)2 and Pb2+ were present at higher
concentrations when the nutrient level was low in the nutrient medium. The
absolute major part of added Pb precipitated as phosphate complexes
(Puigdomenech 1983), which increased with increasing nutrient level and which
were very easily observed by the eye. The distinct effect of varied nutrient
levels on Pb accumulation in I. aquatica leaves and roots is seen in Table 3.
23
Table 3. Lead concentrations (µg/kg DW) in I. aquatica exposed to the same Pb
concentrations but different nutrient levels in the nutrient medium (Paper II).
10 % Hoagland
µM Pb Leaves
120
72 000
100 % Hoagland
Roots
Leaves
Roots
3 590 000
250
3 000
If this equilibrium model gives a true picture of the metal speciation in these
tests, it is likely that the metals were taken up by I. aquatica as Hg(OH)2, Cd2+
and Pb2+. Higher metal concentrations in plants at low, than at high external
nutrient levels have also been shown in Eichhornia crassipes (O´Keeffee and
Hardy 1984) and in Beta vulgaris (Greger et al. 1991).
A lack of correlation between nutrient levels and Hg concentrations in the
roots of I. aquatica in this investigation may be because Hg was added in
relatively low concentrations, compared to Cd and Pb. At higher external Hg
concentrations there would possibly be a negative correlation between nutrient
levels and Hg concentrations in the roots too, because competition would arise
between Hg ions and nutrient cations for binding sites in the cell walls of the
roots.
The growth of untreated plants at the lowest nutrient level (1 % Hoagland
solution), was significantly lower than at higher nutrient levels (Paper II; Figure
1). Mercury is the only metal that plants were exposed to at that low nutrient
level. Consequently, the high Hg concentrations in leaves and stems of I.
aquatica at that nutrient level may also, more or less, be caused by a reduced
growth, which hampers “dilution” of the metal. It is well established that the
concentrations of metals in plants undergo substancial changes according to the
growth season. As a rule, heavy metal concentrations in plants are highest in the
cold season with reduced growth, decreasing rapidly during the period of
maximal growth (Gommes and Muntau 1981).
Concentrations of methyl Hg
The methyl-Hg concentrations in shoots of field cultivated I. aquatica were
quite low, 0.8 – 2.4 µg/kg DW (0.7-6.7 % of total-Hg), in plants from most of
the sampling sites. In plants from two of the sites, though, the methyl-Hg
concentrations amounted to 11 and 97 – 220 µg/kg DW, which make 11 % and
52 - 100 % of total Hg, respectively (Paper I). Methyl-Hg has also been
observed in other investigations in aquatic and terrestrial plants, at
concentrations from 1 to 75 µg/kg DW (Gardner et al.1978, Mortimer 1985,
Heller and Weber 1998). The concentrations of methyl-Hg in field samples of
the submersed Elodea densa from Ottawa river, Canada, constituted 25-30 % of
the concentrations of total-Hg in the shoots and about 10 % in the roots
(Mortimer 1985).
The two Bangkok sites (Paper I) where methyl Hg concentrations in I.
aquatica were highest, were a flooded, former rice field with standing water and
24
a depth of 0.8 to 1 m and one site with slowly moving wastewater on a
university campus, only 0.10 to 0.15 m deep. At these sites the conditions may
have been favourable for microbial methylation of inorganic Hg, which means
static and anoxic conditions (Furutami and Rudd 1980, St Louis et al. 1994).
Increased total- and methyl-Hg concentrations in biota after flooding of soil (as
at the former rice field) have been seen elsewhere (Paterson et al. 1998, Lucotte
et al. 1999, Guimaraes et al. 1998, 2000). The very high methyl-Hg
concentrations in I. aquatica, growing in wastewater on a university campus,
may be the result of uptake of methyl-Hg that was present in the wastewater
because of pollution or bacterial methylation. Another possibility is that methylHg had been formed in the plant, in analogy with the results in paper III, either
by the plant itself or by endophytic bacteria.
We showed (Paper III) that exposure to external inorganic Hg resulted in
increased concentrations of methyl-Hg in the youngest shoots, which had
developed during the test period and consequently had a high metabolic activity.
This was the case even when plants and equipment had been sterilized (Paper
III), which means that the methylation was not a result of external microbial
activity. Similarly, methyl-Hg was accumulated in pea plants, in the seagrass
Posidonia oceanica and in new shoots of I. aquatica when inorganic Hg was
added externally, at sterilized or not sterilized conditions (Gay 1976a, Ferrat et
al. 2002, Greger and Dabrowska pers. comm.).
The methylation of Hg requires the presence of a suitable methyl donor
molecule. The most likely methyl-group donor for bacterial Hg methylation is
considered to be the vitamin B12 derivate methylcobalamin, even though
bacterial Hg methylation also has been observed independently of the vitamin
B12 metabolic pathway (Siciliano and Lean 2002, Ekstrom et al. 2003).
Methylcobalamin is produced by many organisms, such as bacteria (Ridley et
al. 1977, Choi and Bartha 1993, Ullrich et al. 2001), but not by plants
(Marschner 1995). If the methylation process in the plant is performed by the
plant itself, S-adenosyl methionine is a probable methyl donor molecule (Gay
1976b). S-adenosyl methionine is the donor for most metabolic methylations
(Giovanelli et al. 1985). Another possible methyl donor within the plant could
be cell wall pectin (Hamilton et al. 2003).
Treatment with hypochlorite, not followed by water rinsing, (Paper III)
before exposure to inorganic Hg resulted in very high methyl-Hg
concentrations, especially in the youngest shoots, at the same concentration
level as at the site with the highest methyl-Hg concentrations in Bangkok (Paper
I). The reason for the high concentrations might be that the presence of chloride
in some way stimulated methylation in the plant, for instance by chloride
assistance at transalkylation (Kashin et al. 1979, Celo et al. 2006). Yet another
possibility is that stress, caused by the hypochlorite, stimulates the plant to
increased methylation activity (Kim et al. 2007). Hypothetically, some
component in the wastewater similarly may have stimulated the methylation
process in plants at that site.
25
In this study no demethylation of methyl-Hg was observed in I. aquatica.
A period without any addition of external Hg that followed a period of methylHg-accumulation in I. aquatica did not result in reduced methyl-Hg
concentrations (Paper III). The reason for a lack of demethylation could be that
accumuled methyl-Hg concentrations reached a high and toxic level and thus
metabolic reactions were prevented. Degradation of methyl-Hg has been
described in the submerged macrophyte Elodea densa and in fresh water algae
(Benes and Havlik 1979, Czuba and Mortimer 1980, Simon and Boudou 2001).
In biological systems in the nature, methyl-Hg levels quite likely reflect net
methylation rather than actual rates of methyl-Hg synthesis (Pak and Bartha
1998).
Influence of nutrient levels
In this investigation both growth and methyl-Hg concentrations in the
shoots increased, compared to control plants, when I. aquatica was exposed to
HgCl2-spiked soil which was flooded with nutrient medium, but neither when
exposed to HgCl2-spiked nutrient medium (Paper III) (Figure 6).
*
*
*
Figure 6. Growth (g fresh weight d-1) and methyl-Hg concentrations (µg kg-1 dry weight) in
shoots of Ipomoea aquatica exposed or not exposed for seven days to HgCl2spiked soil (10 mg kg-1 fresh weight) or nutrient medium (0.2 µM). Mean values ±
SE, n=3, * = significantly separated from reference plants (p < 0.05)
It may be so, that soil, which is flooded with nutrient solution, offers a
more optimal nutrients access compared with solely nutrient solution. A
generous access to nutrients favours a high metabolic rate and growth and it is
likely that Hg methylation is linked with high metabolic activity since the
methylation process needs energy (Taiz and Zeiger 1998). Formation of methylHg in new shoots of I. aquatica when exposed to HgCl2-spiked medium has
been demonstrated by Greger and Dabrowska (pers. comm.), but there was no
26
influence by the nutrient level on the methyl-Hg formation. A positive
correlation with growth was exhibited by the percentage methyl-Hg of total-Hg
in field sampled Spartina alterniflora, which increased from the first sample in
May, in the beginning of the growth season, to late July and then decreased to
the last sample in October. On the contrary, the Hg(II) concentrations decreased
throughout the growing season (Heller and Weber 1998).
Concentrations in different tissues
This investigation shows that the concentrations of total-Hg, Cd and Pb are
highest in the roots of I. aquatica and that the concentrations of total-Hg and
methyl-Hg are higher in the leaves than in the stems.
Lead concentrations in shoots of I. aquatica, sampled at sites of cultivation
in Bangkok, mostly were highest in the “bottom” parts while Cd concentrations
showed no such trend (paper I). A hypothesis is that Pb has a low mobility
inside the plant because of a big tendency to bind to negative charges in the cell
walls (Malone et al. 1974, Greger 1999) and to phosphate (Brennan and Shelley
1999). The total-Hg concentrations were higher in the leaves than in the stems
(Paper I), and so they often were in plants in climate chamber tests (Paper II,
III) (Table 4, 5).
Table 4. Highest metal concentrations (µg/kg DW) in shoots of Ipomoea aquatica sampled at
sites of cultivation in Bangkok and approximate highest metal concentrations
without detectable toxic effects in I. aquatica exposed to metal enriched nutrient
medium. Mean values.
Highest concentrations in
I. aquatica from Bangkok.
n=3 (Hg, n=4) (Paper I)
Leaves Stems
Shoot
Highest concentrations without
toxic symptoms in I. aquatica in
climate chamber.
n=5 (Cd, n=4) (Paper II)
Leaves
Stems
(Leaves + stems)
Hg
1 440
Cd
-
Pb
-
420
-
800
500
-
60
30 000
60 000
-
350
20 000
50 000
In some of the climate chamber tests (Paper II) Hg probably evaporated
from test solutions and thus unintentionally contaminated the leaves of the
plants by Hg absorption from the air. This probably is the reason for the
comparatively high Hg concentrations in the leaves of the reference plants in the
tests of Paper II. However, in the climate chamber tests with individual
experimental units for each plant, which were equipped with separated shoot
and root compartments, no Hg could be absorbed by leaves from the air (Paper
27
III). In plants from these tests the Hg concentrations also were higher in leaves
than in stems (Table 5). This is in concordance with findings elsewhere where I.
aquatica and E. crassipes accumulated higher concentrations of Hg in the
leaves than in the stems (Suckcharoen 1978, Gonzales et al. 1994). This
phenomenon is divergent from Cd and Pb. Hypothetically, Hg may have a
greater affinity than the other two metals to some solute that facilitates the
translocation to the leaves, e.g. a S-amino acid or an organic acid forming
transport complexes in the xylem stream, or may be complexed to a lesser
extent to structural substances or solutes with which metals will precipitate.
Table 5. Mercury concentrations (µg kg-1 dry wt) within young shoots (new since test start) of
Ipomoea aquatica exposed to HgCl2. Mean value ± SE (Paper III)
days n
10
10
total-Hg
Leaves
Stems
3 141 ± 17 87 ± 12
2
methyl-Hg
Leaves
Stems
8.5 ± 1.4 4.1 ± 0.46
20 ± 9
13 ± 5.1
Remarks
(blends of 2 plants)
Chlorine treated + H2O
Methyl-Hg concentrations were also higher in leaves than in stems (Paper
III). This was the case both for old leaves, i e leaves that were fully developed
before the start of experiments (Figure 6), and for new leaves within the new
shoots that had grown out since the test start (Table 5). These results are in
agreement with other investigations of methyl-Hg in I. aquatica exposed to
inorganic Hg (Greger and Dabrowska pers. comm.). Highest methyl-Hg
accumulation in the youngest tissues, with a high metabolic rate, has also been
found in other plant species (Gay 1976a, Mortimer 1985, Ericksen et al. 2003).
In the roots the concentrations of tot-Hg, Cd and Pb in both untreated and
treated I. aquatica were much higher than in the leaves and stems (Papers II,
III). Concentrations of tot-Hg and Cd in the roots were on the order of ten times,
and of Pb about four times, higher than in the shoots before the appearance of
toxic symptoms (Paper II). It has been shown that a major part of the metal
taken up by the roots is bound to negative charges in the cell wall structure
(Beauford 1977, Wierzbicka 1998, Wang 2004). The capacity of the roots to
accumulate and tolerate higher metal concentrations prevents the metals from
interfering with sensitive metabolic reactions in the shoot. This mechanism has
been seen in other plant species (Coughtrey and Martin 1978, Ernst et al. 1992,
Landberg and Greger 1996, Österås et al. 2000).
Unlike total-Hg, Cd and Pb, methyl-Hg concentrations were lower in the
roots than in the leaves of plants that were untreated or treated with HgCl2spiked soil (Paper III), maybe because methyl-Hg is formed in the leaves.
28
Distribution
Total-Hg, Cd and Pb were mainly distributed (percent of total amount in
the whole plant) to the roots in treated plants (Paper II and III) and with
increasing additions of metal, a larger proportion of the metal was retained in
the roots. This mechanism was most pronounced for total-Hg (Figure 7).
In contrast, the methyl-Hg distribution to roots was always less than 50 %
in plants exposed or not exposed to HgCl2-spiked soil or nutrient medium
(Paper III) (Figure 7).
Figure 7. Distribution (% of the total amount in the whole plant) of total-Hg and methyl-Hg
in Ipomoea aquatica exposed or not exposed for seven days to HgCl2-spiked soil or
nutrient medium. Mean value ± SE, n=3, (10 d) = 10 days, (Paper III).
Influence of nutrient levels
The nutrient level had a minor influence on the distribution (percent of
total amount in the plant) of Hg to the shoot (leaves + stems) and of Pb to the
leaves. The distribution of Cd showed no relation to nutrient levels (Paper II).
In this thesis it was shown that, when I. aquatica was exposed to HgCl2,
the distribution of total-Hg to the shoots (leaves + stems) increased with
decreasing nutrient strength in the nutrient medium, from 2.9 % to 7.2 % at the
highest and the lowest nutrient strength, respectively (Paper II). Similarly, when
I. aquatica was exposed to methyl-HgCl, the distribution to the shoot of methylHg increased when the nutrient strength decreased (Greger and Dabrowska pers.
comm.). A possible reason for increased distribution to the shoot at a low
29
nutrient level may be less competition at cell membranes and loading of the
xylem at the low nutrient level.
The distribution of Pb to leaves (not the whole shoot) decreased in
untreated and treated plants with decreasing nutrient levels. A most likely
reason is the very low Pb uptake and concentrations in I. aquatica at 100 %
nutrient strength in the nutrient medium and very high Pb uptake and
concentrations at a low nutrient strength (Table 3). When the uptake through the
roots is high, a greater proportion of the metal is retained in the root, possibly
because of a low mobility in the plant caused by a strong tendency to bind to the
cell walls and to phosphate (Brennan and Shelley 1999).
Toxic effects
We showed in laboratory tests, that I. aquatica has a capacity to
accumulate in the order of a hundred and a thousand times higher
concentrations, respectively, of Pb and Cd in leaves and stems, than what was
found in field cultivated I. aquatica from Bangkok (Papers I, II) before
detectable toxic symptoms may occur in the plant (Table 4). For Hg, the
comparison indicates that Hg concentrations in field cultivated I. aquatica from
Bangkok occasionally may reach levels that are toxic for the plant, as
documented by increased dry weight to fresh weight ratio (Paper II) and
affected levels of thiol-rich peptides (Paper IV).
Toxic effects at high and low nutrient levels
Low nutrient strength in the medium resulted in the development of metal
induced toxic effects at low metal concentrations in the medium, and in high
metal concentrations in I. aquatica (Paper II). Greger et al. (1991) found a
greater Cd-induced reduction of growth and water uptake in plants at low than
at optimal nutrient levels. In the current investigation, toxic effects were
correlated to Hg, Cd and Pb concentrations in the shoots and to Cd and Pb
concentrations in the roots (Paper II). Correlations between metal-induced toxic
effects and internal metal concentrations have been found for Cd and Cu in
macrophytes, for example duckweed (Lemna trisulca L) ( Heubert and Shay
1991, Heubert et al.1993). No such correlation was found in the planktonic
diatom Thallassiosira pseudonana (Rijstenbil et al. 1998), in two species of
Brassica (Gadapati and Macfie 2006) or in birch (Betula pendula) and pine
(Pinus sylvestris) ( Österås et al.2000). In contrast, an increase in a single
nutrient may cause increased metal-induced toxicity. Nitrate, for instance,
increased the metal-induced toxicity in algae (Rijstenbil et al. 1998), maybe by
causing a nutritional disorder, such as phosphate deficiency which could
impaire the exclusion/export of the metal.
30
Highest recommended human intake of metals related to
concentrations in Ipomoea aquatica
The highest metal concentrations in I. aquatica that was cultivated at sites
in Bangkok were compared with the highest tolerable weekly intake of Hg, Cd
and Pb according to the World Health Organization (1993, 1999) (Paper I)
(Table 6).
The highest recommended consumption of I. aquatica regarding Cd and Pb
would then be 7.5 and 12 kg fresh weight per day, respectively, for adults and
2.5 and 2 kg fresh weight per day, respectively, for children. Regarding the
intake of Hg and methyl-Hg by children and pregnant women there are no
recommendations by WHO, why a dutch document (Sloof et al. 1995) was used
for these types of consumers. According to calculations based on the highest
analysed concentration of methyl Hg in I. aquatica from Bangkok, the highest
recommended consumption of I. aquatica for adults would be 2 kg fresh weight
per day and for children and pregnant women it would be 0.4 kg fresh weight
per day. Interviews of 557 local households about consumption of I. aquatica
have been performed (Wallin 2001). The highest individual consumption was
0.36 kg per day, which is close to the highest recommended consumption of I.
aquatica regarding total- and methyl-Hg.
Table 6. Highest tolerable intake (HTI) of methyl-Hg, total-Hg, Cd and Pb (WHO 1993,
1995), highest concentrations in water spinach, I. aquatica, and the resulting highest
recommended consumption per day of I. aquatica.
HTI (week)-1
Methyl-Hg
Total-Hg
Adults Children
µg
200
40*
300
µg (kg body weight)-1
7
7
50
25
Highest conc.
in I. aquatica
µg(kg FW)-1
14
168
Highest recommended
consumption of I. aquatica
per day (kg FW)
Adults
Children
2.0
0.3
Adult(60 kg)
7.5
12
0.4
Children(20 kg)
2.5
2
Cd
8
Pb
34
*(Sloof et al. 1995). The World Health Organization (WHO 1993, 1995) regarded data
insufficient to state a HTI for pregnant women and small children, why they are
recommended to avoid food that might contain elevated concentrations.
Consequently, Pb and Cd concentrations in I. aquatica pose no apparent
threat to human health. But with regards to Hg, the consumption of I. aquatica
might pose a health risk, particularly to children and foetuses, and in
combination with other dietary souces, such as fish.
31
Conclusions
It is apparent from this work that there is a capacity in Ipomoea aquatica to
take up Hg, Cd and Pb through the roots and translocate them to the shoots
where they may be accumulated at high concentrations. In field cultivated I.
aquatica from some locations total-Hg and methyl-Hg, but not Cd or Pb, were
found at concentrations that might pose a health risk.
The Hg concentrations in I. aquatica from some field sites reached levels
that may be toxic for the plant, shown by reduced levels of thiol-rich peptides
and increased dry weight to fresh weight ratio.
We showed that inorganic Hg can be transformed to organic methyl-Hg in
the young shoots of I. aquatica. Of the Hg that is taken up through the roots, the
most is bound in the roots. Of the comparatively small amount that reaches the
metabolically active parts, such as leaves and young shoots, a part is
transformed to methyl-Hg.
In this thesis it is shown that a high availability to sulphur or nitrogen
induces the formation of thiol-rich peptides in I. aquatica, compounds which
have a detoxifying effect in plants. A high nutrient strength in the medium,
where all included nutrient elements were equally varied, reduced the
concentrations and toxic effects of Hg, Cd and Pb in I. aquatica. This work
indicates that the use of fertilizers is favourable for the use of this plant as a
frequently consumed vegetable as it reduces the risk for increased metal
concentrations in I. aquatica and for negatively affected crop yields.
Suggestions of future research
At root uptake a low translocation of Hg to the shoot is a restricting factor
for the amount of Hg that is available for methyl-Hg formation in the young,
growing parts of the shoots. However, Hg may be taken up directly by the
shoots from water and air. Under such circumstances a considerable amount of
Hg may be available for methylation in the shoots. Further investigations are
needed to elucidate the degree of methyl-Hg formation at such conditions.
There are different kinds of untreated wastewaters discharged into
watercourses from which aquatic food production systems are supplied with
water. Because of the occasionally high concentrations of methyl-Hg in I.
aquatica presented in this thesis and because other food sources may have high
concentrations of methyl-Hg there is a need for monitoring of Hg, especially
methyl-Hg, in different food-stuff in the region.
32
Acknowledgements
There are many persons who I want to acknowledge...
my dear family, my husband Bengt and my children Oscar and Jenny for
your great support and patience
my supervisor Ass Prof Maria Greger for your great support, inspiration
and scientific guidance
my present and former co-supervisors Prof´s Hans Borg and Bengt-Erik
Bengtsson for scientific support and encouragement
all you people at ITMm: Karin E, Karin H, Pia , Ann-Marie, Jörgen, Bosse,
Hanna, Bertil, Marcus, Margareta and others too, for all sorts of valuable
help, analyses and discussions
Britta, Ann-Kristin, Marsha and Magnus for valuable comments on the
manuscript
present and former members of “the metal group” at the Botanical
Institution for help, interesting discussions and cheerful company : Clara,
Johanna, Sylvia, Tommy, Beata, Åsa, Eva, Yaodong , AnnHelen and Lisa
Yaodong and Tommy for all help with experimental and analytical
equipment
Hans L for construction of experimental equipments and Peter and Ingela
for lots of help with my green house cultivations
For permission to republish figures acknowledgements to:
 University of Florida - IFAS - Center for Aquatic & Invasive Plants
 Sinauer Associates, Inc., Publishers
 Springer Science and Business Media
33
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