1

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.................................................................... 7

Background.................................................................... 7 Hg, Cd and Pb effects on human health.................................. 7

Cultivation of Water Spinach (Ipomoea aquatica).............. 8

Metal uptake in aquatic plants.............................................. 9

Availability in the aquatic environment .......................... 9

Accumulation in plants........................................................ 11

Uptake in roots.......................................................... 11

Uptake in plant cells.................................................. 13

Translocation to the shoot......................................... 14

Distribution in the plant............................................. 14

Uptake in leaves........................................................ 14

Effects of metal stress in plant........................................... 15

Toxic effects of Hg, Cd and Pb................................. 15

Aims................................................................................. 17

Comments on materials and methods............................. 18

Results and discussion…………………………...………… 19

Accumulation of Hg, Cd and Pb in Ipomoea aquatica........ 19

Concentrations of total-Hg,Cd and Pb.............................. 19

Influence of nutrient levels ........................................ 22

Concentrations of methyl-Hg............................................. 23

Influence of nutrient levels........................................ 25

Concentrations in different tissues..................................... 26

Distribution........................................................................ 28

Influence of nutrient levels........................................ 28

Toxic effects............................................................................ 29

Toxic effects at high and low nutrient levels............. 29

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 “Itai-

Itai 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)

root

O2

O2 + MeS + 2H2O Men+

+ SO42-

+ 4H+

Men+

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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endodermis

cortex

xylem

endodermis

xylem

phloem

cortex

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

b)

a)

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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-Hg-

or 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).

19

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 12 – 2 590

Methyl-Hg 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.

20

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

Cd

Pb

Area

Reference

“Aquatic macrophytes” (shoot) 35- 50 Ottawa River, Ontario, Can (ref) Mortimer 1985

“Aquatic macrophytes” (shoot) 250 Ottawa River, Ontario, Can (poll) Mortimer 1985

Emersed

Equisetum arvense (shoot) 100-2 400 1 900 - 3 400 Nepisiguit River, N Brunsw, Can Ray, White 1979

Eriocaulon septangulare (leaves) 30 580 2 600 Bentshoe Lake, Ontario, Can Coquery, Welbourn 1995

Heteranthera dubia 100-6 000 Flowing waters, Austria Ledl et al. 1981

Pontederia cordata (shoot) 200 10 000- 11 000 Flowing waters, Austria Mudrock, Capabianco 1979

Floating/Emersed

Nuphar lutea (leaves) 20 River Pappilanjoki, Finland Lodenius 1991

Nuphar lutea (stem) 10 River Pappilanjoki, Finland Lodenius 1991

Nuphar variegatum 6- 36 St Lawrence River, Can Thompson et al. 1999

Nymphea odorata (shoot) 100 - 400 2 000 - 32 000 Flowing waters, Austria Mudrock, Capabianco 1979

Submersed

Elodea canadensis 58-230 St Lawrence River, Can Thompson et al. 1999

Elodea canadensis (shoot) 790 St Lawrence River, Can Désy et al. 2002

Elodea canadensis (shoot) 2 100-3 400 25 000 – 33 000 Flowing waters, Austria Mudrock, Capabianco 1979

Heteranthera dubia (shoot) 560 St Lawrence River, Can Désy et al. 2002

Myriophyllum (shoot) 40 St Lawrence River, Can (ref) Mortimer 1985

Myriophyllum (shoot) 1 600 St Lawrence River, Can (poll) Mortimer 1985

Myriophyllum spicatum (shoot) 700 10 000 The Stockholm area, Swed (ref) Greger, Kautsky 1990, 1993

Myriophyllum spicatum (shoot) 2 500 60 000 The Stockholm area, Swed (poll) Greger, Kautsky 1990, 1993

Myriophyllum spicatum 63-240 St Lawrence River, Can Thompson et al. 1999

Myriophyllum spicatum (shoot) 500 St Lawrence River, Can Désy et al. 2002

Myriophyllum verticulatum (shoot) 500-1 400 8 000 – 31 000 Flowing waters, Austr Mudrock, Capabianco 1979

Potamogeton crispus 80- 85 St Lawrence River, Can Thompson et al. 1999

Potamogeton perfoliatus 40 River Pappilanjoki, Finland Lodenius 1991

Potamogeton sp (shoot) 380 St Lawrence River, Can Désy et al. 2002

Vallisneria americana (shoot) 880 St Lawrence River, Can Désy et al. 2002

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 100 % Hoagland

µM Pb Leaves Roots Leaves Roots

120 72 000 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 methyl-

Hg 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 methyl-

Hg-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 HgCl2-

spiked 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 methyl-

Hg 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)

Highest concentrations without

toxic symptoms in I. aquatica in

climate chamber.

n=5 (Cd, n=4) (Paper II)

Leaves Stems Shoot (Leaves + stems)

Leaves Stems

Hg 1 440 420 - 800 500

Cd - - 60 30 000 60 000

Pb - - 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 total-Hg methyl-Hg Remarks

Leaves Stems Leaves Stems

10 3 141 ± 17 87 ± 12 8.5 ± 1.4 4.1 ± 0.46 (blends of 2 plants)

10 2 20 ± 9 13 ± 5.1 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 HgCl2-

spiked 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 methyl-

Hg 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

Highest conc.

in I. aquatica

µg(kg FW)-1

Highest recommended

consumption of I. aquatica

per day (kg FW)

Adults Children Adults Children

µg

Methyl-Hg 200 40* 14 2.0 0.4

Total-Hg 300 168 0.3

µg (kg body weight)-1

Adult(60 kg) Children(20 kg)

Cd 7 7 8 7.5 2.5

Pb 50 25 34 12 2

*(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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