Ets EDDS-27957-02

Doctoral Thesis ETH No.
16039
The Use of EDDS in Soil
Washing
and
Phytoremediation
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the
degree
of
Doctor of Sciences
presented by
SUSAN TANDY
MSc Environmental
Science, Nottingham University
Born 21st June 1971
citizen of
Great Britain
accepted
on the recommendation of
Prof. Rainer
Schulin,
examiner
Prof. Emmanuel
Frossard,
co-examiner
PD Dr. Bernd
Nowack,
co-examiner
Dr. Satish
Gupta,
co-examiner
2005
Table of Contents
Summary
V
Zusammenfassung
VII
1 Introduction 1
1.1
Objectives
of this
study
2
1.2 References 3
2 Determination of EDDS and other
amino-polycarboxylic
acids
by
HPLC 7
Abstract 7
2.1 Introduction 8
2.2
Experimental
8
2.2.1
Reagents
and chemicals 8
2.2.2 HPLC 9
2.2.3
Sample Preparation
9
2.3 Results and discussion 10
2.3.1 EDDS 10
2.3.2 EDTA and NTA 13
2.3.3
Analyses
14
2.4 Conclusions 15
Acknowledgements
15
2.5 References 15
3 Determination of EDDS
by
HPLC after derivatization with FMOC 19
Abstract 19
3.1 Introduction 20
3.2
Experimental
20
3.2.1
Reagents
and chemicals 20
3.2.2 Derivatization of EDDS 21
3.2.3 HPLC 21
3.2.4 LC/MS 21
3.2.5 Water and soil solution
samples
21
3.2.6 Plant material extraction 22
I
3.3 Results and Discussion 22
3.3.1 Derivatization of EDDS 22
3.3.2 HPLC
separation
24
3.3.3 Identification
by
LC/MS 25
3.3.4
Analyses
28
3.4 Conclusions 30
Acknowledgements
31
3.5 References 31
4 Extraction of
heavy
metals from soils
using biodegradable chelating
agents
35
Abstract 35
4.1 Introduction 36
4.2 Materials and Methods 38
4.2.1 Soils 38
4.2.2
Chelating agents
38
4.2.3 Metal extractions 38
4.2.4
Analytical
methods 41
4.3 Results and Discussion 42
4.3.1 Effect of
pH
on extraction 42
4.3.2 Extraction kinetics 49
4.3.3
Comparison
to other studies 50
4.3.4 Influence of solid
phase speciation
on extraction
yield
52
Acknowledgements
54
4.4 References 55
5 The Influence of SS-EDDS on the
Uptake
of
Heavy
Metals in
Hydroponically
Grown Sunflowers 59
Abstract 59
5.1 Introduction 60
5.2.1 Nutrient Solution 62
5.2.2
Experimental Setup
62
5.2.3
Sample preparation
63
5.2.4 Metal
Analysis
63
5.2.5 EDDS
analysis
64
5.2.6 Chemicals 64
5.2.7
Speciation Modelling
64
5.2.8 Statistical
analysis
64
II
5.3 Results
5.3.1
Hydroponics
solution
speciation
64
5.3.2 Plant
dry weight
65
5.3.3 Root metal
uptake
65
5.3.4 Shoot metal
uptake
66
5.3.5 EDDS
uptake
69
5.4 Discussion 71
5.5 Conclusions 74
Acknowledgements
74
5.6 References 75
6
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
related to the solubilized metal concentration 81
Abstract 81
6.1 Introduction 82
6.2.1 Soils 83
6.2.2
Experimental Setup
84
6.2.3 Metal and EDDS
Analysis
85
6.2.4 Calculation of shoot metal
uptake
after EDDS addition 86
6.2.5 Chemicals 86
6.2.6 Statistical
analysis
86
6.3 Results 87
6.3.1 Plant
dry weight
87
6.3.2 Solubilized Metals 88
6.3.3 Plant metal
uptake
88
6.3.4 EDDS
uptake
90
6.3.5 Metal
uptake
versus EDDS
uptake
93
6.3.6
Speciation
of EDDS 94
6.3.7
Relationship
of soil solution to
plant uptake
94
6.4 Discussion 97
6.4.1
Comparison
of shoot metal concentrations 97
6.4.2 EDDS
uptake
98
6.4.3 Metal
uptake
in the
presence
of EDDS 99
6.4.4 Factors
influencing
chelant assisted
phytoextraction
99
Acknowledgements
101
6.5 References 101
7 Conclusions 107
7.1 EDDS measurement
by
HPLC 107
7.2 Soil
washing using
EDDS 107
7.3 The use of EDDS for chelant assisted
phytoextraction
108
7.4 Outlook and
open questions
108
Acknowledgements
111
Curriculum Vitae 113
IV
Summary
Heavy
metal
pollution
of soil is a
global problem.
Remediation of metal
polluted
soil is a difficult task and in
general expensive.
Two
potential
remediation
techniques
are ex-situ soil
washing
and in-situ
phytoextraction. Phytoextraction using
hyperaccumulator plants
is
usually
limited
by
low biomass whereas metal
uptake
by high
biomass
plants usually
suffers from low
phytoavailability
of the metal. One
strategy
to over come these limitations is to enhance metal
phytoavailability by
the
application
of chelants. EDTA has been the most
commonly
used
chelating agent
for
phytoextraction.
Its downside is its
persistence
in the environment. This leads
to a
high
risk of metal
leaching
to
groundwater.
EDTA has also been
proposed
to
enhance metal extraction in ex-situ soil
washing.
(S,S)-N,N'-ethylenediamine
disuccinic acid
(EDDS)
is a
biodegradable
isomer
of EDTA. It is now used as a commercial substitute for EDTA in
detergents
and is
recommended to
replace
EDTA and NTA in the
tanning process.
The
goal
of this
work was to
investigate
the
potential
of EDDS to serve as an alternative to EDTA or
other
synthetic
chelants in soil
washing
and chelant assisted
phytoextraction.
The first
step
in this
investigation
was the
development
of a HPLC based method
for EDDS
analysis
that was suitable for detection in
water, plant
and soil solution at
trace levels. This was
successfully
achieved
using
fluorescence detection.
In the second
step
EDDS was
compared
with EDTA and other
biodegradable
complexing agents
in batch soil
washing experiments.
For Cu at
pH 7,
the order of
the extraction
efficiency
for
equimolar
ratios of
chelating agent
to metal was EDDS
>
NTA
>
IDSA
>
MGDA
> EDTA. For Zn it was NTA >
EDDS
> EDTA >
MGDA
>
IDSA. The
comparatively
low
efficiency
of EDTA resulted from
competition
between
the
heavy
metals and co-extracted Ca. For Pb the order of extraction was EDTA >
NTA >
EDDS due to the much
stronger complexation
of Pb
by
EDTA
compared
to
EDDS. In
sequential
extractions EDDS extracted metals almost
exclusively
from the
"exchangeable", "mobile", "manganese
oxide" and
organic
fractions
(according
to
the scheme of Zeien and
Brummer).
We concluded that the extraction with EDDS at
pH
7 showed the best
compromise
between extraction
efficiency
for
Cu, Zn,
and Pb
and loss of Ca and Fe from the soil.
The use of EDDS in chelant assisted
phytoextraction
was
investigated
in two
steps
to ascertain whether it was suitable for this
purpose
and also to
glean
an
insight
into
the
processes
that take
place during
chelant assisted
phytoremediation.
The first
step
was a
hydroponics experiment.
The
objective
was to
investigate
if
V
EDDS could enhance
Cu,
Zn or Pb
uptake
from solution
surrounding
the roots It
was also used to look into the mechanism of
uptake
of chelated metals EDDS was
detected in
roots,
shoots and
xylem sap, proving
that it was taken
up
into the shoots
of sunflowers The essential metals Cu and Zn were decreased in shoots in the
presence
of EDDS whereas
uptake
of the non-essential Pb was enhanced These
results
prove
that EDDS does not
always
increase
uptake
of metals from solution
with a
given
total metal concentration Our
hypothesis
is that in the
presence
of
EDDS all three metals were taken
up by
the non-selective
apoplastic pathway
as
the EDDS
complexes,
whereas in the absence of
EDDS,
essential metal
uptake
was selective
along
the
symplastic pathway
In the second
step,
we
performed pot experiments
in which we studied the effect
of EDDS on
phytoextraction
of
Zn,
Cu and Pb from
artificially
contaminated soils
with
single
or dual metal contamination
(Cu, Zn)
and from field contaminated soils
with multi-metal contamination
(Cu, Zn, Pb, Cd)
It was found that Zn
(when present
as the sole
metal), Cu,
and Pb
uptake by
sunflowers was increased
by EDDS,
but in
multi-metal contaminated soil Zn and Cd were not In the
presence
of EDDS a linear
relationship
between Cu and Zn in soil solution and
plant uptake
was
found,
while
in the absence of EDDS a non-linear
relationship
was found This shows that the
metals were taken
up by
two different mechanisms
depending
on whether chelant
was
present
or not
We conclude that in the case of our soils the
solubihzing
of metals
appeared
to be
more
important
in chelant assisted
phytoextraction
than
enhancing
the metal
uptake
mechanism,
as Cu shoot
uptake
was increased
by
EDDS
(and
Zn when it was
the
only contaminating metal) despite
the fact that at
equal
solution concentrations
uptake
is less in the
presence
of chelants than in the absence
The results from this
study
show EDDS is suitable to
replace
EDTA in soil
washing,
whereas the achieved enhancement of
phytoextraction
is not
yet enough
to be viable
for remediation
purposes
VI
Zusammenfassung
Die
Verschmutzung
von Bden mit Schwermetallen ist ein
globales
Problem.
Die
Reinigung
dieser Bden ist ein
schwieriges Unterfangen
und normalerweise
sehr teuer. Zwei
mgliche Reinigungsverfahren
stellen die ex-situ Bodenwsche
und die in-situ
Phytoextraktion
dar.
Phytoextraktion
mit
hyperakkumulierenden
Pflanzen ist normalerweise durch die
geringe
Biomasse der Pflanzen limitiert
whrend die Metallaufnahme durch Pflanzen mit hoher Biomasse von der oftmals
geringen Bioverfgbarkeit
der Metalle im Boden bestimmt wird. Eine
Strategie,
um
diese
Einschrnkung
zu
umgehen,
ist die
Metallverfgbarkeit
durch
Zugabe
von
Komplexbildnern
zu erhhen. EDTA ist der am
hufigsten
fr diese
Anwendung
eingesetzte Komplexbildner.
Sein Nachteil ist seine
lange
Halbwertszeit in der
Umwelt,
welche zum Risiko des Auswaschens der Metalle ins Grundwasser fhrt.
EDTA wurde auch zur ex-situ Bodenwsche
vorgeschlagen.
(S,S)-N,N'-Ethylendiamin
Di-Bernsteinsure
(EDDS)
ist ein
biologisch
abbaubares
Isomer von EDTA. Es wird als Ersatz von EDTA in Waschmitteln verwendet und
als
mglicher
Ersatz von EDTA und NTA im
Gerbprozess vorgeschlagen.
Das Ziel
dieserArbeitwares,
das Potenzial von EDDS als Alternative zu EDTA oder anderen
synthetischen Komplexbildnern
in Hinblick auf Bodenwsche und die
komplexbildn
eruntersttzte
Phytoextraktion
abzuklren.
Der erste Schritt in dieser Studie war die
Entwicklung
einer HPLC-Methode
zur
Analyse
von
EDDS,
welche den Nachweis von
Spurenkonzentrationen
von
EDDS in
Wasser, Bodenlsung
und Pflanzenmaterial erlaubt. Dies konnte mittels
Derivatisierung
und Fluoreszenzdetektion
erfolgreich umgesetzt
werden.
In einem zweiten Schritt wurden
EDDS,
EDTA und
einige
andere
biologisch
abbaubare
Komplexbildner
im Hinblick auf die
Eignung
fr den Einsatz in der
Bodenwsche
verglichen.
Bei
pH
7 nahm die Extraktion von Cu in der Reihe EDDS
>
NTA> IDSA
>
MGDA
> EDTA ab. Fr Zn war die
Reihenfolge
NTA >
EDDS
> EDTA
>
MGDA
>
IDSA. Die relativ tiefe
Extraktionsleistung
von EDTA ist durch Konkurrenz
zwischen den Schwermetallen und dem mitextrahierten Ca
bedingt.
Fr Pb war die
Reihenfolge
EDTA > NTA >
EDDS, bedingt
durch die viel strkere
Komplexierung
von Pb durch EDTA
verglichen
mit EDDS. In
sequentiellen
Extraktionen lste EDDS
die Schwermetalle fast ausschliesslich aus den
"austauschbaren", "mobilen",
"Manganoxid"
und
"organischen"
Fraktionen
(gemss
dem Schema von Zeien und
Brummer). Wirschliessen,
dass die
Behandlung
des Bodens mit EDDS bei
pH
7 den
besten
Kompromiss
zwischen der Extraktion von
Cu,
Zn und Pb und dem Verlust
VII
von Ca und Fe aus dem Boden bietet
Die
Verwendung
von EDDS in
komplexbildnerunterstutzer Phytoextraktion
wurde in zwei Schritten
untersucht,
um
abzuklren,
ob es sich fur diese
Anwendung
eignet
und um Einblicke in die
wichtigsten
Prozesse wahrend der Extraktion zu
gewinnen
Der erste Schritt war ein
Nahrlosungsexpenment,
bei dem es das Ziel
war,
zu
untersuchen ob EDDS die Aufnahme von
Cu,
Zn oder Pb aus der
Losung
erhohen
wurde Dieses
Experiment
wurde auch
verwendet,
um den Mechanismus der
Aufnahme zu untersuchen EDDS wurde in
Wurzeln, Spross
und im
Xylemsaft
gefunden,
was
beweist,
dass es in die Sonnenblume
aufgenommen
wurde
EDDS
verringerte
die Aufnahme der essentiellen Metalle Cu und
Zn,
wahrend die
Aufnahme von Pb erhht wurde Dieses Resultat
zeigt,
dass ein
Komplexbildner
nicht
notwendigerweise
die Aufnahme von Metallen erhht wenn die totale
Metallkonzentration in
Losung
konstant ist Es ist unsere
Hypothese,
dass in
Anwesenheit von EDDS alle drei Metalle durch einen nicht
selektiven, apoplastischen
Weg
als
Komplexe aufgenommen
wurden wahrend in Abwesenheit von EDDS die
selektive Aufnahme von essentiellen Metallen durch einem
symplastischen Weg
stattfand
In einem zweiten Schritt haben wir
Topfexperimente durchgefhrt,
in welchen
wir den Einfluss von EDDS auf die
Phytoextraktion
von
Cu,
Zn und Pb aus
knstlich kontaminierten Boden mit einfacher oder mehrfacher
Metallbelastung
(Cu, Zn)
und aus mehrfach belasteten Boden
(Cu, Zn, Cd, Pb)
von tatschlichen
Belastungsgebieten
untersucht haben Wir
fanden,
dass EDDS die Aufnahme von
Zn
(wenn
es das
einzige
Metall
war),
Cu und Pb in die Sonnenblume
erhhte,
wahrend Zn und Cd in den natrlich belasteten Boden nicht erhht wurden In
Gegenwart
von EDDS besteht ein linearer
Zusammenhang
zwischen der
gelosten
Metallkonzentration und der
Pflanzenaufnahme,
wahrend in Abwesenheit von
EDDS ein nichthnearer
Zusammenhang
besteht Dies
zeigt,
dass die Metalle mit
zwei verschiedenen Mechanismen
aufgenommen werden, abhangig
davon ob ein
Komplexbilder
vorhanden war oder nicht
Wirschliessen,
dass in den untersuchten Boden die LoshchkeitderMetallewahrend
der
Phytoextraktion wichtiger
war als die
Erhhung
der Pflanzenaufnahme durch
den
Komplexbildner
Die Cu Aufnahme aus Boden wurde durch EDDS erhht
(und
Zn wenn es als
einziges
Metall
vorlag),
obwohl bei
gleicher Losungskonzentration
die Aufnahme
geringer
war
Die Resultate dieser Studie
zeigen,
dass EDDS
geeignet ist,
um EDTA in der
Bodenwasche zu
ersetzen,
wahrend die
Erhhung
der
Aufnahmeleistung
bei der
Phytoextraktion
noch nicht
gengend
fur eine
Anwendung
in der
Bodensanierung
ist
VIII
1 Introduction
Heavy
metal
pollution
of soil is a
global problem
Pollution can arise from
many
different sources
including mining
and
smelting processes, fertilizers,
pesticides,
biosohds and automobile emissions
(1)
The clean
up
of these soils
is a difficult task Various in-situ and ex-situ remediation
techniques
have been
employed,
e
g solidification, stabilization,
chemical
treatment,
soil
flushing,
soil
washing, electroremediation, bioleaching
and
phytoremediation (2)
The
problem
with
solidification,
stabilization and chemical inactivation treatment is that the
contamination is not removed and it is unclear for how
long
these methods are
effective, giving
the
possibility
of
problems
in the
long
term future Soil
flushing brings
risks of
ground
water contamination and
requires
the
application
of metal
mobilizing
chemicals
Bioleaching,
when
in-situ,
can also cause
leaching
and contamination of
groundwater
Electroremediation is
expensive
and
only
suited for small areas with
high
contamination under
special
conditions and so has
rarely
been used
Soil
washing
is ex-situ and can take the form of
heap leaching
or batch
washing
It is
generally
carried out with acids or
chelating agents (2)
After
processing
the soil
can be returned to the site Soil
washing
with
chelating agents
is seen as
particularly
promising (3)
EDTA has been the most
widely
used
chelating agent
for chelant
enhanced soil
washing
as it is
very
efficient at
extracting metals,
but
unfortunately
it
has low
biodegradabihty
and so it is
very persistent
in the environment
(4)
This can
lead to a
high
risk of the
remaining
metals
being
leached to
groundwater
when the
soil is returned to the
original
site
(5)
Phytoextraction
is another
promising
remediation
technique
It is seen as a
cost
effective, environmentally friendly
in-situ
technique
for
cleaning up
metal
contaminated land
(6),
which aims to
preserve
soil structure and
fertility
The idea of
this remediation
technique
is that
plants
remove the
pollutant
from soil and transfer it
to
easily harvestable,
above
ground parts (7)
Two main directions to
phytoextraction
have
developed 1)
the use of
hyperaccumulating plants
and
2)
the use of
high
biomass
plants (8) Hyperaccumulation
of
heavy
metals is
generally
limited
by
the
small biomass of the
hyperaccumulating plants (9)
and efficient metal
uptake by
high
biomass
plants
is often limited
by
low
phytoavailability
of the
targeted metals,
in
particular
in neutral or alkaline soils
(8, 10)
Therefore the focus has been switched
to the induced accumulation of metals
by high
biomass
plants
Various authors have
proposed
the use of
chelating agents
for this
purpose,
as reviewed
by
Lasat
(10)
and
Schmidt
(11)
The use of
hyperaccumulator plants
with
chelating agents
has been
1
Chapter
1
ruled out as it has been found that the addition of
chelating agents
reduces metal
uptake
and not increases it
(12).
As with chelant assisted soil
washing,
EDTA has
been the most
commonly
used
complexing agent
for chelant
phytoextraction,
but
as stated before its environmental
persistence
can cause
problems
due to
leaching
(13, 14).
Other
occasionally
used chelants
(DPTA, HEDTA)
are
equally persistent
in
the environment
(4)
or
possible carcinogens (NTA) (15).
1.1
Objectives
of this
study
disuccinic acid
(EDDS)
is a
biodegradable
isomer
of EDTA
(16-18).
It is now used as a commercial substitute for EDTA in
detergents
(19, 20)
and is recommended to
replace
EDTA and NTA in the
tanning process
(21).
EDDS has the
potential
to be a substitute for EDTA in chelant assisted soil
washing
and
phytoremediation,
as it is a
strong
chelator and unlike EDTA
easily
biodegradable.
Some metal
complexes (Cu,
Ni and
Co)
of EDDS have been found to
be
non-biodegradable
in conditions where
they
were isolated from other metals
(17).
The effect of these
complexes
on total EDDS
biodgradation
has been
investigated
by
the authors but will be
published
in a
separate
document.
In order to
apply
EDDS in soil
washing
and
phytoremediation,
it is
important
to know how EDDS solubilizes the metals in
question
and how the metal-EDDS
complexes
are taken
up by
the
plant,
if indeed
they
are. From the
stability
constants
of the
complexes
it was
expected
that Cu and Zn would show
good
extraction but
that it would be much worse for Pb and Cd.
At the start of this work
virtually nothing
had been
published
with
regards
to the
use of EDDS in soil
washing
or
phytoremediation. However,
since the start of this
thesis,
much more has been
published especially
with
regards
to
phytoremediation.
One work has
investigated
ex-situ soil
washing using
EDDS and found it to be
successful. Extraction of
non-target
metals was not
investigated
and EDDS was
not
compared
to
any
of the new
biodegradable complexing agents
on the market
however
(22).
Work has now been
published
from
pot
and column
phytoremediation
experiments using
EDDS
(23-30).
The work has
mainly
focused on Pb extraction
but also the extraction of
Cu, Zn,
Cd and Ni. All the
investigations
have focused on
the increase on
plant
metal
uptake
after the addition of EDDS but not on the details
of this
uptake.
In most cases the
speciation
of soil solution
during
these
experiments
is
missing
and no
relationship
between soluble metals and
plant uptake
have made.
The
uptake
of EDDS into the
plants
has not been
investigated.
With this in mind the main
objectives
of the
study
were:
i)
to
develop
a
simple
HPLC based method for the
analysis
of EDDS in both
soil solution and
plant
material
ii)
to
investigate
the use of EDDS for chelant enhanced soil
washing
as a means
2
Introduction
of
remediating
metal
polluted
soil
iii)
to
investigate
the use of EDDS for chelant enhanced
phytoextraction
of metal
polluted
soils.
Corresponding
to the above
objectives
the
following
studies were conducted:
i) Development
and evaluation of a HPLC
analysis
method for EDDS based on
the conversion of all EDDS
complexes
to
Fe(lll)EDDS
and detection
by
UV-
detector
(Chapter 2).
ii) Development
and evaluation of an
analysis
method for EDDS based
on derivatization with FMOC
reagent
followed
by
HPLC
separation
and
fluorescence detection
(Chapter 3).
iii)
Batch
experiments
to
investigate
the use of EDDS for soil
washing
of
heavy
metal
polluted
soil and
compare
its
performance
to EDTA and other
biodegradable chelating agents (Chapter 4).
iv) Hydroponics experiment
to
investigated
the influence of EDDS on
uptake
of essential
(Cu
and
Zn)
and non-essential
(Pb)
metals
by
sunflowers from
nutrient solution. The
uptake
of EDDS was also
investigated (Chapter 5).
v)
Pot
experiments
with sunflowers to
investigate
the use of EDDS for
phytoextraction
of
heavy
metal contaminated soils. Soils
artificially
contaminated with
single
or combined metals
(Cu, Zn) along
with soils with
multi-metal contamination from the field
(Cu, Zn,
Pb and
Cd)
were
investigated.
In order to
clarify
the
processes occurring,
soil solution and
plant
metals were
measured
along
with EDDS
(Chapter 6).
1.2 References
(1) Sparks,
D. L. Environmental Soil
Chemistry;
2nd
ed.;
Academic Press:
London,
2003.
(2) Mulligan,
C.
N.; Yong,
R.
N.; Gibbs,
B. F. Remediation
technologies
for
metal-contaminated soils and
groundwater:
an evaluation.
Eng.
Geol.
2001, 60,
193-207.
(3) Peters,
R. W Chelate extraction of
heavy
metals from contaminated soils. J.
Hazard. Mater.
1999, 66,
151-210.
(4) Bucheli-Witschel, M.; Egli,
T Environmental fate and microbial
degradation
of
aminopolycarboxylic
acids. FEMS Microbiol. Rev.
2001, 25,
69-106.
3
Chapter
1
(5) Nowack,
B Environmental
chemistry
of
aminopolycarboxylate chelating
agents
Environ Sei Technol
2002,36,4009-4016
(6) Salt,
D E
,
Smith,
R D
,
Raskin,
I
Phytoremediation
Ann Rev Plant
Phys
1998, 49,
643-668
(7) Kumar,
N P B A
,
Dushenkov, V, Motto,
H
,
Raskin,
I
Phytoextraction
The
use of
plants
to remove
heavy
metals from soils Environ Sei Technol
1995, 29,
1232-1238
(8) Salt,
D E
,
Blaylock,
M J
,
Kumar,
N P B
A, Dushenkov, V, Ensley,
B
D
,
Chet,
I
,
Raskin,
I
Phytoremediation
a novel
strategy
for the removal of toxic
metals from the environment
Biotechnology 1995, 13,
468-474
(9) Blaylock,
M J
,
Salt,
D E
,
Dushenkov,
S
,
Zakharova,
O
,
Gussman,
C
,
Kapulnik, Y, Ensley,
B D
,
Raskin,
I Enhanced accumulation of Pb in Indian mustard
by soil-applied chelating agents
Environ Sei Technol
1997,37,860-865
(10) Lasat,
M
Phytoextraction
of toxic metals A review of
biological
mechanisms
J Environ Qual 2002, 31,
109-120
(11) Schmidt,
U
Enhancing phytoextraction
The effect of chemical soil
manipulation
on
mobility, plant accumulation,
and
leaching
of
heavy
metals J
Environ Qual 2003, 32,
1939-1954
(12) Robinson,
B H
,
Brooks,
R
R, Clothier,
B E Soil amendments
affecting
nickel and cobalt
uptake by Berkheya
coddn Potential use for
phytomining
and
phytoremediation
Annals of
Botany 1999, 84,
689-694
(13) Wenzel,
W
W, Unterbrunner, R, Sommer, P, Sacco,
P Chelate-assisted
phytoextraction using
canola
(Brassica napus
L
)
in outdoors
pot
and
lysimeter
experiments
Plant Soil
2003, 249,
83-96
(14) Thayalakumaran, T, Robinson,
B
,
Vogeler,
I
,
Scotter,
D
,
Clothier,
B
,
Percival,
H Plant
uptake
and
leaching
of
copper during
EDTA-enhanced
phytoremediation
of
repacked
and undisturbed soil Plant Soil
2003, 254,
415-423
(15) Ebina, Y, Okada, S, Hamazaki, S, Ogino, F, Li,
J
L, Midorikawa,
O
Nephrotoxicity
and renal-cell-carcinoma after use of iron-nitrilotnacetate and
4
Introduction
aluminum-nitrilotriacetate
complexes
in rats. Journal of the National Cancer Institute
1986, 76,
107-113.
(16) Schowanek, D.; Feijtel,
T. C.
J.; Perkins,
C.
M.; Hartman,
F.
A.; Federle,
T.
W;
Larson,
R. J.
Biodegradation
of
[S,S], [R,R]
and mixed stereoisomers of
ethylene
diamine disuccinic acid
(EDDS),
a transition metal chelator.
Chemosphere 1997,
34,
2375-2391.
(17) Vandevivere, P.; Saveyn, H.; Verstraete, W; Feijtel, W; Schowanek,
D.
Biodegradation
of
metal-[S,S]-EDDS complexes.
Environ. Sei. Technol.
2001, 35,
1765-1770.
(18) Nishikiori, T; Okuyama, A.; Naganawa, H.; Takita, T; Hamada, M.;
Takeuchi, T; Aoyagi, T; Umezawa,
H. Production
by actinomycetes
of
(S,S)-N,N'-
ethylenediamine
disuccinic
acid,
an inhibitor of
phospholipase-C.
J. Antibiot.
1984,
37,
426-427.
(19) Jaworska,
J.
S.; Schowanek, D.; Feijtel,
T C. J. Environmental risk assessment
for trisodium
[SS]-ethylene
diamine
disuccinate,
a
biodegradable
chelator used in
detergent applications. Chemosphere 1999, 38,
3597-3625.
(20) Knepper,
T P.
Synthetic chelating agents
and
compounds exhibiting
complexing properties
in the
aquatic
environment. Trends Anal. Chem.
2003, 22,
708-724.
(21)
EIPPCB
"Integrated pollution prevention
and control
(IPPC):
Reference
document on best available
techniques
for the
tanning
of hides and
skins," European
Commission,
2003.
(22) Vandevivere, P.; Hammes, F.; Verstraete, W; Feijtel, W; Schowanek,
D.
Metal decontamination of
soil,
sediment and
sewage sludge by
means of transition
metal chelate
[S,S]-EDDS.
J. Environ.
Eng. 2001, 127,
802-811.
(23) Kos, B.; Lestan,
D. Influence of
biodegradable (SS-EDDS)
and
nondegradable (EDTA)
chelate and
hydrogel
modified soil water
sorption capacity
on Pb
phytoextracton
and
leaching.
Plant Soil
2003, 253,
403-411.
(24) Kos, B.; Grcman, H.; Lestan,
D.
Phytoextraction
of
lead,
zinc and cadmium
from soil
by
selected
plants.
Plant Soil and Environment
2003, 49,
548-553.
5
Chapter
1
(25) Luo,
C
,
Shen,
Z
,
Lia,
X Enhanced
phytoextraction
of
Cu, Pb,
Zn and Cd
with EDTA and EDDS
Chemosphere 2005, 59,
1-11
(26) Meers,
E
,
Ruttens,A
,
Hopgood,
M J
,
Samson,
D
,Tack,
F M G
Comparison
of EDTA and EDDS as
potential
soil amendments for enhanced
phytoextraction
of
heavy
metals
Chemosphere 2005,58,1011-1022
(27) Kos,
B
,
Lestan,
D Induced
phytoextraction
/ soil
washing
of Lead
using
biodegradable
chelate and
permeable
barriers Environ Sei Technol
2003, 37,
624-629
(28) Grcman,
H
,
Vodnik,
D
,
Velikonja-Bolta,
S
,
Lestan,
D
Ethylenediaminediss
uccinate as a new chelate for
environmentally
safe enhanced lead
phytoextraction
500-506
(29) Kos,
B
,
Lestan,
D Chelator induced
phytoextraction
and in situ soil
washing
of Cu Environ Pollut
2004, 132,
333-339
(30) Kos,
B
,
Lestan,
D Soil
washing
of
Pb,
Zn and Cd
using biodegradable
chelator and
permeable
barriers and induced
phytoextraction by
Cannabis sativa
Plant Soil
2004, 263,
43-51
6
2 Determination of EDDS and other
acids
by
HPLC
Susan
Tandy
Rainer
Schulin,
and Bernd Nowack
Abstract
A new HPLC method for the determination of
ethylenediamine
disuccinic acid
(EDDS)
is
presented.
Free EDDS and
EDDS-complexes undergo
conversion to
the
Fe(lll) complex
in the
presence
of
Fe(lll)CI3.
Fe(lll)EDDS
is
separated by
HPLC
on an ion
exchange
column
using
(NH4)2S04
eluent with detection at 258 nm. The
detection limit is 0.01 uM. EDTA and NTA can also be
analysed by
this method with
slight
alteration to eluent
pH
and
strength.
The EDTA detection limit is 0.25 uM and
NTA 0.1 uM. We
applied
the method to natural waters and soil solution
samples.
A
background
of natural waters resulted in a reduction in EDDS
peak area,
but this
problem
can be over come
by
standard addition
Chapter
2
2.1 Introduction
The
biodegradable chelating agent S,S'-EDDS (ethylenediamine
disuccinic
acid)
has received some attention in the last
years
as a
potential replacement
for EDTA
and other recalcitrant
chelating agents (1), e.g.
in
laundry detergents (2).
The
S,S-
isomer of EDDS is
produced by
some bacteria
(3, 4)
and
fungi (4),
and is
easily
biodegradable,
while the
R,R-
and
S,R-isomers
are not
(5, 6).
There is some interest
in EDDS for its use in the remediation of metal contaminated
land,
both
by
soil
washing
and chelant enhanced
phytoremediation (7-10).
Very
few methods for the
analysis
of EDDS can be found in
literature, probably
due to the fact that it is a 'new
compound'
and
only recently
become
commonly
used. Ammann
(11)
described an IC-ICP-MS method for the detection of metal-
EDDS
complexes.
Other methods are based on the colorimetric
principle
have
very
high
detection limits
(0.1 mM) (5) compared
to the IC-ICP-MS method.
Knepper (12)
mentions that the ISO method for
complexing agents (a
GC based
method) (13)
can
be used for the
analysis
of
EDDS,
but no such use has been documented.
Three
papers
mentioned that HPLC methods have been used for EDDS
analysis,
but the details
given
are not
comprehensive
and
although
the detection limits are
not
given
it seems from their data that
they
would be
relatively high (approximately
10-50
uM) (3, 6, 14).
The methods
rely
on the same basic
principle,
a
separation
using ion-pairing reagents
with
pre-
or
post-column
conversion to the
Fe(lll)
or
Cu(ll)
complex
followed
by
UV detection. Similar methods have been used
extensively
for
the
analysis
of EDTA
(15-20),
NTA
(15), DPTA(15),
and
phosphonates (21).
Some
methods measure
directly
UV active
complexes giving
a
partial speciation
of the
sample
while others use conversion of all
complexes
to the
Fe(lll)
or
Cu(ll)
form
before measurement.
The aim of our work was to
develop
a
simple
HPLC-based method for the
analysis
of EDDS at sub-micromolar concentrations. The basis of this method is both the
HPLC-ICP-MS method for
separation
of metal-EDDS
complexes (11, 22)
and the
pre-column
conversion to
Fe(lll)-complexes
and
subsequent
detection
by
UV
(15,
18).
In addition we have
adapted
the method for the determination of EDTA and
NTA.
2.2
Experimental
2.2.1
Reagents
and chemicals
All chemicals were obtained from Merck unless otherwise stated. Pure water
(Milli-Q system)
was used in all
preparations.
A 0.5 M stock solution of
(NH4)2S04
8
Determination of EDDS and other
acids
by
HPLC
(Fluka)
was
prepared
in
pure
water and used to
prepare
the
working
eluents. 1
mM
Fe(lll)CI3
was
prepared
from the
anhydrous
chemical and dissolved in 1 mM
HCl to
prevent
the
precipitation
of iron. A 1 mM stock standard of
Fe(lll)-[S,S']-
EDDS was
prepared
from
Na3-[S,S']-EDDS
(Procter
and
Gamble)
and
anhydrous
Fe(lll)CI3
in 1 mM HCl. Likewise 1 mM
Fe(lll)NTA
was
prepared
in 1 mM HCl from
Na3NTA
(Fluka)
and
Fe(lll)CI3.
10 mM
Fe(lll)EDTAin
1 mM HCl was
prepared using
NaFe(lll)EDTA (Fluka).
All stock standards were
kept
at
~
4C in the dark to
prevent
photo-degradation
and
biodgradation.
All
working
standards were
prepared
from
the stock standards in 1 mM HCl.
They
were also
kept
~4C and in the dark to
prevent
deterioration.
2.2.2 HPLC
AJasco
high performance liquid chromatographic system (PU-980) equipped
with
851-AS
auto-sampler,
a 200 ul
sample loop
and a UV
spectrophotometric
detector
(UV 970)
set at 258 nm was used. A
pre-instrument degassing
unit was also installed
(Lacoc,
Gastorr
GT102).
The HPLC
separations
were carried out
using
a Dionex Ion
Pac AS11 column
(230
x 4
mm).
Eluent A for the
separation
of EDDS and NTA was
1 mM HCl
(pH 3.0),
and eluent B was 5 mM
(NH4)2S04
(pH 3.3)
for EDDS and 50
mM
(NH4)2S04
(pH 3.3)
for NTA. Eluent A for EDTA was
pure H20
and eluent B was
5 mM
(NH4)2S04(pH
5.3).
The
following gradients
were used:
EDDS,
0-100 %
B in 12
min,
1 min at 100%
B; EDTA,
0
-
50% B in 10
min,
1 min at 50 %
B; NTA,
0
-
100% B in 30
min,
1 min at 100% B. Table 2.1
gives
an overview of the used
chromatographic
conditions.
Table 2.1.
Chromatographic
conditions for the
separation
of
EDDS,
EDTA and
NTA.
Compound
Eluent A Eluent B
gradient
EDDS
1 mMHCI
pH3
5 mM
(NH4)2S04
pH3.3
0-100% Bin 12
minutes
NTA
1 mMHCI
pH3
50 mM
(NH4)2S04
pH3.3
0-100% Bin 30
minutes
EDTA water
5 mM
(NH4)2S04
pH5.3
0-50% B in 10 minutes
2.2.3
Sample Preparation
All
samples
were filtered
through
0.45 urn membrane filters. The
samples
were
adjusted
to
pH
3 with HCl. For most
samples
it was sufficient to add HCl to
give
a
9
Chapter
2
final
sample
concentration 1 mM. Once at
pH 3,
Fe(lll)CI3
in 1 mM HCl was added
to make the
Fe(lll)
concentration in the
sample
10%
greater
than the estimated
concentration of
chelating agent
to be measured. The
sample
was then
thoroughly
mixed and
kept
in the dark at ~4C until
analysed
to
prevent photo-degradation
of
the Fe
complexes.
The effect of ionic
strength
on EDDS
analysis
was tested
using
NaCI to
adjust
the ionic
strength
of
pure
water standards which were later
prepared
for
analysis
as
above.
The effect of different metals on the
analysis
of EDDS was tested
by spiking
1
\M
EDDS standards with 1
\M Cu, Zn, Ni,
Pb or 1 mM Ca. The standards were also
analysed
without metals in the same matrix
(nitrate concentration)
as the
spiked
standards. The
analysis
was carried out with standards
prepared using Fe(lll)EDDS
prior
to metal
spiking
and also with standards
prepared
with
Na3EDDS
prior
to metal
spiking.
2.3 Results and discussion
2.3.1 EDDS
Figure
2.1 shows a
chromatogram
for
Fe(lll)EDDS (10 uM)
in 1 mM HCl
using
a
100 ul
sample injection.
The
analyte peak
is well
separated
from the
reagent peak.
Plots of
peak
area versus the concentration of EDDS were linear from 0.01 uM to 1
uM
{r2
=
0.9994, n=7)
and from 1 uM
to 10 uM
(r2= 1, n=5).
The detection
limit was 0.01 uM
(S/N=3)
When we increased the
uncomplexed Fe(lll) concentration,
peak height
decreased
although
peak
area remained constant. This
only
started to occur however when
the free
(uncomplexed) Fe(lll)
concentration was
greater
than 50-
100 uM. This can be seen in
Figure
2.2 which shows the effect on
peak
height
of excess Fe
(III)
for a 10
|j,M
EDDS standard. The
peak
area
however was not
greatly
affected. Excess
Fe(lll)
concentrations
may
occur in real
samples
where the EDDS concentration is unknown. Therefore it is
important
to
make sure
Fe(lll)
is not added to the
sample
more than 10 times in excess of the
6x104-i
4x104
-
CD
C
tf>
2x104
-
JVj
0 2 4 6 8
Time
(minutes)
Figure
2.1.
Chromatogram
of 10 uM EDDS.
10
acids
by
HPLC
4x10"
3x10-
A.A
CD
CD
<2x105-
CD
CD
-
1 x105.
0
.6x10"
5x104-o
CD
)
^3x104i
CD
C'
-2x104^
1 1 1 1
0 200 400 600 800
Excess Fe
(|liM)
0
1000
Figure
2.2. The effect of excess
Fe(lll)
on the
peak
area
(triangles)
and
height (cir
cles)
of 10 uM EDDS.
4x10-
3x10b-
-5x104 -rj
CD
CD
i_
2 2x105
CD
CD
-
5
1
x1054
0
6x10'
CD
0)
3x104 I
CD
CQ'
h2x104
-
0 20 40 60 80
Ionic
strength
mM
0
100
Figure
2.3. Effect of ionic
strength
of the
sample (adjusted
with
NaCI)
on
peak
area
(triangles)
and
height (circles)
of 10 uM EDDS.
chelating agent.
The ionic
strength
of the
sample
also seems to affect the
peak response.
As can
be seen in
Figure
2.3
increasing
the
sample
ionic
strength (using NaCI)
reduced
the
peak
area and
height
and also broadened the
peak.
A reduction in
peak
area
was observed
beyond
5 mM ionic
strength.
At 50 mM ionic
strength
the
peak
area
was
only
30% of the
original
value. At
higher
ionic
strengths peak
area remained
constant. Peak
height
however started to decrease
immediately
as ionic
strength
11
Chapter
2
was increased and also came to a constant low at 50 mM.
NaN03
was also used
to check the EDDS
peak response
to
increasing
ionic
strength.
The effect on
peak
height
was
exactly
the same as for
NaCI,
but the effect on
peak
area deviated from
that of NaCI.
Up
to 50 mM the
response
of
peak
area with NaCI and
NaN03
was
about the
same,
but at 100 mM ionic
strength
NaN03
reduced the
peak
area even
more than NaCI. This is
probably
due to
NaN03
increasing
the retention time of
the
peak
and
making
it co-elute with a
very
low broad
peak
which
appears
at
high
NaN03
concentrations. We also
investigated
the effect of
phosphate
on EDDS
peak
area and
height.
Peak area was unaffected at
phosphate
concentrations of 10 mM
but showed
nearly
50% reduction at 100 mM. Peak
height
however showed a
steady
increase between 0 and 10 mM
phosphate,
then reached a
plateau
of 160% of the
original peak height.
As
peak
area is
usually
the measured
parameter, only high
concentrations of
phosphate
would effect the measurement of EDDS. It is essential
given
these
findings
that standards be
prepared
in the same matrix as the
samples
so as to avoid
any
matrix effects.
Table 2.2. Influence of metals on
peak
area and
height
due to the conversion of 1
uM EDDS
complexes
into
Fe(lll)EDDS.
Peak area Peak
height
Metal free 100% 100%
Cu 96.9% 96.8%
Zn 100.4% 102.4%
Ni 99.1% 100.6%
Pb 100.2% 102.8%
Ca 97.3% 75.0%
By
the addition of 1
^M Cu, Zn,
Ni or Pb to 1 uM
Na3EDDS
or 1 uM
Fe(lll)EDDS
prior
to
converting
it to
Fe(lll)EDDS
we
proved
that none of these metals affect
the
peak
area of EDDS
(Table 2.2). Adding
1 mM Ca on the other hand reduced
the
peak height
when added to
Na3EDDS
prior
to its conversion into
Fe(lll)EDDS,
but not when added to
Fe(lll)EDDS.
This shows that Ca affects the conversion of
EDDS from the
Na-complex
into the
Fe(lll) complex.
It is not
possible
to remove
interfering
cations from the EDDS
containing solution, by
cation
exchange
as EDDS
is also retained on the cation
exchange
column. As Ca
only
affects the
peak height
and not
peak
area
however,
it should
only
affect the
sensitivity
and not the actual
measurement of EDDS.
12
acids
by
HPLC
8x104 i
6x1O4 H
"cd
|)4x104^
CO
2.3.2 EDTA and NTA
Fe(lll)NTA required
much
stronger
elution conditions than
Fe(lll)EDDS
due to its
neutrality. However,
we
found it can
easily
be
analysed
with
a
gradient
elution with a maximum
concentration of 50 mM NH.SO,.
4 4
Figure
2.4 shows a
chromatogram
for 10
uM
NTA with a ratio of
Fe(lll):
NTA of
10:1, using
a 200
\i\ injection.
The NTA
peak
is well
separated
from
the
reagent peak
and a
plot
of
peak
area versus concentration
gave
a
relationship
that was linear from 0.1 to 10 uM with a correlation coefficient r2
=
0.9948
(n=7).
When we used a 1:1 ratio of
Fe(l 11): NTA,
the
response
to the standard
was low and the
peaks
were broad.
However,
if the ratio was increased to 10:1
then the
peaks
were
sharper,
the
response higher
and a detection limit of 0.1 uM
was achieved. Unlike
Fe(lll)EDDS, increasing
the free
Fe(lll)
concentration did not
reduce
peak height
even at
high
free
Fe(lll)
concentrations
(Figure 2.5).
120
100
-
80
60
2x104
-
I
'
I
0 2 4 6 8
Time
(minutes)
Figure
2.4.
Chromatogram
of 10 uM NTA.
CD
.c
CD
CD
.
40 4
20
0
i
I
0 200 400 600 800 1000
Excess Fe
(u.M)
Figure
2.5. Reduction in
peak height
of 10 uM EDTA
(diamonds)
and NTA
(squares)
as a function of excess free
Fe(lll).
Peak
height
is
expressed
as a
percentage
of the
peak height
of 10 uM
Fe(lll)EDTAor Fe(lll)NTA
without excess
Fe(lll).
In our first
trials,
we ran
Fe(lll)EDTA
with eluents of the same
pH
as NTA and
EDDS. However the detection limit was not
sufficiently
low and in a bid to
improve
this,
we used
larger sample injections.
This in itself
brought
new
problems
due to an
13
Chapter
2
2x1 Obi
_
1x10bH
CD
g,8x104-
4x1O4 H
increased size of the
reagent peak
and its co-elution with the EDTA
peak.
In order to circumvent these
problems
it was
necessary
to use
eluents of a
higher pH,
so
increasing
the retention time of the EDTA
peak
and
preventing
it from
co-eluting
with
the
reagent peak
even when
large
sample injections
were used
(100 \i\).
Figure
2.6 shows a
chromatogram
for 10
uM Fe(lll)EDTA using
100
jLtl sample injection,
H20
and 5 mM
NH4S04
(pH 5.3)
as eluents. The
relationship
between
peak
area and EDTA concentration was linear from 0.25 to 1
|j,M
with a correlation coefficient r2
=
0.998
(n=4),
from 1 to 10 uM with a correlation
coefficient r2 =1
(n=5)
and from 10 to 100
|j,M
with a correlation coefficient r2
=
0.9999
(n=5).
Like
EDDS,
EDTA
peaks
decreased in
height
when
uncomplexed Fe(lll)
was
in the
system, although
this was a
very
small effect
(15%
reduction of
peak height
at an excess
Fe(lll)
concentration of 1000
|j,M) compared
to EDDS
(70% reduction)
(Figure 2.5).
2 4 6
Time
(minutes)
Figure
2.6.
Chromatogram
of 10 uM EDTA.
1x10J-i
_
8x10'
-
CD
2>6x102H
CO
4x102
1
I
'
I
'
I
'
I
0 12 3 4
Time
(minutes)
a)
4x10' -i
3x10J
-
c)2x103H
1x103

CD
c
a
CO
1
I
'
I
'
I
'
I
'
I
0 12 3 4 5
Time
(minutes)
Figure
2.7.
Chromatograms
of EDDS
a)
0.1
uM, b)
1 uM in
pure
water
(thin line)
and
drinking
water
(thick line)
2.3.3
Analyses
Drinking
water
samples spiked
with
Fe(lll)EDDS
where
analysed along
with
pure
water standards. The
drinking
water
samples
showed
peak
areas of ~50-60% and
peak heights
of 72-78% of the standards made in
pure
water
(Figure 2.7).
This shows
14
acids
by
HPLC
that if
drinking
water
(or
a natural water with a similar
matrix)
is to be
analysed
then
the standards should be made
up
in
drinking
water that is EDDS-free. Soil extract
(1
mM
P042")
was acidified
(pH 3)
and had
Fe(lll)CI3
added before
being spiked
with
Fe(lll)EDDS.
These
samples
showed that in the
range
0.1-100 uM
EDDS, although
the
peak
area was
comparable
to that of the
pure
water
standards,
the
peak height
was
only
half of
this,
thus
increasing
the detection limit in this matrix
by
a factor
of two. It is not clear whether this is due to excess Fe in the
samples,
as the soil
extract was made to be 139.8 uM
Fe(lll) prior
to
spiking
with
Fe(lll)EDDS
or due to
the ionic
strength
of the soil extract. In section 2.3.1 it was shown that
phosphate
at
this concentration increased
peak height slightly
not reduced
it,
so
phosphate
is not
responsible
for the reduction in
peak height
in this case.
2.4 Conclusions
As demonstrated
above,
the conversion of
EDDS,
NTA and EDTA
complexes
into the
respective Fe(lll)
forms
prior
to HPLC
separation
on a Dionex Ion PacAS11
column makes it
possible
to detect all three
compounds
to sub-micromolar levels
by
UV detection.
Acknowledgements
We thank Diederik Schowanek from Procter & Gamble for
providing S,S-EDDS.
This work was funded in
part by
the Federal Office for Education and Science within
COST Action 837 and the Swiss National Science Foundation in the framework of
the Swiss
Priority Program
Environment.
2.5 References
(1) Nowack,
B. Environmental
chemistry
of
agents.
2002, 36,
4009-4016.
T. C.
J.; Perkins,
C.
M.; Hartman,
F.
A.; Federle,
T.
W;
Larson,
R. J.
Biodegradation
of
[S,S], [R,R]
ethylene
(EDDS),
Chemosphere 1997,
34,
2375-2391.
(3) Takahashi, R.; Yamayoshi, K.; Fujimoto, N.; Suzuki,
M. Production of
(S,S)-
ethylenediallaine-N,N'-disuccinic
acid from
ethylenediamine
and fumaric acid
by
15
Chapter
2
bacteria. Biosci. Biotech. Biochem.
1999, 63,
1269-1273.
(4) Nishikiori, T.; Okuyama, A.; Naganawa, H.; Takita, T.; Hamada, M.;
H. Production
by actinomycetes
of
(S,S)-N,N'-
ethylenediamine
disuccinic
acid,
an inhibitor of
phospholipase-C.
J. Antibiot.
1984,
37,
426-427.
D.
Biodegradation
of
2001, 35,
1765-1770.
(6) Takahashi, R.; Fujimoto, N.; Suzuki, M.; Endo,
T.
Biodegradabilities
of
ethylenediamine-N,N'-disuccunic
acid
(EDDS)and
other
chelating agents.
Biosci.
Biotech. Biochem.
1997, 61,
1957-1959.
D.
soil,
sediment and
sewage sludge by
means of transition
metal chelate
[S,S]-EDDS.
J. Environ.
Eng. 2001, 127,
802-811.
(8) Tandy, S.; Bossart, K.; Mueller, R.; Ritschel, J.; Hauser, L; Schulin, R.;
Nowack,
B. Extraction of
heavy
metals from soils
agents.
2004, 38,
937-944.
(9) Grcman, H.; Vodnik, D.; Velikonja-Bolta, S.; Lestan,
D.
Ethylenediamine
dissuccinateas a new
chelateforenvironmentally
safe enhanced lead
phytoextraction.
J. Environ.Qual. 2003, 32,
500-506.
D. Influence of
and
chelate and
hydrogel
modified soil water
sortpion capacity
on Pb
phytoextracton
and
leaching.
Plant Soil
2003, 253,
403-411.
(11) Ammann,
A. A. Determination of
strong binding
chelators and their metal
complexes by anion-exchange chromatography
and
inductively coupled plasma
mass
spectrometry.
J.
Chromatogr.
A
2002, 947,
205-216.
(12) Knepper,
T P.
and
in the
aquatic
2003, 22,
708-724.
16
acids
by
HPLC
(13)
International Standards
Organization
Water
Quality
-
Determination of
six
complexing agents
-
Gas
chromatographic method, IS016588,
ISO
Geneva,
Switzerland,
2002
(14) Metsannne,
S
,
Tuhkanen, T, Aksela,
R
Photodegradation
of
ethylenedia
minetetraacetic acid
(EDTA)
and
ethylenediamine
disuccinic acid
(EDDS)
within
natural UV radiation
range Chemosphere 2001,45,949-955
(15) Geschke,
R
,Zehnnger,
M Anew method forthe determination of
complexing
agents
in river water
using
HPLC Fresen J Anal Chem
1997, 357,
773-776
(16) Buchberger, W, Haddad,
P
R, Alexander,
P W
Separation
of metal
complexes
of
ethylenediaminetetraacetic
acid in environmental water
samples by
ion
chromatography
and with UV and
Potentiometrie
detection J
Chromatogr
A
1991, 558,
181-186
(17) Deacon, M, Smyth,
M
R, Tuinstra,
L M T
Chromatographic separation
of metal chelates
present
in commercial fertilisers II
Development
of an
ion-pair
chromatographic separation
for the simultaneous determination of the
Fe(lll)
chelates of
EDTA, DPTA, HEEDTA, EDDHA,
EDDHMA and the
Cu(ll),
Zn
(II)
and
Mn(ll)
chelates of EDTA J
Chromatogr
A
1994, 659,
349-357
(18) Nowack,
B
,
Kan,
F G
,
Hilger,
S U
,
Sigg,
L Determination of dissolved
and adsorbed EDTA
species
in water and sediments
by
HPLC Anal Chem
1996,
68,
561-566
(19) Epstein,
A L
,
Gussman,
C D
,
Blaylock,
M J
,
Yermiyahu,
U
,
Huang,
J
W, Kapulnik, Y, Orser,
C S EDTA and Pb-EDTA accumulation in Brassica
juncea
grown
in Pb-amended soil Plant Soil
1999, 208,
87-94
(20) Bedsworth,
W
W, Sedlak,
S L HPLC Determination of
heavy
metal
complexes
of EDTA in the
presence
of
organic
matter
by
HPLC J
Chromatogr
A
2001, 905,
157-162
(21) Nowack,
B Determination of
phosphonates
in natural waters
by ion-pair
high-performance liquid chromatography
J
Chromatogr
A
1997, 773,
139-146
(22) Ammann,
A A
Speciation
of
heavy
metals in environmental water
by
ion
chromatography coupled
to ICP-MS Anal Bioanal Chem
2002, 372,
448-452
17
3 Determination of EDDS
by
HPLC after derivatization with FMOC
Susan
Tandy
Rainer
Schulin,
Marc J. F. Suter and Bernd Nowack
Journal of
Chromatography A,
in
press
Abstract
The
paper
describes a new HPLC method for the determ ination of
ethylenediam
ine
disuccinic acid
(EDDS).
EDDS is derivatized with FMOC
reagent
followed
by
HPLC
separation
on a
reversed-phase
column. The eluents consist of
phosphate
buffer
at
pH
6.8 and acetonitrile.
Separation
was carried out
using gradient
elution. The
FMOC-EDDS derivative is detected with a fluorescence detector with an excitation
wavelength
of 265 nm and an emission
wavelength
of 313 nm. The detection limit is
0.01 uM. The method is
applicable
to the determination of the
compound
in
water,
soil solution and
plant
material at trace levels.
19
Chapter
3
3.1 Introduction
SS-ethylenediamine-N,N'-disuccinic
acid
(EDDS)
is a
naturally occurring,
biodegradable complexing agent (1). Recently
it has been used
commercially
in
detergents (2, 3)
to
replace EDTA,
which is found to be too recalcitrant in the
environment
(4).
There has also been some interest in it for remediation of metal
contaminated
soils,
both
by
soil
washing
and chelant enhanced
phytoextraction (5-
8).
The colorimetric detection of EDDS has been described but this method has a
very high
detection limit
(0.1 mM) (1). Knepper (3)
mentions that the GC-based ISO
method for
complexing agents (9)
can be used for the
analysis
of EDDS but no
such use has been documented. In three
investigations
HPLC methods have been
used for the
analysis
of EDDS but the details
given
are not
comprehensive (10-12).
Although
the detection limits are not
given,
it seems from the data that
they
would be
relatively high.
These methods are based on the
photometric
detection of CuEDDS
or
Fe(lll)EDDS complexes.
A HPLC method based on the
photometric
detection of
Fe(lll)EDDS
has been described in detail but it is not suitable for use with
complex
matrices at trace levels of EDDS
(13).
One method has been described
using
IC-
ICP-MS for the detection of metal-EDDS
complexes (14).
This method is suitable for
trace
analysis
in natural waters but
requires
the use of an ICP-MS for detection and
is therefore not suitable for routine
analysis.
The aim of this work was to
develop
a HPLC based
analytical
method for EDDS
that is
applicable
to a broad
range
of
sample types
and has a detection limit suitable
for
analysis
at sub-m icromolar concentrations. To achieve this
goal
we chose a FMOC
(9-fluorenyl-methyl
chloroform
ate)
derivatization followed
by
HPLC
separation
and
fluorescence detection. Fluorescence detection has
advantages
over UV
detection,
in that it
gives
low detection limits and
high sensitivity
due to low interferences.
FMOC is a standard
reagent
for the determination of amino and imino acids
(15,
16).
FMOC has also been used for the derivatization of
aminophosphonates (17),
aminopolyphosphonates (18)
and for
glyphosate
and its
degradation product
aminomethylphosphonicacid (19, 20).
3.2
Experimental
3.2.1
Reagents
and chemicals
Water was obtained from a MilliQ
system (Millipore).
All chemicals were obtained
from Merck
(Switzerland)
and were
analytical grade
if not otherwise
specified.
All
solvents were LiChrosolv
grade. S,S-EDDS
was obtained from Procter & Gamble
20
Determination of EDDS
by
(Belgium).
A 1 M borate buffer was
prepared
from boric acid
adjusted
with sodium
hydroxide
to
pH
6.2. A 0.1 M EDTA buffer was
prepared by dissolving
Na2H2EDTA
in water and
adjusting
the
pH
with NaOH to 8 or 11.5. The metals were used in
their nitrate forms. The FMOC
reagent
was
prepared by dissolving
155
mg
of 9-
fluorenylmethyl
chloroformate
(FMOC-chloride, puriss; Fluka, Switzerland)
in 40 mL
acetone to
give
a concentration of 15 mM. It is
important
to
prepare
the FMOC
reagent freshly
each time it is used.
3.2.2 Derivatization of EDDS
0.2 ml EDTA buffer
(pH 11.5)
was added to 0.8 ml of
sample.
This was heated for 3
hours at 90C. After
cooling,
1.0 ml of the FMOC
reagent
was added and the
sample
was allowed to react for 30 minutes at room
temperature.
2 ml of dichloromethane
were then
added,
the
sample
was
shaken, centrifuged
and 50 ul of the
aqueous
layer injected
into the HPLC.
3.2.3 HPLC
AJasco
high-performance liquid Chromatograph (PU-980; Jasco, Japan) equipped
with a fluorescence detector
(821-FP
or
FP-2020), using
265 nm as excitation and
313 nm as emission
wavelength,
and an
autosampler
851 -AS were used. An
injection
volume of 50 ul was used. The HPLC
separations
were
performed
on a
Lichrospher
100
RP-18,
5 urn column
(Merck,
12.5 cm
length,
4 mm
diameter).
Some
preliminary
work was carried out on a PLRP-S
polymer
reversed
phase
C18 column
(Polymer
Laboratories,
15 cm
length,
4 mm
diameter).
The
aqueous
mobile
phase
consisted
of 0.05 M
NaH2P04/
0.05 M
Na2HP04
with a
pH
of 6.8. The
following gradient
elution was used: 0-6 minutes from 10% acetonitrile
-
20%,
6-8 minutes from 20
to 80%
acetonitrile,
8-11 minutes at
80%,
11-12 minutes from 80 to
10%,
then 8
minutes
re-equilibration
at 10%. The flow rate was 1 ml/min at room
temperature.
The eluents were
degassed
online
(Gastorr GT102,
FLOM
Corporation, Japan).
3.2.4 LC/MS
LC/MS was
performed
on an API4000 LC/MS/MS
(Applied Biosystems, Rotkreuz,
Switzerland) using electrospray
in the
negative
ion mode.
Chromatography
was
performed
with a 0.1 M
NH4-acetate
buffer at
pH 7, using
a
Lichrospher
100
RP-18,
5 urn 125 x 4 mm column
(Merck).
The
following gradient
elution with acetonitrile
was used: 0 -10 minutes from 0 to
80%,
1 minute at
80%,
then in 1 minute to 0% and
re-equilibration
for 6 minutes. 50 ul
samples (100
uM
FMOC-EDDS)
were
injected.
3.2.5 Water and soil solution
samples
Tap
water
samples
were taken from the non-chlorinated normal domestic
supply.
21
Chapter
3
Its calcium content was 2 mM and the
magnesium
content 0.7 mM.
Soil solution was collected
using
Rhizon Flex soil moisture
samplers (Rhizosphere
Research
Products, Wageningen,
The
Netherlands)
from two
top
soils. The first soil
was a
non-calcareous, acidic, sandy
loam with a
pH
of 5.5
(in
0.01 M
CaCI2),
the
second soil was a
non-calcareous,
near neutral loam with a
pH
of 6.6
(in
0.01 M
CaCI2).
Both soils were
agricultural
in
origin
and from Northern Switzerland.
3.2.6 Plant material extraction
The
plant
material
originated
from a
hydroponic experiment using
sunflowers to
investigate
the
uptake
of
heavy
metals and EDDS from nutrient solution
(21)
and
also a
pot experiment investigating
the use of EDDS for
enhancing phytoremediation.
Dried
(40C) ground plant
material from both roots and shoots were extracted in
pure
water
(10 mg/10 ml) by
sonication with a
micro-tip
sonic
probe
for one minute.
The
samples
were
kept
on ice
during
sonication to
prevent heating. They
were then
centrifuged
and filtered
(0.45 urn)
before derivatization.
Xylem sap samples
were
collected
by decapitating
the
plants
and
collecting
the
xylem sap
for 2-3 hours. The
samples
were diluted
immediately
before derivatization
by adding
800 ul of
pure
water
(sample weight
4-140
ug).
3.3 Results and Discussion
3.3.1 Derivatization of EDDS
The derivatization of amino acids
by
FMOC with borate buffer at
pH
8 and at
room
temperature
is
complete
within 30 seconds
(15, 16).
The derivatization of
EDDS, however,
is much slower. A reaction time of less than one minute at room
temperature
is not sufficient for a
complete
derivatization of EDDS. After
heating
for 10 minutes at 60C a maximal conversion to the derivative was achieved.
However, longer heating
times reduced the
peak
area
again.
We found that at room
temperature
maximal derivatization of EDDS with FMOC occurred at a reaction
time of 30
minutes, yielding
the same maximal
peak
area as
heating
for 10 minutes
at 60C
(Figure 3.1a).
The effect of
pH
on derivatization at room
temperature
is
shown in
Figure
3.1 b. It can be seen that the
peak
area increased
exponentially
with
increasing pH.
An EDTA buffer with
pH
11.5 was therefore chosen for the
analysis
of
all natural
samples.
Some method
development
was also carried out
using
an EDTA
buffer with
pH
8 or a borate buffer with
pH
8.
Metals
present
in the
sample may
inhibit the derivatization of EDDS.
Attempts
to remove the cations
by passing
the
sample through
a cation
exchange
column
in the H+ form were not successful because EDDS was also retained. Addition of
22
by
1.2x10
S
8x10
2x10
30 40 60 70
Reaction Time
(min)
10 11
pH
Figure
3.1. Influence of reaction time
(a)
and EDTA-buffer
pH (b)
on the derivati
zation of 1 uM EDDS with FMOC at room
temperature.
the
chelating agent
EDTA in excess of all metals
(10 mM)
resulted in a maximal
conversion of EDDS to the FMOC derivative in most cases. Concentrations of Ca2+
up
to 1 mM and of
N03" up
to 0.1 mM did not affect the derivatization of 1 uM
EDDS
(Table 3.1).
The addition of 10 uM
Zn(ll), Cu(ll), Pb(ll),
as nitrates was also
investigated
but showed no effect. 10 uM
Fe(lll)
reduced the EDDS
peak
area
by
14%,
while 10 uM Ni reduced it
by
91%. In further tests with concentrations of
Fe(lll)
and Ni
up
to 100
uM,
Ni
produced
the same reduction whatever its
concentration,
while
Fe(lll)'s
effect increased with
increasing
concentrations
(Table 3.1). Heating
1
uM EDDS and 10 uM Ni with EDTA buffer at
pH
8 for 3 hours at 90C before
adding
the FMOC
reagent produced
the same
signal
as 1 uM EDDS in the absence of
23
Chapter
3
Table 3.1. Influence of metals on EDDS derivatization
by
FMOC. Conditions: 1 uM
EDDS,
addition of EDTA-buffer
(pH 8),
30 minutes reaction with FMOC at room
temperature.
Conditions
Relative Peak Area
Unheated Heated1
free 1.00 1.00
1 mM Ca2+ 0.96
100 uM
N03-
1.00
10 uM
Cu, Zn,
Pb 0.98-1.00
10uMFe 0.86
10 uM Ni 0.09 0.99
lOOuMFe 0.69
100 uM Ni 0.09
1
heated for 3 hours at 90C after the addition of EDTA buffer and before FMOC
addition
Ni with and without
heating.
The same
experiments
carried out
using
EDTA buffer
at
pH
11.5
produced
enhanced
signals,
due to a more efficient derivatization at
higher pH.
NiEDTA is a
complex
that is known to react
very slowly. Heating greatly
increases the reaction rate
(22).
As EDDS is an isomer of
EDTA,
the same can be
assumed for NiEDDS. EDDS
complexes
must
yield
their metals to
EDTA,
in order
for the free EDDS to be derivatized
by
the FMOC
reagent. Heating
accelerates this
rate
limiting step.
Repeated
measurement of a derivatized
sample
indicated that the derivative was
stable for at least 18
days
when stored at 4C in the dark.
3.3.2 HPLC
separation
Figure
3.2 shows a
chromatogram
of 10 uM EDDS in
pure
water derivatized in
EDTA-buffer. The EDDS
peak
is well
separated
from the
reagent peak (elution
time
9
minutes)
and additional
peaks originating
from
impurities
in the EDTA
(elution
time between 7 and 9
minutes).
Some batches of EDTA also
gave
a small
peak
originating
from
impurities,
or
degradation products emerging
over
time,
that
elutes at the same time as EDDS. Careful
testing
of the
purity
of the used EDTA
batch is therefore
necessary
in order to ensure that it is free of this interference. A
solvent blank
sample
should also be derivatized with
every
calibration to check for
degradation
of the EDTA buffer over time.
For standards made in
pure
water the
relationship
between the
peak
area and
24
by
_
1x10
CO
g>
8x105
CO
g
6x10
c
CD =
o
4x106
CD
2x10-|
i 1 1 r
8 10 12 14
Time
(minutes)
Figure
3.2.
Chromatogram
of 10 uM EDDS in
pure
water.
the concentration of EDDS was linear from 0.01 to 10 uM
(fluorescence
detector
gain *10)
with a correlation coefficient r2 of 0.9952
(n=10).
The
relationship
was also
linear from 1 to 30 uM
(gain *1)
with a correlation coefficient r2 of 0.9910
(n=6).
The
detection limit is 0.01 uM
(S/N
=
3).
3.3.3 Identification
by
LC/MS
Figure
3.3 shows a
chromatogram
for EDDS derivatized in borate buffer without
addition of EDTA
(PLRP-S polymer
RP-C18
column).
Borate instead of EDTA
CO
I
9X105-
CD
1
6x105-
1
2
li
o
CO
CD c
fc
3x105-
3
LL
0-
c
-
u.
I I I I
) 2 4 6 8 1
Time
(rr
lini
jtes)
0
Figure
3.3.
Chromatogram
of 1 uM EDDS derivatized with FMOC in borate buffer
at
pH 7.7,
PLRP-S
polymer
RP-C18 column
(see
section 3.3.3. for
details).
25
Chapter
3
buffer was used in LC/MS
analysis
because the derivatization in EDTA buffer
gives
additional
overlapping peaks
which obscure the EDDS
signals (peaks
2 to
4)
and
thus
complicate
structure
assignment.
The EDDS standard used
yielded
4 distinct
peaks.
The first
peak
consists of a
compound
with a molecular ion of m/z 513 which
corresponds
to a
singly
derivatized
FMOC-EDDS
(see
Scheme 3.1
).
MS/MS of this ion
gave fragments
at m/z 495
(-H20),
397
(loss
of maleic
acid),
317
(loss
of
fluorenyl-methanol),
291
(EDDS;
loss
of
FMOC),
273
(291
minus water and
cyclization),
229
(elimination
of
C02
from
273,
one of three
possible
structures is
shown),
157
(loss
of maleic acid from
273).
-FMOC
r'Y*
x
291
I;
Y
COOH
229
NH
HOCK
'
>"-N^
COOH
273
HN J
495
Scheme 3.1. MS/MS
fragmentation pattern
of the
negatively charged
molecular
ion m/z 513 of
peak
1 in
Figure
3.3.
The m/z of the molecular ion of
peak
2 was
495, indicating
that EDDS had
undergone cyclization
before or
during derivatization,
as described
by Kolleganov
et al.
(23).
Elimination of FMOC from this ion
yields
m/z
273,
followed
by
loss of
C02
(one
of three
possible
structures is
shown)
or maleic acid
(see
Scheme
3.2).
Peak 3
corresponds
to a
deprotonated
molecular ion with a m/z of 354. Its
fragmentation pattern
indicates
FMOC-aspartate.
The
S,S'-EDDS
used had been
synthesized
from
L-aspartic
acid and
1,2-dibromoethane.
This
peak
thus was
likely
an
impurity
in the EDDS standard. The
major
masses were m/z 158
(elimination
of
fluorenyl-methanol)
and 165
(formation
of the
fluorenyl anion).
Scheme 3.3 shows
26
by
^Y
r
SN COOH
H
291
^NH
?^y
^
EDDS
COOH
273
495
-FMOC
\
-co2
COOH
229
r
:-^Y
U
COOH
273
I
Y^NH
HN^I
157
Scheme 3.2.
Fragmentation pattern
of the molecular ion m/z 495 of
peak
2 in
Figure
3.3.
H(XX,^N^1N112
+
TOO
Aspartic
acid "9
FMOC
|j~a
o
-FMOC-OH
jj
*"~
O NH
M
O^
^^
coo
h
(Anii
HOOC
vAa
354
0*0
165
Scheme 3.3.
Fragmentation pattern
of molecular ion m/z 354 of
peak
3 in
Figure
3.3.
the
proposed fragmentation pattern.
Peak 4 had a molecular ion of m/z
735,
which
corresponds
to the derivatization of
both imine
groups
of EDDS. The
fragmentation yielded
m/z 513
(elimination
of one
FMOC)
and further all the masses observed in Peak 1
(Scheme 3.1).
27
Chapter
3
Based on the data
presented,
the first
peak
observed in the
chromatogram
was
used for
quantifying EDDS,
since it
clearly corresponds
to the derivatized
EDDS,
while the other
peaks
could be
assigned
to reaction
side-products.
3.3.4
Analyses
Both
tap
water and two
types
of soil solution were
spiked
with EDDS over
a concentration
range
of 0.01
-
10 uM and the results
compared
to
pure
water
standards.
Tap
water was found to reduce the
peak
area
by
5%. The ten times
diluted soil solutions both
gave
a 7% reduction in
peak
area and the undiluted soil
solutions a 9-10% reduction. For soil solution or other natural water
samples
we
have therefore
always prepared
the standards in the same EDDS-free matrix as
the
samples.
Where an EDDS-free matrix is not
available,
standard addition has to
be used for the
samples.
In addition real soil solution
samples
from a soil
washing
experiment
with EDDS were
successfully quantified (undiluted
and diluted 10 and
50
times). Figure
3.4 shows an undiluted soil solution
sample containing
0.75 uM of
EDDS.
CO
CO
CD
O
aj 5x104
o
CO
l_
o
u-
0
012345678
Time
(minutes)
Figure
3.4.
Chromatogram
of an undiluted soil solution
sample containing
0.75 uM
EDDS.
There is
only
one other detailed
report
on the
analysis
of EDDS in natural waters
[14].
This method is based on the ion
chromatographic separation
of
Fe(lll)EDDS
and UV-detection. In distilled water the detection limits of both methods are the
same. The
Fe(lll)EDDS method, however,
suffers from matrix effects
by major
ions
(e.g. chloride, sulfate, phosphate).
The observed
peak broadening
and
peak
area reduction results in a reduced
sensitivity
in natural waters which limits the
applicability
of the method to well defined matrices.
28
by
CO
O)
CO
CD
O
CD
O
CO
CD
i_
O
"3
CO
D)
CO
CD
O
CD
O
CO
CD
i_
O
3
3x10
2x104-
1 x10
8x10-
6x10-
4x10-
2x10-
0-
0
Time
(minutes)
^mple
EDDS
EDDS
spike Kr
2
b)
8
Time
(minutes)
Figure
3.5.
Chromatogram
of
(a)
an extract of a low concentration shoot
sample
(0.19
uM
EDDS) spiked
with 0.2 uM EDDS and
(b)
an extract of a
high
concentra
tion root
sample (6.43
uM
EDDS)
with
spike (2.5
uM
EDDS).
Using plants grown
in the
presence
of EDDS
(see
section
3.2.6)
it was found that
extracts ofshoots and roots could be
successfully analysed
after FMOC-derivatization
(Figure 3.5). Sub-samples
were also
spiked
with EDDS
prior
to derivatization in order
to
help identify
the EDDS
peak among
the
plant
matrix
peaks
at low concentrations.
Figure
3.5a shows a
chromatogram
of a shoot extract with an EDDS concentration
of 0.19 uM from the
hydroponics experiment
and also the
sample spiked
with 0.2
uM EDDS which
gave
a
recovery
of 91%. The actual shoot concentration was 183
umol/kg. Figure
3.5b shows a root extract from a
pot experiment
where much
higher
concentrations of EDDS were found. The extract concentration was 6.43 uM EDDS
29
Chapter
3
sample
CO
w 4x104-
EDDS
spike
EDDS
C
o
CD
$
2x104-
Matrix
peak
! I
o
/
^W
J_
^
/\ _-/
0-
i i i
0 2 4 6
Time
(minutes)
8
Figure
3.6.
Chromatogram
of
plant xylem sap
with
spike (0.5
uM
EDDS).
and the
spike
concentration was 2.5 uM with a
recovery
of 97%. The actual root
concentration was 4791
umol/kg.
Plant
xylem sap samples
were also
analysed.
Some interference from the
matrix,
which
produced
a
partially co-eluting peak
with the EDDS
peak,
could not
be overcome
by changing
the
gradient.
The
spike recovery
was between 30 and
90 % for these
samples.
Small
sample
volumes
(few ul)
in some cases
may
have
led to inaccuracies due to the
large
dilution factors
required
to be able to
analyse
the
samples. Figure
3.6 shows a
sample
and
corresponding spiked sample
with a
concentration of about 0.23 uM EDDS and a
spike
of 0.5 uM EDDS. In this case the
spike recovery
was 47%.
Xylem sap
and
plant
material
analysis
was not
possible using
the
Fe(lll)EDDS
method
[14]
due to
co-eluting compounds
and a
large
reduction in
peak
area and
excessive
peak broadening.
3.4 Conclusions
The results show that the
chelating agent
EDDS can be derivatized
using
FMOC
to
give
derivatives suitable for
separation by reversed-phase
HPLC. The method
is
applicable
to the determination of the
compound
in
water,
soil solution and
plant
30
by
Acknowledgements
We thank Ren
Schnenberger
for the
help
with the HPLC-MS measurements
and Diederik Schowanek from Procter & Gamble for
providing S,S-EDDS.
This work
was funded in
part by
the Federal Office for Education and Science within COST
Action 837 and the Swiss National Science Foundation in the framework of the
Swiss
Priority Program
Environment.
3.5 References
D.
Biodegradation
of
2001, 35,
1765-1770.
(2) Jaworska,
J.
T. C. J. Environmental risk assessment
for trisodium
[SS]-ethylene
diamine
disuccinate,
a
biodegradable
chelator used in
3597-3625.
(3) Knepper,
T P.
and
in the
aquatic
2003, 22,
708-724.
(4) Nowack,
B. Environmental
chemistry
of
agents.
2002, 36,
4009-4016.
Nowack,
B. Extraction of
heavy
metals from soils
agents.
2004, 38,
937-944.
D.
Ethylenediamine
safe enhanced lead
phytoextraction.
J. Environ. Qual. 2003, 32,
500-506.
D. Influence of
and
chelate and
hydrogel
modified soil water
sorption capacity
on Pb
phytoextracton
and
leaching.
Plant Soil
2003, 253,
403-411.
D.
soil,
sediment and
sewage sludge by
means of transition
31
Chapter
3
metal chelate
[S,S]-EDDS.
J. Environ.
Eng. 2001, 127,
802-811.
(9)
International Standards
Organization.
Water
Quality
-
Determination of
six
complexing agents
-
Gas
chromatographic method, IS016588,
ISO:
Geneva,
Switzerland,
2002.
(10) Metsrinne, S.; Tuhkanen, T.; Aksela,
R.
Photodegradation
of
ethylenedia
minetetraacetic acid
(EDTA)
and
ethylenediamine
disuccinic acid
(EDDS)
within
natural UV radiation
range. Chemosphere 2001, 45,
949-955.
T.
Biodegradabilities
of
ethylenediamine-N,
N'-disuccunic acid
(EDDS)
and other
chelating agents.
Biosci.
Biotech. Biochem.
1997, 61,
1957-1959.
(12) Takahashi, R.; Yamayoshi, K.; Fujimoto, N.; Suzuki,
M. Production of
(S,S)-
ethylenediallaine-N,N
'
-disuccinic acid from
ethylenediamine
and fumaric acid
by
bacteria. Biosci. Biotech. Biochem.
1999, 63,
1269-1273.
(13) Tandy, S.,
Ph.D.
Dissertation;
The Use of EDDS in Soil
Washing
and
Phytoremediation.
Swiss Federal Institute of
Techology, Zrich,
Switzerland. Diss.
ETH Nr. 16039. 2005.
(14) Ammann,
A. A. Determination of
strong binding
chelators and their metal
complexes by anion-exchange chromatography
and
inductively coupled plasma
mass
spectrometry.
J.
Chromatogr.
A
2002, 947,
205-216.
(15) Einarsson, S.; Josefsson, B.; Lagerkvist,
S. Determination of amino-acids
with
9-fluorenylmethyl
chloroformate and
reversed-phase high-performance liquid-
chromatography.
J.
Chromatogr. 1983, 282,
609-618.
(16) Gustavsson, B.; Betner,
I.
Fully
automated amino-acid
analysis
for
protein
and
peptide hydrolysates by precolumn
derivatization with
9-fluorenyl
methylchloroformate
and 1-aminoadamantane. J.
Chromatogr. 1990, 507,
67-77.
(17) Huber,
J.
W; Calabrese,
K. L. Derivatization of
aminophosphonic
acids for
HPLC
analysis.
J.
Liquid Chromatogr. 1985, 8,
1989-2001.
(18) Nowack,
B. Determination of
phosphonic
acid breakdown
products by high
performance liquid chromatography
after derivatization. J.
Chromatogr.
A
2002,
32
by
942,
185-190.
(19) Glass,
R. L.
Liquid-chromatographic
determination of
glyphosate
in fortified
soil and water
samples.
Journal of
Agricultural
and Food
Chemistry 1983, 31,
280-
283.
(20) Sancho,
J.
V; Hernandez, F.; Lopez,
F.
J.; Hogendoorn,
E.
A.; Dijkman, E.;
vanZoonen,
P.
Rapid
determination of
glufosinate, glyphosate
and
aminomethyl
phosphonic
acid in environmental water
samples using precolumn fluorogenic
labeling
and
coupled-column liquid chromatography.
J.
Chromatogr.
A
1996,
737.
(21 ) Wenger, K.; Tandy, S.; Nowack,
B. Effects of
chelating agents
on trace metal
speciation
and
bioavailability
In
Biogeochemistry
of
chelating agents; Nowack, B.,
Vanbriesen, J., Eds.;
American Chemical
Society,
ACS
Symposium Series, 2005,
Vol.
910, pp
204-224.
(22) Nowack, B.; Kari,
F.
G.; Hilger,
S.
U.; Sigg,
L. Determination of dissolved
and adsorbed EDTA
species
in water and sediments
by
HPLC. Anal. Chem.
1996,
68,
561-566.
(23) Kolleganov, M.; Kolleganova,
I.
G.; Mitrofanova,
N.
D.; Martynenko,
L.
I.; Nazarov,
P.
P.; Spitsyn,
V I. Influence of
cyclization
of
N,N'-ethylenediamine
disuccinic acid on its
complex forming properties.
Bull. Acad. Sei. USSR Div. Chem
Sei.
1983, 32,
1167-1175.
33
4 Extraction of
heavy
metals from soils
using biodegradable
chelating agents
Susan
Tandy,
Karin
Bossart,
Roland
Mueller,
Jens
Ritschel,
Lukas
Hauser,
Rainer
Schulin and Bernd Nowack
Environmental Science &
Technology
38
(2004),
937-944
Abstract
Metal
pollution
of soils is
widespread
across the
globe,
and the clean
up
of these
soils is a difficult task. One
possible
remediation
techniques
is ex-situ soil
washing
using chelating agents. Ethylenediaminetetraacetic
acid
(EDTA)
is a
very
effective
chelating agent
for this
purpose,
but has the
disadvantage
that it is
quite persistent
in
the environment due to its low
biodegradability.
The aim of ourwork was to
investigate
the
biodegradable chelating agents [S,S]-ethylenediaminedisuccinic
acid
(EDDS),
iminodisuccinic acid
(IDSA), methylglycine
diacetic acid
(MGDA)
and nitrilotriacetic
acid
(NTA)
as
potential
alternatives and
compare
them with EDTA for effectiveness.
Kinetic
experiments
showed for all metals and soils that 24 hours was the
optimum
extraction time.
Longer
times
only gave
minor additional benefits for
heavy
metal
extraction but an unwanted increase in iron mobilization. For Cu at
pH
7 the order of
the extraction
efficiency
for
equimolar
ratios of
chelating agent
to metal was EDDS
> NTA >
IDSA
>
MGDA
> EDTA and for Zn it was NTA >
EDDS
> EDTA >
MGDA
>
IDSA. The
comparatively
low
efficiency
competition
between
the
heavy
metals and co-extracted Ca. For Pb the order of extraction was EDTA
> NTA >
EDDS,
due to the much
of Pb
by
EDTA
compared
to EDDS. At
higher
concentration of
complexing agent
less difference between
the
agents
was found and less
pH dependence.
There was an increase in
heavy
metal extraction with
decreasing pH
but this was offset
by
an increase in Ca and Fe
extraction. In
sequential
extractions EDDS extracted metals almost
exclusively
from
the
exchangeable,
mobile and Mn-oxide fractions. We conclude that the extraction
with EDDS at
pH
7 showed the best
compromise
between extraction
efficiency
for
Cu,
Zn and Pb and loss of Ca and Fe from the soil.
35
Chapter
4
4.1 Introduction
Metal
pollution
of soils is
widespread
across the
globe,
and the clean
up
of these
soils is a difficult task. Various in-situ and ex-situ remediation
techniques
have been
employed, e.g. solidification, stabilization, flotation,
soil
washing, electroremediation,
bioleaching
and
phytoremediation (1 ).
Soil
washing
includes the
physical separation
of the
clay
and silt fraction
containing
the
majority
of the metals due to their
high
specific adsorption capacity,
as well as the extraction of metals
by
mineral acids
or
chelating agents.
A
particularly promising technique
is ex-situ soil
washing
with
chelating agents (2).
The soil is removed from the
site,
treated in a closed reactor
with the
chelating agent
and returned to the site after
separation
of the extraction
solution that now contains the extracted
heavy
metals. The
advantage
of the method
is the
high potential
extraction
efficiency
and the
specificity
for
heavy
metals. In
order to
keep
treatment costs low it is
necessary
to achieve a
clean-up
so that the
soil can be re-used and it should be
possible
to recover and re-use the
chelating
agent
for further extraction
cycles.
There are
many
factors to consider when
comparing
studies of
chelating agent
assisted soil
washing
in order to decide whether the
chelating agent
is suitable for
field scale decontamination of
polluted
sites. Ratio of
chelating agent
to toxic
metals,
pH, quantity
of
major
cations extracted and source of contamination
(artificial
or
anthropogenic)
are the most
important
ones. Most studies of chelant-assisted soil
washing
have found that a ratio > 1 between chelant and toxic metal is
required
to
give good
toxic metal extraction
(3-7).
When the ratio is increased so does the
extracted fraction of metal until the extraction
efficiency
levels off
(4-7). Many
studies
however do not
give
the chelant: metal ratio used. In some cases this ratio can be
calculated from the concentration and volume of
chelating agent
and the mass of
soil
(8-14),
but in others even this information is
missing.
One reason
why
an excess
of
chelating agent
is needed is that
major
cations in the soil such as
Mn, Mg,
Fe
and Ca
along
with toxic metals
present
in smaller amounts
compete
with the metals
being studied,
for the
chelating agent
and are extracted too
(7, 14, 15).
This is both
important
from the
point
of view that it reduces the extraction of the
target
metals
and that it extracts
major
cations which are
important
as
plant
nutrients and for
maintaining
soil structure in future re-use of the decontaminated soil.
Another crucial factor to be considered in
comparing
studies on
chelating agent
extraction is the
pH
of the extraction solution. While extraction was
investigated
at various
pH
values in some studies
(3, 5, 7, 14),
some
only
stated the
pH
of the
solution
(10, 11, 13),
while others did not consider
pH
at all
(6, 8, 9, 12).
In
general,
the lower the
pH
of the
chelating agent solution,
the
greater
the extraction
efficiency
of the toxic metals.
36
Extraction of
heavy
metals from soils
using biodegradable chelating agents
The source of the metal and its form can also affect the extraction
efficiency
especially
for Pb
(16)
Furthermore it has been found that
significantly larger
extraction efficiencies are obtained when
chelating agents
are
applied
to
artificially
contaminated soils than to soils with field contamination
(7,17)
Added soluble metals
quickly
transfer to the
exchangeable
fraction and more
slowly
but still within the first
hour to the carbonate and
organic
matter fractions
(18)
Inclusion in
precipitates
and diffusion into
micropores
takes
place
at much slower rates
(19)
In
recently
contaminated soils the metals are
generally
in a much more accessible form than
in soils that have been contaminated
years ago
This
important
factor needs to be
considered when
chelating agents
are evaluated for field use
Many
studies have
compared EDTAtootherchelating agents,
acids and surfactants
and found it better suited than or
equal
to its
competitors
for the extraction of toxic
metals from soils
(3-5, 9, 11)
Between 45 and 100%
Pb,
54
-
100% Zn and 47
-
98% Cu were extracted from various contaminated soils
by
EDTA
(3-11, 14)
The
maximum extraction efficiencies of other
chelating agents
for Pb were found to be the
following
63% for ADA
(N-2(acetamido)iminodiacetic acid),
60% for
NTA,
58% for
PDA(pyndine-2,6,dicarboxyhc acid),
11% for
DPTA(diethylenetnamine pentaacetic
acid)
and 0% for citric acid
(3-5, 9)
For Zn maximum extraction efficiencies were
reported
as 41% with
DPTA,
12% with citric acid
(8, 9, 14),
and for Cu
they
were
32% with DPTA and 11% with citric acid
(9, 12, 14)
So far EDTA has been the most
widely
used
chelating agent
for these
processes
due to its
high
extraction
efficiency
It is
very
effective in
mobilizing metals,
but
unfortunately,
due to its low
biodegradabihty (20),
it is also
very persistent
in the
environment This can cause a rather
high
risk of metal
leaching
to the
groundwater
(21) NTA,
which is
easily biodegradable,
is under
scrutiny
due to
possible
adverse
health effects
(22) Recently
the
easily biodegradable chelating agent
SS-EDDS
(S,S-ethylenediaminedisuccinic acid) (23)
has been
proposed
as a safe and
environmentally benign replacement
for EDTA in soil
washing (24)
and for chelant-
enhanced
phytoremediation (25)
The aim of this
study
was to
investigate
SS-EDDS and other
chelating agents
that can be
potentially
used in soil
washing
and that are less
persistent
in the
environment than EDTA and to
compare
them with
commonly
used
chelating
agents
for effectiveness Extraction
experiments
were carried out under controlled
conditions
(pH
and chelant toxic metal
ratio) using
soil from contaminated sites
requiring clean-up
or other treatment
according
to Swiss
legislation
37
Chapter
4
4.2 Materials and Methods
4.2.1 Soils
The soil materials used in this
study
were taken from two
polluted
sites in
Switzerland. Two soils were taken from an area in Dornach which has been
heavily
contaminated with
Cu,
Zn and Cd for about a
century by particulate
emissions from
an
adjacent
brass smelter
(26).
The two soils differed in their carbonate content: one
was a Calcaric
Regosol (Dornach 1)
the other a non-calcareous
Regosol (Dornach
2) (27).
Both soils had
very
similar total metal contents. The third soil
(Rafz)
a
Haplic
Luvisol, originated
from an
agricultural
field in Northern
Switzerland,
which had been
contaminated with
Zn, Pb,
and Cd due to
sewage sludge applications (27).
The soil
samples
were taken from the
top
20
cm,
dried at 40 C and sieved to <
2mm. Soil
properties
are
displayed
in Table 4.1. The two Dornach soils contained
almost the same Cu and Zn concentrations but differed in
pH according
to their
difference in carbonate content. The molar sum of the
heavy
metals
Zn, Cu, Pb,
Cd and Ni in all three soils was between 18.6 and 20.1 mmol
kg-1.
The total metal
content of the three soils was therefore almost the same.
4.2.2
Chelating agents
EDTA was used as a reference
compound
to which the other
chelating agents
were
compared.
It was used as the
Na2-EDTAsalt
(Merck).
NTAwas used as
Na3NTA
(Fluka). S,S-ethylenediaminedisuccinic
acid
(S,S-EDDS,
in this article referred to as
EDDS),
was obtained from Procter & Gamble. EDDS is used as a
replacement
for
EDTA in
laundry detergents (23).
The
S,S-isomer
is
easily biodegradable
while the
R,R-
and
S,R-isomers
are not
(28).
We therefore used the
pure S,S'
enantiomer.
Iminodisuccinic acid
(IDSA)
was obtained from
Bayer, Leverkusen, Germany.
Manufactured in a
green process avoiding
toxic chemicals and traded under the
name
Baypure
CX
100,
it is used in
detergents (29). Methylglycinediacetic
acid
(MGDA)
was obtained from
BASF, Ludwigshafen, Germany. MGDA,
which is traded
under the name Trilon
M,
is
easily biodegradable (30).
The structures of the
chelating
agents
used in this
study
are shown in Table 4.2.
4.2.3 Metal extractions
Chelating agents
were
applied
in two concentrations: 0.4 mM and 4 mM. Metal
extractions were carried out in batch
experiments
at a solid: solution ratio of 1:50.
Thus,
two ratios between chelant and the sum of the total concentrations of
Cu,
Zn, Pb,
and Ni in the soil were achieved:
equimolar (20
umol
g_1 soil)
and 10 times
of that
(10:1) (200
umol
g_1 soil).
For kinetic
experiments
8
g
soil were
suspended
38
Extraction of
heavy
metals from soils
to
'-4'
CD
Q.
O
O
C/D
1-
n
.J
to
CD (0
4 ^
O
S
CD
S
3
o
fi
to
o
c
to
(/)
o
O
O
to
O
o
O)
to _
o
E
E
CO
ai
CD
in
CO
m
CD
CM
CD
CD
CM
CD
CO
CO
CO
CD
CO
CD
CO
m
CD
en
ai
m
CD
m
a>
CM
CM
o
CM
CO
CM
CO
CM
CD
CO
CD
a>
CD
m
o
CO
CD
r^ CO ^
m r^ co
CD
CO
CD
o CM m
r^ CD m
n
l
CO CO
CO
c
1 a:
o o
Q Q
39
Chapter
4
Table 4.2.
Abbreviation,
structures and
log
K values for metal
complexation
of the
used
chelating agents1)
abbreviation structure
logK
Ca
logK
Cu
logK
Zn
logK
Fe(lll)
LogK
Pb
EDTA
HOOCl
^"^ ^^
N^ ^COOH
^COOH
10.65 18.78 16.5 25.1 17.9
NTA
HOOC
HOOCv^ ^N\ ^COOH
6.3 12.7 10.45 15.9 11.34
EDDS
COOH
HOOC Y^
^^
N COOH
H
COOH
4.58 18.4 13.4 22.0 12.7
IDSA
H
HOOC^ /N\ ^COOH
HOOC COOH
4.3 12.7 9.88 15.22> 9.75
MGDA
HOOL\
HOOC.
_/N\ ^-CK,
COOH
6.97 13.88 10.98 16.53> 12.1
1)
log
K values are for 0.1 M ionic
strength
from ref.
(34)
2)A.
Mitschker, BASF, personal
communication.
3>
from ref.
(30).
in 400 ml 0.01 M
NaN03
as a
background electrolyte.
In the case of the non-
calcareous soils
(soil
Dornach 2 and
Rafz)
the
pH
was
adjusted
with 1 M
HN03
or 1 M NaOH two
days
before the addition of
chelating agent.
With the calcareous
soil Dornach 1 the
experiments
were carried out at the
pH
that was established in
suspension (pH
7.5
-
8.0)
without
any adjustment.
The
suspensions
were shaken
at room
temperature
and
sampled
after various time intervals.
Samples
used for
metal
analysis
were
centrifuged
for 15 minutes at 3000
rpm
and
passed through
0.45 urn filters
(Sartorius). Samples
used for iron
analysis
were
additionally
filtered
through
0.05 urn filters. The
pH
was measured in
suspension
and was
readjusted
if
necessary.
To inhibit microbial
degradation
the biocide sodium azide
(NaN3)
was
added in some
experiments (concentration
1
g I"1).
Variation of
pH
was
only
studied with the non-calcareous soils. The
experiments
were carried out in the same
way
as described
above, except
that for each batch
40
Extraction of
heavy
metals from soils
only
0.8
g
soil was
suspended
in 40 ml solution and the reaction time was fixed at
24 h.
Chelating agents
were added at two concentrations: 0.4 mM and 4 mM.
Subsamples
of 2
g
of soil from the treatments with 20 umol
g_1
EDDS for 24
hours at
pH
7 were also
analyzed
in
duplicate by sequential
extraction
following
the scheme of Zeien and Brummer before and after the treatment
(31, 32).
The
operational
definition of the fractions obtained with this scheme and the
compositions
of the extraction solutions are
given
in Table 4.3. All
suspensions
were
centrifuged
at 2500
rpm
for 10 minutes and
passed through
Schleicher & Schll 790 1/2
paper
filters before metal
analysis.
Table 4.3. Chemical
interpretation
and
operational
definition of the fractions within
the
sequential
extraction scheme
(for
2
g
of soil and 50 ml
solution) (31, 32).
Fraction chemical
interpretation
extraction conditions duration
F1 mobile 1 M
NH4N03
24 hours
F2
easily
mobilizable
1MNH4-acetate(pH6)
24 hours
F3 bound to Mn oxides
0.1
MNH2OH-HCI
+ 1
MNH4-
acetate
(pH 6)
30 min.
F4
bound to
organic
matter
0.025 M
NH4EDTA(pH4.6)
90 min.
F5
bound to
amorphous
Fe
oxides
0.2 M
NH4-oxalate
(pH 3.25)
4 hours in the
dark
F6
bound to
crystalline
0.1 M ascorbic acid + 0.2 M 30 min. at
Fe oxides
NH4-oxalate
(pH 3.25)
96 C
F7 residual fraction
X-ray
fluorescence
analysis
To evaluate the
reproducibility
of the results
triplicate
extractions with and without
EDDS have been
performed
with all three soils at
pH
4 and 7. The
average
standard
deviation for Cu was
4.9%,
for Pb
3.2%,
for Zn 5.4% for the EDDS extractions
and 8.3% without
EDDS,
for Fe 4.0% and for Ca 9.9%. We can therefore
assign
an
average
error of about 5 % to all data
except
for Ca where the error is
10%.
4.2.4
Analytical
methods
The carbonate content was measured
by
volumetric
analysis
of the evolved
C02
41
Chapter
4
after addition of HCl to the soil. The
pH
was measured at a solid: solution ratio of 1:2.5
in 0.01 M
CaCI2,
the
organic
matter content was determined
gravimetrically following
oxidation with
H202,
the
grain
size fractions
by
the
pipetting method,
and the total
metal contents of the soil
by X-ray
fluorescence
(Bruker
D4 Endeavour/
Germany).
The
pH
of the metal extraction solutions was measured with
pH-electrodes
in the
suspension.
The dissolved
Cu, Zn, Pb, Fe, Mn, Mg,
and Ca concentrations in the
extraction solutions were measured with a flame-AAS
(Varian AA400).
4.3 Results and Discussion
4.3.1 Effect of
pH
on extraction
In the absence of
chelating agents
less than 20% Cu and Pb and about 40% of
Zn were extracted at
pH
3
(Figures
4.1
-
4.3).
At
pH
7 the extraction of
Cu,
Zn and Pb amounted to less than 1% of their
total concentration.
Already
at an
equimolar
ratio between chelant and metal Cu
extraction was
greatly
enhanced
by
all tested
chelating agents.
At
pH
4 the order
of the extraction
efficiency
was EDDS
> EDTA >
MGDA
> NTA >
IDSA and at
pH
7
EDDS
> NTA> IDSA
>
MGDA
> EDTA. At
pH
4 EDDS and IDSAdid not enhance Zn-
extraction
compared
to the controls without
chelating agent,
due to their
very
weak
complexes
at this
pH,
but
EDTA, NTA,
and MGDA were able to mobilize Zn. At
pH
7 the order of extraction
efficiency
was NTA >
EDDS
> EDTA >
MGDA >IDSA. Pb
was
analyzed only
for treatments of Rafz soil with
EDTA,
NTA and EDDS. At both
pH
4 and 7 the extraction
efficiency
was EDTA > NTA >
EDDS
although
at
pH
7 the
extraction
efficiency
of EDDS was much closer to that of NTA and EDTA than at
pH
4
(Figure 4.3).
At a chelant: metal ratio of 10 the
pH dependence
of the extraction and the
differences between the
compounds
was much less
pronounced (Figures
4.1
-4.3).
At this concentration EDTAwas the most effective
compound
for
Cu,
Zn and Pb over
the whole
pH-range
studied.
At the low chelant: metal ratio neither EDDS or EDTAaffected the Ca concentration
in solution
compared
to the treatment without chelant
(Figure 4.4).
The Ca
concentration was determined
by
ion
exchange
in the 0.01 M
NaN03
medium. At
the
high
chelant: metal ratio of 10
significantly
more Ca was extracted than in the
absence of chelant. About 20% of the total Ca in the soil was extracted
by
EDTA and
EDDS at
pH
7 at the
high
ratio
compared
to about 10% at the low ratio.
The Fe concentration was
very
low in the absence of chelant. Both EDTA and
EDDS mobilized
significant
concentrations of Fe
(Figure 4.4).
The extracted iron
was in the form of an
Fe(lll)-complex
because
any
extracted
Fe(ll)-complex
would
42
Extraction of
heavy
metals from soils
100
0)
o
(0
1-
X
0
3
T5
(1)
u
W
k.
+->
x
0
3
X no chelant
EDTA
EDDS
MGDA
IDSA
NTA
~X X X X X ^= =x=
7 8
PH
Figure
4.1. Extraction of Cu from the non-calcareous soil Dornach 2 as a function
of
pH
with 0.4 mM
(ratio
chelant: metal
1)
and 4 mM chelant
(ratio
chelant: metal
10).
Conditions: 20
g
I"1
soil,
0.01 M NaN03.
have been
immediately
oxidized to the
Fe(lll)-complex by oxygen (33).
Whereas
the EDTA extractable Fe
dropped
to
very
low levels at
pH
7 and
above,
EDDS was
still able to extract
relatively high
concentrations of Fe. At the
high
chelant: metal
ratio EDTA extracted about 2% of the iron in the soil at
pH 7,
EDDS
only
0.25 %.
Fe extraction is
important
for chelate
speciation, although
it affected
only
a small
fraction of the total iron in the soil.
At
pH
4 the order of Cu extraction
roughly
followed the decrease in the
stability
constants of the
Cu-complex,
but not at
pH
7
(Table 4.2).
The order of extraction
efficiency
of Cu and Zn at
pH
7 was more
closely
related to the
stability
constant of
the
Ca-complex.
To understand this behavior we
performed speciation
calculations.
Reactions considered were
complexation
with
Ca, Mg, Fe, Mn,
Zn and Cu. The
results are
presented
in
Figure
4.5. Two
important
differences are obvious: Where
43
Chapter
4
100
o
80
o
+
(0 60
1_
+
X
0
40
c
N
20
0
+
03
i-
+>
X
o
c
N
chelant: metal
=
1
pH
Figure
4.2. Extraction of Zn from soil Dornach 2 as a function of
pH
with 0.4 mM
(ratio
chelant: metal
1)
and 4 mM chelant
(ratio
chelant: metal
10).
Conditions: 20
g
I"1
soil,
0.01 M NaN03.
as CaEDTA resulted as the
major species
at
pH 8,
CaEDDS was found to be
only
a
marginal species
at that
pH. Competition
between
heavy
metals and Ca thus
appears
to be an
important
factor for extraction with EDTA but not with EDDS and
this results in a decrease in extraction
efficiency
for EDTA
compared
to EDDS.
A
very
small fraction of EDTA and about 20-35% of EDDS were
predicted
to be
in the
free, uncomplexed
form. Part of this fraction could also be
complexed
to
metals not included in the calculations
(e.g.
Ni or
Pb).
The
Fe-complex
is the
major
species
for both EDTA and EDDS at low
pH.
Due to the
Ca-competition,
FeEDTA
decreases with
increasing pH
while FeEDDS is also a relevant
species
at neutral
pH.
These results contradict the statement of Vandevivere et al.
(24)
that Fe
may
be
neglected
when
speciating
EDDS in soil
suspension.
These authors based their
44
Extraction of
heavy
metals from soils
0
+->
U
(
s_
+>
X
0
.Q
0_
D
0
+
U
i-
+
X
0
.Q
Q_
100-
80j
60
40-
20-
0-
100-
80-
60-
40-
20-I
0
chelant: metal
=
1
chelant: metal
=
10
X no chelant
-EDTA
CAEDDS
^^NTA
>Xi XX X
XiXX-
5 6
pH
8 9
Figure
4.3. Extraction of Pb from soil Rafz as a function of
pH
with 0.4 mM
(ratio
chelant: metal
1)
and 4 mM
EDTA,
EDDS and NTA
(ratio
chelant: metal
10).
Con
ditions: 20
g
h1
soil,
0.01 M NaN03.
statement on a
comparison
of the
log
K values for
heavy
metals and
Fe(lll),
but did
not
provide
measurements of dissolved Fe. Our results show that Ca and Fe have to
be taken into account in chelant-assisted extraction of
heavy
metals from soils. The
competition
between the two metals and the
target pollutant
metals for the available
chelating agents
is
particular important
at the low chelant: metal ratio of 1.
The extraction of Pb at the low chelant: metal ratio seems to
depend mainly
on
the
stability
constants of the Pb
complexes (log
K EDTA
17.9,
EDDS 12.7 and NTA
11.3) apart
from Ca
competition
in the case of EDTA at
high pH (Figure 4.5).
At low
concentrations of
chelating agent
Pb extraction showed a
very strong dependence
on
pH (Figure 4.3).
EDTA shows
very strong
extraction of Pb
up
to
pH
6 due to its
high log
K
value,
after this however the extraction
efficiency
reduces
by
about 50%
45
Chapter
4
pH
Figure
4.4. Concentration of Ca and Fe in the extraction solution from soil Dor
nach 2 with 0.4
(ratio
chelant: metal
1)
and 4 mM chelant. Conditions: 20
g
I"1
soil,
0.01 M NaN03.
due to
competition
for EDTA from Ca.
Although
NTA has a lower
log
K value than
EDDS it extracts much
greater
amounts of Pb than EDDS at
pH's
below
pH
7. This is
due to the much
higher
conditional
log
K value of NTA at low
pH
values which is due
to the much smaller second
pKa
value of NTA
(2.48) compared
to EDDS
(6.84) (34).
NTA too suffers due to Ca
competition
at
pH's
above 7
although
to a much smaller
extent than
EDTA, showing
a reduction in extraction
efficiency.
So at
pH
values
below 7 EDDS was not suitable for Pb
extraction,
but above
pH
7 it
performed
as
well as EDTA and better than NTA due to the above mentioned factors.
At a chelant: metal ratio of 10 most of the EDTA and EDDS were
present
in
uncomplexed
form
according
to the
speciation
calculations. About 25% of EDTA
was
present
as CaEDTA.
Uncomplexed
EDDS
always
accounted for about 90% of
46
Extraction of
heavy
metals from soils
V)
0
"5
0
Q.
LU
0
"5
0
Q.
(A
U>
Q
LU
pH
Figure
4.5. Calculated
speciation
of EDTA and EDDS in the extraction solution
from soil Dornach 2 at a chelant: metal ratio of 1.
total EDDS. Thus there was
always enough
free EDDS to extract further metals. For
EDTAthis was
only
the case at the
high
ratio.
However,
metals are not
only
extracted
by
the free
ligand
but also
by
metal
complexes.
A metal in a chelate
may
not be at
equilibrium
and can be
exchanged
with another metal. CuEDTA for
example
was
able to extract Zn
efficiently
from a river
sediment, although
the rate was
only
20%
of that of CaEDTA
(35).
Our results
suggest
that EDDS is a better metal extractant for Cu and Zn than
EDTA at
pH
values above 6 at low chelant: metal ratio because it forms
only
a
weak
Ca-complex.
In addition it has the
advantage
that it is
readily biodegradable
as classified
by
the modified Sturm test OECD 301B
(36). Any
residual EDDS that
remains in the soil after extraction will
rapidly
be
degraded
and
poses
little risk
with
respect
to
leaching
of metals to the
groundwater.
Chelates are
only weakly
47
Chapter
4
100
o
0
+
U
3_
X
0
3
O
73
0
+j
U
(0
>_
*j
X
0
c
N
"D
0
+>
U
(0
s_
+J
X
0
.Q
_
time
(hours)
Figure
4.6. Extraction kinetics of
Cu, Zn,
and Pb
by
EDDS from the three soils
at
pH
7. Conditions: chelant: metal ratio
1,
20
g
I"1
soil,
0.01 M NaN03. The "no
chelant" data are for soil Dornach 1
(Cu
and
Zn)
and for soil Rafz
(Pb).
adsorbed at neutral
pH (37, 38)
and are therefore
easily
leached. NTA extracted
a similar amount of Zn as EDDS at neutral
pH
but
significantly
less Cu. IDSA and
MGDAwere less efficient at low
concentrations,
but
performed
more or less as well
48
Extraction of
heavy
metals from soils
as EDDS at
high
chelant: metal ratios.
We conclude that the extraction with EDDS at
pH
7
gave
the best
compromise
between extraction
efficiency
for
Cu,
Zn and Pb and loss of Ca and Fe from the soil.
In order to extract
polluting
metals from soil with near-neutral
pH,
no acidification is
necessary
and unwanted extraction of
major
ions is minimal.
4.3.2 Extraction kinetics
The results
presented
so far refer to 24 hours extraction time.
Figure
4.6 shows
the kinetics of
Cu, Zn,
and Pb extraction from the three soils
using
EDDS at
pH
7
up
to a reaction time of 48 hours. Zn and Pb extraction exhibited a fast initial
step
followed
by
a much slower release of metals. Extraction of Cu was slower than that
of Pb orZn. The extraction kinetics of MGDA and EDTA were
comparable
to
EDDS,
only
the extracted amounts were smaller
(data
not
shown).
EDDS extracted different
amounts from the three
soils,
a fact that will be discussed later in more detail.
IDSA first increased the dissolved
Zn,
but after 30 hours the concentrations
decreased
again (Figure 4.7).
The same trend could be seen for Cu. The
hypothesis
that this was caused
by
microbial
degradation
of the
ligand
and
subsequent
immobilization of the released metals was tested
by conducting experiments
in the
u
cu
(0
1_
+>
X
CD
C
N
40-
0.
-- EDDS
-O- EDDS + NaN
- IDSA
IDSA+ NaN
1 1 1
0 20 40 60
time
(hours)
80 100
Figure
4.7. Extraction of Zn from soil Dornach 2 with EDDS at
pH
4.5 and with
IDSA at
pH
7 in the absence and
presence
of the biocide sodium azide. Condi
tions: chelant: metal ratio
1,
20
g
h1
soil,
0.01 M NaN03.
presence
of the biocide sodium azide.
Indeed,
no re-immobilization of solubilized Cu
and Zn was observed with IDSA in the
presence
of the biocide. For the efficient use
of IDSA and
any
other
rapidly biodegradable chelating agent
therefore the extraction
time must be
optimized
in order to
gain
the maximum extraction before the start of
49
Chapter
4
biodgradation.
At
pH 4.5,
Zn extraction
by
EDDS also showed a
very rapid
initial
increase,
and
then a
gradual
decrease in dissolved Zn concentration with time
(Figure 4.7).
This
decrease,
which was not observed at
pH 7,
also occurred in the
presence
of sodium
azide. It cannot be
explained, therefore, by biodgradation.
We
suspect
that it was
caused
by
the dissolution of iron oxides
by
ZnEDDS.
Metal-ligand complexes
are
able to dissolve oxides in a similar manner as the free
ligands, although
at a slower
rate
(39).
The mobilization of Zn
by
free EDDS at
pH
4 is
very rapid showing
an
exponential
increase before the first
sampling
at 1 hour
(Figure 4.7). Subsequently,
the
newly
formed ZnEDDS
slowly
dissolves Fe-oxides.
Equilibrium
calculations show
that about 70% of the EDDS in solution could have been
present
as
Fe-complex
if
the
system
would have been at
equilibrium
with
hydrous
ferric oxide. The observed
FeEDDS concentration
(as
a
percentage
of the total
chelating agent concentration)
was 28% after 8 hours and 36% after 24 hours
only gradually increasing up
to
48 hours
(Figure 4.8).
Dissolution of iron oxides either
by
the free
ligand
or metal
complexes
therefore continued
during
the whole
period
of the
experiment.
The kinetic
experiments
showed for all metals and soils that 24 hours was the
optimum
extraction time with the time
period
from 24 to 48 hours
only giving
minor
additional benefits.
c
re
a>
u
75
+
Q
X
Q.
E
o
o
0
60-
50-
40-
30-
20-
10-
0i
EDTA
pH
7
EDTA
pH
4.5
EDDSpH7
EDDS
pH
4.5
20 30
time
(hours)
Figure
4.8. Extraction of Fe from Dornach soil 2 as a function of time at
pH
4.5
and
pH
7 with EDDS and EDTA. Conditions: chelant: metal ratio
1,
20
g
I"1
soil,
0.01 M NaN03.
4.3.3
Comparison
to other studies
The factor of
primary importance
in chelant-assisted metal extraction is the ratio
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Chapter
4
concentration of the
applied
extractant
solution,
which is
usually specified,
is of
little use if the metal concentrations of the soils and the solid: solution ratio are not
known.
In order to
compare
our results with those of other
authors,
we
compiled
data
obtained under the
following
conditions: The
pollution
occurred in the field and not
by
fresh addition of metals in the
laboratory,
the
pH during
extraction was around
7,
the extraction time was 24 hours and the ratio chelant: metal was at least 1. Table
4.4 summarizes the results of this
compilation.
Extraction of Zn
spans
a
range
from
17 to
63%,
with most data
ranging
between 30 and 35%.
Thus,
Zn extraction was
quite
weak for most
soils,
and an increased chelant: metal ratio did not result in a
significant
increase in extraction
efficiency.
Cu extraction varied between 28 and
100%,
with most data
ranging
between 40 and 80% and Pb between 23 and
96%,
with most data
ranging
between 30 and 89%. It is obvious that a
higher
ratio resulted
in a more effective extraction of these two metals and that without consideration of
this
parameter, comparison
of data from different studies has little
meaning.
The extraction efficiencies obtained with EDDS in this
study
for Cu are
among
the best
observed so far at a low
chelating agent:
metal ratio. Zn extraction at
pH
7 was about
the same as observed for EDTA in our
study
and
comparable
to other efficiencies
reported
in the literature for low
chelating agent:
metal ratios. Pb extraction
by
EDDS
was lower than for EDTA in most
cases,
but at the
high
ratio it exceeded some
extraction efficiencies
given
in literature for
high
EDTA concentrations. The use of
a low ratio of EDDS at neutral
pH
is therefore
very promising,
and we can
expect
results
comparable
to or better than other
yields reported
in the literature. This is
especially good considering
that SS-EDDS was found to be
readily biodegradable
(40)
and EDTA was not
(20).
4.3.4 Influence of solid
phase speciation
on extraction
yield
Sequential
extractions can
give
the information needed to
explain
different
extraction efficiencies for different metals.
Figure
4.9 shows the distribution of Cu
and Zn fractions in soil Dornach 2 before and after the extraction with EDDS. Cu
was
mainly
found in the
"organic" fraction,
while about 60% of the Zn was found in
iron oxide and residual fractions. The
applied
EDDS solution extracted these metals
mainly
from the first 4
fractions,
the
exchangeable, mobile,
Mn-oxides and
organic
fraction. Table 4.5 shows the relative reduction of the
respective
metal fractions
by
EDDS.
For Cu and Zn the first 3 fractions were reduced
by 71, 80,
and 75 % on
average,
the
organic
Zn fraction
by 36%,
and the
organic
Cu fraction
by
58%. The iron-
oxide and residual fractions were not or
only marginally
reduced. The much better
extractability
of Cu
compared
to Zn
by chelating agents
can therefore be
explained
52
Extraction of
heavy
metals from soils
before after extraction
Figure
4.9.
Sequential
extraction of Cu and Zn in soil Dornach 2 before and after
extraction with EDDS.
by
the
presence
of
larger weakly
bound fractions. The maximum extraction
efficiency
(pH 7)
at
high
chelant: metal ratio for soil Dornach 2 was 84% for Cu and 48% for
Zn
(EDTA),
while the first 4 fractions of the
sequential
extraction amounted to 79%
for Cu and 41% forZn. A similar result also holds for Pb in soil Rafz. The maximum
53
Chapter
4
Table 4.5. Reduction of metal fractions in the soils
by
extraction with EDDS
(in %),
according
to the Zeien and Brummer scheme
(31, 32).
metal soil exch. mobile Mn-Ox
organic
am-Fe
cryst.
Fe res.
Zn Dornach 1 73 69 65 26 2 -5 -32
Dornach 2 86 83 68 44 20 1 -33
Rafz 92 80 64 40 8 -23 -25
Cu Dornach 1 63 84 87 46 12 -14 -32
Dornach 2 40 86 91 69 33 16 -32
Pb Rafz 16 37 36 17 0 -3 -11
extraction
efficiency (pH 7)
is 95%
(EDTA),
which is also the
percentage
of Pb in the
first 4 fractions of the
sequential
extraction. Van Benschoten et al.
(13)
found similar
results for 7 soils from sites
polluted
with Pb. The Pb not removed was found in the
Fe-oxide,
sulfide and residual fractions. When
looking
at the extractants used in the
sequential extraction,
this result is not
surprising
since fraction 4 is determined
by
extraction with 25 mM EDTA
(31, 32).
Table 4.1 shows the
properties
of each soil. These
properties
influence the
distribution of the metals between the different fractions as classified
by sequential
extraction. For
example
soil Dornach 1 is a calcareous soil with a
high pH,
which
is
likely
to make the metals less labile and less
easily
extractable. Soil Dornach 2
has little carbonate and a neutral
pH,
while the soil Rafz soil has a acidic
pH
with
a low
clay
content
making
the metals more
easily
available. This can be seen in
the distribution of Zn in these 3 soils. 31% Zn in soil Dornach 1 is found in the
first four fractions while 41% is found in soil Dornach 2 and 57% in soil Rafz. The
different extraction efficiencies for different metal obtained
by
the
chelating agents
are therefore
simply
due to the different distribution of the metal fractions. Extraction
by chelating agents
is therefore feasible for soils
containing
a
large
fraction of metals
in the first 4 fractions of the
sequential
extraction
procedure applied here,
but will not
be an efficient treatment for soils
containing
a
high percentage
of metals in
strongly
bound fractions.
Acknowledgements
We thank Werner
Attinger
and Anna Grnwald for
help
with the soil
sampling
and
54
Extraction of
heavy
metals from soils
the chemical
analyses
and Diederik Schowanek from Procter & Gamble for
providing
S,S-EDDS.
part by
the Federal Office for Education and
Science within COSTAction 837 and the Swiss National Science Foundation in the
framework of the Swiss
Priority Program
Environment.
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in Bden. Mitt. Dtsch. Bodenkundl. Ges
1989, 59,
505-510.
(32) Zeien,
H. Chemische Extraktionen zur
Bestimmung
der
Bindungsformen
von
Schwermetallen in Bden
Friedrich-Wilhelms-Universitt,
1995.
(33) Zang, V; EvanEldik,
R. Kinetics and mechanism of the autoxidation of
iron(ll)
induced
through
chelation
by ethylenediaminetetraacetate
and related
ligands.
Inorg.
Chem.
1990,29,1705-1711.
(34) Martell,
A.
E.; Smith,
R.
M.; Motekaitis,
R. J. NIST
critically
selected
stability
constants of metal
complexes,
Version 6.0. NIST
Gaithersburg,
MD
20899, USA,
2001.
(35) Nowack, B.; Kari,
F.
G.; Kruger,
H. G. The remobilization of metals from iron
oxides and sediments
by
metal-EDTA
complexes.
Water Air Soil Poll.
2001, 125,
243-257.
(36) Jaworska,
J.
for
trisodium(S,S)-ethylenediamine disuccinate,
a
biodegradable
chelator used in
3597-3625.
(37) Nowack, B.; Ltzenkirchen, J.; Behra, P.; Sigg,
L.
Modeling
the
adsorption
of
metal-EDTA
complexes
onto oxides. Environ. Sei. Technol.
1996, 30,
2397-2405.
(38) Nowack, B.; Sigg,
L.
Adsorption
of EDTA and metal-EDTA
complexes
to
goethite.
J. Colloid. Interface Sei.
1996, 177,
106-121.
(39) Nowack, B.; Sigg,
L. Dissolution of
iron(lll)(hydr)oxides by
metal-EDTA
complexes.
Geochem. Cosmochim. Ac.
1997, 61,
951-963.
T.
Biodegradabilities
of
ethylenediamine-N,N'-disuccinic
acid
(EDDS)
and other
chelating agents.
Biosci.
57
Chapter
4
Biotech Biochem
1997, 61,
1957-1959
(41) Theodoratus, P, Papassiopi,
N
,
Georgoudis, T, Kontopoulos,
A Selective
removal of lead from calcareous
polluted
soils
using
the Ca-EDTA salt Water Air
SoilPollut
2000,722,351-368
(42) Hong,
P K
A, Li,
C
,
Banerji,
S
K, Regmi,
T
Extraction, recovery,
and
biostabihty
of EDTAfor remediation of
heavy
metal-contaminated soil J Soil Contam
1999, 8,
81-103
(43) Yu,
J
,
Klarup,
D Extraction kinetics of
copper, zinc,
iron and
manganese
from contaminated sediment
using
disodium
ethylenediaminetetraacetate
Water
Air Soil Poll
1994,75,205-225
(44) Barona,
A
,
Aranguiz,
I
,
Elias,
A Metal associations in soils before and after
EDTA extractive decontamination
implications
for the effectiveness of further clean
up strategies
Environ Pollut
2001, 773,79-85
(45) Linn,
J H
,
Elliott,
H A Mobilization of Cu and Zn in contaminated soil
by
nitnlotnacetic acid WaterAir Soil Pollut
1988,37,449-458
58
5 The Influence of SS-EDDS on the
Uptake
of
Heavy
Metals in
Hydroponically
Grown Sunflowers.
Susan
Tandy,
Rainer Schulin and Bernd Nowack
Submitted to
Chemosphere
Abstract
Phytoextraction
is an
environmentally friendly
in-situ
technique
for
cleaning up
metal contaminated land.
Unfortunately,
efficient metal
uptake by
remediation
plants
is often limited
by
low
phytoavailability
of the
targeted
metals. Chelant assisted
phytoextraction
has been
proposed
to
improve
the
efficiency
of
phytoextraction.
Phytoremediation
involves several
subsequent steps:
transfer of metals from the
bulk soil to the root
surfaces, uptake
into the roots and translocation to the shoots.
Nutrient solution
experiments
address the latter two
steps.
In this context we
investigated
the influence of the
biodegradable chelating agent
SS-EDDS on
uptake
of essential
(Cu
and
Zn)
and non-essential
(Pb)
metals
by
sunflowers from nutrient
solution. EDDS was detected in shoots and
xylem sap
for the first
time, proving
that it is taken
up
into the above
ground
biomass of
plants.
The essential metals
Cu and Zn were decreased in shoots in the
presence
of EDDS whereas
uptake
of
the non-essential Pb was enhanced. We
suggest
that in the
presence
of EDDS all
three metals were taken
up by
the non-selective
apoplastic pathway
as the EDDS
complexes,
whereas in the absence of EDDS essential metal
uptake
was
primarily
selective
along
the
symplastic pathway.
This shows that
synthetic chelating agents
do not
necessarily
increase
uptake
of
heavy metals,
when soluble concentrations
are
equal
in the
presence
and absence of chelates.
59
Chapter
5
5.1 Introduction
Heavy
metal
pollution
of soil is a
widespread global problem.
The clean
up
of
metal contaminated land
by
traditional
physiochemical
methods can be
very costly
and also destructive to the soil.
Phytoextraction
is an
environmentally friendly
in-situ
technique
for
cleaning up
metal contaminated land
(1),
which aims to
preserve
soil
structure and
fertility.
Chelant
(ligand)
assisted
phytoextraction
has been
proposed
to
improve
the
efficiency
of conventional
phytoextraction
of metal
polluted
soils
by
solubilizing target
metals from soil
(2, 3)
and
making
them more available for
plant
uptake
and translocation to the shoots.
EDTA
(ethylenediamine
tetraacetic
acid)
has been the most
commonly
used
chelating agent
for this
purpose (4-10),
but due to its
persistence
in the environment
(11,12)
it is now considered unsuitable for field use. Other
such as NTA
(nitrilotriacetate) (13-15),
have also
occasionally
been used.
disuccinic acid
(EDDS)
is a
biodegradable
structural
isomer of EDTA
(16-18).
Other stereoisomers of
ethylenediamine
disuccinic acid
are either
non-biodegradable (R,R)
or
only partically degradeable (R,S, S,R)
so
the SS-isomer is
generally
used
(16).
It is now used as a commercial substitute for
EDTA in
detergents (19, 20)
and has the
potential
to be a substitute for EDTA in
chelant assisted
phytoremediation,
as it is a
strong
chelator and unlike
EDTA,
it is
easily biodegradable.
EDDS can
readily
solubilize metals from soil and at
pH
7 it
was shown to be better at
solubilizing
Cu and Zn than EDTA at
equimolar
ratios of
chelating agent
to metals
(21).
Several
papers
have
recently
been
published
on the
use of EDDS in chelant enhanced
phytoremediation
of
Pb, Zn,
Cu and Cd in soil
(22-27).
Several
processes
are involved in
phytoremediation:
solubilization of the metals
from soil and transfer to the
roots, uptake
of the metals into the roots and translocation
to the above
ground
biomass.
Ideally
chelants would enhance all of these
steps.
Hydroponics experiments
can be used to
investigate
the latter two
processes.
Early
work used
hydroponics experiments
to
investigate
trace element
plant
nutrition. Therefore low metal
(<
20
uM)
and chelant concentrations were
employed
in these studies. It was found that EDTA or DPTA
produced
Zn
deficiency
in corn
(Zea mays)
and
barley (Hordeum vulgare) (28, 29).
Also Ni
uptake by
beans
(Phaseolus vulgaris)
was not
improved by
DTPA
(30).
These observations were
seen as evidence that EDTA and DPTA
complexes
are not taken
up by plants.
Other
investigators
have studied the influence of
high
concentrations of chelants
(>
50
uM)
on metal
uptake, mainly
on the
phytoextraction
of Pb. 0.25 mM EDTA
was the threshold for
rapid
shoot
uptake
of Pb and at
higher
concentrations
(0.75
mM)
a maximum shoot Pb concentration of 56 mmol
kg-1
could be reached in Indian
60
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
mustard
(Brassica juncea),
a 400 fold increase on the control
(31 ).
This led to
drying
of shoot matter and the formation of necrotic lesions however. Increases in shoot Pb
uptake
in the
presence
of chelants
mostly
fall in the
range
2-10 times the control
and 0.9-11 mmol
kg-1
shoot Pb concentration
(32-34).
Although chelating agents
have been seen to increase
plant
shoot
uptake
of Pb
in most
cases,
the
uptake
of Cu and Zn seems
mainly
to be unaffected or even
decreased with chelants.
Very
little work has been carried out on chelant effects on
Cu
uptake
and none on Zn at elevated metal concentrations in solution. In a
toxicity
study
carried out
using
wheat
(Triticum aestivum)
it was found that at 30 uM Cu in
hydroponic solution,
leaf concentrations were similar in
plants grown
in chelated
and non-chelated solutions
(35).
When the concentration was increased to 50 uM
for
CuS04and
60 uM for
CuEDTA, however,
the concentration was
higher (43 mg
kg-1)
in leaves from
plants grown
in the absence of chelates
(chelate
denotes the
ligand-metal complex,
chelant the
ligand itself)
than those
grown
in the
presence
of
chelates Cu
(~
30
mg kg-1).
On the other hand Cu shoot concentrations were found
to increase when tobacco
(Nicotiana tabacum)
was
grown
in a solution of 0.126 mM
Cu and 0.5 mM NTA
compared
to a solution of 0.038 mM free Cu in the absence of
NTA
(36).
Direct methods have been used to measure EDTA inside
plants,
either as a
complex (4, 37-39)
or as total EDTA
(10, 31, 40).
These
investigations prove
that
the EDTA is taken
up
from solutions
containing high
concentrations.
There are indications that metal-chelant
complexes
are taken
up along
an
apoplastic pathway (41 ).
This means that the
complexes pass through
the free
space
of the roots which is made
up
of root cell walls and water filled intercellular
spaces
in the root cortex and which is continuous with the
surrounding
soil solution
(42).
Explanations
of the translocation of
from roots to shoots
are confounded with the
problem
that
prior
to
reaching
the
xylem
the
complexes
meet the
Casparian strip,
a
highly
suberized band that halts
apoplastic
flow and
forces them to cross the cell membranes of the endoderm is
(42).
As most chelates
are
charged, large
in size and have no known
specific transporters,
it is
unlikely they
can
pass through
the cell membrane. However the
Casparian strip
is not a
perfect
barrier. At the root
tips
it is not
fully
formed
(41, 43),
and where lateral roots
protrude
from the main root
system
the
Casparian strip
can be
disrupted.
It has been found
in fact that at such
places components
from the
surrounding
solution can enter the
stele which houses the
xylem
without
passing through
a cell membrane
(43, 44).
Furthermore some
species
have a small number of cells in the endodermis called
passage
cells remain unsuberized. It is assumed that
they
allow
entry
of water and
solutes into the
xylem (45).
investigate
the influence of the
chelating agent
61
Chapter
5
EDDS on metal
uptake
of essential
(Cu
and
Zn)
and non-essential
(Pb)
metals
by
sunflowers
(Helianthus
annuus cv.
Iregi)
from nutrient solution. It was
envisaged
due to the evidence mentioned
previously
that Pb shoot
uptake
would increase
with the addition of EDDS
although
the use of a different chelant could lead to
alternative results. With
regards
to Cu and Zn
uptake,
it was considered an
open
question
to whether
they
would be increased in the
presence
of EDDS or not. We
hypothesised
that EDDS would be taken
up
into the
xylem
and shoots as seen for
EDTA. To
investigate
this we measured both metal and chelant in sunflower roots
and shoots. This is also the first
step
to
investigate
if EDDS can be substituted for
EDTA in
phytoextraction
of
heavy
metals.
5.2.1 Nutrient Solution
Seedlings
of Helianthus annuus
(cv Iregi)
were
grown
in aerated 10%
strength
modified
Hoagland
solution for three weeks before
they
were
exposed
to metal
and chelator solutions
(Table 5.1).
The
Hoagland
solution was modified
by using
FeS04-7H20
instead of
NaFe(lll)EDTA
to avoid interference between another
complexing agent
and EDDS
(Table 5.1).
5.2.2
Experimental Setup
After three weeks of
growth
the
plants
were transferred into the
experimental
solution,
in which all micronutrients other than the metal under
study
and
KH2P04
were omitted to avoid
competition
between metals and
precipitation
of Cu and Pb
phosphate (Table 5.1). Single
metals
(Cu
126
uM,
Zn 122
uM,
Pb 125
uM)
were
added alone or in combination with EDDS
(500 uM).
EDDS was therefore in excess
of the treatment metals. For each treatment four
replicate plants
were
placed
in
individual 1 I brown covered
pots containing
the treatment solution. The
experiment
was carried out for six d in a
growth
chamber on a 16 h
(21 C)
/ 8 h
(15 C) day/
night cycle.
Due to the lack of
P042"
in the
experimental solution,
there was no loss
of
Cu,
Zn or Pb due to
precipitation.
At harvest the shoots were cut 1 cm above the roots and removed.
Xylem sap
was collected at
atmospheric pressure
from the cut stem for 2-3 h after
cutting
and
frozen until
analysis.
Shoot and root
samples
were
analysed
for metal and EDDS
concentration after
sample preparation. Xylem sap
was
only analysed
for EDDS
due to the small
sample
size. The initial
experimental
solutions were filtered and
also
analysed
for metal concentrations.
62
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
Table 5.1. Nutrient solution
composition, pre-experimental
and
experimental.
The
pre-experimental
solution was used to
grow
the
plants prior
to
uptake experiment
and the
experimental
solution was used as a
during
the
uptake experiment
with the
addition of the treatments.
Pre-experiment Experimental
Nutrients Nutrient solution Nutrient solution
(uM)
without P
(uM)
Ca(N03)-4H20
400 400
MgSCy7H20
200 200
KH2P04
100
-
KN03
500 500
FeS04-7H20
10 10
H3BO3
10
-
MnS04H20
2
-
ZnS04-7H20
0.2
-
CuSCy5H20
0.2
-
Na2MoCy2H20
0.1
-
NaCI 20
-
MES
-
2000
pH1
6.0 6.0
1
Unit not uM
5.2.3
Sample preparation
After harvest the shoots and roots were washed with deionised water and dried at
40C until constant
weight. High temperatures
can lead to
degradation
or
cyclization
of EDDS
(46)
and were therefore avoided. The oven-dried
plant
material was
ground
in a titanium mill.
5.2.4 Metal
Analysis
Plant
samples
of between 100 and 250
mg
were microwave
digested
in 5 ml
HN03
(65%),
2 ml
H202
(30%),
and 2 ml
H20
for metal
analysis.
The
digested samples
were diluted to 25 ml in
Millipore
water.
Digests
and filtered nutrient solutions were
analysed
for
Cu, Zn, Pb, Fe, Ca, Mg,
K
by
Flame-AAS
(Varian, SpectraAA 220FS)
and Cu and Pb
by
GF-AAS
(Varian,
SpectraAA
300 with
GTA96)
when
samples
were below the detection limit of the
63
Chapter
5
Flame-AAS. Standards were
prepared
in 0.1M
HN03
5.2.5 EDDS
analysis
Dried
plant
material was extracted with
pure
water
(10 mg
10
ml-1) by
sonication
with a
micro-tip
sonic
probe
for one minute. The
samples
were surrounded
by
ice
during
sonication to
were then
centrifuged
and filtered
(0.45 urn). Xylem sap samples
were diluted to 800 ul with distilled water. EDDS
derivatization and
analysis
was carried out as described
by Tandy
et al
(47).
This
method involves the derivatization of EDDS
by
FMOC
(fluorenylmethyl chloroformate)
followed
by
HPLC
(Jasco PU-980) separation using
a
Lichrospher
100
RP-18,
5
urn column and fluorescence detection
(Jasco 821-FP). Deviating
from the
original
method,
derivatization was carried out at
pH
8 instead of
pH
11.5. Each
sample
was
also
spiked
with an EDDS standard in order to
help identify
the EDDS
peak
at low
concentrations from the matrix
peaks.
5.2.6 Chemicals
All chemicals were obtained from Merck unless stated otherwise and were
analytical grade
or HPLC
grade
for the solvents. EDDS was obtained from Proctor
and Gamble
(Belgium)
as the
SS-Na3EDDS
salt.
2-morpholinoethane-sulfonic
acid
monohydrate (MES), (analytical grade)
and
fluorenylmethyl
chloroformate
(FMOC-
chloride), (puriss)
were obtained from Fluka. All solutions were made with
high purity
water
(Millipore, Bedford, MA).
5.2.7
Speciation Modelling
Chemical
speciation
calculations on the initial
experimental
treatment solutions
were
performed
with the
program
ChemEQL
(48). Stability
constants were taken
from
(49-51).
5.2.8 Statistical
analysis
All statistical
analysis (ANOVA)
was carried out with
Systat
10.2 on
log
transformed
data
(52).
Differences at P < 0.05 level were considered
statistically significant.
5.3 Results
5.3.1
Hydroponics
solution
speciation
Chemical
speciation modelling
was carried out to
give
an
insight
into the
speciation
of the
hydroponics
solution
although
this does not
necessarily give
the transitional
status of chemical
species
in solution. In the metal
only
treatments 95%
Cu,
97%
64
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
Zn and 91% Pb were calculated to be in the free form. The
remaining
metals were
present
as
complexes
with
sulphate (Cu 2.6%,
Zn
3%,
Pb 6
%), hydroxide (Cu
2%,
Pb
1.4%)
and nitrate
(Cu 0.4%,
Pb
1.6%).
In the
presence
of EDDS free metal
concentrations were
negligible (Cu
4.24
x
10"15,
Zn 3.84
x
10"10,
Pb 3.87
x 10"9 M
respectively),
as almost all the metals were bound to the chelator. All EDDS treatment
solutions had an excess of EDDS. In the metal-EDDS treatment solutions between
22
-
25% of the EDDS was in the
Cu,
Zn or Pb-EDDS2"
complex
and 68
-
71 % in the
H2EDDS2"
form. Between 92 and 94% of the EDDS
complexes
were in the divalent
anionic form whether it was
H2EDDS2"
or Metal-EDDS2".
5.3.2 Plant
dry weight
Figure
5.1 shows that 500 uM EDDS did not reduce shoot or root biomass
compared
to the control. Cu and Pb reduced shoot
(Cu
P
<0.008,
Pb P
=
0.077),
and root
biomass,
where as Zn had a
significant
adverse effect
only
on root
growth.
All roots showed brown discolouration in the metal
treatments,
where as roots were
creamy
white in the controls. This discolouration has also been noted for
leguminous
plants exposed
to
Pb(N03)
and is a
sign
of intense suberification
(53).
The reduction
in root
dry weight by
the metal
only
treatments was
significant
at the P < 0.001 level
compared
to the control and EDDS
only
treatments. In all cases EDDS alleviated
the observed
symptoms
of the shoot and root
toxicity
when added to the
Cu,
Zn or
Pb solutions. The
strongest
effect of metal in combination with EDDS was a
slight
reduction in root
growth (in comparison
to the
control)
in the Pb/EDDS treatment.
This effect was not
very significant
however
(P
=
0.831).
This shows that EDDS
was not
phytotoxic
at the
applied
concentrations. Similar results have been found in
other studies for EDTA. Shoots of Indian mustard for
example only
showed
toxicity
symptoms
at EDTA concentrations
greater
than 500 uM
(31).
5.3.3 Root metal
uptake
Figure
5.2 shows root
uptake
of
Cu,
Zn and Pb in the absence and
presence
of
EDDS in sunflower. Cu and Zn in the roots that were not
exposed
to the
respective
metal treatment came from
uptake
of these micro-nutrients in the
pre-experimental
nutrient solution. Pb in the non-Pb treatments is within
background
concentrations
due to environmental contamination or shows the detection limit.
Root
uptake
of Cu in the Cu treatment was more than 350 times that in the Cu/
EDDS treatment
(P <0.001),
Zn
uptake
in the Zn treatment was 77 times
greater
than in the Zn/EDDS treatment
(P <0.001),
and Pb
uptake
in the Pb treatment was
26 times that in the Pb/EDDS treatment
(P <0.05).
These
findings
tie in with the
dry
weight
results and
symptoms
of metal
toxicity.
Reduction in root
growth
occurred
where the
Cu,
Zn and Pb root concentrations were
highest
in the metal
only
65
Chapter
5
Control Cu Zn Pb
0.4
o)
0.3
0.2 J
-lJ
0
b)
:fe
Control Cu Zn Pb
Figure
5.1.
Dry weight
of sunflower shoots
(a)
and roots
(b)
at
harvest,
in the ab
sence of EDDS
(diagonal striped bars)
and
presence
of EDDS
(white bars).
Results
shown are the mean
values,
the error bars are standard error.
treatments,
while the metal-EDDS treatments had smaller root metal concentrations
and no
toxicity symptoms.
5.3.4 Shoot metal
uptake
Cu shoot
uptake
in the Cu treatment was twice that in the Cu/EDDS treatment
(P
<
0.05) (Figure 5.3).
Zn
uptake
was 10 times
larger
for the Zn treatment
(P
<0.001
)
than for the Zn/EDDS treatment. In
contrast,
Pb shoot
uptake
from the Pb treatment
was
very
low and
significantly
smaller
(22 times,
P
<0.001)
than from the Pb/EDDS
treatment. A similar decrease in Zn shoot
uptake
from solution has been observed
66
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
Control Cu Zn Pb
J*
"
E
c
N
Control Cu
10
.Q
Q_
105-
_
c)
104-
103-
//////'//'/
,//,/</<
W'',',
/'W,
~^-n
102-
101-
/ / /
1
1
-\r-
i
/
z /
//
1
1
Control Cu Zn Pb
Figure
5.2. Root
uptake
of Cu
(a),
Zn
(b)
and Pb
(c)
from nutrient solution in the
absence
and
presence (white bars)
of EDDS. Results shown
are mean
values,
error bars
represent
standard errors.
67
Chapter
5
1000
O 200
Control Cu Zn Pb
20000
15000-
10000-
^
5000
0
Control Cu Zn Pb
_Q
0_
Control Cu Zn Pb
Figure
5.3. Shoot
uptake
of Cu
(a),
Zn
(b)
and Pb
(c)
from nutrient solution in
the absence
and
presence (white bars)
of EDDS. Results
shown are mean values, error bars are standard error.
68
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
by
other authors in the
presence
of EDTA but at low metal concentrations
(28).
In our
work,
the shoot
uptake
of Pb
by
sunflowers from Pb-EDDS solution was
less than those found
by
other authors for other
plant species
from Pb-EDTA solution
(31 -34).
One reason
may
be that Pb forms a weaker
complex
with EDDS
(log K12.7)
than with EDTA
(log
K
18) (50).
Another factor is that most other studies used Pb-
EDTA concentrations much
greater
than our Pb-EDDS concentration. The smallest
Pb-EDTA solution concentration at which Pb accumulation in shoots was observed
was four times
(500 uM)
that used in our
experiment (125 uM).
5.3.5 EDDS
uptake
if)
Q
Q
LU
600
400-
200-
EDDS Cu/EDDS Zn/EDDS Pb/EDDS
if)
LU
6000
4000-
2000-
i i
EDDS Cu/EDDS Zn/EDDS Pb/EDDS
Figure
5.4. Shoot
(a)
and root
(b) uptake
of EDDS from treatments
containing
EDDS. Results shown are mean
values,
error bars are standard error.
69
Chapter
5
Total EDDS was
analysed
for in the
plant samples.
EDDS was detected in
extracted shoot and root material and in
xylem sap.
This is the first time that EDDS
uptake by plants
has been observed. Shoot and root concentrations of EDDS were
not
significantly
different between the different EDDS treatments
(P
=
0.404
-
1.0).
EDDS in the roots was an order of
magnitude greater
than in the shoots
(P
< 0.001
)
(Figure 5.4).
The same
phenomenon
has been observed in
experiments
with EDTA.
Ten times more EDTA was found in the roots than in the shoots of Swiss Chard
(40).
EDDS in the
xylem sap
could not be
quantified accurately
due to an
interfering
partially co-eluting peak (Figure 5.5). Spike recovery
had
poor reproducibility
and
varied between 11 and 91%.
However,
the EDDS
peak
can
obviously
be seen. The
measured EDDS values in
xylem sap
fell within the
range
0.1
-
30 uM with an
average
value of 6.5 uM. The
presence
of EDDS in the shoots and the
xylem sap proves
that EDDS is taken
up by
the
plant
and translocated to the above
ground
biomass.
The EDDS concentration in the
xylem sap corresponds
to about 1 % of the EDDS
concentration in the nutrient solution. Measured concentrations of EDTA in
xylem
sap (the only
similar
ligand
where
xylem sap
concentrations have been
measured)
ranged
between 5 uM and 900 uM
(4, 10, 37, 38)
for different
plant species
and
different conditions. The EDDS concentrations measured in the sunflowers in our
investigations
fall in to the lower
part
of this
range.
8x104T
"c
&6x104-
<D
c 4x104-
<D
O
CO
2x104-
u_
0^
0
Figure
5.5.
Chromatogram
of
xylem sap sample (continuous line)
and
spiked sample
(dashed line)
measured after derivatization
by
HPLC with fluorescence detection.
Only
Vassil
(31 )
has carried out measurement of EDTA in shoots of
plants grown
in
hydroponics
solutions so far. However it is not
possible
to
directly compare
his
results with
ours,
as he used much
greater
concentrations of EDTA and Pb than we
70
sample
EDDS
EDDS
spike J
Matrix Peak
12 3 4 5
Time
(minutes)
6 7
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
used.
We calculated the amount of metals taken
up by
shoots
during exposure
to the
treatment solution
by subtracting
the
pre-experimental
metal
uptake (metals
in the
EDDS
treatment)
from the total metal concentration in the shoots in the metal-EDDS
treatments. We found
approximately equal
amounts of EDDS and metal taken
up
into
the shoots for the Zn/EDDS and Cu/EDDS treatments
(Figure 5.6).
However there
was about five times more EDDS than Pb in the shoots in the Pb/EDDS treatment
(Figure 5.6).
600-
"5
400-
200-
0-
3.
AAA
AAA,
AA
, AA
- AAA
r///'-.
y//
:
V

/// :
if)
, ///
A
; ///
/^:
, ///
a
; ///
*
;
///
/^:
, ///
a
;
///
/^:
; ///
/^:
Q
Q
LU
1
O
"05
y
AAA
AAA
AAA
A
/
, AAA
y
AAA
AAA
0
a
'
AAA
A
'
AAA
:
^
, A
AAA:
/// :
-/"fr-AAA.
':AAA ^
'
1
r
'" A
Cu/EDDS Zn/EDDS Pb/EDDS
Figure
5.6. Metal and EDDS
uptake
from metal-EDDS treatments into shoots
during
the
experimental
time.
Metal, diagonal striped bars;
EDDS white bars. Results shown
are mean
values,
error bars are standard error.
5.4 Discussion
The results
suggest
that the observed decrease in Cu and Zn
uptake
in the
presence
of EDDS is
directly
related to the reduction in the free metal concentration
in solution. This fits with what is known about Cu and Zn
uptake by plants by
the
symplastic pathway, involving
the
uptake
into cells via the cell membrane. Several
Zn
transporters
and one Cu
transporter
in
plants
have been identified for the
uptake
of these metals
through
the cell membrane
(54).
The
dependence
of this selective
uptake
on free metal concentration is a
basicassumption
of the FIAM
(Free
Ion
Activity
Model) (55, 56).
The reduction in
toxicity symptoms
in metal-EDDS treatments also
suggests
that
toxicity
is
closely
related to the free metal
concentrations,
therefore
agreeing
with FIAM.
The effect of EDDS on Pb
uptake by
shoots is in conflict with this
picture:
almost
no
uptake
occurred in the absence of EDDS and there was a clear increase in
uptake
71
Chapter
5
in the
presence
of EDDS. No selective
uptake
mechanism for Pb is known and no
specific
Pb
transporters
have been found
(54).
The
uptake
of Pb in the
presence
of
EDDS is most
likely
in the form of Pb-EDDS
complexes through
another
pathway.
There has been no record of
uptake
of
large synthetic
chelants into cells. However
the
presence
of EDDS in the
xylem sap
and shoot material shows it must have been
transported through
the
plant.
The
similarity
of Pb to EDDS ratios in the shoots and
in solution
suggests
a
passive uptake
mechanism.
Uptake
seems to have been the same for
H2EDDS2"
(free EDDS)
and metal-
EDDS2- if treatments are
compared,
so there was little or no
selectivity
due to matrix
interactions or transfer
by
selective channels
through
cell membranes. In view of
what is known from the literature it is most
likely
that this
uptake
was non-selective
and
primarily
or
completely apoplastic.
Although
we found no
significant
difference between
H2EDDS2"
and metal-
EDDS2-uptake
into the
shoots,
the ratios between root and shoot
suggest
there is
interaction between
EDDS-complexes
and the root matrix. A
rough
calculation of
EDDS concentration in the free
space
of the roots
(approximately
10 % fresh root
volume in
young
roots is free
space (42)),
shows that the total EDDS concentration
in the
apoplast
was three to four that of the
surrounding
solution.
Therefore,
either
a
large
fraction of the
negatively charged
EDDS
complexes
were adsorbed to the
cell walls
(e.g. binding
to anion
exchange
sites
(43))
or EDDS was taken
up
into the
root cells as well.
The fact that EDDS reduced Pb concentrations in the roots does not contradict
but is consistent with this
picture.
Metals measured in the roots were not
necessarily
internalized within cells. To a
large
extent
they may
have been
simply
bound to the cell
walls. It is known that metal cations bind to extracellular cation
exchange
sites in the
roots
apart
from
being
stored within the cells
(42). Cu,
Zn and Pb-EDDS
complexes
are
negatively charged
and hence do not bind to these cation
exchange
sites. This
explains
the
greatly
reduced root concentrations of metals in the
presence
of the
chelant. The root Cu and Zn
present
in treatments without these metal additions had
most
likely
been taken
up
from the
pre-experimental
nutrient solution.
Interestingly
Pb concentrations were much
greater
in the Pb/EDDS treatment
than Cu and Zn concentrations in the
respective
metal-EDDS treatments. PbEDDS
is a weaker
complex (log
K
=
12.7)
than CuEDDS
(log
K
=
18.4)
or ZnEDDS
(log
K
=
13.4) (50).
Thus we
presume
that in the roots the cation
exchange
sites
compete
with EDDS for Pb and
split
the
complex
so more Pb is bound to these sites.
In contrast to the Pb/EDDS
treatment,
Zn or Cu/EDDS shoot
uptake
of metal and
EDDS were almost the same and
quite
different from the solution ratios. Pb/EDDS
on the other hand showed more EDDS
uptake
than Pb. As mentioned before the
ratio was similar to that in solution
(free
EDDS was
present
as well as
Pb-EDDS).
If
72
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
only
metal-EDDS was taken
up,
then no EDDS would be found in the shoots of the
EDDS control treatment which was not the case. It seems therefore that free-EDDS
was taken
up by
the shoots and was
complexed
later
by
Cu and Zn. This means
that extra Cu and Zn was
sequestered
and
transported
between the treatment
solution and shoots. If this had come from the metal adsorbed to the roots from the
pre-experimental solution,
then there should be more Cu and Zn in the
EDDS-only
treatment shoots than in the control shoots without
EDDS,
which can not be seen.
A more
likely explanation
is that the trace levels of free Cu and Zn in the treatment
solutions were
very efficiently
taken
up by
a selective or active
uptake mechanism,
then chelated
by
the free EDDS after
passing
into the
xylem
and
transported
to the
shoots. Pb does not have a selective
uptake
mechanism so trace amounts of free
Pb could not be taken
up
in this manner.
In
summary,
the differences in
uptake
between the essential and non-essential
metals and the effects of EDDS on their
uptake
can be
explained by
two
parallel
pathways:
a selective
symplastic pathway
and a non-selective
apoplastic pathway.
Figure
5.7 shows a schematic
demonstrating
this. Selective
uptake
of Cu and Zn
is
very
efficient and maximum shoot concentrations are reached at low dissolved
metals concentration.
Uptake
of Pb into the shoots is
very
low under these conditions
as
long
as the cation
exchange capacity
of the roots is not exceeded.
Apoplastic
(passive) uptake
of
metal-complexes
is a function of the
complex
concentration in
the
surrounding
solution. The scheme shows that under the
hypothetical
conditions
of our
experiment, represented by
the vertical
line,
Pb
uptake
is
increased,
while
~y~
_C/)
CO
-1
CD
C
CO
Experimental
Concentration
Selective
uptake (Cu, Zn)
y
y
y
y
y
y
y
y
y
Uptake
with chelant
(Cu, Zn, Pb)
Pb
uptake
without chelant
\
y.
Solution Metals
Figure
5.7.
Hypothetical
schematic of selective and non-selective
uptake
of metals
by plant
shoots.
73
Chapter
5
Cu and Zn
uptake
is decreased. The scheme also
suggests
that
higher
chelate
concentrations should lead to increased Cu and Zn
uptake.
The discussion shows that it is
important
to look at both chelant and metal
uptake
in roots and shoots to be able to see the full
picture. Only
Vassil
(31 )
has carried this
out for
so
far,
with EDTA and Pb.
They
used much
greater
concentrations of both than our
experiment
but showed when the ratio of Pb:EDTA
was 1:3 in solution it was 1:1.5 in the shoots. He also identified Pb-EDTA in the
xylem sap, supporting
the
theory
of
apoplastic transport
of metal-chelants into the
xylem
followed
by transport
to the shoots.
5.5 Conclusions
Most work on
phytoextraction only
considers the final
result,
whether
plant
metal
uptake
was increased.
Although
EDDS has been found to increase
uptake
of several
metals
including
Cu and Pb from soil it is
important
to find out more about the
processes
involved
(22, 24). Investigations
into chelant assisted
phytoremediation
should be considered in three
parts;
solubilization of the metals from soil and transfer
to the
roots, uptake
of the metals into the roots and translocation to the above
ground
biomass. Nutrient solution
experiments only
address the last two
parts
of
the
issue,
whether the
chelating agent
increases the root
uptake
and translocation
of the metal to the shoots. These
experiments
alone can't be transferred
directly
to
phytoextraction
in the field. The other
important part
is the solubilization of metals
from soil in order to increase the
availability
of metal to the roots. In this case the
chelating agent
increases the metal concentration in soil solution with
regards
to
untreated soil solution. From this work it is clear that EDDS increases
uptake
and
translocation in shoots of Pb
but,
it
may
not
necessarily
increase the accumulation
of Cu or Zn when total metal concentrations in solution are
equal.
Acknowledgements
We thank Diederik Schowanek from Procter & Gamble for
providing S,S-EDDS
and Kathrin
Wenger
for her
help
in the
experimental design.
This work was funded
in
part by
the Federal Office for Education and Science within COST Action 837
and the Swiss National Science Foundation in the framework of the Swiss
Priority
Program
Environment.
74
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
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78
Metal
uptake
from
hydroponics by
sunflowers in the
presence
of EDDS
(42) Marschner,
H. Mineral Nutrition of
HigherPlants;
Academic Press: San
Diego,
USA,
1986.
(43) Haynes,
R. J. Ion
exchange properties
of roots and ionic interactions within
the root
apoplasm:
Their role in ion accumulation
by plants.
Bot. Rev.
1980, 46,
75-
99.
(44) Haussling, M.; Jrns,
C.
A.; Lehmbecker, G.; Hecht-Buchholz, C; Marschner,
H. Ion and water
uptake
in relation to root
development
in
Norway Spruce (Picea
abies
(L) Karst).
J. Plant
Physiol. 1988, 133,
486-491.
(45) Clarkson,
D. T Root structure and sites of ion
uptake
In Plant Roots: The
Hidden
Half;
2nd
ed.; Waisel, Y, Eshel, A., Kafkafi, U., Eds.;
Marcel Dekker Inc.:
New
York, U.S.A., 1996; pp
483-510.
I.
G.; Mitrofanova,
N.
D.; Martynenko,
L.
I.;
Nazarov,
P.
P.; Spitsyn,
V I. Influence of
cyclization
of
N,N' -ethylenediaminedisuc
cinic acid on its
Bull. Acad. Sei. USSR Div. Chem Sei.
1983, 32,
1167-1175.
(47) Tandy, S.; Schulin, R.; Suter,
M. J.
F.; Nowack,
B. Determination of
[S,S']-
ethylenediamine
disuccinic acid
(EDDS) by high performance liquid chromatography
after derivatization with FMOC. J.
Chromatogr.
A 2005.
(48) Mller,
B.
ChemEQL,
V 2.0.
Eidgenssische
Anstalt fr
Wasserversorgung:
Dbendorf, Switzerland,
1996.
(49) Smith,
R.
M.; Martell,
A. E. Critical
Stability Constants;
Plenum Press: New
York, 1976;
Vol. 1-5.
(50) Martell,
A.
E.; Smith,
R.
M.; Motekaitis,
R. J. NIST
Critically
Selected
Stability
Constants of Metal
Complexes,
V6.0. NIST:
Gaithersburg, USA,
2001.
(51) Orama, M.; Hyvonen, H.; Saarinen, H.;Aksela,
R.
Complexation
of
[S,S]and
mixed stereoisomers of
N,N'-ethylenediaminedisuccinic
acid
(EDDS)
with
Fe(lll),
Cu(ll), Zn(ll)
and Mn
(II)
ions in
aqueous
solution. J. Chem.
Soc,
Dalton Trans.
2002, 24,
4644-4648.
(52) Systat Systat,
10.2.
Systat
Software Inc.:
Richmond, California, USA.,
2002.
79
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5
(53) Piechalak,
A
,
Tomaszewska,
B
,
Baralkiewicz,
D
,
Malecka,
A Accumulation
and detoxification of lead ions in
legumes Phytochem 2002, 60,
153-162
(54) Clemens,
S
,
Palmgren,
M G
,
Kramer,
U A
long way
ahead
understanding
and
engineering plant
metal accumulation Trends Plant Sei
2002, 7,
309-315
(55) Campbell,
P G C Interactions between trace metals and
aquatic organisms
a
critique
of the free-ion
activity
model In Metal
Speciation
and
Bioavailability
in
Aquatic Systems
,
2 ed
,
Tessier,
A
,
Turner,
D R
,
Eds
,
Wiley Chichester, 1999, pp
45-102
(56) Morel,
F M M
,
Hering,
J G
Complexation
In
Principles
and
Applications
of
Aquatic Chemistry,
2nd ed
,
Wiley
New
York, 1993, pp
321-420
80
6
Uptake
of metals
during
chelant-assisted
phytoextraction
with
EDDS related to the solubilized metal concentration
Susan
Tandy,
Rainer Schulin and Bernd Nowack
Submitted to Environmental Science and
Technology
Abstract
The use of chelants to enhance
phytoextraction
is one method
being
trialled to
make
phytoextraction
efficient
enough
to be used as a remediation
technique
for
heavy
metal
pollution
in the field. We
performed pot experiments
with sunflowers in
order to
investigate
the use of the
SS-EDDS for this
purpose.
Soil was contaminated with Cu
(360 mg kg-1)
and Zn
(530 mg kg-1)
in order
to
investigate single
and combined metals and mimic aerial contamination from a
metal smelter. In a second
experiment,
field contaminated soils with
multiple (Cu,
Zn,
Pb and
Cd)
metals were used. In order to
clarify
the
situation,
soil solution and
plant
metals were measured
along
with EDDS. 10 mmol EDDS
kg-1
soil increased
soil solution metals
greatly
for Cu
(x
840
-
4260)
and Pb
(x
100
-
315),
and to a
lesser extent for Zn
(x
23
-
50)
and Cd
(x2.5
-
38).
It was found that Zn
(when
present
as the sole
metal),
Cu and Pb
uptake by
by EDDS,
but in multi-metal contaminated
soil,
Zn and Cd were not. EDDS was observed in
the sunflower roots and shoots
proving
that it was taken
up by
the
plants.
A linear
relationship
between Cu and Zn in soil solution in the
presence
of EDDS and
plant
uptake
was
found, indicating
the
great importance
to measure and
report
dissolved
metal concentrations in
phytoextraction
studies. Our
study
shows that the main
factor
determining
whether metal
uptake
increases in the
presence
of chelants
is,
besides the solubilized metal
concentration,
the different efficiencies of metal
uptake
for different metals in the absence of chelants.
81
Chapter
6
6.1 Introduction
Heavy
metal contamination of soil is a
global problem.
The remediation of
heavy
metal
polluted
soils
by
traditional
physicochemical
methods is
generally
expensive
and the
physical
and
biological properties
of the soil can be
damaged
(1). Phytoextraction
is seen as a cost
in-situ
remediation
technique,
which strives to maintain soil
fertility
and structure
(2).
Three
main directions to
phytoextraction
have
developed:
1
)
the use of
hyperaccumulating
plants, 2)
the use of
high
biomass
plants
and
3)
the use of
high
biomass
plants
with induced accumulation of metals
(3).
As
hyperaccumulation
of
heavy
metals
is
generally
limited
by
the small biomass of the
hyperaccumulating plants (4),
the
focus has been switched to the induced accumulation of metals
using
chelants
by
high
biomass
plants (5).
The most studied metal for chelant enhanced
phytoremediation
is Pb due to its
high toxicity, widespread
distribution and its low
phytoavailability
in soils
(4, 6).
While
increases in shoot concentration
up
to a factor of 348 have been observed for
Pb,
the increase was
generally
lower for
Cu,
Zn and Cd
(7-10).
EDTA has been the most
commonly
used
chelating agent
for chelant enhanced
phytoextraction (4, 6, 7, 11-15) although
other chelants such as H EDTA
(6, 16),
DTPA(13, 16, 17)
and
NTA(13, 18)
have also been used.
EDTA,
HEDTAand DTPA
are
very environmentally
stable
(19)
and are therefore not ideal for chelant assisted
phytoremediation.
Slow
degradation
increases the risk of
leaching
mobilized metals
due to the weak interaction of chelates with mineral surfaces
(20, 21).
disuccinic acid
(EDDS)
is an isomer of EDTA but
is
easily biodegradable (22, 23).
We found that EDDS was better than EDTA at
solubilizing
Cu and Zn from soils at
pH
7 at
equimolar
ratios of
chelating agent
to
metals
(24).
A few studies have
recently
been carried out
using
EDDS for chelant-
assisted
phytoextraction
in
pot
or column
experiments, mainly
for Pb
(7, 25-28),
but
also for
Cu,
Zn and Cd
(27-30).
We have shown
previously
that EDDS increases the
uptake
of Pb from
hydroponic
solution
compared
to in the absence of EDDS but this
was not true for the essential metals Cu and Zn
(31 ).
The effect of chelants on
phytoextraction
was first
thought
to consist
merely
of
mobilizing
metals from the soil which increased the bioavailable fraction. The
uptake
of chelates was considered irrelevant.
However,
EDTA and EDDS have been
measured in
plants
either as individual
complexes (11, 32-34)
or as total EDTA
(12,
35, 36)
or EDDS
(31) proving
that
chelating agents
can be taken
up by plants
into
their shoots. It has been
proposed
that chelates
pass through
the roots to the
xylem
via a
fully apoplastic pathway (11, 37, 38) through
the root free
space (39).
Chelants
can cross the barrier of the
Casparian strip
where it is not
fully
formed at the root
82
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
tip, damaged by
lateral roots or
through passage cells,
to reach the
xylem
and from
there to be
transported
to the shoots
(37, 40, 41).
If chelants are taken
up by
this mechanism
then,
as it is
passive
in nature and
driven
by transpiration,
the
relationship
between soil solution metal
(complexed
to
chelants)
and shoot metal
uptake
should be linear. This has indeed been found for
Pb
(6, 14),
whereas Lai and Chen
(10)
observed a non-linear
relationship
for Pb and
Zn.
Uncomplexed
essential metals however do not take this route.
They
travel
through
the roots
by
a selective
symplastic pathway
where
they
enter the cells and travel
from cell to cell in the
cytoplasm
via
plasmodesmata.
It is known Cu and Zn have
transporters
to
help
them
pass through
cell membranes
(42).
Carrier mediated ion
transport through
membranes is
subject
to control
by
saturation kinetics
assuming
the number of carriers
(binding sites)
in the membrane is limited
(39).
The soil solution concentration of
complexed
metals is therefore an
important
parameter
to be measured and
reported
in chelant-assisted
phytoextraction
studies.
However, they
have
only occasionally
been measured
(6, 10, 11, 14, 43). Although
some work has been carried out with EDDS
previously,
either no measurement of
soil solution metals have been taken
(7, 25-28, 30),
or when
taken, they
were not
linked to the shoot metal concentrations
(29).
investigate
if EDDS could be used for chelant assisted
phytoextraction
and to
investigate
the link between soil solution metal concentration
and shoot metal concentration. We wished to
investigate
if the solubilization of
metals from soil
by
EDDS was more
important
than the effect EDDS has on
uptake
of metals
by
the
plant
from solution. We also wanted to
investigate
the
uptake
of
EDDS
by
the
plants.
Sunflowers were used as
they
are a
high
biomass
plant
with
reported
metal tolerance and accumulation
potential
in the field
(18).
Given the
literature reviewed it was
expected
that the metals studied would be increased in
plant
shoots
by
the addition of EDDS to soil as a linear function of the solubilized
metal concentration.
6.2.1 Soils
A
moist, (84
%
dry weight (DW)) loamy topsoil
from an
agricultural
field in
Birr,
northwest Switzerland
(Soil
1
)
was 2 mm sieved and for each treatment 20
kg (DW)
of soil were contaminated with either
Cu,
Zn or both. For the Cu treatment 9.02
g
Cu(ll)0 powder
was added to the soil and
thoroughly
mixed to
give
an addition of
360
mg/kg
Cu. The Zn treatment had 13.02
g Zn(ll)0 powder
added
(530 mg/kg Zn)
83
Chapter
6
and the ZnCu had both
Cu(ll)0
and
Zn(ll)0 powder
added
together
in the
quantities
mentioned before. The soils were then stored in
large
closed
plastic
containers in a
cool
dry place
for 7 months. Oxides were used as the
contaminating
material to
try
to mimic the
type
of contamination found in Soils 2 and 3
(see below). Periodically
the soils were mixed and
samples
taken to assess the extractable
metals,
which
had reached a
steady
state at the start of the
experiment.
The characteristics for
Soil 1 and the different treatments can be seen in Table 6.1.
Table 6.1. Initial soil characteristics.
Soil Treatment
pH
Corg
CaC03
Cu Zn Pb Cd Ni
(CaCI2)
% %
mg/kg mg/kg mg/kg mg/kg mg/kg
1 Control 6.42 0.94 0.16 26 94 30 < 0.5 25.1
1 Cu 6.38 0.94 0.16 430 94 29 < 0.5 25.9
1 Zn 6.30 0.94 0.16 25 596 28 < 0.5 24.8
1 ZnCu 6.32 0.94 0.16 409 594 28 < 0.5 26.4
2 5.65 2.73 0.48 537 786 66 2.07 46
3 6.87 1.79 1.74 111 191 39 0.53 32
4 5.07 1.24 0.49 52 515 386 0.7 21
Three field contaminated soils were taken from contaminated sites in northwest
Switzerland. The soil characteristics can be seen in Table 6.1. Soils 2 and 3 were
cultivated soils taken from the
municipality
of Dornach which had been contaminated
with
Cu,
Zn and Cd from an
adjacent
brass smelter. Soil 2 was a
heavily
contaminated
topsoil
of a non-calcareous
Regosol (Kirschgarten),
while Soil 3 was a
lightly
contaminated
topsoil
of a Calcaric
Regosol (Mattenweg).
Soil 4 was the
topsoil
of a
Haplic
Luvisol taken from in the
vicinity
of the
village Rafz,
north of
Zrich,
from an
agricultural
field contaminated with
Zn,
Pb and Cd from
sewage sludge applications.
All soils were dried at 40 C and sieved to < 2 mm
prior
to use.
6.2.2
Experimental Setup
A disk of fine
nylon
mesh
(60 urn)
was
placed
in the bottom of each
pot
and
1
kg (DW)
of soil was added to each
pot.
A Rhizon Flex soil moisture
sampler
84
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
(Rhizosphere
Research
Products, Wageningen, Netherlands)
was
placed
at 45
angle through
the soil. The soil was fertilized with 130
mg
N
kg-1
soil
(NH4N03),
164
mg kg-1
K
(KH2P04),
130
mg kg-1
P
(KH2P04)
and 42
mg kg-1 Mg
(MgS04.H20)
given
in the form of 100 ml nutrient solution
per pot.
Each
pot
was moistened to about 60%
of the water
holding capacity (WHC) by adding
ultra
pure
water
(Millipore, Bedford,
MA).
The
pots
were
kept
at about this water content
throughout
the
experiment.
The
experiment
was carried out in a
growth
chamber on a 16 hr
(21 C)
/ 8 hr
(16 C)
day/night cycle.
For each treatment and soil
type,
6
replicate pots
were
planted
with sunflowers
Helianthus annuus
(cv Iregi)
and 6
replicates
were left bare. In each
planted pot
5 seeds were
planted initially.
One week after
germination
four
seedlings
were
removed
leaving
1
seedling per pot.
After 3 weeks of
growth (4
weeks after
planting)
200 ml of either ultra
pure
water
(3 replicates)
or EDDS
(pH 7.12,
50
mM,
to
give
10
mmol
kg-1) (3 replicates)
was added to each
pot (bare
and
planted).
After 24 hours
20 ml of soil solution was extracted from the bare
pots
via the Rhizon Flex
samplers.
After 3 more
days (total
4
days)
100 ml water was added to the bare
pots
and after 2
hours the soil solution was
again
extracted. Planted
pots
were watered
according
to
the needs of the
plants,
i.e. as much as
necessary
to
keep
the soil moist.
Samples
were
refrigerated
until
analysis.
The
plants
were harvested 5
days
after the addition of EDDS
by cutting
the stem
1 cm above the soil. The shoots were washed with deionised water and dried at
40C. The
temperature
was limited because EDDS can
degrade
or
cyclize
at
high
temperatures (44).
The soil from the
planted pots
was sieved
(2 mm)
in order to collect the roots.
Although
it was not
possible
to collect all the
roots, especially
the
very
fine
ones,
it
is
thought
that an
equal proportion
of roots were collected from each
pot.
The roots
were well washed with deionised water and dried as the shoot
samples.
The oven-
dried
plant
material was
ground
in a titanium mill.
6.2.3 Metal and EDDS
Analysis
Plant
samples
were microwave
digested
in 5 ml
HN03
(65%),
2 ml
H202
(30%),
and 2 ml
H20.
The
digested samples
were diluted to 25 ml in
Millipore
water.
Digests
and soil solution
samples
were
analysed
for metals
by
Flame-AAS
(Varian,
SpectraAA220FS)
and GF-AAS
(Varian, SpectraAA300
with
GTA96) (Soil 1)
or
by
ICP-OES
(Varian,
Vista-MPX CCS
simultaneous) (Soil
2
-
4).
Soil solution results
were normalized to a
gravimetric
soil water content of 30%. This was done because
the four soils had different water
holding capacities
and because in the
planted pots
it was difficult to control soil water content due to
plant
water
consumption.
NaN03
extractable soil metal concentrations were determined
by
extraction of
85
Chapter
6
the soil for 2 hours with 0.1 M
NaN03
followed
by
filtration
(45).
Total metal
analysis
of the soil was carried out
by X-ray
fluorescence
spectroscopy (Spectro
X-lab
2000,
Germany).
For EDDS
analysis,
dried
plant
material was extracted with
pure
water
(10 mg
/ 10
ml) by
sonication with a
micro-tip
sonic
probe
for one minute. The
samples
were surrounded
by
ice
during
sonication to
were then
centrifuged
and filtered
(0.45 urn).
EDDS derivatization and
analysis
was carried out
as described
by Tandy
et al.
(46).
This method involves the derivatization of EDDS
by
FMOC
(Fluorenylmethyl chloroformate, puriss, Fluka)
followed
by separation by
HPLC
(Jasco PU-980)
and fluorescence detection
(Jasco 821-FP).
6.2.4 Calculation of shoot metal
uptake
after EDDS addition
For
comparing
shoot metal and EDDS
uptake
after the addition of
EDDS,
the
metal
uptake
was calculated
by subtracting
the
pre-addition
metal
uptake
from the
total metal content. To do this we summed
up
the concentrations of
Cu,
Zn and
Fe
(Soil 1)
and
Cu, Zn, Pb, Cd,
Ni and Fe
(Soils
2
-
4)
in the
plant
shoots for each
treatment. 80 % of the value from the treatments in which
only
water was added
(H20
treatments)
was then subtracted from the
corresponding
EDDS treatment.
Only
80 % was subtracted as the
plants
were
grown
for 26
days
in
total,
21
days
prior
to the addition of EDDS and 5
days
afterwards.
For
investigating
the mechanism of metal
uptake
in the
presence
and absence
of
EDDS,
the shoot metal
uptake
for the 5
days
after EDDS addition was used. For
EDDS treatments it was calculated as above but
using
individual metal values and
not the sum of the different metals. For the
H20
treatments 20% of the summed
metal value was considered as
uptake
in the last
part
of the
experiment.
6.2.5 Chemicals
All chemicals were obtained from Merck unless stated otherwise and were
analytical grade
or HPLC
grade
for the solvents. SS-EDDS
(Octaquest E30)
was
obtained from
Octel,
Cheshire for the
experiments
and from Proctor and Gamble
(Belgium)
as the
Na3EDDS
salt for the EDDS
analysis.
All solutions were made with
high purity
water
(Millipore, Bedford, MA).
6.2.6 Statistical
analysis
All statistical
analyses
were carried out with
Systat
10.2
(47).
ANOVA was
carried out on
log
transformed data
except
for
dry weight analysis.
Differences were
considered
significant
if
p
= < 0.05.
86
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
6.3 Results
6.3.1 Plant
dry weight
Plant shoots showed adverse effects to the addition of EDDS. Two
days
after
the addition of EDDS to the soil the shoots started to show
signs
of
toxicity
and
by
3
days
were necrotic. EDDS also seemed to reduce the shoot
dry weight (Table
6.2).
In
previous hydroponics investigations
no
signs
of
toxicity
were seen for 500
uM EDDS
(31).
In the
pot experiments
the concentrations of EDDS in soil solution
were around 25
mM,
about 50 times
greater
than in the
hydroponics experiment.
Necrosis and loss of
dry weight
has been noted
previously
for smaller additions
(5
mmol
kg-1)
of EDDS
(28)
and also for EDTA
(25, 26, 36).
Shoot
dry weight
was not
adversely
affected
by
the Cu treatment and
only
showed a small reduction in the Zn treatment
compared
to the control
(Table 6.2),
but was
greatly
reduced in the ZnCu treatment however
(p
=
0.002).
The root
dry
weight
showed a similar trend to shoot
dry weight (Table 6.2)
but with even
greater
Table 6.2. Shoot and root biomass of sunflowers.
Soil Treatment
Shoot
dry weight
(g)
SE
Root
dry weight
(g)
SE
Control
(1)
H20
7.50 0.96 0.24 0.43
Control
(1)
EDDS 4.52 0.46 0.66 0.20
Cu(1)
H20
8.00 0.58 2.05 0.04
Cu(1)
EDDS 4.76 0.11 0.78 0.04
Zn(1)
H20
4.69 1.70 1.04 0.20
Zn(1)
EDDS 2.82 0.39 0.60 0.06
ZnCu(1)
H20
1.59 0.29 0.58 0.14
ZnCu(1)
EDDS 1.44 0.46 0.46 0.16
2
H20
0.44 0.22 0.14 0.06
2 EDDS 0.53 0.10 0.14 0.03
3
H20
8.32 0.89 2.52 0.12
3 EDDS 5.24 0.73 1.57 0.30
4
H20
0.47 0.32 0.22 0.15
4 EDDS 0.36 0.12 0.10 0.02
87
Chapter
6
reductions due to Zn
(p
=
0.016),
ZnCu
(p
=
0.001
),
and EDDS
(p
=
0.001
) compared
to the control. Both shoot and root
dry weights
were much smaller on the
heavily
contaminated Soils 2 and 4 than on the
lightly
contaminated Soil 3
(p
=
<0.001
)
and
were even less than the ZnCu treatment of Soil 1. As the Soils 2 and 4 had multi-
metal contamination it was
expected
that the
dry weight
would be similar to the
ZnCu treatment. While the
NaN03
extractable Cu concentrations were similar in the
ZnCu treated Soil 1 and Soils 2 and 4 in the absence of EDDS
(results
not
shown),
the
respective
Zn values were between 4-8 fold
greater
in Soils 2 and 4 than in
ZnCu treated Soil 1.
Therefore,
we
suspect
that the
high availability
of Zn was the
cause for the low
growth
of sunflowers in Soils 2 and 4.
6.3.2 Solubilized Metals
Soil solution Cu and Zn were both
significantly
increased one
day (Figure
6.1a
and
b)
and 4
days
after the addition of 10 mmol
kg-1
EDDS to Soil 1
(p
=
<0.001).
Soil solution was
only
extracted from the bare
pots
because it was not
possible
to
extract solution from the
planted pots
after 1
day
due to water
consumption
of the
plants.
In the second
sampling
3
days later,
it was
possible
to extract some solution
from some
planted pots
2 hours after
adding
water to the soil. After
estimating
the
water content of the
planted pots
based on the fresh
weight
of the
plants
at harvest
on
day 5,
the soil solution metal concentrations from
planted
and bare
pots
were
compared using
ANOVA.
Only
the Cu concentrations differed
significantly
between
bare and
planted pots
for the control and EDDS treatments. All other treatments
and metals
proved
to be not
significantly
different due to the
large
variation
among
replicate samples.
In
light
of
this,
we feel the use of soil solution from the bare
pots
is
justified.
The addition of EDDS to soil
dramatically
enhanced the soil solution concentrations
for Cu
(x
840
-
4260),
Zn
(x
23
-
50 for Soils
1,
2 and
4,
x
8000 for Soil
3),
Pb
(x
100
-
315)
and Cd
(x
2.5
-
38) (Figure 6.1).
Soluble Fe increased from around the
detection limit
(0.4
or 1.8 uM
depending
on the
analysis method)
to between 1000
-
3350 uM
upon
EDDS addition
depending
on the soil. The
pH
of soil solutions in the
EDDS treatments can be seen in Table 6.3.
6.3.3 Plant metal
uptake
Shoot Cu
uptake
was
significantly
enhanced
by
EDDS in all treatments but
especially
in the Cu
(x
11
)
and ZnCu
(x 8)
treatments
(p
= < 0.001
) (Figure 6.2a).
Cu
uptake
from the ZnCuEDDS treatment did seem to be less than from the CuEDDS
treatment.
Zn shoot
uptake
was
only
enhanced
by
EDDS in the ZnEDDS treatment
(x 1.7)
88
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
O
_<D
-Q
O
if)
10000
1000
100-
10-
1-
0.1
a)
10000
V T
Control
Cu
ZnCu
b)
r~"
10000
M000
^
3.
100 N
ID
O
if)
M0
1
0.1
Control Zn ZnCu
zl
ID
_^
o
c/)
iooo4
100
10
1
0.1
c) d)
r**
-1000
MOO
N
Soil 2 Soil 3 Soil 4
10000
M0
1
0.1
100 -r
zL
ID
_
ID
o
if)
10
-
1
-
0.1
-
0.01
e)
f)
MO
-1
rt
0
ID
o
C/3
O
ID
O
if)
-0.1
.2
0.01
Figure
6.1. Soil solution concentration of
heavy
metals. Soil 1 Cu
(a)
and Zn
(b)
and Soils 2
-
4 Cu
(c),
Zn
(d),
Pb
(e)
and Cd
(f),
24 hours after addition of
H20
(diagonal stripped bars)
or EDDS
(white bars).
Error bars are standard errors.
89
Chapter
6
Table 6.3. Soil solution
pH
of EDDS treatments 4
day
after EDDS
addition,
bare
pots.
Soil
Control
(1)
Cu(1)
Zn(1)
ZnCu(1)
Soil 2
Soil 3
Soil 4
Treatment
PH
EDDS 6.56 0.08
EDDS 6.20 0.03
EDDS 6.46 0.07
EDDS 6.21 0.20
EDDS 5.48 0.09
EDDS 7.36 0.01
EDDS 5.28 0.13
(p
=
0.035)
and not in the ZnCuEDDS treatment
compared
to the
respective
metal
only
treatments
(Figure 6.2b).
In the absence of
EDDS,
Zn
uptake
from the ZnCu
treatment was much
greater
than from Zn
treatment,
while in the
presence
of EDDS
Zn
uptake
in these two treatments was not
significantly
different
(p
=
1.0).
Fe shoot
uptake
was increased
by
between 2.8 and 5.1 times
(p
=
<0.001) (2341
-
3379
umol/kg)
in the
presence
of EDDS.
In the field contaminated soils shoot Cu was enhanced
by
EDDS in all treatments
(Figure 6.2c).
Zn shoot
uptake
saw no enhancement in the
presence
of EDDS in
soils that were
heavily
contaminated with Zn.
Uptake
was
only
enhanced in Soil
3,
which was
lightly
contaminated
(x 3, p
= <
0.001).
Pb shoot
uptake
was increased
by
EDDS
(x 4.3, p
=
0.025)
in Soil
4,
the
only
soil
substantially
contaminated with
Pb
(Figure 6.2e).
Cd shoot
uptake
saw no
significant
enhancement in the
presence
of EDDS
(Figure 2f).
Root metal
uptake
was
generally
not enhanced
by
the addition of EDDS to soil
(Table 6.4).
This is
probably
due to the fact that in the
presence
of
EDDS,
dissolved
metals are
primarily present
in the form of metal-EDDS2-
complexes
which are
unable to bind to cation
exchange
sites in the roots unlike free metal cations
(e.g.
Zn2+)
that are found in unadulterated soil solution.
6.3.4 EDDS
uptake
EDDS was detected in shoots and roots of sunflowers from all treatments where
EDDS was added to the
soil, showing
that it was taken
up
into the above
ground
biomass of the
plants.
The concentrations of EDDS in the shoots and roots were
not
significantly
different between
treatments,
but in most cases root EDDS was
90
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
3x10
^ 2x10-
"
E
zL
=5 1 X103-J
o
0-
a)
r**"t
2x10"
M.5X10V
Hx104

-5x10
3 C
N
Control Cu
Zn ZnCu
Control Cu
Zn ZnCu
5x10c
7
4x10M
CD
.*:
-5
3x10 4
E
zL
=3
o
2x10
-
1 x103-
0
C)
rt.
d)
fi
,^r*i
i
-3x10"
V2
x104 V
CD
^2x104 "5
E
4
i
N
-1 x10
^5x10"
0
80^
, -, ,-40
CD
.*:
E
.Q
Q_
60-
40-
20-
0
e)
*r^
f)
-30
-20
M0
0
cd
"5
E
T3
O
Figure
6.2. Shoot
uptake
in Soil 1 of Cu
(a)
and Zn
(b)
and in Soils 2
-
4 of Cu
(c),
Zn
(d),
Pb
(e)
and Cd
(f,)
in the absence
and
presence
(white bars)
of EDDS. Error bars are standard errors.
91
Chapter
6
Table 6.4. Root metal concentrations.
Soil Treatment
Cu
mg/kg
Zn
mg/kg
Pb
mg/kg
Cd
mg/kg
SE SE SE SE
Control
(1)
Control
(1)
Cu(1)
Cu(1)
Zn(1)
Zn(1)
ZnCu(1)
ZnCu(1)
H20
EDDS
H20
EDDS
H20
EDDS
H20
EDDS
219 26 884 110
326 9 1675 199
3747 265 926 73
5581 572 1290 36
283 14 12549 2455
272 18 13222 810
2941 220 10444 1102
2883 292 9872 344
2
2
3
3
4
4
H20
EDDS
H20
EDDS
H20
EDDS
6531 855 18074 695
7180 889 12093 644
1023 105 1048 169
1975 240 2338 123
869 102 22975 3474 483
36
2102 577 15558 2901
77313
55 6.7 74 5.4
54 4.5 72 9.4
21 4.0 < 8.8 0.05
34 2.0 24 1.2
483
36
< 79 32
77313
< 61 17
2x10
f 1.5x104J
Q
Q
3
lu
5x103
0
111 iii
#ppp
MME
Iiiii
pllll
.l.
iilj
liiis
illtl
liiii
liiii
mm
Ml
lilt
m
mi
-^piiii
PPP
_Ufi_
m
M
IIIII
IIIII
l.
EDDS CuEDDS ZnEDDS ZnCuEDDS
Figure
6.3. EDDS
uptake
in shoots
(white bars)
and roots
(shaded bars)
from Soil
1. Error bars are standard errors.
92
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
greater
than shoot EDDS
(Figure 6.3).
The difference between roots and shoots
was not as
great
as
previously
seen from
hydroponics
solutions
(31).
There lower
concentrations of EDDS were
applied (0.5 mM)
and the difference between roots
and shoots was at least an order of
magnitude.
However the
magnitude
of increase
of EDDS concentrations in shoots
compared
to the
was
similar to the
magnitude
of increased EDDS in the
solution,
but the increase in root
EDDS did not follow this trend.
2x10
en
1.5x10'
E
1x104-|
LU
^ 5x103J
CD
-i>
Q)
5
o
a)
r^ n
EDDS CuEDDS ZnEDDS ZnCuEDDS
2x10
1.5x104-]
o
1 x104J
Q
LU
75
5x103J
-i
0
*
b)
n 1
Soil 2 Soil3 Soil 4
Figure
6.4. Metal
(diagonal stripped bars)
and EDDS
(white bars) uptake
in shoots
from EDDS treatments after the addition of EDDS.
a)
Soil
1, b)
Soils 2
-
4. Error bars
are standard errors.
6.3.5 Metal
uptake
versus EDDS
uptake
In the control treatment with
EDDS,
more EDDS was
present
in the shoots than
93
Chapter
6
metals
(Figure 6.4a).
For the CuEDDS and ZnCuEDDS treatments
equal
amounts of
metals were found in the shoots
compared
to EDDS. This is the same as seen for Cu
and Zn in
hydroponics experiments (31).
In the ZnEDDS treatment there was more
metal in the shoots than
EDDS,
a
phenomenon
that hasn't been encountered before.
In all field contaminated soils
roughly equal quantities
of metal and EDDS were
taken
up (Figure 6.4b).
6.3.6
Speciation
of EDDS
We have determined the
speciation
of EDDS for
day
4 of the
experiments
with Soil 1.
We assumed that all
Cu,
Zn and Fe in solution were
complexed
with EDDS. EDDS not
complexed
tothese metals
ranged
between
35and70%(Table6.5).
Model calculations
by
Hauser et al.
(48)
have shown that
Ca, Mg
and Mn are the main additional ions
that
complex
EDDS in addition to
heavy
metals. The EDDS treatment had the
largest
free EDDS concentration followed
by
the CuEDDS treatment and then the treatments
containing
Zn. CuEDDS
ranged
between 27
-
31 % in the treatments with added
Cu and 1.7-2.1 % in the non-Cu treatments. Likewise ZnEDDS was 1.2
-
2.5 % in
non-Zn treatments. ZnEDDS was
greater
in the ZnEDDS treatment
(50 %)
than in
the ZnCuEDDS treatment
(28%) probably
due to the increased
competition
with Cu
for EDDS in the dual metal treatment. FeEDDS decreased with
increasing heavy
metal contamination.
Table 6.5.
Speciation
of soil solution taken 4
days
after EDDS addition from Soil 1.
Values
presented
as a
percentage
of the total EDDS concentration.
Treatment "Free" EDDS1 CuEDDS ZnEDDS FeEDDS
EDDS 70 2.9 2.1 0.5 2.5 0.4 25 2.0
CuEDDS 56 7.6 31 7.2 1.2 0.2 12 0.8
ZnEDDS 35 1.2 1.7 0.1 50 0.8 14 0.4
ZnCuEDDS 38 1.5 27 2.8 28 3.2 7.2 0.8
1
Free EDDS and
complexes
with
Ca, Mg,
and Mn
6.3.7
Relationship
of soil solution to
plant uptake
Figures
6.5 and 6.6 show the
relationships
between metal
uptake by
sunflowers
and the measured solubilized metal concentration in soil solution. In
addition,
the
data from the
of
Tandy
et al.
(31 )
is included. In the absence
94
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
S 600J
3
o
o
CO
25 50 75 100 125
Total Soluble Cu uM
150
5000
'co
4000-
2000 4000 6000 8000 10000
Total Soluble Cu uM
Figure
6.5.
Uptake
of Cu over 5
days,
in the absence
(a)
and
presence (b)
of EDDS.
Black diamonds
pot experiments, open
diamonds
hydroponics experiment (ref.
(31)).
of
EDDS,
shoot accumulation of Cu and Zn shows a
strong
increase with
increasing
concentrations in soil solution and then saturation
(Fig
6.5a and
6.6a).
This behaviour
is
typical
for carrier driven
transport
and can be described
by
a Michaelis-Menten
type equation (39).
The
parameter
values
giving
the best fits for
experimental
data
are
given
in Table 6.6. For Zn the
hydroponics
data is not included in the
regression
as it can be seen to be much
greater
than for the
pot experiments.
In soil solution not
all Zn will be in the 'free' form which would be taken
up by
a selective mechanism.
In the
hydroponics
solution most metal was in the 'free' form.
A
completely
different
relationship
was obtained for the
uptake
in the
presence
of
EDDS
(Figure
6.5b and
6.6b).
In this case the
dependence
on metal concentration
95
Chapter
6
18000
100 200
Total Soluble Zn uM
300
10000-
co
8000-
6000-
N
4000J
o
o
if)
2000-
2000 4000 6000 8000 10000
Total Soluble Zn uM
Figure
6.6.
Uptake
of Zn
duringover
5
days,
in the absence
(a)
and
presence (b)
of EDDS. Black diamonds
pot experiment, open
diamonds
hydroponics experi
ment
(ref. (31)).
in solution was linear. The
parameters
of the lines are
given
in Table 6.6. It was not
possible
to establish the
analogous relationship
for Pb as
only
one treatment
gave
shoot concentrations above the detection limit. Fe also showed a linear
relationship
in the
presence
of
EDDS, except
for an
exceptionally high uptake
of Fe in the EDDS
treatment of Soil 2.
Uptake
of Cu was different
compared
to Zn and Fe in the
presence
of
EDDS,
as can be seen from the
slopes (a)
in Table 6.6. All have linear
relationships
with
soil solution
concentrations,
but the
uptake
of Zn and Fe was twice that of Cu.
This difference was
significant.
In
no
significant
influence
96
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
Table 6.6. Constants for
uptake
of Cu and Zn
by
selective
(non-complexed metal)
and
non-selective
(complexed metal)
mechanisms. The metal
uptake
for
H20
treatments
was fitted to a Michaelis-Menten
type equation (39)
max Cs
S= shoot uptake, S
=
maximum shoot concentration
\J
'
max
K+Cs Cs
=
concentration of metal
ion,
K-
constant, equal
to the ion concentration
giving
half the
maximum shoot
uptake.
The increase of the lvalue reflects the
affinity
of the carrier sites for the metal.
The metal
uptake
in the
presence
of chelates was modelled with a linear model:
metal
uptake .
,
=
a
*
metal
,,
(in umol
kg-1 and uM).
~
shoot solution
x
~
a
~
'
Metal Mechanism
S
max
(pmol kg-1)
K
(pM)
a R2
Cu Selective 817 49 0.99
Zn Selective 3329 15.95

0.74
Cu Non-selective 0.47 0.98
Zn Non-selective

0.97 0.90
Fe Non-selective

1.38 0.741
1
with outlier removed.
of the EDDS
speciation
on chelate
uptake
was found
(31).
In the
pot experiment
however,
ZnEDDS and
Fe(lll)EDDS
were taken
up
more efficient than
CuEDDS,
a
phenomenon
that needs further
investigation.
6.4 Discussion
6.4.1
Comparison
of shoot metal concentrations
Metal accumulation in the shoots fitted within the broad
range
for concentrations
of metals in
plants
in the
presence
of
chelating agents.
For
sunflowers,
the increase
in Cu and actual concentrations in our
experiment
was much
greater
than in other
experiments (18, 29).
For
experiments
with EDDS our results were also similar or
better than others
(29, 30), except
for one
experiment
where the soil was
artificially
contaminated and corn and beans were
used,
which seem to have an
extraordinary
97
Chapter
6
ability
to take
up
metals
(28).
For Zn we obtained a lower increase than in other studies with sunflowers
(18,
29), although
the actual shoot concentrations were
higher.
The same was true when
we
compared
our shoot concentrations to those in other
experiments
conducted
with EDDS
(7, 27-29).
This
may
be due to the fact that ZnEDDS is a much weaker
complex
than ZnEDTA
(49)
which was used in the
majority
of other studies. In the
other EDDS studies soil Zn was
always
much
greater
than Cu with which it must
compete
for EDDS and which has a
higher stability
with EDDS. Ourfield contaminated
soils also had
very high
bioavailable Zn.
Pb accumulation was lower than in other sunflower
experiments (6, 15-17)
and
other EDDS
experiments (7, 25-28).
PbEDDS is weaker
by
5 orders of
magnitude
than
PbEDTA,
so Pb will be much worse at
competing
with other metals for EDDS
than for EDTA. Our Pb contaminated soil also was contaminated with Zn to a similar
degree,
while other EDDS studies and most studies on
phytoextraction
of Pb used
soil that had Pb as the main contaminate. No increase in Cd accumulation was
observed and is
explainable by
the weak interaction of EDDS with Cd
(log
K
=
10.8)
(49).
Despite
the
strong
enhancement
by
EDDS the maximum accumulation of Cu in
shoots was
only
0.02 % and for Pb 0.0011 % DW. For
phytoextraction
to be viable
the shoots need to accumulate in the order of 1 %. Thus these results are still far
from the mark.
6.4.2 EDDS
uptake
The measurement of EDDS in roots and shoots
clearly proves
that substantial
uptake
of EDDS had taken
place.
Whereas the root concentration was similar to that
observed in
hydroponics (about
4000
umol/kg) (31 ),
the shoot concentration in the
pot
was about 30 times
higher
than in
hydroponics. However,
the EDDS concentration
in the soil solution was also about a factor of 50
higher
in the
pot experiments.
This
suggests
that shoot
uptake
increases in
proportion
to the dissolved
EDDS,
while
the root EDDS follows a
saturation-type
behaviour with
relatively strong binding
of
EDDS to the root cells
(31).
The fact that accumulation of metals and EDDS in the shoots was
approximately
equal
in most EDDS treatments indicates that metals were taken
up
in the
complexed
form or that all EDDS was
complexed
once inside the
plant. Uptake
of free EDDS
that later reacted with metals within the
plant
is
suggested by
the
speciation
calculations. In soil
solution,
35-70% of the EDDS was not bound to
Cu,
Zn or Fe. A
similar behaviour was found in
hydroponics
where inside the
plant equal
amount of
metal and EDDS were found
despite
the fact that in solution most of the EDDS was
present
in
uncomplexed
form.
Only
in the
EDDS-only
control treatment of Soil 1 was
98
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
the metal
uptake
less than the EDDS
uptake.
The
majority
of EDDS in solution of
that treatment was
uncomplexed
due to the low levels of metals in the soil. It seems
therefore that free-EDDS was taken
up by
the shoots and was
complexed
later
by
Cu,
Zn or other metals. This means that extra metals must have been
sequestered
and
transported
from the soil solution to the location where
complexation
took
place.
This could have come from the metals adsorbed and taken
up
into the roots in the
time before EDDS was added.
Alternatively,
trace levels of free
Cu,
Zn or Fe in
the soil solutions could have been taken
up very efficiently by
a selective
uptake
mechanism,
as
suggested by previous hydroponics experiments (31).
6.4.3 Metal
uptake
in the
presence
of EDDS
It is difficult to
compare
our observed metal concentrations in the
presence
of
chelants to literature values because soil solution metal concentrations are
hardly
ever
reported.
Differences in
uptake
after addition of chelants
may
be related in
part
to the
varying extractability
of metals and their
varying
concentrations in soil
solution.
The linear
relationship
between chelate
uptake
and soil solution concentrations
for Cu and Zn is a new
finding
for EDDS.
Although
one EDDS
study
carried out
soil solution metal
analysis, they
did not made the link
directly
to
plant uptake (29).
This
linearity
and the
presence
of EDDS in the sunflower shoots are evidence that
the chelates are indeed taken
up by
the
passive apoplastic pathway
into the
xylem
and are then
transported
to the
plant
shoots. One
previous study
found a non-linear
relationship
between Zn shoot content and soluble Zn in the
presence
of EDTA.
However the shoot concentration was not corrected for Zn taken
up
before the
addition of EDTA to soil
(10).
Two other studies have also found linear
relationships
between Pb
uptake
and soil solution concentrations in the
presence
of chelants
(6,
14).
As
expected
metal
uptake
of Cu and Zn from soil solution not
containing chelating
agents
did not follow a linear
relationship,
but saturation limited kinetics which could
be described
by
a Michaelis-Menten
type equation.
The different kinetics
strongly
suggests
that the
uptake
of essential metals in the absence and
presence
of
chelating
agents
is dominated
by
different mechanisms.
6.4.4 Factors
influencing
chelant assisted
phytoextraction
Obviously speciation
of metals and
competition
for the chelants in the soil
solution
play
an
important
role in
determining
the
uptake
of
contaminating
metals
from multi-metal contaminated soils. There is now evidence in literature
suggesting
that the effects of chelants such as EDDS on metal
uptake
can be
explained by
a shift of the main
transport
route from the
symplastic pathway (selective uptake)
99
Chapter
6
to the
apoplastic pathway (non-selective uptake) (11, 37, 38). Figure
6.7 shows a
schematic
illustrating
this
hypothesis
for the
uptake
of Cu and Zn. In the absence of
EDDS
uptake mainly
occurs
by
a selective
uptake
mechanism.
By adding EDDS,
the Zn soil solution concentration is increased but the
plant uptake
decreases. This
is because free Zn concentrations decrease and thus selective
uptake
is
greatly
reduced and the unselective
uptake
of metal-EDDS
complexes (dashed line)
is less
efficient than the selective mechanism. Cu
uptake
in the absence of EDDS
(dash-
dot
line) although selective,
is less efficient than for Zn.
By adding
EDDS the solution
concentration increases to a level above the
intercept
of the two lines
representing
the two different mechanisms of Cu
uptake.
This means that shoot Cu
uptake
in
the
presence
of EDDS is
greater
than in the absence. Another factor
adding
to this
situation is that the soil solution concentration of Cu in the absence of EDDS
(dot
on
the Cu
line)
is much less than for
Zn, making
the increase in solution on the addition
of EDDS
greater
than for Zn.
A hurdle for
phytoremediation
is the
growth
of the
plant prior
to chelant addition.
CD
CO
CO
-I
CD
co
Soil Solution Metal
Figure
6.7. Schematic
illustrating
how the effects of EDDS on Cu and Zn
uptake
into
plant
shoots can be
explained by
the
change
from selective
uptake
of metals to
non-selective
uptake
of metal-EDDS
complexes.
Solid
line,
selective
uptake
of
Zn;
dot-dash
line,
selective
uptake
of
Cu;
dashed line unselective
uptake
of Cu and Zn
bound to EDDS. The black and
open
circles
represent
solution concentrations of Zn
and Cu
respectively,
in the absence of EDDS and the shaded area
represents
metal
concentrations in the
presence
of EDDS.
100
Uptake
of metals
during
chelant-assisted
phytoextraction
with EDDS
Chelants
may
decrease the
growth
of a
plant.
This need not be a
problem
if
they
are
added
shortly
before harvest. A
high
biomass at the
point
of addition is
important
in order to
provide
a
high transpirational
flow for metal-chelate
uptake.
In Soil 1
Cu and Zn in combination were more toxic to the
plants
than alone at the same
concentrations. In Soils 2 and 4 the co-contamination of Cu and Zn
coupled
with
the
highly
available form of Zn reduced sunflower
growth
even more. However it
is
possible
that once the sunflowers reach a mature
stage
their biomass would no
longer
be affected. It was found for sunflowers
growing
on the Aznalcollar mine
spill
in
Spain,
that
although
shoot and root biomass of
seedlings
was reduced this was
not the case for sunflowers at the mature
stage (50).
If this was not the case then
only moderately
contaminated soils
(such
as Soil
3)
or soils with low
bioavailability
of the
contaminating
metals could be decontaminated in this
way.
Acknowledgements
We thank Diederik Schowanek from Procter & Gamble and
Raj
Patel from
Octel,
U.K. for
providing
the
S,S-EDDS.
part by
the Federal Office
for Education and Science within COST Action 837 and the Swiss National Science
Foundation in the framework of the Swiss
Priority Program
Environment. We also
thank Madeleine
Gnthardt-Goerg
from WSLfor
providing
soil from the Cell-to-Tree
framework and Werner
Attinger,
Anna Grnwald and Ren Saladin for their technical
assistance.
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823-826
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B
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B
,
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leaching
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EDTA-enhanced
phytoremediation
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2003, 254,
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W
W, Unterbrunner, R, Sommer, P, Sacco,
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phytoextraction using
canola
(Brassica napus
L
)
in outdoors
pot
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lysimeter
experiments
Plant Soil
2003, 249,
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D
,
Feijtel,
T C J
,
Perkins,
C M
,
Hartman,
F A
,
Federle,
T
W,
Larson,
R J
Biodegradation
of
[S,S], [R,R]
ethylene
(EDDS),
a transition metal chelator
Chemosphere 1997,
34,
2375-2391
(23) Vandevivere, P, Saveyn, H, Verstraete, W, Feijtel, W, Schowanek,
D
Biodegradation
of
metal-[S,S]-EDDS complexes
Environ Sei Technol
2001,35,
1765-1770
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S
,
Bossart, K, Mueller,
R
,
Ritschel,
J
,
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L
,
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R
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103
Chapter
6
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B. Extraction of
heavy
metals from soils
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phytoextraction
/ soil
washing
of Lead
using
biodegradable
chelate and
permeable
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D. Influence of
and
chelate and
hydrogel
modified soil water
sorption capacity
on Pb
phytoextracton
and
leaching.
Plant Soil
2003, 253,
403-411.
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Ethylenediaminediss
uccinate as a new chelate for
environmentally
safe enhanced lead
phytoextraction.
500-506.
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X. Enhanced
phytoextraction
of
Cu, Pb,
Zn and Cd
with EDTA and EDDS.
1-11.
(29) Meers, E.; Ruttens, A.; Hopgood,
M.
J.; Samson, D.;Tack,
F. M. G.
Comparison
of EDTA and EDDS as
potential
phytoextraction
of
heavy
metals.
1011-1022.
D. Chelator induced
phytoextraction
and in situ soil
washing
of Cu. Environ. Pollut.
2004, 132,
333-339.
(31) Tandy, S.; Schulin, R.; Nowack,
B. The influence of SS-EDDS on the
uptake
of
heavy
metals in
hydroponically grown
sunflowers.
Chemosphere
submitted.
(32) Collins,
R.
N.; Onisko,
B.
C; McLaughlin,
M.
J.; Merrington,
G. Determination
of metal-EDTA
complexes
in soil solution and
plant xylem by
ion
chromatography-
electospray
mass
spectrometry.
2001, 35,
2589-2598.
(33) Schaider,
L.
A.; Sedlak,
S. L.
Uptake
of metal-EDTA
complexes by
Brassica
juncea: implications
for the free ion
activity
model and
phytoremediation.
Abstracts
of
Papers
of the American Chemical
Society 2003, 226,
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J.; Hazemann,
J. Accumulation forms of Zn and Pb in Phaseolus
vulgaris
in the
104
Uptake
of metals
during
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with EDDS
presence
and absence of EDTA. Environ. Sei. Technol.
2001, 35,
2854-2859.
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P.
F.; McLaughlin,
M.
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D.
P.; Owens, G.; South,
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uptake
of
C-14-EDTA, C-14-Citrate,
and C-14-Histidine from chelator-
buffered and conventional
hydroponic
solutions. Plant Soil
2003, 253,
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A.
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and accumulation
by
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Phytoremediation
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versus
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1919-1926.
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G.; Mitrofanova,
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FAL Extraktion von Schwermetallen mit Natriumnitrat
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In Schweizerische
105
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Referenzmethoden der
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FAL, RAC,
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(46) Tandy, S.; Schulin, R.; Suter,
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B. Determination of
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ethylenediamine
disuccinic acid
(EDDS) by high performance liquid chromatography
after derivatization with FMOC. J.
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two
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2003, 307,
239-257.
106
7 Conclusions
The main
goals
of this
study
were
(i)
to
develop
a
simple
HPLC based method for
the
analysis
of EDDS in both soil solution and
plant material, (ii)
to
investigate
the
use of EDDS for chelant enhanced soil
washing
as a means of
remediating
metal
polluted soil, (iii)
to
investigate
the use of EDDS for chelant enhanced
phytoextraction
of metal
polluted
soils.
7.1 EDDS measurement
by
HPLC
A method for the
analysis
of EDDS
by
HPLC was
developed.
It was based on
the derivatization of EDDS with FMOC
reagent
followed
by
HPLC
separation
on
a
reversed-phase
column
using phosphate
buffer and acetonitrile in a
gradient
elution. The FMOC-EDDS derivative was detected
by
fluorescence detection. A
low detection limit
(0.01 uM)
was achieved and the method was
applicable
to the
determination of the EDDS in
water,
soil solution and
plant
This method was used in the later
phytoextraction
studies.
7.2 Soil
washing using
EDDS
EDDS was
compared
to
IDSA, MGDA,
NTA and EDTA in batch soil
washing
experiments.
For Cu at
pH 7,
the order of the extraction
efficiency
for
equimolar
ratios of
chelating agent
to metal was EDDS
> NTA >
IDSA
>
MGDA
> EDTA and for
Zn it was NTA >
EDDS
> EDTA >
MGDA
>
IDSA. The
comparatively
low
efficiency
competition
between the
heavy
metals and co-extracted
Ca. For Pb the order of extraction was EDTA
> NTA >
EDDS due to the much
of Pb
by
EDTA
compared
to EDDS. At
higher
concentrations
of
complexing agent,
less difference between the
agents
was found and less
pH
dependence.
There was an increase in
heavy
metal extraction with
decreasing pH,
but this was offset
by
an increase in Ca and Fe extraction. In
sequential
extractions
EDDS extracted metals almost
exclusively
from the
'exchangeable', 'mobile',
'Mn-
oxide' and
organic
fractions
according
to the scheme of Zeien and Brummer.
The extraction with EDDS at
pH
7 and
equimolar
concentrations to the
polluting
metals showed the best
compromise
between extraction
efficiency
for
Cu, Zn,
and
Pb and loss of Ca and Fe from the soil. At this
pH
EDDS was more efficient than
EDTA for extraction of Cu and Zn and could be used as a
biodegradable
substitute
to EDTA for real scale remediation of
polluted
soil.
107
Chapter
7
7.3 The use of EDDS for chelant assisted
phytoextraction
The use of EDDS in chelant assisted
phytoextraction
was
investigated
in two
steps
to ascertain not
only
was it suitable for this
purpose
but also to
glean
an
insight
into the
processes
that take
place during
chelant assisted
phytoremediation
and their relative
importance.
In the first
step
a
was used to
investigate
if EDDS could
enhance
Cu,
Zn or Pb
uptake
if solution concentrations were
equal.
It was also used
to look into the mechanism of
uptake
of chelated metals. EDDS was detected in
shoots and
xylem sap, proving
that it is taken
up
into the above
ground
biomass of
plants.
The essential metals Cu and Zn were decreased in shoots in the
presence
of EDDS whereas
uptake
of the non-essential Pb was enhanced. We
suggest
that in
the
presence
of EDDS all three metals were taken
up by
the non-selective
apoplastic
pathway
as the EDDS
complexes,
whereas in the absence of
EDDS,
essential metal
uptake
was selective
along
the
symplastic pathway.
From this we can conclude that
only uptake
of metals without efficient selective
uptake
mechanisms are increased
by
EDDS when soluble metal
concentrations,
with and without
chelants,
are
equal.
In the second
step
we tested EDDS in
artificially
contaminated soils with
single
or dual metal contamination
(Cu, Zn), along
with field contaminated soils with multi-
metal contamination
(Cu, Zn, Pb, Cd).
EDDS increased soil solution metals
greatly
for Cu and Pb and to a lesser extent for Zn and Cd. It was found that Zn
(when
present
as the sole
metal), Cu,
and Pb
uptake by
by
EDDS,
but in multi-metal contaminated soil Zn and Cd were not. EDDS was observed
in the sunflower roots and shoots
proving
that it was taken
up by
the
plants.
A linear
relationship
between Cu and Zn in soil solution in the
presence
of EDDS and
plant
uptake
was found. This
proved
to be different from the non-linear
relationship
found
in the absence of EDDS and shows that the metals were taken
up by
two different
mechanisms
depending
on whether chelant was
present
or not.
It can be seen from these results that in the case of our soils the
solubilizing
of
metals
appeared
to be the more
important step
in chelant assisted
phytoextraction,
as
Cu shoot
uptake
was increased
by
EDDS
(and
Zn when it was the
only contaminating
metal) despite
the fact that at
equal
solution concentrations
uptake
is less in the
presence
of chelants than in the absence.
Despite
the
strong
enhancement
by
EDDS the maximum accumulation of Cu in
shoots was
only
0.02 % and for Pb 0.0011 % DW. For
phytoextraction
to be viable
the shoots need to accumulate in the order of 1 %. Thus these results are still far
from the mark.
7.4 Outlook and
open questions
The results of this
study
show that EDDS can be used
effectively
for soil
washing
108
Conclusions
to remediate
heavy
metal contaminated soils As not all of the
contaminating
metals
are
removed,
it is
important
to establish if
any
of this residual metal is bioavailable
and to see how soil
washing
affects the soil
quality
Although
EDDS did enhance Cu and Pb
uptake by
sunflowers the shoot
concentrations were not
great enough
to make chelant enhanced
phytoextraction
with EDDS viable It
may
be
possible
that other
plant species
or
older, larger plants
can take
up greater quantities
of chelated metals The
optimal
concentration of
EDDS for
phytoextraction
and the
possible leaching
of
heavy
metals after EDDS
addition should also be
investigated
If these issues could be addressed then it would
result in a cost
in-situ method that
preserves
soil
structure and
fertility
109
Acknowledgements
I would like to thank Prof Rainer Schuhn and Dr Bernd Nowack for
giving
me
the
opportunity
to
carry
out
my
PhD thesis and for their
advice, help
and
guidance
during
this time
I would also like to thank
my friends, family
and
colleagues
for their
help
and
support
which enabled me to
carry
out and finish this work I would
especially
like
to thank Anna
Grunwald,
Ren
Saladin,
Wernie
Attinger
and
Bjorn
Studer for their
technical
help
in the
laboratory
and field
Lastly
I would like to thank Diederik Schowanek from Procter & Gamble and
Raj
Patel from
Octel,
U K for
providing
the
S,S-EDDS
and the funders of this
work,
the Federal Office for Education and Science within COST Action 837 and the
Swiss National Science Foundation in the framework of the Swiss
Priority Program
Environment
111
Curriculum Vitae
Surname
Tandy
First Name Susan
Date of Birth 21.06.1971
Place of Birth
Leamington Spa,
United
Kingdom
Nationality
British
School Education
1982
-
87 Alcester
High School, Warwickshire,
U.K.
1987
-
89 Alcester Grammar
School, Warwickshire,
U.K.
Higher
Education
1989
-
92 BSc.
(Hons) Chemistry, University
of Wales
Cardiff,
U.K.
1997
-
98 MSc. Environmental
Science, Nottingham University,
U.K.
Occupation
Sept
1992
-
Sept
1997 Scientist and Senior
Scientist,
Southern Water
Laboratories, Medway Towns, Kent,
U.K.
March 1999
-
Sept
2001 Research
Scientist,
Rothamsted
Research, Harpenden,
Hertforshire,
U.K.
October 2001
-
present
PhD studies and
teaching assistant,
Soil Protection
Group,
Institute of Terrestrial
Ecology,
Swiss Federal
Institute of
Technology (ETH) Zurich,
Switzerland.

Ets EDDS-27957-02

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