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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

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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

2.3.3Analyses 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

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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 283.4 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

4.2.4Analytical 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

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5.3 Results

5.3.1 Hydroponics solution speciation 64

5.3.2 Plant dryweight 65

5.3.3 Root metal uptake 655.3.4 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 dryweight 87

6.3.2 Solubilized Metals 88

6.3.3 Plant metal uptake 88


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


6.4.3 Metal uptake in the presence of EDDS 99

6.4.4 Factors influencing chelant assisted phytoextraction 99

Acknowledgements 101

6.5 References 101

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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

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Summary

Heavy metal pollution of soil is a global problem. Remediation of metal polluted

soil is a difficult task and in general expensive. Twopotential 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 thedevelopment of a HPLC based methodfor 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 forequimolar 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 intwo steps

to ascertain whether it was suitable forthis 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

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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 andxylem 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 Theseresults 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 potexperiments 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 thisstudy show EDDS is suitable to replace EDTA in soilwashing,

whereas the achieved enhancement ofphytoextraction is notyetenough to beviable

for remediation purposes

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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 einbiologisch 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 alsAlternative 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 istdurch 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

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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 dieseAnwendung

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 nichtselektiven, 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 Wirfanden, 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 Bodendie 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 Bodensanierungist

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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 ofproblems in the long term future Soil flushing brings

risks ofground 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 ofheap 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 Soilwashing with chelating agents is seen as particularlypromising (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 ofthe 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

proposedthe use of chelatingagents forthis purpose, as reviewed by Lasat(10) and

Schmidt (11) The use of hyperaccumulator plants with chelating agents has been

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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 equallypersistent inthe environment (4) or possible carcinogens (NTA) (15).

1.1 Objectives of this study

(S,S)-N,N'-ethylenediamine 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

easilybiodegradable. Some metal complexes (Cu, Ni andCo) 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 potand 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

ofthis uptake. In most cases the speciation ofsoil 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 soilwashing as a means

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Introduction

of remediating metal polluted soil

iii) to investigate the use of EDDS forchelant 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-metalcontamination from thefield(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 ofheavy 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.

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(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 oftoxic metals A review ofbiological 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

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Introduction

aluminum-nitrilotriacetate complexes in rats. Journalof 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 ethylenediamine 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 forthe 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.

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(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 Comparisonof 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

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Vodnik, D

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Velikonja-Bolta, S

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Lestan, D

Ethylenediaminedissuccinate as a new chelate for environmentally safe enhanced lead phytoextraction

J Environ Qual 2003, 32, 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

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2 Determination of EDDS and other amino-polycarboxylic 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

o n a n 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

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Determination of EDDS and other amino-polycarboxylic 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 chromatographicsystem (PU-980) equippedwith

851-AS auto-sampler, a 200 ul sample loop and a UV spectrophotometric detector

(UV 970) set at258 nm was used. A pre-instrument degassing unit was also installed

(Lacoc, GastorrGT102). 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 Afor 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

EDDS1 mMHCI

pH3

5 mM (NH4)2S04

pH3.3

0-100% Bin 12

minutes

NTA1 mMHCI

pH3

50 mM (NH4)2S04

pH3.3

0-100% Bin 30

minutes

EDTA water5 mM (NH4)2S04

pH5.30-50% 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

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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 foranalysis 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

priorto metal

spiking and also with standards

prepared with

Na3EDDS priorto 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 -CDC

tf> 2x104 -

JVj

0 2 4 6 8

Time (minutes)

Figure 2.1. Chromatogram of 10 uM EDDS.

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4x10"

3x10-

A.A

CD

CD

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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 evenmore 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 ofphosphate on EDDS peak

area and height. Peak area was unaffected at phosphate concentrations of 10 mM

butshowed nearly 50% reduction at 100 mM. Peakheight 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

phosphatewould 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.

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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 witha 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 NTApeak is well separated from

the reagent peak and a plot of peak

area versus concentrationgave

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 ofthe

peak height of 10 uM Fe(lll)EDTAor Fe(lll)NTAwithout 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 largersample injections. This in itself brought new problems due to an

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Chapter 2

2x1Obi

_

1x10bHCD

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 increasingthe 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, EDTApeaks 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>6x102HCO

4x102

1I

' I ' I ' I

0 12 3 4

Time (minutes)

a)

4x10' -i

3x10J -

c)2x103H

1x103

CDc

a

CO

1I

'

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)

2.3.3Analyses

Drinking watersamples spiked with Fe(lll)EDDS where analysed along with pure

water standards. The drinking water samples showed peak areas of ~50-60% and

peakheights of 72-78% ofthe standards made in pure water(Figure2.7). This shows

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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 heightwas 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 forthe 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 aminopolycarboxylate chelating

agents. Environ. Sei. Technol. 2002, 36, 4009-4016.

(2) 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.

(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

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Chapter 2

bacteria. Biosci. Biotech. Biochem. 1999, 63, 1269-1273.

(4) 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.



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.




(8) Tandy, S.; Bossart, K.; Mueller, R.; Ritschel, J.; Hauser, L; Schulin, R.;

Nowack, B. Extraction of heavy metals from soils using biodegradable chelating


(9) Grcman, H.; Vodnik, D.; Velikonja-Bolta, S.; Lestan, D. Ethylenediamine

dissuccinateasa new chelateforenvironmentallysafe enhanced leadphytoextraction.

J. Environ.Qual. 2003, 32, 500-506.


nondegradable (EDTA) chelate and hydrogel modified soil water sortpion capacity


(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.



708-724.

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(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 ethylenediaminetetraacetic 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 ofcomplexing

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 byion 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

chromatographycoupled to ICP-MS Anal Bioanal Chem 2002, 372, 448-452

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3 Determination of EDDS by HPLC after derivatization with FMOC

Susan Tandy Rainer Schulin, Marc J. F. Suterand Bernd Nowack

Journal ofChromatographyA, in press

Abstract

The paperdescribes a new HPLC methodfor thedeterm

ination of ethylenediam inedisuccinic 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.

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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 theenvironment (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 notgiven, it seems from the data thatthey 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 ofsample types and has a detection limit suitable

foranalysis at sub-m icromolarconcentrations. Toachieve thisgoal 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

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Determination of EDDS by HPLC after derivatization with FMOC

(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 ofsample. This was heated for3

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

aqueouslayer injected into the HPLC.

3.2.3 HPLC

AJascohigh-performance liquidChromatograph(PU-980; Jasco, Japan)equipped

with a fluorescence detector (821-FP or FP-2020), using 265 nm as excitation and

313 nm as emissionwavelength, and an autosampler 851 -A S 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 to80%, 1 minute at80%, 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 watersamples were taken from the non-chlorinated normal domestic supply.

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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 potexperiment investigating the use of EDDS forenhancing 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 bufferwith pH 11.5 was therefore chosen forthe 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

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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 addingthe FMOC reagent produced the same signal as 1 uM EDDS in the absence of

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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 PeakArea

Unheated Heated1

free 1.00 1.00

1 m M 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

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_

1x10

CO

g> 8x105CO

g 6x10c

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

lio

CO

CD c

fc 3x105-3

LL

0-

c

-

u.I I I I

) 2 4 6 8 1

Time (rrlinijtes)

0

Figure 3.3. Chromatogram of 1 uM EDDS derivatized with FMOC in borate bufferat pH 7.7, PLRP-S polymer RP-C18 column (see section 3.3.3. fordetails).

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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 derivatizedFMOC-EDDS (see Scheme 3.1 ). MS/MS of this ion gavefragments at m/z 495

(-H20), 397 (loss of maleic acid), 317 (loss offluorenyl-methanol), 291 (EDDS; loss

ofFMOC), 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* x291

I;

YCOOH

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/z273, 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

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^Yr

SN COOHH

291

^NH

?^y^

EDDS

COOH

273

495

-FMOC\-co2

COOH

229

r

:-^YU

COOH

273

IY^NHHN^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

Asparticacid "9

FMOC |j~ao

-FMOC-OH jj*"~

O NH

M

O^ ^^ coo

h (AniiHOOC vAa

354

0*0165

Scheme 3.3. Fragmentation pattern of molecular ion m/z 354 of peak 3 in Figure3.3.

the proposed fragmentation pattern.

Peak 4 had a molecular ion of m/z735, 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).

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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.

3.3.4Analyses

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

CDO

aj 5x104oCOl_

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.

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CO

O)

CO

CDO

CDO

CO

CDi_

O"3

CO

D)

CO

CDO

CDO

CO

CDi_

O3

3x10

2x104-

1 x10

8x10-

6x10-

4x10-

2x10-

0-

0

Time (minutes)

^mple EDDSEDDS 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 besuccessfully analysed after FMOC-derivatization

(Figure3.5). Sub-samples were also spiked with EDDS priorto 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

potexperimentwhere much

higherconcentrations of EDDS were found. The extract concentration was 6.43 uM EDDS

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Chapter 3

sampleCO

w 4x104-

EDDS spike EDDS

Co

CD

$ 2x104-

Matrix peak ! I

o / ^WJ_

^ /\ _-/

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

material at trace levels.

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Acknowledgements

We thank Ren Schnenberger for the help with the HPLC-MS measurements

and Diederik Schowanek from Procter & Gamble forproviding 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



1765-1770.

(2) 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.



708-724.

(4) Nowack, B. Environmental chemistry of aminopolycarboxylate chelating


(5) Tandy, S.; Bossart, K.; Mueller, R.; Ritschel, J.; Hauser, L; Schulin, R.;

Nowack, B. Extraction of heavy metals from soils using biodegradable chelating


(6) Grcman, H.; Vodnik, D.; Velikonja-Bolta, S.; Lestan, D. Ethylenediamine

dissuccinateas a new chelateforenvironmentallysafe enhanced leadphytoextraction.

J. Environ. Qual. 2003, 32, 500-506.


nondegradable (EDTA) chelate and hydrogel modified soil water sorption capacity




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Chapter 3


(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.

(11) 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.

(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,

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942, 185-190.

(19) Glass, R. L. Liquid-chromatographic determination of glyphosate in fortified

soil and water samples. Journal ofAgricultural 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 ofchelating 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 EDTAspecies 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 complexforming properties. Bull. Acad. Sei. USSR Div. Chem

Sei. 1983, 32, 1167-1175.

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4 Extraction ofheavy 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

chelatingagent for this purpose, but has thedisadvantage 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),

iminodisuccinicacid (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 atpH 7 the order of

the extraction efficiency forequimolar 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 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 EDTAcomparedto 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.

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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, soilwashing, electroremediation,

bioleaching and phytoremediation (1 ) . Soilwashing 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 ofthe 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 forfurther 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 ofpolluted sites. Ratio ofchelating 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

extractedfraction 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 Caalong 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

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