Modeling Cyanide

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Modeling Cyanide Uptake by Willows for Phytoremediation

Joseph T. Bushey

B.S., Johns Hopkins University, Baltimore, MD, 1995 Stanford University, Stanford, CA, 1996

A dissertation submitted in partial fulfillment

of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING CARNEGIE INSTITUTE OF TECHNOLOGY

CARNEGIE MELLON UNIVERSITY

Pittsburgh, Pennsylvania May 15,2003

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UMI Number 3084715

Copyright 2003 by Bushey, Joseph T.

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Carnegie Mellon UniversityCARNEGIE INSTITUTE OF TECHNOLOGY

THESIS

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF . Doctor of Philosophy ____

t i t l e Modeling Cyanide Uptake bv Willows for Phvtoremediation

PRESENTED BY Josep h T. Bushev

ACCEPTED BY THE DEPARTMENT OF

Civil and Environmental Engineering

APPROVED BY THE COLLEGE COUNCIL

9 / F 5 Y 2 . 0 0 5

S'J/f/jop z

€ ~ - ! 6-€>5

DATE

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ACKNOWLEDGEMENTS

Special thanks to my Mom & Dad, who have always been there for me & given so much of

themselves. A simple “thank you” cannot express how much I feel indebted to the two of you.

I would like to give my sincerest thanks to my advisor. Dr. David Dzombak, for his tutelage,

insight, support, and patience over the last four years. He has provided me with an invaluable

asset through his shared knowledge, experience, and example. I would also like to thank Dr.

Stephen Ebbs for his continued guidance and insight with respect to plant physiology.

Financial support for this project was provided by ALCOA, Inc., The Gas Technology Institute,

New York Gas Group, and Niagara Mohawk Power Corporation and organized by The RETEC

Group, Inc. I particularly wish to acknowledge the helpful comments and insight provided by S.

Geiger, R. Ghosh, and D. Nakles of The RETEC Group, by S. Drop of Alcoa, Inc., by L.

Weinstein of the Boyce Thompson Institute, and by E. Neuhauser of Niagara Mohawk.

I am grateful to Dominic Boccelli and Ki-Joo Kim for their modeling and optimization guidance

as well as their friendship.

Finally, I would like to thank my family and friends who have supported me throughout the past

few years. Without them, I would not be who I am today. Particular thanks to my brother Jon,

Chad Bumsted, and Gonzalo Pizarro for many a late-night chat; to Wei Tang for her patience and

humor, and to the rest of those who made my time at CMU so enjoyable.

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ABSTRACT

The potential for phytoremediation of cyanide-contaminated groundwater with willow trees was

investigated in this research. The objectives were to investigate the uptake and metabolism of

dissolved free cyanide and iron cyanide by willow and to determine the major plant processes

governing iron cyanide fate in the willow plant. Hydroponic uptake experiments were performed

to demonstrate the uptake and fate of lsN-!abeled CN~ and FefCNV** solutions containing 2 ppm

cyanide. A novel extraction method was developed and used to analyze tissue cyanide content to

separate cyanide uptake from metabolism. Willow was observed to take up and metabolize both

free cyanide and ferrocyanide, with faster rates for free cyanide. Metabolism of the cyanide

species by willow was demonstrated by the difference between measured cyanide species and

cyanogenic-,sN concentrations in extracted plant tissue.

A process model was constructed to represent the physiological processes affecting the cyanide

mass transfer and transformation processes in the willow plant. The model was fitted to theexperimental hydroponic data to obtain the optimal parameter values and to examine the

importance of the various processes. Consistent with the experimental observations, the uptake

and metabolic rate constants were higher for free cyanide than for ferrocyanide. Also, free

cyanide volatilization and root cell wall adsorption did not affect cyanide fate. Active uptake

was applicable for free cyanide, but did not apply to ferrocyanide uptake. To achieve the

observed solution cyanide concentration profiles, the plant must actively take up free cyanide

while ferrocyanide must be excluded from entering the root. Predicted assimilate concentrations

for the root and stem tissue were significantly underestimated. Predicted and actual tissue

cyanide and leaf assimilate concentrations were of identical magnitude. In order to match the

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root, stem, and leaf assimilate concentrations in the plant, a means of removing assimilate from

leaf tissue is required. This suggests that phloem redistribution may be important for

determining uptake of cyanide from solution and fate within the willow plant.

Calculations pertaining to the applicability of ferrocyanide phytoremediation to the field-scale

were conducted using the uptake rate data from the hydroponic experiments and operating

parameters for an existing wetland treatment system. A conservative estimate ignoring photo

dissociation, surface volatilization, and biodegradation showed that a typical wetland system

could remove an influent concentration of up to 0.2 ppm as CN of FefCNfo4" via plant uptake.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................... ii

ABSTRACT .................................................................................................................................... iii

LIST OF TABLES ......................................................................................................................... x

LIST OF FIGURES ....................................................................................................................... xiii

1. INTRODUCTION .................................................................................................................... I

1.1 Objectives ...................................................................................................................... 3

12 Organization of Thesis ................................................................................................. 6

13 References ..................................................................................................................... 6

2. BACKGROUND ....................................................................................................................... 8

2.1 Cyanide Chemistry ....................................................................................................... 8

2.2 Anthropogenic Cyanide Sources .................................................................................. 10

23 Cyanide in Nature ......................................................................................................... 11

2.4 Cyanide Toxicity ............................................................................................................ 12

2.4.1 Animals ............................................................................................................ 12

2.4.2 Plants ................................................................................................................ 13

IS Natural Cyanide Cycle .................................................................................................. 14

2.6 References .................................................................................................................... 14

3. PLANT TISSUE EXTRACTION METHOD FOR COMPLEXED AND FREE

CYANIDE..................................................................................................................................25

3.1 Introduction ................................................................................................................. 26

32 Methods ......................................................................................................................... 30

3.2.1 Solvent Selection ............................................................................................. 31

3.2.2 Sample Spike Recovery ................................................................................... 33

3.2.3 Control Tissue ................................................................................................. 34

33 Results ........................................................................................................................... 34

3.3.1 Solvent Selection ............................................................................................. 34

3.3.2 Sample Spike Recovery ................................................................................... 35

3.3.3 Control Tissue ................................................................................................. 36

3.4 Discussion ...................................................................................................................... 36

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3.5 Acknowledgements ....................................................................................................... 40

3.6 References ..................................................................................................................... 40

4. TRANSPORT AND METABOLISM OF FREE CYANIDE AND IRON CYANIDE

COMPLEXES BY WILLOW .................................................................................................. 49

4.1 Introduction .................................................................................................................. 50

4.2 Materials and Methods ................................................................................................ 54

4.2.1 Willow Propagation. ........................................................................................ 54

4.2.2 Ferrocyanide Biodegradation Assay ................................................................ 56

4.2.3 Cvanide Uptake bv Willow ............................................................................. 57

4.2.4 Ferrocyanide Sorption to Roots ....................................................................... 59

4.2.5 Analytical Procedures ...................................................................................... 60

43 Results ........................................................................................................................... 63

4.3.1 Willow Growth and Water Relations .............................................................. 63

4.3.2 Solution pH. pe. and Cvanide Speciation ........................................................ 64

4.3.3 i5N Content of Willow Tissue ......................................................................... 65

4.3.4 Iron Cvanide Root Sorption Versus Root Uptake ........................................... 66

4.3.5 Cvanide Content of Willow Tissue ................................................................. 66

4.3.6 Mass Balance ................................................................................................... 68

4.4 Discussion ...................................................................................................................... 68


4.6 References ..................................................................................................................... 71

5. MODEL FOR CYANIDE UPTAKE BY WILLOW: MODEL DEVELOPMENT 88

5.1 Introduction .................................................................................................................. 89

5.2 Model Structure ............................................................................................................ 93

5.2.1 Model Compartments ...................................................................................... 94

5.2.2 Transfer and Reaction Processes ..................................................................... 95

5 3 Mass Balance Equations .............................................................................................. 99

5.4 Model Capabilities and Solution Technique .............................................................. 103

5.5 Summary and Conclusions...........................................................................................105


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5.7 References .................................................................................................................. 106

6. MODEL FOR CYANIDE UPTAKE BY WILLOW: APPLICATION TO

EXPERIMENTAL DATA AND CALIBRATION ............................................................... 117

6.1 Introduction ................................................................................................................ 118

6J Model Parameter Optimization Technique Overview ........................................... 120

6-3 Model Parameter Estimation ................................................................................... 123

6.3.1 Parameters for System Control Loss .............................................................. 124

6.3.2 Parameters for Cvanide and Ferrocyanide Uptake and Mass Transfer

in the Willow Plants .................................................................................... 126

6.3.3 13-Parameters Model .................................................................................... 129

6.4 Optimal Model Fits of Experimental Data ............................................................... 130

6.5 Model Variability ...................................................................................................... 131

6.6 Discussion .................................................................................................................... 135

6.7 Summary and Conclusions ........................................................................................ 140

6.8 Acknowledgements .................................................................................................... 143

6.9 References ................................................................................................................... 143

7. CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK ......................... 165

7.1 Major Findings ........................................................................................................... 166

7.1.1 Plant Tissue Extraction Method for Complexed and Free Cvanide ............... 166

7.1.2 Transport and Metabolism of Free Cvanide and Iron Cvanide

Complexes bv Willow ................................................................................. 167

7.1.3 Model for Cvanide Uptake bv Willow: Model Development ........................ 168

7.1.4 Model for Cvanide Uptake bv Willow: Application to Experimental

Data and Calibration .................................................................................... 169

7.2 Engineering Applications .......................................................................................... 171

1 3 Future Considerations ............................................................................................... 172

7.4 References ................................................................................................................. 178

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APPENDICES

A. FERROCYANIDE ADSORPTION ON ALUMINUM OXIDES ....................................... 182

A.1 Introduction .................................................................................................................. 183

A.2 Materials and Methods ................................................................................................ 185

A3 Results and Discussion ................................................................................................. 189

A.4 Conclusions ................................................................................................................... 192

A.5 Acknowledgements ....................................................................................................... 193

A.6 References ..................................................................................................................... 194

B. CHERRY TREE SAMPLING FOR CYANIDE ..................................................................... 205

B.1 Materials and Methods ................................................................................................ 206

B.2 Results ........................................................................................................................... 206

B.3 References ..................................................................................................................... 207

C. PEA UPTAKE STUDY ............................................................................................................. 210

C.1 Methods ........................................................................................................................ 210

CJ, Results ........................................................................................................................... 211

C J Summary and Conclusions .......................................................................................... 213

C.4 References .................................................................................................................... 215

D. HYDROPONIC SYSTEM DESIGN FOR STUDYING CYANIDE UPTAKE .................... 219

D.I Introduction ................................................................................................................. 220

D.2 Cyanide Chemistry of Hydroponic Test Solu tion .................................................... 221

D.2.1 Methods .......................................................................................................... 222

D.2.2 Results ............................................................................................................ 224

D J Hydroponic System Development ............................................................................... 226

D.3.1 System Criteria ................................................................................................ 226

D.3.2 System Design ................................................................................................. 227

D.3.3 Hydroponic System Testing ............................................................................ 228

D.4 Volatilization of Cyanide by Plant Tissues ................................................................ 229

D.5 Summary and Conclusions .......................................................................................... 230

D.6 References .................................................................................................................... 231

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E. SET 1 HYDROPONIC UPTAKE STUDY ............................................................................. 241

E.1 Materials and M ethods .............................................................................................. 241

E.1.1 Experimental Overview .................................................................................. 241

E1.2 Harvest and Analytical Procedures ................................................................. 243

E.1.3 Data Analysis .................................................................................................. 243

E2 Results ........................................................................................................................... 244

E.2.1 Willow Growth and Water Relations. ............................................................. 244

E.2.2 l5N Content of Willow Tissues ....................................................................... 244

E.2.3 Solution Cvanide Analyses ............................................................................. 245

EJ Discussion ..................................................................................................................... 247

E.4 References ..................................................................................................................... 248

F. ASSIMILATION OF CYANOGENIC NITROGEN INTO AMINO ACIDS BY

WILLOW ................................................................................................................................... 259

F.l Introduction .................................................................................................................. 260

F.2 Materials and Methods ................................................................................................ 260

FJ Results ........................................................................................................................... 261

F.4 Summary and Conclusions ......................................................................................... 262

F.5 Reference ...................................................................................................................... 263

G. FORTRAN CODES FOR SYSTEM MODELS ..................................................................... 267

G .l Control Hydroponic System Simulation Code.........................................................267

G.2 Plant Uptake Hydroponic System Code - 17-Parameter Model ............................ 272

H. EXPERIMENTAL DATA ........................................................................................................ 287

H.1 Extraction of Cyanide from Plant Tissue .................................................................. 287

H i Hydroponic Uptake Study .......................................................................................... 291

H.2.1 Solution ................................................................................................................ 291

H.2.2 Plant Tissue .......................................................................................................... 302

H.2.3 Stripped Tissue ..................................................................................................... 308

HJ Model Output Distributions ...................................................................................... 310

H.4 Ferrocyanide Adsorption to Aluminum Oxides ....................................................... 336

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LIST OF TABLES

CHAPTER 3

Table 3.1 Free cyanide and ferrocyanide recovery with optimal extraction method ............ 43

Table 3.2 Control willow tissue total and free cyanide concentration .................................. 44

CHAPTER 4

Table 4.1 Composition of nutrient solutions ........................................................................ 77

Table 4 3 Solution cyanide content in hydroponic experiment with time ............................ 78

Table 4.3 Cyanide concentration and speciation in willow tissues ...................................... 79

Table 4.4 Mass balance for ferrocyanide and free cyanide in hydroponic systems .............. 80

CHAPTER 5

Table 5.1 Definitions and units for willow plant-cyanide model parameters ...................... 110

Table 5 J l Parameters solved for within the plant uptake model ......................................... 111

Table 5.3 Input parameter set for a simplified, representative uptake model solution 112

Table 5.4 Compartmental cyanide calculated from a representative input parameter set.... 113

CHAPTER 6

Table 6.1 Input parameter values for the plant uptake model .............................................. 146

Table 6^(a) Input experimental solution concentrations for the cyanide uptake model ...... 147

(b) Input experimental tissue concentrations for the cyanide uptake model 147

Table 6 3 Process variables determined through fitting hydroponic uptake data ................. 148

Table 6.4 Predicted tissue cyanide concentrations obtained with optimal parameters 149

Table 6 ^ Replicate and measurement error used in the generation of random samples ISO

Table 6.6 Mean and standard error for each parameters resulting from data uncertainty.... 151

Table 6.7 Mean and standard error for fraction cyanide in specified compartment 152

Table 6.8 Correlation coefficients for output parameter distributions in variability study.. 153

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APPENDIX A

Table A.1 Solid properties for aluminum and iron oxides ................................................... 197

Table A.2 Regression results for ferrocyanide adsorbed concentration versus pH .............. 198

APPENDIX B

Table B.1 Cyanide concentration in cherry tree soil and leaf tissue samples ...................... 209

APPENDIX C

Table C.1 Solid properts ...................................................................................................... 216

Table C.2 Regression rbed cons pH .................................................................................... 217

Table C.3 Regression reide adsous pH ................................................................................ 218

APPENDIX D

Table D.l Recommended hydroponic nutrient solution ....................................................... 233

APPENDIX E

Table E.1 Set 1 tissue cyanide speciation ............................................................................ 249

APPENDIX F

Table F .l Atom % of l5N in the amino acid fractions from exposed willow tissue ............. 264

APPENDIX H

Table H.I.1 Free cyanide recovery in MeOH: NaOH spike solutions ................................. 287

Table H. 1.2(a) Cyanide recovery from spike solutions with and without tissue for

chloroform: NaOH mixtures .................................................................................... 288

Table H.1.2(b) Cyanide recovery from tissue subjected to chloroform: NaOH extraction 289

Table H.13 Cyanide recovery from free cyanide spike samples in hexane: NaOH and 2-

octanol: NaOH mixtures ........................................................................................... 290

Table H.2.1.1 Individual cyanide concentration (a) and mass (b) replicate data for

hydroponic uptake experiment ................................................................................. 291

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Table H J .I .2 Hydroponic solution volume ......................................................................... 299

Table H.2.1.3 Hydroponic solution pH ................................................................................ 300

Table H.2.1.4 Hydroponic solution pe ................................................................................. 301

Table HA2.1 Hydroponic plant tissue mass ........................................................................ 302

Table H 7-2.2 Hydroponic plant tissue water content .......................................................... 303

Table H-2-2J3 Hydroponic plant root (a), stem (b), and leaf (c) tissue >SN enrichment ....... 304

Table H-2.2.4 Set 2 tissue cyanide speciation after 20-day exposure ................................... 307

Table H 2 J .1 Stripped root solution cyanide concentration (a) and normalized average

total ferrocyanide concentration ................................................................................ 308

Tab le H J.1 Set of optimal adjustable parameter output sets for 17-parameter model ........ 310

Table H J 2 Set of optimal adjustable parameter output sets for 13-parameter model ........ 312

Table H.3.2 Set of optimal adjustable parameter output sets for 13-parameter model with

arithmetically-averaged data ..................................................................................... 312

Table H J J Set of optimal adjustable parameter output sets for 13-parameter model with

geometrically-averaged data ...................................................................................... 316

Table H.3.4 Set of optimal adjustable parameter output sets for 13-parameter model with

geometrically-averaged data and the inclusion of replicate uncertainty ................... 320

Table H J i Set of optimal adjustable parameter output sets for 13-parameter model with

geometrically-averaged data and the inclusion of measurement and replicate

uncertainty ................................................................................................................. 327

Table H.4.1 Ferrocyanide (1 ppm) adsorption to 2.0 g/L g-ALC^,,, .................................... 336

Table HA2 Ferrocyanide (1 ppm) adsorption to 0.6 g/L g -A L O ^ .................................... 338

Table H A3 Ferrocyanide (1 ppm) adsorption to 0.3 and 1.2 g/L g-AhO^s) ...................... 340

Table H.4.4 Ferrocyanide (0.75 ppm) adsorption to 1.2 g/L g-ALO^) ............................... 342

Table H AS Ferrocyanide (1 ppm) adsorption to various solid doses of Al(OH) 3(s)............. 343

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LIST OF FIGURES

CHAPTER 2

Figure 2.1 Some common cyanogenic glycosides ................................................................ 20

Figure 23 Conceptual diagram pathways of cyanide cycling in general plant metabolism. 21

Figure 23 Cyanide content of some common plants ........................................................... 22

Figure 2.4 Assimilatory reactions for cyanide within plants ................................................ 23

Figure 23 Natural cyanide cycle in the environment ........................................................... 24

CHAPTER 3

Figure 3.1 Recovery of free cyanide with methanol inclusion in the solvent matrix ........... 46

Figure 33 Investigation of 2.5 M NaOH/chloroform solution KCN-spike samples ........... 47

Figure 33 Free cyanide recovery from KCN-spike solutions .............................................. 48

CHAPTER4

Figure 4.1 l5N enrichment ratios for willow roots, stems, and leaves .................................. 83

Figure 43 15N content of willow roots, stems, and leaves ................................................... 84

Figure 43 Relationship between sorption and uptake for stripped willow roots ................. 85

Figure 4.4 Total tissue cyanide concentrations for KCN-treated plants ............................... 86

Figure 43 Total tissue cyanide concentrations for ferrocyanide-treated plants ................... 87

CHAPTER 5

Figure 5.1 Schematic of plant compartmentalized model ................................................... 115

Figure 5 3 Cyanide concentration profiles for a representative parameter input set 116

CHAPTER 6

Figure 6.1 Predicted solution cyanide concentrations for optimal parameter values 157

Figure 63 Predicted solution total cyanide mass profile for optimal parameter values 158

Figure 63 Variability in the mass fraction of initial cyanide remaining in solution 159

Figure 6.4 Variability in the mass fraction of initial cyanide dose assimilated .................... 160

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Figure &5 /W CVversus KmCN for parameter output sets for simulated input data sets ....... 161

Figure 6.6 / w FC versus K„FC for parameter output sets for simulated input data sets ....... 162

Figure 6.7 e / c versus Y rooi for parameter output sets for simulated input data se ts 163

Figure 6 £ Solution concentration comparison for “Flow Only” versus “Active Uptake” .. 164

CHAPTER 7

Figure 7.1 Effectiveness of a wetland phytoremediation system .......................................... 180

Figure 7.2 The diagram of potential loss processes for cyanide in a wetland system...........181

APPENDIX A

Figure A .l Ferrocyanide equilibrium pH-dependent sorption edge on y-AFO^ s,................ 200

Figure A.2 Ferrocyanide equilibrium pH-dependent sorption edge on Al(OH) 3.............. 201

Figure A 3 Adsorbed ferrocyanide concentration versus pH for y-AhC^s, ......................... 202

Figure A.4 Adsorbed ferrocyanide concentration versus pH Al(OH> 3 (s).............................. 203

Figure A.5 Adsorbed ferrocyanide concentration versus pH a-FeOOH(S) ........................... 204

APPENDIX D

Figure D.l Solubility limitations for ferrocyanide addition to the hydroponic solution ...... 236

Figure D.2 Predicted equilibrium percentage of dissolved cyanide in the free form ........... 237

Figure D 3 Reactor system for uptake experiments ............................................................. 238

Figure D.4 Schematic of the hydroponic system .................................................................. 239

Figure D.5 Ferri- and ferrocyanide sorption to hydroponic system materials ...................... 240

APPENDIX E

Figure E.I Daily and cumulative transpiration for cyanide-exposed willows ..................... 252

Figure E.2 Biomass of treated willows after 7-day exposure ............................................... 253

Figure E J Water content of exposed willow plants ........................................................... 254

Figure E.4 Enrichment of 1SN in root and leaf tissue of willow exposed for 7 days ............ 255

Figure E.5 l5N concentration in exposed root and leaf tissue .............................................. 256

Figure E.6 Cyanide species distribution in the ferrocyanide treatment solution .................. 257

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Figure E.7 Cyanide species distribution in the free cyanide treatment solution .................. 258

APPENDIX F

Figure F.l >SN content of the amino acid fraction from exposed willow plants .................. 266

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CHAPTER 1

INTRODUCTION*1*

Cyanide contamination of groundwater and soils has been observed at many manufacturing sites

including those relating to former manufactured gas plants (Theis et al., 1994) and to aluminum

production (Dzombak et al.. 1996). Solids that were used for cleaning sulfur and other gaseous

pollutants from manufactured gas became contaminated when cyanide solids formed on the iron

oxides due to the presence of low levels of cyanide in the waste stream. For aluminum production, cyanide solids formed on the pot-liners as a result of the reaction at the carbon

cathode (Haupin. 1987). These MGP site "oxide box" residuals and aluminum smelting spent

pot-liners were often used as fill. Over time, the cyanide solids (in the form of Prussian Blue

[Fe4 (Fe(CN) 6 )3 ] and Turbull’s Blue [Fe 3 (Fe(CN) 6 );]) associated with the solids dissolve and

leach into groundwater (Dzombak et al.. 1993; Ghosh et al.. 1999) as soluble iron cyanide or free

cyanide. The free form is acutely toxic (ATDSR. 1999). Although the stronglv-complexed iron

cyanides are less toxic, these compounds have been shown to photo-dissociate to free cyanide

(Meeussen et al. 1992) under specific laboratory conditions. Release of free cyanide to the

environment may have significant impacts on water quality and aquatic life. A recent example is

the release of free cyanide-contaminated mine water into Romania’s Tisza River, a tributary of

the Danube River, in February 2000 (NY Times, 2000). This spill sterilized the river for several

miles, disrupting the fishing industry in the region.

Modified from the final project report submitted October 17,2002 to Niagara Mohawk Power Corporation. TheGas Technology Institute. ALCOA. Inc.. and The New York Gas Group. Report coauthored with Stephen Ebbs.David Dzombak. Rajat Ghosh, and Stephen Geiger.

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Cyanide is produced via multiple plant pathways and exists as part of a natural cycle in nature.

Both plants, and some animals that they interact with, have developed pathways to incorporate

cyanide into metabolic functions, minimize cyanide toxicity, or use cyanide for their own

advantage (Seigler, 1991).

Phytoremediation, the use of vegetation as a remediation strategy for cleaning up contaminated

waste, can be a practical and cost-effective method for remediating shallow contamination in

groundwater (Schnoor, 2002) and the soil vadose zone. Phytoremediation strategies are less

invasive and have much lower capital and long-term operating costs compared with typical

groundwater treatment technologies such as pump-and-treat. Phytoremediation has potential for

in situ treatment of cyanide-contaminated groundwater, through exploitation of the existing

assimilatory pathways for cyanide within plants, using the natural cyanide cycle to remediate

cyanide-contaminated groundwater and soil.

Willow is well suited for phytoremediation applications compared w ith other plants because it is

a phreatophyte (a plant that sends a root to groundwater) with a high biomass production and a

high transpiration rate (Schnoor, 2002). Recent hydroponic results involving the examination of

ferrocyanide uptake by willows (Reeves, 2000) indicated that that willow potentially could

remove cyanide from solution. Reeves (2000) provided evidence that the cyanogenic N atom

from iron cyanides accumulated in willow leaves, suggesting a possible pathway for iron cyanide

uptake and assimilation in the plant. However, concerns about precipitation and speciation of

iron cyanide in the hydroponic solution, an inability to close the mass balance of iron cyanide in

the hydroponic system, and limited knowledge of the ultimate fate of iron cyanide within the

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plant prevented drawing definitive conclusions about the extent and magnitude of uptake.

Additional studies with tighter controls were required to monitor potential, undesirable cyanide

losses from the system including but not limited to iron cyanide solid precipitation, iron cyanide

adsorption, biodegradation, and free cyanide volatilization. Although each of these removal

processes is important for assessing the overall performance efficiency for remedial systems,

each adversely affects the examination of the uptake of cyanide from solution by willows. The

potential effect of ferrocyanide adsorption to metal oxides was examined separately and is

presented in Appendix A.

Additional background information on cyanide chemistry, toxicity, and interaction with plants

and animals is given in Chapter 2.

1.1 Objectives

The overall objective of this research was to demonstrate the potential for phytoremediation of

dissolved iron cyanide by willow plants and to develop a physiologically-based model to identify

important processes affecting free cyanide and ferrocyanide fate within the system. Particular

objectives were to:

i. Develop a method for the extraction and measurement of total cyanide and free

cyanide from plant tissue and determine recovery from spiked solutions

ii. Demonstrate uptake of free cyanide and ferrocyanide from hydroponic solution and

characterize cyanide fate within the plant-solution system

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iii. Construct a physiologically-based model to describe the mass transfer and fate of

free cyanide and ferrocyanide within the willow plant

iv. Fit the plant uptake model to the hydroponic study observations and assess the

importance of mass transfer processes for free cyanide and ferrocyanide within the

plant-solution system

The first objective was to develop a method for the extraction and measurement of plant tissue

cyanide content in order to assess cyanide fate within the plant. A typical method o f determining

chemical uptake during hydroponic experiments is to measure the uptake of a stable-isotope (e.g.

I5N) from solution. Measurement of the increased isotope content does not distinguish between

tissue cyanide content and assimilated product. Comparison of the cyanide content with the total

uptake provides a measure of assimilation. Solution sample spikes of free cyanide and

ferrocyanide were used to examine the effect of methanol, chloroform. 2-octanol, and hexane

inclusion in the solvent matrix with NaOH on cyanide recovery and scan for possible

interference with the cyanide analytical technique. Untreated willow tissue root, stem, and leaf

were assessed for background cyanide content. Exposed tissue cyanide content was measured

using the extraction technique and compared with the tissue 15N concentrations.

The second objective was to demonstrate the uptake of free cyanide and ferrocyanide by the

willow plant. This work was performed collaboratively with Dr. Stephen Ebbs and students at

Southern Illinois University Carbondale (SIUC). The experiments were designed jointly and

conducted at SIUC, with water and tissue sample cyanide analyses at Carnegie Mellon

University. A well-controlled hydroponic system was constructed carefully to control the

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cyanide speciation within the solution and to minimize losses other than through the plant. Four

replicates of unplanted and planted solutions containing either KCN or Fe(CN)64‘ were sampled

for 20 days before sacrificing the plant tissue. Solutions were analyzed for total cyanide and free

cyanide while tissue was analyzed for total cyanide, free cyanide, and ,5N. The measurement of

tissue cyanide content was required to help interpret whether assimilation had occurred. The

solution and tissue concentrations were used to calculate a mass balance on both systems.

The third objective was to construct a model for the plant-solution system that represented the

physiological processes affecting cyanide fate within the system. Not all of the relevant

processes could be measured experimentally in the hydroponic study. Modeling provides a

method for estimating the parameter values for plant processes from the observations of the data

and, ultimately, for extension of the laboratory data to the field. A series of equations

representing the mass balances for free cyanide, ferrocyanide, and assimilated product formed

the model. Advection. diffusion in solution, plant-mediated dissociation and assimilation, active

uptake, cell wall adsorption, and volatilization were the mass transfer processes included. The

model contained 17 unknown parameters and consisted of 27 ordinary differential equations.

The fourth objective was to fit the plant uptake model to the hydroponic study observations and

assess the importance of the various cyanide mass transfer and transformation processes. A large

number of optimization runs (n = 500) with different initial values of parameter values was

required in order to find a global minimum when fitting the model predictions with the

arithmetically-averaged data. Each set of model output parameters was optimized based upon

comparison of the model results with the data. Optimal parameter values and the predicted

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fractional compart mental partitioning of initial cyanide mass were examined to assess process

importance. After review of initial data fitting results, the model was restructured with four less

parameters and the resulting 13-parameter model was fit to the arithmetically-averaged and

geometrically-averaged data. Uncertainty in optimal parameter values was assessed by

propagating the data uncertainty through the model. Data residuals were used to generate a

simulated, bootstrapped set of observations. Distributions of optimal parameter values were

generated by fitting the simulated data. The variability in the parameter values and predicted

compartmental concentrations was characterized.

1.2 Organization of Thesis

This work is arranged as a collection of independent contributions (Chapters 3 through 6). Each

is self-contained with an individual abstract, introduction, and summary. The final chapter joins

and summarizes the work from the individual papers and lists the major contributions to the

knowledge base and future recommendations. Information supplemental to the work presented

in Chapters 3 through 6 is provided in the appendices.

1J References

Agency for Toxic Substances and Disease Registry. (1997) Toxicological Profile for Cyanide. U.S. Dep. Health Human Serv., Public Health Serv., Atlanta, GA.

Dzombak, D.A.; Ali, M.A.; and Dobbs, C.L. (1993) “Evaluation of Subsurface Fate/Transport

of Chemical Species in Spent Potlining Leachate.” Division Report No. 08-93-350, Analytical

Chemistry Division, Aluminum Company of America, Alcoa Center, PA 15069.

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Dzombak, D.A.; Dobbs, C.L.; Culleiton, CJ.; Smith, J.R.; and Krause, D. (1996) “Removal of

Cyanide from Spent Potlining Leachate by Iron Cyanide Precipitation.” Proceedings: Water

Environment Federation, 60* Annual Conference & Exposition. Dallas, TX, October 5-9, 19%.

Ghosh, R.S.; Dzombak, D.A.: Luthy, R.G.; and Nakles, D.V. (1999) “Subsurface Fate and

Transport of Cyanide Species at a Manufactured Gas Plant Site.” Water Environ. Res. 71,1205.

Haupin, W.E. (1987) “Environmental Considerations.” Crit. Rev. Appl. Chem. 20:176.

Meeussen, J.L.; Keizer. M.G.: and de Haan, F.A.M. (1992) “Chemical Stability and

Decomposition Rate of Iron Cyanide Complexes in Soil Solutions.” Environ. Sci. Technol. 26,

511.

NY Times. (2000) “Cyanide Spill Kills Danube Fish.” February 14, 2000. p. A8.

Reeves, M. (2000). Treatment o f Fluoride and Iron Cyanides Using Willow: A Greenhouse

Feasibility Study. Master’s Thesis, Cornell University, January 2000.

Schnoor, J.L. (2002) Phytoremediation: Technology Evaluation Report TE-02-01. Ground-

Water Remediation Technologies Analysis Center, Pittsburgh, PA.

Seigler, D.S. (1991) “Cyanide and Cyanogenic Glycosides”, In: Rosenthal, G.A.; and

Berenbaum, M.A. eds. Herbivores: Their Interactions with Secondary Plant Metabolites, Vol. I:

The Chemical Participants , Academic Press, San Diego, CA.

Theis, T.L.; Young, T.C.; Huang, M.; and Knutsen, K.C. (1994) “Leachate Characteristics andComposition of Cyanide-Bearing Wastes from Manufactured Gas Plants.” Environ. Sci. Tech.

28:1,99.

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CHAPTER 2

B a c k g r o u n d cyan ide c h e m i s t r y (1)

2.1 Cyanide Chemistry

Cyanide occurs in many different aqueous chemical forms. The three common cyanide

distinctions are:

• free cyanide (HCN and CN),

• weakly-complexed or weak-acid dissociable (WAD) cyanide, and

• strongly complexed cyanide.

Free cyanide is volatile (pKa 9.2), mobile, and acutely toxic (ATDSR, 1997). WAD cyanide

compounds such as those with copper [Cu(CN)x‘ '] and zinc [Zn(CN)y~y] are weakly complexed

with cyanide, while gold, cobalt, and iron-cyanide complexes such as ferrocyanide [Fe(CN)64 ]

and ferricyanide [Fe(CN)63 ] are strongly-complexed. The strongly-complexed iron cyanides

represent a very common form occurring in groundwater systems at contaminated sites and are

also less toxic than free cyanide (Shifrin et al., 1996; Ghosh et al„ 1999b). The hazard

associated with complexed cyanides arises from dissociation to free cyanide upon exposure to

UV light (Meeussen et al., 1992; Young, 1995) such as occurs when groundwater discharges at

the surface. Exposure to UV light decreases the half-life for ferrocyanide in solution from

approximately 33 years (Ghosh et al., 1999b) to approximately 7 hours (Meeussen et al., 1992),

assuming that the dissociation rate is independent of the increasing free cyanide concentration.

a> Coauthored with Stephen Ebbs. David Dzombak, Rajat Ghosh, and Ed Neuhauser

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Sorption to and interaction with soil are important for cyanide, particularly in complexed forms.

Research indicates that both ferro- and ferricyanide complexes adsorb onto aluminum and iron

oxides especially in acidic conditions (Alesii and Fuller, 1976: Cheng and Huang, 19%; Theis

and West, 1986: Young and Theis, 1997; Appendix A). Free cyanide is less sorptive (Theis and

West, 1986) with soil association increasing with organic carbon content (Chatwin et al., 1988).

Iron-cyanide solid formation as Prussian Blue [Fe4(Fe(CN)6) 3 (s)] or Turnbull’s Blue

[Fej(Fe(CN) 6 )2 (s)] serves as another important mechanism of iron cyanide removal from solution

particularly in aqueous systems containing excess iron (Ghosh et al., 1999c). Iron cyanide

solubility increases with both pH and pe (Meeussen et al., 1994; Ghosh et al., 1999a) with iron-

cyanide as the prevalent dissolved cyanide form at high pH when excess iron is present (Ghosh

et al., 1999a: 1999c).

Dissolved cyanide speciation is strongly dependent on the pH, pe. and relative concentrations of

metal and cyanide in solution. Metal complexation dominates cyanide speciation at neutral to

alkaline PH values, as cyanide competes successfully with hydroxide for complexation with

metals. Increasing the solution pe (e.g. with the addition of O^,) influences cyanide chemistry

by favoring the more oxidized metal valence state (i.e. Fe3+ over Fe2*), altering the binding

affinities of the associated metal cations with which cyanide complexes. At pH and pe

conditions favoring complexation, cyanide will bind preferentially to form strong complexes,

such as those with iron, gold, and cobalt, followed by weak complexes such as those with zinc

and copper. However, solution cyanide speciation depends on the specific solution composition,

particularly the relative concentration of dissolved metals, with the chemical complexity

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preventing a more detailed general discussion. Ghosh et al. (1999a), Meeussen et al. (1992), and

Shifrin et al. (1996) provide a more complete discussion of solution cyanide chemistry.

Cyanide speciation, sorption to soil, and precipitation will affect the bioavailability of cyanide

under field conditions. For phytoremediation to be successful, the contaminant of interest must

be in a soluble form available for uptake into the plant. An understanding of the soil cyanide

chemistry is necessary for optimizing the availability and treatment of cyanide with willows.

2.2 Anthropogenic Cyanide Sources

Cyanide contamination of groundwater and surface water is common at manufactured gas plant

(MGP) sites (Theis et al., 1994), spent potlining (SPL) from aluminum production (Dzombak et

al., 19%), and gold mining. For MGP sites, product gas streams from the coal carbonization

process were purified by passage through boxes containing rusted iron filings/ores and other

forms of iron-containing solids to remove selected impurities, notably H;S and HCN. The sulfur

and cyanide were removed from the gas through a combination of reactions and sorption with the

solid media. For SPL facilities, cyanide was produced on the carbon cathode in the aluminum

reduction cell presumably due to nitrogen from the air diffusing in and reacting with sodium and

hot carbon (Haupin, 1987). Spent solids from both MGP sites and SPL facilities, including those

containing cyanide, were managed both onsite and offsite, depending upon site-specific

conditions and circumstances. At some sites, the use of these solid residues as HU led to

groundwater impacts, which resulted from the leaching of cyanide compounds from these solids

into infiltrating rainwater or directly into groundwater (Dzombak et al., 1993; Ghosh et al.,

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1999b). For mining, cyanide is used to extract small amounts of precious metals from ore

because of the binding capabilities and solubility of metal-cyanide complexes. The

contaminated process water has been stored behind tailing dams such as the one that broke

spilling cyanide-laden water down the Danube River. Cyanide is also used as a raw material

during chemical production of nylon, plastics, pesticides, fire retardants, cosmetics, and

pharmaceuticals. Cyanide is a common anti-caking agent in road salt (Paschka et al., 1999) and

also as a filler or dye in printing inks and pottery glazes.

1 3 Cyanide in Nature

Anthropogenic activities are not the only source of cyanide release into nature as plants already

contain production pathways for cyanide and cyanide derivatives. At least 2,650 plant species

from more than 550 genera and 130 families can produce cyanogenic glycosides (Figure 2.1),

including many food sources such as cassava and sorghum (Seigler, 1998). While largely used

as a defense mechanism by releasing free cyanide during tissue rupture (Taiz and Zeiger, 1998),

cyanogenic glycosides are also used as a nitrogen source in young plant tissue as determined

through comparison of hydrolysis and assimilation enzyme activity (Selmar et al., 1988; 1990).

Some insects have adapted to the cyanogenic potential of specific plants for their own benefit.

The heliconius butterfly has developed detoxification mechanisms to gain a feeding monopoly

(Engler et al., 2000) while the eastern tent caterpillar accumulates cyanogenic chemicals for its

own defense. The poisoning effect of the caterpillars was suspected in recent incidents involving

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the death of foals at Kentucky horse farms (NY Times, 2001), leading to a characterization of

cherry tree cyanide content (Appendix B).

Cyanide is also produced within plants during the production of ethylene (Mizutani et al., 1987;

Seigler, 1998). Cyanide is released upon conversion of 1-aminocyclopropane-l-carboxylic acid

(ACC) to ethylene by ACC oxidase (Figure 2.2). The production of cyanide as a by-product

during ethylene synthesis provides evidence that the distribution of cyanide within plants

exceeds those containing cyanogenic glycosides as ethylene is a ubiquitous plant hormone. The

cyanide concentration in plants due to both pathways is shown in Figure 2.3.

2.4 Cyanide Toxicity

2.4.1 Animals

The free cyanide species exhibit acute toxicity towards humans and animals through inhalation

and ingestion (ATSDR. 1997). Free cyanide binds with cytochrome oxidase in red blood cells,

prevent O: from binding and reaching cells. Cytochrome oxidase binds O: through Fe3+and Cu*

cofactors. Cyanide has a stronger affinity for the cofactors compared with (K The drinking

water MCL is 0.2 mg/L as free cyanide while the U.S. water quality criteria for free cyanide are

22 pg/L acute and 5 pg/L chronic (ATSDR, 1997).

Animals detoxify cyanide poisoning via reaction with the enzyme rhodanese or through the

formation of cyanomethemoglobin. Rhodanese catalyzes the conversion of low-levels of

cyanide to thiocyanate in the presence of sulfur donor groups. The thiocyanate is then excreted

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2^ Natural Cyanide Cycle

Cyanide does not accumulate within plant tissue. Both plants and animals possess mechanisms

for the detoxification of cyanide. Cyanide produced within plant tissue or present in animals is

recycled in the nitrogen cycle either by plant or microbiological breakdown (Figure 2.5). Plants,

such as willow, function in the cycle to extract cyanide species from the soil and groundwater as

well as to assist in converting the cyanide into biologically acceptable nitrogen sources. The

absence of cyanide accumulation supports the detoxification of cyanide produced in nature. The

same is also true for anthropogenically-produced cyanide. Dissolved cyanide enters groundwater

and surface water, either directly or from dissolution of cyanide solids. The cyanide can then

enter the natural cycle and be converted to nitrogenous products.

2.6 References

Agency for Toxic Substances and Disease Registry. (1997) Toxicological Profile fo r Cyanide.

U.S. Dep. Health Human Serv., Public Health Serv., Atlanta, GA.

Alesii, B.A., and Fuller, W.H. (1976) “The Mobility of Three Cyanide Forms in Soils.” Proc.

Haz. Waste Res. Symp., EPA-600/9-76-015, U.S. EPA, Cincinnati, Ohio.

Agency for Toxic Substances and Disease Registry. (1997) Toxicological Profile for Cyanide.

U.S. Dep. Health Human Serv., Public Health Serv., Atlanta, GA.

Chatwin, T.D.; Zhang, J.; and Gridley, G.M. (1988) “Natural Mechanisms in Soil to Mitigate

Cyanide Releases.” Superfund '88: Proc.


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Cheng, W.P.; and Huang, C. (1996) “Adsorption Characteristics of Iron Cyanide Complex on y-

AI2O 3 .” J. Colloid Interface Sci. 181:627

Dzombak, D.A.; Ali. M.A.: and Dobbs, C.L (1993) “Evaluation of Subsurface Fate/Transport

of Chemical Species in Spent Potlining Leachate.” Division Report No. 08-93-350, Analytical

Chemistry Division, Aluminum Company of America, Alcoa Center, PA 15069.

Dzombak, D.A.; Dobbs, C.L: Culleiton, C.J.; Smith, J.R.; and Krause, D. (1996) “Removal of

Cyanide from Spent Potlining Leachate by Iron Cyanide Precipitation.” Proceedings: Water

Environment Federation, 6&h Annual Conference & Exposition. Dallas. TX, October 5-9. 1996.

Elias, M.; Sudhakaran, P.R.; and Nambisan, B. (1997) “Purification and Characterisation of P-

Cyanoalanine Synthase from Cassava Tissues.” Phytochemistry. 46:469.

Engler, H.S.: Spencer, K.C.; and Gilbert, L.E. (2000) “Insect Metabolism: Preventing Cyanide

Release from Leaves.” Nature. 406:144.

Ghosh, R.S.; Dzombak. D.A.; and Luthy, R.G. (1999a) “Equilibrium Precipitation and

Dissolution of Iron Cyanide solids in Water.” Environ. Eng. Sci. 16:293.

Ghosh, R.S.; Dzombak, D.A.; Luthy, R.G.; and Nackles, D.V. (1999b) “Subsurface Fate and

Transport of Cyanide Species at a Manufactured Gas Plant Site.” Water Environ. Res. 71:1205.

Ghosh, R.S.; Dzombak, D.A.; Luthy, R.G.; and Smith, J.R. (1999c) “In Situ Treatment of

Cyanide-Contaminated Groundwater by Iron Cyanide Precipitation.” Water Environ. Res. 71:

1217.

Gonzalez-Meler, M.A.; Ribas-Carbo, M.; Giles, L; and Siedow, J.N. (1999) “The Effect of

Growth and Measurement Temperature on the Activity of the alternative Respiratory Pathway.”

Plant Physiol. 120:765.

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Grossmann, K. (1996) “A Role for Cyanide, Derived from Ethylene Biosysnthesis, in the

Development o f Stress Symptoms.” Physiol. Plant. 97:772.

Haupin, W.E. (1987) “Environmental Considerations.” Crit. Rev. Appl. Chem. 20:176.

Koster, H.W. (2001) Risk Assessment o f Historical Soil Contamination with Cyanides: Origin.

Potential Human Exposure and Evaluation o f Intervention Values. RIVM report 711701019.

Rijksinstituut voor Volksgezondheid en Milieu (National Institute of Public Health and the

Environment). Bilthoven, The Netherlands.

Meeussen, J.L.; Keizer. M.G.: and de Haan. F.A.M. (1992) “Chemical Stability and

Decomposition Rate of Iron Cyanide Complexes in Soil Solutions.” Environ. Sci. Technol.

26:511.

Meeussen, J.L.; Keizer, M.G.; van Riemsdijk, W.H.; and de Haan, F.A.M. (1994) “Solubility of

Cyanide in Contaminated Soils.” J. Environ. Qual. 23:785.

Mizutani, F.; Hirota, R.; and Kadoya, K. (1987) “Cyanide Metabolism Linked with Ethylene

Biosynthesis in Ripening Apple Fruit.” J. Japan. Soc. Hort. Sci. 56:31.

NY Times. (2001) “Cyanide Possible Cause of Deaths.” May 25, 2001. p. D7.

Paschka, M.G.: Ghosh, R.S.; and Dzombak, D.A. (1999) “Potential Water Quality Effects from

Iron Cyanide Anti-Caking Agents in Road Salt.” Water Environ. Res. 71:1235.

Selmar, D.; Lieberei, R.; and Biehl, B. (1988) “Mobilization and Utilization of CyanogenicGlycosides: The Linustatin Pathway.” Plant. Physiol. 86:711.

Selmar, D.; Grocholewski, S.; and Seigler, D.S. (1990) “Cyanogenic Lipids: Utilization During

Seedling Development of Ungnadia speciosa." Plant. Physiol. 93:631.

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Shifrin, N.S.; Beck, B.D.; Gauthier, T.D.; Chapnick, S.D.: and Goodman, G. (19%)

“Chemistry, Toxicology, and Human Health Risk of Cyanide Compounds in Soils at former

Manufactured Gas Plant Sites.” Regul. Toxicol. Pharmacol. 23:106.

Seigler, D.S. (1998) Plant Secondary Metabolism. Kluwer Academic Publishers, Boston.

Taiz, L, and Zeiger, E. (1998) Plant Physiology, 2nd Ed. Sinauer Associates, Inc., Sunderland,

MA.

Theis, T.L.; and West, M J. (1986) “Effects of Cyanide Complexation on Adsorption of Trace

Metals at the Surface of Goethite.” Environ. Technol. Lett. 7:309.

Theis. T.L.: Young, T.C.; Huang, M.: and Knutsen, K.C. (1994) “Leachate Characteristics and

Composition of Cyanide-Bearing Wastes from Manufactured Gas Plants.” Environ. Sci. Tech.

28: 99.

Tittle, F.L.: Goudey. J.S.: and Spencer, M.S. (1990) “Effect of 2,4-Dichlorophenoxyacetic Acid

on Endogenous Cyanide, p-Cyanoalanine Synthase Activity, and Ethylene Evolution in

Seedlings of Soybean and Barley.” Plant Physiol. 94:1143.

Yip, W.; and Yang. S.F. (1988) “Cyanide Metabolism in Relation to Ethylene Production in

Plant Tissues.” Plant Physiol. 8 8 , 473.

Young, T.C. (1995) Issues Pertaining to Environmental Transport, Fate, and Biotic Exposure

to Complex Cyanides in Surface HzO: Research Needs fo r Mathematical Modeling. Alcoa

Technical Center Report, Pittsburgh, PA.

Young, T.C.; and Theis, T.L. (1997) “Exposure Assessment and Fate of Cyanides in Surface

Waters.” Proc. Water Environ. Fed. 7(fh Annu. Conf. Exposition. Chicago, 111., 3:167.

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Figure 23 Cyanide content of some common plants. Note that the figure is not to scale. Units

are same as mg/kg for comparison with some data from willow uptake study.

Figure 2.4 Assimilatory reactions for cyanide within plants. Ferrocyanide may be dissociated

prior to assimilation.

Figure 23 Natural cyanide cycle in the environment. Cyanide is broken down within the plant

or by microorganisms. Anthropogenic sources released into soil and groundwater for conversion

within the soil column or uptake into plants.

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N=C O-gfucose CMH O .- C C !«

HO* OK M Ono

M O ' O H

Amygdalin LinamarinOHDhurrin

Figure 2.1

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zu£

so3t/1

3 -i': j °o

oc

JUoC / 2


F i g u r e

2 . 2

29


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F r e e C y a n i d e C o n c e n t r a t i o n

Source: ATSDR, 1997

Figure 2.3

produced with permission of the copyright owner. Further reproduction prohibited without permission.


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CH-jSH

CHNH2

COOH

Cvstene

+ HCN

CHjCN

CHNH2

ICOOH

Cyanoalanine

H>S

CONH2

: h 2

CHNH-.

ICOOH

Asparagine

FeiCN)**Plant Assimilation

Mediated

■> CN -► Amino Acids

Figure 2.4

23

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Plant cyanogenic glycosides

Cyanogenic N incorporal into plant

Insects feeding On leaves

Decay of insects and leaves releases free cyanide

ami no-acids ■1t

i

FFC used as fill Soil bacteria and fungi convert CN* to nitrate

rand ammonia

roeen I

Lracna l e

V Plant source of nil

(free cyanide, FelCN nitrate and ammonia)

>groundwater

Figure 2.5

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CHAPTER 3

P l a n t t i s s u e e x t r a c t i o n m e t h o d f o r

COMPLEXED AND FREE CYANIDE*!)

A b s t r a c t

A method for measurement of the cyanide speciation and concentration within plant tissue

was developed to study uptake and movement of cyanide species separately from cyanide

metabolism and metabolite movement by a willow plant (SalLx eriocephala var. Michaux).

Spike recoveries from solutions with and without plant tissue, using various solvent

combinations, and background control tissue contributions were investigated to obtain an

accurate and precise extraction method for measurement of complexed and free cyanide

concentrations within plant tissue. The optimum extraction technique involved the freezing

of plant tissue with liquid nitrogen to facilitate homogenization prior to extraction.Homogenized willow tissue samples. 1 to 1.5 g-FW. were re-ground under liquid nitrogen

followed by grinding in slurry with 2.5 M NaOH. The slurry' was brought to 100 mL volume,

sonicated for five minutes, extracted in the dark for sixteen hours, and analyzed without

filtration for total and free cyanide by acid distillation and microdiffusion respectively.

Sample tissue extraction controls found recoveries of 89% and 100% for 100 ppb CNt as

KCN and KaFefCNfo spiked -in willow tissue slurries. Methanol, hexane, and 2-octanol

inclusion in the solvent matrix with 2.5 M NaOH interfered with the cyanide analytical

technique while chloroform reacted with NaOH and free cyanide in solution. Filtration was

' 1' Coauthored with Stephen Ebbs and David Dzombak

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not included due to increased cyanide loss, and analysis of control tissue showed minimal

release of cyanide or interference of plant tissue with the cyanide analytical method. Tissue

cyanide concentrations from hydroponically-exposed tissue using the optimal extraction

method agreed with tissue cyanide stable isotope (I 5 N) results.

Keywords: cyanide, ferrocyanide. extraction, plant analysis, plant concentration, willow.

Salix eriocephala var. Michaux

Abbreviations: CN - cyanide: CNT - total cyanide: FW - fresh weight: GC-ECD - gas

chromatography-electron capture detector: MeOH - methanol: NaOH - sodium hydroxide

3.1 Introduction

Measurement of cyanide within plant tissue is important for evaluation of phytoremediation

of cyanide in soil and groundwater (Chapter 4) and also for assessing routes of cyanide

toxicity to both plant and animals. For a phytoremediation system, cyanide must be taken up

from solution and assimilated within plant tissue as plant tissue containing cyanide,

particularly in the free form, can be toxic if consumed (ATSDR. 1997: Koster. 2001). The

fate of the cyanide and the toxic risk associated within the plant tissue must be considered.

Therefore, the removal of solution cyanide together with evidence of assimilation within plant

tissue determines remediation effectiveness.

Cyanide occurs naturally in plant tissue due to the breakdown of cyanogenic glycosides

(Selmar et al.. 1990) as well as cyanide release during ethylene synthesis (Grossman and

Kwiatkowski. 1995: Yip and Yang. 1988), but can also occur due to uptake from

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contaminated water and soil. Previous methods for cyanide determination in plant tissue have

utilized various solvent extraction techniques with an emphasis on the determination of

cyanide release potential, primarily from the breakdown of cyanogenic glycosides, rather than

cyanide speciation and concentration.

Some studies of plant tissue analysis for cyanide have employed extraction methods similar to

the conventional distillation (APHA/AWWA/WEF. 1998) and microdiffusion (ASTM. 1998)

analytical techniques with colorimetric determination. Howe and Noble (1985) digested plant

tissue samples directly in a distillation apparatus with MgCN and NaH;POj prior to color

development using chloramine-T and pyridine barbituric acid reagent. Tittle et al. (1990)

acid-digested tissue in a distillation unit with analysis of the liberated free cyanide both

colorimetrically and by gas chromotography after bromination. Mizutani et al. (1987) also

utilized bromination for analysis of cyanide in apple samples ground under distilled water.

Forensics analysis for chemical poisoning has provided additional examples for cyanide

analysis of biological samples. The analytical methods for total and free cyanide content of

animal tissue are modified distillation (Nolte and Dasgupta. 1996) and microdiffusion

(Swanson and Krasseit. 1994) techniques. Analytical concerns for animal tissues are similar

to those for plants in that the samples must be preserved to prevent cyanide release prior to

analysis and the samples contain high amounts of various organics that have the potential to

interfere with cyanide detection.

None of the reported techniques for cyanide analysis in plant tissue have explored the issues

of cyanide recovery during the extraction process or plant tissue interference. Many of the

plant studies have been concerned only with free cyanide concentration and release potential,

primarily from cyanogenic glycosides. As such, clean-up of samples is directed towards

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purifying the cyanogenic glycoside rather than removing cyanide analytical interferences.

Regarding cyanide speciation. only Howe and Noble (1985) addressed total cyanide

concentration.

Breakdown of the plant tissue in a plant sample and the analytical interference issues that can

result are of concern for cyanide analysis. Yet. tissue destruction is necessary to bring about

complete release to solution of plant cyanide content. Typical methods for stripping organic

material from plant tissue involve a methanol/chloroform (2:1 v/v) soak for three days (Cohen

et al.. 1998: Hart et al.. 1998). However, breakdown products from the destruction of plant

tissue, particularly organics or sulfides, can interfere with the cyanide detection technique

(APHA/AWWA/WEF. 1998). Also, the release of naturally-occurring cyanide or cyanide-

reactive compounds into solution can artificially elevate cyanide concentration measurements

for plant tissue exposed to external cyanide sources. Cyanogenic glycosides are one

classification of naturally-occurring compounds that release cyanide upon enzymatic

hydrolysis, at neutral pH. to form the highly volatile HCNlg>. for the purpose of protection

versus herbivory or as a nitrogen source in seedling development. Glycosidic cyanide can be

released via hydrolysis following plant tissue extraction in a polar organic solvent such as

methanol or ethanol (Forslund and Jonsson. 1997; Kobaisy et al.. 1996: Selmar et al.. 1990).

Extreme pH conditions, such as those used for preserving and analyzing aqueous cyanide

samples, disable the functionality of the hydrolytic enzyme and prevent release of cyanide,

thereby minimizing interference (Halkier and Moller, 1990; Lechtenberg et al.. 1994).

Analytical methods used to determine cyanide content after extraction involve capturing

released cyanide on picrate paper (Jacobs et al., 1996) according to the Feigl-Anger method

(Aikman et al., 1996), the use of NaOH-soaked paper (Grossman and Kwiatkowski, 1995), or

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the bromination of cyanide trapped in caustic solution (Mizutani et al.. 1987: Tittle et al..

1990). Picrate paper is calibrated based upon the color change of the paper while cyanide

trapped on NaOH-soaked paper is extracted and analyzed via gas chromatography. Analysis

is performed in a closed flask to capture released cyanide gas. Recovery was only 16% for

the NaOH-soaked paper (Grossman and Kwiatkowski. 199S) and the analytical precision of

picrate paper is limited by the discernment of the color range relative to controls, particularly

compared to detection ability with aqueous cyanide analyses (APHA/AWWA/WEF. 1998).

The bromination technique involves additional sample handling, increasing the potential for

free cyanide loss. Another limitation of all three analyses is that only free cyanide is targeted

unless the sample is acid-distilled prior to analysis.

Detection limitations and sample preservation were a concern for developing a methodology

to extract and analyze cyanide within willow plant tissue. The analysis of cyanide in an

aqueous sample by extraction and distillation (APHA/AWWA/WEF. 1998) and

microdiffusion (ASTM. 1998) offers precision, a lower detection limit, and the ability to

measure complexed cyanide. Development of an extraction method yielding a solution that

can be analyzed successfully with these analytical techniques was the focus of this study. To

preserve the initial tissue cyanide speciation. it is necessary that the extracted cyanide be

preserved in basic solution with limited additional cleanup of the extraction solution to

minimize the potential for free cyanide losses. The investigation and method development of

tissue analysis for cyanide was structured with these limitations in mind. The objective of the

investigation was to develop a method that minimized cyanide losses from the system while

providing an accurate and precise measure of cyanide content and speciation within plant

tissue. Experiments involving spike recoveries from solutions with and without plant tissue.

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using various solvent mixtures, and background control tissue contributions were conducted

to obtain the optimal extraction method.

3.2 Methods

Solvent combinations were examined to assess the potential interference of solvent w ith the

cyanide analytical technique using solution spike samples without tissue. Un-exposed willow

tissue was utilized to assess background cyanide concentrations in willow and to determine

cyanide recovery from solution spike samples. Willow growth and treatment is described in

Chapter 4. as is the cyanide concentration and speciation of exposed willow tissue obtained in

support of hydroponic experiments. Exposed pea tissue was used for some preliminary

solvent testing as a surrogate for willow tissue to assess the effect of solvent combinations on

extraction. Pea tissue samples were grown for preliminary testing due to their common use

and rapid growth (Appendix C) until the willow crop reached maturity and willow control

tissue became available.

Exposed willow and pea tissue were used to examine the effects of tissue homogenization

prior to extraction. Preparation of plant tissue prior to extraction is important for obtaining

uniform, consistent results as determined by the sample replicate standard deviation.

Extraction tests were performed on plant tissue samples with (willow) and without (pea)

grinding under liquid nitrogen (i.e., sample homogenization) prior to extraction in 2.5 M

NaOH. Grinding of plant tissue under liquid nitrogen increases recovery of tissue content by

rupturing cells (Halkier and Moller, 1990; Lechtenberg et al.. 1994) and improves

measurement precision w'hile freezing minimizes volatilization losses. Willow and pea root

tissue taken from plants exposed to 2 ppm CNT as K 4 Fe(CN ) 6 for 20 and 7 days, respectively.

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were extracted in 2.5 M NaOH with and without homogenization. The results demonstrated

that the inclusion of liquid nitrogen grinding significantly reduced the standard deviation of

specific tissue total cyanide content replicates from 27 to 10% (p 4/water (1:1 v/v) to

overcome the additional buffering capacity of the 2.5 M NaOH extract solution relative to the

conventional 1.6 g L ' 1 NaOH concentration used in the analytical methods cited.

3.2.1 Solvent Selection

Solvent choice is important for maximizing tissue breakdown and cyanide recovery without

adversely affecting cyanide analysis. Analysis of solids for cyanide content involves a 16-

hour leach in 2.5 M NaOH (APHA/AWWA/WEF, 1998). The combination of both extreme

base concentration during extraction and high acid concentration during the distillation, as

employed in solid extraction, provides a wide pH range to break apart plant tissue while

preventing enzymatic action during extraction and distillation. Previous literature on cyanide

extraction from plants discusses the use of caustic solution for tissue extraction, and also the

use of methanol (CH 3OH) and chloroform (CHC13) for tissue breakdown by attacking tissue

with both a polar and an organic compound (Cohen et al., 1998; Hart et al., 1998). Each of

these solvents was examined in this study. Hexane (C 6 Hu) and 2-octanol (2-CsHitOH) were

also examined to broaden the range of solvent polarity based upon a recommended method

for preventing fatty acid interference in cyanide analysis (APHA/AWWA/WEF, 1998).

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Results obtained with caustic (2.5 M NaOH) were used as the baseline for comparison.

Alternative solvents were investigated against the baseline recovery using cyanide-spiked

solutions without tissue. Some preliminary investigations were performed with

hydroponically-exposed pea tissue. Caustic was always included in the solvent matrix to

maintain a high pH in the extract and minimize volatilization losses during extraction. The

various solvent matrices were assessed based upon cyanide recovery and the extent of

interference with the cyanide analytical technique relative to the baseline levels.

Methanol in combination with 2.5 M NaOH was the first solvent matrix examined. Concerns

about methanol interference with cyanide colorimetric analysis suggested evaporation of the

Documents

Modeling Cyanide