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.
All rights reserved.
UMI*UMI Microform 3084715
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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
□ATE
DEPARTMENT HEAD 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
4.5 Acknowledgements ....................................................................................................... 71
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
5.6 Acknowledgements ....................................................................................................... 106
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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
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F i g u r e
2 . 2
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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
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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
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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
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