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
UMI Number 3084715
Copyright 2003 by
Bushey, Joseph T.
All rights reserved.
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CARNEGIE INSTITUTE OF TECHNOLOGY
TH ESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE O F .
Doctor of Philosophy____
t it l e
Modeling Cyanide Uptake bv Willows for Phvtoremediation
PRESENTED BY
Joseph T. Bushev
ACCEPTED BY THE DEPARTMENT OF
Civil and Environmental Engineering

9 / F 5 Y 2 .0 0 5
DATE
S'J/f/jop z
ATE
~ -! 6->5
DEPARTMENT HEAD
DATE
APPROVED BY THE COLLEGE COUNCIL
DATE
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.
ii
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 the
experimental 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
iii
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.
iv
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
I S Natural Cyanide Cycle..................................................................................................14
2.6 References.................................................................................................................... 14
3. PLANT TISSUE EXTRACTION METHOD FOR COMPLEXED AND FREE
CYANIDE..................................................................................................................................25
3.1 Introduction................................................................................................................. 26
3 2 Methods......................................................................................................................... 30
3.2.1 Solvent Selection............................................................................................. 31
3.2.2 Sample Spike Recovery................................................................................... 33
3.2.3 Control Tissue................................................................................................. 34
3 3 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
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
vi
Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission.
5.7 References..................................................................................................................106
6. MODEL FOR CYANIDE UPTAKE BY WILLOW: APPLICATION TO
EXPERIMENTAL DATA AND CALIBRATION............................................................... 117
6.1 Introduction................................................................................................................ 118
6 J 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
V ll
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 STUDYINGCYANIDE UPTAKE.................... 219
D.I Introduction................................................................................................................. 220
D.2 Cyanide Chemistry of Hydroponic Test Solution.................................................... 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
viii
%
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
E 2 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
E J 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
F J 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 C ode.........................................................267
G.2 Plant Uptake Hydroponic System Code - 17-Parameter M odel............................ 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
H J Model Output Distributions...................................................................................... 310
H.4 Ferrocyanide Adsorption to Aluminum O xides.......................................................336
ix
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 43 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 5Jl 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
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 2octanol: NaOH mixtures...........................................................................................290
Table H.2.1.1 Individual cyanide concentration (a) and mass (b) replicate data for
hydroponic uptake experiment.................................................................................291
xi
Table HJ .I .2 Hydroponic solution volume.........................................................................299

Table H.2.1.3 Hydroponic solution pH ................................................................................300
Table H.2.1.4 Hydroponic solution p e .................................................................................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
Table 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 H A 2 Ferrocyanide (1 ppm) adsorption to 0.6 g/L g -A L O ^ ....................................338
Table H A 3 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 tovarious solid doses of Al(OH)3 (s).............343
xii
LIST OF FIGURES
CHAPTER 2
Figure 2.1 Some common cyanogenic glycosides................................................................20
Figure 2 3 Conceptual diagram pathways of cyanide cycling in general plant metabolism. 21
Figure 2 3 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 3 3 Investigation of 2.5 M NaOH/chloroform solution KCN-spike samples........... 47
Figure 3 3 Free cyanide recovery from KCN-spike solutions.............................................. 48
CHAPTER4
Figure 4.1 l5N enrichment ratios for willow roots, stems, and leaves.................................. 83
Figure 4 3 15N content of willow roots, stems, and leaves................................................... 84
Figure 4 3 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 6 3 Predicted solution total cyanide mass profile for optimal parameter values
158
Figure 6 3 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
xiii
Figure &5 / W CVversus KmCNfor parameter output sets for simulated input data sets....... 161
Figure 6.6 / w FC versus KFCfor parameter output sets for simulated input data sets....... 162
Figure 6.7 e / c versus Yrooi for parameter output sets for simulated input data sets
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 <s>.............. 201
Figure A3 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 D3 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
xiv
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
XV
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 Turbulls Blue [Fe3 (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 Romanias 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. The
Gas Technology Institute. ALCOA. Inc.. and The New York Gas Group. Report coauthored with Stephen Ebbs.
David Dzombak. Rajat Ghosh, and Stephen Geiger.
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 with 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
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
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 of 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
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
fractional compartmental 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.
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. Masters Thesis, Cornell University, January 2000.
Schnoor, J.L. (2002) Phytoremediation: Technology Evaluation Report TE-02-01. GroundWater 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 and
Composition of Cyanide-Bearing Wastes from Manufactured Gas Plants. Environ. Sci. Tech.
28:1,99.
CHAPTER 2
Ba c k g r o u n d
c y a n id e c h e m ist 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
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 Turnbulls 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 ironcyanide 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
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.,
10
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
11
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
12
in urine (ATSDR, 1997). Reaction of cyanide with methemoglobin forms cyanomethemoglobin

which is eventually purged in urine. Methemoglobin forms from the reaction of hemoglobin
with an oxidant (e.g. nitrate). A common antidote for cyanide poisoning is sodium thiosulfate
(N a 2 S 2 0 3 ).
2.4.2
Plants
Cyanide inhibits many metalloenzymes and can also form an inhibitory compound upon reaction
with a Schiff base intermediate. Such enzymes are involved in major plant metabolic pathways,
including photosynthesis and respiration (Grossmann, 1996). The interference of cyanide with
proteins disrupts cellular function affecting ATP production, ion uptake, and phloem transport
(Koster, 2001). Plants contain an alternative cyanide-resistant respiration pathway that operates
during exposure to elevated cyanide levels, minimizing the impact of cyanide on plant energetics
and metabolism (Gonzalez-Meler et al., 1999).
Plants have developed assimilatory pathways to reduce the detrimental effects of cyanide.
Evidence of such a reaction to offset endogenous free cyanide production has been suggested
through reaction with cysteine to form 3-cyanoalanine and asparagine as given in Figure 2.4
(Seigler, 1998; Elias et al., 1997). fi-cyanoalanine synthase activity increases with ethylene
production to prevent an elevation of cyanide concentrations within plant tissue (Mizutani et al.,
1987; Tittle et al., 1990; Yip and Yang, 1988). Assimilation of cyanide also provides a means of
biologically recovering the carbon and nitrogen and directing those atoms back into the amino
acid pool from which cyanide is ultimately derived (Figure 2.2). The potential also exists for
plant assimilation of Fe(CN)6v beginning with complex dissociation into iron and free cyanide.
13
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 for Cyanide.
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 fo r Cyanide.
Chatwin, T.D.; Zhang, J.; and Gridley, G.M. (1988) Natural Mechanisms in Soil to Mitigate
Cyanide Releases. Superfund '88: Proc. <fh Nat. Conf. November 28-30, 1988. Washington,
DC, 467.
14
Cheng, W.P.; and Huang, C. (1996) Adsorption Characteristics of Iron Cyanide Complex on yAI2 O 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 PCyanoalanine 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.
15
Grossmann, K.
(1996) A Role for Cyanide, Derived from Ethylene Biosysnthesis, in the
Development of 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)
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 Cyanogenic
Glycosides: 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.
16
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
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.
17
Figure Legends
Figure 2.1 Some common cyanogenic glycosides. Upon tissue rupture, enzymatic breakdown
releases free cyanide.
Figure 2 2 A conceptual diagram showing the two common pathways of cyanide cycling in the
general plant metabolism. Plants synthesize all 20 of the amino acids required for protein
synthesis and metabolism (1). Following its synthesis, methionine is diverted for other uses
besides protein synthesis (2). Methionine can serves a precursor (3) for 1-aminocyclopropane-lcarboxylic acid (ACC), which is converted to ethylene with the concomitant synthesis of HCN
(4). In some plants, aromatic amino acids such as tyrosine and phenylalanine, as well as some
nonpolar amino acids (5), are used to synthesize cyanogenic glycosides (6) that are stored in
leaves and seeds. Cyanogenic glycosides are cleaved to release HCN (7) during pathogenic
attack or by the seed embryo to release CN as a source of C and N for the amino acids needed for
growth. At low to moderate concentrations, the cyanoalanine pathway and other enzymes (i.e.rhodanese) (8) convert the cyanide and utilize the C and N directly to synthesize more amino
acids. Ethylene, produced during periods of plant stress, enhances the activity of this pathway,
increasing the efficiency of CN recovery. Plants would experience cyanide toxicity when CN
levels are maintained for long periods of time at concentrations higher than those that can be
effectively detoxified by (8). Iron cyanide phytoremediation and assimilation are likely involved
with this pathway (dotted arrows) because Fe(CN)6x' can dissociate in the external media to
release cyanide (9) that is transported and assimilated by the plant, or, as suggested by the data in
this study, assimilated directly by the plant after Fe(CN)6 X uptake (10).
18
Figure 2 3 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 2 3 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.
19
N=C
HO.
O-gfucose
CM
- C
HO*
OK
MO
no
M O'
OH
A m ygdalin
OH
Linamarin
Dhurrin
Figure 2.1
20
C !
z
u
so
3
t/1
3 -i'
:
29
oc
JU
o
Figure 2.2
C /2
Free Cyanide Concentration
S ource: ATSDR, 1997
Figure 2.3
CONH2
CH-jSH
CHjCN
:h2
CHNH2
CHNH2
CHNH-.
+ HCN
H>S
COOH
COOH
COOH
Cvstene
Cyanoalanine
Asparagine
Plant
FeiCN)**
>
CN
Assimilation
-
Amino Acids
Mediated
Figure 2.4
23
Plant cyanogenic
glycosides
Insects feeding
On leaves
Cyanogenic N
incorporal
into plant
amino-acids
Decay of insects and leaves

releases free cyanide
1
FFC used as fill Soil bacteria and fungi
convert CN* to nitrate
L ra c n a le
r and ammonia
V
I
Plant source of nil roeen
(free cyanide, FelCN
nitrate and ammonia)
t
i>
groundwater
Figure 2.5
24
CHAPTER 3
Plant
t is s u e e x t r a c t io n m e t h o d f o r
COMPLEXED AND FREE CYANIDE*!)
A bstract
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
25
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 (I5 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
26
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
27
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 cyanidereactive 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
28
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.
29
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 with 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 K4 Fe(CN) 6 for 20 and 7 days, respectively.
30
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<0.0185).
Statistical
analysis was assessed using a t-test to determine p. the probability of the results being the
same.
Sample extract was analyzed for total cyanide by distillation (APHA/AWWA/WEF. 1998)
and free cyanide by microdiffusion (ASTM. 1998). with cyanide content determined
colorimetrically. The pH was reduced in each method by additional H;SC>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 16hour 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 (CH3 OH) 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 (C6 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).
31
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
methanol prior to distillation. To examine the effect of evaporation in addition to potential
methanol spectrophotometric interference, eight 50 ppb CNt as KCN sample spikes were
prepared: two NaOH controls and six volumetric flasks containing 30 mL caustic brought to
100 mL volume with methanol. Three of the MeOH: NaOH samples were evaporated
overnight in an oven at 60C while the others were sealed and mixed for 16 hours. Aliquots
were drawn from the caustic portion of each sample and analyzed for total cyanide before and
after distillation.
Testing of chloroform as an extraction enhancer was performed using extraction of cyanidespiked solvent mixtures. As caustic-chloroform mixtures can be explosive (McKetta and
Cunningham, 1976), flasks for samples containing NaOH-chloroform mixtures were left open
during mixing but kept closed during sonication due to increased volatilization potential
caused by sample heating. Spike samples containing 70 mg L' 1 CNT as KCN without tissue
were prepared for 2.5 M NaOH and for 2.5 M NaOH/chloroform (35:15 v/v).
32
After
sonication and a 16-hour extraction in the dark, the caustic solution was withdrawn and
analyzed for total cyanide.
Hexane and 2-octanol were examined using 2.5 M NaOH/Hexane and 2.5 M NaOH/2Octanol (4:1 v/v) mixtures in 50 mL centrifuge tubes. The ratio was chosen based on the
recommended
value
for
preventing
fatty
acid
interference
during distillation
(APHA/AWWA/WEF. 1998). Sample spike mixtures of 50 ppb CNTas KCN were prepared
for each solvent combination, including a 2.5 M NaOH control. The tubes were sealed,
covered to prevent light intrusion, sonicated for 2 0 minutes, and rotated for 16 hours prior to
total cyanide analysis without filtration of the caustic phase.
3.2.2 Sample Spike Recovery

Once solvent selection studies concluded, cyanide-spiked solutions were used to determine
the cyanide recovery w'ith the optimal solvent, to investigate filtration, and to examine the
presence of plant tissue. Tissue-water slurry spikes were utilized as tissue samples with
known quantities of cyanide are not available. Sets of 200 mL volumetric flasks spiked with
100 ppb CNt as KCN and K^FefCNfo were prepared with and without homogenized, un
exposed. willow plant root tissue (-800 mg-FW). Two samples were included for each
cyanide species-tissue combination with each sample analyzed for total cyanide before and
after filtration with a 0.2 pm Millipore Type HN filter. Therefore, four conditions existed for
each cyanide species with duplicates run for each condition. All samples were subjected to a
five-minute sonication followed by a 16-hour extraction in the dark under constant mixing.
Control flasks containing 100 ppb KCN (undistilled) and KiFe(CN) 6 (distilled) as CN and not
subjected to the extraction procedure were used for recovery comparison.
33
3.2.3 Control Tissue

The cyanide concentration and speciation in willow root stem, and leaf tissue un-exposed to
KCN and KtFefCNfo hydroponic solutions were measured as plant tissue could contribute to
the background cyanide level as well as release organic compounds that interfere with the
detection methods. The root stem, and leaf unexposed tissue samples for each of four
replicates within a hydroponic uptake study (Chapter 4) were homogenized and -1700 mgFW of tissue was subjected to the optimal extraction method and analyzed for total cyanide
and free cyanide.
Two tissue extractions were performed for each tissue within each
replicate.
3 J Results
The results obtained in the solvent selection, spike recovery, and control tissue tests are
summarized here. All original, unreduced extraction data are available in Appendix H.l.
3.3.1
Solvent Selection
The results of methanol (MeOH) inclusion in the solvent matrix are given in Figure 3.1 for 52
ppb CNt as free cyanide (KCN) solution check standards.
Elevated cyanide levels for
MeOH: NaOH relative to NaOH were measured in both un-distilled and distilled samples
without evaporation, although the increase was not significant as determined by a t-test
(p<0.312). MeOH interference with spectrophotometric cyanide detection was of greater
concern than the slightly elevated cyanide sample results for each trial. The presence of
methanol in the NaOH trap for methanol-containing samples, evident by foaming,
substantiated concerns over interference. The presence of MeOH in the trap solutions
magnified the elevation of distilled sample spike cyanide concentrations over the course of
34
subsequent distillations. Attempts to evaporate the methanol prior to distillation reduced free
cyanide recovery significantly (p<0.0001) from 47 ppb to <13 ppb while not removing the
methanol completely. As expected, cyanide concentrations were consistently higher for un
distilled samples compared to distilled samples for all three treatments.
Results obtained with the extraction matrix containing chloroform are given in Figure 3.2.
where total cyanide concentrations in KCN solution check samples are presented. Check
sample results with KCN solutions show a disappearance of free cyanide from solvent
matrices containing chloroform (initial total cyanide. CNt. -60 ppb) to non-detectable levels.
Chloroform reacts with the free cyanide. Chloroform also reacts violently with NaOH.
releasing gas and causing pressure to increase to dangerous levels within the extraction vessel
and free cyanide loss.
Results from tests of 2.5 M NaOH/hexane and 2.5 M NaOH/2-octanol (4:1 v/v) solvent
mixtures for tissue extraction are given in Figure 3.3. The control 2.5 M NaOH served as the
basis for comparison with 37 ppb as CN. Total cyanide for NaOH/hexane and NaOH/2octanol solvent check samples without tissue increased to 45 and 40 ppb. As only one sample
was performed per solvent mixture, the concentration changes within the solution cannot be
evaluated for significance.
However, foaming in the distillation unit for all samples
containing hexane and 2 -octanoI was more excessive than that with methanol and indicated
potential interference of the organic solvent with the cyanide analytical method.
3.3.2 Sample Spike Recovery

Cyanide spike sample results are given in Table 3.1 for 2.5 M NaOH extractions from willow
root tissue slurry spiked with 100 ppb CNT as KCN and KoFefCNfo. as well as from control
35
samples containing no root tissue. As expected due to the relative volatility, KCN samples
exhibited a lower recovery than IQFetCNfo samples. Filtration decreased sample cyanide
recovery, particularly for KCN spike samples. Recovery in filtered. KCN-spike samples
decreased 4% (p<0.31) for samples with plant tissue and 5% (p<0.13) for those without
tissue. For KtFefCNfo samples, recovery decreased 5% (p<0.12) for filtration of solution
check samples without tissue. The presence of plant tissue also decreased recovery for
unfiltered KCN-spike samples from 96 to 84% (p<0.0007). All free cyanide recoveries are
based upon results for un-distilied spike samples without tissue while those for ferrocyanide
are based upon distilled samples.
3.3.3 Control Tissue

Results for analysis of 2.S M NaOH extracts of homogenized (i.e.. ground under liquid
nitrogen), unexposed willow root. stem, and leaf tissue are given in Table 3.2 for samples
grown for a hydroponic willow uptake study (Chapter 4). Tissue extractions and analysis
yielded low background cyanide concentrations for all three tissues.
Stem and leaf
background total and free cyanide concentrations were near non-detect and not significant
(p<0.112 and 0.161 for stem and leaf) at 0.04 mg kg-fresh weight' 1 (mg kg-FW'1) as CN.
Values were higher for root tissue, measuring 0.46 and 0.22 mg kg-FW' 1 as CN for total and
free cyanide, respectively, but still were not significant (p<0.180) as the deviation was also
higher.
3.4 Discussion
The objective was to develop an accurate, precise technique for the measurement of plant
tissue cyanide content and speciation. Tissue extract solution was analyzed for total cyanide
36
by the digestion-distillation method (APHA/AWWA/WEF. 1998) and free cyanide by the

microdiffusion method (ASTM. 1998). Analysis of both total and free (diffusible) cyanide
species provides information on tissue sample cyanide speciation.
Solvent choice is
important for maximizing tissue breakdown and cyanide recovery during extraction without
adversely affecting cyanide analysis. The baseline for comparison was extraction in 2.5 M
NaOH after homogenization with grinding under liquid nitrogen, as a high pH in the extract
solution was required to prevent free cyanide loss and maintain speciation.
Methanol,
chloroform, hexane, and 2-octanol were examined as potential additions to 2.5 M NaOH in
the extraction matrix. Slight changes in cyanide recovery were found for methanol, hexane,
and 2-octanol (Figures 3.1 and 3.3).
Chloroform extraction without tissue resulted in
complete loss of free cyanide (Figure 3.2). All of the organic solvents interfered with the
spectrophotometric analysis of cyanide after distillation when added in addition to NaOH.
The 1.6 g L 1 NaOH cyanide traps used in the distillation method foamed for samples
containing the alternative solvents with increasing interference with each subsequent
distillation. Hexane and 2-octanol interfered with the total cyanide analysis to a greater
extent than methanol due to higher volatility and lower solubility. Attempts to remove
methanol by evaporation prior to distillation significantly reduced cyanide recovery (Figure
3.1).
Previous studies on plant extraction for cyanide suggested that adding organic solvents to 2.5
M NaOH could increase recovery.
However, organic solvents interfered with the total
cyanide analytical technique without significantly increasing recovery from plant tissue. The
removal of free cyanide in chloroform-containing check samples and the hazards posed by the
violent nature of mixing chloroform and caustic (McKetta and Cunningham, 1976) justify the
exclusion of chloroform in the solvent matrix. Also, extraction of cyanide with 2.5 M NaOH
37
from hydroponic uptake study plant tissue yielded results indicating uptake consistent with
that calculated from cyanide stable isotope tracer (ISN) results (Chapter 4). Therefore. 2.5 M
NaOH was determined to be the optimal solvent for extraction of plant tissue for analysis of
free cyanide and total cyanide.
Recoveries of cyanide with 2.5 M NaOH extraction of plant-water slurries spiked with KCN
and KtFetCNfo were used to examine whether to filter tissue samples prior to distillation and
also to determine cyanide recovery during the extraction procedure (Table 3.1). Although
ferrocyanide has been shown to sorb to plant tissue at pH
(Chapter 4). sorption of
ferrocyanide did not change recovery from tissue-containing samples.
For free cyanide,
recovery significantly decreased in tissue-containing samples. While partitioning of free

cyanide to plant tissue was possible, a more likely cause of the losses was volatilization
during the extraction process. Filtration only increased the potential for additional cyanide
loss and was not included in the final extraction procedure. Recoveries from unfiltered
samples containing plant tissue were 84% for KCN and 100% for K^FefCNfo (Table 3.1).
These values do not account for recovery of cyanide in the standard distillation technique,
which is approximately 95% for free cyanide and 98-100% for ferrocyanide samples (results
not given here). Therefore, the adjusted extraction recovery was 89% for free-cyanideexposed plant tissue. These recoveries are significantly higher than those reported for some
alternative cyanide analytical methods using treated papers (Grossman and Kwiatkowski,
1995) or a modified microdiffusion technique (Yip and Yang, 1988).
Analysis of control tissue is important for assessing the background contribution to total and
fiee cyanide tissue concentrations. Complete tissue breakdown and release of organics could
interfere with cyanide analysis. Additional contributions could also occur from the release of
38
endogenous cyanide, possibly from cyanogenic glycosides or ethylene production.
The
background levels were consistent with those previously reported for plant tissue free (Tittle
et al.. 1990; Yip and Yang. 1988) and total (Howe and Noble, 1985) cyanide. The low
cyanide concentrations in the root. stem, and leaf control tissue (Table 3.2) for both total and
free cyanide relative to exposed tissue (Chapter 4) negate concern over tissue breakdown and
endogenous cyanide levels producing significant interferences.
The optimal tissue extraction method. 2.5 M NaOH extraction of samples homogenized by
grinding under liquid nitrogen, provided reproducible results and high total and free cyanide
spike recoveries for willow plant tissue. Results for hydroponically-exposed tissue agreed
with tissue stable isotope tracer (>SN) uptake data (Chapter 4). Preserved (frozen) plant
tissue samples were homogenized by grinding under liquid nitrogen in a ceramic crucible. A
weighed amount of homogenized sample (approximately 1-1.5 g-FW) was re-ground under
liquid nitrogen prior to grinding under 2.5 M NaOH to a fine paste. The sample was rinsed
from the crucible and brought to volume with 2.5 M NaOH in a 100 mL volumetric flask.
Following a five-minute sonication. the sealed flask was covered with aluminum foil to
prevent light intrusion and subjected to a 16-hour extraction with constant mixing. After
extraction, volumes of the tissue slurry were pipetted off and analyzed, without filtration, for
total cyanide by distillation (APHA/AWWA/WEF, 1998) and free cyanide by microdiffusion
(ASTM, 1998) with the pH adjusted to the desired value by adding additional H;S(Vwater
( 1 : 1 v/v).
39
3.5 Acknowledgements
This research was supported by a grant from the ALCOA. Inc.. The Gas Technology Institute.
N'iagara-Mohawk Power Company, and the New York Gas Group to Carnegie Mellon
University and Southern Illinois University Carbondale. The authors wish to acknowledge
the helpful comments and insight provided by S. Geiger. R. Ghosh, and D. Nakles of The
RETEC Group, and by S. Drop of Alcoa. E. Neuhauser of Niagara Mohawk, and L.
Weinstein of the Boyce Thompson Institute.
3.6 References
Agency for Toxic Substances and Disease Registry (ATSDR). (1997) Toxicological Profile
for Cyanide. U.S. Dep. Health Human Serv.. Public Health Serv.. Atlanta. GA.
Aikman. K.: Bergman. D.: Ebinger. J.; and Seigler. D. (1996) Variation of Cyanogenesis in
Some Plant Species of the Midwestern United States.
Biochemical Systematics and
Ecology. 24. 637.

APHA/AWWA/WEF.
(1998)
Standard Methods for the Examination of Water and
Wastewater. 20th ed., American Public Health Association, American Water Works
Association, and Water Environment Federation, Washington, D.C.
ASTM. (1998) Standard Test Method for Determination of Free Cyanide in Water and
Wastewater by Microdiffusion. Designation D4282-95, 1998 Annual Book of ASTM
Standards, Vol. 14.02, American Society for Testing and Materials, Philadelphia, PA.
Cohen, C.K.; Fox, T.C.; Garvin, DP.; and Kocian, L.V. (1998)
The Role of Iron-
Deficiency Stress Responses in Stimulating Heavy Metal Transport in Plants. Plant Physiol.
116, 1063.
40
Forsiund. K. and Jonsson. L. (1997) Cyanogenic Glycosides and Their Metabolic Enzymes
in Barley, in Relation to Nitrogen Levels. Physiologia Plantarum. 101. 367.
Grossman. K. and Kwiatkowski, J. (1995) Evidence for a Causative Role of Cyanide,
Derived from Ethylene Biosynthesis, in the Herbicidal Mode of Action of Quinclorac in
Barnyard Grass. Pesticide Biochemistry and Physiology. 51. 150.
Halkier. B.A. and Moller. B.L. (1990) The Biosynthesis of Cyanogenic Glucosides in
Higher Plants. The Journal of Biological Chemistry. 265. 21114.
Hart. JJ.; Norvell. W.J.; Welch. R.M.; Sullivan. L.A.; and Kocian. L.V.
(1998)
Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread

and Durum Wheat Cultivars. Plant Physiol. 118, 219.
Howe. M. and Noble. D. (1985) Effect of Cyanide Residue on Vegetation Bordering a
Black Hills Stream. Proc. S. D. Acad. Sci. 64, 112.
Jacobs, K.A.; Santamour. F.S. Jr.: Johnson, G.R.; and Dirr, M.A. (1996) Differential
Resistance to Entomosporium Leafspot Disease and Hydrogen Cyanide Potential in
Photinia." J. Environ. Tech. 14. 154.
Kobaisy. M.; Oomah, B.D.: and Mazza. G.
(1996)
Determination of Cyanogenic
Glycosides in Flaxseed by Barbituric Acid - Pyridine. Pyridine - Pyrazolone, and HighPerformance Liquid Chromatography Methods. J. Agric. Food Chem. 44, 3178.
Koster, H.W. (2001) Risk Assessment of Historical Soil Contamination with Cyanides:
Origin. Potential Human Exposure and Evaluation of Intervention Values. RIVM report
711701019, Rijksinstituut voor Volksgezondheid en Milieu (National Institute of Public
Health and the Environment), Bilthoven, The Netherlands.
Lechtenberg, M.; Nahrstedt, A.; Wray, V.; and Fronczek, F.R. (1994) Cyanoglucosides
from Osmaronia cerasiformis (rosaceae)." Phytochemistry. 37, 1039.
41
McKetta. JJ. and Cunningham, W.A., eds. (1976) Encyclopedia of Chemical Processing
and Design. M. Dekkar. New York, NY.
Mizutani, F.; Hirota, R.; and Kadoya. K. (1987) Cyanide Metabolism Linked with Ethylene
Biosysnthesis in Ripening Apple Fruit. J. Japan. Soc. Hort. Sci. 56. 31.
Nolte. K.B. and Dasgupta. A. (1996) Prevention of Occupational Cyanide Exposure in
Autopsy Prosectors. Journal of Forensic Sciences. 41, 146.
Selmar. D.; Grocholewski. S.: and Seigler, D.S. (1990) Cyanogenic Lipids. Plant Physiol.
93. 631.
Swanson. J.R. and Krasselt. W.G. (1994) An Acetonitrile-Related Death. Journal of
Forensic Sciences. 39. 271.
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.
42
Table 3.1 Free cyanide and ferrocyanide recovery with optimal extraction method. Recovery
evaluated using 100 ppb CNTaqueous spike samples (KCN and IQFe(CN)6) with and without
control root tissue added. Filtration performed with 0.2 pm Millipore Type HN filter.
Approximately 800 mg-FW of homogenized willow control tissue added to selected flasks.
Total cyanide concentrations measured by distillation according to Standard Methods 4500CN- C/E (APHA/AWWA/WEF. 1998).
Recovery determined relative to un-extracted,
distilled (KtFe(CN)6) and undistilled (KCN) check standards. Boldface values represent
recovery using the optimum extraction method for unfiltered, tissue-containing samples.
KCN
No Tissue
KFe(CN) 6
Tissue
No Tissue
Filter
% Recovery
95.5
90.7
83.5
80.0
1 0 0 .0
95.0
Standard Error
1.5
3.3
1.5
6 .0
0 .0
3.5
Tissue
N
100.5 101.0
1.5
1.0
43
Table 3.2 Control willow tissue total and free cyanide concentrations (mg kg-FW" 1 as CN).
Detection limits for total and free cyanide in the extract solution are
and 2.5 ppb as CN.
Approximately 1700 mg-FW of homogenized tissue extracted in 100 mL 2.5 M NaOH (n =

4). Based upon total and free cyanide measured by distillation (APHA/AWWA/WEF. 1998)
and microdiffusion (ASTM. 1998).
Total Cyanide
Free Cyanide
(mg CN kg-FW 1)
(mg CN kg-FW1)
Average
0.46
0.22
Standard Error
0.43
0.13
Average
0.04
0.08
Standard Error
0.03
0.03
Average
0.04
0.03
Standard Error
0.04
0.005
Root
Stem
Leaf
44
Figure Legends
Figure 3.1 Recovery of free cyanide with methanol inclusion in the solvent matrix. 2.5 M
NaOH (30 mL) solution containing approximately 50 ppm CNT (added as KCN) brought to
volume (100 mL) with methanol (MeOH) without plant tissue in 125 mL Erlenmeyer flasks.
Non-evaporated (NE) samples closed until distillation. Samples evaporated (EV) at 60C for
18 hours but methanol still present. Total CN measured by spectrophotometric methods
with/without
distillation
according
to
Standard
Methods
4500-CN-C/E
(APHA/AWWA/WEF. 1998).
Figure 3.2 Investigation of 2.5 M NaOH/chloroform (CHC13) (35:15 v/v) for 100 mL
solution KCN-spike samples without tissue. Samples sonicated, extracted for 16 hours with
mixing in the dark, filtered, and analyzed for total CN according to Standard Methods 4500CN- C/E (APHA/AWWA/WEF. 1998). Chloroform-containing samples left un-covered
during extraction to prevent pressure accumulation.
Concentrations for chloroform-
containing samples (NaOH/CHCL) reflect detection limit for total cyanide analysis. 1 pg L' 1
asCN.
Figure 3 3 Free cyanide recovery from KCN-spike solutions extracted with 2.5 M
NaOH/hexane and 2.5 M NaOH/2-Octanol (4:1 v/v). Recovery compared with base solvent
NaOH solution. Covered samples sonicated and mixed in the dark for 16 hours in 50 mL
centrifuge tubes prior to total CN analysis by distillation according to Standard Methods
4500-CN-C/E (APHA/AWWA/WEF. 1998).
45
/////A
Undistilled
Distilled
lig L 1as CN
99
10% NaOH
29
McOH/NaOH (NE) MeOH/NaOH (EV)
Extraction Procedure
Figure 3.1
46
m m NaOH
NaOH:CCl3H
70
/////A
*ig L 1as CN
60
50
10
5
0
? ///////////,
Filtered
'///////////A
Unfiltered
Figure 3.2
47
fig L 1as CN
NaOH
NaOH/Hexane NaOH/2-Octanol
Figure 3.3
C H A PTER 4
T r an spo r t
a n d m e t a b o l is m o f f r e e c y a n id e a n d i r o n c y a n id e
COMPLEXES BY WILLOW(1K2)
Abstrac t
Cyanide compounds are contaminants of growing importance that could be remediated

biologically via phytoremediation, provided plants possess suitable mechanisms for managing
these pollutants without toxicity. The transport and metabolism of two cyanide compounds,
potassium cyanide and potassium ferrocyanide. by willow iSalix eriocephala L. var. Michaux)
were compared using a hydroponic system that preserved cyanide speciation and solubility. The
cyanide compounds were labeled with 15N to quantify transport while a novel tissue extraction
procedure was used to relate tissue l5N to cyanide content and speciation.
These analyses
revealed that while little free cy anide was detected in the aerial tissues of plants exposed to either
of these two cyanide compounds, significant enrichments in 15N were observed, suggesting
transport and subsequent metabolism of free cyanide as well as ferrocyanide. The results for
ferrocyanide are of interest because this molecule is resistant to microbial degradation and if
oxidized to feiTicyanide is purportedly membrane impermeable. Nevertheless, these results and
mass balance calculations for tissue 1SN and solution cyanide confirming 100% recovery for the
added ferrocyanide were suggestive of ferrocyanide uptake and metabolism. This study provides
new' information describing the biological transport and metabolism of these two cyanide
Coauthors: Stephen Ebbs (lead author). Suzanne Poston. Dylan Kosma. Maria Samiotakis. and David Dzombak
Accepted for publication in Plant. Cell and Environment
49
compounds in plants. Moreover, the data also suggest that phytoremediation of cyanide may be
possible and ecologically safe due to the lack of cyanide bioaccumulation in aerial tissues.
Key words:
cyanide, ferrocyanide, iron cyanide, phytoremediation, willow
Abbreviations: CMU - Carnegie Mellon University; CN - free cyanide; CNt - total cyanide as
CN; EDDHA -
ethylenediamine o-dihvdroxyphenylacetic acid; HDPE -
high density
polyethylene; MC - methanol:chloroform; MES - n-morpholinoethanesulfonic acid; ORP oxidation-reduction potential; SIUC - Southern Illinois University Carbondale; TRIS - Trizma
base; WAD - weak acid dissociable
4.1 Introduction
Although regarded as a highly toxic compound and a potent metabolic inhibitor, free cyanide is
involved in several common plant biochemical pathways. For example, cyanogenic glycosides
are produced by >1.000 plant species to deter herbivory and to provide a source of N for
germinating seeds (see Seigler, 1991 for a review). Glucosinolates in Brassicaceae are broken
down to produce isothiocyanates and nitriles, which contribute to the strong odors of cabbage
and mustards.
Hydrogen cyanide is also produced during the synthesis of the plant stress
hormone ethylene (e.g., Yip and Yang, 1998).
Cyanide is also used extensively in industry and can be a prominent environmental contaminant
in some aqueous and terrestrial systems.
Major sources of cyanide are mining discharges,
50
organic chemical synthesis, plastics synthesis, electroplating, metal and aluminum works, and
the manufactured gas industry (ATSDR, 1997). Some iron cyanide solids, such as Prussian blue
[Fe4 (Fe(CN)6 )3 ] and Turnbulls blue [FejCFetCN^):], have been used commercially in dyes,
inks, pharmaceuticals, and cosmetics, but are also significant industrial byproducts. Because of
this ubiquitous industrial use, cyanide has been among the top 30 compounds on the CERCLA
Priority List of Hazardous Substances since 1995 (ATSDR. 1997; 1999; 2001). Cyanide in the
environment can be present as free or simple cyanide salts (i.e. CN or KCN, respectively) or as
cyanates and thiocyanates (OCN or SCN\ respectively), but is most often present as cyanide
complexes with metal cations such as Fe, Ni. and Zn at industrial sites (Meeussen et al.. 1992;
Theis et al.. 1994; Dzombak et al.. 1996). Iron cyanides such as ferrocyanide [Fe(CN)64] are
strongly complexed, stable cyanide species, which are common in contaminated aqueous
environmental matrices.
These aqueous complexes dissociate extremely slowly under dark
conditions (Ghosh et al., 1999a; 1999b). but are photosensitive, dissociating to release free
cyanide upon exposure to strong UV light (Broderius and Smith, 1980; Meeussen et al., 1992).
The release of cyanide from these compounds may pose both a human and ecological risk.
While several studies have shown that free cyanide is rapidly biodegraded by microorganisms
(e.g., Knowles and Bunch, 1986), iron cyanide complexes tend to be resistant to microbial
degradation (Aronstein et al., 1994). There have been reports of microbial (Cherryholmes et al.,
1983; Dursun et al., 1999) and fungal (Barclay et al., 1998a; Barclay et al., 1998b)
biodegradation of metal-cyanide complexes, but this has only been observed during in vitro
studies for which conditions are well-controlled using strains isolated from contaminated sites.
Procedurally, such studies are complicated to conduct, due primarily to the photosensitivity of
51
the iron cyanide complexes and the difficulty in maintaining cyanide speciation in solution over
time.
Nevertheless, these studies suggest that biological processes may contribute to the
degradation of iron cyanide complexes in the environment.
Phytoremediation, the use of plants for environmental remediation, may provide another
opportunity to remediate cyanide and iron cyanide contamination, provided that these
compounds can be transported and assimilated by plants. Recent studies demonstrating transport
of organic contaminants and metai-chelate complexes by plants (Burken and Schnoor, 1997;
Thompson et al., 1998; Vassil et al., 1998; Epstein et al., 1999; Thompson et al., 1999), possibly
via ATP binding cassette (ABC) transporters (Maser et al. 2001) or other transport mechanisms,
do not exclude the possibility of ferrocyanide transport.
Moreover, a mitochondrial inner
membrane anion channel has been shown to transport ferrocyanide as well as a variety of other
anions (Beavis and Vercesi, 1992).
Plants are also inherently more resistant to low
concentrations of free cyanide, due to the presence of the alternative oxidase in the mitochondrial
electron transport chain, and to endogenous plant cyanide-detoxifying enzymes such as cyanase
(Aichi et al.. 1998), rhodanese (Hatzfeld and Saito, 2000), and cyanoalanine synthase (Hasegawa
et al., 1994; 1995). Finally, recent surveys of an iron cyanide contaminated site have identified
willow (Salix eriocephala L. var. Michaux) trees thriving in close proximity to iron cyanide
contamination (Reeves, 2000). The presence of willow in proximity to this contamination raises
the possibility that this clone possesses one or more mechanisms for transporting and
metabolizing iron cyanide contaminants, a contention supported by recent studies (Reeves,
2000). Given the hydraulic control of soil water levels provided by willow, and the rapid rate of
biomass production by this species, a willow clone capable of phytoremediating iron cyanide
52
complexes could have significant utility for the remediation of sites contaminated with cyanide
compounds, provided the cyanide is assimilated by the plant, rather than being bioaccumulated.
Additional empirical knowledge concerning the transport and fate of environmentally relevant
chemical cyanide species would also be gained.
The present work had three primary objectives. The first was to describe the plant uptake and
transport of free cyanide (CN). This provided a baseline for the second objective, a
characterization of ferrocyanide uptake and transport. Cyanide, as either KCN or ferrocyanide.
was provided at equivalent concentrations of total cyanide (CNt) to provide a basis for
comparing the two treatments. The third objective was to draw inferences about the metabolism
of cyanide from KCN and ferrocyanide, principally to determine if free cyanide from the latter
compound was retained in plant tissues or assimilated. The data from KCN served as a positive
control since transport and metabolism of free cyanide by plants, including willow- (Trapp et al..
2001) has been previously demonstrated. Microbes (Kunz et al., 1998) and plants (Selmaret al.,
1988; 1990) have been shown to use cyanide as a nitrogen source. Prior to assimilation by
plants, nitrogen-containing compounds are converted to ammonium (Williams and Miller, 2001),
requiring less effort the closer the oxidation state of the original source is to ammonium.
The plant utilized in this study was a willow (Salix eriocephala L. var. Michaux) clone
reportedly able to transport ferrocyanide (Reeves, 2000). The experiments described here used
both a stable isotope (,5 N) label to trace movement of the nitrogen atom from the cyanide
molecule and corroborating chemical analyses to describe tissue and solution cyanide content
53
and speciation. The objective of these experiments and subsequent analyses was to provide
evidence transport and metabolism of both free cyanide and iron cyanide complexes.
42 Materials and Methods
4.2.1
Willow Propagation
Whips of willow (Salix eriocephala L. var. Michaux) were collected by Dr. Ebbs during winter
dormancy from a single tree growing near iron cyanide contamination at a former manufactured
gas plant site and propagated at Southern Illinois University Carbondale (SIUC). Cuttings were
taken from individual branches with obvious lateral buds and a diameter of no more than
10
mm
and stored in bundles at 4C. Prior to use, cuttings were gradually warmed to room temperature.
Defrosted cuttings were cut into smaller sections with at least 2-3 lateral buds present and a total
length of >40 nm. Cut willow sections were surface sterilized in Physan 20 (Maril Products,
Tustin, CA) for 20 min. according to the manufacturer instructions and established in sterile
polypropylene centrifuge tubes containing sterile deionized water and surface-sterilized
polyethylene beads, supported with autoclaved foam stoppers. The tubes were placed in a
Plexiglas chamber with additive humidity at ambient temperature to encourage bud break. The
interior surface of the chamber and the benchtop had been surface sterilized with 70% ethanol
and the vaporizer continuously supplied with sterile deionized water. Relative humidity within
the chamber varied between ambient and near
1 0 0
%, which proved adequate as bud break and
plant growth were maintained with a <10% failure rate. Plants showing fungal contamination
were removed from the humidity chamber to prevent cross contamination. The cut sections were
54
grown under these conditions until root development was apparent, at which time they were
transferred under sterile conditions to a basal autoclaved nutrient solution (Table 4.1).
When a shoot of 50-100 mm had developed, individual willows were transferred to pots
containing 2 L of autoclaved nutrient solution (Table 4.1) for biomass development. Pots were
placed in a Phytotron under controlled conditions (25C + 2C, natural illumination
supplemented with sodium vapor lamps on a 16 hr photoperiod) until plants reached a height of
400-500 mm. The nutrient solution was replaced with fresh sterile solution as necessary (2-3
times per week). Solution changes were performed under sterile conditions. Nutrient solutions
were screened for microbial contamination throughout the growth period along with parallel pots
containing no plants. Microbial contamination of the pots without plants was rare. However,
maintaining sterile conditions in the pots containing willow plants proved an insurmountable
task.
Despite repeated efforts and modifications of the surface sterilization procedure,
contamination developed in nearly all pots with plants. Microbial contamination often did not
appear until several weeks into the growth period, and was generally at a low level. Since the
nutrient solutions in the pot were changed frequently during this period of the biomass
development, with the frequency of the changes increasing as the plant grew larger, microbial
growth w'as minimized. A biodegradation study using a minimal media approach similar to that
in Silva-Avalos et al. (1990) was conducted at SIUC with the mixed culture isolated from these
pots to determine whether these microorganisms were capable of biodegrading iron cyanide.
55
4.2.2
Ferrocyanide Biodegradation Assay
Aliquots of nutrient solution from six hydroponic pots compromised by microorganisms were
collected. Microorganisms were isolated by centrifugation and cultured in AC broth according
to standard protocols (Difco, Sparks, MD, USA). Cultures were incubated in a shaking water
bath at 37C for 48 hr. A subculture (1/50 dilution) was established from the primary culture at
late log phase (as estimated by optical density) and grown under the above conditions for 36 hr.
Three cultures, each from a different hydroponic pot. showing the most vigorous and presumably
diverse or more competitive microbial growth, as determined by optical density, were selected
for study.
The biodegradation assay utilized a minimal medium approach similar to Silva-Avalos et al.
(1990) to determine if the microorganisms isolated from the hydroponic culture systems were
capable of biodegrading iron cyanide compounds. The minimal medium consisted of 10 mmol
m glucose as the carbon source. 2 mg L 1 CNT (added as potassium ferrocyanide) as the
nitrogen source, and 10 mmol m MES-TRIS to buffer the solution at pH 6.0. Aliquots of this
medium were transferred to brown HPDE bottles and incubated in a shaking water bath at 37C
for 2 hr with the cap loosely fitted to allow for gas exchange. An inoculum from one of the three
selected cultures was introduced into the minimal medium with four replicates. Control samples
consisted of the minimal medium with a sterile culture medium inoculation. The bottles were
cultured at 37 in a shaking water bath for 14 days, with samples taken at the inception of the
study and at the termination of the experiment. Samples were preserved with 1.6 g L 1 NaOH
prior to analysis for total cyanide content by Carnegie Mellon University (CMU).
56
4.2.3
Cyanide Uptake by Willow
A temporal replicate block design was used for the willow uptake study at SIUC, with each
block representing one complete replicate (four replicates in total). Each replicate consisted of
six pots in three pairs. One pot in each pair received a willow plant while the second did not.
The pots without plants were included to account for physical and chemical processes
influencing cyanide speciation and concentration. The three treatments included a control (no
cyanide), a ferrocyanide (added as potassium ferrocyanide) treatment, and a free cyanide (added
as KCN) treatment. The free cyanide (KCN) and ferrocyanide treatments were added such that
the total cyanide concentration (CNt) in each system was 2 mg L'1. allowing a more direct
comparison of the two treatments. Both the ferrocyanide and KCN were 15N labeled (Cambridge
Isotopes, Andover. MA. USA) at every nitrogen atom and added at 1007c by mass (i.e. all
ferrocyanide or free cyanide was CI5 N). Experiments used a modification of the basal nutrient
solution to ensure that the desired cyanide speciation and solubility was maintained during the
course of the experiment (Table 4.1). This modified nutrient solution for the cyanide treatments
was developed by CMU using MINEQL* (Schecher and McAvoy. 1994) with the most recent
thermodynamic data (Sehmel, 1989; Ghosh et al., 1999b) in cooperation with SIUC. Preparation
of the treatment solutions was carried out under reduced light to preclude light-mediated
dissociation of ferrocyanide.
Also, iron was not included in the nutrient media, thereby
preventing the solution phase precipitation of iron cyanide complexes, which would have
otherwise affected the overall cyanide mass balance. Despite the absence of an iron source in the
pots with plants, there was no evidence of iron deficiency (chlorosis, decrease in biomass) in the
plants at any time throughout the study.
57
When willow plants reached the indicated size, they were transferred to a hydroponic system
specifically developed for this research. This system used a polyethylene bag liner pinched
within the lid of a black polyethylene pail, forming an airtight seal. This seal helped decrease
evaporative loss from the pots. The point where the plant passed through the lid was sealed with
a thick layer of
10
% agarose and parafilm to minimize evaporation of solution or escape of
cyanide volatilized from the nutrient solution. This film also served as a barrier to prevent entry
of microbes or insects into the pots. Care was taken to ensure that the agarose had cooled
sufficiently to avoid heat killing phloem tissue in the willow stems.
Treatments were initiated by transferring willow plants under sterile, low-light conditions to the
treatment solutions. After the willow cuttings were established in the system and after a period
of slight mixing, the pH and pe (log Redox potential) of the treatment solution were determined.
The pe value was calculated from the oxidation-reduction potential (ORP) of the solution, while
the ORP was determined using a double junction gold disk ORP electrode (Cole-Parmer, Vernon
Hills. IL, USA). An aliquot of the solution was withdrawn from a sampling port in the pot, and
analyzed for cyanide content and speciation (see below).
Plants were transferred to the
aforementioned Phytotron for a 20-day exposure period. The solutions were not aerated during
this period because the introduction of air into the system could alter cyanide speciation in
solution by shifting redox states. The tree from which the cuttings used in this study were taken
is in a low area that floods annually, so the conditions imposed here were not expected to
adversely impact plant growth. Studies have also demonstrated that willow (Salix viminalis L)
can sustain root growth for 20 days under waterlogged conditions (Jackson and Atwood, 1996).
58
Although decreases in root growth were observed in that study beyond this period, the existing
roots tips resumed growth when plants were removed from waterlogged conditions.
Aliquots were withdrawn from each pot at each time point and analyzed by CMU for cyanide
content and speciation. At the termination of the experiment, roots were rinsed with copious
amounts of deionized water to remove residual nutrient solution and blotted dry. Shoots were
separated into stem and leaf tissues, and the fresh weight of all tissues determined. Each tissue
was then separated into two subsamples, one frozen at -80C and the other dried at 60C to
constant mass. The frozen subsample of the root, stem, and leaf tissue from each plant was used
by CMU to determine tissue cyanide content. The dried subsample was ground to a fine powder
and the dry weight determined. The water content (in 7c) of each tissue was determined from the
fresh and dry weight values.
4.2.4
Ferrocyanide Sorption to Roots
To aid in distinguishing root uptake from root sorption, a sorption experiment with willow root
cell wall skeletons was performed at SIUC. Willows of the same size and age as those used in
the uptake experiment described above were transferred to a
2 :1
(v/v) solution of
methanolxhloroform (MC) for three days. This treatment strips the biotic material from the
roots, leaving a cell wall skeleton that can be used to examine sorption processes at the root cell
wall level in isolation from biological processes. The results obtained from such experiments are
semi-quantitative in nature because the morphological and biochemical effects on the cell walls
have not been fully characterized.
Nevertheless, the results provide general information
59
concerning the sorption capacity of the ceil wall for the ion of interest, as was done for Zn2+
sorption (Lasat et al., 1996).
After the three-day MC treatment, the plants were removed and the roots excised. As the MC
treatment kills the plant, the shoot material exerts no influence on physical processes at the root
cell wall level and can be discarded. The excised roots were rinsed with deionized water, blotted
dry, and the fresh weight determined. The excised roots were transferred to an ,5 N-ferrocyanide
treatment solution identical to that used for the uptake studies with intact willow plants. The
sorption experiments used amber glass jars covered with aluminum foil to exclude light and
minimize ferrocyanide photodissociation. The fresh weight:nutrient solution volume ratio used
(2 g FW:L) for this experiment was similar to that in the uptake studies (1.2 g FW:L). After the
excised roots were introduced into the treatment solution, the jars were placed in the dark at
ambient temperature. Solution samples were taken over a similar time course and treated as
described above. Solution pH and pe values were also determined during the 20-day duration of
the experiment. At the termination of the experiment, the roots were harvested as described
above for the intact plants, except that the roots from this experiment were not separated into
subsamples.
4.2.5
Analytical Procedures
The dried tissue samples were prepared for 1SN analysis by transferring approximately 5 mg of
dried ground tissue to 8x5 mm tin capsules (Elemental Microanalysis Ltd, Manchester, MA,
USA).
Tissue samples were analyzed by the Stable Isotope Facility at the University of
Califomia-Davis for 15N analysis, which utilizes a Europa Scientific Integra Isotope Ratio Mass
60
Spectrometer (PDZ Europa, Cheshire, UK). These analyses also provided total N data for each
sample. Standards for analysis were provided by the analytical facility.
Tissue samples were analyzed for cyanide speciation and content by CMU using the procedure
described in Chapter 3. Briefly, leaf or root tissues were frozen in liquid N? and ground to a fine
powder. Approximately I g (fresh weight) of the ground sample was quickly separated and
reground using liquid nitrogen and then under 10% NaOH. The sample was rinsed into a 100
mL volumetric flask and brought to volume with the caustic solution.
The samples were
sonicated for 5 minutes and then extracted with mixing in the dark for 16 hours. The unfiltered
extract was analyzed for total cyanide and free cyanide (see below for methods). The results
were corrected for extraction recoveries of 89% and
1 0 0
% for free cyanide and ferrocyanide,
respectively, accounting for a distillation recovery of 95% for free cyanide.
Solution samples and tissue extracts were analyzed by CMU for total cyanide and weak acid
dissociable (WAD or weakly complexed) cyanide by distillation (Standard Methods 4500-CN
C/E and 4500-CN' I/E respectively - A PH A /A WWA/WEF, 1998), and free cyanide by
microdiffusion (ASTM D 4282-95 - ASTM, 1998). Limit of detection was 1 fig L 1 for total and
WAD and 2.5 ng L' 1 for free cyanide.
Transpiration was determined on a daily basis over the 20-day exposure period, and as
cumulative transpiration. Analysis of the ISN data used the equations described in Shearer and
Kohl (1993). Results were expressed initially in terms of the enrichment ratio (5ISN /oo), which
describes the abundance of 15N relative to naturally occurring levels. Uptake and transport of
61
cyanogenic ISN would be expected to increase this enrichment ratio, as illustrated by Reeves
(2000). The enrichment ratio is determined from the Equation 4.1
<515N = Rsample ^standard x I000/O0

^standard
(4.1)
where R for both the sample and standard is determined with Equation 4.2.
15N
R=
(4.2)
14N
The i5N data were converted by CMU to an estimate of total CN (CNt), assuming that all l5N
remained in that form. The background (control tissue) ISN was subtracted out to obtain an
estimate of the excess 1SN as CN present in the tissue due to the uptake of labeled solution
cyanide. The corrected value provided a theoretical predicted cyanide content to which the
extraction data from fresh tissue could be compared. The l5N reflects the total amount of labeled
cyanide taken up, while the extraction value provides a means of quantifying the amount present
as free or complexed cyanide. In addition, the l5N concentration in the tissue (dry weight basis)
was calculated using the total N in the sample and atom percent ISN. For the root sorption
experiment, the 1SN results for the excised roots were taken as an estimate of root sorption while
the >5N results from the intact plants from the uptake study were assumed to represent
uptake+sorption. The pool of I5N in the root symplasm, indicative of uptake, was estimated as
the difference between the mean 1SN concentrations between the intact and excised roots.
62
A mass balance was performed by CMU for both the ferrocyanide and potassium cyanide
treatments. The factors in the mass balance included initial cyanide in solution, residual cyanide
in solution after the 20-day uptake period, tissue cyanide (estimated from the 1SN data), and
cyanide loss from control pots. Control pots account for losses from the solution that may have
occurred during the course of the experiment, such as during sampling. Microbial uptake of
solution cyanide would have been accounted for during the analysis of the unfiltered solution
samples. However, microbial mineralization of either cyanide species in solution could not be
accounted for due to concern over mass losses of l5N in the stable isotope analytical preparation
of residual uptake solution. Cyanide volatilization was not considered in the mass balance, as
previous experiments with pea (Pisum sativum) and willow provided no evidence of
volatilization following treatment with ferrocyanide at the concentration used here (Ebbs et al..
unpublished results). Statistical comparisons of the data from this study used the Student's t-test
and ANOVA (Snedecor and Cochran, 1967).
4 J Results
4.3.1
Willow Growth and Water Relations
Willow plants, both control and treated, showed no adverse effects at any point in this study.
Treatment with 2 mg L 1 CNt as ferrocyanide or KCN had no adverse effect on transpiration,
tissue water content, or biomass production, as these values were not significantly different from
controls (data not shown). Over the 20-d time period, plant growth was comparable to that for
plants in aerated nutrient solutions in the presence of iron (data not shown). There were no
63
visual symptoms indicative of cyanide toxicity, physiological stress, or mineral nutrient

deficiency, including iron, from the growth conditions employed.
4.3.2
Solution p H. pe. and Cyanide Speciation
The solution pH values remained stable during the treatment period, with no significant
difference across the various treatments and no significant change with time (data not shown).
The solution pe values differed depending upon the cyanide treatment. Solution pe values for the
control and -plant ferrocyanide treatment were >6 .0 . approximately one unit higher than the
+plant ferrocyanide treatment pe. Solution pe values of 3.0 were observed for the +plant and plant KCN treatments. These values did not differ significantly from the initial value over the
20-day period of the experiment in any treatment (data not shown). The stability of the pe over
the
2 0
-day period, coupled with the lack of any physiological stress, suggests that anoxic
conditions did not develop in the root zone during this experiment and that plants were not
experiencing hypoxia. This could not be confirmed, however, as measurements of the dissolved
oxygen content of the hydroponic system would compromise the seal and perhaps promote loss
of cyanide to the vapor phase.
The nutrient solution used in the uptake experiment maintained the desired cyanide speciation
for both the KCN and ferrocyanide treatments (Table 4.2). In the KCN treatment, the free and
WAD cyanide measurements corroborated the total CN measurements, demonstrating that
cyanide was predominantly present in free form in this system. Similarly, for the ferrocyanide
treatment, cyanide was present predominantly (>95%) as complexed CN.
For the KCN
treatment, the CN content of the solution in the +plant treatment showed a significant decrease
64
on a mass basis with time (P<0.001). This decrease was significantly larger relative to the
corresponding -plant treatment as determined by a t-test on the regressed slopes for the two
treatments (P<0.001). For the ferrocyanide treatment, free cyanide mass increased from below
detection to 2 % of the initial cyanide mass in the pots without plants with an initial rate of about
18 fig day'1. The biodegradation assay demonstrated no microbial growth and no significant
change in solution ferrocyanide levels (data not shown). Thus, the increase in free cyanide is
likely the result of ferrocyanide dissociation in solution over time. Free cyanide mass exhibited
an initial increase followed by a decrease in the pots with plants (Table 4.2).
4.3.3
i5N Content of Willow Tissue
Analysis of the willow tissues revealed significant enrichments in 15N following exposure to
either l5 N-Iabeled KCN or ferrocyanide. Enrichment ratios are a standard means of presenting
stable isotope data (Shearer and Kohl, 1993). In this case, the enrichment ratio expresses the
degree to which the treatment increased the percentage of N as l5N with respect to the total
amount of N present in the control and sample. Enrichments of 250 to >22,000-fold were
observed, depending upon the tissue (Figure 4.1). The enrichments were larger in the KCN
treatment than for the ferrocyanide treatment, and within a treatment were greater in roots that in
shoots. Nevertheless, there was no significant difference between the total tissue N content of
plants from the control, ferrocyanide, and KCN treatments (data not shown). Given the total N
content of the tissues (data not shown) and that the natural abundance of ISN is approximately
0.367%, the 15N concentration in control plants was 75 - 150 pg g' 1 DW. The highest l5N
concentrations were detected in roots, with an 15N concentration >635 pg g 1 DW in
ferrocyanide-treated roots and an 15N concentration >2,500 pg g' 1 DW in KCN-treated roots.
65
The total ,5N concentrations in tissues are presented without a correction for background ,SN
because the available data do not allow ISN from the labeled cyanide pool to be distinguished
from the considerably smaller pool of naturally occurring I5N in the nitrogen compounds of the
nutrient solution (i.e. NO3 , NH4 *). The l5N concentration in tissues differed significantly among
the three treatments for all three tissues, root, stem, and leaf (P<0.001; Figure 4.2), with KCNtreated plants containing higher l5N concentrations than plants treated with ferrocyanide.
4.3.4
Iron Cyanide Root Sorption Versus Root Uptake
The sorption experiment with stripped willow roots demonstrated that the cell wall-sorbed
ferrocyanide (as estimated from the ISN concentration) accounted for approximately 23% of the
total iron cyanide associated with roots (Figure 4.3). Monitoring of the solutions during this
experiment (data not shown) revealed the same preservation of iron cyanide speciation described
above for the ferrocyanide treatment (Table 4.2), indicating that solution conditions were similar
to those from the experiment with intact willow plants. The fraction representing presumed
ferrocyanide uptake was estimated as the difference between the l5N concentration in roots of the
intact willow plants (Figure 4.2) and the stripped willow roots.
4.3.5 Cyanide Content of Willow Tissue

Tissue extracts of untreated control plants (Table 4.3) revealed low concentrations of cyanide in
root, stem, and leaf tissue (0.46,0.04, and 0.04 pg g' 1 FW, respectively). The background levels
agree with cyanide values previously reported for plant tissue (Howe and Noble, 1985; Yip and
Yang, 1988; Tittle et al 1990). For the KCN treatment, leaf and root concentrations in plants
exposed to KCN were not significantly different from the control values. The cyanide extracted
66
from tissues of KCN-treated plants was significantly lower (P <0.01) than that predicted by the
,5N content for each tissue (Figure 4.4). The predicted CN content is a conceptual value that
represents the total cyanide content that would be expected if the increased ISN from the labeled
cyanide compounds were retained as cyanide in the tissue. For each tissue, the predicted cyanide
was >400-fold greater than the extractable cyanide. The cyanide present in roots and stems of
plants treated with KCN was present in the free form, with a root cyanide concentration of -1.1
pg CN g ' FW. Conversely. KCN-treated leaf cyanide was present as an unidentified complex.
For the ferrocyanide-treated plants, a slightly different pattern emerged. Biochemical analyses of
the willow tissue extracts following ferrocyanide exposure revealed a significant accumulation of
total cyanide in roots (P<0.01, Table 4.3). Overall, the concentration of total cyanide was
significantly higher in the ferrocyanide-treated plants compared with that in KCN-treated plants
for root (P <0.01) and stem (P <0.05) but not in leaves. Leaf concentrations of total cyanide
were significantly higher than background levels (P <0.01), but were still less than 1 pg CN g 1
FW. All plant tissues contained cyanide in a complexed form (>96% for aerial tissue, >99.5%
for roots). A significantly higher (P<0.05) amount of complexed cyanide was measured in leaf
tissue as compared to the KCN-treated plants and to controls.
Without further analysis,
including dual uptake studies, the speciation of the cyanide complex in tissue cannot be
determined.
The presence of complexed cyanide could be the result of parent compound
transport to the leaf tissue or cyanide complexation with existing metals within the plant tissue.
While the cyanide concentration in roots was in agreement with that predicted by the ISN
analysis, both stem and leaf tissue extracts contained significantly less cyanide (P <0.05) than
predicted from the 15N data (Figure 4.5), with the difference between the observed and predicted
67
values >30-fold for each tissue. The presumed distribution of this complexed cyanide within the
root tissue, with respect to external sorption to root cell walls versus uptake into the root, can be
inferred from the results of the sorption study (Figure 4.3). The agreement of cyanide extraction
and ISN analyses suggests intact ferrocyanide uptake into the plant, rather than the plant iron
scavenging. Additional experimentation to determine the uptake processes would be necessary.
4.3.6
Mass Balance
The mass balance for the ferrocyanide and KCN systems (Table 4.4) was calculated using the
tissue 15N and the solution total cyanide data. The mass closure on the ferrocyanide was 101%
while recovery from the KCN treatment was significantly less at 60% (P<0.001). Within the
mass balance, ferrocyanide remained predominantly in the hydroponic solution with
approximately 8% of the initial mass moving into the plant tissue. The cyanide mass moving
into plant tissue for the KCN treatment was larger (38%). In the absence of a plant, losses of
l5N-cyanide from the ferrocyanide- and KCN-treated hydroponic systems (i.e. control losses)
were not significantly different. This suggests that the mass balance for the KCN treatment did
not account for a significant l5N-cyanide loss process (e.g. volatilization).
However, the
complete closure of the ferrocyanide mass balance suggests that this same process evidently did
not influence I5N-cyanide fate in that treatment.
4.4 Discussion
Considerable effort was expended to design a hydroponic solution and system for this study.
This necessity was due in part to the difficulty in working with cyanide but also to minimize
68
concerns that the results from the ferrocyanide experiments were artifacts stemming from
limitations of the experimental design.
Preserving cyanide speciation in solution was the
principal concern. The hydroponic solutions modeled with MINEQL* (Table 4.1) provided the
best possible conditions in terms of cyanide solubility and speciation. At equilibrium, formation
of cyanide solids was predicted in the solution. However, the absence of cyanide solid formation
in preliminary solution investigations displayed that the slow ferrocyanide dissociation in the
absence of light would maintain solution cyanide speciation.
Since in the absence of light
ferrocyanide dissociates extremely slowly to free cyanide over time (Ghosh et al., 1999a;
Chapter 2), care was taken to maintain the hydroponic system under complete dark conditions.
Cyanide remained principally (>95^) in complexed form in the ferrocyanide treatment with an
estimated initial free cyanide formation rate of 18 pg d'1. This resulted in a significant increase
in free cyanide in the ferrocyanide treatment solution in the absence of a plant (Table 4.2).
While the increase was significant, free cyanide still represented <7* of the total cyanide in
solution after 20 days. In the ferrocyanide treatment in the presence of a plant, free cyanide
increased initially, but then decreased. This could indicate uptake of free cyanide. Nonetheless,
if it is assumed that the dissociation of ferrocyanide in solution is independent of plant uptake of
free cyanide and continued at a rate of 18 |ig d '1 for the 20 day period, then a conservative
estimate of the total free cyanide uptake would be calculated as 0.36 mg CN. As this value is
significantly less than (P<0.01, t-test) the total amount determined in the tissue by ISN (0.79 mg),
this suggests uptake of ferrocyanide by willow roots. The sorption study and the tissue analyses
offer additional data in support of ferrocyanide uptake. Although complexed in the leaf tissues,
the total cyanide content was much lower than predicted from the 15N data (Figure 4.5), an
observation that also implied in vivo metabolism of ferrocyanide.
69
As expected, results from the KCN treatment provided clear evidence of plant transport and
metabolism of free cyanide. The cyanide concentration used (2 mg L'1 CNx) was clearly not
phytotoxic, as transpiration, water content, and biomass showed no significant difference with
time and across treatments. Despite a significant enrichment in the ISN content of the tissues
(Figures 4.1 and 4.2), negligible cyanide was detected in any plant tissue, consistent with the
results of Trapp et al. (2001). The cyanide detected in roots was present as free cyanide, while
the majority of the leaf cyanide was in complexed form, but not necessarily ferrocyanide.
Determining the subsequent steps in metabolism of free cyanide and ferrocyanide will require a
more thorough biochemical analysis of the nitrogenous compounds formed in these plants after
exposure to l5N-labeled cyanide, seeking specifically for those showing l5N enrichment. Such
efforts will also aid in determining which of the plant cyanide assimilatory pathways are
involved in the metabolism observed in this study.
Transport of cyanide and ferrocyanide by plants, coupled with the potential metabolism of these
compounds suggests the possible application of phytoremediation to these contaminants. Since
the cyanide content of aerial tissues was considerably less than that predicted by the stable
isotope data, cyanide bioaccumulation may not pose an ecological risk. However, the inability to
close the mass balance for the KCN treatment suggests that other processes may occur that relate
to the biological fate of cyanide. Although preliminary experiments (Ebbs et al., unpublished
results) suggested that willows did not volatilize cyanide, Trapp et al. (2001) provided results to
the contrary, but concluded that this route was of minor importance compared to the amount of
cyanide metabolized by the plant.
Whether the pathway of metabolism in the KCN- and
ferrocyanide-treated plant is the same has yet to be determined. Additional efforts are underway
70
to more fully elucidate the potential pathways of cyanide metabolism for these compounds, the
importance of cyanide assimilation to plant nitrogen metabolism, and the efficacy of cyanide
phytoremediation.
This research was supported by a grant from the Alcoa, Inc., The Gas Technology Institute,
Niagara-Mohawk Power Company, and the New York Gas Group to Southern Illinois University
Carbondale and Carnegie Mellon University. The authors 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 for Plant Research, and
E. Neuhauser of Niagara Mohawk.
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and Solids for the EPA-MINTEQ Geochemical Code. Report submitted to USEPA. PNL-6835.
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Glycosides: The Linustatin Pathway.'* Plant. Physiol. 86:711.
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Sources to a Common Sink and Use of Isotope Discrimination. In: Knowles, R. & Blackburn,
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by Hybrid Poplar Trees. Environ. Sci. Tech. 32,975.
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Hexahydro-1.3,5-trinitro-1.3.5-
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76
Table 4.1 Composition of the two nutrient solutions used in this research, showing the total
concentration of macronutrients and micronutrients.
Willows are grown in "basal nutrient
solution to increase biomass while the +Cyamde" nutrient solution represents the treatment
solution with 2 mg L'1 total CN (CNt) as either KCN or ferrocyanide. Iron is omitted from the
cyanide treatment solution to prevent the formation of iron cyanide precipitates. All solutions are
buffered to pH 6.0 with 1 mM MES-TRIS.
Mineral Nutrient
Solution
Macronutrients,
mM
K1 Ca1 pb Mgc
Cld
Basal
6.0
4.0
0.10
1.0
50.0 12.5
1.0
1.0
0.5
0.1
0.1
5.0
+Cyanide
a vr:. ____i*
6.0
4.0
0.02
1.0
50.0 12.5 0.16

J r.___ -
1.0
0.5
0.1
0.1
Dibasic ammonium phosphate salt

c Sulfate salt
Be
Micronutrients,
UM
Mnc Znc Cuc Nic
e Acid form
f EDDHA chelate
77
Moe Fel
Table 4.2 Change in the cyanide content of the four replicate hydroponic solutions over time, expressed on a mass (mg) basis for the
ferrocyanide and KCN treatments in the absence (-Plant) or presence (+Plunt) of a willow plant. Expression of these data on a mass
rather than concentration basis corrects for the cvapotranspirntion observed from each system. Weak acid dissociable (WAD) cyanide
represents weak cyanide complexes in solution while free cyanide represents cyanide present as the CN anion. Strongly complexed
(i.e. ferrocyanide) cyanide is represented by the difference between the total cyanide in solution and the WAD cyanide. "ND" denotes
concentrations below the limit of cyanide detection. Data represent the mean of four replicates for all except the day 20 duta (/i=3).
Ferrocyanide, mg
-Plant
KCN, mg
+I1anl
-Plant
+Plant
Days
Total
WAD
Free
Total
WAD
Free
Total
WAD
Free
Total
WAD
Free
9.67
0.16
ND
9.75
0.20
ND
9.51
9.59
9.00
9.28
9.37
8.94
8.48
0.29
0.09
8.33
0.29
0.07
8.75
9.05
8.21
6.59
6.82
6.50
10
7.95
0.27
0.13
8.05
0.24
0.08
8.42
8.52
8.81
3.50
3.61
3.57
15
7.61
0.30
0.15
7.70
0.27
0.06
8.21
8.32
8.47
0.98
1.13
1.13
20
8.09
0.36
0.19
7.41
0.26
0.03
8.13
8.31
8.38
0.56
0.69
0.69
78
Table 4 J Cyanide concentration and speciation in willow tissues after a 20-day exposure to
KCN or ferrocyanide1. The cyanide concentrations were adjusted to reflect 98% recovery of
complexed cyanide and 95% recovery of free cyanide in total cyanide analysis during distillation
as well as 11% free cyanide losses during 16-hr extraction in 10% NaOH.
Treated tissue
concentrations also account for control cyanide concentrations in plant tissues.
Root
s.e.
Control,
pg CN g 1 FW
Free
Total
0.46
0.22
0.14
0.50
Ferrocvanide.
Ub Cn V 'F W
Free
Total
0.19
128
0.19
21.4
Stem
s.e.
0.04
0.03
0.05
0.03
0.14
0.09
0.04
0.05
0.04
0.04
0.04
0.05
Leaf
s.e.
0.04
0.04
0.01
0.01
0.76
0.17
0.01
0.01
0.23
0.18
0.01
0.01
KCN,
ue CN e'1 FW
Total
Free
1.12
1.66
0.72
1.0
1 The detection limits were 0.01 pg g 1 for total cyanide and 0.015 pg g '1 for free cyanide.
Values represent the mean and standard error (n = 4).
79
Table 4.4 Mass balance for ferrocyanide and free cyanide in the hydroponic systems containing
willow plants. Data represent mean values and standard error for the replicates calculated on a
mass basis for CN. based upon analysis of the initial (day 0) and residual (day 20) solution and
the predicted CN mass in the plant tissues derived from the concentration data in Figure 4.2. All
values except Recovery are expressed in milligrams as CN and represent the mean of four
replicates. Control loss represents a correction factor used to account for CN losses from the
-plant systems. The control losses from the ferrocyanide and KCN treatments do not differ
significantly at a=0.05.
Mass in solution,
ms
Mass in tissue,
mg
Treatment
Initial
Residual
Root
Stem
Leaf
Ferrocyanide
s.e.
9.76
0.14
7.17
0.46
0.44
0.07
0.06
0.02
0.29
0.07
Control
Loss, mg
1.90
0.55
KCN
s.e.
9.28
0.30
0.59
0.46
1.36
0.24
0.97
0.19
1.19
0.19
1.50
0.16
Recover},
80
101
5
60
5
Figure Legends
Figure 4.1 I5N enrichment ratios for willow roots, steins, and leaves following a 20-day
exposure to 2 mg L 1 total CN as either potassium cyanide or potassium ferrocyanide. with error
bars representing the standard error of the mean (n = 4). Enrichment ratios are calculated based
upon dry weight l5N values.
Figure 4.2 I5N content of willow roots, stems, and leaves following a 20-day exposure to 2 mg
L'1 total CN as either potassium cyanide or potassium ferrocyanide. with error bars representing
the standard error of the mean (n = 4). For a given tissue, each treatment is significantly
different from the other treatments in that group (P<0.001).
Figure 4 J Relationship between sorption and uptake for willow' roots exposed for 22 days to the
compound ferrocyanide. Sorption" refers to all physical processes by which ferrocyanide may
associate with cell walls and refers to the experiment with stripped willow roots. Sorption +
Uptake, which refers to the results with intact plants (root, ferrocyanide treatment from Figure
4.2). includes all processes, both physical and biologically-mediated, that involve association of
cyanogenic N (as ,5N) with roots.
Uptake, the biological processes only, is obtained by
subtracting sorption from Sorption + Uptake (mean values for each). Since the sorption and
intact root data are not paired, the calculated uptake does not have associated error bars. All
values include background 1SN content.
81
Figure 4.4 Total cyanide concentrations in tissues from willow, either from extraction and
analysis (CN measured) or predicted based upon conversion of l5N content to CN concentration
and adjusted to a fresh weight basis (CN predicted). Data are from willows receiving the free
cyanide (KCN) treatment. Error bars correspond to the standard error of the mean (n = 4).
Detection limit for total cyanide was approximately 0.1 pg g '
Figure 4.5 Total cyanide concentrations in tissues from willow, either from extraction and
analysis (CNj measured) or predicted based upon conversion of I5N content to cyanide and
adjusted to a fresh weight basis (CN predicted).
ferrocyanide [Fe(CN)6] treatment.
Data are from willows receiving the
The total concentration. CNt. shown here includes the
cyanide adsorbed onto the exterior root surface and the cyanide inside the root (see Figure 4.3).
The i5N content was converted to total cyanide content as in Figure 4.4. Error bars represent the
standard error of the mean (n = 4). The detection limit for total cyanide was approximately 0.1
pg g
82
30000
6000
25000
.20000
5000-L
7500
a>
5000
L.
N /, KCN-trcatcd plants
7000
IT .
- 2500
VI
e
I
KCN
Ferrocvanide
Treatment
Figure 4.1
83
3000
N content, jig g 1 DW
2500
Control
l.
I Ferrocyanide
m m KCN
750
500
250
I
Stem
Root
Leaf
Plant tissue
Figure 4.2
84
800
Sorption
i
2 Sorption + uptake
I Uptake
I'SNJ, (ig g'1 DW
600
400
200
Figure 4.3
85
ng CN g ' FW from
K CN
750
500 -
total CN measured
total CN predicted
250 -
0
Root
Stem
Leaf
Tissue
Figure 4.4
86
Hg CN g'1 FW from ferrocyanide
160.0
120.0
I total CN measured
] total CN predicted
80.0 -
40.0
1.0
0.5 0.0
Stem
Root
Leaf
Tissue
Figure 4.5
87
CHAPTERS
MODEL FOR CYANIDE UPTAKE BY WILLOW: MODEL DEVELOPMENT*!)
A b stra c t
A model for cyanide species uptake by willow (SalLx eriocephala L. var. Michaux) plants was
developed to quantitatively interpret data from hydroponic experiments (Chapter 4) and to aid in
the development of field studies that extend the initial laboratory research. The experimental
data have demonstrated the potential for cyanide phytoremediation. A process model will be
useful to augment efforts to determine which plant processes contribute to cyanide transport and
metabolism in willow.
Further, modeling can be used to target and optimize specific
physiological parameters important to field-scale phytoremediation efforts. The objective of the

model development was to gain insight into the relative role of different processes with respect to
dissolved free cyanide and iron-complexed cyanide transport and assimilation in plants, and to
determine rates at which these processes occur within the willow plant under the experimental
conditions.
A physiologically-based model describing plant uptake, transport, and metabolism of cyanide

species was developed to reflect the physiological processes that influence the movement of
cyanide into and throughout the plant.
Plant organs (root, stem, leaf) were considered for
compartments to correspond to the data specificity used to calibrate the model (Chapter 3). More
111 Coauthored with Stephen Ebbs and David Dzombak
88
specific physiological data would be needed to describe more detailed intercellular processes,
which would add complexity to the model.
Mass balances around each compartment were
developed with kinetic representations for the mass transfer processes for cyanide movement.
The mass balance equations were combined to form a model describing the fate of cyanide
compounds within plant-water systems.
Keywords: compartmentalized, cyanide, ferrocyanide, modeling, uptake.
Abbreviations: A - assimilate/metabolite; BDF - backward difference formulation; CN - free

cyanide; CNT - total cyanide; FC - ferrocyanide; CN - free cyanide within ferrocyanide
treatmen; M-M - Michaelis-Menton kinetics; RCF - root concentration factor. Tot CN - total
cyanide in the KCN system; TSCF - transpiration stream concentration factor
5.1 Introduction
Solutes in soil water can move into plants by several mechanisms. Mass flow with water and
diffusive flux both serve to move ions and organic chemicals towards plant roots.
Anions,
especially larger solutes, are impeded from passive flow into root cells by the membrane
electrochemical gradient (Marschner, 1995; Ryan et al., 2001) while lipophilic organic
compounds can diffuse across the root cell membrane and partition to the transpiration stream
(Marschner, 1995). Once inside the roots, solutes can remain unchanged, become bound to a
ligand, or be modified by reactions associated with detoxification, degradation, or assimilation.
Both the unchanged and/or chemically-modified solutes can be stored in the root in specific cells
89
or cellular compartments (e.g., vacuoles), exuded back into the rhizosphere, or transported to
aerial tissues through the xylem in the transpiration stream.
Solutes can undergo similar
modification in the leaves or be directed to specific cellular compartments. Solutes mobile in the
phloem can be redistributed laterally to other shoot tissues or basipetally to roots.
Volatile
compounds can also be released from leaf or root tissues.
Various models have been developed to describe the movement and fate of compounds,
particularly organics, in plants (Burken and Schnoor, 1997; Trapp et al., 1994; Trapp and
MacFarlane, 1995; Lindstrom et al., 1991). Each model describes the movement of chemical
between well-mixed compartments within the plant representing root, stem, and shoot (leaf)
sections. The transpiration stream often is the dominant mass transfer mode and is considered to
flow at constant rate. Some models include the potential for partial reflux with the assimilation
stream in the phloem (Lindstrom et al., 1991; Trapp and McFarlane. 1995; Trapp et al., 1994).
The relative proportion of solute entering plant tissue passively via water movement usually is
described using a transpiration stream concentration factor, TSCF (Briggs et al., 1987; Schnoor,
1997; Burken and Schnoor, 1998). TSCF is a proportionality constant relating the chemical
concentrations in the transpiration stream and soil water (or hydroponic solution). Lindstrom et
al. (1991) treated organic contaminant uptake similarly using a partitioning efficiency.
Interaction with the root system can be treated either separately (Burken and Schnoor, 1998) or in
conjunction with (Lindstrom et al., 1991; Trapp et al. 1994) uptake into the transpiration stream.
When separated, root partitioning is described by a similar relationship to the TSCF termed the
root concentration factor, RCF (Briggs et al., 1987; Schnoor, 1997; Burken and Schnoor, 1998).
90
The RCF does not distinguish between chemical uptake into root tissue and sorption onto root
issue. Lindstrom et al. (1991) and Trapp et al. (1994) considered movement through the root
prior to transfer to the shoot, with root accumulation that portion of chemical reflected at the
endodermis. Diffusive chemical transfer from the roots to the solution could occur if chemical
accumulated in root tissue, or if an active transport process selectively exuded solutes. The root
interstitial space offers a low resistance route from the bulk solution to the endodermis and
provides open space through which water can flow (Nobel, 1999 - p.374).
Although
recognized as an important compartment for solute uptake, only Lindstrom et al. (1991) treated
plant root interstitial space separately.
Tissue sorption reactions are treated similarly to soil sorption with both equilibrium (Davis et al..
1993; Lindstrom et al., 1991) and rate-limited partitioning (Burken and Schnoor. 1997) to root
tissue reported. Diffusive movement is included for transfer to roots (Trapp et al., 1994) and
between all plant compartments (Lindstrom et al., 1991). Each of the models considers firstorder chemical metabolism within plant tissue for all plant compartments (Lindstrom et al., 1991:
Trapp et al.; 1994) or all compartments except for stem tissue (Burken and Schnoor, 1997).
Other considerations included for the mass balance are volatilization (Trapp et al., 1994), storage
(Lindstrom et al., 1991), plant growth (Trapp et al., 1994), and multiple leaf and stem sections
(Lindstrom et al., 1991). The use of first-order loss and transfer processes results in a linear
system of equations describing uptake and fate within the plant.
The system of equations
representing chemical mass balances is solved using matrix techniques (Lindstrom et al., 1991)
or numerical simulation with data fitting of parameters (Burken and Schnoor, 1998: Trapp et al.,
1994).
91
Ions such as free cyanide (CN") and ferrocyanide (FefCN^4") behave differently from organics
and require different considerations. None of the plant-level phytoremediation models have
considered active uptake, mainly because modeling of the transport and metabolism of lipophilic
organic chemicals does not requires this consideration.
Active uptake against the
electrochemical gradient, often described by Michaelis-Menten kinetics (M-M), has been shown
to be important in the movement of nutrients and ionic chemicals into plant tissue (Ben-Asher,
1994; Marschner. 1986; Nye and Tinker, 1977) and was included in two chemical migration
models (Ben-Asher, 1994; Somma et al., 1998). Plant uptake of inorganic contaminants such as
heavy metals has been shown to fit the saturation kinetics typically used to represent proteinmediated uptake (Cohen et al., 1998; Lasat et al., 2000; Hart et al., 1998). While previous
studies have speculated that transport of ferrocyanide may require active chemical uptake
(Reeves, 2000), others have provided evidence of passive transport through channels, but this
was restricted to mitochondria (Beavis & Vercesi 1992).
The objective of the current work was to construct a model for free cyanide and ferrocyanide
uptake from hydroponic solution into willow plants that reflects the relevant physiological
processes involved. The ionic nature of cyanide species is suggestive of active uptake across cell
plasma membranes. In the developed model, mass transfer processes such as advection and
diffusion are assumed to govern the movement between compartments with active uptake
included for species transfer into the plant root interior. Adsorption and metabolism are included
to account for removal processes within the various plant compartments. The overall objective
was to simulate uptake of cyanide from solution and movement through the willow plant with
time, to gain insight into the relative importance of particular transport and metabolic processes.
92
5.2 Model Structure
The plant uptake model was constructed using a compartmentalized representation of the plantsoil-water system as depicted in Figure 5.1. The compartmentalized approach is similar to that
employed in previous plant uptake models (Burken and Schnoor, 1997; Lindstrom et al., 1991;
Trapp and McFarlane, 1995). A hydroponic system (Chapter 4) was used as the basis for Figure
5.1, as data from a hydroponic system were used to calibrate the model (Chapter 6). Five
primary compartments were designated - solution, plant root, plant stem, plant leaf, and air. Root
tissue was separated further to account for processes that might influence chemical fate and
movement within the root zone. A control volume was assumed around each compartment with
mass balances derived from the various mass fluxes for each compartment. In this specific
system, cyanide solid precipitation, plant growth, and phloem redistribution were not included
due to, respectively, the lack of cyanide precipitation found in preliminary cyanide hydroponic
solution experiments, the minimal growth over the short length of the experiment, and the
experimental data (Chapter 4) suggesting that metabolism of cyanide occurs in roots, preceding
the possible redistribution of cyanide via the phloem. Additional physiological and biochemical
studies would be required to integrate these components into the model.
The assumptions to develop the model were based upon the available data. The goal was to
balance the complexity of cyanide transport and metabolism in plants with the data obtained
from willow. Assumptions, such as those regarding cyanide precipitation, plant growth, and
phloem redistribution, are needed to reduce the number of variables within the model. Another
limitation is that of data specificity. The available data was collected at the tissue level and
93
provides little information
about the processes
associated with specific subceliular
compartments. Thus the model is constrained by available data. Increasing the number of
unknown process parameters does not provide any benefits as sufficient data is not available for
an improved model fit. The information gained from solving such a model is also more difficult
to relate to particular processes. A smaller model is advantageous because the lesser number of
equations and unknowns reflects the data specificity and strengthens the relationship between
processes and results while representing physiological and metabolic processes.
5.2.1
Model Compartments
Due to the importance of the root tissue in controlling chemical uptake, the root compartment
was separated into sections inside and outside the endodermis, a highly-suberized cell layer that
regulates solute movement into the vascular bundle of the root. The root interstitial compartment
was kept to reflect the active uptake of ions at the membrane level. Maintaining the interstitial
compartment also permits the possibility of a chemical concentrating within the inter-cellular
space in the root or the root cell vacuoles due to an uptake process slower than movement of
chemical into the compartment with the transpiration water. Although the water free space in the
root accounts for only a small percent of the root tissue volume, this compartment must be
crossed if water and chemicals are to enter the root (Lindstrom et al., 1991; Nye and Tinker,
1977).
The stem and leaf tissues were each treated as single compartments. Stem tissue acts primarily
as a conduit for flow between the root and leaf tissue (Burken and Schnoor, 1997) and serves as a
storage organ only in specific plant species. Excluding phloem redistribution, the only process
94
that affects movement from the collective leaf tissue to other compartments is volatilization.
Neglected reactions could be incorporated into the assumed first-order rate coefficients for the
modeled processes of advective transfer with the transpiration stream and plant-mediated
metabolism.
5.2.2 Transfer and Reaction Processes

By aggregating the plant into stem, leaf, and interior and exterior root zones, rate coefficients
could be obtained based upon the entire tissue mass and the cyanide concentration within the
respective tissues. Cyanide mass balances for each compartment were developed considering
advection, diffusion, active transport, and metabolic reaction (Figure 5.1).
Each of the
compartments, including the hydroponic solution, was assumed to be well-mixed with

concentration gradients only between compartments. Flow was considered positive entering a
compartment. With this sign convention, loss functions were considered negative. Symbols for
the various model parameters are defined in Table 5.1
Passive mass transfer via advection was included for the movement between all compartments
except for movement into the root interior from the intracellular space. Advection with the
transpiration stream from compartment i as Q,tiCl was considered a passive process in the current
context because this process is governed primarily by physical rather than biological factors.
Transpiration was fit to experimental data (Chapter 4) and considered constant throughout the
plant. Advective flow impedance was not included for passage through the root wall from
solution. A conductance efficiency {e'g) was included to describe species partitioning from root
tissue into the stem advective transpiration stream.
95
Diffusive mass transfer was included for movement between the hydroponic solution (2) and the
interstitial space (J) as Dr(C2 - C<), where D,=DA 2j/Ax23- In the case of ferrocyanide, the
inclusion of diffusive mass transfer was important to allow for concentration redistribution
between the two compartments if uptake into the root did not occur as rapidly as advective
transport into the interstitial space.
Previous investigation speculated active uptake of cyanide species into plants (Reeves, 2000).
Ionic species do not partition past the root endodermis as organic chemicals can. The ionic
nature, particularly for negatively-charged ions that move against the electrochemical potential,
requires mediation by a plant membrane protein. Others have provided evidence of passive
transport through channels, but this was restricted to the inner membrane of mitochondria under
specific pH conditions (Beavis & Vercesi 1992). Active transfer processes were incorporated to
reflect movement against the electrochemical gradient using Michaelis-Menten kinetics (M-M),
pfuLxVfC'i/iK',+C'}), for uptake from the interstitial space into the root interior where a
membrane was crossed (Nobel. 1999). The mathematical structure of M-M can still account for
linear uptake by selecting Kmm C .t.
Other chemical considerations included ferrocyanide dissociation and free cyanide assimilation.
Ferrocyanide dissociation was included for the hydroponic solution and interior root and leaf
tissues (Equation S.l). Stem tissue was assumed to act solely as a conduit for flow between root
and leaf tissue. Direct metabolism of ferrocyanide was not included. Metabolism was assumed
to occur via non-reversible, plant-mediated ferrocyanide dissociation followed by assimilation of
96
the free form.
Solution ferrocyanide dissociation was considered reversible and kinetically-
controiled with rate constants determined from control solution data.
Fe{CN)*' 4 F e + 6CAT
(51)
Plant-mediated ferrocyanide dissociation (yi5,) and free cyanide assimilation (A.5,) were modeled
as first-order, irreversible reactions. Separate dissociation and assimilation rates were assumed
for root interior and leaf sections as the reactive processes could be different within the
respective plant tissues due to plant physiology, including light-mediated ferrocyanide
dissociation within leaf tissue. Ferrocyanide dissociation in water is known to be accelerated in
the presence of light, with the rate dependent on the solution conditions (Meeussen et al., 1992).
Biological metabolism in the hydroponic solution, both due to the plant and/or microbial
contamination, was assumed to be negligible. Biodegradation of ferrocyanide, if occurring at all.
is limited (Aronstein et al., 1994). A few studies have claimed complexed cyanide removal but
few have exercised suitable controls for comparison (Cherryholmes et al.. 1985: Dursun et al.,
1999). Additionally, ferrocyanide biodegradation studies conducted in conjunction with the
hydroponic uptake study were negative (Chapter 4).
Mass balance equations were also included to account for the transfer of assimilation products
within the various willow compartments and the metabolism to different chemical forms
(experimentally, conversion of 15N-labeled cyanide compounds, Chapter 4). Conversion was
modeled as the rate of metabolism from free cyanide within the compartment while transfer
occurred via advection as with the cyanide species, including an efficiency coefficient for
movement from the root to the stem tissue. Redistribution in the phloem of the assimilated
97
nitrogen was ignored in the current model due to solution difficulty associated with the additional
variables and limited data. Physically, redistribution in the phloem introduces a bi-directional
process that cannot be separated experimentally.
Adsorptive processes in the interstitial space and hydroponic solutions were assumed to be firstorder. reversible reactions (Equation 5.2). Cyanide species in the root interstitial space could
partition to sites within the cell wall structure of the root tissue. Previous metal uptake studies
have shown such partitioning to be significant (Hart et al., 1998; Lasat et al.. 2000). Sorption
was included in both systems as free cyanide sorbs to carbonaceous materials (Chatwin et al.,
1988) and ferrocyanide readily adsorbs to charged sites, particularly oxides (Appendix A).
Control losses within the hydroponic solution were also modeled with first-order equilibrium
adsorption.
While the actual loss process may not have been adsorption, the goal was to
represent empirically the generalized physical processes that are involved in the disappearance
within the control, unplanted solutions.
(5.2)
Preliminary investigation during the hydroponic experiments suggested that free cyanide
volatilization was negligible (Chapter 4).
Nevertheless, the potential for free cyanide
volatilization from the leaf tissue, along with that for assimilated product, was included as a firstorder air/water partitioning (Hj'S?*). Rapid dispersion within the surrounding air kept the
airborne concentration for both species at zero.
98
S 3 Mass Balance Equations
Considering the transfer and reaction processes described, the mass balances for the various
compartments were constructed for each of two willow plant hydroponic systems: one with
ferrocyanide in the hydroponic solution and the other with free cyanide. In ferrocyanide-treated
systems, the ferrocyanide. dissociated free cyanide, and metabolite systems are connected via
conversions within plant tissue and solution.
For free cyanide treatment, the equations and
reaction processes associated with ferrocyanide were not included. The five sets of equations,
two for the free cyanide system and three for the ferrocyanide treatment, must be solved
simultaneously. The mass balance expressions derived from the mass transfer and reaction
components for each compartment were written for each of the three species within each system.
The units associated with each of the balances were pmoles of chemical species i per day.
The basic mass balance equations are provided below. The number headings for each subsection
reflect the compartmental designations within the equations and figures. Symbols are defined in
Table 5.1.
1. C ontrol loss - Control losses within the hydroponic solution were identical for both
cyanide species regardless of the treatment system. The reversible, adsorptive losses
were based upon the surface area of the hydroponic system (A).
99
(5.3)
A l- = + a 'C ,-p 'cX |

dt
c c *
2. H y d ro po n ic
solution
- For the ferrocyanide system, the free cyanide and feirocyanide
balances within the hydroponic solution differ by the sign of the dissociation reaction
terms.
Each includes advective and diffusive transfer as well as control losses and
ferrocyanide reversible dissociation. For the ferrocyanide system, v equals +1 for the
ferrocyanide balance and -6 for the free cyanide balance. For the free cyanide system, v
equals 0. Volumetric change in the hydroponic solution was accounted for in the mass
balance equation, adding a differential term.
dt
=-QnC -D ;(C -Q -flfc C
= V^
' dt
-v**K<^c +rVXT6Cf')
+C^
(54)
dt
Dissolved ferrous iron concentration for the ferrocyanide system was represented for the
reversible dissociation reaction assuming no additional iron interactions, including plant
uptake, within the system.
^ ^ = U C f f - r K C r 'i C f ' = K ^ + C f ' ^
dt
..................................dt
l55)
dt
3. ROOT interstitial space - The balances around the root interstitial space were the same
for both chemical species.
Each accounted for advective and diffusive transfer in
addition to active uptake and cell wall sorption.
100
(5.6)
The interstitial volume (V was defined as:

^R im * ~ & u Q Rim*
^3
4.
r o o t T ft i w ai
PR u *
u Q R tm *
i - Cell wall adsorption/desorption in Equation 5.6 was represented by:
dS*
-c
qt - = + a C , - P 5,
dt
5.
(5.7)
rem a in der of the root
(5.8)
- Balances for ferrocyanide. free cyanide, and assimilate in both
systems were also performed around the remainder of root. Active uptake, advection. and
reactive terms are included, n equals y? for ferrocyanide. A> for free cyanide, and
assimilate.
for
Note that r is +6 for the free cyanide balance within the ferrocyanide
treatment system and that the associated concentration is that for ferrocyanide within the
remainder of the root,
r equals 0 for ferrocyanide. the free cyanide system, and
assimilate, n niax equals 0 for assimilate.

dSf _ [
dt
r+ c,
effp walerQlnS 5
+ TYsS f c - n S ,
0,
(5.9)
0 .was used to account for conversion between mass and volume and was adjusted to
account for the fraction of water in the interstitial space. Using the total mass of the root
and the definition of 0 , the water fraction of /, the water fraction for the rest of the root
was calculated.
101
(5.10)
<?*. = Rx + ?5 + P~u"rVy
(5.11)
e ,= M
%
Q
_ H zORon( _ PwaerV', + H zOs
to ~
~
Q R oot
Root
(5.13)
_ g ugfcf
Rut* ~
6.
stem
(5.12)
Qr,**
- The change in mass with the stem for all mass balances was due to mass transfer
within the transpiration stream. Inflow from the root contained a factor for the efficiency
of transfer while movement to leaf tissue was assumed to be 100*7c as the stem acts as an
efficient chemical conduit.
dS,_ e'tP^MnSs
qb dt
e5
7.
leaf
P . rQ,St
(5.14)
06
- The balances for ferrocyanide, free cyanide, and assimilate in both systems were
performed around the leaf tissue. Species entered via advection with loss processes
included for reaction and volatilization, o) equals ft for ferrocyanide, fA7 +Wcv/-) for free
cyanide in both systems, and (-X7 + ^ 7) for assimilate. Note that ^ is +6 for the free
cyanide balance within the ferrocyanide treatment system and that the associated
concentration is that for ferrocyanide within the leaf, q equals 0 for ferrocyanide, the free
cyanide system, and assimilate.
The mass of cyanide and assimilate volatilized from leaf tissue needs to be calculated to
close the mass balance for each system.
d(MassVui) _ [ //:5 .
dt
(5.16)
For comparison of model predictions with hydroponic system experimental data, it is useful to
group together the concentrations of cyanide and conversion products in the various root
compartments. The cyanide and conversion product mass in the root interstitial space, sorbed to
cell wall, and in the rest of root can be combined to calculate whole-root concentrations. For free
cyanide and ferrocyanide concentrations in root, all root compartments had to be considered.
C
Root
_ (^3^3 +
) /
/ ft
<5 1 7 >
As assimilated product was not present in either the interstitial space or cell wall of the root, only
the rest of the root needed to be considered.
c*
Rottr
= (^ 5 ^ )/
/
<5 l 8 >
/ /i
HRtKit
5.4 Model Capabilities and Solution Technique
The resulting model was solved for an input set of the 17 unknown variables (Table 5.2) using
Gears BDF technique. The forward model calculates the species concentration profiles with
time within each of the seven compartments. Using the compartment size, the mov ement of the
initial cyanide mass through the ferrocyanide and free cyanide hydroponic systems can be
103
determined at any time during the experiment. The model also tracks the volatilization of free
cyanide and assimilated product, allowing for losses from the system. An objective function
assessing the relative fit to the hydroponic data was included consisting of the sum of the square
difference in the log values between the data and predicted concentrations. An example of the
model output is given for the input variables in Table 5.3. The fraction of the initial cyanide
mass present in each of the compartments as ferrocyanide (FC), free cyanide (CN) and assimilate
(A) are given for day 20 for the ferrocyanide and free cyanide systems (Table 5.4). The solution
concentration profiles are also given for ferrocyanide and free cyanide within the ferrocyanide
system and total cyanide within the free cyanide system (Figure 5.2).
Model calibration,
involving optimization and uncertainty characterization associated with the model based upon
fitting with hydroponic data, is described in Chapter 6.
The goal of developing the model is to gain insight into and assist in interpreting hydroponic
uptake data for the uptake and assimilation of free and ferrocyanide with willows. Solution of
the model will provide important information regarding which are the key processes for cyanide
movement as the unknowns within the model centered upon the rate constants involved in the
mass transfer processes describing the fate of cyanide within the willow system. Unlike previous
models for organics that treated uptake into plant tissue as a partitioning process, the movement
of cyanide species through the Casparian strip of the root tissue was represented by active uptake
kinetics.
A balance in the model complexity between the inclusion of relevant plant
physiological processes, such as active uptake, and the data available for calibrating the model
was maintained.
Contaminant transfer into plant tissue was modeled to represent plant
physiology by describing chemical movement through the root interstitial space prior to uptake
104
into the root and transfer to the aerial tissue as in Lindstrom et al. (1991) and in contrasts to the
use of the TSCF and RCF. Additionally, the plant uptake model recognizes the ability of the
plant to regulate ionic movement into cellular tissue and the relative importance of diffusion
versus transpiration for movement between plant compartments. To reflect these particulars,
diffusion was only included for the solution-interstitial space interaction rather than between all
compartments.
The maximum level of complexity was achieved that included important transfer process but did
not prevent the system solution from having meaning. The resulting model consisting of seven
compartments and 17 unknowns was a reduction compared with a previous model developed by
Lindstrom et al. (1991) that contained 24 compartments and 24 parameters.
Larger models
require the estimation of more input parameters to compensate for the inclusion of added
processes and resulting over-specification of the chemical fate within the plant system. Model
applicability decreases as the number of unknown parameters increases.
S i Summary and Conclusions
A compartmentally-based mathematical model was constructed for the uptake and movement of
cyanide species in a willow plant. The objective was to build a mathematical representation that
reflected plant physiological processes for the movement of cyanide ions within the plant root,
stem, and leaf compartments. The mathematical model consisted of a set of mass balance
equations for each compartment accounting for diffusive, advective, and active-uptake mass
transfer as well as adsorptive, dissociation, and metabolism reaction terms.
Movement of
105
metabolites was also accounted for in the model while phloem transport, growth dilution, and
solution cyanide precipitation were not considered.
The mathematical model development produced a well-defined concept of uptake and movement
throughout the plant-water system. Active uptake was included for the movement of cyanide
species into plant tissue at the endodermis. Also, the ability of plant cells to regulate uptake into
tissue was reflected in the exclusion of diffusion except for mass transfer between the solution
and root interstitial space.
The model was developed to encompass as many known plant
processes as possible while accounting for the limitations of the data available for model
calibration. Physiological processes were included and grouped to reflect mass transfer without
over-specifying the system, resulting in a seven-compartment model containing 17 unknown
parameters.
This research was supported by a grant from ALCOA, Inc., The Gas Technology Institute,
Niagara-Mohawk Power Company, and the New York Gas Group to Carnegie Mellon
University.
5.7 References
Aronstein, B.N.; Maka, A.; and Srivastava, V.J. (1994) Chemical and Biological Removal of
Cyanides from Aqueous and Soil-Containing Systems. Appl. Microbiol. Biotechnol. 41, 700.
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Beavis, A.D. and Vercesi. A.E. (1992) Anion Uniport in Plant Mitochondria is Mediated by a
Mg:+-Insensitive Inner Membrane Anion Channel. J. of Biological Chemistry. 267,3079.
Ben-Asher. J. (1994) Simplified Model of Integrated Water and Solute Uptake by Salts- and
Selenium-Accumulating Plants. Soil Sci. Soc. Am. J. 58,1012.
Briggs, G.G.; Rigitano. R.L.O.; and Bromilow. R.H.
(1987)
Physio-Chemical Factors
Affecting Uptake by Roots and Translocation to Shoots of Weak Acids in Barley. Pestic. Sci.
19. 101.
Burken, J.G.. and Schnoor. J.L. (1997) "Uptake and Metabolism of Atrazine by Poplar Trees.
Environ. Sci. Tech. 31:5. 1399.
Burken. J.G.. and Schnoor. J.L.
(1998)
Predictive Relationships for Uptake of Organic
Contaminants by Hybrid Poplar Trees. Environ. Sci. Tech. 32:21. 3379.
Chatwin. T.D.; Zhang, J.: and Gridley, G.M. (1988) Natural Mechanisms in Soil to Mitigate
Cyanide Releases. Superfnnd '88: Proc. 9fh Nat. Conf. November 28-30. 1988. Washington.
DC, 467-473.
Cherryholmes, K.L.: Comils, W.J.; McDonald. D.B: and Splinter, R.C. (1985) Biological
Degradation of Complex Iron Cyanides in Natural Aquatic Systems. Aquatic Toxicology and
Hazard Assessment: Seventh Symposium. ASTM STP 854. R.D. Cardwell, R. Purdy, and R.C.
Bahner, Eds., American Society for Testing and Materials, Philadelphia, PA, 502.
Cohen, C.K.; Fox, T.C.; Garvin, D.F.; and Kocian, L.V. (1998) The Role of Iron-Deficiency
Stress Responses in Stimulating Heavy Metal Transport in Plants. Plant Physiol. 116,1063.
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Davis, L.C.: Erickson, L.E.: Lee. E; Shimp. J.F.; and Tracy. J.C. (1993) Modeling the Effects
of Plants on the Bioremediation of Contaminated Soil and Ground Water. Environ. Prog. 12:1.
67.
Dursun. A.Y.: Qalik. A.: and Aksu. L (1999) Degradation of Ferrous(II) Cyanide Complex
Ions by Pseudomonas fluorescens." 34.901.
Han. J.J.; Welch. R.M.: Norvell. W.A.; Sullivan. LA.: and Kocian. LV.
(1998)
Characterization of Cadmium Binding. Uptake, and Translocation in Intact Seedlings of Bread

and Durum Wheat Cultivars. Plant Physiol. 116. 1413.
Lasat M.M.: Pence. N.S.: Garvin, D.F.: Ebbs. S.D: and Kocian. L.V.
(2000)
"Molecular
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112, 1715.
Lindstrom, F.T.: Boersma. L.; and McFarlane. C. (1991) Mathematical Model of Plant Uptake
and Translocation of Organic Chemicals: Development of the Model. J. Environ. Qual. 20.
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Marschner. H. (1986) The Mineral Nutrition of Higher Plants. Academic Press. London.
Meeussen, J.C.L.; Keizer, M.G.; and de Haan. F.A.M.
(1992)
Decomposition Rate of Iron Cyanide Complexes in Soil Solutions. Environ. Sci. Tech. 26, 511.
Nobel, P.S. (1999) Physicochemical and Envrionmental Plant Physiology, 2nd ed. Academic
Press, New York.
Nye, P.H.; and Tinker, P.B. (1977) Solute Movement in the Soil-Root System. University of
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Reeves, M. (2000) Treatment o f Fluoride and Iron Cyanides Using Willow: A Greenhouse
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(1991)
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Trapp. S.; McFarlane, C.: and Matthies. M. (1994) Model for Uptake of Xenobiotics into
Plants: Validation with Bromacil Experiments. Environ. Tox. Chem. 13:3,413.
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Organic Chemical Processes. Lewis Publishers, Boca Raton, FL.
109
Table 5.1 Definitions and units for willow plant-cyanide model parameters.
C, - volume-based cyanide concentration in compartment i [kg/L solution]

Q<t>- time-dependent transpiration (advective) flow [L/day]
V,volume compartment i [L]
t - time [day]
k - ferrocyanide solution dissociation rate constant [day1]
r - ferrocyanide solution complexation rate constant [(L/pmol CN)6/day]
A - hydroponic surface area for control losses [m*]
a*c - rate constant of control adsorption loss of species * [L/day]
0*c - rate constant of control desorption gain of species * [m2/day]
X| - cyanide concentration in control loss compartment [pmol/nr]
D' - cyanide diffusion coefficient for species * [dm/day]
A:j - interfacial area between solution and interstitial space [dm:]
A xzj - diffusion length between solution and interstitial space [dm]
D \ - diffusive cyanide mass transfer coefficient for species * [L/day]
a* -rate constant for cell wall adsorption of species * [L/day]
0* -rate constant for cell wall desorption of species * [kg/day]
5, - mass-based species concentration in compartment i [pmol/kg soil]
U max - maximum active uptake rate for species * [kg/L-day]
K m- half-saturation parameter for active cyanide uptake of species * [kg/L]
qi - plant mass of compartment i [kg]
a IS- fraction of root volume that is interstitial space [L/L]
PRoot - density of root tissue = pwaur [kg/L]
Yi - ferrocyanide plant-mediated dissociation rate constant in i [day1]
A, - free cyanide assimilation rate constant in i [day1]
e 'ff- advection efficiency for species * from interior root to stem [-]
@ - water fraction in i [kg/kg FW]
HzOi - Mass HjO in i [kg]
H ' tvolatilization rate constant from leaves [kg/day]
Massvoi - mass of species * lost from system through volatilization [pmol CN]
Abbreviations for species in system (*): CN - free cyanide; FC - ferrocyanide; A - assimilated
product; Fe - ferrous iron. Unless otherwise noted, concentrations in equation are those
for which the balance was constructed.
Table 5.2 Parameters solved for within the plant uptake model. Abbreviations are: CN - free
cyanide, FC - ferrocyanide. and A - assimilate.
Process
Symbol
Number of Parameters
Cell wall adsorption/desorption
aCN a PC pCN pPC
Active uptake
mcn.
k cs. k J *
Transfer efficiency
CN FC A
eff ' eff eff
Plant-mediated ferrocyanide dissociation
YRooi*YLaf
Free cyanide plant assimilation
^Rock*^Leaf
Volatilization from leaves
H uaF .H ua*
ill
Table 5 3 Input parameter set for a simplified, representative solution to the plant uptake model.
The parameter values were chosen to reflect those that provide a reasonable solution compared
with hydroponic data and were not intended to test the model. Abbreviations are: CN - free
cyanide, FC - ferrocyanide, and A - assimilate.
Symbol
Description
Value
Units
Meg
Meg
Ferrocyanide Parameters
c fc
Cell wall adsorption
5.0
ffc
Cell wall desorption
1.0
e /C
FC
Umax
K FC
Transfer efficiency
0.005
750
YLraf
Maximum active uptake rate constant
pmol/pmol
0.005
0.01
pmol/L-day
pmol/L
kg/day
kg/day
Cell wall adsorption
0.1
L/kg
Cell wall desorption
0.5
L/kg
Transfer efficiency
0.10
0.25
pmol/pmol
pmol/L-day
pmol/L
kg/day
Leaf assimilation constant
0.10
kg/day
Volatilization rate constant
0.0005
kg/day
0.25
0.01
pmol/pmol
kg/day
Half-saturation constant
Root dissociation constant
Leaf dissociation constant
Free Cyanide Parameters

cP
/F
e /*
u cs
f*max
K CS
^Root
A-Uaf
CN
ttLeaf
it

Root assimilation constant
50000
20
Assimilate Parameters
eg
H uat
Transfer efficiency
Table 5.4 Percent of the initial cyanide mass in each of the compartments after Day 20 as
calculated for the ferrocyanide and free cyanide systems from a simplified, representative input
parameter set (Table 5.3). The circled numbers (e.g. (D) reflect the corresponding compartment
mass balance designation within the text and figures. The initial total cyanide mass in the
ferrocyanide and free cyanide systems was 371 and 357 pmoles as CN. Values below 0.01% for
the fractional mass within a compartment are listed as 0".
Abbreviations are: CN - free
cyanide, FC - ferrocyanide. A - assimilate, and NA - not applicable.
FC
CN
Control losses
20.5
0.06
NA
Hydroponic solution
66.4
0.28
NA
Root interstitial space
0.02
NA
Root cell wall
0.02
NA
Remainder (interior) of root
0.39
0.01
0.04
0.02
Leaf
Volatilization
0.13
0.02
0.94
NA
0.02
11.2
Total
87.4
0.39
12.2
Control losses
NA
3.93
NA
Hydroponic solution
NA
15.4
NA
Root interstitial space
NA
NA
Root cell wall
NA
NA
Remainder (interior) of root
NA
0.04
0.38
Stem
NA
0.01
0.20
Leaf
Volatilization
NA
0.01
3.86
NA
0.01
76.2
Total
NA
19.4
80.6
Compartment
Fenrocvanide System
Stem
Free Cyanide System
113
Figure Legends
Figure 5.1 Schematic of plant compartmentalized model depicts major compartments. The
circled numbers (e.g. CD) reflect the corresponding compartment mass balance designation within
the text. The root interior represents fraction of root inside the endodermis while the interstitial
space represents the root tissue outside the endodermis. Transfer and reaction processes affecting
the movement of a chemical species within a hydroponic system including solution and plant
compartments. Transfer processes include advection (A), inefficient advection (Aetf). diffusion
(D). and volatilization (VO. Reactive processes include active uptake (U), reversible, un-mediated
ferrocyanide dissociation (it), irreversible, plant-mediated ferrocyanide dissociation (y), and
plant-mediated free cyanide assimilation (A).
Figure 5.2 Solution cyanide concentration profiles obtained for a representative parameter input
set. For the ferrocyanide system, the ferrocyanide (FC) and free cyanide (CN) solution profiles
are given. Only the total cyanide (Tot CN) profile is shown for the free cyanide system. FC and
Tot CN are given in (a) while CN for the ferrocyanide system is plotted in (b).
114
Air
Root Interior
Cell Wall
t V
Interstitial Space
Aeff
Root
t i A .D
Control jS
Losses
Stem
CN- _ k
Fe(CN)64
> Solution
Figure 5.1
115
100
Tot CN
FC
80
Z
U
0
9
s
S
10
15
20
15
20
Day
CN
0.6
Z
u
03
SB
0.4
10
Day
Figure 5.2
116
C H A PTER 6
MODEL FOR CYANIDE UPTAKE BY WILLOW: APPLICATION TO

EXPERIMENTAL DATA AND CALIBRATION*!)
ABSTRACT
Application of a plant uptake model to describe hydroponic system data for the uptake of free
cyanide and ferrocyanide by willow provided an assessment of optimal mass transfer parameter
values and their associated uncertainty.
Modeling provided a method for assessing rates of
various plant processes as well as the importance of parameters relative to phytoremediation with
particular interest in assessing potential phytoremediation field-scale applications. The system of
equations representing the compartmental mass balances for free cyanide and ferrocyanide were
solved numerically for 500 initial parameter values to obtain an optimal solution for the complex
decision surface. The effect of data variability was assessed by propagating through the model
the uncertainty contained in 500 realizations of simulated data randomly-generated by
bootstrapping of the residuals.
Optimization results showed that free cyanide volatilization and cyanide cell wall adsorption
were negligible. Free cyanide volatilization accounted for <0.01% of the initial cyanide mass
within each system. The root cell wall fraction was also <0.01% for free cyanide and while
larger for ferrocyanide, was still negligible at <0.04%. Of the 13 parameters in the final model
Coauthored with Mitchell Small. David Dzombak. and Stephen Ebbs
117
formulation, only the maximum uptake rate constant for free cyanide
) and the assimilate
volatilization constant (Huaf) showed significant correlation with the objective function. Both
parameters also had a low coefficient of variation with respect to data uncertainty. Active uptake
kinetics are justified for free cyanide but not for ferrocyanide, for which the non-convergent, low
KmfC value does not support active uptake. Examination of ferrocyanide uptake as a first-order
process with the transpiration stream was also not sufficient to describe the solution
concentrations. Uptake for ferrocyanide likely occurs as a combination of the two processes.
The predicted optimal solution and tissue concentrations fit the data sufficiently except for the
stem and leaf tissue assimilate concentrations, particularly in the free cyanide system. The low
predicted values for these compartments combined with the slightly underestimated removal of
free cyanide from the KCN solution suggest a potential importance for redistribution of
assimilates in the phloem.
Keywords: compartmentalized, cyanide, ferrocyanide, modeling, optimization: uptake.
Abbreviations: A - assimilate/metabolite; CN - cyanide; CNt - total cyanide; COV coefficient of variation; FC - ferrocyanide; ROCC - rank order correlation coefficient
6.1 Introduction
In the context of chemical fate within a plant-solution system, uptake, transfer efficiency,
metabolism, and volatilization rate constants can be significant mass transfer processes. A model
is necessary to consider the integrated effect of the various processes that affect chemical fate and
118
transport in plants.
Parameter estimation through modeling provides insight into individual
processes that are difficult to measure. Model calibration via comparison with experimental
data, in this case from the hydroponic uptake experiments, provides a means to obtain an
estimation of each of the individual parameters and the relative importance of the various
processes.
Uncertainty analysis can also be conducted using the model to determine the
uncertainty associated with each of the parameter estimates given variability and error in the
experimental data.
A model for plant uptake and assimilation of ionic solutes (Chapter 5) was applied to describe
and help interpret the experimental data obtained from a hydroponic study of free cyanide and
ferrocyanide uptake by willow (Chapter 4). The goal of the model investigation was to gain
insight into the processes that govern free cyanide and ferrocyanide uptake and metabolism
within a piant-solution system. The individual parameters obtained through the model fitting of
hydroponic data, such as the active uptake rate, can be adjusted to a field-scale estimate for the
purpose of applying phytoremediation towards removing free cyanide and ferrocyanide from
groundwater.
Specific objectives of the modeling study were to optimize the unknown
parameters for the plant uptake model, obtain a measure of the uncertainty associated with the
model due to the data, and to assess the importance of the transfer processes within the model
relative to the fate of cyanide within the system. Parameters for plant process rates constants
were investigated by fitting hydroponic uptake data with the model predictions. Bootstrapping of
the residuals of the data was used to generate a set of simulated data for which the uncertainty
was propagated through the model to assess the variability in the output.
119
6.2 Model and Parameter Optimization Technique Overview
The model describes the physiological and chemical processes governing a plant-solution system
using a set of mass balances around each compartment (Chapter 5).
The set of ordinary
differential equations that constitutes the model is too complex to solve analytically. As is the
situation with many physical systems involving chemical kinetic reactions proceeding towards
equilibrium, the model experiences stiffness as the derivatives approach completeness on
different time scales. Gear's backward difference formulation numerical solution technique, e.g.
the FORTRAN code DIVPAG developed by Visual Numerics**/ (1997), can handle stiff
problems represented by large differences in the gradient values for the ODEs. As the method is
implicit, a series of non-linear equations must be solved for each time step.
The solution to the differential equations can then be optimized by evaluating an objective
function value for each solution of the ODEs. The minimum of the objective function is found
by successive quadratic programming including constraints on the process variables.
Optimization is obtained by solving a general non-linear programming problem with a successive
quadratic programming algorithm and a finite-difference gradient. An objective function, such as
the difference in the square of the log values, is minimized subject to a set of non-linear equality
and inequality constraints on the variables in the system. The method was based on the iterative
solution of quadratic programming subproblems.
Each successive sub-problem is obtained
through a quadratic approximation of the Lagrangian and linearization of the constraints. A line
search is then used to find a new point for searching (Arora, 1986).
120
The numerical methods described above were used to optimize the solution to the set of ODEs
comprising the plant model.
Optimization was performed using the general approach of
randomly sampling the parameter space to obtain the best fit with respect to the objective
function.
Error analysis on the optimal solution was necessary to determine the uncertainty associated with
the optimized model parameter values.
Many methods of conducting error analysis and
evaluating the importance of parameters and processes within the model require a measure of
uncertainty associated with model solutions. Regionalized sensitivity analysis (RSA) assesses
model structure, including parameters and process importance, by classifying each model
simulation as good or bad according to a criterion function (Spear et al., 1994; Spear and
Homberger, 1980; Wade et al.. 2001). Comparison of the parameter conditional probabilities
given each classification determines the parameter sensitivity. Bayes Monte Carlo (BMC), an
alternative method for assessing and reducing model characterization, also requires a measure of
likelihood associated with a given model outcome (Sohn et al., 2000). Neither the RSA nor the
BMC was applicable, as the model did not provide a posterior measure of uncertainty associated
with the parameters. A measure of posterior uncertainty in the model was necessary prior to
analyzing parameter importance.
The uncertainty in the input parameters can be propagated through the model to obtain measure
of the output parameter uncertainty (Maddalena et al., 2002).
As the distribution for the
parameters was indeterminate, the data uncertainty was used as a surrogate. The measurement
and replicate error were used to generate a simulated set of input data for the model via
121
parametric bootstrapping of residuals (Efron and Tibshirani, 1993).
Bootstrapping is a re
sampling method for statistical inference commonly used to generate confidence intervals or
variance of an estimator.
The critical concept of the bootstrap is that a variable distribution can be approximated from
estimates from a repeated sampling from an approximation of the unknown population
(Chemick, 1999; Davison and Hinkley. 1997). The population within the bootstrap can be
parametric or nonparametric. Parametric assumes a distribution using the likelihood estimates
for the unknown parameters while nonparametric sampling is performed using the empirical
distribution of the observed values. For problems that involve temporally or spatially correlated
data observations, such as that for the hydroponic study focused on in this work (Chapter 5), two
general approaches exist (Efron and Tibshirani, 1993; Chemick. 1999; Davison and Hinkley.
1997).
Bootstrapping can be performed using the observations (case re-sampling) or the
residuals (error re-sampling) from Equation 6.1

Yt = X , P + et
(6-1)
where K, is the observation and e, is the residual. Error re-sampling using the residuals is a threestep process. First, e, is calculated for each observation. Next, the bootstrap sample of residuals
is drawn with replacement from the residual distribution based on the observed residuals.
Finally, the bootstrapped observation set is constructed from the randomly-generated residuals
and the predicted value for each observation according to Equation 6.1 and input to the model to
generate the output parameter distributions having propagated the uncertainty in the data through
the model.
122
6 J Model Parameter Estimation
The model was applied to obtain rate constants for plant processes based upon measured plant
cyanide species concentrations in a three-week, willow-plant hydroponic uptake experiment with
free cyanide and ferrocyanide (Chapter 4). Solution concentrations of free cyanide and total
cyanide were measured every 5 days from 0 to 20 days for both free cyanide- and ferrocyanidecontaining pots (Table 6.2). After 20 days, the plant tissue was harvested and root, stem, and leaf
sections were analyzed for free cyanide, complexed cyanide (presumably ferrocyanide), and l5N
content. The complexed cyanide in the leaf tissue of ferrocyanide-treated plants was assumed to
be parent compound. The cyanide could potentially be complexed with metal cations other than
iron that were present in the plant tissue, such as zinc or copper, but could not be present as an
organocyanide. Arithmetic averaging over four replicates was used to calculate the data for
model fitting.
Geometric averaging using logarithms of the replicate data (Table 6.2) was
included for comparison with error analysis as a means of eliminating the generation of negative
concentrations. In calculating the geometric averages, non-detect values were reassessed as half
of the detection limit for the particular analytical technique.
Data values were lower for
geometric calculations, particularly for plant tissue free cyanide and solution Day 10, 15, and 20
free cyanide, all of which had low individual replicate concentrations, as the geometric
calculations decreased the domination by large replicate values, introduced detectable values of
assimilated root cyanide (Table 6.2 b), and produced more representative values.
A number of the plant physical parameters were set using measured values from the hydroponic
experiments or reported literature values. Model parameter values that were fixed are given in
123
Table 6.1. Mass and water content for root, stem, and leaf tissues were obtained for both
treatment systems. Hydroponic liquid volume and plant transpiration data as a function of time
were available. Second-order quadratic curves were fit to the pot liquid volume and plant
transpiration data for the planted treatment systems. Evaporation from the control pots was
ignored due to the small volume change in the unplanted solutions relative to planted pots.
To reduce complexity, parameters that could be obtained from previous, related studies were set
within the model. Additional parameters were calculated using mass conservation relationships.
Reported diffusion coefficients in water. D \ of 1.25 x 105 cm: s 1 for free cyanide (Sun et al..
1996) and 0.63 x 105 cm:/s for ferrocyanide (Adams. 1969) were used. The effective diffusion
coefficients for transfer from solution to interstitial space, D \ = D'A zjJAxzj, of 14.4 and 7.26 L
dayrespectively, were calculated by approximating the interfacial area (A;.j = 1000 cm2) and
the distance between (Ar:. = 0.05 cm) the solution and the interstitial space as reported for
soybean plants (Boersma et al.. 1991). A fraction of root interstitial space value of 0.112 was
chosen based upon the value in Boersma et al. (1991). The water fraction for the interior of the
root was chosen as 0.89 for consistency with plant tissue water fraction measured in the other
hydroponic tissues.
6.3.1 Parameters for System Control Losses

Control data from the unplanted ferrocyanide and free-cyanide systems were used to assess
control losses and to determine the rate of ferrocyanide dissociation within the ferrocyanide
hydroponic system. Cyanide losses were modeled as a first-order, reversible process adsorption
process for free cyanide and ferrocyanide within the hydroponic solution system based upon the
124
adsorbent surface area. A, in contact with the hydroponic solution. The hydroponic pot surface
area was calculated as 0.129 n r from the dimensions used in the hydroponic system from
Chapter 4. Reversible, stoichiometric dissociation of ferrocyanide was included to account for
the potential uptake of free cyanide within the ferrocyanide system. The solution volume was
assumed to be constant in the control pots as relatively small evaporative loss occurred over the
20-day experiment.
Control loss models were constructed for each cyanide system with the free cyanide system
model consisting of two differential equations, representing free cyanide in the hydroponic
solution and the control loss compartment, and the ferrocyanide system model consisting of four
differential equations, representing the free cyanide and ferrocyanide in the hydroponic solution
and the control loss compartment. Mass balance constraints were included for both systems.
The free cyanide system first-order adsorption rate constants were obtained and then used as
inputs into the ferrocyanide system model to obtain the ferrocyanide adsorption and
dissociation rate constants.
Each system of equations was solved using DIVPAG (Visual
Numerics/f.i/, 1997), a double-precision Gears BDF technique for solving initial value problems
for ordinary differential equations. The differential equation solutions were optimized with a
successive quadratic programming algorithm similar to that described in (Arora, 1989). Each
model was fit to the arithmetically-averaged control data given in Table 6.2 by minimizing the
sum of the relative squared error between the actual and predicted data. The values for the loss
and dissociation constants are given in Table 6.1.
125
6.3.2
Parameters for Cyanide and Ferrocyanide Uptake and Mass Transfer in the Willow Plants
The ferrocyanide and free cyanide uptake model was solved using DIVPAG (Visual Numerics*.*,
1997)
for the concentrations within the various compartments developed in Chapter 5. The
solution to the differential equation was optimized with a sequential quadratic programming
routine as described for the control solutions. Model parameters were allowed to vary over a
predetermined range.
Each individual model solution was assessed based on an objective
function consisting of the sum of the squares between solution and tissue experimental and
predicted values. Initial, Day 0. concentrations were set within the model and not included in the
calculation of the objective function. Logarithmic values were used in the objective function to
prevent bias tow ards larger values in the data. Each of the data points was weighted equally as a
preference for any of the data relative to the rest did not exist. Weighting could also be used to
adjust the fitting of the data relative to the magnitude of the data, similar to using the logarithmic
values. Within the optimization program, constraints were included for:
i.
The cyanide mass balance on the ferrocyanide system, including assimilated I5N
ii.
The cyanide mass balance on the free cyanide system, including assimilated l5N
iii.
Day 20 ferrocyanide root cell wall concentration equality based upon root adsorption
results (Chapter 4)
iv.
Free cyanide cell wall adsorption is less than that for ferrocyanide.
The uptake and mass transfer parameters (Table 6.3) for the model were adjusted within the
optimization program to search for the solution set that minimized the objective function. The
potential range for each parameter was established so as not to constrain the parameter
variability. Some of the parameters were bounded with more certainty due to natural values,
126
such as those for transiocation efficiency (0 to I). Remaining bounds were established based
upon preliminary model optimization testing.
With the level of complexity included in the model relative to the number of data points and
constraints, the optimization solution was found to be dependent on the initial parameter values
specified to being the optimization. The objective function surface was complex with many local
minima. To search for the global minimum, repeated optimization runs were performed with
different sets of initial parameter values. In order to sample the input space adequately. 500
initial variable sets were generated from uniform parameter distributions using a Monte-Carlo
sampling routine and run with a fixed optimization perturbation setting and tolerance. This
approach was similar to that employed by Maddalena et al. (2002) to assess model uncertainty.
Of the 500 trials. 25 viable solutions were found after removing those solutions with parameters
within 0.08 log units from a bound or more than 5% error associated with the ferrocyanide
sorption constraint.
Each of the individual parameter value sets was plotted versus the objective value to assess
potential convergence to a global minimum optimum solution. As the value of the objective
function decreased, cc/ f f c>fimaxCN, and k u af approached constant values. Other variables, such
as
Yro,,', e / c, Kncs, kRoot, e / s , e f , and HUaf S fell within a reduced range. KmfC, Yua/.
a F \ f f N, and H uat showed no relationship with the objective function.
To investigate
parameter trends further, the output parameter distributions, as determined by Blom ROCC
plotting (Looney and Gulledge, 1985). were compared for a significant change relative to the
uniform input distribution using the Kolmogorov-Smimov test of distribution (Ang and Tang,
127
1975). Eleven parameters differed significantly at the 5% level from the uniform distribution, as
assessed by the largest probability difference, suggesting a significant impact on model
optimization. f f c. K ^ . yRlMM, e / c, c f s , and HUa? did not differ significantly from the uniform
distribution.
Of these. Yu.** and e / c displayed a trend versus the objective function while
a fc/ f f c approached a constant, implying significance for each.
Examination of trends in optimal parameter values versus the objective function predicted
cyanide species distributions provided evidence to support reduction in model processes and
parameters. In an effort to reduce the complexity of the model, descriptions for ferrocyanide
adsorption, free cyanide adsorption, and free cyanide volatilization were fixed or removed from
the model. Adsorptive and desorptive mass flow for both cyanide species to root cell wall were
in equilibrium and the cell wall free cyanide adsorbed mass was less than 0.001% of the initial
system cyanide loading.
As a measure of ferrocyanide adsorption was obtained separately
(Chapter 4) and represented approximately 23% of the total root ferrocyanide. the process was
described using equilibrium kinetics rather than complete removal of the process. Free cyanide
adsorption was removed completely.
Volatilized free cyanide was also a small percentage,
<0.01%, of the initial system cyanide loading and was also removed. Within a phytoremediation
system, the volatilized free cyanide would be significantly less than the OSHA permissible
exposure limit of 10 ppm for hydrogen cyanide. Thus, the number of model process parameters
was reduced from 17 to 13. The remaining 13 process variables significantly contributed to
model optimization. Reducing the number of parameters from 17 to 13 did not change the
objective function for the optimal solution.
128
6.3.3
13-Parameter Model
The 13-parameter model was fit to the experimental data using the same optimization technique
as for the 17-parameter model. Again, 500 sets of initial parameter values were used. Parameter
bounds were adjusted to reflect the range observed for the 17-parameter solutions. The 500
initial parameter value sets were used to fit to both the arithmetically-averaged and
geometrically-averaged data.
The model parameter optimal solutions (Table 6.3) differed depending on the averaging
technique for the data.
Optimization of arithmetically-averaged data produced 50 viable
parameter solution sets while geometrically-averaged data produced 54 sets. Each parameter
within the two groups was plotted versus the objective function. For both averaging approaches.
cP /p r* [inuir , and fit
approached constant values, jXnmr , Yr***>i* Yi *t ^m *^Root*
and
eg exhibited decreased variability with the objective function, while e / c. KmFC, and e g S
displayed little or no trend. For arithmetic averaging, only the distribution for K,FC was not
significantly different (a = 0.05) from the initial uniform distribution. Kolmogorov-Smimov
testing again was used to assess differences in the parameter output distributions between the two
averaging approaches. A significant difference in the </c/ f f c , egFC. Yua/- Kmcs, a Ruo k Uag and
eg distributions was observed, showing the importance of the processes related to these variables
in determining cyanide fate within the willow plant. The major changes in the optimal solution
were related to the plant dissociation, y and assimilation, A rate constants. Increased root and
decreased leaf metabolic constants for plant-mediated reactions involving both cyanide species
reflected the changes in the data caused by the change in averaging technique.
129
Also, the
significantly different parameter distributions reflected the lower tissue cyanide concentrations
(except for root tissue ferrocyanide) obtained by geometric averaging.
As expected, the optimal parameter values indicated that free cyanide active uptake, transfer
efficiency, and tissue metabolic rate constants were higher than were those for ferrocyanide. The
half-saturation constant for ferrocyanide, KnFC. was 1/20* of the respective concentration for
active uptake kinetics, suggesting that saturation kinetics were not applicable for ferrocyanide.
The free cyanide constant. Kmcs. was larger. The root free cyanide assimilation and ferrocyanide
metabolic constants. A*,,,, and
were higher than those for leaf tissues. A^, and yuaf.
6.4 Optimal Model Fits of Experimental Data
Cyanide species distributions were calculated for the experimental system using the optimal
parameter values. Except for root and stem assimilated cyanide, predicted tissue concentrations
(Table 6.4) agreed with the data for the cyanide content and speciation. The model transfer and
metabolic processes included to describe free cyanide and ferrocyanide fate within willow tissue
closely resembled the experimental results and are, therefore, sufficient for representing the
system. The contributions to the objective function due to the differences between the predicted
and actual solution concentrations were small relative to that contributed by the tissue. While the
solution concentration (Figure 6.1) and total mass (Figure 6.2) predictions were acceptable
relative to the data and were not a major source of error, the model predictions underestimated
free cyanide uptake and overestimated ferrocyanide removal. However, the major contributions
to the objective function, the measure of error associated with the optimal fit of the data, were
130
three tissue assimilate concentrations. Plant tissue predicted concentrations (Table 6.4) differed
from the data in the assimilate concentrations for root (KCN system) and stem (KCN and
Fe(CN)6+ systems). The tissue cyanide and the tissue assimilate leaf concentration predictions
agreed with the data. Weighting the tissue assimilate concentrations more during data fitting
may provide a better fit with the data. A more likely suggestion is the inclusion of phloem
redistribution as evidenced by the underestimation of assimilate within the root and stem tissue
combined with the potential for slightly increased uptake of free cyanide from solution. Phloem
redistribution could be an important mass transfer process for determining cyanide fate and
requires further investigation.
6.5 Model Variability
A measure of parameter uncertainty was unavailable to assess the variability caused by the
model, as the error on each parameter for each model solution was not calculated within the
optimization routine. In order to assess model variability, the uncertainty associated with the
hydroponic study data w as used in a parametric bootstrap technique on the residuals (Efron and
Tibshirani, 1993) to obtain a measure of the parameter and model output distributions. The
importance of measurement error relative to the error between replicates was examined. All
calculations were conducted in log space for generation of random data to prevent negative
concentrations.
The replicate error. eRep, was determined by examining the difference associated with each
replicate relative to the geometric average for each measured value within a replicate.
131
In
determining the distributions, solution cyanide was separated into Day 0, Fe(CN)6'1' - total
cyanide, KCN - total cyanide, and Fe(CN)6'1' - free cyanide. Tissue was separated into total
cyanide, free cyanide, and l5N. regardless of treatment. All six distributions were normal with /i,
= 0 and the standard error of the mean given in Table 6.5. The bootstrapped observation data
sets (n = 500) were constructed from the randomly-generated gep using Monte-Carlo sampling
techniques and used to calculate the model input data for each of four replicates.
Tissue
background concentrations and the adjustment to obtain the complexed cyanide from the total
and free measurements were included. Calculated negative values for plant tissue were set at
0.005 mg/kg FW as CN. one-half the detection limit for plant tissue. The individual, randomlygenerated replicate values were averaged to obtain the data values for fitting with the model.
Measurement error, , was calculated from the difference between the duplicate measurements,
with the second subtracted from the first. Solution cyanide ydirt were separated into total
cyanide, high free cyanide concentrations, and low free cyanide concentrations (<0.30 ppm as
CN). Tissue
was separated into ferrocyanide-treated root total cyanide, high total cyanide
concentrations, low total cyanide concentrations (<0.15 ppm as CN), high free cyanide
concentrations, and low free cyanide concentrations (<0.30 ppm as CN).
was assumed
negligible for ISN measurements. All distributions were normal (as assessed by a KolmogorovSmimov test of the distribution) except those for low free cyanide, which were affected by the
proximity to detection limits. For all y^g distributions, /ix 0 as the first cyanide measurement
tends to be higher than the second. The means of the true values for the two measurements were
assumed to be different, allowing the assumption nyd,g=0 and m to be related to yd,g (Equations
2 and 3). <Xaware given in Table 6.7.
132
(6.2)
= {My ~Py, )+ f l
and
(6.3)
& eM ~ G y
The bootstrap replicate observation sets were the basis for calculations involving the
bootstrapping of the measurement error.
Two randomly-generated
from Monte-Carlo
sampling techniques were averaged and used to adjust the replicate basis value to reflect
measurement uncertainty. Model entry values were calculated as before from the randomlygenerated measured values of each of four replicates. Calculated negative values were corrected
using one-half the detection limit, 0.005 mg/kg FW as CN for plant tissue. 0.0005 mg/L as CN
for solution complexed cyanide, and 0.00125 mg/L as CN for solution free cyanide.
The relative effect of measurement error with respect to replicate error was investigated by
comparing the resulting parameter value distributions from fitting of 500 randomly-generated
data sets for each case. The initial parameter value set for all 500 trials was the optimal solution
obtained from fitting the geometric mean data (Table 6.3). The 500 input data sets provided
sufficient variability in optimal solutions, negating the need to solve each data set for multiple
sets of input parameter distributions. Replicate error produced 96 viable solutions while the
inclusion of measurement error produced 119 viable solutions. The distributions for each of the
model parameters were assessed using Kolmogorov-Smimov testing to determine if
measurement error significantly affected the solutions. Assessment of the distributions showed
that three variable distributions, e g S, ATmcv, and Aâ/, differed significantly (a=0.05), reflecting
the larger measurement variability for total cyanide in KCN-treated plant tissue.
133
As measurement error proved significant, the output distributions for the 119 viable solutions
from bootstrapped replicate and measurement error were assessed for parameter and data
uncertainty. The mean and standard error for the thirteen parameters (Table 6.6) were used to
assess significant changes relative to the input values (optimal solution obtained for log-averaged
data). The output parameter distributions obtained using bootstrapped data were not comparable
with those obtained by varying the initial parameter set. In comparing the output distributions
from bootstrapping, only e tfs and kuaf were not significantly different (t-test. a=0.05) from the
optimal solution values obtained from the input parameter investigation. The effect of the data
uncertainty on the variability in cyanide concentrations within the plant hydroponic system was
examined for the distribution of the fraction of the initial cyanide mass remaining as cyanide for
control losses, in solution, plant root tissue, and aerial plant sections for both treatments at the
end of the experiment (Day 20). The distribution of the initial cyanide mass assimilated or
released from the system was assessed as well. All quantities are important assessments of the
effectiveness and practicality of using phvtoremediation for the treatment of cyanidecontaminated soil and groundwater. The uncertainty in the fraction of the initial cyanide mass
within the compartments was low with coefficient of variation (COV)<0.09 (Table 6.7). For the
ferrocyanide system, 66% of the initial cyanide mass remained in solution (Figure 6.3) with 20%
accounted for by control losses and approximately 5% each in plant root tissue and volatilized as
assimilate. Only 1% was assimilated (Figure 6.4). In the free cyanide system, 86% of the initial
mass was assimilated (Figure 6.4) with only 10% remaining in solution (Figure 6.3) and 3%
accounted for by control losses.
134
Correlation analysis of the output parameter distributions displayed a number of significant

relationships among the variables (Table 6.8). Significant positive correlation existed between
/ W FC and Yi but not for /W tCV and A,. / w * and Km for each cyanide species were highly
positively correlated while a significant negative correlation existed between
and / w CV-
As ferrocyanide uptake increased, less free cyanide was required to be taken up to achieve the
assimilated product concentrations within the plant tissue. The correlation also exists for the
plant-mediated reaction constants and transfer efficiencies.
Increasing eg
was correlated
significantly with increased y, and lower KmFC to compensate for the accumulation in tissue due
to the increased transfer of ferrocyanide. The increased plant-mediated leaf dissociation was
significantly correlated with reduced free cyanide uptake parameters.
Significant negative
correlation existed between Yr,h and e g S as well as Yuaf and k RlMU. Yuar was correlated positively
with Xua/ to prevent excess buildup of free cyanide in leaf tissue. Increased kua/ compensated
for increased e g S.
between Huat and ^
To maintain the mass balance, significant negative correlation existed

1
. kRlM, and e g . Figures 6.5. 6.6, and 6.7 display the parameter plots
for the three highest correlations: Kmcs versus / w CV (p = 0.74), K ,/c versus nnuJ c (p = 0.64),
and Yroo, versus e / c (p = 0.70).
6.6 Discussion
Fitting of the 17-parameter model developed in Chapter 5 to experimental data exposed the
inclusion of some unnecessary parameters. The minimum number of process parameters was
sought as the goal of the model was to simplify the model to reflect plant physiology without
135
over-representing transfer processes. Many of the model output parameter distributions showed
a decrease in variability with objective function values or a significant difference from the
uniform parameter input distribution. Among those that did not show either were o fs , KnFC, and
HuafA- However, the magnitude of the mass of assimilate volatilized was significant as was the
contribution of KFC relative to the ferrocyanide concentration within the active uptake kinetic
representation. Equilibrium conditions existed for cell wall adsorption processes. Analysis of
the mass fraction adsorbed showed that the cell wall fraction for the both cyanide species
(<0.04%) and the free cyanide mass released via volatilization (<0.01%) were negligible relative
to the total cyanide in the system and were not significant in determining cyanide fate. The rate
of free cyanide volatilization was significantly less than the rate of free cyanide assimilation with
plant tissue.
Ferrocyanide cell wall adsorption was kept in the model because separate
experiments (Chapter 4) provided a measure of ferrocyanide adsorption and showed the sorbed
fraction to be a significant portion of root tissue ferrocyanide.
Evaluation of the 13-parameter model with geometrically-averaged data showed that / w / ' and
H uat approached constant values, nnuJ c, Yro.* Yuaf. Kmcs,
AUaf- and eg variability
declined, and egCS, e / c, and KmFC lacked a trend as the objective function decreased. The
presence of a trend in parameter values relative to objective function values suggests relevancy
for a mass transfer processes as relating to cyanide fate within the hydroponic system.
Examination of parameter distribution COV within the uncertainty analysis provided additional
comparisons to assess relevancy.
136
Model sensitivity was assessed by examining the effects of data uncertainty on the mean
parameter estimates. Parameter significance was assessed through the coefficients of variation
(COV) relative to the standard error on the mean. Small values reflect parameter rigidity to input
variance and elevated confidence in the parameter estimates of the mean. The COV for / w CV
was 0.002 while the remaining parameters were <0.03 except for KmFC for which COVSE was
0.66.
With respect to the output concentration distributions within the free cyanide and
ferrocyanide systems, the assimilate volatilized within the free cyanide system and the total
assimilated cyanide within the ferrocyanide system were significantly different from the mass
fractions predicted with the optimal. 13-parameter solution as determined using the geometric
mean data. The rest of the cyanide concentration mean estimates were not significantly different
at the 5% level.
The solution and tissue concentrations predicted from the optimal fit of the 13-parameter model
with the geometrically-averaged data suggest that an important mass transfer process, phloem
redistribution of assimilated cyanide, was excluded from the model. Free cyanide removal from
KCN solution was underestimated while that for ferrocyanide solution was overestimated (Figure
6.1). Tissue concentrations (Table 6.4) were underestimated for free cyanide, particularly for
assimilate within root and stem tissue. The model matches the assimilate tissue concentration
and the amount of assimilate volatilized. In order to do so without removing too much from the
system, the amount within plant root and stem tissue was kept below measured values. Without
the inclusion of a recycling mechanism, assimilate nitrogen (as assessed by measuring tissue 1SN
content) could not concentrate within the plant nitrogen cycle. Phloem redistribution would
allow for assimilated nitrogen from the leaf tissue to be returned to root and stem tissue,
137
increasing the concentration with time in both tissues as more accumulates within the system.
Movement within the phloem was not included in the current model to minimize the inclusion of
adjustable parameters. Given the tissue assimilate concentration results, inclusion of nitrogen
cycling appears to be an important process to include in assessing cyanide fate within willows.
Additional investigation of the recycled water fraction of water and the effective transfer
efficiency between the leaf tissue and phloem tissue for assimilate is necessary for inclusion into
the model.
The fact that the optimal Yum was less than
could signify insignificance of light-mediated
ferrocyanide dissociation in the leaf tissue. Light-induced leaf dissociation would produce a
higher dissociation constant in leaf tissue compared with root. Therefore, the results suggest that
photo-dissociation does not occur. However. Yuaf contains a cluster around a higher value for the
varied input results. The optimal solution had a value of 1.39x105 kg/day compared with the
alternative optimum has a value of 3.16x10' kg/dav, which wras higher than the optimal
2.85.x 10-4 kg/day.
of
A higher value of yuaf would result from the inclusion of phloem
redistribution, necessitating re-examination of the importance of light-induced leaf ferrocyanide

dissociation.
The results pertaining to the significance of KmFC suggest that active uptake kinetics may not be
applicable for ferrocyanide uptake into plants. KmCN proved more significant and was highly
correlated with / v
with both displaying reduced variability with respect to lower objective
function values in the varying input parameter trials. The ratio of KmFC to that of the ferrocyanide
interstitial concentration ranged from 0.04 to 0.075 while the same ratio for the free cyanide
138
system ranged from 0.14 to 0.9. Active uptake kinetics were appropriate for the free cyanide
system, suggesting the potential for plant use of cyanogenic-N as a nitrogen source. The small
half-saturation constant. KmFC. relative to interstitial ferrocyanide concentration and the lack of a
trend versus the objective function in the varying input parameter trials suggest that fenocyanide
uptake does not occur via saturation kinetics.
The model was modified in order to examine the effect of active uptake versus passive uptake
with the transpiration stream as in models presented for organi c s (Boersma et al.. 1991: Burken
and Schnoor. 1997; Trapp et al.. 1994).
Uptake of ferrocyanide could occur with the
transpiration stream or as a combination of active and passive flow rather than strictly due to sitelimited saturation kinetics. Beavis and Bercesi (1992) have shown passive movement through
anion channels in the mitochondrial inner membrane of potato. Leakage is also possible due to
uptake at the root tips or through damaged membrane sections.
Each of these two uptake
processes, anion channels and leakage, would be represented with first-order kinetics. For the
current examination, a first-order process was substituted for the saturation kinetics with the firstorder rate constant equal to the transpiration rate. A partitioning factor was not included for the
investigation.
Results from examining the potential for only passive uptake show that ferrocyanide must be
excluded at the root membrane as transfer to root tissue with the transpiration stream
underestimated the concentration effect witnessed in the solution (Figure 6.8). A membrane
process occurs that limits uptake and prevents ferrocyanide from freely entering plant tissue. The
plant-mediated process could still be first order with respect to the concentration in the interstitial
139
space explaining the results for KmFC. Analysis of the free cyanide system also supports plantmediated uptake as results showed that uptake only with the transpiration stream, even if
unimpeded, underestimated the removal of cyanide from solution (Figure 6.8).
The plant
actively takes up the free cyanide at an elevated rate compared with transpiration flow.
Combined with the Kmcy results, saturation kinetics were valid for the uptake of free cyanide.
6.7 Summary and Conclusions
A plant uptake model for ferrocyanide and free cyanide fate within plants reflecting the mass
balance equations within the systems (Chapter 5) was applied to describe hydroponic uptake data
(Chapter 4). Fitting of parameters provided an assessment of the uptake and assimilation rates as
well as a determination of the influential mass transfer processes involved in the uptake and
movement of cyanide.
As many parameters as possible were obtained from plant system
property measurements, literature values, or control data for input into the model. The resulting
17-parameter model was solved numerically and optimized using a general sequential quadratic
programming routine for 500 randomly-generated initial parameter value sets to sample the
parameter space. Initial data fitting revealed four parameters and that did not influence the
parameter fitting significantly. These four parameters were removed and the model was re
applied to the data using the same approach for geometrically-averaged data.
Variability in the optimal model parameter values due to data uncertainty was examined using
500 randomly-generated data sets accounting for replicate and measurement error. Parameter
140
trends with respect to the objective function, parameter output distributions, and optimal fits with
respect to input data were assessed to determine important mass transfer processes.
Simulations with the plant uptake model could describe the observed trends in the hydroponic
data for free cyanide and ferrocyanide uptake by willow.
Initial trials showed that cyanide
adsorption to root cell wall tissue and free cyanide volatilization from leaf tissue were not
significant cyanide sinks with less than 0.04% of the initial mass present within any of the
relevant compartments within the 17-parameter model. These processes could be included in
future models using the parameters obtained in the solution of the 17-parameter model. Free
cyanide volatilization potential may be important under specific field conditions. The modeling
indicated that diffusion and advection both influenced chemical movement between the bulk
solution and the root interstitial space, as an accumulation occurred for both cyanide species
within the interstitial space. As expected, free cyanide uptake and assimilation rate constants
were higher compared with ferrocyanide uptake and assimilation rate constants.
The optimal model solution for fitting the hydroponic data sufficiently predicted the tissue
cyanide concentrations and the leaf assimilate concentrations.
Although the model
underestimated free cyanide uptake from solution and overestimated ferrocyanide uptake from
solution slightly, the predicted concentrations were still acceptable. The principle contributions
to the error were the result of the root and leaf tissue assimilate concentrations. A particular
disparity existed in root and stem assimilate concentrations for the free cyanide system. The
deficiency in the assimilate concentrations for root and stem suggest that phloem transport be
examined.
Phloem transport was not considered but could redistribute assimilated product
141
within plant root and stem tissue to reflect experimental data. Additional considerations such as
plant growth effects and cyanide solid precipitation can also be added to the model.
Modeling of the data suggests that uptake of ferrocyanide may not occur via an active process.
Examination of potential uptake with the transpiration stream showed that ferrocyanide was
excluded at the endodermis. Conversely, a plant-mediated uptake process was required to reflect
the hydroponic solution data for free cyanide. Saturation kinetics were valid for free cyanide but
may not be necessary for ferrocyanide. Representation of such processes, exclusion as well as
first-order uptake, were captured within the active uptake kinetics used to represent the system.
However, an alternative representation such as the combination of saturation and first-order
kinetics as described in Somma et al. (1998).
Analysis of the parameter trends relative to the objective function for the acceptable solutions
from the 500 input initial parameter sets showed that / w CN and Hua* had a low COV in
response to data uncertainty. Neither was affected by variations in either the data or initial
parameter set. suggestive of parameter significance. The data uncertainty analysis provided a
measure of uncertainty for the rest of the 13 parameters as well as compartmental cyanide
concentrations. Each of these uncertainties was characterized by a COV. However, none of the
results provided sufficient evidence regarding the relative importance of particular processes in
describing the system. Further investigation of the uncertainty data, possibly with regionalized
sensitivity analysis, is required to assess process and parameter importance. Validation of the
model is also necessary using a second set of plant uptake data. The optimal parameters obtained
142
using the current set of data (Chapter 4), including the associated uncertainty, can be used to
predict free cyanide and ferrocyanide fate within a similar system.
&8 Acknowledgements
University.
The authors would also like to express sincere appreciation for the invaluable
modeling advice provided by Dominic Boccelli and Ki-joo Kim.
6.9 References
Adams, R.N. (1969) Electrochemistry at Solid Electrodes. Marcel Dekker, Inc., New York,
NY, 220.
Ang, A.H-S. and Tang, W.H.
(1975)
Probability Concepts in Engineering Planning and
Design. Wiley, New York.
Arora, J.S. (1989) Introduction to Optimum Design. McGraw-Hill, New York, NY.
Boersma, L.; McFarlane, C.; and Lindstrom, F.T. (1991) Mathematical Model of Plant Uptake
and Translocation of Organic Chemicals: Application to Experiments. J. Envir. Qual. 20:137.
Environ. Sci. Tech. 31:5, 1399.
Chemick, M.R. (1999) Bootstrap Methods, A Practitioners Guide. Wiley, New York.
143
Davison, A.C., and Hinkley, D.V.
(1997)
Bootstrap Methods and Their Application.
Cambridge University Press, Cambridge.
Efron, B., and Tibshirani, RJ. (1993) An Introduction to the Bootstrap. Chapman and Hall,
New York.
Looney, S.W. and Gulledge, Jr., T.R. (1985) Use of the Correlation Coefficient with Normal
Probability Plots. The American Statistitian. 39, 75.
Maddalena, R.L.; McKone, T.E.: and Kado, N.Y. (2002) Exposure Chamber Measurements of
Mass Transfer and Partitioning at the Plant/Air Interface. Environ. Sci. Technol. 36:16, 3577.
Meeussen. J.C.L.; Keizer. M.G.: and de Haan, F.A.M.
(1992)
Decomposition Rate of Iron Cyanide Complexes in Soil Solutions. Environ. Sci. Tech. 26, 511.
Sohn, M.D.; Small, M.J.; and Pantazidou, M.
(2000)
Reducing Uncertainty in Site
Characterization Using Bayes Monte Carlo Methods. J. Environ. Eng. 126:10. 893.
Somma, F: Hopmans, J.W.; and Clausnitzer, V.
(1998)
Transient Three-Dimensional
Modeling of Soil Water and Solute Transport with Simultaneous Root Growth, Root Water and
Nutrient Uptake. Plant and Soil. 202, 281.
Spear, R.C.; Grieb, T.M.; and Shang, N. (1994) Parameter Uncertainty and Interaction in
Complex Environmental Models. Water Resour. Res. 30:11,3159.
Spear, R.C., and Homberger, G.M. (1980) Eutrophication in Peel Inlet, II Identification of
Critical Uncertainties Via Generalized Sensitivity Analysis. Water Res. 14,43.
144
Sun, X.; Guan, Y.C.; and Han, K.N. (1996) Electrochemical Behavior of the dissolution of
Gold-Silver Alloys in Cyanide Solutions.
Metallurgical and Materials Transactions B.
27B355.
Taylor. J.R. (1982) An Introduction to Error Analysis: The Study of Uncertainties in Physical
Measurements. University Science Books, Mill Valley, CA. 182-184.
Trapp, S.; McFarlane, C.; and Matthies, M. (1994) Model for Uptake of Xenobiotics into
Plants: Validation with Bromacil Experiments. Environ. Tox. Chem. 13:3.413.
Visual Numerics/?.i/. (1997) Chapter 5: Differential Equations. IMSL Fortran Subroutines for
Mathematical Applications: Math Library Volumes I and 2. Visual Numerics, Inc. Houston.
TX.
Wade, A.J.: Homberger, G.M.: Whitehead, P.G.; Jarvie, H.P.: and Flynn, N.
(2001) On
Modeling the Mechanisms that Control In-stream Phosphorus, Macrophyte, and Epiphyte
Dynamics: An Assessment of a New Model Using General Sensitivity Analysis. Water Resour.
Res. 37:11.2777.
145
Table 6.1 Parameter values used as input for the plant uptake model for free cyanide and
ferrocyanide by willow. Variables and equation set up for uptake model defined in Chapter 5.
Symbol
Parameter Description
Parameter Value
Units
Joint Parameters
A'
F ro r
-
Gis
2a
FeRq1
ViRt1
Stq1
ThSt*
Folq1
D 7b
Vfe3
FeQ3
SbFe4
DsFe4
SbFe4
DsFe4
CNRq'
ThRtC1
StqC1
ThStC'
FolqC1
d
7 c
VCN3
CNQ3
SbCN*
DsCN*
0.129
0.89
0.112
Control adsorbent area

Interior root water content
Fraction root interstitial space
0.00514
Total root mass
0.884
Total root water content
0.00928
Stem mass
Stem water content
0.767
Leaf mass
0.01375
Diffusion rate constant
7.26
5.1858-0.0028 r-0.0843 t
Ferrocyanide solution volume
Transpiration rate
0.09888+0.00017 t*+0.00202 t
Control sorption rate constant
0.135
Control desorption rate constant
0.0495
Dissociation rate constant
0.001915
0.0187
Complexation rate constant
nr
kg/kg
UL
Kg
UL
Kg
UL
Kg
L/day
L
L/day
L/day
kg/day
l/day
(L/pmoI)6/day

0.00384
Total root mass
Total root water content
0.894
0.00862
Stem mass
Stem water content
0.735
Leaf mass
0.01212
14.4
Diffusion rate constant
5.2056-0.0002 r-0.I209t
Free cyanide solution volume
Transpiration rate
0.0978-0.0004 r+0.0076 1
Control sorption rate constant
0.139
Control desorption rate constant
0.110
Kg
UL
Kg
UL
Kg
L/day
L
L/day
L/day
kg/day
1Measured during hydroponic uptake experiment

2 Obtained from the literature. Boersma et al. (1991) J. Environ. Qual. 20,137. bAdams. (1969)
Electrochemistry at Solid Interface. cSun et al. (1996) Metallurgical and Materials Transactions
B. 27B.355.
3Fit to experimental data.
4Attained from control system optimization.
146
Table 6.2 Experimental solution (a) and tissue (b) concentrations used to fit the cyanide uptake
model. The values were obtained from 20-day hydroponic uptake results with willow for 2 ppm
as CNt solution of KCN and potassium ferrocyanide (FCiFefCN^). All data was the average of
four replicates except for all Day 20 solution and Day 0 FC-CN solution values (n = 3).
(a) Solution Concentration Data1*2

Arithmetically-Averaged
Planted
Control
KCN
Geometrically-Averaged
KtFefCN),
Planted
K*Fe(CN)4
KCN
KCN
KtFefCN)*
Day
CN
FC
FC-CN
CN
FC
FC-CN
CN
FC
FC-CN
70.38
11.75
0.0
68.63
11.93
0.0
68.55
12.02
0.043
65.38
10.52
0.654
55.37
11.34
0.540
55.21
11.32
0.551
10
63.85
9.951
1.000
34.11
12.50
0.733
14.09
12.47
0.542
15
63.08
9.601
1.154
11.05
14.95
0.640
3.85
14.86
0.498
20
63.08
10.28
1.462
7.44
20.27
0.387
1.58
20.04
0.372
'Units are p.mol/L

'Abbreviations: CN - free cyanide, FC - ferrocyanide, FC-CN - free cyanide ferrocyanide
system, and A - assimilated cyanide
(b) Tissue Concentration Data1-2

Geometrically-A veraged
Arithmetically-Averaged
KCN
KjFefCN)*
KCN
K|Fe(CN)
Tissue
CN
FC
FC-CN
CN
FC
FC-CN
Root
43.08
18688
819.3
7.31
0.0
13.52
17906
794.3
2.07
1.39
Stem
1.54
4422
0.64
1.54
308.8
0.57
4121
0.22
0.41
245.5
Leaf
8.85
3810
4.81
0.38
824.6
2.32
3741
4.42
0.23
783.4
'Units are pmol/kg-fresh-weight

'Abbreviations: CN - free cyanide, FC - ferrocyanide, FC-CN - free cyanide ferrocyanide
system, and A - assimilated cyanide
147
Table 63 .
Process variables determined through fitting of hydroponic uptake data for free
cyanide and ferrocyanide.
Some values constrained within possible range.
Plant physical
parameter values fixed and given in Table 6.1. Parameters eliminated from the 17-parameter
model include o fs/0 :s (free cyanide cell wall adsorption/desorption) and HuafS (leaf free
cyanide volatilization).
Parameter Value
Symbol
a fc/ $ c
FC
eg
u FC
f*max
K FC
YRool
YU af
CN
eg
HmaxCS
Kmcs
^R oot
^Leaf
eg
Hua?
Arithmetic
Averaging
Geometric
Averaging
Units
Ratio of cell wall adsorption/desorption
3.49
Transfer efficiency
2.67 x 1(T*
755
0.374
1.77 x 10~*
9.45 x 103
3.10
1.11 x 10"*
535
0.503
2.85 x 10"*
1.39 x 105
Meg
pmol/pmol
pmol/L-day
|xmol/L
kg/day
kg/day
0.115
54550
15.0
0.878
0.093
pmol/pmol
pmol/L-day
jimol/L
kg/day
kg/day
Description
Free Cvanide Parameters

Transfer efficiency
0.072
49740
25.2
0.145
0.361
Transfer efficiency
0.106
0.083
2.37 x I O'3 2.35 x 103
148
pmol/pmol
kg/day
Table 6.4 Predicted tissue cyanide concentrations (pmol/kg-fresh-weight as CN for CN and A

and as F eC ^ 4* for FC) obtained with optimal parameter values from fitting of geometricallyaveraged input data set (Measured). For the ferrocyanide system (FefCNfo4*), the ferrocyanide
(FC), free cyanide (CN), and assimilate (A) values are given. Only the free (total) cyanide (CN)
and assimilate (A) concentrations are shown for the free cyanide system (KCN). Std Err
reflects the standard error associated with each data point (n = 4).
FeCN*4Leaf
KCN
FeCNt4*
Stem
KCN
FeCN*4Root
Model
Measured (Std Err)
FC
12.4
4.42(1.25)
CN
0.574
0.229(1.19)
661
783(1.20)
CN
1.33
2.32 (2.88)
5595
3741 (1.11)
FC
0.070
0.223 (3.04)
CN
0.1%
0.410(2.13)
6.48
245(1.51)
CN
0.984
0.570(1.91)
75.5
4121 (1.24)
FC
639
794(1.16)
CN
1.75
2.07 (3.38)
77.1
1.39 (7.2)
CN
9.32
13.5 (3.12)
939
17906(1.2)
KCN
Table 6.5 The replicate and measurement error used in the generation of random samples for
error analysis.
Replicate error reflected as the standard error on the mean (SEmcu) in the
difference between the replicate value and the geometric average.
Measurement error
distribution calculated using the difference between the two measurement values, y^g, and
reflected by the standard deviation, a E = <Jydifl^2. For measurement error, Low/High Free CN
solution split at 0.30 ppm, Low/High Total CN tissue split at 0.15 ppm, and Low/High Free CN
tissue split at 0.30 ppm. All values calculated in log-space. n is the number of samples within
each set.
Replicate SEMcan (/)*
Measurement <JE(n)2
Solution fumol/L as CN)
Solution (umol/L as CN)
Total C N -F C
0.011(14)
Total C N -C N
Free C N -F C
0.183(15)
0.082(14)
Day 0 - Total & Free
0.009(11)
Total CN
High Free CN
Low Free CN
1.696 (74)
4.109(30)
0.0904 (42)
Tissue (umol/ke- fresh-weisht as CN)
Tissue (umol/ke-fresh-weight as CN)

Total CN
Free CN
0.068 (36)
0.042 (36)
FC Root Total CN
Low Total CN
257.8 (4)
0.954(16)
l5N
0.033 (29)
High Total CN
Low Free CN
13.2(15)
0.380 (24)
High Free CN
,SN
8.51 (9)
Assumed Negligible
Total CN - total cyanide; Free CN - free cyanide; Day 0 - ALL (total and free cyanide for both
systems) initial measurements; FC - ferrocyanide system; CN - KCN system
Total CN - total cyanide; Free CN - free cyanide; FC - ferrocyanide system
150
Table 6.6 The mean (Log Xopt) and standard error. (SEtog x) for each of the 13 model parameters,
expressed as logarithms, resulting from uncertainty in the input data (n = 119). The log of the
optimal solution for each parameter was included for comparison with the distribution from the
uncertainty investigation.
Symbol
d c/ f f c
FC
eff
n axFC
b*m
K fC
YRihii
Yljtaf
CN
eff
u cv
K cs
I'm
*
A-Rihh
^Leaf
eff
Huat
Description
LogX
SEugx
Log
Ferrocvanide Parameters
Ratio of cell wall adsorption/desorption
0.510
Transfer efficiency
-3.7%
2.839
-0.112
-3.669
-4.272
0.004
0.037
0.029
0.073
0.048
0.091
0.492
-3.953
2.728
-0.299
-3.546
-4.858

Transfer efficiency
-0.966
4.669
0.980
-0.115
-1.003
0.024
0.008
0.022
0.022
0.030
-0.940
4.737
1.175
-0.056
-1.031
Transfer efficiency
-2.485
-0.873
0.036
0.026
-2.630
-1.080
151
Table 6.7 The mean and standard error (n = 119) for the fraction of initial cyanide mass present
in specified compartments after 20 days. The fractions for the optimal solution were included for
comparison with the distribution from the uncertainty investigation. The average total cyanide
mass within each of the systems was 375 and 357 pmole as CN for the ferrocyanide and free
cyanide systems.
Compartment
^Ferrocyanide System
X
SEX
Control losses
Solution
Root tissue
Aerial tissue
0.203
0.656
0.067
3.4x103
1.1x103
4.3x 103
4.4x103
1.5x10"*
Plant tissue
Volatilized
0.020
0.051
1.2x 103
3.8xl03
1.000
Free Cyanide System

X
SEx
Xqq,
Cvanide
0.206
0.670
0.052
2.8xlO'3
0.032
0.106
1.6x1 O'4
1.2x10"*
1.0x103
6.9x10 5
1.4x105
2.5x105
0.027
0.077
1.1x10
6.9x10
Assimilate
0.026
0.044
0.175
0.686
0.010
0.012
0.202
0.694
Total
1.000
1.000
152
1.000
Table 6.8 Correlation coefficients were determined for the output parameter distributions of the variability study including
measurement and replicate error (/ = 119). The objective function (OF) values were also included to assess the effect of the variable
on solution optimality. Values were calculated using ExcclOs Data Analysis toolpack. Significant associations were determined
according to Taylor (1982) arc in bold.
Tn
CN
c t(/p<
FC
FC
0.59
0.04
0.09
0.19
0.64
0.09
0.70
0.27
0.32
0.47
0.30
0.17
0.33
0.13
0.37
0.12
-0.06
-0.06
0.02
-0.26
-0.08
-0.24
-0.01
-0.09
-0.06
-0.12
'Hunt
0.02
-0.12
-0.15
0.16
0.20
0.10
-0.17
0.09
CN
CN
0.21
0.32
0.29
0.07
-0.08
-0.33
-0.22
0.08
-0.12
-0.33
-0.15
0.07
0.74
-0.14
0.17
0.24
-0.25
0.09
0.16
0.04
0.02
0.10
0.51
0.39
-0.05
-0.10
-0.12
0.12
0.10
0.27
-0.18
0.22
0.22
-0.27
-0.29
15.1
0.04
Figure Legends
Figure 6.1 Predicted solution cyanide concentration profiles obtained with optimal parameter
values fitted to geometrically-averaged input data set.
For the ferrocyanide system, the
ferrocyanide (FC) and free cyanide (CN) solution profiles are given. Only the total cyanide (Tot
CN) profile is shown for the free cyanide system. Curves (e.g .------- ) represent model results
while symbols (e.g. ) reflect data points used to calibrate the model. The FC and Tot CN
comparison is given in (a) while that for free cyanide within the ferrocyanide system (CN) is
plotted for (b). Error bars reflect the standard error of the four data replicates.
Figure 6.2 Predicted solution total cyanide mass profiles obtained with optimal parameter values
fitted to geometrically-averaged input data set for the ferrocyanide system (FC) and free cyanide
(Tot CN) systems. Curves (e.g.-----) represent model results while symbols (e.g. ) reflect data
points used to calibrate the model. Error bars reflect the standard error of the four data replicates.
Figure 6 3 The variability in the mass fraction of the initial cyanide dose remaining in solution at
Day 20 was calculated based upon the uncertainty in the experimental concentration data used in
model fitting. Only the amount of mass in solution as either complexed or free cyanide was
considered. Control losses from the solution were not included.
The mass fractions were
obtained for the ferrocyanide (FC) and KCN (CN) systems from the model output (n = 119) with
the randomly-generated, simulated input data.
154
Figure 6.4 The variability in the mass fraction of the initial cyanide dose assimilated was
calculated based upon the uncertainty in the experimental concentration data used in model
fitting. The assimilated fraction included the mass assimilated present in the plant root, stem,
and leaf tissue as well as the assimilate mass volatilized at Day 20. The mass fractions were
obtained for the ferrocyanide (FC) and KCN (CN) systems from the model output (n = 119) with
the randomly-generated, simulated input data.
Figure 6.5
was correlated significantly with Kmcs (p = 0.74). Data for the plot was
obtained by bootstrapping the residuals to obtain a set of 500 randomly-generated, simulated

input data sets. The error associated with the data propagated through the model to produce
output parameter distributions.
Figure 6.6 Hnut/ C was correlated significantly with KmFC (p = 0.64). Data for the plot was
obtained by bootstrapping the residuals to obtain a set of 500 randomly-generated, simulated
input data sets. The error associated with the data propagated through the model to produce
output parameter distributions.
Figure 6.7 e / c was correlated significantly with Yr,m (p = 0.70). Data for the plot was obtained
by bootstrapping the residuals to obtain a set of 500 randomly-generated, simulated input data
sets.
The error associated with the data propagated through the model to produce output
parameter distributions.
155
Figure 6.8 Cyanide uptake inside the endodermis was compared for transfer with the
transpiration stream (Flow Only) and with active uptake for the ferrocyanide (FC) and free
cyanide (CN) systems. Fate within the willow plant was ignored. The model was run in the
forward mode using the optimal parameter values for c f c/ f t c, Umax'. and K. A selectivity
coefficient (0 to 1) was not included for transfer into plant tissue with the transpiration stream.
The optimal fit with active uptake was better than that for flow only.
156
Tot CN (modd)
FC (modd)
Tot CN (data)
FC (data)
120
100
u
a
c
i
^
60
40 20
10
15
20
15
20
Day
2.0
Z
U
X
CN (modd)
CN (data)
1.5
SB
1.0
0.0
10
Day
Figure 6.1
157
400
Umol as CN
300
200
100
Tol CN (model)
FC (model)
Tot CN (data)
FC (data)
0
0
10
15
Day
Figure 6.2
158
20
FC
CN
0.4
03
0.2
0.0
0.0
0.2
0.6
0.4
0.8
Fraction of Initial Cyanide Mass in Solution
Figure 6.3
159
1.0
FC
0.4
03
0.1
0.0
0.0
0.2
0.6
0.4
0.8
Fraction of Initial Cyanide Mass Assimilated
Figure 6.4
160
1.0
CN
max
Figure 6.5
161
Si
u max FC
Figure 6.6
162
-1
-2
-3
-5
FC
Figure 6.7
163
Solution Concentration (jimol/L as CN)
100
80 -
O'
60
40
CN
20
CN-Fl ow O nly
v - FC-FIow Only
10
15
Day
Figure 6.8
20
C H A PTER 7
C o n c l u sio n s
and
R ec o m m e n d a t io n s fo r
fu t u r e w ork
This research addressed the general topic of the phytoremediation of cyanides, particularly iron
cyanides, by willow. The project was separated into four major components, described in the
four main chapters of the thesis. As outlined in Chapter 1, the primary issues that this research
addressed were:
i.
What is the best extraction method for the determination of cyanide content and
speciation in plant tissue to provide reliability and reduce interferences? Can
cyanide content be measured with certainty?
ii.
What is the fate of free cyanide and iron cyanide within a willow plant-solution
system? Do willow plants take up iron cyanides and, if so. is complexed cyanide
metabolized?
iii.
Can a physiologically-based plant uptake model be constructed to represent the

hydroponic system?
iv.
Can fitting of a process model to experimental data provide insight into which mass
transfer and transformation processes are important for cyanide species in the
willow plant? Which are negligible? And what is the uncertainty associated with
the model?
This chapter summarizes the major findings of the research, particularly regarding these four sets
of questions. The engineering applications are addressed as pertaining to the practicality of the
165
phytoremediation of iron cyanide using willows. Recommendations for the continuation of the
research are included.
7.1 Major Findings
7.1.1
Plant Tissue Extraction Method for Complexed and Free Cyanide
The investigation of the extraction method examined spike recoveries of cyanide species from
solution with and without plant tissue using various solvent combinations. Methanol, 2-octanoI,
and hexane interfered with the cyanide analytical method (APHA/AWWA/WEF, 1998) when
included with NaOH in the solvent matrix while chloroform reacted with NaOH and free cyanide
in sample spikes. NaOH (1.6 g/L) was chosen as the solvent for the extraction. Plant tissue was
homogenized by grinding under liquid nitrogen prior to measurement to decrease measurement
variability and increase precision. Following homogenization, a known amount of tissue was re
ground under NaOH and brought to 100 mL volume. The slurry was sonicated, extracted in the
dark for 16 hours under constant mixing, and analyzed for free and total cyanide without
filtration. Recoveries were 89% and 100% for 100 ppb CNt spike solutions added as KCN and
K+FefCNV
Background contributions to the cyanide measurement in plant tissue were
quantified and not significant for stem and leaf tissue at 0.04 mg/kg-fresh-weight as CN. Root
contributions were higher, measuring 0.46 and 0.22 mg/kg-fresh-weight as CN for total and free
cyanide, but were still not significant. Extraction results from exposed tissue were in agreement
with stable isotope results (Chapter 4). The method developed is the first reported for extraction
of cyanide species from plant tissue.
166
7.1.2
Transport and Metabolism of Free Cvanide and Iron Cyanide Complexes bv Willow
The hydroponic solution and tissue extraction results, combined with I5N analysis of tissues,
indicate uptake and assimilation of ferrocyanide and free cyanide by willow plants. The tissue
extraction results show the presence of complexed cyanide within leaf tissue. Cyanide could be
present due to parent transfer to leaf tissue or complexation with existing metals with the aerial
tissue. Comparison of tissue extraction results with the tissue ISN results shows a discrepancy
between the complexed cyanide and the amount of cyanogenic nitrogen in the plant tissue for
stem and leaf. The root tissue results suggest that the cyanogenic nitrogen present in the root
tissue remains in the complexed form, likely iron cyanide. Further investigation of the sorption
of ferrocyanide to stripped root tissue revealed that 18% of cyanide is sorbed to cell wall material
while the remaining 82% is transported across the cell membrane into the root cells. The uptake
of complexed cyanide into the root along with the difference between cyanogenic nitrogen and
extracted cyanide in aerial tissues suggests that ferrocyanide is being broken down by the plant
and assimilated. The free cyanide results, in agreement with previous studies, support the
assimilation of free cyanide by plants as solution cyanide concentration decreased significantly
and the discrepancy between extracted cyanide and 15N results existed for all KCN-exposed
tissues.
As expected, the uptake and metabolic rates for free cyanide were much higher than the
corresponding constants for ferrocyanide. The applicability of active uptake kinetics supports
the plant uptake of cyanide as a nitrogen source from the surrounding solution.
For the
ferrocyanide system, dissolved FetCN^4" remained in the hydroponic solution while almost all
of the cyanide in the KCN treatment was removed. The increased uptake and assimilation for
167
the free form may explain the discrepancy in the mass balance results. Only 60% of the initial
cyanide in the KCN system was recovered while 100% of that in the ferrocyanide system was
recovered.
Free cyanide volatilization was monitored during the design of the hydroponic
experiments and found to be negligible.
7.1.3
Model for Cyanide Uptake bv Willow: Model Development
The objective of the model was to represent the processes that affect cyanide fate within the
plant-solution system without over-specifying the system.
A balance between the model
complexity and the correspondence with the scale and amount of available data is necessary for
model utility.
A physiologically-based model was developed from mass balances for free
cyanide, ferrocyanide, and assimilate around each compartment in an idealized plant

representation. Model compartments were the solution and root, stem, and leaf tissue. To
include active uptake and represent the ability of the plant to regulate uptake into tissue, the root
was separated into the interstitial space (outside the endodermis) and the root interior.
Advection, diffusion in solution, cell wall adsorption, plant-mediated dissociation and
assimilation, active uptake across the root endodermis, and free cyanide and assimilate
volatilization represented the mass transfer and reaction processes included in the model.
Control losses were accounted for using the solution data control hydroponic pots not containing
plants.
A first-order, reversible loss process was assumed.
volume were represented as quadratic functions of time.
Transpiration and the solution
The physiologically-based model
included descriptions of the uptake, movement, and reactions involving cyanogenic nitrogen
within the plant water system. The model considers seven compartments and consists of 27 mass
balance equations and 17 adjustable parameters.
168
7.1.4
Model for Cyanide Uptake bv Willow: Application to Experimental Data and Calibration
The model was fitted to the experimental hydroponic data to gain insight into parameter
importance. An optimization algorithm was employed to determine optimal parameter values.
Optimal solutions were found to be dependent on initial parameter values, reflecting local
minima, so randomly-generated sets of initial parameter values (n = 500) were used to sample
the decision space. The resulting sets of optimal parameter values were evaluated versus the
objective function and also relative to the original assumed uniform distribution. The maximum
uptake rate for free cyanide,
and the ratio of ferrocyanide cell wall adsorption to
desorption, cc/ f f c', approached constant values, indicating importance relative to the model
outcome.
None of the other parameters displayed a significant importance relative to the
objective function. However, adsorbed free cyanide in the cell wall compartment constituted
<0.01% of the initial cyanide in the system at Day 20 while ferrocyanide adsorption represented
only 0.04%. Free cyanide volatilization accounted for <0.01% of the cyanide mass. Cell wall
adsorption and free cyanide volatilization are not important processes affecting cyanide fate in
willow.
Free cyanide sorption and volatilization were removed from consideration, three
parameters, while ferrocyanide adsorption was changed to an equilibrium relationship from the
rate-dependent process, a reduction of one parameter.
Subsequent analysis with the reduced model (13 parameters) provided equally good fits of the
hydroponic experimental data.
An uncertainty analysis with the 13-parameter model was
conducted to investigate the influence of data variability on the uncertainty in the optimal
parameter values.
observations.
This analysis was conducted by bootstrapping the residuals of the data
The sensitivity of the predicted cyanide compartment concentrations to the
169
variability in the simulated data was characterized by the mean and standard error (n = 119).
Only e f N and A ^ were not significantly different (a = 0.05) from the mean predicted by the
optimal, 13-parameter solution. All other process parameters were significantly sensitive to the
uncertainty reflected by the data variability. The data uncertainty was also reflected in the
significant variability associated with the compartmental mass fractions of cyanide.
As expected, the uptake and plant-mediated metabolic rates for free cyanide were higher than
those predicted for ferrocyanide. Free cyanide active uptake rate constant estimates support the
inclusion of active uptake kinetics, supporting plant use of cyanogenic-N as a nitrogen source.
Those for ferrocyanide do not. Active uptake kinetics may not be applicable for ferrocyanide. A
check of the uptake being only a function of the transpiration flow showed that free cyanide
required plant-mediation for uptake and ferrocyanide required exclusion at the endodermis rather
than unimpeded flow with the transpiration stream. A combination of the two is likely necessary
for ferrocyanide.
Predicted tissue concentrations for the optimal solution underestimated the assimilate
concentration in root (KCN system) and stem (both systems) tissue. Combined with the under
prediction of free cyanide uptake, these results suggest potential for phloem redistribution. Re
circulation of assimilated product back to the root tissue from the leaves would allow for the
buildup of higher concentrations in the root and stem such that the leaf does not act as a sink.
170
7.2 Engineering Implications*!)
A simple engineering assessment was performed for phytoremediation using the uptake data
from the hydroponic experiments. The size and planting density of an existing pilot wetland
system were used to determine the critical concentration that the wetland could handle. Each
plant was assumed to be 1 kg fresh weight. Plant spacing was I 'x l and the planting density in
the wetland was 75% of the plan area. The uptake for ferrocyanide and free cyanide was
calculated from the total amount of cyanide taken up over the 20-day experiment. Assimilate
concentrations were considered to provide better estimates as the cyanide assimilated is
completely remediated and not just being stored with the root tissue. The critical concentration
was calculated by a simple balance on the cyanide within the system. The calculations were
based on an assumed, fixed planting density. The planting density could be adjusted relative to
the assumed
kg fresh weight per ft* of treatment area, which is not necessarily appropriate fora
willow plant. Additionally, without the inclusion of surface volatilization, the wetland system
can be considered to be either a submerged or surface treatment.
The results (Figure 7.1) show that the particular test wetland could handle a ferrocyanide input
concentration less than 0.2 ppm as CN and a free cyanide input concentration of 1.2 ppm as CN
for a hydraulic residence time (HRT) of 5.1 days through the 3,487,800 L volume. These values
are in the range of water concentrations present at many contaminated sites. The estimated
maximum input concentrations are conservative, in that cyanide uptake by plants is only one of
many loss processes for cyanide within a wetland system as depicted in Figure 7.2. Cyanide can
also be potentially removed from the system by volatilization, adsorption, absorption,
10 with Rajat Ghosh
171
precipitation, or biodegradation. In addition, the removal of ferrocyanide may be accelerated by

photo-dissociation, converting cyanide into a much more reactive form. If possible within the
wetland loading flow constraints, the HRT could be increased to provide more cyanide removal
from the system.
The critical uptake of ferrocyanide within the treatment system ignores the potential for photo
dissociation. If photo-dissociation were considered, the potential uptake would be similar to that
for the free cyanide uptake. The potential ferrocyanide removal would be limited by free
cyanide plant uptake, not photo-dissociation.
The estimation also assumes continual treatment. Seasonal dependencies can be accounted for
by scaling the absolute mass of cyanide removed by the system with the fraction of time the
system is active. During winter dormancy, the abiotic loss processes (i.e. adsorption and solid
precipitation) would dominate removal of cyanide from the aqueous phase.
7 J Future Considerations
The data obtained indicate ferrocyanide uptake and conversion within willow plant tissue.
Additional testing of the plant fractions to determine the fate of cyanogenic nitrogen within the
willow would provide additional evidence for ferrocyanide uptake and assimilation. This study
found that willow plants have the ability to take up complexed cyanide and to mediate the
dissociation of the complexed cyanide to obtain the cyanogenic nitrogen. The volatilization of
dissociated free cyanide appears to be very low (below detection) and the treated aerial plant
172
tissue does not accumulate cyanide. Both findings support phytoremediation with willow as a
promising remediation technique for ferrocyanide-contaminated systems.
Analysis of the amino acid fraction within plant root, stem, and leaf tissue did not account for all
of the assimilated cyanide (Chapter 4). A significant increase was measured for KCN-exposed
tissues with a non-significant increase observed for FefCN^-exposed tissues. Even for the
KCN-exposed tissues, however, the amount of cyanogenic ,5N present in amino acids was 0.1%
of the total tissue content. This suggests that the assimilated ferrocyanide and free cyanide is
either past the amino acid fraction (and in proteins) or has yet to reach the amino acid fraction.
Further testing of the plant tissues would be beneficial to determine the process by which the
plant is assimilating the cyanide. Analysis of the various nitrogen pools within the plant is
required to locate the fate of cyanide within the willow. Additional investigation of the uptake
processes for both free cyanide and ferrocyanide would elucidate the ability of the plant to use
the cyanogenic N or Fe as a nutrient source.
The mass balance for the free cyanide system only recovered 60% of the initial cyanide added to
the system.
A process, assumed to be assimilate volatilization for the process model, was
missing from the mass balance. To represent the system accurately, all of the relevant processes
need to be known and measured, enabling closure of the mass balance. Free cyanide was not
detected being released from leaf tissue. Future work should examine the potential loss of
cyanogenic nitrogen from the system, e.g. via assimilate volatilization. Free cyanide may be an
important process under specific field conditions and can be included in the model to assess the
potential exposure due to release from plant tissue via volatilization.
173
The important interactions impacting the uptake of chemicals into the plant occur at the root
level. In the current analysis, the root was examined at the same scale as the other tissues. In
order to focus on the critical processes, they need to be separated and examined individually.
Root-level experiments separating the root from the rest of the plant would allow delineation of
individual processes within the root. In pulse-trace experiments, a pulse of labeled cyanide
would be injected into solution and then the movement of the chemical would be traced as it
moves through the plant root system.
By examining the labeled concentrations within the
various compartments, the time until breakthrough, and the shape of the concentration vs. time,
the rates of chemical movement between the various compartments could be determined. The
usefulness would be that the movement of chemical into and out of compartments such as the
root could be singled out for determination within the model. A pulse-trace test with >5N would
permit determination of the movement within the apoplasm, symplasm, and shoot (Lasat et al.,
19%). Other root-level experiments could be carried out at low temperature to study the transfer
across the root wall and the movement through the apoplasm. Low temperatures minimize the
active uptake of chemical into cells in an attempt to remove biological transfer processes from
the system (Hart et al., 1998). Again, l5N content in the stem xylem would be collected and
measured to determine the flux through the root. The final root-level experiment would involve
the most active section of the root concerning uptake of water and nutrients, the root tip. The tip
is comprised of the first 5 mm from the tip where the xylem and Caspian strip are not fully
developed. The issue is whether the root tips account for a disproportionate amount of water and
nutrient flow into the plant compared with the rest of the root. If the root tip accounts for the
majority of nutrient uptake, the issues pertaining to apoplastic and symplastic flow are
negligible. Nutrients will move passively with the water flow into the root and move towards the
174
beginnings of the root xylem as it forms. Passive leakage at the root tips would be particularly
important for the uptake of ferrocyanide based upon the larger molecular size and charge.
Investigation of uptake at the root level may provide insight into whether ferrocyanide is taken
up actively and can be described by saturation kinetics. Additional modeling may help answer
that question. Just like in the laboratory experiments, the model could be separated into two
sections. Uptake of cyanide when the root is separated from the rest of the plant is a function of
just the active uptake parameters.
Separating the root tissue specifically targets the uptake
parameters as the only unknown parameters affecting cyanide removal from solution.
Another extension of the model would be to include phloem redistribution. As cyanogenic

nitrogen is assimilated, the fraction of 1SN in the regular plant nitrogen cycle increases. The
chance of losses from the system occurring increases with the amount of cyanide assimilated.
The stable isotope is diluted across all of the plant compartments. In the model as currently
formulated, assimilated nitrogen can only continue to concentrate in the leaf tissue. Movement
of i5N from the leaf towards the root tissue releases the pressure within the model to fit the leaf
tissue assimilate concentration. With the addition of a route for assimilate to leave the leaf
compartment, the movement into the compartment could also increase. This would serve to
increase the amount of cyanide assimilated, which is then distributed throughout the system. As
all of the tissue compartments are included in the model, the increased assimilate will
preferentially distribute to the root and stem tissue. The inclusion of phloem re-distribution
affects the assessment of the leaf ferrocyanide metabolism. Increasing assimilate re-circulation
175
would allow leaf metabolism to increase, suggesting the potential for photo-dissociation of
ferrocyanide within leaf tissue.
Additional processes are important for field-scale applicability of the model to a

phytoremediation system. Phloem re-distribution and free cyanide volatilization were addressed.
Plant growth and cyanide solution chemistry are also important. Plant growth serves to dilute the
existing concentrations within the plant tissue over time. Increasing plant size also increases the
transpiration rate within the treatment system. A separate measure of respective plant growth
under similar conditions to the actual system is required prior to inclusion in the model. Cyanide
speciation, particularly with respect to the actual chemistry at the site of interest, needs to be
adjusted for the existing conditions.
Cyanide precipitation, compiexation, and adsorption
(Appendix A) within the groundwater add abiotic loss processes that affect cyanide fate within
the system.
Each cyanide species interacts with plants differently.
The respective uptake
parameters for each of the species present at the contaminated site must be investigated
separately. Rate constants for cyanide species uptake as well as cyanide solid formation and
adsorption are also necessary.
Active uptake processes and phloem redistribution are two processes within the model that can
be examined more closely using the available data to determine the level of importance
associated with each of the model processes. Regionalized sensitivity analysis (RSA) provides a
means to separate each parameter distribution into two conditional distributions of the
parameters given the output result of the model (Spear et al., 1994). The two conditional
parameter distributions for the good and bad sets are compared to determine if a significant
176
difference exists, signifying parameter importance. Unnecessary parameters are eliminated from
the model.
The cyanide uptake model has been calibrated using the existing data. Additional analysis such
as RSA can further define the parameters related to cyanide fate in a phytoremediation system.
Validation of the model must be performed using a second set of uptake data prior to field-scale
application. Extension of model to the field scale involves the addition of groundwater flow and
cyanide solution chemistry within the groundwater. The loss processes that were ignored in the
Section 7.2 evaluation must be included to assess cyanide fate at the field scale.
The final recommendation for future work is a field-scale test application. Without evidence of
removal on a large scale under environmental conditions, the true effectiveness of
phytoremediation of cyanide remains in doubt. Laboratory results and experimental calculations
based on laboratory results strongly suggest that willows are able to take up and convert
ferrocyanide to assimilation products. Field-scale testing will also provide information on the
cumulative effect of the removal processes as depicted in Figure 7.2. Unforeseen benefits may
occur as part of utilizing the natural cyanide cycle. Incorporated in this is the development of the
ecosystem around the remediation facility. The general wellness of the ecosystem increases the
population affecting the natural cyanide cycle, increasing opportunities for removal.
177
7.4 References
Hart. JJ.; Norvell, W.A.; Welch, R.M.; Sullivan, L.A.; and Kochian, L.V.
(1998)
Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread and
Durum Wheat Cultivars. Plant Physiol. 118,219.
Lasat. M.M.: Baker, AJ.M.; and Kochian, L.V. (1996) Physiological Characterization of Root
Zn2+ Absorption and Translocation to Shoots in Zn Hyperaccumulator and Nonaccumulator
Species of Thlaspi." Plant Physiol. 112, 1715.
Spear, R.C.: Grieb, T.M.; and Shang, N. (1994) Parameter Uncertainty and Interaction in
Complex Environmental Models. Water Resour. Res. 30, 3159.
178
Figure Legends
Figure 7.1 Effectiveness of a wetland phytoremediation system based upon the uptake from
hydroponic uptake experiments with willows (Chapter 4). The y-axis is showing the total
amount of cyanide taken up by the plants normalized by the total amount of cyanide entering the
system. The critical value is 1.0 - the point at which the plants are taking up everything that
enters the system. The uptake calculated from the total amount accumulated in plant tissue over
the 20-day experiment. The critical concentration is based on a mass balance on the system.
Assumptions include (1) l x l plant spacing; (2) 75% surface coverage; and (3) each plant mass
is 1 kg fresh weight.
Figure 7.2 The diagram of a wetland phytoremediation system showing potential loss processes
for ferrocyanide and free cyanide. Potential loss processes include: Volatilization of free
cyanide; Uptake by plants; Biodegradation; Sorption to soil; Adsorption on
Fe(OH)3 s); and Formation of metal-cyanide solid. Ferrocyanide can also undergo photo
dissociation.
179
Total CN Uptake/Assimilation by Plants

Total CN Entering System
2.0
Ferrocyanide Uptake
* * * Ferrocyanide Assimilation
Free Cyanide Uptake/Assimilation
' Limit for Process Effectiveness = 1.0
IS
OS -
0.0
0.0
0.1
0.2
0J
0.9
1.0
1.1
Inflow Concentration (m g L*1 as CN)
Figure 7.1
180
1.2
1J
APPENDIX A
F e r r o c y a n id e ADSORPTION ON ALUMINUM OXIDES*1}
A b st r a c t
Ferrocyanide (Fe(CN)6*') adsorption on y-aJumina (Y-Al2 C>3<s)) and gibbsite (AI(OH)3 <sl) was
investigated over a wide pH range and various solid loadings.
Batch experiments were
performed using 100 mL solutions (I = 0.01 M NaCl) dosed with 1.0 mg L 1 Fe(CN)6'1' as CN.
Equilibrium adsorption-pH edges were developed for 0.3,0.6. 1.2, and 2.0 g/L y-A1;0?(S, and 25
g/L A1(OH)3 (s). Ferrocyanide adsorption increased as pH decreased, consistent with the general
pH dependence for adsorption of anions on oxide minerals.
Ferrocyanide adsorption on
A1(OH)3 ,s) was approximately 300 times lower than Y-ANC^s, due to the higher surface reactivity
of the y-A1:0 3 (s,. The adsorbed cyanide versus pH relationship was consistent for each at the
variable adsorbate-adsorbent ratios used. Ferrocyanide adsorption on y-AFO^, was significantly
greater than previously reported data for goethite (FeOOH<s,) and both Y-A1 : 0 3 (S, and FeOOH,s,
adsorbed ferrocyanide to a greater extent than Al(OH)3 <s).
The investigation showed that
ferrocyanide could adsorb significantly on aluminum oxides spanning a range of crystallinity and
properties, with the extent of adsorption highly dependent on pH, the solid cry stalline structure,
and associated surface reactivity.
(1>Coauthored with Dave Dzombak
182
Key words: adsorption, aluminum oxide, cyanide, ferrocyanide, gibbsite, iron oxide, adsorptive
concentration
Abbreviations: Al(OH>3 (S>- gibbsite; CN - cyanide; y-ANO^s) - gamma aluminum oxide; aFeOOH - goethite; LOI - loss on ignition; CNx - total cyanide
A .l Introduction
Adsorption on mineral surfaces often controls ion mobility in soil systems (Sposito, 1984) and
also reduces ion bioavailability and uptake efficiency by plants. Phytoremediation, the use of
plants for soil and groundwater remediation purposes, has been shown to have the ability to
remove chemicals from the groundwater in the plant root zone (Burken and Schnoor, 1997:
Thompson et al., 1999; Schnoor, 2002) including lead chelates (Vassil et al., 1998; Epstein et al..
1999) and ferrocyanide (Chapter 4).
Adsorption onto minerals in soil reduces the aqueous
concentration in the root vicinity and limits the ability of the plant to assist in reducing pollutant
mass in contaminated soil and groundwater (Schnoor, 2002).
Adsorption can also produce
contaminant-laden site soils containing adsorbed ions, providing a continual pollutant source via
chemical desorption.
Cyanide contamination of groundwater and soils is a problem at numerous industrial sites

including those relating to former manufactured gas plants (Theis et al., 1994) and to aluminum
production (Dzombak et al., 1996). Cyanide has often been observed to be mobile in soils and
aquifers (Alesii and Fuller, 1976; Ghosh et al., 1999a), but cyanide adsorption has been shown to
183
depend on the particular cyanide species and the mineral surface. In particular, previous research
indicates that ferro- and ferricyanide complexes, common cyanide forms in environmental
contamination scenarios (Ghosh et al., 1999b), can sorb on aluminum and iron oxides (Alesii and
Fuller, 1976; Renneit and Mansfeldt, 2002) but do not adsorb onto sand and gravel material at
neutral pH (Ghosh et al., 1999a). Adsorption experiments with individual aluminum (Cheng and
Huang, 1996; Cheng et al., 1999) and iron (Theis and West, 1986; Theis et al., 1988; Young and
Theis, 1997; Rennert and Mansfeldt, 2001) oxides have shown ferro- and ferricyanide cyanide
species adsorption, especially under acidic conditions.
The ferro- and ferricyanide species
(F e ^ N )^ and FefCNfo3') are anions that can adsorb onto oxidic minerals, with maximum
adsorption at low pH and a lower extent of adsorption at high pH. Iron cyanide adsorption
studies have been limited to equilibrium partitioning on y-AI:C)3(5) (Cheng and Huang, 1996;
Cheng et al., 1999), the iron oxide goethite (Theis and West, 1986; Theis et al., 1988; Young and
Theis, 1997; Rennert and Mansfeldt, 2001), sand and gravel (Ghosh et al., 1999a), activated
carbon (Theis and Young. 1998), and a few selected whole soils (Alesii and Fuller, 1976;
Rennert and Mansfeldt, 2002).
The focus in most studies has been on aqueous species
interactions with individual soil components.
Cyanide adsorption onto soils is dependent on cyanide speciation and soil mineralogy. Natural
soils contain a mixture of many minerals varying greatly in physical characteristics. Aluminum
oxides are ubiquitous soil components and important adsorbents in soil systems.
Gibbsite
(Al(OH)3 <S)) is the most common form of aluminum oxide found in soils (Sposito, 1984).
Calcination of the Al(OH)3 (s) alters the crystallinity to produce the more adsorptive y- -Al:0 3 (s,,
or activated alumina. In addition to changing the crystallinity, calcination also affects adsorption
184
by altering properties such as pore size, particle diameter, and surface area (Hudson, 1987;
Rouquerol et al., 1999) that can significantly affect the number of surface sites and surface site
availability (Morel and Hering, 1993; Levenspiel, 1972).
There are naturally-occurring
aluminum oxides intermediate in the surface properties between gibbsite and activated alumina.
The objectives of this study were to examine the sorption of ferrocyanide to two aluminum oxide
solids and to compare the sorptive properties of the investigated solids with previously reported
results based on solid mineralogy and solid surface properties. The study examined adsorption of
ferrocyanide to Al(OH)3 ,s) and a y-AhChu, solid with a larger particle diameter than previously
examined (Cheng and Huang, 19%). Batch equilibrium adsorption studies were conducted with
clean aluminum solids at pH ranging from 4 - 10. Solution samples were monitored to obtain a
measure of the adsorbed ferrocyanide fraction and to compare adsorbed concentrations between
the A1(OH)3 ,s) and y-A1 ;C>3 <s), providing a measure of the relative importance of crystalline
structure on ferrocyanide adsorption to aluminum oxide.
Comparison of the experimental
aluminum oxide data with the adsorptive results from previous studies for y-AhO^, (Cheng and
Huang, 1996) and goethite, a-FeOOH<S) (Rennert and Mansfeldt, 2001) show the relative
capabilities of a range of oxide minerals to adsorb ferrocyanide.
A 2 Materials and Methods
Batch studies were carried out to examine the adsorption of ferrocyanide (FetCNfo4') on two
aluminum solids, y-ALOs^ (28 - 48 mesh, 91.5% AI2 O 3 ; 8.0% LOI) and Al(OH)3 (s) (100 - 200
mesh, 65% AI2 O3 ; 34.7% LOI) were obtained from Alcoa World Chemicals (Port Allen, LA
185
facility). The particle size chosen for y-AhGte) was consistent with previous Ottawa sand
adsorption experiments (Ghosh et al., 1999a) but larger than that used in a previous y-AIzO^s)
study (Cheng and Huang, 1996) to allow for separation by settling. The small particle size for
Al(OH)3 <S), compared with that for the y-AbO^s), still settled sufficiently for gravity separation
(Table A.l). The y-AbO^s, was rinsed at least 4 times with deionized water followed by drying
overnight at 105C prior to use. The Al(OH)3 (s, was not washed prior to adsorption experiments,
as investigations showed no benefit with respect to sorption and mass loss of solid during
washing was a concern.
Stock 100 ppm Fe(CN)6'1' solution as CN was prepared by adding 270.7 mg K4 Fe(CN)6- 3H ;0 to
de-gassed with N2 ,g), 0.01 M NaCl electrolyte solution. Other than the initial degassing, no
additional attempt was made to exclude carbonate from the system. All sorption experiments
were conducted in 125 mL Erlenmeyer flasks with the desired amount of aluminum solid added
to 100 mL of electrolyte solution and permitted to equilibrate for at least 30 minutes prior to pH
adjustment. The pH of each flask was adjusted by drop-wise addition of 2 % HC1 or 0.02 M
NaOH to obtain a pH range of 4 to 10. The solutions were not buffered so as to not affect
adsorption to the aluminum solids.
At least two flasks were included for each pH value.
Following pH adjustment, the samples were shaken for 30 minutes at 90 to 160 rpm on an Orbit
Shaker from Lab-Line Industries, Inc. (Melrose Park, IL). After pH equilibration, 1 mL of 100
ppm as CN stock FefCNje4" solution was added to each flask providing an initial concentration of
1.0 ppm as CNf.
All Erlenmeyer flasks were covered with aluminum foil to prevent light
intrusion, sealed with rubber stoppers, and shaken at 90 to 160 rpm at 20"C (=2.0) with initial
time-course samples taken after approximately four minutes. After 33 hours, an 8 mL sample
186
pipetted from each flask, adjusted to pH >11 using 300 g L 1 NaOH, and stored in the dark at 4C
until analysis. Preliminary time-course investigations determined 33 hours to be an appropriate
adsorption equilibration time. For both solids, physical settling for 5 to 10 minutes before
sampling was sufficient to prevent solids in the cyanide samples. The samples were analyzed for
total cyanide (CNt) according to Standard Methods 4500-CN* C/E (APHA/AWWA/WEF, 1998).
The amount of FefCNfo4' adsorbed was calculated from the CNt mass in the solution at the
sampling time and the solid dose. After drawing off the samples, the pH of each flask was
measured using an Accumet model 15 meter with a standard combination pH probe (Fisher
Scientific, Pittsburgh, PA). The flasks were gently hand-agitated and given 60 seconds for
reading stabilization prior to pH measurement. The pH of the un-buffered solutions edged
towards the solid isoelectric point over the experiment but this did not prevent obtaining a stable
sample pH reading nor maintaining a sufficient pH range.
Control sets of three Erlenmeyer flasks each were examined at pH 3.7 and 9.5 with 1.0 ppm
FefCNfo4- as CN without aluminum solids to determine ferrocyanide losses from sources other
than adsorption to AhO**). CNt concentrations remained constant in the high pH control series
with recovery slightly above 100%. The low pH series exhibited a linear loss with time reaching
5% at 33.6 hours.
CNt losses were thus small and ignored in interpreting results of the
adsorption experiments. FefCNfo4' adsorption to aluminum solids occurs at low pH values and
reduces the proportion of dissolved FefCNfo4 to much less than 100% of the original CNt. With
the fraction of dissolved CN significantly reduced, the fraction of CN lost was considered to be
negligible ( 5 %) at the pH values where significant losses were observed in the controls. This
187
assumption agrees with previously reported losses from solution that have been shown to be
small excepting at very acidic pH values (Meeussen et al., 1992).
Adsorption curves for FefCNV*' were determined as a function of pH for Y-AhCbu) and
AI(OH>3(S,. Adsorption edges were produced for solid doses of 0.3, 0.6, 1.2 and 2.0 g/L yALQfcs) added to
mg/L Fe(CN)6'1' as
CN t -
As
Fe(CNV^ adsorption onto Al(OH>3 (s> had not
been examined previously, an appropriate solid dose needed to be determined for the pHdependent adsorption experiments. Three 125 mL Erlenmeyer flasks each were prepared as
previously described for 0.2, 0.6, 1.25, 7.5, 25, 75, and 200 g/L Al(OH)3 <s,. Each flask was
adjusted to an acidic pH value with the drop-wise addition of 2% (v/v) HC1 to maximize
FefCNfo4* adsorption. An Al(OH)3 (s) solid dose of 25 g/L was chosen to maximize adsorption
without experiencing mixing and pH concerns associated with higher solid doses. Experiments
to determine the adsorption edge for 1 mg/L Fe(CN)64 as CNT per 25 g L1 Al(OH)3 <s) were then
completed.
The solution Fe(CN)64' data for y-AhC^s) and Al(OH>3 (s> experiments were convened to
adsorbed concentration as a function of pH for comparison purposes. When convened, the data
for both solids collapsed to the same relationship as a function of pH regardless of the solid dose.
Previously reponed Fe(CN)6^ adsorption data (Cheng and Huang, 19%; Rennert and Mansfeldt,
2001) were also converted to compare the sorbed concentrations on y-AliC^s* and AI(OH)3(s)
with cited values for FefCNV*' on y-AhC^s) and a-FeOOH(s). The solid properties are provided
in Table A.I.
188
A J Results and Discussion
While the adsorptive characteristics of the y-ALO^s) and Al(OH}j(S) solids differed in both the
range of the pH edge and the adsorptive capacities, each displayed the typical adsorptive edge for
metal-oxide adsorption of anions (Morel and Hering, 1993). For the adsorption edges, the
concentration of cyanide remaining in solution was normalized by the initial cyanide
concentration in the flasks. As expected, y-AIO ^ s) and AlfOH)^, solids adsorbed ferrocyanide
at low pH with adsorption decreasing as the pH increased (Figures A.l and A.2).
Equilibrium adsorption edges for Fe(CN)64' adsorption to y-AhO^s, are given for adsorbateadsorbent ratios of 3.3, 1.7, 0.83, and 0.50 mg Fe(CN)64' as CN per g y-AhOj^ (Figure A.l).
The adsorption edge for y-Al:0 3 ,S) began at pH 7 and proceeds through pH 10. The slope of the
adsorption edge increased as the adsorbate-adsorbent ratio decreased.
The solid adsorbing
potential increased relative to the amount of cyanide present in solution while the pH at which
the adsorbed fraction asymptotically approached zero was maintained.
The adsorption edge for Al(OH)3 tS) was broader and ranged from pH 4 to 7 with a tail of low
values at higher pH (Figure A.2). The adsorptive capacity for a 25 g/L Al(OH)3 (s, dose was not
sufficient to adsorb all of the 1.0 ppm FefCNfo4' as CN added to the system, even under acidic
conditions. Adsorption only reached 48% at pH 4 and was <10% for pH >7. Although the solid
did not adsorb all of the cyanide in the system, the adsorption edge was as steep as that for 0.50
mg Fe(CN)64" as CN per g y-AkOs^), decreasing approximately 30% within less than a pH unit.
The incomplete adsorption and location along the pH curve of the adsorption edge resulted from
189
25 g/L Al(OH>3 (S) being a high adsorbate-adsorbent ratio for the solid adsorption potential.
Results obtained with higher Al(OH>j(S, doses of 75 and 200 g AKOH)*** L'1 while investigating
the optimal solid dose suggest an increased adsorption relative to pH. This would shift the
adsorption edge towards a higher pH range more typical for aluminum oxide.
However,
concerns over maintaining a wide enough pH range in the un-buffered solutions and inefficient
solid mixing prevented selection of a higher AI(OH>3 <s) dose.
Ferrocyanide adsorbed concentrations varied significantly between the two aluminum solids. For
y-Al2 0 3 (s), Fe(CN)64' adsorption reached 3.2 mg as CN g under adsorptive conditions (pH 6)
and decreased linearly with pH to 0.1 mg as CN g 1 by pH 9.5 (Figure A.3). The Fe(CN>64'
adsorption to Al(OH)3 <s, was 300 times lower than that for y-AhO^s, at pH 6. The adsorbed
concentration ranged from 0.002 mg as CN g'1 for pH >7 to 0.034 mg as CN g 1 for pH 3.5
(Figure A.4).
The adsorbed concentration remained constant over the pH range 7 - 10.
However, these values are very low and largely reflected variability in the low-level results for
the cyanide analytical technique. In calculating the sorptive capacity from the adsorption edges,
values were discarded for all data except for along the adsorption edge. An excess of sorptive
capacity existed at low pH as the adsorbed fraction asymptotically approached 100%.
The observed Al(OH)3<S) and y-AliCbts) adsorbed concentrations for ferrocyanide compared with
those previously reported for y-AhOs^) (Cheng and Huang, 1996) and a-FeOOH(S) (Rennert and
Mansfeldt, 2001). The adsorbed concentrations reported for y-AhC^s) (Cheng and Huang, 1996)
were plotted on Figure A.3 with the experimental y-AbC^s) results. The adsorbed concentrations
were significantly higher than those for the experimental y-AliC^s) reaching 10 mg CN per g y-
190
AlO^s) at pH 6. The results for a-FeOOH(s) were significantly below that for the experimental
Y-Al2 <>3<S) with an adsorbed concentration of 3 mg CN per g a-FeOOH(S) at pH 4 compared with
pH 6 for Y-AItQ**, (Figure A.5). Adsorbed concentrations for both solids approached zero at pH
9.5 and, as with the experimental aluminum oxide solids, the adsorbed concentrations were linear
with respect to pH. The initial solid loadings of CN on the solids differed (Table A.l) with 0.5 3.3 mg as CN g'1 for experimental Y-AhO**), 0.005 - 5.0 mg as CN g'1 for experimental
AI(OH)3 <S), 3.1-21 mg as CN g'1 for Y-AIÔ^s, (Cheng and Huang, 1996), and 3.9 mg as CN g 1
for a-FeOOH(S). However, the linearity of the adsorbed concentration-pH relationship was
applicable for each solid regardless of the initial dose, making comparison of the various results
possible. Regression analysis was performed for the ferrocyanide adsorption concentration data
for all four solids (Table A.2). At -0.810 mg g 1 and 7.58 mg g'1 respectively, the experimental
Y-AIjO^s, slope and intercept were significantly lower than those for Cheng and Huang (1996) yAlO^s,, -1.183 mg g'1 and 9.13 mg g'1 (P < 0.001; t-test), and significantly higher than those for
a-FeOOH<s), -0.520 mg g 1 and 5.07 mg g 1(P < 0.001; t-test).
The experimental adsorption results for Fe(CN)64' to the aluminum oxides, y-AbOs^ and
A1(OH)3(s),
were expected as Y-Ab0 3 <s) activated alumina was a calcined product designed to
have high sorptive capabilities. Gibbsite has a lower density of reactive surface sites compared
to Y-A12 0 3 (s) as water is incorporated in the Gibbsite molecular structure. Hydrogen bonding
occurs between the hydroxyls in the molecular layers and lowers the surface charge (Rouquerol
et al., 1999).
The calcination process used to produce Y-AbC^s) (T >1000C) alters the
molecular arrangement of Gibbsite by driving off water and creating gaps in the structure
191
(Hudson, 1987; Rouquerol et al., 2001). The physical alterations for y-AljO^s) are reflected in
the increased surface area (Table 1) and higher surface charge accompanying the shift in
crystalline structure.
The differences in the pH adsorption profiles with previously reported values for y-AhO^j) and
a-FeOOH,S) (Figures A.3 and A.5) are smaller compared with those between the two
experimental aluminum oxide solids. The reported ferrocyanide adsorptive ability of goethite
was lower than that for y-ANO^s, while the experimental y-AbOj^, adsorption was lower than
that previously reported.
Overall, ferrocyanide adsorbs to the greater extent on y-AljO^s,
followed by a-FeOOH^, and then AI(OH)3 (s). Differences in solid properties such as surface
area, particle size, and pore size affect the ability of the solid particles to adsorb FefCNfo4" in
addition to the crystallinity. Decreasing particle diameter or increasing the solid surface area
increases the adsorption potential of a solid. The differences in FefCNfo4" adsorption reflect the
differences in the solid surface properties, particularly between the experimental and previously
studied (Cheng and Huang, 1996) y-AhC^s) (Table A.l).
The particle diameter for the
experimental y-Al2 0 3 (s) was over 300 times smaller than that previously studied.
A.4 Conclusions
The experimental results show that adsorption of ferrocyanide (FefCN^4*) onto aluminum oxides
can be substantial, especially at pH<7, but varies significantly depending upon solid crystallinity.
Experiments conducted with y-AIiO^sj displayed adsorption edges for FefCNfe4* consistent with
those previously reported for anions on metal oxides as 100% adsorbed at pH <7 decreasing to
192
0% for pH >10. Decreasing the adsorbate-adsorbent ratio from 3.3 to 0.5 mg FefCN^4 as CN
per g y-AljO^s, shifted the edge towards higher pH. The adsorption percentage for experiments
with an initial loading ratio of 0.5 mg FefCNV*" as CNT per g AlfOH^s, only achieved 48%
adsorption at pH 4 with the adsorption edge ranging from pH 4 to 7. The ferrocyanide adsorbed
concentrations on AKOH)**) were 300 times lower than those for y-ANO^) at 0.01 versus 3.2 mg
Fe(CN)64' as CN g 1 for pH 6 . The reduced surface binding capability for Al(OH>3<s) resulted
from the increased interior hydroxylation and reduced surface charge reactivity associated with
Gibbsite.
Comparison of ferrocyanide adsorption on Y-A1i0 3 <s, and Al(OH)3 ,s) with previously reported
data for a-FeOOH<s) enables evaluation of the relative importance of these oxide minerals as
ferrocyanide adsorbents. While the adsorbate-adsorbent ratio varied among the solid studies, the
relationship between the adsorbed concentration and pH remained constant for each of the solids
regardless of the ratio. The adsorptive differences reflected the respective crystalline structures
as Fe(CN)64 adsorption on y-AhC^, was greater than on a-FeOOH<s>, with both solids adsorbing
Fe(CN)64' to a much greater extent than Al(OH)3 (s).
A.5 Acknowledgements
University. The authors wish to acknowledge the helpful comments and insight provided by S.
Geiger, R. Ghosh, and D. Nakles of The RETEC Group.
193
A.6 References
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Haz- Waste Res. Symp., EPA-60Q/9-76-015, U.S. EPA, Cincinnati, p213.
APHA/AWWA/WEF. (1998) Standard Methods for the Examination o f Water and Wastewater,
20* ed American Public Health Association, American Water Works Association, and Water
Environ. Sci. Tech. 31,1399.
Cheng, W.P., and Huang, C. (1996) Adsorption Characteristics of Iron-Cyanide Complex on yAI2O3.
J. Colloid Interface Sci. 181,627.
Cheng, W.P., Huang, C., and Pan, J.R. (1999) Adsorption Behavior of Iron-Cyanide onto yAI2 O3 Interface: A Coagulation Approach. J. Colloid Interface Sci. 213, 204.
Dzombak, D.A., Dobbs, C.L., Culleiton, CJ., Smith, J.R., and Krause, D. (1996) Proceedings:
Water Environment Federation, 6 $ h Annual Conference & Exposition. Dallas, TX, October 5-9,
Removal of Cyanide from Spent Potlining Leachate by Iron Cyanide Precipitation.
Epstein A.L., Gussman CD., Blaylock M J., Yermiyahu U., Huang J.W., Kapulnik Y., and Orser
C.S. (1999) EDTA and Pb-EDTA Accumulation in Brassica juncea Grown in Pb-Amended
Soil. Plant and Soil. 208,87.
Ghosh, R.S., Dzombak, D.A., Luthy, R.G., and Nakles, D.V. (1999a) Subsurface Fate and
Transport of Cyanide Species at a Manufactured-Gas Plant Site. Water Environ. Res. 71, 1205.
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Ghosh, R.S., Dzombak, D.A., and Luthy, R.G. (1999b) Equilibrium Precipitation and
Dissolution of Iron Cyanide Solids in Water. Environ. Eng. Sci. 16, 293.
Hudson, L.K. (1987) in Critical Reports on Applied Chemistry, Volume 20: Production of
Aluminum and Alumina (A.R. Burkin Ed.), p. 11. John Wiley and Sons, Inc., New York.
Levenspiel, O., (1972) Chemical Reaction Engineering, 2'/ Edition, p. 460. John Wiley and
Sons, Inc., New York.
Meeussen, J.L., Keizer, M.G., and de Haan, F.A.M. (1992) Chemical Stability and
Decomposition Rate of Iron Cyanide Complexes in Soil Solution. Environ. Sci. Technol. 26,
511.
Morel, F.M.M., and Hering, J.G. (1993) Principles and Applications o f Aquatic Chemistry, p.
509. John Wiley and Sons, Inc., New York.
Rennert, T., and Mansfeldt, T. (2002) Sorption of Iron-Cyanide Complexes in Soils. Soil Sci.
Am. J. 6 6 ,437.
Rennert, T., and Mansfeldt, T. (2001) Sorption of Iron-Cyanide Complexes on Goethite.

European J. Soil Sci. 52, 121.
Rouquerol, F., Rouquerol, J., and Sing, K. (1999) Adsorption by Powders and Porous Solids, p.
311. Academic Press, New York.
Schnoor, J.L. (2002) Phytoremediation: Technology Evaluation Report TE-02-01. GroundWater Remediation Technologies Analysis Center, Pittsburgh.
Sposito, G. (1984) The Surface Chemistry of Soils. Oxford University Press, New York.
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Theis, T.L., Young, T.C., Huang, M., and Knutsen, K.C. (1994) Leachate Characteristics and
28,99.
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., Iyer, R., and Kaul, L.W. (1988) Kinetic Studies of Cadmium and Ferricyanide
Adsorption on Goethite. Environ. Sci. Tech. 22, 1013.
Theis, T.L., and Young, T.C. (1998) Research on the Environmental Chemistry o f Cyanide.
Clarkson University, Potsdam, NY.
Thompson, P.L., Ramer, L.A., and Schnoor J.L.
(1999)
Environmental Toxicology -
Hexahydro-1,3,5-trinitro-l,3,5-triazine Translocation in Poplar Trees. Environ. Tox. Chem. 18,

279.
Vassil, A.D., Kapulnik, Y., Raskin, I., and Salt, D.E. (1998) The Role of EDTA in Lead
Transport and Accumulation by Indian Mustard. Plant Physiol. 117,447.
Young, T.C., and Theis, T.L. (1997) Proc. Water Environ. Fed. 7(fh Annu. Conf. Exposition. 3,
167.
196
Table A.1 Solid properties for aluminum and iron oxides investigated and compared. Product
specifications for experimental Al(OH>3 <s) and y-ANO^s) obtained from Alcoa, Inc. Referenced
information obtained either from cited paper or direct communication with the author(<>.
Property
Experimental
Cheng & Huang

(1996)
Rennert &
Mansfeldt
(2001)
Solid
Al(OH) 3
Y-A12 0 3
Y-AI2 O 3
FeOOH
Supplier
Alcoa, Inc.
Alcoa, Inc.
Japan Aerosol,
Co.
Produced in
laboratory
Pre-treated
No
Yes - Deionized
water
Y es-0.01 N
NaOH
Pressure
Filtration
Equilibration Time
(hr)
33
33
24
24
Filtered
No
No
Yes
(0.45 nm)
Yes
(0.45 pm)
0.3 - 2.0
2.5*
10
Solid Dose
(g L )
0 .2
2 0 0
Cyanide Dose
(mg L' 1 as CN)
1 .0
1 .0
7.8 - 62.4
39
Sorbate-Adsorbent
(mg g' 1 as CN)
0.005-5.0
0.5 - 3.3
3.12-21.0
3.9
dp
(pm)
75 -1 5 0
300-600
<I
NA
Surface Area
(m2 g'1)
0 .1
350
118
30
197
Table A.2 Regression results for ferrocyanide adsorbed concentration versus pH. Std Err
represents the standard error of the intercept (A) and slope (B) respectively. Regression for
Al(OH>}(s) obtained from data for pH <7.
Solid
Y-A1203
Cheng & Huang, 1995
Experimental
Al(OH> 3
Experimental
cc-FeOOH
Rennert & Mansfeldt, 2001
Intercept Std Eita
Slope
Std Errs
r*
9.131
7.580
0.814
0.513
-1.183
-0.810
0.139
0.061
0.923
0.877
0.057
0.006
-0.0083
0 .0 0 1 0
0.905
5.071
0.255
-0.520
0.040
0.971
198
Figure Legends
Figure A.1 Equilibrium pH-dependent adsorption edges at 33 hours for 1.0 ppm K4 Fe(CN> 6 as
CN and the respective experimental y-AhO**) solid doses in 100 mL 0.01 M NaCl solution.
Error bars reflect the standard error of replicates.
Figure A.2 Equilibrium pH-dependent adsorption edge at 33 hours for 1.0 ppm KiFefCN^ as
CN and 25 g Al(OH)3 <s) L 1 in 100 mL 0.01 M NaCI solution. Error bars reflect the standard
error of replicates.
Figure A J Adsorbed ferrocyanide concentration as a function of pH for experimental y-ANO^s,

and y-Al2 0 3 (s) studied by Cheng and Huang (1996). The adsorbate-adsorbent ratio ranged from
0.5 - 3.3 mg as CN g' 1 for experimental Y-AI2 C>3 (S) and 3.1 - 21 mg as CN g 1 for y-AljC^s)
(Cheng and Huang, 1996).
Figure A.4 Adsorbed ferrocyanide concentration as a function of pH for Al(OH)3 <s). The
adsorbate-adsorbent ratio range was 0.005 - 5.0 mg as CN g '1.
Figure A.5 Adsorbed ferrocyanide concentration as a function of pH for a-FeOOH(S) (data from
Rennert and Mansfeldt, 2001). The adsorbate-adsorbent ratio was 3.9 mg as CN g 1.
199
100
<
p-
75 -
u
s
50
s
es
>*
25 *
u
V
lam
03 g/L
o 0.6 g/L
1.2 g/L
V
2.0 g/L
&
<v
f
<1
<
T-
pH
Figure A.1
200
10
% Ferrocyanide Adsorbed to Al(OH)
100
80
60
pH
Figure A.2
201
10
eo
IS
eo
i-
10
Experimental
Cheng & Huang (9)
cp
c0
1
a
8
co
4)
o
es
L.
U
C
St
pH
Figure A.3
202
10
eo
BO
s
m
0.03 -
o
<
e
e
0.02
1
5
<
0.01
*9s
>
La
0.00
PH
Figure A.4
203
10
Ferrocyanide Adsorbed on a-FeOOH(s) (mg g*1)
0
6
pH
Figure A.5
204
10
A PPEN D IX B
C h e r r y T r e e C y a n id e S a m p l in g
Cyanide analyses were performed on cherry tree leaf samples and on surface soil collected from
beneath the cherry tree to determine potential contributions of cyanide to the surrounding
environment. Numerous plants, including cherry, have been shown to produce cyanide, either
directly or as a cyanogenic glycoside (ATSDR, 1997; Koster, 2001; Seigler, 1991). Cherry was
selected as an example of a cyanide-producing plant (Howe and Noble, 1985) due to recent
events involving foal abortions at Kentucky horse farms (Ohio State University, 2001;
Townsend, 2001). Foliage from such plants poses a possible source for cyanide contamination
either from direct consumption of leaves or through contributions to the soil cyanide levels after
decay.
Cherry leaf and surface soil samples from beneath the tree were analyzed for total and free
cyanide. The soil samples did not show a significant increase versus control soil samples.
Results may have been influenced by recent fertilizer application. However, as all soil cyanide
concentrations were low, fertilizer effect on total cyanide was discounted.
Leaf samples
analyzed by the technique developed in Section 4 detected low levels of cyanide. The 0.76
mg/kg wet weight concentration was slightly above the background level of 0.4 mg/kg wet
weight measured for the control willow plants (not exposed to any cyanide). With 0.36 mg/kg
205
wet weight increase, and assuming a range of 3 to 30 kg (wet weight) of total leaf mass, a cherry
tree would contribute approximately 1*10 mg total cyanide per year to the surroundings.
B.1 Materials and Methods
The leaf and soil samples were acquired from soil beneath a cherry tree in Southeastern
Pennsylvania when leaf litter was appreciable and presumably had an effect on the quality of the
soil. The cherry tree samples were taken on 11/11/01 from a sloped, grassy, residential yard.
Soil samples were collected from T upslope of the trunk, 5 downslope of the trunk, and from
another part of the yard unaffected by cherry leaf deposition. Leaf samples were taken fresh
from the tree. Each sample was preserved in airtight plastic containers and protected from light.
The leaf samples were crushed under liquid nitrogen and extracted in either 10% NaOH or
deionized water. Deionized water was used in place of enzyme-inhibiting caustic to determine
whether enzymatic breakdown of cyanogenic compounds was occurring.
An additional
extraction was also performed on chopped, fresh leaf tissue without crushing under liquid
nitrogen and with extraction in deionized water. Soil extraction was conducted with 10% NaOH
for 16 hours. All extracts were performed in duplicate and analyzed for total and free cyanide.
B.2 Results
The measured cyanide concentrations in the cherry tree soil sample and leaf samples are given in
Table B .l. The levels of cyanide in the soil samples were less than 1 ppm and close to that
measured in the control with most of the cyanide in the free form. A complicating factor,
206
discovered after the sampling, was the later knowledge of an application of general lawn
fertilizer approximately one month prior to sampling. Regardless, the samples still did not
exceed the background level as the control area was also fertilized. For the leaf tissue, only
extraction with 10% NaOH resulted in detectable cyanide. The concentration of 0.76 ppm was
only slightly larger than the background level of 0.40 ppm measured for willow leaves. The
samples were taken at a time reflecting potential effect of leaf litter on soil cyanide levels. With
0.36 mg/kg CNt in leaf tissue versus control values, the amount of total cyanide that cherry leaf
tissue would contribute to the surrounding soil is approximately 1-10 mg CNT assuming 3 to 30
kg (wet weight) of total leaf mass on the tree.
B J References
Agency for Toxic Substances and Disease Registry. (1997) Toxicological Profile fo r Cyanide.
Howe, M.; and Noble, D. (1985) Effect of Cyanide Residue on Vegetation Bordering a Black
Hills Stream. Proc. S. D. Acad. Sci. 64, 112.
Koster, H.W. (2001) Risk Assessment of Historical Soil Contamination with Cyanides: Origin,
Potential Human Exposure and Evaluation o f Intervention Values. RTVM report 711701019.
Rijksinstituut voor Volksgezondheid en Milieu (National Institute of Public Health and the
Environment). Bilthoven, The Netherlands.
Ohio State University. (2001). What Killed the Foals? Ornamental Plants Annual Reports
and Research Reviews 2001. Special Circular 186-02.
http://ohioline.osu.edu/sc 186/sc 186 20b.html.
207
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.
Townsend, L. (2001). Farm Contingency plant for MRLS Risk Reduction for Kentucky Horse
Farms: Eastern Tent Caterpillar Recommendations.
University of Kentucky Entomology.
http://www.ukv.edu/Agriculture/Entomoloev/bugaleit.htm.
208
Table B.1 Cyanide concentration in cherry tree soil and leaf tissue samples (mg/kg wet weight).
Approximately 2500 mg soil (wet weight) extracted in 100 mL 10% NaOH for 16 hrs. prior to
extract analysis using distillation and microdiffusion for total and free cyanide respectively.
Tissue ground with liquid nitrogen prior to extraction.
Soil
Leaf
Sample Location
Depth
Control
Surface
0.716
0.299
7 Upslope
Surface
0.357
0.262
T Upslope
3-7
0.872
0.270
5 Downslope
Surface
0.5%
0.289
Pretreatment
Extraction
Ground Under
Liquid Ni
Ground Under
Liquid N:
Chopped
Total Cyanide Free Cyanide
Total Cyanide Free Cyanide
NaOH
0.757
0.069
DI Water
NT)
0.059
DI Water
ND
0.041
209
APPENDIX C
P ea U pta k e Stu d y
Some preliminary cyanide uptake studies were conducted in December 2000 to test the
performance of the hydroponic experiment protocols at Southern Illinois University Carbondale
(SIUC) and the analytical protocols of Carnegie Mellon University (CMU). The common use
and rapid growth of pea plants in hydroponic systems were the reasons for their use in the initial
study. The pea uptake studies were performed to examine the uptake and distribution of free
cyanide and iron-complexed cyanide in pea plant tissue. The study was also conducted to
examine the effectiveness of caustic extraction in determining the cyanide content of plant tissue.
C.l Method
The plants were grown at SIUC with both aqueous and tissue analyses conducted at CMU. The
plants were grown from seed in a basal nutrient solution for three weeks prior to treatment. Four
plants were grown in each pot, with three pots each dedicated to solutions of ferrocyanide and
free cyanide containing 2 ppm as cyanide. The hydroponic solutions for pots 1-3 contained free
cyanide added as KCN while the solutions for pots 4-6 contained iron-complexed cyanide added
as potassium ferrocyanide, ICtFe(CN)6. The complete hydroponic solution chemistry was the
same as that for the willow uptake studies given in Table 3.1. The dark pots prevented light
intrusion to minimize conversion of ferrocyanide to free cyanide. Upon cyanide addition, the
210
pots were gently agitated for a half hour, after which the initial aqueous sample was drawn.
Aqueous samples were obtained from the hydroponic solution at 0, 4, and 7 days. Tissue
samples were taken at the end of the 7-day experiment, separated into root, stem, and leaf
components, and frozen for preservation prior to shipment to CMU. The root, leaf, and stem
tissue samples from the four plants grown in pot 1 and those grown in pot 4 were each combined
prior to shipment to form a composite sample for each tissue for both pots. These composite
tissue samples were used to gauge the free and complexed concentrations of cyanide within the
plant tissue relative to the detection limits of the analytical methods. Tissue leaching was
conducted using approximately 600 mg of tissue per 250 mL of 10% NaOH extract solution.
Plant tissue was extracted un-ground for 16 hours in 10% NaOH solution with constant mixing.
The extract was sonicated for 20 minutes before extraction and filtered prior to analysis using a
0.45 pm type HN filter. Aqueous samples and the tissue leachate were tested for free cyanide by
microdiffusion ASTM D 4282-95 (ASTM, 1998) and WAD and total cyanide by distillation
according to Standard Methods 4500-CNC/E and 4500-CN' I/E (APHA/AWWA/WEF, 1998).
C.2Results
The results of the aqueous sample analyses for the pea plant studies are given in Table C.l. Free
cyanide was removed from solution while iron-complexed cyanide remained relatively constant
over the experiment. The distribution of cyanide species in the samples reflected the added
species as solution for pots 1-3 contained free cyanide while pots 4-6 had high complexed
cyanide contents and low WAD and free concentrations. Approximately 2 mg out of 4 mg total
mass of free cyanide (i.e., 50%) was removed from the hydroponic solutions of pots 1-3.
211
Cyanide mass in the pea plant root and leaf tissue samples are given in Table C.2. Analysis of
root and leaf tissue leachate of the composite tissue samples from pots 1 and 4 identified
measurable quantities of total cyanide. However, the root and leaf tissue leachate total cyanide
concentrations were low enough such that the root, leaf, and stem tissues from the individual
plants in the remaining pots (2, 3, 5, and 6) were also combined to form composite tissue
samples of each respective tissue. Therefore, each root and leaf tissue sample analyzed for all
pots was a combined representation of the tissue from the four replicate plants grown in each pot.
The analysis of the pea plant tissue was not performed according to the method developed in
Section 4. Pea tissue was not homogenized, ground under liquid nitrogen, or ground under
caustic prior to extraction. The free, WAD, and total cyanide concentration results for tissue
extracts for pots 1 and 4 were significantly higher than the rest. Root total cyanide was higher for
ferrocyanide-treated pots.
KCN-exposed roots also contained significant concentrations of
cyanide. For the root tissue, the tissue cyanide speciation reflected the added form of cyanide.
Leaf concentrations were also higher for ferrocyanide-treated plants.
concentrations were much lower compared with the root tissue.
However, the
The higher cyanide
concentrations and water contents of the root tissue and visible evidence of hydroponic solution
frozen on the root tissue samples was suggestive that some of the cyanide detected in the root
tissue from pots 1-6 may have been contained in excess hydroponic solution on the exterior of
the root tissue.
The cyanide concentrations in the leaf tissue were very small. Control samples of pea root and
leaf tissue were extracted to determine if the cyanide levels detected are above the background
levels associated with the breakdown of plant root, leaf, and stem tissue under such harsh
212
conditions as 10% caustic leach. Control pea root concentrations were non-detect while control
pea leaf background measured only 0.26 ppm - wet weight. Background concentrations for the
willow root and leaf tissue were also significantly lower than the total cyanide results for the pea
root tissue. However, the background leaf level was comparable to the leaf tissue results for all
but 2 plants. Therefore, the cyanide concentrations measured in the pea root tissue can be
assumed to be significant while pea leaf tissue is close to background levels.
C J Summary and Conclusions
The results suggest that free cyanide was more readily taken up compared with iron-complexed
cyanide and that pea plant mediation of iron-complexed cyanide uptake is uncertain.
The
relatively constant ferrocyanide concentration in the hydroponic solution does not equate to zero
uptake of ferrocyanide by the pea plants as ferrocyanide-exposed tissue contained higher cyanide
levels compared with those exposed to KCN, though there was small cyanide mass in plant tissue
relative to hydroponic solution in both cases. Such a result could be caused by either the short
experimental time frame (7 days) or different uptake characteristics of pea plants compared to
willow. Detection of uptake could also be influenced by the low tissue concentrations relative to
hydroponic solution concentrations. The specific results obtained in the pea plant studies are not
critical, as these studies were conducted only for testing of experimental and analytical protocols.
Previous hydroponic studies by Kreitinger (1998) and Reeves (2000) have already shown the
ability of willow to remove iron cyanide from hydroponic solutions. Further, Reeves (2000)
demonstrated, with the use of 15N-labeled iron cyanide, assimilation of iron cyanide into willow
plant tissue.
213
The cyanide levels detected in the plant do not account for the total cyanide mass removal from
the hydroponic systems. A comparison of the cyanide mass in tissue and lost from solution over
the 7-day experiment is given in Table C.3. The largest amount present in the plant tissue was in
the root tissue from pot 4 at 0.2 mg. The total cyanide concentration in the 2 L solution for pot 4
decreased from 3.7 to 2.9 mg as cyanide for a total mass removal of 1.6 mg. The free cyanide
hydroponic solutions exhibited decreases in cyanide mass of 2.0 mg with cyanide content in the
tissue for pot 1 accounting for only 0.04 mg of cyanide. Pots I and 4 reflected the highest
concentrations measured in the plant tissue. The majority of tissue cyanide mass was contained
in the root tissue. Stem tissue was not analyzed. However, the other tissue results suggest that
the stem will not contain significant quantities of cyanide.
Solution cyanide loss is greater for the KCN-treated pots. However, tissue concentrations for
these pots are less than the ferrocyanide-treated pots.
This suggests that free cyanide is
metabolized within the pea plant tissue. The pea plants may also metabolize ferrocyanide. The
variability associated with the solution and tissue concentration results prevents the drawing of
conclusions concerning uptake. However, the presence of cyanide within the plant tissue for
ferrocyanide treatment suggests that ferrocyanide is being assimilated by the pea plant, but at a
much slower rate compared with free cyanide.
214
C.4References
APHA/AWWA/WEF. (1998) Standard Methods for the Examination of Water and Wastewater,
20* ed, American Public Health Association, American Water Works Association, and Water
ASTM. (1998)
Standard Test Method for Determination of Free Cyanide in Water and
Kreitinger, J.P.
(1998)
Final Report: Investigation o f Phytoremediation of the
Hexacyanoferrate Ion by Plants. Final Report submitted by the Boyce Thompson Institute for
Plant Research and Remediation Technologies, Inc., to Alcoa. March 17, 1998. Ithaca, NY.
Reeves, M. (2000). Treatment of Fluoride and Iron Cyanides Using Willow: A Greenhouse
215
Table C.1 Pea plant hydroponic solution cyanide concentration and speciation.'~
12^ mM ItiFeiCN)*
75 mM KCN
Cyanide Concentration (ppm)
Cyanide Concentration (ppm)
Free
Sample3
Total
Free
WAD
WAD
Sample3 Total
1-1
1-2
1-3
2.391
1.926
1.473
2.507
1.972
1.543
2.577
1.679
1.462
4-1
4-2
4-3
1.855
1.557
1.639
0.028
0.028
0.026
0.012
0.015
0.012
2-1
2-2
2-3
2.216
1.415
1.159
2.332
1.601
1.182
2.263
1.218
1.045
5-1
5-2
5-3
1.606
1.606
1.772
0.008
0.040
0.069
0.006
0.004
0.016
3-1
3-2
3-3
1.833
1.333
0.904
1.879
1.740
0.892
1.827
1.160
0.705
6-1
6-2
6-3
1.142
1.241
1.371
0.026
0.055
0.037
0.012
0.006
0.008
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CN'C/E and 4500-CN I/E. and Free Cyanide
as per ASTM D 4282-95.
: ND = Not Detectable. Detection Limit for Total and WAD Cyanides = 0.001 ppm. for Free Cyanide =2.50 ppb.
NA = Not Applicable
3 Samples are listed according to pot and time sequence. The pot is given first with the time sequence numbered for
0 Days - 1 . 4 Days - 2. and 7 Days - 3. Therefore, sample 5-2 is for Pot 5 at day 4.
216
Table C.2 Pea plant tissue cyanide content after 7-day exposure.
Pea
Pot
Tissue Cyanide Concentration (ppm-wet wt)u

Leaf
Root
Free
WAD
Total
WAD
Free
Total
1
2
3
4
5
6
13.9
3.4
3.8
64.8
25.2
28.4
9.4
3.1
3.0
5.2
0.5
0.1
6.3
4.0
3.1
0.1
0.2
ND
3.8
0.3
ND
9.8
0.6
0.3
1.4
0.5
ND
1.0
0.1
0.3
ND
0.3
ND
ND
ND
ND
Control
ND
NA
NA
0.26
NA
NA
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CN'C/E and 4500-CN' I/E. and Free Cyanide
as per ASTM D 4282-95. ND = non-detect; NA = not applicable
: ND = Not Detectable. Detection Limit for Total and WAD Cyanides = 0.001 ppm. for Free Cyanide =2.50 ppb.
3 Tissue samples for each pot are a composite o f 4 plants. Samples extracted with 250 mL of 10**- NaOH solution.
217
Table C J Mass balance on cyanide in pea plant uptake study.
Pea
Pot
1
2
3
Root
0.029
0.009
0.012
4
5
6
0.221
0.092
0.105
Mass Cyanide (mg)1

Tissue
Solution
Total
Loss
Leaf
0.041
0.012
2.062
0.010
0.001
2.293
0.012
1.998
ND
0.042
0.003
0.002
0.263
0.095
0.106
0.831
0.101
-0.124
1 For solution loss, initial and final cyanide mass calculated from the total cyanide concentration and multiplying by
the volume o f the aqueous solution. Cyanide mass in pea tissue calculated by multiplying concentration (ppm-wet
weight) by the wet weight o f the tissue. Initial volume adjusted by the transpiration for the free (154 mL) and
complexed (244 mL) assuming linear removal o f water from the system.
218
APPENDIX D
HYDROPONIC SOLUTION AND SYSTEM DEVELOPMENT
Study of the geochemical and biological fate of cyanide is made difficult by the complex
chemistry of cyanide in water. Free cyanide (as HCN) is potentially volatile and represents both
a health risk and a potential loss from the system mass balance. In addition, cyanide (as CN or
HCN) is subject to microbial degradation, which may lead to additional unaccounted losses of
CN from solution. Cyanide can also complex with cations or other constituents in solution,
forming complexes and precipitates that vary in solubility.
resistance to biodegradation.
This complexation provides
Although highly resistant to microbial attack, iron cyanide
complexes are photosensitive and release free cyanide upon photodissociation for microbial
degradation, plant uptake, or chemical interactions with ions in solution.
Prior to conducting the hydroponic uptake experiments, the solution chemistry and system setup
required evaluation to optimize the potential for monitoring uptake of cyanide from solution and
to control cyanide speciation in solution. The hydroponic solution chemistry was examined with
the aid of a chemical equilibrium model, MINEQL+ (Schecher and McAvoy, 1994), to maintain
a concentration of 2 ppm as total cyanide dissolved in the complexed form over the duration of
the experiment. The hydroponic solution chemistry eventually chosen for the iron cyanide
experiments is predicted by equilibrium calculations to form cyanide solids at the conditions
given in the experiment. However, the slow dissociation kinetics for dissolved ferrocyanide in
219
the dark inhibit solid formation and enable a maintenance of 2 ppm CNt in the solution over the
duration of the experiment.
Additional concerns with the hydroponic system design and
functionality were investigated individually to assess their effectiveness. The design of the
hydroponic experimental system, including the use of black pots with bag liners fitted with
sampling ports, was selected to minimize volatilization and photodissociation losses while
accounting for transpiration and the need to obtain samples. Leak tests, chemical trial runs, and
apparatus sorption experiments were conducted to check potential for volatilization and solute
losses that were found to be small. Checks of plant-mediated volatilization of cyanide using
picrate paper and alkaline traps also indicated non-detectable volatilization of cyanide.
D.l Introduction
Since the previous studies by Kreitinger (1998) and Reeves (2000) involved an initial
investigation into iron cyanide uptake by plants, each used a relatively simple hydroponic
technique. The studies based on these methods generated results suggestive of iron cyanide
uptake. Reeves (2000), for example, observed a 500- to 750-fold increase in I5N enrichment in
willow leaves following FeCN exposure. However, since these studies did not control or analyze
for cyanide speciation, microbial contamination, and light exposure, it is not clear if this
enrichment represents plant uptake of FeCN or plant uptake of some dissociation or degradation
product of FeCN.
Confirmation was necessary to evaluate the efficacy of iron cyanide
phytoremediation. The ability to definitively state the occurrence of cyanide uptake requires an
experimental approach that controls the variables described above so that the observed effects
can be more confidently attributed to plant uptake and metabolism.
220
The two primary elements required to achieve the necessary level of control involved a nutrient
solution in which cyanide speciation and solubility was controlled and a hydroponic uptake
system that minimized variables during the experiments. Initial equilibrium model formulations
were conducted using MINEQL+ (Scherer and McAvoy, 1994) to examine the stability of
cyanide within the proposed hydroponic system. Solution sampling was also investigated to
confirm the modeling results. Investigations were also carried out to determine the effect of
other potential cyanide losses from the system. Included in these were cyanide volatilization and
biodegradation.
D.2Cyanide Chemistry of Hydroponic Test Solution
The initial and recommended compositions of the hydroponic test solution employed in the
ferrocyanide hydroponic phytoremediation tests with willow are presented in Table D.l. The
recommended composition was selected to mimic the site conditions at the Harbor Point, NY
site, maintain cyanide in the complexed form within solution, provide the necessary nutrients and
pH buffers for plant growth, and also to deter cyanide precipitation and complexation. With the
aid of chemical equilibrium modeling using MINEQL+ (Schecher and McAvoy, 1994), the form
and concentration of the nutrient and buffer reagent salts were selected such that cyanide would
remain in the form FefCNV*" without significant complexation or precipitation. The prevention
of chemical precipitation during hydroponic studies was desired to insure that precipitation does
not contribute to the loss of cyanide from the system. Solid precipitation would complicate the
hydroponic solution chemistry and cause difficulty in distinguishing between cyanide uptake and
cyanide solid formation and sorption on the root tissue. Prevention of the formation of any other
221
solids, such as metal carbonates and hydroxides, was also of concern as precipitation decreases
nutrient availability and coats the willow roots potentially reducing cyanide uptake. Model
simulations investigated the effects of chemical species, chemical concentration, pH, and pe on
cyanide speciation, cyanide solid formation, and additional solid formation.
investigation examined the effect of
CN t
The main
and Fej on dissolved cyanide speciation and on the
formation of cyanide solids. Additional studies examined the effect of FeHEDTA addition on
the formation of hematite and chemical concentration reduction to prevent additional solid
formation.
D.2.1 Methods
Chemical equilibrium modeling calculations were performed to assess chemical speciation in the
proposed nutrient solution, and for adjusted nutrient solution recipes. These simulations were
conducted using MINEQL+ (Schecher and McAvoy, 1994), with the thermodynamic data from
Sehmel (1989) and Ghosh et al. ( 1999b) for the iron cyanide chemistry.
The addition of 6.0 x 10-4 M TRIS with 1.0 x 10' 3 M MES as a buffer to the basic hydroponic
solution achieved the desired pH of 6.0. The pH and pe of the system were adjusted as necessary
by explicitly setting values within MINEQL+. The total cyanide and iron concentrations were
fixed at different values. Cyanide was incorporated in the model as either as KCN or K4 Fe(CN)6 .
The Cl* and K+ varied with the amount and speciation of the iron and cyanide additions but did
not significantly alter the chemistry of the system.
222
Simulation of the initially proposed solution with 1.0 x 10 s M FeHEDTA and 4 x 10~* M KCN
showed that EDTA complexed a large portion of Ca, Zn, Ni, and Mn. Neither cyanide solid
formed.
However, hematite (FeOOH(S)), hydroxyapatite (CasOHCPCL^s)), and MnHPO^s)
formed. Both hydroxyapatite and MnHPO^s) formation were controlled predominantly by the
amount of total phosphorus in the system. Hematite accounted for 94% of the Fe(m) while over
99% of the cyanide was predicted to occur as HCN. Lowering the cyanide addition increased
EDTA4" complexation with Fe(III) but barely reduced the formation of hematite.
The effects of adjusting the cyanide chemical additions were investigated without EDTAîn the
solution to prevent complexation of the iron and other metals. KtFe(CN) 6 and KCN addition
ranged from 3.0 x 10' 5 to 0.1 M and 7.5 x 10 s to 0.001 M respectively. FeCl3 addition was
varied between 1.0 x 10 10 to 0.1 M when included as the source of Fej in an attempt to shift the
cyanide speciation in the system toward iron-cyanide complexes. Representative pH values were
6
, 7, and
with the pe varied between 3 and 12 for each pH setting. For each pH/pe setting, the
cyanide addition was adjusted over a range to establish the critical point for the formation of
cyanide solids. The cyanide speciation was also determined for this range.
The effect of EDTA4* on the formation of hematite, cyanide complexation, and metals
complexation was examined by increasing the addition of HtFDTA up to values > 1.0 x 10 2 M.
Suppression of other solids, notably hydroxyapatite and MnHP0 3 (s), was investigated by altering
NH4H1PO4, Ca(N 0 3 )2 , and/or MnS0 4 from the suggested values until the point where other
solids did not form.
223
D.2.2 Results
The trial simulations described previously resulted in the identification of a hydroponic solution
recipe that would yield the desired solution chemistry while meeting most of the specified
constraints.
The recommended hydroponic solution recipe is given in Table D.l.
Model
simulations indicated that for pH 6 -8 , equilibrium solubility of iron cyanides in the recommended
hydroponic solution given in Table D.l can only be maintained under conditions that are more
oxic, pe = 4-9, than those typically encountered in groundwater at MGP sites, pe = 2-4.
MINEQL+ calculations indicated that maintenance of dissolved cyanide concentrations similar to
the field range of 1-5 ppm is possible under specific pH and pe conditions as depicted in Figure
D.l.
Potassium ferrocyanide, [JCiFefCNfo], is the only iron source added to the system.
However, as shown in Figure D.2, the majority of the dissolved cyanide within the system is
predicted to be present in the free form except at pH 7 at high pe values or pH 8 . Additional
iron, an element normally included in hydroponic culture solutions, was eliminated from the
nutrient solution to help preclude the formation of cyanide solids.
Chemical equilibrium modeling of the hydroponic solution using MINEQL+ suggested that at a
total cyanide concentration of 2 ppm, precipitation might occur if the pe decreased sufficiently.
In initial experiments, the pe of the recommended hydroponic solution in Table D.l was already
below the critical pe for the formation of iron-cyanide solids. Also, as shown in Figure D .l, the
predominant form of the dissolved cyanide in solution is the free form rather than the desired
ferrocyanide. However, considering the short time frame of the planned hydroponic studies and
the very slow dissociation kinetics of ferrocyanide in the dark, this was deemed not a significant
problem. Dissociation of ferro- and ferricyanide occurs very slowly in the dark (Ghosh et al.,
224
1998; Meeussen et al., 1992a) so solid formation should not occur as the equilibrium conditions
predicted by MINEQL+ will not be reached. Possible solid formation in the hydroponic solution
is further complicated by the variability reported in some of the equilibrium constants for cyanide
reactions (Ghosh et al., 1999a; Meeussen et al., 1992b). Preliminary laboratory tests of the
proposed hydroponic solution given in Table D.l verified the prevention of iron cyanide
precipitation due to the slow dissociation kinetics.
[EDTA]t> I x 10' 2 M inhibited hematite formation (for [KCN] = 7.5 x 10' 5 M and [FeCls] = 1.0
x 10 3 M) by complexation with the Fe(III) but did not alter the primary form of dissolved
cyanide. The high relative EDTA concentration necessary to prevent hematite formation resulted
in the complexation of all of the metals in the system. The addition of such high levels of
EDTA4" altered the metal chemistry of the system without altering the chemical speciation of
cyanide to favor the complexed form.
Lowering total PO43' below the initially proposed value of 1 x lO- 4 M in the solution prevented
both hydroxyapatite and MnHPO^S) from forming. Hydroxyapatite remained dissolved with a
decrease to 1.9 x 10' 5 M while MnHPO^s) dissolved at 3.0 x 10- 6 M. Lowering the calcium and
manganese also functioned to prevent the respective formation of each solid and can be done to
an extent without changing the dissolved metal concentration. In the initially proposed solution,
3% of [Ca]T and 87% of [Mn]r was predicted to occur in solids. The total dissolved metal
concentrations did not significantly change upon decreasing [Ca]j to 3.9 x
1 0 '3
M and [Mn]r to
1.6 x 10*7 M to avoid solid formation.
225
DJHydroponk System Development
D.3.1 System Criteria

The hydroponic system for these uptake experiments had to meet several specific criteria if it was
to effectively address the study questions. The system had to provide an airtight root zone (to
preclude loss of cyanide to the vapor phase) and completely restrict entry of light (to prevent
photodissociation). The loss of solution due to transpiration had to be accounted for to preclude
formation of headspace in the pot that might also facilitate CN movement into the vapor phase.
However, the root zone had to be accessible so that the nutrient solution could be sampled. An
additional consideration was the potential for cyanide volatilization by plant tissues.
Researchers, such as Burken and Schnoor (1998), have developed systems that meet several, but
not all, of these criteria as shown by Figure D.3. This system provides the root enclosure and
sampling capability, but not the control of light and solution level. The latter limitation can be
addressed with a polyethylene (PE) bag liner approach. The PE liner is placed in a container
with an airtight seal. As the plant decreases the solution volume during evapotranspiration, a
negative pressure is created which collapses the bag liner and maintains the solution level. This
approach typically uses a screw-type lid and glass jar to create the seal, a system with drawbacks
for the proposed research. Drawing on the merits of the above systems, a hybrid system was
developed to meet the necessary criteria.
226
D.3.2 System Design

The system utilized solid black plastic buckets with airtight snap-on lids. These pots have been
used previously for hydroponic experiments because they completely restrict light from the root
zone. The lids form an airtight seal that prevents entry or egress of air or solution. At several
points during the development of the system, submerged leak tests were performed to insure that
the system provided the isolated environment required for the experiments. In all instances
during design and testing, the system successfully maintained air-tight conditions to a depth of
20-30 cm. The pot lid was modified to provide a single large opening for a willow plant and two
smaller openings for sampling. One of the smaller openings served as an injections/sampling
port for a hypodermic syringe. The second smaller opening was fitted with a stopcock for larger
volume sampling. The fittings used in these smaller openings did not compromise the pot seal.
Willow plants were supported in the larger opening with a black plastic canister exactly the
diameter of the opening. The position of the willow was maintained in the canister with a foam
identi-plug. Prior to any given experiment, the space around the willow stem inside the canister
was sealed using a thick layer of 5% agarose (cooled to room temperature to avoid heat killing
cells in the willow stem) and a layer of parafilm to slow desiccation of the gel. This high
concentration agarose became almost plastic-like with time and provided a highly effective seal
as long as the pot was handled gently. Short-term shallow submergence tests (1-2 min with pot
lid slightly submerged) with the fully assembled system containing a willow again showed that
the integrity of the system was maintained. The negative tension generated in the system was
also evident.
When solution samples were withdrawn for analysis, there was a noticeable
resistance to solution removal, indicative of a negative pressure within the plant root zone. A
schematic of the complete system is shown in Figure D.4.
227
In the initial system design, solution sampling was to be provided by a tandem in-line flow cell
fitted with a pH (P-27003-22, Cole-Parmer Instrument Co, Vernon Hills, IL) and ORP electrode
(P-27003-60, Cole-Parmer Instrument Co, Vernon Hills, IL), providing a low-flow means of
talcing these measurements. A multiple stopcock system was envisioned, with a peristaltic pump
directing solution flow through this cell to preclude the need to open the pot. Pharmed tubing
was to be employed because it restricts UV light and does not sorb iron cyanide species (see
Section 3.3.3). This approach was considered before the PE bag liner had been integrated into
the system design and was subsequently abandoned. Extensive testing revealed that a stable
ORP reading could not be maintained within the cell as the readings fluctuated significantly even
at extremely low rates of solution flow. While it initially appeared that the electrode was faulty,
later comparisons to another ORP electrode (P-27006-25, Cole-Parmer Instrument Co, Vernon
Hills, IL) revealed that the electrode was functioning normally, but did not provide the same
readings, despite careful calibration of the electrodes. Given these and other problems associated
with this sampling approach, the introduction of the bag liner system, and time constraints, it was
decided that it would be more practical to collect solution samples by withdrawing a small
volume of solution from the syringe port to determine the solution pH and pe. This approach
was already in practice with this system as it provided a means to recover solution samples from
the system for the CN analyses. Thus, at each sampling point, a parallel aliquot was withdrawn
from the pot and used for pH and pe determination.
D.3.3 Hydroponic System Testing

As described above, submergence tests were used to demonstrate the seals in the system.
Samples of all materials used in the system were sent to Carnegie Mellon and tested for their
228
ability to sorb feni- and ferrocyanide, Fe(CN>6 3' and FefCN)*4'. The results as given in Figure
D.5 demonstrated that none of the components showed significant interactions with the iron
cyanide species and would therefore not affect the solution chemistry of the dissolved iron
cyanide.
The nutrient solution modeled by Carnegie Mellon (Section 3.2) was tested
experimentally in this system to insure that iron cyanides remain in soluble form.
No
precipitation on pot walls or plant roots was observed in the system. This result is in contrast to
those obtained in experiments with higher added total cyanide by Kreitinger (1998) and Reeves
(2000), who saw extensive precipitation of iron cyanides on all interior surfaces.
Solution
analyses of test solutions for the current study indicated that the added iron cyanides remain
complexed and that there were only slight shifts (<0.5 units) in pH and pe, regardless of whether
cyanide was added as potassium cyanide (75 nM) or ferrocyanide (12.5 jiM). Some dissociation
of ferrocyanide was observed, but this represented only a small fraction of the total ferrocyanide
added.
D.4Volatilizatlon of Cyanide by Plant Tissues
Three separate experiments were conducted to determine whether CN may be volatilized from
plants treated with cyanide or iron cyanide. Cyanide volatilization represents a regulatory issue
for phytoremediation of cyanide that may have to be addressed before field trials can be
performed. In one of these experiments, peas were used as a surrogate species, mainly due to the
ease with which they can be cultured hydroponically. In this experiment, a single primary leaf
was enclosed and picrate papers were used to monitor for volatilized cyanide. Alkaline picrate
undergoes a color change from yellow to red in the presence of cyanide. This red pigment can be
229
extracted and quantified colorimetrically. However, no evidence of cyanide volatilization was

obtained during a 10-day treatment period with cyanide or ferrocyanide. In a separate study with
willow, the entire willow shoot was enclosed and positive pressure used to direct the air volume
of the chamber into an alkaline gas trap. There was no evidence of cyanide volatilization.
However, technical difficulties associated with these enclosures left the data suspect, so
repetition of this experiment was warranted. In the third experiment, only the terminal 10-15 cm
of the shoot was enclosed, as this young tissue represents the most biologically active in the
plant. An alkaline picrate paper was placed inside for the duration of the exposure. While a
slight color change was observed over time, this did not appear to be due to CN volatilization but
rather to aging of the paper while in direct sunlight. To test this hypothesis, a parallel set of
picrate papers were exposed to sun under similar conditions in the absence of the plant. These
papers showed a similar color change. Absorbance of the eluted pigments was not significantly
different, indicating that the color change was not due to the presence of CN.
D^Summary and Conclusions
The hydroponic solution chemistry was modeled to select a recipe that will mimic cyanide
chemistry in solution, and also will prevent unnecessary cyanide loss from the system. Cyanide
was maintained in the complex form at a concentration representative of that at MGP sites.
Solution equilibrium modeling with MINEQL+ predicted cyanide solid formation at 2 ppm CNt
when added as ferrocyanide. However, the slow dissociation kinetics of ferrocyanide prevented
solid formation as corroborated with solution testing. The prevention of light enabled a cyanide
230
concentration of 2 ppm to be maintained in solution as complex cyanide over the duration of the
experiment.
Additional testing during the development of the hydroponic system design was aimed at
minimizing the potential for cyanide losses and optimizing detection of plant uptake of cyanide
from solution. Leak tests, solution trial runs, experimental apparatus sorption testing, and tests to
assess plant volatilization of cyanide were performed to investigate the effectiveness of the
hydroponic design.
All proved negative for cyanide loss from solution.
To prevent
photodissociation and account for transpiration, black pots with a bag liner were used.
D.6References
Burken J.G.; and Schnoor, J.L.
(1998).
Predictive Relationships for Uptake of Organic
Contaminants by Hybrid Poplar Trees. Environ Sci Technol. 32:3379.
Ghosh, R.S. (1998) Geochemistry, Transport and Treatment of Cyanide. Ph.D. Dissertation,
Carnegie Mellon University. Pittsburgh, PA.
Ghosh, R.S.; Dzombak, D.A.; Luthy, R.G.; and Nakles, D.V.
(1999a)
Equilibrium
Precipitation and Dissolution of Iron Cyanide solids in Water. Environ. Eng. Sci. 16:4, 293.
Ghosh, R.S.; Dzombak, D.A.; Luthy, R.G.; and Nakles, D.V. (1999b) Subsurface Fate and
Transport of Cyanide Species at a Manufactured Gas Plant Site. Water Environ. Res. 71:6,
1205.
231
Kreitinger, J.P.
(1998)
Final Report: Investigation o f Phytoremediation of the
Hexacyanoferrate Ion by Plants. Final Report submitted by the Boyce Thompson Institute for
Plant Research and Remediation Technologies, Inc., to Alcoa. March 17,1998. Ithaca, NY.
Meeussen, J.L.; Keizer, M.G.; and de Haan, F.A.M.
(1992a)
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. (1992b) Dissolution
Behavior of Iron Cyanide (Prussian Blue) in Contaminated Soils. Environ. Sci. Technol. 26:9,
1832.
Reeves, M. (2000). Treatment o f Fluoride and Iron Cyanides Using Willow: A Greenhouse
Schecher, W.D.; and McAvoy, D.C. (1994) MINEQL+ Version 3: A Chemical Equilibrium
Program fo r Personal Computers. The Proctor and Gamble Company.
Sehmel, G.A.
(1989)
Cyanide and Antimony Thermodynamic Database for the Aqueous
Species and Solids for the EPA-MINTEQ Geochemical Code. Report submitted to USEPA
under a service agreement with US Department of Energy.
232
Table D .l Recommended concentrations for the hydroponic nutrient solution.
The
recommended cyanide concentration is high enough for formation of iron cyanide solids, but
under dark conditions in the hydroponic pots, solid formation is inhibited due to slow
dissociation of ferrocyanide. Initial values given by S. Ebbs were based on conventional nutrient
recipes for hydroponic experiments.
Nutrient
KNOj
Ca(N0 3 ) 2
NH4 H2 PO4
MgS04
KC1
H3 BO3
MnS04
ZnS0 4
CuS0 4
H2 Mo04
NiS0 4
MES
TRIS
FeHEDTA
ICtFeCCN^
KCN
Concentration [M]
Initial
Recommended
6 .0 x 1 0 3
6 .0 x 1 0 *
3.9 x 10 3
4 x 10 3
1.9 x 10*
1 x 1 0 "*
1 .0 x 1 0 3
1 .0 x 1 0 3
5.0 x 10 5
5.0 x 10 5
1.25 x 10*
1.25 x 10*
1 .6 x 1 0 7
1 x 1 0 *
I.Ox 1 0 *
1 .0 x 1 0 *
5.0 x 10' 7
5.0 x 10 7
1 . 0 x 1 0 '7
1 .0 x 1 0 7
1
. 0 x 1 0 7
1 .0 x 1 0 7
1 .0 x 1 0 3
1 .0 x 1 0 3
6 . 0 x 1 0 "*
Added to pH 6.0
None
1 -1 0 x 1 0 *
1.25 x 10*
At suggested value
7.5 x 10*
233
Figure Legends
Figure D.l Solubility limitations for ferrocyanide addition to hydroponic solution. Critical
concentration of ICtFe(CN) 6 addition dependent on pH and pe of system. Calculated using
MINEQL+, a chemical equilibrium-modeling program (Schecher and McAvoy, 1993).
Figure D.2 Percentage of total dissolved cyanide present in the free form. Calculated using
MINEQL+, a chemical equilibrium-modeling program (Schecher and McAvoy, 1993).
Figure D 3 Reactor system for uptake experiments with hybrid poplar (Populus deltoides x
nigra, DN34). A septa separates the upper and lower chambers, supports the cutting, and seals
the system (after Burken and Schnoor 1998).
Figure D.4 Schematic of the hydroponic system used for l5 N-Iabeled cyanide uptake studies,
showing the arrangement of the willow plants, sampling port, and polyethylene (PE) bag liner.
The insert supporting the willow plant is also sealed to prevent entry of air. Only one of the two
sampling ports is shown here.
234
Figure D.5 Results from the ferri- and ferrocyanide sorption test conducted with materials used
in the hydroponic uptake system. Material included various tube fittings (BK = black: Wt =
white; F/F = female-female connector, M/F = male-female connector), disks cut from the pot
material, the Tygon (clear) and Pharmed (white) tubing involved, and the control solution. Batch
sorption tests were employed; reaction time = 11.5 hours; CN0 = 0.65 ppm as CN.
235
Log([CN]ertt (ppm))
1
4
P
- - pH 6 pH 7 pH 8
Figure D. 1
10
100
80
60
*(N0] %
40
20
0
5
10
11
_________ pe_________
- - pH 6 pH 7
pH 8 1
Figure D.2
12
Aerial Compartment'
: Air Inlet
VOC Pretrap
!Mininert sampling
! and feeding valve :
Root Compartment
Figure D.3
238
Sampling port
Airtight pot lid
Edges of PE bag,
sealed with pot lid
Figure D.4
239
W ///S /S Y /////////////////////////S
' / // / / / // / '////////////////////A
V ////////////////////////////////.
- V
O
TT
04
y y ////////////////////////////////.
v /////////////////////////////.
V //X ///////////////////////////A
m
04
(uidd) ii|N0l
Figure D.5
APPENDIX E
S e t 1 H y d r o po n ic U pt a k e S tudy
The first run of the hydroponic study. Set 1, provided results that supported the project
hypotheses regarding ferrocyanide uptake by willow. This was the first experiment in which the
ISN-Iabeled ferrocyanide was used. The results demonstrated a modest enrichment of 15N in both
the root and leaf tissues. This experiment also demonstrated that the desired solution conditions
(cyanide speciation. pH. and pe) were generally maintained. However, the solution analyses also
demonstrated the need to make revisions to the experimental protocols as evidence suggested
that the bioavailability of ferrocyanide may have been lower than expected, due to local
complexation and/or precipitation of the added ferrocyanide caused by mixing problems. A
revised protocol was developed from these observations (Chapter 4), which successfully
overcame the problems noted.
E.1 Materials and Methods
H. 1.1 Experimental Overview

A replicate block design was utilized for the first uptake experiment with willow, with three
replicates for each of the cyanide and iron cyanide treatments. Plants growing under greenhouse
conditions served as the control tissue. The l5N content of plant tissues is very constant, as is the
atmospheric l5N content, so specific controls were deemed unnecessary for this experiment. For
241
each of the cyanide treatment replicates, plants were transferred from the hydroponic pots into
the experimental system described in Appendix D. The roots were rinsed with copious amounts
of sterile deionized water to remove any sorbed nutrient solution. After the excess water had
drained from the roots, the plants were transferred to 5 L pots lined with a polyethylene bag. The
treatment solution was then added to the bag liner containing the plant. The polyethylene bag
was used to prevent the formation of headspace, which might facilitate movement of cyanide into
the vapor phase. When pinched in the pot lid, this bag formed an airtight seal. As the willows
transpire, the bag pulls inward, maintaining the solution level. Once the pot had been sealed, the
cyanide treatment was imposed. The cyanide content of the two treatments was adjusted to the
same initial cyanide concentration, 2 mg/L, with potassium cyanide (KCN) or potassium
ferrocyanide (ICtFelCNfo) and labeled with 25% I5N by mass as KCI5N and K4 Fe(CI5N)6,
respectively. Cyanide treatments were introduced as a 5 mL injection into the 5 L of solution in
the pot, providing a 1:1,000 dilution with respect to the desired final cyanide concentration.
Approximately 15 min. after the introduction of the cyanide treatment, a sample of the nutrient
solution was withdrawn from each pot and preserved with NaOH in brown HDPE bottles. The
solution pH and pe were also measured. The plants were moved to the greenhouse for a sevenday exposure period, with additional solution samples withdrawn through the sampling port on
day 4 and at harvest. Additional pH and pe measurements were made on day 4 and day 7 as well.
Pots were weighed over the seven-day interval to track transpiration.
242
E.1.2 Harvest and Analytical Procedures

At harvest, plants were separated into root and leaf tissues and the fresh weight determined.
Each tissue from a single plant was subdivided into three subsamples, with two frozen at -80 C
and the third dried to constant mass at 60 C. A frozen subsample of the root and leaf tissue from
each plant was sent to Carnegie Mellon University for extraction and analysis of cyanide content
and speciation using the method described in Chapter 3. The second frozen subsample was held
in reserve. The dried subsample was prepared for 15N analysis using the procedures described in
Chapter 4.
The difference in fresh weight and dry weight between this sample was used to
calculate the water content of the root tissue. Solution samples were analyzed for total and WAD
cyanide by distillation (APHA/AWWA/WEF, 1998) and free cyanide by microdiffusion (ASTM,
1998).
E.1.3 Data Analysis

Transpiration was expressed on a daily basis over the one-week exposure period and as
cumulative transpiration. Analysis of the ,5N data proceeded as described in Appendix H.2.3.
Additional calculations were required in this case to account for the >SN contributed by the
labeled cyanide compound, given that this label was added as a proportion of 25% by mass.
Results were expressed in terms of the enrichment ratio (8 ,5N /oo) in the tissue (Shearer and
Cole, 1993) and as 1SN concentration in the plant tissue (mg t5N kg DW 1). The results from the
tissue cyanide analyses and 1SN analyses were converted to similar units for comparison.
243
EL2Results
E.2.1 Willow Growth and Water Relations

Transpiration was variable but largely unaffected by either the ferrocyanide or KCN treatments
(Figure E.1). Transpiration can be taken as a crude measure of plant physiological status. The
increase in cumulative transpiration with time suggests that the willow plants were suffering no
overt physiological stress from the two cyanide treatments.
KCN-treated plants generally had
lower daily and cumulative rates of transpiration but this difference was not significant.
Transpiration from control pots containing no plants was negligible, indicating that the
hydroponic system maintained a consistent seal during the experiment. As shown in Figure E.2,
the similarity in tissue biomass between the two plants indicates that there was no growth
stimulation or inhibition from either treatment. Water content of roots was variable but it was
unclear if this was the result of the treatments (Figure E.3).
E.2.2 i5N Content of Willow Tissues

The cyanide treatments increased the ISN content of the willow tissues. The enrichment ratio in
roots increased to 450 and 870 for the ferrocyanide and KCN treatments respectively (Figure
E.4). Although larger, the enrichment ratio for KCN-treated roots was more variable and was
therefore not significantly greater than that for ferrocyanide-treated roots. These enrichments
resulted from an increased in the root atom% of I5N in the tissue to 0.54 and 0.69% for the
ferrocyanide and KCN treatments, respectively. The background atom% for ISN is -0.37%. The
increase in the enrichment ratio in leaves was less dramatic, an order of magnitude lower than
that observed for roots. The enrichment ratio for 15N in the leaves of KCN-treated plants was
244
five-fold greater than that for the ferrocyanide-treated plants. The impact of this enrichment on
,SN content is more clearly visible if the data are expressed in terms of I5N concentration (Figure
E.5).
The i5N concentration in willow roots increased by four- to five-fold, although not
significantly, following both ferrocyanide and KCN treatments. No increase was evident in
willow leaves in either treatment. There was also no relationship between the cyanide treatment
and the total N content of the tissues (data not shown).
The i5N content was converted to a total cyanide basis for comparison with the Set 1 tissue
extraction data. The comparison is given in Table C.l.
Results for the tissue extraction
exhibited a high variability, particularly for the root tissue. Total cyanide extraction results were
significant for ferrocyanide-exposed root tissue and were of the same magnitude to predicted
cyanide concentrations from ,5N data. Root levels for KCN-exposure and ferrocyanide-exposed
leaf tissue contained small amounts of extractable cyanide.
For each of these tissues, the
predicted ,SN results were substantially higher than extraction values. Leaf extraction values
were non-detectable. All tissue cyanide reflected the original speciation of the solution.
A mass balance on the total cyanide in the hydroponic system was not performed on the Set 1
data due to the problems associated with the initial introduction of cyanide into the system. This
prevented an accurate measure of the initial cyanide mass in solution.
E.2.3 Solution Cvanide Analyses

Solution analyses confirmed that the nutrient solutions, designed with the aid of chemical
modeling, preserved cyanide speciation over the course of the experiment. The distribution of
245
chemical species (WAD and free) as a function of the total cyanide remained consistent within a
given treatment. For the ferrocyanide treatment, <5% of the added ferrocyanide was detected as
WAD or free cyanide, demonstrating that little dissociation occurred over the 7-day period
(Figure E.6).
As expected, all of the KCN remained in the free form (Figure E.7).
An
unexpected observation was that the initial total cyanide concentration on day 0 for both
treatments was 150-200% higher than the desired 2 ppm value. Values on day 4 and 7 were
more representative (i.e. <2 ppm CNj), assuming that the willow plants readily transported
cyanide from KCN solution and transported ferrocyanide from solution more slowly.
The
calculations used to determine the mass of ferrocyanide and KCN needed to reach 2 ppm CNj as
cyanide were reviewed and determined to be correct.
The abnormally high total cyanide concentrations in the pots on day 0 were of concern. The
chemical speciation modeling done for the hydroponic solution indicated that iron cyanide solids
could precipitate if the concentration far exceeded the target value of 2 ppm as cyanide. This
solubility limit formed the basis for selecting this concentration for study. The routinely high
initial concentrations of cyanide may have rendered much of the ferrocyanide unavailable for
uptake, further reducing the possible accumulation of ISN by ferrocyanide-exposed willow plants.
One speculation was that the injection of the cyanide treatment as 5 mL of l,000x solution may
have created this problem. Recall that this 5 mL volume was introduced by injection, with the
first solution sample taken from the pot 15-20 min. later. Given the solubility limit of cyanide
compounds in the ionic nutrient solution used, one concern was that there may have been local
precipitation of the added cyanide in the solution near the injection port. Since the solution
246
samples for analysis were also drawn from this port, the aliquots may have picked up precipitate
along with solution, skewing the concentration higher. Additionally, diffusion limitations may
have prevented the cyanide from becoming uniformly distributed in the nutrient solution. The
treatment was imposed after plants had already been established in the systems. This limited the
degree of agitation that the pots could be subjected to. With little agitation, a relatively short
time period between injection and aliquot removal, and the possible local precipitation around
the injection port, there may simply have been insufficient time for the cyanide to disperse
throughout the pot. Thus, the day 0 samples may not have provided an accurate representation of
the solution cyanide content. Considering that the day 4 and 7 solution samples fell below the 2
ppm total cyanide target and that there was not a significant uptake of 1SN or cyanide by the
willow plants, the most likely explanation is that the day 0 samples provided erroneous results.
IL3 Discussion
The Set 1 results demonstrated that the nutrient solution designed for this experiment maintained
the cyanide speciation necessary to accurately assess whether willow can accumulate
ferrocyanide.
Additionally, the steady transpiration observed during the exposure period
suggested that the willow plants were not experiencing physiological stress. Given the presence
of cyanide-resistant respiration in plant cells and the presence of detoxification mechanisms like
the cyanoalanine pathway, this is not surprising. The dramatic increase in 1SN enrichment in
plant tissues, although insignificant in terms of total 1SN concentration, suggested that uptake of
cyanogenic nitrogen occurred but that the duration of the experiment was too short to observe a
significant shift in l5N content. Additionally, the 25% loading rate for the stable isotope label
247
made accurate quantification of differences difficult. Longer exposures at a higher loading rate
were required to increase the signal for l5N accumulation.
These modifications to the
experimental design were adopted for the second series of willow uptake experiments (see
Chapter 4).
A series of solution mixing experiments conducted after Set 1 demonstrated that with more
vigorous mixing, the addition of a larger volume of more dilute cyanide solution, and a longer
waiting period before initial sampling, yielded more representative values for the initial total
cyanide concentration.
Subsequent experiments (Chapter 4) utilized this modified sample
introduction approach and alleviated the problems observed in the Set 1 experiments.
E.4 References
APHA/AWWA/WEF. (1998) Standard Methods for the Examination o f Water and Wastewater,
2&x ed., American Public Health Association, American Water Works Association, and Water
ASTM.
(1998) Standard Test Method for Determination of Free Cyanide in Water and
Shearer G.; and Cole, D.H. (1993) Natural Abundance of 1SN: Fractional Contribution of Two
Sources to a Common Sink and Use of Isotope Discrimination. In Knowles R; and Blackburn,
T.H., eds. Nitrogen Isotope Techniques. San Diego, CA, Academic Press, p. 89.
248
Table E.1 Set 1 Tissue Cyanide Speciation After 7-Day Exposure (mg/kg FW) for Ferrocyanideexposed (FeCN) and KCN-exposed Tissue.1'2
Sample
l-l Root
2-2 Root
3-2 Root
1-3 Root
2-4 Root
3-4 Root
Root Control6
1-1 Leaf
2-2 Leaf
3-2 Leaf
1-3 Leaf
2-4 Leaf
3-4 Leaf
Leaf Control6
Cyanide
Exposure
FeCN
KCN
None
FeCN
KCN
None
Total Cyanide
Total
Free Cyanide
via N
Cyanide
(mgfltg)4
(ms/kg)3
(mg/kg)5
30.9
0.02
29.3
35.9
75.0
0.04
188.4
92.4
0.09
88.0
4.2
2.0
21.6
ND
0.2
18.6
4.9
7.8
NA
0.9
ND
0.01
ND
0.21
0.19
0.15
0.37
ND
0.6
1.4
1.8
ND
0.1
ND
0.4
23.1
ND
25.6
2.7
ND
21.3
NA
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CNT7E and 4500-CN- I/E
(APHA/AWWA/WEF. 1998), and Free Cyanide by microdiffusion as per ASTM D 4282-95 (ASTM. 1998)
* All reported concentrations are averages of at least duplicate analyses. ND = Not Detectable, NA = Not
Applicable. Detection Limit for Total and WAD Cyanides = 0.1 mg/kg, for Free Cyanide = 0.25 mg/kg.
3 Cyanide concentrations adjusted to reflect 98% recovery of complexed cyanide and 95% recovery of free
cyanide in total cyanide analysis during distillation. Concentrations also corrected for 11% free cyanide
losses during extraction.
4 Cyanide concentrations adjusted to reflect 11% loss of free cyanide during extraction.
5 Cyanide concentrations calculated from ISN data using 81% H2O content. 25% 1SN labeling, and sample
mass.
6 Root and leaf concentrations are measured concentrations as remaining values adjusted to reflect
respective cyanide levels in control tissue.
249
Figure Legends
Figure E.1 Daily (closed symbols) and cumulative (open symbols) transpiration for willows
exposed to 2 ppm CNT for 7 days as either potassium ferrocyanide (FeCN) or as potassium
cyanide (KCN). Error bars reflect the standard error of the mean (n = 2-4).
Figure E.2 Biomass of willow tissues from plants treated with 2 ppm CNt as potassium
ferrocyanide or potassium cyanide for 7 days. A specific control treatment was not included in
this ran, so comparable data on control biomass is not available.
Figure E J Water content of willow roots from control plants or plants exposed to 2 ppm CNt as
either potassium ferrocyanide (FeCN) or potassium cyanide (KCN) for 7 days (n = 2-4).
Figure E.4 Enrichment of l5N in root and leaf tissues of willow exposed 7 days to 15N-labeIed
ferrocyanide (FeCN) or KCN. Error bars reflect the standard error of the mean (rt = 2-4).
Figure E.5 l5N concentration in root and leaf tissues of willow exposed 7 days to l5N-labeled
ferrocyanide (FeCN) or KCN. Tissue lsN concentrations in the FeCN and KCN treatments
account for both background >SN and the increase in 1SN due to the presence of the labeled
compounds. Error bars reflect the standard error of the mean (n = 2-4).
250
Figure E.6 Distribution of cyanide chemical species in the ferrocyanide (FeCN) treatment
solution over the course of the 7-day exposure period. Data are expressed in terms of total
cyanide concentration, rather than as ferrocyanide concentrations, to provide a more direct
comparison between this treatment and the KCN treatment (Figure E.7).
The mass of
ferrocyanide added to the pots should have provided a final solution concentration of 2 ppm CNt
at day 0. A possible explanation for the difference observed between this value and the total
cyanide concentration here is discussed in the text.
Note that WAD and free cyanide
concentrations represent <5% of the total cyanide in solution at each time point, indicating that
speciation remains predominantly as complexed iron cyanide. Error bars denote the standard
error of the mean (n - 2-3).
Figure E.7 Distribution of cyanide chemical species in the KCN treatment solution over the
course of the 7-day exposure period. Data are expressed in terms of total cyanide concentration.
As for the ferrocyanide treatment, the mass of KCN added to the pots should have provided a
final solution concentration of 2 ppm CNt at day 0. Note that as expected, the KCN treatment
provided all cyanide as free cyanide. Error bars denote the standard error of the mean (n = 2-3).
251
800
Daily transpiration, FeCN treatment
Daily transpiration. KCN treatment
Cumulative transpiration. FeCN treatment
Cumulative transpiration, KCN treatment
E
1
s*
400 -
2
i_
a
^ 200 -
Days
Figure E.1
252
FeCN treatment
KCN treatment
Fresh weight, g
12 -
4 -
leaf
stem
root
Plant tissue
Figure E.2
253
control
FeCN
KCN
Treatment
Figure E 3
254
i
i FeCN treatment
KCN Treatment
Root
Leaf
Willow tissue
Figure E.4
Tissue i5N concentration, mg kg DW*1
400
control
FeCN treatment
KCN treatment
300 -
200
100
Root
Leaf
Willow tissue
Figure E.5
256
6.00 Total CN
WAD CN
Free CN
CN concentration, ppm
4.00 -
2.00
0.10 H
0.05 H
M S L
0.00
0
1. 1:
4
Days of treatment
Figure E.6
257
CN concentration, ppm
4H
Total CN
WAD CN
wmm Free CN
3-
1-
Days of treatment
Figure E.7
258
APPENDIX F
A s s im il a t io n o f c y a n o g e n ic n it r o g e n in t o a m in o a c id s b y w il l o w 11}
Results from the lsN-labeled free cyanide and ferrocyanide hydroponic uptake studies with
willow (Chapter 4) provide clear evidence that cyanogenic nitrogen from iron cyanide complexes
is transported to the aerial tissues of willow. These uptake results do not, however, indicate the
biochemical fate of the nitrogen.
The discrepancy between tissue I5N and extraction
concentrations is strongly suggestive of assimilation as I5N is not present as cyanide. To assess

the extent of cyanogenic nitrogen assimilation, analyses of the amino acid fraction of the 15N in
the willow tissues from the cyanide uptake studies were performed.
Measurements of ISN amino acids in willow plant tissue revealed a significant increase in the
amino acid fraction of free cyanide-treated plants and a non-significant increase for ferrocyanidetreated plants. While definitely suggestive that both free cyanide and ferrocyanide are being
assimilated, the amino acid pool contained only a fraction of the total 15N in the plant tissues,
indicating that the assimilation product is either metabolically beyond the amino acid stage and
has been incorporated into proteins and other compounds, or occurs as a metabolite prior to the
amino acid pool.
Additional research is needed to determine the specific biochemical
compounds containing the I5N. Nevertheless, the data provided additional evidence in support of
cyanide phytoremediation, demonstrating that willows assimilate cyanide harmlessly into their
general metabolic cycles.
( Authored by Stephen Ebbs; Work conducted at SIUC
259
F.l Introduction
The hydroponic uptake studies showed that cyanogenic nitrogen was being taken up into the
willow plant and being assimilated within the plant tissue. As illustrated in Figure 2.2 and
described in Chapter 2, plant pathways involving cyanide converge into a common pathway
converting cyanide to amino acids. Based upon the data in the previous section, a logical
assumption is that evidence of assimilation could be obtained by analyzing the ISN content of the
amino acid pool. Such data would provide additional information pertaining to the safety of iron
cyanide phytoremediation and the biological reuse of cyanide for growth and development.
The objective of the amino acid fraction analysis was to provide further information concerning
the ultimate fate of cyanogenic nitrogen within plant tissue. Evidence revealing the inclusion of
cyanide into the existing plant metabolic pathways demonstrates a removal rather than
displacement of cyanide regulatory risk. Inclusion of ,5N into the amino acid fraction of plant
tissue would provide information regarding the plant assimilation pathway for cyanide.
F.2 Materials and Methods
Amino acids were isolated from the reserved tissue retained from the experiment described in
Chapter 4. Amino acids were separated using ion chromatography into two fractions (acidic and
basic/neutral amino acids), using a method modified from Wood et al. (1996). Plant tissues were
extracted in 10 mL methanol at 4 C in the dark for 24 hours. The extract was mixed with 5 mL
chloroform and 6 mL water and allowed to phase separate for 2.5 hours at room temperature.
260
The aqueous phase was decanted and dried overnight under a stream of air. The dried extract
was re-dissolved in 2 mL sterile water and the basic/neutral and acidic amino acids separated
using a Dowex-l-acetate column. Basic/neutral amino acids were eluted with water and the
acidic amino acids were eluted with 2 M acetic acid
The two fractions recovered from the Dowex-l-acetate columns were dried under a stream of air.
Each sample was re-dissolved in 1 mL water and applied individually to Dowex-50-H columns.
Amino acids were eluted with 6 M NFLOH and dried overnight under a stream of air. The dried
fractions were re-suspended in 60% methanol and transferred to microcentrifuge tubes.
Subsamples of the amino acid fractions were analyzed for total N and l5N content by Isotope
Ratio Mass Spectrometry (IRMS) at the University of Califomia-Davis Stable Isotope Facility.
Given the complexity of this isolation protocol, only one replicate was completed by the end of
the project.
F J Results
Analysis of the amino acid fraction isolated from willow tissues treated with potassium cyanide
(KCN) and ferrocyanide ([Fe(CN)6]4) revealed an increase in the atom% of lsN in the leaf and
root amino acid fraction from plants treated with either KCN or [Fe(CN)6]4'. The increase in
atom% was greater for roots compared to leaves and greater for KCN treatment as compared to
[Fe(CN)6]'l treatment (Table F.I). The increase in the atom% of [Fe(CN)6]4 treated roots still
represents a substantial increase in ISN.
A similar pattern is evident when the results are
converted to 15N concentration (Figure F.I). However, these concentrations are 1,000-fold less
261
than the tissue ISN concentrations shown in Figure 3.2. Although there is considerable ISN in the
root amino acid fraction of the KCN treatment, the amount still represents only a small fraction
of the total ISN in the plant tissue. Combined with the discrepancy between tissue ISN and
extraction results (Figures 4.4 and 4.5), the amino acid results suggest that assimilation of free
cyanide and ferrocyanide is either far beyond amino acids metabolically (i.e.- proteins and other
amino acid derivatives) or in a metabolite prior to the amino acid pool. The former seems a
more plausible explanation and could be addressed by isolating and analyzing the protein
fraction for ,5N. This falls outside the current scope of work but would provide more tangible
evidence of assimilation and the ecological safety of the phytoremediation approach. It would
also allow the biochemical fate of the cyanogenic N to be more completely described.
F.4 Summary and Conclusions
The ability of willow plants to assimilate free cyanide and ferrocyanide species in the form of
amino acids was investigated by separation and analysis of ISN fractions in plant tissues from
l5N-labeIed-cyanide uptake experiments. Amino acids in aqueous plant extract solutions were
isolated by ion chromatography, and analyzed for >SN content. Increases in the amino acid
fraction atom% of 15N and l5N concentration were substantial for both the KCN and [FefCNfo]4
treatments.
Root results were more pronounced than those for leaves.
This supports the
contention that much of the cyanide assimilation occurring in this species is occurring at the root
level. More importantly, it can be inferred that after 20 days of exposure to KCN or [FefCN^]4',
most cyanide within willow tissue has already passed through the amino acid pool into general
plant metabolism. This conclusion is tentative at this point but could be more conclusively
262
demonstrated with a simple array of additional biochemical analyses. These analyses would
more clearly described the fate of cyanide in willows and provide more evidence to satisfy
regulatory concerns.
Also, the additional information might improve our understanding of
cyanide metabolism in willows so as to potentially increase the future applicability of cyanide

phytoremediation at impacted sites.
F i Reference
Wood. A.J.; Saneoka, H.; Rhodes, D.; Joly, RJ.; and Goldsbrough, P.B. (1996) Betaine
Aldehyde Dehydrogenase in Sorghum. Molecular Cloning and Expression of Two Related
Genes. Plant Physiol. 110: 1301.
263
Table F.I The atom% of >SN in the amino acid fractions from willow tissues treated with 2 ppm
CNt as lsN-labeled potassium ferrocyanide ([FefCN^J4") or potassium cyanide (KCN). Data are
for a single replicate. The atom% for the control treatment represents the natural abundance of
ISN in the environment and in plant tissues.
Treatment
Tissue
No CN Added
[FefCN)*]4-
KCN
Root
0.38
0.79
5.90
Leaf
0.38
0.46
0.93
264
Figure Legend
Figure F.I l5N content of the amino acid fraction from willow roots and leaves following a 20day exposure to 2 mg L '1 total cyanide as either potassium cyanide (KCN) or ferrocyanide
([FefCN^]4*).
Data represent the 15N concentration in the tissue from the single replicate
analyzed. Note that the units for the y-axis are in jig kg D W 1 rather than mg kg D W 1 as shown
in Figure 4.2, demonstrating the low concentration of ISN in the amino acid pool.
265
15N concentrations, fig /kg-DW
uoo
] Control
1000
W Z7A Fe(CN),
885883 KCN
200
100
0
root
lea f
Tissue
Figure F. 1
266
*
A PPEN D IX G
Fo r t r a n C o d es
fo r sy stem
Models
G.l Control Hydroponic System Simulation Code

Control Hydroponic System Simulation Code fo r Ferrocyanide Solution
Joseph T. Bushey
C
C
C
C
SUBROUTINE OBJFI
C
IMPLICIT REAL*8 (A-H.O-Z)
INTEGER MXNC, MXNV, NDP, J, N
PARAMETER (MXNC=50, MXNV=40, NDP=8, J=5, N=4)
NDP - # OF DATA POINTS
J - # OF DIFF. EQNS.
N - # OF DECISION VARIABLES, XD
COMMON/OPTVAR1/ OPTVAR(MXNV). CNS(MXNC), OBJFUN
COMMON/OptStiff/ XD(N),YY(5),Vcont,YoFeCN,CNSorb,CNDe,Asorb
COMMON/CONC/ CC(8), CD(8)
C
C
C
C
&
C
C
DATA CD(1),CD(2),CD(3),CD(4),CD(5),CD(6),CD(7),CD(8)/10.52,9.951,
9.601,10.28,0.654,1.000.1.154,1.462/
DO 1=1,N
XD(I)=OPTVAR(I)
ENDDO
Call for Stiff ODE solution program
CALLCALLStiff
C
C
Set objective function value, F

OBJFUN = 0.0
DO 1=1,NDP
OBJFUN=OBJFUN-K((CC(I)-CD(I)))/(CD(D))**2
write(*,9514) OBJFUN
ENDDO
9514 format(2x, fl2.10)
C
C
CNS( 1) - mass balance on CN in FeCN system
267
&
CNS(l) = Asorb*(6.*YY(l)+YY(3))+Vcom*(6.*YY(2)+YY(4))
-6.*Vcont*YoFeCN
C
RETURN
END
u u u
BEGINS ODE STIFF SOLVER ************************

SUBROUTINE CALLS tiff
u
u
u
IMPLICIT REAL*8 (A-H,0-Z)

INTEGER NPARAM, J
PARAMETER (NPARAM=50, J=5)
J : NUMBER OF ODEs
DIMENSION Y(J), PARAM(NPARAM)
SPECIFICATIONS FOR LOCAL VARIABLES
INTEGER IDO, EN D . NOUT
u
u
SPECIFICATIONS FOR SUBROUTINES

EXTERNAL DIVPAG, DSET, FNCODE, FCNJac
u
COMMON/CONC/ CC(8), CD(8)
u
u
OPEN( 11,FILE='Opt-StiffCon_DisFeCN.DAT.STATUS='UN KNOWN)

SET INITIAL CONDITIONS
IDO = 1
T = 0.0
Y(l) = 0.00
Y(2)= 11.75
Y(3) = 0.00
Y(4) = 0.00
Y(5) = 0.00
u u u u
WRITE (11,99999)
WRITE (11,'(I6,6F12.4)') END, T, Y
SET ERROR TOLERANCE
TOL = 0.00005
C
C
SET PARAM TO DEFAULT

CALL DSET (NPARAM, 0.0, PARAM, 1)
SELECT ABSOLUTE ERROR CONTROL, Reset # of MaxSteps, Select Gear
PARAM(10)= 1.0
PARAM(4) = 1000.
268
PARAM(12) = 2.
PARAM(13) = 1.
C
10
IDO = 1
IEND = 0
CONTINUE
IEND = IEND + 1
TEND = IEND
CALL DIVPAG (IDO, J, FNCODE, FCNJac, A, T, TEND, TOL, PARAM, Y)
IF(T .EQ. 5.0) CC(1)=Y(2)
IF(T .EQ. 5.0) CC(5)=Y(4)
IF a EQ. 10.0) CC(2)=Y(2)
IF(T .EQ. 10.0) CC(6)=Y(4)
IF(T .EQ. 15.0) CC(3)=Y(2)
I F a EQ- 15.0) CC(7)=Y(4)
I F a EQ. 20.0) CC(4)=Y(2)
I F a EQ. 20.0) CC(8)=Y(4)
IF(IEND .LE. 20) THEN

WRITE (NOUT,'(I6,6F 12.4)) IEND. T. Y
WRITE (1 l,(I6,6F12.4)') IEND, T, Y
Final call to release
IF (IEND .EQ. 20) IDO = 3
GO TO 10
ENDIF
C
99999 FORMAT(4X,TEND',5X,Time',9X,Y 1' 11X, Y2* 11X,'Y3 11X.
&
Y4* 11X,Y5 11X)
C
CLOSE(U)
RETURN
END
C
SUBROUTINE FNCODE (J, T, Y, YP)
C
J : NUMBER OFODEs
C
N : NUMBER OF DESIGN VARIABLES
DIMENSION Y(J), YP(J)
COMMON/OptStiff/ XD(4), YY (5), Vcont, YoFeCN,CNSorb,CNDe,Asorb
C
VCont=5.10
Asorb=0.129
YoFeCN=11.75
CNSorb=0.136
CNDe=0.0361
269
uuuuu
ODEs
FeCN system
YP( 1)=-(XD(2)*Y( 1))/Asorb+(XD( 1)*'Y(2))/Asorb
YP(2)=-(XD( 1yVCont)* Y(2)+<XD(2)/VCont)* Y(1 )-XD(3)*Y(2)
& +XD(4)*(Y(4)**6)*Y(5)
u u u uuu uu u
Need to include a term for FeCN breakdown to free CN in control to account for rate of
dissociation - forward and backward
FeCN = Fe + 6CN
Ignore sorptive competition between cyanide species and Fe2+ complexation
Free CN system
YP(3)=-(CNDe*Y(3))/Asorb+(CNSorb*Y(4))/Asorb
YP(4)=-(CNSorh/VCont)*Y(4)+(CNDe/VCont)*Y(3)+6.*XD(3)*Y(2)
& -6.*XD(4)*(Y(4)**6)*Y(5)
YP(5)=+XD(3)*Y(2)-XD(4)*(Y(4)**6)*Y(5)
u
DO K=1 J
YY(K)=Y(K)
ENDDO
u
RETURN
END
uuu
Subroutine to calculate Jacobian for ODE solver
SUBROUTINE FCNJac (J, T, Y, DYPDY)

Specifications for arguments
INTEGER J
DIMENSION Y(J), DYPDY(J,*)
COMMON/OptStiff/ XD(4),YY(5),Vcont,YoFeCN,CNSorb,CNDe,Asorb
EXTERNAL DSET
uuu
Clear array to zero

CALL DSET(J**2,0.0, DYPDY, I)
DYPD Y( 1,1 )=-(XD(2))/Asorb
DYPD Y( 1,2)=+(XD( 1))/Asorb
270
DYPDY(2,1)=+(XD(2yVCont)
DYPDY(2^)=-(XD( 1VVCont)-XD(3)
DYPDY(2,4)=+6.*XD(4)*(Y(4)**5)*Y(5)
DYPDY(2,7)=+XD(4)*(Y(4)**6)
DYPDY(3,3)=-CNDe/Asorb
D YPDY(3,4)=+CNSorb/Asorb
DYPDY(4,2)=46.*XD(3)
DYPDY(4,3)=+CNDe/VCont
DYPDY(4,4)=-CNSorta/VCont-36.*XD(4)*(Y(4)**5)*Y(5)
DYPDY(4,5)=-6.*XD(4)*(Y(4)**6)
DYPDY(5,2)=+XD(3)
DYPDY(5,4)=-6.*XD(4)*(Y(4)**5)*Y(5)
DYPDY(5,5)=-XD(4)*(Y(4)**6)
Return
End
271
GJ. Plant Uptake Hydroponic System Code - 17-Parameter Model

Solution of hydroponic uptake system differential equations using stiff equations solver
Joseph T. Bushey
C
C
C
C
SUBROUTINE OBJFI
C
fc> 9? * fc*
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C

INTEGER MXNC, MXNV, NDP, J, N
PARAMETER (MXNC=50, MXNV=40, NDP=27, J=32, N=I7)
NDP - # OF DATA POINTS
J - # OF DIFF. EQNS.
N - # OF DECISION VARIABLES, XD
COMMON/OPTVAR1/ OPTVAR(MXNV), CNS(MXNC), OBJFUN
COMMON/OptStiff/ XD(I7), VFeo, VCNo, YoFe, YoCN, YZFe, YZCNFe,
YZCNAm, YZFeAm, YZCN, VRIFe, ThStC, DsFe, FeRq, ThRt, Stq,
ThSt, Folq, SbCN, DsCN, CNRq, ThRtC, StqC, FolqC, VRICN,
Asorb, RhWt, SbFe, VFe, VCN, YY(32), FeFor, FeBack, DFeCN,
DCN, CWq, RORq, CWqC, RORqC, Fror, Fis, CNvoI, CNQ, FeQ,
dVFedt, dVCNdt
COMMON/CONC/ CC(27), CD(27), EqCon
LISTING OF PARAMETERS:
FeCN parameters
all tissue weights and per tissue rates given on fresh wt. basis
SbFe - control sorption rate [L/day]
DsFe - control desorption rate [mA2/day]
FeRq - total root mass [kg]
ThRt - total root water fraction [kg/kg]
Stq - stem mass [kg]
ThSt - stem water fraction [kg/kg]
Folq - leaf mass [kg]
FeQ - water flow rate [L/day]
VFe - hydroponic system bucket volume [L]
dVFedt - change in bucket volume [L/day]
VRIFe - volume of interstitial space [L]
YZFe - measured root FeCNtot [umol/kg]
YZFeAm - measured root C 15Ntot [umol as CN/kg]
YZFCN - measured root CNtot from dissociation of FeCN [umol/kg]
FeFor - Forward dissociation rate constant [ l/day]
FeBack - Backward dissociation rate constant [(L/umol)A6/day]
272
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
DFeCN - diffusion mass transfer coefficient from solution to interstitial space [L/day]
CWq - mass cell wall [kg]
RORq - mass rest of root (interstitial + biotic inside) [kg]
CN parameters (same units as FeCN)
SbCN - control sorption rate
DsCN - control desorption rate
CNRq - total root mass
ThRtC - total root water fraction
StqC - stem mass
ThStC - stem water fraction
FoIqC - leaf water fraction
CNQ - water flow rate
VCN - hydroponic system bucket volume
dVCNdt - change in bucket volume
VRICN - volume of interstitial spacec
YZCN - measured root CNtot
YZCNAm - measured root C 15Ntot
DCN - diffusion mass transfer coefficient from solution to interstitial space
CWqC - mass cell wall
RORqC - mass rest of root (interstitial + biotic inside)
Joint/general parameters
Asorb - control sorption surface area [mA2]
RhWt - density of water [kg/L]
Fror - water fraction for rest of root [kg/kg]
Fis - fraction of root that is interstitial space [L/L]
CNvol - volatilization rate of cyanide from leaf tissue [kg/day]
DESIGN VARIABLES:
Assume no assimilation, biological function, or sorption within stem. Stem acts as
perfect transport mechanism.
FeCN
XD(1) - sorption to cell wall [L/day]
XD(2) - desorption from cell wall [kg/day]
XD(3) - maximum active uptake rate [umoL/L-day]
XD(4) - half-saturation constant for active uptake [umol/L]
XD(5) - first order dissociation rate for rest of root [kg/day]
XD(6) - transfer efficiency for FeCN movement from root to stem [umol/umol]
XD(7) - first order dissociation rate for leaf [kg/day]
273
u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u
CN
XD(8) - sorption to cell wall [L/day]
XD(9) - desorption from cell wall [kg/day]
XD(10) - maximum active uptake rate [umol/L-day]
XD(11) - half-saturation constant for active uptake [umol/L]
XD(12) - first order assimilation rate for rest of root [kg/day]
XD(13) - transfer efficiency for CN movement from root to stem [umol/umol]
XD(14) - first order assimilation rate for leaf [kg/day]
Joint
Water fraction for Rest-of-Root, Transfer efficiency of metabolites for Root-Stern and
Stem-Leaf, and Volatilization rate of metabolites and CN same for FeCN and CN
system. Water fraction calculated from experimental data.
XD(15) - transfer efficiency for assimilated products between root and stem [umol/umol]
XD(16) - volatilization rate for assimilated products from leaf [kg/day]
XD(I7) - volatilization rate for free cyanide from leaf [kg/day]
Compartements: Used for development of the set of differential equations representing

mass flow through the plant.
Y( 1-3,11-13,25-27,32) [umol/L]
Y(4-10,14-20,28-31) [umol/kg-FW]
Y(2l-24) [umol]
FeCN
Y(l) - Sorption phase for control loss to apparatus
Y(2) - Hydroponic solution concentration
Y(3) - Interstitial space concentration
Y(4) - Root sorptive component
Y(5) - Interior section of root tissue inside endodermis - in xylem of root
Y(6) - Amount in stem tissue
Y(7) - Amount present in leaf tissue
Y(8) - Assimilated form in root tissue
Y(9) - Assimilated form in stem tissue
Y(10) - Assimilated form in leaf tissue
CN
Y(11) - Sorption phase for control loss to apparatus
274
o o n n n o n n n n o n n o n o n n n o n n n n n o o n o n o n n n n n n n n o

Y(18) - Assimilated form in root tissue
Y(I9) - Assimilated form in stem tissue
Y(20) - Assimilated form in leaf tissue
Volatilization losses - written in terms of total mass volatilized from t=0
Y(21) - Assimilated form from FeCN system
Y(22) - Assimilated form from CN system
Y(23) - Free CN from FeCN system
Y(24) - Free CN from CN system
Dissociated Free CN in FeCN system
Y(25) - Sorption phase for control loss to apparatus
Y(32) - Fe2+ in hydroponic solution - assume no interaction with plant/sorption
CD(l)-(4) FeCN solution concentrations; (5)-(8) CN solution concentrations;
(9)-(I2) CN dissociation from FeCN solution concentrations; (13)-(15) FeCN
within plant tissue; (16)-(18) Free CN from FeCN in tissue; (19)-(20)
Assimilated CN within FeCN system; (21)-(23) CN from CN in tissue; (24)-(26)
Assimilated CN within CN system; (27) Assim. Root Cone, for FeCN system
Free CN subtracted from total CN to obtain FeCN; Total subtracted from 15N data to
obtain assimilated amount; total CN values used for free CN system; root
assimilated for FeCN system treated as 5% level of significance from
experimental data as 15N level lower than FeCN
Input data from hydroponic results.
&
&
&
&
DATA CD( 1),CD(2),CD(3),CD(4),CD(5),CD(6),CD(7),CD(8),CD(9),

CD( 10),CD( 11),CD( 12),CD( 13),CD( 14),CD( 15),CD( 16),CD( 17),
CD( 18),CD( 19),CD(20),CD(21),CD(22),CD(23),CD(24),CD(25),
CD(26), CD(27)/11.34, 12.50,14.95, 20.27,55.37,34.11, 11.05,
7.44,0.540,0.733,0.640,0.387, 819.3,0.64,4.81,7.31,1.54,0.38,
275
&
308.8, 824.6,43.08,1.54,8.85, 18688.0,4422.0,3810.0,0.00/
uuu
Calculate optimization variables from log values

DO 1=1,N
XD(I)=10**(OPTVAR(R)
ENDDO
uu
Call for Stiff ODE solution program
CALLCALLStiff
uu
Set objective function value, OBJFUN
OPEN( 11,FILE=Op-Stf-Pnt.DAT\ACCESS=APPEND')
OBJFUN = 0.0
u u u u
ACD = 0.459 based upon 95% confidence interval from experimental data that
assimilated root content for FeCN system was 0.0
9514
C
C
C
C
C
C
C
C
C
&
&
&
&
&
&
DO 1=1,NDP
IF (CD(I). GT. 0.0) ACD=logl0(CD(I))
IF (CD(I). LE. 0.0) ACD=0.459
IF (CC(I). GT. 0.0) ACC=logl0(CC(I))
IF (CC(I). LE. 0.0) ACC=0.0
OBJFUN=OBJFUN+(ACC-ACD)**2
write(*,9514) I, OBJFUN, CC(I), CD(I)
write( 11,9514) I, OBJFUN, CC(I), CD(I)
ENDDO
format(2x, 12, 2x, f7.4,2(2x, fl0.4))
Constraints:
(1) = Mass balance for FeCN in plant-water system
(2) = Mass balance for CN in plant-water system
(3) = FeCN sorption equal to 61.7
(4) = Ratio of sorption to cell wall higher for FeCN
YY(l-7) in umol FeCN while rest as CN
CNS(l) = 6*(VFeo*YoFe-YY(l)*Asorb-YY(2)*VFe-YY(3)* VRIFe
- YY(4)*CWq-YY(5)*RORq-YY(6)*Stq-YY(7)*Folq)
- YY(8)*RORq-YY(9)*Stq-YY( 10)*Folq-YY(21)-YY(23)
-YY(25)*Asorb-YY(26)*VFe-YY(27)*VRIFe-YY(28)*CWq
-YY(29)*RORq-YY(30)*Stq-YY(31)*Folq
CNS(2) = VCNo*YoCN-YY(l l)*Asorb-YY(12)*VCN
- YY(13)* VRICN-YY(!4)*CWqC-YY (15)*RORqC-YY(16)*StqC
- YY(17)*FolqC-YY(18)*RORqC-YY( 19)*StqC-YY(20)*FolqC-YY(22)
276
&
-YY(24)
uuu
No need to adjust for dissociation of FeCN as amount of free CN small;

sorption due to FeCN
CNS(3) = EqCon-61.7
CNS(4) = XD(8)/XD(9)-XD( 1)/XD(2)
u
RETURN
END
u u u
BEGINS ODE STIFF SOLVER ************************

SUBROUTINE CALLStiff
u
u u u u

INTEGER NPARAM, J
PARAMETER (NPARAM=50, J=32)
J : NUMBER OFODEs
DIMENSION Y(J)
REAL* 8 PARAM(NPARAM)
SPECIFICATIONS FOR LOCAL VARIABLES
INTEGER IDO, IEND, NOUT
SPECIFICATIONS FOR SUBROUTINES
EXTERNAL DIVPAG, DSET, FNCODE, FCNJac
COMMON/CONC/ CC(27), CD(27), EqCon
COMMON/ROOTCONC/ a YZFE, aYZCNFE, aYZCNAM. a YZFEAM. aYZCN
u u u
OPEN( 11 ,FILE='Op-Stf-Pnt.DAT,STATUS=,UNKNOWN')
SET INITIAL CONDITIONS
IDO= 1
T = 0.0
Y(l) = 0.00
Y(2)= 11.93
Y(3) = 0.00
Y(4) = 0.00
Y(5) = 0.00
Y(6) = 0.00
Y(7) = 0.00
Y(8) = 0.00
Y(9) = 0.00
Y(10) = 0.00
Y(11) = 0.00
Y(12) = 68.63
277
c
c
Y(13 = 0.00
Y(14 = 0.00
Y(15 = 0.00
Y(16 = 0.00
Y(17 = 0.00
Y(18 = 0.00
Y(I9 = 0.00
Y(20 = 0.00
Y(21 = 0.00
Y(22 = 0.00
Y(23 = 0.00
Y(24 = 0.00
Y(25 = 0.00
Y(26 = 0.00
Y(27 = 0.00
Y(28 = 0.00
Y(29 ) = 0.00
Y(30 ) = 0.00
Y(3l ) = 0.00
Y(32) = 0.00
WRITE (11,99999)
WRITE ( l l / f t e ^ F U T ) ) END, T, Y
c
c
c
c
c
c
10
c
c
SET ERROR TOLERANCE

TOL = 0.0005
SET PARAM TO DEFAULT
CALL DSET (NPARAM, 0.0, PARAM, 1)
SELECT ABSOLUTE ERROR CONTROL, Reset # of MaxSteps, Select Gear
PARAM(IO) =1.0
PARAM(4) = 200000.
PARAM(12) = 2.
PARAM(13) = 1.
IDO = 1
IEND = 0
CONTINUE
IEND = EN D + 1
TEND = EN D
CALL DIVPAG (IDO, J, FNCODE, FCNJac, A, T, TEND, TOL, PARAM, Y)
Solution Data for FeCN, CN, and CN from FeCN dissociation
E (T .EQ. 5.0) CC(1)=Y(2)
278
C
C
C
IF(T .EQ. 5.0) CC(5)=Y(12)

IF(T .EQ. 5.0) CC(9)=Y(26)
IF(T .EQ. 10.0) CC(2)=Y(2)
IF(T EQ. 10.0) CC(6)=Y(12)
IF(T .EQ. 10.0) CC(10)=Y(26)
IF(T .EQ. 15.0) CC(3)=Y(2)
IF(T .EQ. 15.0) CC(7)=Y(12)
IF(T EQ. 15.0) CC(11)=Y(26)
IF(T EQ. 20.0) CC(4)=Y(2)
IF(T .EQ. 20.0) CC(8)=Y(12)
IF(T .EQ. 20.0) CC(12)=Y(26)
Tissue Data
IF(T .EQ. 20.0) CC(13)=aYZFe
I F a EQ. 20.0) CC(14)=Y(6)
I F a EQ. 20.0) CC(15)=Y(7)
IF a -EQ. 20.0) CC( 16)=aYZCNFe
I F a -EQ. 20.0) CC(17)=Y(30)
I F a -EQ. 20.0) CC(18)=Y(31)
I F a -EQ. 20.0) CC(19)=Y(9)
IF a -EQ. 20.0) CC(20)=Y(10)
IF a -EQ. 20.0) CC(21)=aYZCN
I F a -EQ. 20.0) CC(22)=Y(16)
I F a -EQ. 20.0) CC(23)=Y(17)
I F a -EQ. 20.0) CC(24)=aYZCNAm
I F a -EQ. 20.0) CC(25)=Y(19)
I F a -EQ. 20.0) CC(26)=Y(20)
IF a -EQ. 20.0) CC(27)=aYZFeAm
Root sorption treated as equality to stress data from additional stripped
root experiments
IF a -EQ. 20.0) EqCon=Y(4)
IF(IEND .LE. 20) THEN

WRITE (NOUT,(I6,33F 12.7)') IEND, T, Y
WRITE (11,'(I6,33F12.5)') IEND, T, Y
Final call to release
IF (IEND .EQ. 20) IDO = 3
GOTO 10
ENDIF
C
FORMAT(4X,TEND',5X,Time',9X,'Y 1' 9X, 'Y2' 9X, Y3' 9X,
*Y4 9X, 'Y5' 9X, Y6 9X, T 7 9X, Y8 9X,
Y9 10X, T 10 10X, T i l ' 10X, 'Y12' 10X, Y139X,
'Y14' 9X, Y15 9X, Y16 10X, 'Y17' 10X, Y18' 9X,
'Y19' 9X, Y20' 10X, 'Y21' 10X, T22' 9X, Y239X,
279
&
&
Y24' 9X, 'Y25' 10X, T 26 10X, Y27 9X, Y28 9X,

T29* 9X, T30 10X, T 3 1* 10X, Y32 9X)
C
CLOSE(ll)
RETURN
END
C
C
C
&
&
&
&
&
C
C
C
C
C
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C
SUBROUTINE FNCODE (J, T, Y, YP)

J: NUMBER OF ODEs
N : NUMBER OF DESIGN VARIABLES
IMPLICIT REAL* 8 (A-H,OZ)
DIMENSION Y(J), YP(J)
COMMON/OptStiff/ XD(17), VFeo. VCNo, YoFe, YoCN, YZFe, YZCNFe,
DCN, CWq, RORq, CWqC, RORqC, Fror, Fis, CNvol, CNQ, FeQ,
dVFedt, dVCNdt
COMMON/ROOTCONC/ aYZFE, aYZCNFE, aYZCNAM, aYZFEAM, a YZCN
ODEs & Equations for them
Flow and volume equations obtained by least squares fitting of experimental data
FeQ=0.00017*(T**2)+0.00202*T+0.09888
VFe=+5.1858-0.0028*(T**2)-0.0843*T
dVFedt=-2.0*0.0028*T-0.0843
CNQ=+0.0978-0.0004*(T**2)+0.0076*T
VCN=+5.2056-0.0002*(T**2)-0.1209*T
dVCNdt=-2.0*0.0002*T-0.1209
Joint/general parameters
RhWt=I.O
Asorb=0.129
Fis=0.112
Fror=0.89
CNvoI=0.00001
FeCN parameters
SbFe=0.196
DsFe=0.02875
FeRq=+(5.14)/1000.
ThRt=0.884
Stq=+(9.28)/1000.
ThSt=0.767
Folq=+(13.75)/1000.
280
VFeo=5.1858
YoFe=11.93
FeFor=0.001915
FeBack=0.0187
DFeCN=7.26
Assumes D*=DA/delx where A=1000 cmA2 & delx=0.05 cm
VRIFe=Fis*FeRq/RhWt
RORq=FeRq*(ThRt-Fis)/Fror
CWq=FeRq*( l-Fis)-RORq
C
C
CN parameters
SbCN=0.136
DsCN=0.0361
CNRq=+<3.84)/1000.
ThRtC=0.894
StqC=+(8.62V1000.
ThStC=0.735
FoIqC=+{ 12.12 V1000.
VCNo=5.2056
YoCN=68.63
DCN=14.4
Assumes D*=DA/delx where A=1000 cmA2 & delx=0.05 cm
VRICN=Fis*CNRq/RhWt
RORqC=CNRq*(ThRtC-Fis)/Fror
CWqC=CNRq*( 1-Fis)-RORqC
C
C
C
C
C
C
C
C
ODEs
FeCN system
Need to include a term for FeCN breakdown to free CN in control to account for rate of
dissociation - forward and backward.Plant-mediated FeCN dissociation first order
FeCN = Fe + 6CN
&
&
&
&
YP( 1)=-DsFe*Y( IVAsorb+SbFe* Y(2VAsorb

YP(2)=-(SbFe/VFe)*Y(2)+(DsFe/VFe)*Y( 1)-(FeQ/VFe)*Y(2)
-(dVFedt/VFe)*Y(2)-(DFeCN/VFe)*(Y(2)-Y(3))-FeFor*Y(2)
+FeBack*(Y(26)**6)*Y(32)
YP(3)=-(XD(l)/VRIFe)*Y(3)+(XD(2)/VRIFe)*Y(4)+(FeQ/VRIFe)*Y(2)
+(DFeCNAlUFe)*(Y(2)-Y(3))-(XD(3)*Y(3V(XD(4)+Y(3)))
YP(4)=+(XD( 1VCWq)*Y(3)-(XD(2VCWq)* Y(4)
YP(5)=+(XD(3)*VRIFe*Y(3y(RORq*(XD(4)+Y(3))))-XD(5)*Y(5)/RORq
-(XD(6)*RhWt*FeQ/(RORq*Fror)*Y(5))
YP(6)=+(RhWt*FeQ/Stq)*(XD(6)*Y(5yFror-Y(6yThSt)
YP(7)=+(RhWt*FeQ/Folq)*(Y(6)/ThSt)-XD(7)*Y(7yFolq
281
u u u u u
Need to account for assimilated product for free cyanide convention within
FeCN system; FeCN dissoc. releases 6x free cyanide but assimilated CN
produces 1:1 amount of assimilation product on molar basis of CN
YP(8)=+XD(12)*Y(29)/RORq-XD(15)*RhWt*FeQ*Y(8)/(RORq*Fror)
YP(9)=+(RhWt*FeQ/Stq)*(XD(15)*Y(8)/Fror-Y(9)/ThSt)
YP( 10)=+{RhWt*FeQ/FoIq)*(Y(9yThSt)-XD( 16)*Y( lOVFolq
&
+XD(14)*Y(31)/Folq
uuu
Free CN system - NOT DISSOCIATED CN in FeCN SYSTEM

YP( 11 )=-DsCN* Y( 11 )/Asorb+SbCN* Y( 12)/Asorb
YP( 12)=-(SbCN/VCN)*Y( 12)+(DsCN/VCN)* Y(I I MCNQ/VCN)*Y( 12)
&
-(dVCNdt/VCN)*Y( 12)-(DCN/VCN)*(Y( 12)-Y( 13))
YP( 13)=-(XD(8 WRICN)* Y( 13)+(XD(9)/VRICN)* Y( 14)+CNQ* Y( 12)/VRICN
&
+(DCN/VRICN)*(Y( 12)-Y( 13)HXD( 10)*Y( 13)/(XD( 11 )+Y(13)))
YP( I4)=+(XD(8)/CWqC)*Y( 13HXD(9)/CWqC)* Y( 14)
YP( 15)=+(XD( 10)*VRICN*Y( 13)/(RORqC*(XD( 11 )+Y( 13))))
&
-XD( 12)* Y( 15VRORqC-XD( 13)*RhWt*CNQ* Y( 15)/(Fror*RORqC)
YP(16)=+(RhWt*CNQ/StqC)*(XD(13)*Y(l5VFror-Y(I6yThStC)
YP( 17)=+(Rh Wt*CNQ/FolqC)*(Y( 16)/ThStC)-XD( 14)*Y( 17)/FoIqC
&
-XD( 17)* Y( 17)/FolqC
YP( 18)=+XD( 12)* Y( 15)/RORqC-XD( 15)*RhWt*CNQ* Y( 18)/(RORqC*Fror)
YP( 19)=+(RhWt*CNQ/StqC)*(XD( 15)* Y( 18yFror-Y( 19VThStC)
YP(20)=+(RhWt*CNQ/FolqC)*(Y( !9VThStC)+XD( 14)*Y( 17)/FolqC
&
-XD(16)*Y(20)/FolqC
u u u u u u
To account for volatilization losses, include two additional differential equations.

One for each species, YP(21) and YP(22) are the change in mass of assimilated
product volatilized for each system. Set equal to +XD(16)*Y(10) and +XD(16)*Y(20)
Total mass volatilized from t=0
YP(2l)=+XD(16)*Y(10)
YP(22)=+XD( 16)* Y(20)
u u u u u
C
C
To account for volatilization losses of CN, include terms for both CN system and
CN from dissociation in FeCN system.
Total mass volatilized from t=0
YP(23)=+XD(17)*Y(17)
YP(24)=+XD( 17)*Y(31)
Equations to account for CN from dissociation within FeCN system.
Need to combine assimilated product for free and ferrocyanide conversion within
282
UU
FeCN system
4 4
4 4
4 4
4
YP(25)=-DsCN*Y(25)/Asorb+SbCN*Y(26VAsorb
YP(26)=-(SbCN/VFe)*Y(26)+(DsCN/VFe)*Y(25HFeQ/VFe)*Y(26)
-(dVFedt/VFe)* Y(26MDCN/VFe)*(Y(26)-Y(27))+6.*FeFor* Y(2)
-6.*FeBack*(Y(26)**6)*Y(32)
YP(27)=-{XD(8yVRIFe)*Y(27)+(XD(9VVRIFe)*Y(28)
+{FeQAOUFe)*Y(26)+(DCNAaUFe)*(Y(26)-Y(27))
-(XD( 10)*Y(27)/(XD( 11)+Y(27)))
YP(28)=+<XD(8yCWq)*Y(27HXD(9yCWq)*Y(28)
YP(29)=+(XD( 10)* VRIFe* Y(27y(RORq*(XD(l I)+Y(27))))
-XD(12)*Y(29yRORq-(XD(13)*RhWt*FeQ*Y(29y(RORq*Fror))
+6.*XD(5)*Y(5yRORq
YP(30)=+(RhWt*FeQ/Stq)*(XD( 13)* Y(29)/Fror-Y(30)/ThSt)
YP(31)=+(RhWt*FeQ/FoIq)*( Y(30)/ThSt)-XD( 14)*Y(31)/Folq
-XD( 17)* Y(31yFolq+6.*XD(7)*Y(7)/Folq
UUU
Fe2+ Aqueous concentration from FeCN dissociation neglect plant interactions

YP(32)=+6.*(FeFor*Y(2)-FeBack*(Y(26)**6)*Y(32))
UUUU
Need to adjust for the concentration measured in the total root

agglomerate root compartments for CN and assimilated CN
YZFe=(VRIFe* Y(3)+CWq*Y(4)+RORq*Y(5))/FeRq
YZCNFe=(VRIFe*Y(27)+CWq*Y(28)+RORq*Y(29))/FeRq
YZFeAm=RORq*Y(8)/FeRq
YZCN=(VRICN*Y(13)+CWqC*Y( !4)+RORqC* Y( 15)yCNRq
YZCNAm=RORqC*Y( 18yCNRq
U
aYZFe=YZFe
aYZCNFe=YZCNFe
aYZFeAm=YZFeAm
aYZCN=YZCN
aYZCNAm=YZCNAm
U
DO K=1 J
YY(K)=Y(K)
ENDDO
U
RETURN
END
UUU
Subroutine to calculate Jacobian for ODE solver
283
* > fc>
fc>
C
C
C
SUBROUTINE FCNJac (J, T, Y, DYPDY)

Specifications for arguments
INTEGER J
DIMENSION Y(J), DYPDY(J,*)
COMMON/OptStiff/ XD(17), VFeo, VCNo, YoFe, YoCN, YZFe, YZCNFe,
DCN, CWq, RORq, CWqC, RORqC, Fror, Fis, CNvol, CNQ, FeQ,
dVFedt, dVCNdt
EXTERNAL DSET
Clear array to zero
CALLDSET(J**2,0.0, DYPDY, I)
DYPDY( 1,1 )=-DsFe/Asorb

DYPDY( 1,2)=+SbFe/Asorb
DYPDY(2,1)=+DsFe/VFe
DYPDY(2,2)=-SbFeATe-FeQ/VFe-dVFedtArFe-DFeCN/VFe-FeFor
DYPDY(2,3)=+DFeCNATe
DYPDY(2,26)=+6.*FeBack*(Y(26)**5)*Y(32)
DYPDY(2,32)=+FeBack*( Y(26)**6)
DYPDY(3,2)=+FeQ/VRIFe+DFeCNA^RIFe
DYPDY(3,3)=-(XD( 1)yVRIFe-DFeCNATUFe
&
-(XD(3)*XD(4))/((XD(4)+Y(3))**2)
DYPDY(3,4)=+(XD(2))/VRIFe
DYPDY(4,3)=+(XD( 1))/CWq
DYPDY(4,4)=-(XD(2))/CWq
DYPDY(5,3)=+(XD(3)*XD(4)*VRIFe/(RORq*(XD(4)+Y(3))**2))
DYPDY(5,5)=-XD(5)/RORq-XD(6)*RhWt*FeQ/(RORq*Fror)
DYPDY(6,5)=+(XD(6)*RhWt*FeQ/Stq)/Fror
DYPDY(6,6)=-(RhWt*FeQ/StqVThSt
DYPDY(7,6)=+(RhWt*FeQ/FoIq)/ThSt
DYPDY(7,7)=-(XD(7))/Folq
DYPDY(8,8)=-XD(15)*RhWt*FeQ/(RORq*Fror)
DYPDY(8,29)=+XD( 12VRORq
DYPD Y(9,8)=+(Rh Wt*FeQ/Stq)*XD( 15)/Fror
DYPDY(9,9)=-(Rh Wt*FeQ/StqVThSt
DYPDY( 10,9)=+(RhWt*FeQ/Folq)/ThSt
DYPDY( 10,10)=-XD( 16)/FoIq
DYPDY( 10,31 )=+XD( 14VFolq
DYPDY( 11,11 )=-DsCN/Asorb
DYPDY( 11,12)=+SbCN/Asorb
284
DYPDY( 12,11 )=+DsCN/VCN

DYPDY( 12,12)=-SbCN/VCN-CNQ/VCN-(dVCNdt/VCN)-(DCN/VCN)
DYPDY(12,13)=+(DCN/VCN)
DYPDY( 13,12)=-KJNQ/VRICN+{DCN/VRICN)
DYPDY( 13,13)=-(XD(8yVRICNHDCNATUCN)
&
-XD( 10)*XD( 11)/((XD( 11)+Y(13))**2)
DYPDY( 13,14)=+(XD(9VVRICN)
DYPDY( 14,13)=+{XD(8VCWqC)
DYPDY( 14,14)=-(XD(9yCWqC)
DYPDY(15,13)=+(XD(10)*VRICN*XD(1 l)/(RORqC*(XD(l 1)+Y(13))**2))
DYPDY( 15,15)=-XD( 12yRORqC-XD( 13 )*RhWt*CNQ/(Fror*RORqC)
DYPDY( 16,15)=+{XD( 13)*RhWt*CNQ/StqC)/Fror
DYPDY( 16,16)=-(RhWt*CNQ/StqCyThStC
DYPDY( 17,16)=+(Rh Wt*CNQ/FolqC)/ThStC
DYPDY( 17,17)=-XD( 14yFo!qC-XD( 17)/FolqC
DYPDY( 18,15)=+XD( 12)/R0RqC
DYPDY( 18,18)=-XD( 15)*RhWt*CNQ/(RORqC*Fror)
DYPDY( 19,18)=+(Rh Wt*CNQ/StqC)*(XD( 15yFror)
DYPDY( 19,19)=-(RhWt*CNQ/StqC)/ThStC
DYPDY(20,17)=+XD( 14yFolqC
DYPDY(20,19)=-KRhWt*CNQ/FolqC)/ThStC
DYPDY(20,20)=-XD( 16yFoIqC
DYPDY(21,10)=+XD( 16)
DYPDY(22,20)=+XD( 16)
DYPDY(23,17)=+XD(17)
DYPDY(24,31 )=+XD( 17)
DYPDY(25,25)=-DsCN/Asorb
DYPDY(25,26)=+SbCN/Asorb
DYPDY(26,25)=+(DsCNA^Fe)
DYPDY(26,26)=-(SbCNATe)-(FeQATe)-(dVFedtATe)-(DCN/VFe)
&
-36.*FeBack*(Y(26)**5)*Y(32)
DYPDY(26,27)=+(DCNATe)
DYPDY(26,2)=+6.*FeFor
DYPDY(26,32)=-6.*FeBack*(Y(26)**6)
DYPDY(27,26)=+(FeQ/VRIFe)+(DCN/VRIFe)
DYPDY(27,27)=-(XD(8)/VRIFe)-(DCN/VRIFe)
&
-XD( 10)*XD( 11y((XD( 11)+Y(27))**2)
DYPDY(27,28)=+(XD(9yVRIFe)
DYPDY(28^7)=+{XD(8yCWq)
DYPDY(28,28)=-(XD(9yCWq)
DYPDY(29,27)=+XD( 10)*VRIFe*XD( 11 y(RORq*(XD( 1i)+Y(27))**2)
DYPDY(29,29)=-XD( 12)/RORq-(XD( 13)*RhWt*FeQ/(RORq*Fror))
DYPDY(29,5)=+6.*XD(5yRORq
DYPDY(30,29)=-KXD(13)*RhWt*FeQ/StqyFror
DYPDY(30,30)=-(RhWt*FeQ/StqyThSt
285
DYPDY(31,30)=-KRhWt*FeQ/Folq)/ThSt
DYPDY(3131 )=-XD< l4)/Folq-XD( 17)/Folq
DYPDY(31,7)=+6.*XD(7yFoIq
DYPDY(32,2)=+6.*FeFor
DYPDY(32,26)=-36.*FeBack*(Y(26)**5)*Y(32)
DYPDY(3232)=-6.*FeBack*(Y(26)**6)
Return
End
286
A PPEN D IX H
EXPERIMENTAL DATA
H.1
Extraction of Cyanide From Plant Tissue
Table H.1.1 Free cyanide recovery from KCN spike samples in methanol: NaOH extraction
mixture (30/70 - v/v).
Solvent
HH
i1vf\CL
/c M
ptan im
MeOH: NaOH
MeOH: NaOH
with
Evaporation
RItratlon
N
N
Y
Y
Y
Y
N
N
N
Y
Y
N
N
N
Cyanide
Average
Concentration Concentration
(ppb)
(me/L)
0.0519
51.9
0.0519
0.0476
46.3
0.0450
0.0462
47.6
0.0490
0.0533
54.4
0.0550
0.0547
0.0121
12.2
0.0124
0.0141
13.8
0.0135
0.0138
287
Table H .lJ(a) Cyanide recovery from spike samples in chloroform: NaOH extraction mixture
(15/35 - v/v) and NaOH. Tissue samples spiked with 10960 mg/L tissue slurry, made from pea
root tissue exposed to 2 ppm CNt as ferrocyanide for 7 days. Solution samples spiked with
KCN. Chi - chloroform; NA - not applicable.
Solvent
Tissue
NaOH
NaOH:
Chi
NaOH
NaOH:
Chi
NaOH:
Chi
N
N
NaOH
NaOH
1-Day
2-Day
Solution
Tissue
Tissue
Solution
Cyanide
Cyanide
Cyanide
Cyanide
Filtration
Concentration Concentration Concentration Concentration
(mg/L)
(mg/kg)
(mg/kg)
(mg/L)
31.0
32.8
Y
31.8
31.8
44.6
39.4
NA
NA
Y
44.3
38.3
31.8
31.7
N
31.4
31.5
0.049
0.060
vI
0.063
0.058
N
0.066
0.064
0.067
0.066
NA
NA
0.001
0.001
Y
0.001
0.001
0.001
0.001
N
0.001
0.001
288
Table H.1.2(b) Cyanide recovery from tissue samples in chloroform: NaOH extraction mixture
(15/35 - v/v) and NaOH. Pea root tissue samples exposed to 2 ppm CNj as ferrocyanide or free
cyanide for 7 days.
Ferrocyanide
Solvent
NaOH: Chi
NaOH
NaOH
Cyanide
Concentration
(mg/kg)
143.2
141.1
53.8
50.9
87.5
86.5
Free Cyanide
Average
Concentration
(mg/kg)
Cyanide
Concentration
(mg/kg)
Average
Concentration
(mg/kg)
142.1
6.2
5.7
5.9
53.8
3.6
3.2
87.5
2.8
289
Table H .1J Cyanide recovery from free cyanide spike samples in hexane: NaOH (1/4 - v/v), 2octanol: NaOH (1/4 - v/v), and 1.6 g/L NaOH. Pea leaf tissue, un-exposed to cyanide, was
included in one sample of each of the alternative solvent mixtures.
Solvent
Tissue
NaOH Control
NaOH + hexane
NaOH + 2-octanol
NaOH + hexane
NaOH + 2-octanol
No
Yes
Yes
No
No
Solution Cyanide Concentration

(ppb)
36.8
26.7
31.2
45.2
40.1
290
HJ
Hydroponic Uptake Study
H.2.1 Solution
Table H.2.1.1 Individual Cyanide Concentration Replicate Data for Hydroponic Experiment
(a) Cyanide Concentration and Speciation {mg as CN/L)1"
Replicate 1
Total3 WAD4 Free
Days
Cyanide Plant
1.79
0.02
NA
0
0.09
0.02
1.37
5
FeCN
N
0.02
1.44
0.02
10
0.03
1.34
0.02
15
0.02
NA
1.81
0
1.59
0.08
0.01
5
v
FeCN
I
0.76
0.75
10
0.68
0.004
0.03
2.01
15
1.69
1.71
NA
0
1.67
1.64
1.72
5
CN
N
1.59
1.71
1.59
10
1.54
1.59
1.62
15
1.70
NA
1 .6 6
0
1.29
1.14
1.26
5
v
CN
I
0.04
0.02
10
ND
0.30
0.19
0.22
15
291
Table H.2.1.1 Individual Cyanide Concentration Replicate Data for Hydroponic Experiment
(a) Cyanide Concentration and Speciation (mg as CN/L)12 (cont.)

Replicate 2
Free
Total3 WAD4
Days
Cyanide Plant
ND
1.94
0.01
0
0.02
1.84
0.03
5
1.67
0.03
0.03
N
FeCN
10
0.03
1.59
0.08
15
0.04
1.67
0.09
20
0.04
ND
1.92
0
0.02
0.09
1.92
5
0.03
2.15
0.05
Y
FeCN
10
0.03
2.53
0.08
15
0.11
0.01
3.67
20
1.74
1.89
1.92
0
1.61
1.77
1.75
5
1.74
1.69
N
1.62
CN
10
1.86
1.71
1.78
15
1.67
1.69
1.75
20
1.81
1.89
1.90
0
1.57
1.61
1.35
5
1.21
1.12
1.15
CN
Y
10
0.01
ND
ND
15
0.03
0.01
ND
20
292
Table H.2-1.1 Individual Cyanide Concentration Replicate Data for Hydroponic Experiment
(a) Cyanide Concentration and Speciation (mg as

Replicate 3
Total3 WAD4
Days
Cyanide Plant
1.90
0.05
0
1.78
0.05
5
1.63
0.05
N
10
FeCN
1.59
0.06
15
1.62
0.06
21
1.90
0.06
0
1.90
0.03
5
1.86
0.06
Y
10
FeCN
2.52
0.10
15
3.13
0.16
21
1.83
1.80
0
1.73
1.79
5
1.71
N
1.70
10
CN
1.57
1.60
15
1.60
1.66
21
1.75
1.74
0
1.48
1.43
5
Y
1.13
1.19
CN
10
0.05
0.12
15
0.002
0.01
21
CN/L)1' (com.)
Free
ND
0.02
0.03
0.03
0.03
ND
0.004
0.004
0.01
0.004
1.55
2.00
1.77
1.71
1.72
1.60
1.50
1.13
0.14
0.01
293
Table H-2-1,1 Individual Cyanide Concentration Replicate Data for Hydroponic Experiment
(a) Cyanide Concentration and Speciation (mg as CN/L)12 (com.)

Replicate 4
Days
Total3 WAD4 Free
Cyanide Plant
0.04
0.01
1.70
0
0.01
1.64
0.05
5
0.02
1.57
0.03
10
N
FeCN
0.07
0.03
1.59
15
0.04
0.06
1.63
20
ND
1.87
0.03
0
0.04
0.02
1.74
5
0.06
0.02
1.89
10
Y
FeCN
0.02
0.10
2.32
15
0.07
0.02
20
2.68
1.94
1.90
1.90
0
1.84
1.81
1.69
5
1.74
1.75
1.74
10
CN
N
1.70
1.58
1.71
15
1.68
1.60
1.64
20
1.87
1.75
0
1.83
1.61
1.68
1.46
5
1.24
1.31
1.30
10
CN
Y
0.89
0.99
15
0.91
0.69
0.68
20
0.58
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CN'C/E and 4500-CN I/E. and
Free Cyanide as per ASTM D 4282-95. Bold indicates outlier.
2 All reported concentrations are averages of at least duplicate analyses. ND = Not Detectable. NA = Not
Applicable. Detection Limit for Total and WAD Cyanides = 0.001 ppm, for Free Cyanide = 2.50 ppb.
cyanide in total cyanide analysis.
4 Cyanide concentrations adjusted to reflect 100% recovery of complexed cyanide and 98% recovery of
free cyanide in WAD cyanide analysis.
294
Table H.2.1.1 Individual Cyanide Mass Replicate Data for Hydroponic Experiment
(b) Cyanide Mass (mg as CN)1

Replicate I
Total
Days
Cyanide Plant
9.86
0
6.99
5
N
FeCN
7.27
10
6.63
15
9.44
0
7.20
5
Y
FeCN
10
2.79
6.44
15
8.77
0
8.40
5
N
CN
8.00
10
7.68
15
8.65
0
5.75
5
vI
CN
10
ND
0.67
15
WAD
0.11
0.48
0.10
0.10
0.11
0.38
3.07
0.10
8.88
8.53
8.02
7.92
8.83
5.92
0.08
0.80
Free
NA
0.08
0.11
0.14
NA
0.07
3.12
0.01
NA
8.78
8.59
8.08
NA
5.23
0.18
1.09
295
(b) Cyanide Mass (mg as CN)1 (cont.)

Replicate 2
Total
Days
Cyanide Plant
10.10
0
9.40
5
8.33
N
10
FeCN
7.91
15
8.15
20
9.99
0
8.92
5
8.49
Y
10
FeCN
8.31
15
7.94
20
9.85
0
8.95
5
8.11
N
CN
10
8.48
15
8.20
20
9.85
0
7.30
5
4.41
Y
CN
10
ND
15
ND
20
WAD
0.05
0.16
0.16
0.41
0.46
0.22
0.43
0.20
0.27
0.25
9.99
9.05
8.45
8.86
8.27
9.88
7.52
4.53
0.03
0.03
Free
ND
0.10
0.16
0.16
0.20
ND
0.08
0.13
0.10
0.01
9.03
8.23
8.70
9.25
8.56
9.41
6.30
4.77
ND
0.08
296
(b) Cyanide Mass (mg as CN)1 (cont.)

Replicate 3
Total
Days
Cyanide Plant
9.88
0
9.13
5
8.26
10
N
FeCN
7.91
15
7.96
20
9.88
0
8.78
5
7.56
10
Y
FeCN
7.90
15
6.52
20
9.51
0
8.87
5
8.57
N
10
CN
7.94
15
7.83
20
9.11
0
6.59
5
4.34
Y
10
CN
0.17
15
0.01
20
WAD
0.27
0.27
0.26
0.31
0.31
0.32
0.14
0.25
0.33
0.32
9.38
9.20
8.63
7.81
8.11
9.05
6.38
4.57
0.36
0.03
Free
ND
0.12
0.16
0.16
0.15
ND
0.02
0.02
0.03
0.01
8.06
10.25
8.93
8.48
8.41
8.33
6.67
4.36
0.41
0.02
297
(b) Cyanide Mass (mg as CN)1(com.)

Replicate 4
Total
Days
Cyanide Plant
8.83
0
8.41
5
7.96
FeCN
N
10
8.01
15
8.16
20
9.71
0
8.41
5
8.08
FeCN
Y
10
8.13
15
7.76
20
9.91
0
8.77
5
9.00
CN
N
10
8.74
15
8.35
20
9.51
0
6.74
5
CN
5.25
Y
10
3.08
15
1.68
20
WAD
0.22
0.27
0.16
0.37
0.31
0.16
0.20
0.27
0.36
0.21
10.10
9.44
9.00
8.69
8.56
9.71
7.47
5.26
3.35
2.01
Free
ND
0.05
0.11
0.16
0.21
ND
0.10
0.09
0.07
0.06
9.90
9.57
9.03
8.09
8.16
9.09
7.80
4.99
3.03
1.97
1 CN mass obtained by multiplying concentration data by hydroponic volume.

ND = Not Detectable, NA = Not Applicable. Bold indicates oudier.
298
Table IL2.1.2 Hydroponic Solution Volume (L).
Replicate
4,4
Average
Plant
Pot
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
0
5.22
5.22
5.50
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.20
5.22
5.20
5.20
5.20
5.20
5.20
5.21
5.21
5.28
5.20
5.20
5.20
Day
10
5.08
4.15
5.04
4.10
5.04
4.4!
5.05
3.75
5.00
3.94
5.01
3.95
5.05
4.13
5.05
4.07
5.05
3.85
5.20
4.27
5.07
4.28
5.17
4.03
5.10
4.08
5.04
4.10
5.07
4.06
5
5.16
4.63
5.11
4.52
5.12
4.57
5.12
4.60
5.12
4.64
5.11
4.66
5.13
4.66
5.12
4.62
5.13
4.46
5.20
4.87
5.11
4.83
5.20
4.63
5.15
4.69
5.11
4.65
5.14
4.58
15
5.00
3.56
4.%
3.21
4.98
3.60
4.97
3.36
4.97
3.29
4.97
2.96
4.98
3.33
4.97
3.14
4.97
3.06
5.18
3.50
5.03
3.50
5.12
3.39
5.03
3.44
4.98
3.29
5.01
3.25
20
4.94
2.89
4.90
2.39
4.93
3.12
4.89
1.59
4.89
2.16
4.90
2.57
4.92
2.46
4.90
2.08
4.90
2.37
5.15
2.90
4.99
2.89
5.09
2.90
4.98
2.46
4.92
2.38
4.95
2.74
299
Table H.2.1-3 Hydroponic Solution pH.1
Pot
Replicate
1
PM
0
5
10
15
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
1
5.8
5.8
5.9
5.9
5.9
5.9
6.0
5.8
6.1
6.0
6.1
5.9
NA
5.9
6.1
6.1
6.0
6.1
6.0
2
5.8
5.7
5.8
5.9
5.9
5.8
6.0
5.7
5.5
6.1
5.8
5.8
NA
5.7
6.2
6.1
6.0
6.1
6.1
3
5.8
5.9
6.0
6.0
5.7
5.9
6.2
6.0
5.9
6.1
6.0
6.0
NA
6.0
6.2
6.1
6.0
6.0
6.1
4
5.8
5.8
6.0
5.9
5.7
5.7
6.1
5.5
5.1
6.2
5.8
5.7
NA
5.8
6.8
6.0
5.9
5.8
5.8
5
6.0
6.0
6.1
6.0
6.0
6.1
6.3
6.2
6.9
6.3
6.1
6.1
NA
6.1
6.3
6.3
6.2
6.2
6.2
6
6.1
6.0
6.1
6.1
6.1
6.0
6.2
5.8
6.9
6.3
6.0
6.0
NA
5.9
7.4
6.0
5.8
5.8
6.1
1NA = not available
300
Table H.2.1.4 Hydroponic Solution pe.1
Pot
Replicate
1
Pot
0
5
10
15
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
1
6.78
7.12
7.10
7.39
7.02
7.14
7.20
7.39
7.18
7.38
7.65
7.17
NA
7.12
6.95
7.22
6.19
6.20
6.52
2
6.78
7.14
7.07
7.26
7.02
7.13
6.99
7.00
6.97
7.26
7.54
7.01
NA
6.17
6.62
7.09
6.22
5.90
6.03
3
6.28
6.79
6.79
6.89
6.35
7.42
7.48
7.45
7.66
6.34
7.32
7.32
NA
7.36
6.39
7.03
6.08
6.16
6.23
4
6.30
6.09
5.83
5.69
6.19
6.40
6.30
6.39
6.57
NA
6.27
6.43
NA
6.20
6.25
6.24
5.32
5.19
4.94
S
3.10
3.06
3.15
3.31
3.07
3.05
3.20
3.21
3.21
2.95
2.99
3.24
NA
3.24
3.16
2.99
2.38
2.44
2.34
6
3.09
3.21
NA
NA
3.09
3.24
3.10
3.07
3.03
2.85
3.24
3.25
NA
3.03
2.95
3.27
2.85
2.88
3.14
1NA = not available
301
H.2.2 Plant Tissue
Table H ^ 2 .I Hydroponic Plant Tissue Mass (g).
Replicate
l3
4
Average
Plant
Tissue
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Treatment2
FeCN
Control
13.50
10.40
4.04
3.13
9.00
6.00
14.40
16.60
6.80
3.75
9.40
10.50
12.49
13.35
7.64
5.51
9.44
9.95
9.90
9.40
2.26
2.92
6.40
5.20
13.75
13.16
5.14
5.15
9.28
8.82
CN
9.80
2.03
6.00
12.80
2.40
9.12
13.76
7.10
10.73
11.50
1.85
9.45
12.12
3.84
8.62
1Biomass measured on fresh weight basis after exposure for 20 days to 2 ppm as cyanide ferrocyanide and free
cyanide solutions.
: Treatment designations - ferrocyanide - FeCN and free cyanide - CN. Control unexposed tissue.
5 Replicate I only exposed for 15 days.
302
Table H 2 1 ? Hydroponic Plant Tissue Water Content (%).'
Replicate
l3
4
Average
Plant
Tissue
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Leaf
Root
Stem
Control
79.55
89.09
73.20
76.76
88.24
75.61
74.21
89.74
78.38
77.00
90.11
69.57
76.88
89.29
74.19
Treatment2
FeCN
78.52
88.71
78.91
75.93
87.78
75.37
75.58
89.02
73.93
78.57
87.88
79.38
77.15
88.35
76.90
CN
75.65
88.46
71.48
75.86
88.00
76.56
74.71
90.59
73.01
77.00
88.24
73.98
75.81
88.82
73.76
1Biomass measured by difference between fresh and dry weight on a fraction of willow tissue after exposure for 20
days to 2 ppm as cyanide.
2Treatment designations - ferrocyanide - FeCN and free cyanide - CN. Control unexposed tissue.
3 Replicate 1 only exposed for 15 days.
303
Table H ^ 1 3 Hydroponic Solution Plant l5N Enrichment Data and Calculations
(a) Root Tissue
u
u
z
u
s
o
U
Z
rj
z
u
Replicate
Tissue
Mass
(mg)
N
(Mg)
N
(%)
Atom%
1SN
Atom
% 14n
R2
52
,5n
(mg/kg)2
1
2
3
4
I
2
3
4
1
2
3
4
4.2
4.8
5.0
2.8
4.8
4.4
5.0
5.6
6.9
6.4
5.0
2.8
132.4
124.9
146.3
80.5
177.3
136.9
112.4
125.6
276.3
155.6
161.3
90.4
3.15
2.60
2.93
2.88
3.69
3.11
2.25
2.24
4.0
2.4
3.2
3.2
0.3663
0.3664
0.3696
0.3654
1.6567
2.5646
1.9059
3.1332
6.8342
10.1054
5.4218
9.3264
99.6337
99.6336
99.6304
99.6346
98.3433
97.4354
98.0941
96.8668
93.1658
89.8946
94.5783
90.6736
0.0037
0.0037
0.0037
0.0037
0.0168
0.0263
0.0194
0.0323
0.0734
0.1124
0.0573
0.1029
NA
NA
NA
NA
3567
6135
4267
7768
18885
29473
14540
26882
115
95
108
105
612
798
428
703
2737
2457
1749
3011
Replicate
D W ISN
(mg/kg)
1
2
3
4
Average
1
2
3
4
1
2
3
4
115.47
95.33
108.15
105.04
106.00
612
798
428
703
2737
2457
1749
3011
Fractional
W ater
Content
<%/100)
0.8909
0.8824
0.8974
0.9011
0.89
0.887
0.878
0.890
0.879
0.885
0.880
0.906
0.882
15n
(mg/kg)3
fw
12.60
11.22
11.09
10.39
11.32
69.09
97.53
47.05
85.18
315.77
294.83
164.62
354.25
,5n
(mg/kg)3
Control
(mg/kg)3
22.7
20.2
20.0
18.7
20.4
124.4
175.5
84.7
153.3
568.4
530.7
296.3
637.6
NA
NA
NA
NA
NA
104.0
155.2
64.3
132.9
548.0
510.3
275.9
617.3
fw c
c 15n
dw
304
Hydroponic Solution Plant l5N Enrichment Data and Calculations, (cont.)1
Table H
(b) Stem Tissue
e
0
U
'W
u
Z
u
Replicate
Tissue
Mass
(mg)
N
(Mg)
N
(% )
Atom%
ISN
Atom
% I4N
R2
82
I5n
(mg/kg)2
1
2
3
4
1
2
3
4
1
2
3
4
5.5
5.3
5.0
5.1
5.8
6.4
5.0
3.7
5.5
5.0
5.0
3.9
53.L
56.5
52.9
54.6
81.8
54.4
46.3
58.3
82.1
30.8
46.5
44.1
0.97
1.07
1.06
1.07
1.41
0.85
0.93
1.58
1.5
0.6
0.9
1.1
0.3699
0.3687
0.3803
0.3677
0.4553
0.5190
0.5246
0.5179
2.3436
3.3881
2.1083
3.1883
99.6301
99.6313
99.6197
99.6323
99.5447
99.4811
99.4754
99.4821
97.6564
96.6120
97.8917
96.8117
0.0037
0.0037
0.0038
0.0037
0.0046
0.0052
0.0053
0.0052
0.0240
0.0351
0.0215
0.0329
NA
NA
NA
NA
240
414
429
411
5505
8506
4838
7927
35.7
39.3
40.2
39.4
64.2
44.1
48.6
81.6
350
209
196
361
Replicate
i
B
e
U
z
z
u
1
2
3
4
Average
1
2
3
4
1
2
3
4
,5n
(mg/kg)
dw
35.71
39.30
40.23
39.36
38.65
64.21
44.11
48.58
81.60
350
209
196
361
Fractional
W ater
Content
(%/100)
0.732
0.756
0.784
0.696
0.74
0.789
0.754
0.739
0.794
0.715
0.766
0.730
0.740
I5n
(mg/kg)3
fw
9.57
9.58
8.70
11.98
9.96
13.54
10.86
12.66
16.83
99.77
48.91
52.91
93.81
dw
I5n
(mg/kg)3
C,5N Control
(mg/kg)3
17.2
17.3
15.7
21.6
17.9
24.4
19.6
22.8
30.3
179.6
88.0
95.2
168.9
NA
NA
NA
NA
NA
6.4
1.6
4.9
12.4
161.7
70.1
77.3
150.9
fw c
305
Table H 2 1 3 Hydroponic Solution Plant ,5N Enrichment Data and Calculations, (cont.)1
Tissue
Replicate Mass
(mg)
2
s
0
U
Z
U
I
e
6
zrj
i
z
u
1
2
3
4
1
2
3
4
1
2
3
4
4.4
5.2
5.0
4.0
6.7
5.3
5.0
4.7
7.6
5.7
5.0
6.7
N
(Mg)
N
(%)
Atom%
i5N
Atom
% 14n
R2
52
DW I5N
(mg/kg)2
173.4
193.2
149.1
150.9
316.5
185.6
204.8
183.4
264.7
170.1
134.1
248.5
3.94
3.72
2.98
3.77
4.72
3.50
4.10
3.90
3.5
3.0
2.7
3.7
0.3682
0.3669
0.3686
0.3672
0.4575
0.4964
0.4593
0.4320
1.0653
1.3272
1.3618
0.8215
99.6318
99.6331
99.6314
99.6328
99.5425
99.5036
99.5407
99.5680
98.9347
98.6728
98.6382
99.1785
0.0037
0.0037
0.0037
0.0037
0.0046
0.0050
0.0046
0.0043
0.0108
0.0135
0.0138
0.0083
NA
NA
NA
NA
246
352
251
176
1919
2646
2742
1245
145
136
110
139
216
174
188
169
371
396
365
305
Replicate
DW 1SN
(mg/kg)
1
2
3
4
Average
1
2
3
4
I
2
3
4
145
136
110
139
132.46
216
174
188
169
371
396
365
305
Fractional
W ater
Content
(%/100)
0.795
0.768
0.742
0.770
0.77
0.785
0.759
0.756
0.786
0.757
0.759
0.747
0.770
,5n
(mg/kg)3
fw
29.68
31.68
28.35
31.86
30.39
46.43
41.85
45.94
36.12
90.34
95.60
92.38
70.08
,5n
(mg/kg)3
C ISN Control
(mg/kg)3
53.4
57.0
51.0
57.3
54.7
83.6
75.3
82.7
65.0
162.6
172.1
166.3
126.1
NA
NA
NA
NA
NA
28.9
20.6
28.0
10.3
107.9
117.4
111.6
71.4
fw c
1NA = not applicable

2Equations 5.2,5.3, and 5.4 used for calculating R, 5, and DW l5N.
3Calculated using equation 5 J . FW C ,SN with control value for respective tissue subtracted off.
306
Table H .2^.4 Set 2 Tissue Cyanide Speciation After 20-Day Exposure (mg/kg FW)12.
Control4
Ferrocyanide
Total CN
Replicate7 Total3 Free4 Total3 Free4 via i5N*
104
ND
0.06 0.02
112
1
155
0.67
ND
139
0.32
2
0.07
64.3
0.04
ND
87.6
3
fie
133
174
ND
1.75 0.52
4
114
0.19
Average 0.46
128
0.22
6.45
0.05
ND
1
0.09
0.32
1.63
0.17
ND
0.01
ND
2
eB
4.87
{
ND
0.04
ND
3
0.15
OS
12.4
0.11
ND
4
0.08
0.20
0.14
0.04
6.33
Average 0.04
0.05
28.9
0.01 0.04
ND
1
0.88
2
20.6
0.03
0.72
ND
0.02
tmm
9
28.0
0.15
1.07
ND
3
0.03
2
10.3
0.01
ND
4
0.02
0.38
21.9
Average 0.04
0.01
0.01
0.76
Free Cyanide
Total CN
Total3 Free4 via i5N5
548
1.61
0.51
510
0.20
1.96
276
0.01
0.18
617
2.67
4.00
488
1.66
1.12
162
ND
ND
0.17
70.1
ND
77.3
0.14
ND
151
ND
ND
115
0.04
0.04
108
0.01
ND
117
0.04
0.25
112
ND
ND
71.4
0.01
0.65
102.1
0.01
0.23
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CN'C/E and 4500-CN' I/E, and
Free Cyanide as per ASTM D 4282-95.
2 All reported concentrations are averages of at least duplicate analyses. ND = Not Detectable, NA = Not
Applicable. Detection Limit for Total and WAD Cyanides = 0.01 mg/kg. for Free Cyanide = 0.015 mg/kg.
3Cyanide concentrations adjusted to reflect 98% recovery o f complexed cyanide and 95% recovery of free
cyanide in total cyanide analysis during distillation. Concentrations also corrected for 11% free cyanide
losses during extraction.
4 Cyanide concentrations adjusted to reflect 11% loss o f free cyanide during extraction.
5Cyanide concentrations calculated from ISN data for H:0 content, 100% l5N labeling, and sample
mass.
6 Root and leaf concentrations are measured concentrations as remaining values adjusted to reflect
respective cyanide levels in control tissue.
7Replicate 1 tissues exposed for 15 days.
307
H.2.3 Stripped Root Tissue
Table H 23.1 Stripped Root Solution Data
(a) Cyanide Concentration and Speciation (ppm as CN)12

WAD4
Free
Total3
Day
Replicate
2.044
ND
NA
0
NA
0.02
1.740
5
NA
1.729
0.03
I
10
1.694
NA
0.03
15
NA
0.03
1.661
20
2.074
ND
NA
0
NA
0.02
1.751
5
2
1.807
NA
0.03
10
0.04
NA
15
1.733
0.04
1.697
NA
20
2.047
0.012
0
NA
1.826
NA
0.02
5
1.778
NA
3
10
0.03
1.718
NA
0.03
15
1.661
NA
0.04
20
0.019
0
2.050
NA
2.384
NA
5
0.02
4
NA
10
0.03
1.796
1.715
NA
0.04
15
1.694
NA
0.04
20
0
1.968
ND
NA
0.01
5
2.016
NA
1.714
5
NA
10
0.03
15
1.646
NA
0.03
1.577
20
NA
0.04
1Test Methods: Total and WAD as per APHA Standard Methods 4500-CN'C/E and 4500-CN" I/E, and
Free Cyanide as per ASTM D 4282-95. Tissue stripped prior to sorption experiment using a 3-day soak in
chIoroform:methanoI.
2 All reported concentrations are averages of at least duplicate analyses. ND = Not Detectable, NA = Not
Applicable. Detection Limit for Total and WAD Cyanides = 0.001 ppm, for Free Cyanide = 2 JO ppb.
cyanide in total cyanide analysis.
4 Cyanide concentrations adjusted to reflect 100% recovery of complexed cyanide and 98% recovery of
free cyanide in WAD cyanide analysis.
Table H.2-3.1 Stripped Root Solution Data (cont.)
(b) Normalized Average Total Ferrocyanide Concentration (as CN)

Biologically Active
Stripped Tissue
Day
1.000
1.000
0
0.904
0.954
5
0.861
0.866
10
0.834
0.835
15
0.895
0.814
20
Table H23J1 Stripped Root Tissue Data1

15N Enrichment
Replicate
Tissue
Mass
(mg)
N
(Mg)
N
(%>
Atom%
15n
Atom
% WN
R1
81
I5n
(mg/kg)2
1
2
3
4
5
Average
5.2
5.2
6.0
4.3
5.8
5.3
113.6
107.7
143.4
100.6
133.3
119.7
2.2
2.1
2.4
2.3
2.3
2.3
0.7000
0.6030
0.6244
0.6260
0.5510
0.6209
99.300
99.397
99.376
99.374
99.449
99.379
0.00705
0.00607
0.00628
0.00630
0.00554
0.00625
865
605
663
667
466
653
153
125
149
146
127
140
Replicate
DW l5N
(mg/kg)
1
2
3
4
5
Average
153
125
149
146
127
140
Fractional
Water
Content
(%/100)
0.86
0.83
0.84
0.83
0.85
0.841
dw
F W ,SN
(mg/kg)3
F W C ISN
(mg/kg)3
CISN Control
(mg/kg)3
21.14
21.32
24.07
24.94
19.28
22.15
38.0
38.4
43.3
44.9
34.7
39.87
17.7
18.0
22.9
24.5
14.3
19.5
1Equations S.2 and S.3 used for calculating R and 5.

2Calculated using equation S.4
1Calculated using equation 5.5. FW C ISN with control value of 20.39 subtracted off.
309
-o
<u
50
S'
O ' IT,
01
C
pi
-5.729
i f 0 0 50 rp
" <o
O P m*
fN p
CP
fN 5 0 fN O 5 0 fN
m P
fN
cp
in O ' 9 00 I f
o fN1 rp1 fN1 i f
o fN
1 o 1
o
o
00
c.
_o
.0
to
3
"E
-o'
a
a
E
M
B
Q.
0
3
1
s
GO
fp
S
2
s
H
ir;
ir ,
0
O
O m
00
iTi 00 O
O1
. n O '
00 r** r -
w N
m in p
fN fn 00 ir ; 00
. 0
fN rn
pi
1 1 1 1 o
IT',
fN
O
r0
pi
p
O' 3
m ir; p
50
O
1 1 1
f^. r i fN
50
p 00 P i n
O ' Jn f ir;
fN fN
n N S00
. M
N fN
' C ^'f O
^ i00
0 P , N 2 5 00
O
CN
fN
f^
00
MMq
^
O
O
N
W
N
o
n
O
O
O
O
3
N
O
fN
50
s
C/5
u>
rp o
fN cn
1 t
O1
CN iTi
o n
pi 0
CN
1
>
cuo
^ 00 p fN
p Np 0
rn r n _
O fN p i in
fN
p sc
*
O
fN o
in fN
p Oi m g
pi
p 2
p
BO
^ .
o -t -
O
ir * t
O1
'f
*f
00
CN
5 ' 0- 5* f nN ur bi _S^ o noo oo OoO oN00

o
^n SPrn r i0O
0 0 CN -
_3
fN g
cn c p
o
p
"ip w
1 n
* t* *
>
-o
u00
05
u2
if
50

>
fN pn Tf fN
O
in
3
pn
p
i
rp
m fN O
o o
05
PI
fl
pi
OO O
' 0 T* : 0 ,'m ; 0
Pv
O
O1 f
^
o
4
1 *
1l
~ w
1
1
fN Tf
p
05
fN P I 5
Tf O
s Ê
m O'
* o 9 9
3
O'
3
O'
00
i
1
fN
fN
1
O'
S m
iT cn
O
o
1
1 o
1
P
fN
1
01
-4.735
-4.996
-3.785
M
5
u
-4.956
BO
-3.727
.3
IT'i sO
rs
3
p;
00
P
(N
8 N o
8
2
00
00
pi
1E!
3
Q.
rn
tr\
pp
u
f
o
't
2 5 nP N^P I O
fN
fN fN
fN -: fN 00
5 0 00 m
O ' 00
i n 'f s
"" c n
O 5 0 fN
1
p
50 50
o
1 o N-
pi
p
m
2 8 On'
00
rf OI
I O1
O ' 0 0 1 in r~
CN
CN
50 9
PO t n f P
1 O N
1
1 1
50
PI
pi
vq
i n 00 O ' p i CN
t n 00
i n pn O ' fN 0 0 f p 00 SO
00
f p 00 5 <n p i 0 0 5 0 c n
n
in
00
fN fN
wm cp o p-i
pn
ssO os P| |* pcn
o o * - 9 9 O1
CP
00 00
OO
p
3
-3.999
-5.689
O
b.
E
a
O ' 00
CP 50
0 <T
CN
1
1
-4.502
00
O'
O1
pi
14
15
16
-C
50
O S
p i If
pi
p 00 in 05 O 05
50 50 m m m
9 't- in
*
~
! rn
if n p
1
1 l
MB
50
m,
in
o
00 in
oa
u
00
n- 05 P I
i f P fN 00 fN fN 00
m 50
00
00 P
if O
00 rp m O p 0
s 00 p pn
rn
VO 05
i f 0 00 p i fN 00
I f ' f fN
pi
o 1 p i o
1
1 pp1 1 1 o o i f 1 1 o
2a
o.
u(A
O in p O ' p p
0
O ' p m m rp
O p
fN
r p * 0 O ' in
p i pp1 c p1 p 1i if o
fN m
. fN 00 fN
O 5 0 r0 p0 O '
if
cp 00
3 fN $ 00 50 pp
O SO
pp 5 0 s

p1
i P P I rp
1
1
i f o i n p i o
50
W
s3
cp
fN 0
o
1 rp
Q
u
8
E
rp
-2.636
"5
o
c
CS
a
if
p
fp
501
-4.628
_>
50 p
m
m m
o 1' O*
-5.413
r p fN Q pp 0 0 fN 00 00
O' i f c p
O1fN
if p
O
pp
fN 3 r p CP
. Tf O
S P p m
00 O fN c p r p 0
S
o o r p I f o o 1
fN f p
1
>
-5.020
so
-4.748
00
Table H.3.1 Set of optimal adjustable parameter output distribution sets for the 17-parameter model. Data arithmetically averaged
P
n O
.
in Q
O _ir i im
H I- 2J ? (N
nn ^
O
m
cn
o' o'
o'
o'
i
fsj o
w
i i
fN
^NO
in
rO
fN
fN NO tr*.
i
1
i
m
O'
N O r i - - N
^ C t ^ - C
' Nn
o c i 5 0 ' i ^ NO
' C -
r
n
i
n > 0 ' 0 0 ' ' 0 - S 0 ' O n O f n inn o
fN
fN fn rn o o
i
1
i
r-~ fN 't
00 r . J o ' r ^ ( N
O
tT O n m
O'
fN fN
>
O
mm
O
o
00
.3
3
C
U
>
ob
at
U
J3
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H.4
Ferrocyanide Adsorption to Aluminum Oxides
Table H.4.1 Solution concentration of ferrocyanide in a system with yAKO^s, for an adsorption
study. Initial concentration was 1 mg/L ferrocyanide as CN and the solid dose was 2.0 g/L yAI2 0 3 <s). Blanks represent erroneous pH readings. ND - non-detect; DL = 0.001 ppm.
Time
(hr)
0.05
0.05
2.13
2.13
7.95
7.95
25.07
25.07
0.05
0.05
2.13
2.13
7.95
7.95
25.07
25.07
0.05
0.05
2.13
2.13
7.95
7.95
25.07
25.07
0.05
0.05
0.05
2.13
2.13
7.95
pH
3.71
4.12
3.69
3.69
3.73
3.74
3.80
9.59
9.65
9.68
9.73
9.51
9.73
9.73
4.00
4.20
4.11
4.63
5.74
5.85
6.60
6.23
6.20
6.20
6.15
6.76
6.62
7.37
Concentration
mg/L as CN
1.02
1.04
1.04
1.03
1.03
1.04
0.99
0.98
1.02
1.02
1.04
1.04
1.04
1.04
1.00
1.02
0.95
0.98
0.60
0.43
0.03
0.01
0.00
0.01
0.73
0.67
0.97
0.53
0.38
0.06
Time
(hr)
0.07
8.12
8.12
25.13
25.13
33.65
33.65
0.07
8.12
8.12
25.13
25.13
33.65
33.65
8.12
8.12
25.13
25.13
33.65
33.65
8.12
8.12
25.13
25.13
33.65
33.65
33.65
33.65
0.07
8.12
pH
4.35
6.70
6.53
6.61
6.54
7.75
7.66
6.62
7.84
7.79
7.68
7.81
8.13
8.20
8.24
8.31
8.30
8.29
8.52
8.62
8.70
8.72
8.62
8.65
8.86
8.88
9.84
9.36
Concentration
mg/L as CN
0.98
0.04
0.02
ND
ND
0.00
0.00
1.01
0.08
0.11
0.02
0.02
0.02
0.00
0.19
0.20
0.08
0.10
0.10
0.09
0.36
0.41
0.31
0.32
0.24
0.27
0.41
0.44
1.02
0.86
Table H.4.1 Solution concentration of ferrocyanide in a system with Y-ALQks) for an adsorption
study. Initial concentration was 1 mg/L ferrocyanide as CN and the solid dose was 2.0 g/L yAliO^s). Blanks represent erroneous pH readings. ND - non-detect; DL = 0.001 ppm. (cont.)
Time
(hr)
7.95
25.07
25.07
0.05
0.05
2.13
2.13
7.95
7.95
25.07
25.07
pH
7.35
7.72
7.60
9.29
9.38
9.36
9.23
8.75
8.74
8.79
8.92
Concentration
mg/L as CN
0.07
0.01
0.02
1.00
0.98
0.91
0.91
0.58
0.61
0.57
0.57
Tune
(hr)
8.12
25.13
25.13
33.65
33.65
8.12
8.12
25.13
25.13
33.65
33.65
pH
9.35
9.32
9.24
9.27
9.24
9.67
9.68
9.61
9.56
9.64
9.60
Concentration
mg/L as CN
0.86
0.88
0.81
0.79
0.78
0.98
0.96
0.96
0.95
0.91
0.93
337
Table H.4.2 Solution concentration of ferrocyanide in a system with y-AhO^s, for an adsorption
study. Initial concentration was 1 mg/L fenocyanide as CN and the solid dose was 0.6 g/L y
AljO^s). Blanks represent erroneous measurements. ND - non-detect; DL = 0.001 ppm.
Time
(hr)
0.12
25.97
25.97
25.97
33.52
33.52
0.12
33.52
33.52
25.97
25.97
25.97
33.52
33.52
25.97
25.97
25.97
33.52
33.52
8.10
8.10
25.97
25.97
25.97
33.52
33.52
0.12
33.52
33.52
8.10
pH
4.05
6.30
6.23
6.14
6.62
6.30
6.21
6.92
6.%
6.96
7.34
7.43
7.29
7.06
8.00
8.38
Concentration
mg/L as CN
1.00
ND
ND
0.003
0.00
0.01
1.00
0.03
0.02
0.00
0.07
0.08
0.04
0.42
0.44
8.17
8.13
8.89
8.89
0.28
0.28
0.83
0.80
8.65
8.65
8.59
8.69
9.98
9.41
9.36
10.05
0.56
0.61
0.55
0.56
1.03
0.95
0.91
1.02
Time
(hr)
0.05
8.05
8.05
8.05
25.55
25.55
8.05
8.05
8.05
0.05
0.05
8.05
8.05
8.05
25.55
25.55
8.05
8.05
8.05
8.05
8.05
8.05
8.05
8.05
8.05
0.05
8.05
8.05
8.05
25.55
pH
6.58
6.98
6.56
6.57
6.67
6.65
6.08
5.%
5.94
4.08
4.08
5.12
5.13
5.13
5.61
5.69
4.10
3.96
4.02
7.20
7.20
7.24
7.60
7.72
7.81
9.46
9.29
9.27
9.27
8.21
Concentration
mg/L as CN
0.97
0.22
0.39
0.24
0.03
0.05
0.16
0.25
0.25
0.96
0.15
0.17
0.15
0.00
0.01
0.20
0.23
0.18
0.31
0.33
0.40
0.55
0.44
0.56
1.00
0.92
0.94
0.90
0.48
338
Table H.4.2 Solution concentration of ferrocyanide in a system with y-AliO^s) for an adsorption
study. Initial concentration was 1 mg/L ferrocyanide as CN and the solid dose was 0.6 g/L yA l^ * ). Blanks represent erroneous pH readings. ND - non-detect; DL = 0.001 ppm. (cont.)
Time
(hr)
8.10
25.97
25.97
25.97
pH
10.07
9.91
9.87
9.89
Concentration
me/L as CN
1.00
0.97
0.94
0.95
Time
(hr)
25.55
33.52
33.52
pH
9.24
9.72
9.79
Concentration
mg/L as CN
0.93
1.00
0.97
339
Table H .4J Solution concentration of ferrocyanide in equilibrium with y-AhC^,) for 0.3 and 1.2
g/L solid doses.
Initial concentration was 1 mg/L ferrocyanide as CN.
Blanks represent
erroneous measurements. ND - non-detect: DL = 0.001 ppm.
pH
6.60
6.16
5.89
6.02
6.12
6.10
6.29
6.48
6.38
7.10
7.54
8.09
8.36
8.48
8.96
9.17
9.09
8.57
9.42
9.45
9.49
9.60
9.62
9.60
0.3 g/L
Concentration
mg/L as CN
0.034
0.030
0.023
0.096
0.089
0.063
0.203
0.199
0.181
0.551
0.653
0.785
0.796
0.789
0.910
0.917
0.892
0.631
1.005
0.914
0.994
0.972
0.991
0.976
pH
5.20
5.40
5.55
5.79
5.79
5.82
6.32
6.36
6.28
6.69
6.82
6.89
7.46
7.51
7.62
8.12
8.26
8.40
8.61
8.59
8.82
5.96
6.08
6.11
8.79
8.85
9.03
9.39
9.51
1.2 g/L
Concentration
mg/L as CN
0.005
0.009
0.005
0.005
0.009
0.013
0.036
0.029
0.032
0.032
0.025
0.036
0.083
0.111
0.169
0.415
0.415
0.500
0.525
0.525
0.661
0.009
0.012
0.012
0.806
0.752
0.836
0.937
0.925
340
Table H .43 Solution concentration of ferrocyanide in equilibrium with y-AIjO**) for 0.3 and 1.2
g/L solid doses.
Initial concentration was 1 mg/L ferrocyanide as CN.
Blanks represent
erroneous measurements. ND - non-detect: DL = 0.001 ppm. (cont.)
pH
0.3 g/L
Concentration
mg/L as CN
pH
9.44
8.81
8.89
8.93
1.2 g/L
Concentration
mg/L as CN
0.904
0.468
0.521
0.530
341
Table H.4.4 Solution concentration of ferrocyanide in equilibrium with y-AhO^s) 1.2 g/L solid
doses. Initial concentration was 0.75 mg/L ferrocyanide as CN. Blanks represent erroneous
measurements. ND - non-detect; DL = 0.001 ppm.
pH
6.59
6.56
6.12
6.46
6.66
6.88
7.24
7.70
7.98
8.03
8.54
8.42
1.2 g/L
Concentration
mg/L as CN
0.045
0.049
0.030
0.023
0.027
0.049
0.045
0.082
0.100
0.089
0.269
0.243
342
Table HAS Solution concentration for equilibrium (33 hr) adsorption of ferrocyanide to
A1(OH>3<s)
for various solid doses. Initial concentration was 1 mg/L fenocyanide as CN. Blanks
represent erroneous measurements. ND - non-detect; DL = 0.001 ppm.
Dose
(e/L)
0.0
0.0
0.0
0.2
0.2
0.2
0.6
0.6
0.6
1.25
1.25
1.25
2.0
2.0
2.0
7.5
7.5
7.5
25
25
25
75
75
75
200
200
200
75
75
75
pH
3.24
3.34
3.29
3.31
3.37
3.37
3.37
3.37
NA
3.46
3.44
NA
3.44
3.44
NA
3.50
3.50
NA
4.33
4.28
NA
6.99
6.78
NA
8.97
8.91
NA
5.71
5.83
5.85
Concentration
me/L as CN
0.965
1.315
1.033
0.971
0.912
0.924
1.193
0.969
0.958
0.928
1.205
0.963
0.955
0.917
0.961
0.804
0.779
0.838
0.556
0.591
0.588
0.787
0.795
0.791
1.021
1.014
0.994
0.562
0.525
0.558
Dose
(g/L)
200
200
200
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
pH
6.36
6.41
6.43
3.79
4.08
3.99
6.57
6.22
5.95
6.01
6.05
6.09
6.21
6.23
6.30
8.02
8.32
8.41
8.91
8.97
8.99
9.19
9.19
9.16
9.45
9.47
9.42
9.58
9.59
9.59
Concentration
mg/L as CN
0.741
0.675
0.679
0.511
0.558
0.503
0.740
0.731
0.675
0.806
0.819
0.819
0.899
0.903
0.928
0.998
0.980
0.969
0.969
0.969
0.983
0.998
0.976
0.994
0.976
0.972
0.961
0.987
0.969
0.998

Modeling Cyanide

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ProQ uest Information and Learning

Modeling Cyanide Uptake by Willows for Phytoremediation

for the degree of

UMI Number 3084715

ProQuest Information and Learning Company

Carnegie Mellon University

FOR THE DEGREE O F .

Modeling Cyanide Uptake bv Willows for Phvtoremediation

ACCEPTED BY THE DEPARTMENT OF

Civil and Environmental Engineering

APPROVED BY THE COLLEGE COUNCIL

This suggests that phloem redistribution may be important for

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

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

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

Table HJ .I .2 Hydroponic solution volume.........................................................................299

Figure 6 3 Variability in the mass fraction of initial cyanide remaining in solution

Release of free cyanide to the

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

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

Solution sample spikes of free cyanide and

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

Not all of the relevant

processes could be measured experimentally in the hydroponic study.

Uncertainty in optimal parameter values was assessed by

1.2 Organization of Thesis

Meeussen, J.L.; Keizer. M.G.: and de Haan, F.A.M.

Chemical Stability and

(1991) Cyanide and Cyanogenic Glycosides, In:

Rosenthal, G.A.; and

2.1 Cyanide Chemistry

Cyanide occurs in many different aqueous chemical forms.

The three common cyanide

free cyanide (HCN and CN),

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

strongly complexed cyanide.

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

2.2 Anthropogenic Cyanide Sources

At some sites, the use of these solid residues as HU led to

2.4 Cyanide Toxicity

Rhodanese catalyzes the conversion of low-levels of

in urine (ATSDR, 1997). Reaction of cyanide with methemoglobin forms cyanomethemoglobin

2^ Natural Cyanide Cycle

Ghosh, R.S.; Dzombak. D.A.; and Luthy, R.G.

Equilibrium Precipitation and

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

(1996) A Role for Cyanide, Derived from Ethylene Biosysnthesis, in the

Development of Stress Symptoms. Physiol. Plant. 97:772.

Meeussen, J.L.; Keizer. M.G.: and de Haan. F.A.M.

Chemical Stability and

Free Cyanide Concentration

S ource: ATSDR, 1997

Decay of insects and leaves

COMPLEXED AND FREE CYANIDE*!)

' 1' Coauthored with Stephen Ebbs and David Dzombak

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

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

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

Cyanogenic glycosides are one

classification of naturally-occurring compounds that release cyanide upon enzymatic

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

Some preliminary investigations were performed with