MINE RECLAMATION AND NITROGEN CYCLING; APPLICATION OF
MINE SUBSOIL, SOIL AMENDMENTS AND NATIVE VEGETATION AS
TOOLS FOR EFFECTIVE ECOSYSTEM RECLAMATION.
by
PIOTR GRZEGORZ DZUMEK

A thesis submitted in partial fulfillment of the requirements for the degree of:
MASTER OF SCIENCE IN ENVIRONMENTAL SCIENCE

Thesis Examining Committee:
Lauchlan Fraser (PhD), Professor and Thesis Supervisor,
Department of Natural Resource Sciences, Thompson Rivers University.
Jonathan Van Hamme (PhD), Professor and Committee Member,
Department of Biological Sciences, Thompson Rivers University.
John Karakatsoulis (PhD), Associate Teaching Professor and Committee Member,
Department of Natural Resource Sciences, Thompson Rivers University
Eric Bottos (PhD), Assistant Professor and Committee Member,
Department of Biological Sciences, Thompson Rivers University
Jerzy Szwagrzyk (PhD), Professor and External Examiner,
Department of Forestry, University of Agriculture in Krakow

© Piotr Grzegorz Dzumek 2021

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Abstract
Mining is a strong contributor to Canadian and British Columbian economies. Mining
yields raw materials without which human society would not be able to function. Also, this
industry is a source of income for thousands of households. However, because of the large
scale of operation, mining has an impact on landscapes and ecosystems including fragile and
unique ones such as grasslands. This highlights the need for an effective and ecologicallybased reclamation strategy. The reclamation of mine lands is a challenge because mine soils
following mining activity are often deprived of organic matter, nutrient-poor, possess adverse
physicochemical properties, and can be contaminated by heavy metals. Due to the large size,
the capping of reclaimed land by topsoil is often not possible, the attention of reclamation
practitioners has focused towards subsoil which is generated as a waste product and often
stockpiled in large quantities. It is an open question whether mine subsoil can play a role of
starting substrate for mine lands ecological reclamation. This is the overarching question of
this thesis: can subsoil be transformed into operational topsoil? It is hypothesized that this
process will not be successful if the proper nitrogen cycling is not fully restored as nitrogen is
one of six elements called biogenic and is intrinsic to all living organisms. The objectives of
this thesis were to investigate (1) whether subsoils collected from New Afton New Gold and
Teck Highland Valley Copper mines were suitable to sustain a viable vegetation cover, (2)
whether an application of biochar, woodchips, biosolids (nitrogen-rich) and a mixture thereof
ameliorate subsoil futures and help in subsoil transformation, (3) whether nitrogen influences
revegetation and what transformation this element passes on the way of its cycling during the
early stages of reclamation. Two potting experiments were conducted to address the research
objectives. The first one took place in the controlled conditions of a greenhouse and worked
with native in BC graminoids and legume (nitrogen fixer). The second one was conducted in
open-air conditions and worked with three native shrubby species including one nonleguminous nitrogen fixer.
The results indicated that none of the analyzed mine subsoils, due to their low fertility
and adverse physicochemical properties, was capable of sustaining viable vegetation when
unamended. The situation changed when subsoils were amended by biosolids or a mixture.
An addition of 25% of woodchips or 5% of biochar did not help. The vegetation response to
nutrient input related to biosolids application was positive in the case of both mines, however,
productivity on Teck Highland Valley Copper mine subsoil was significantly larger than
productivity on New Afton New Gold mine subsoil in both experiments (experiment 1

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Kruskal-Wallis p < 2.2e-16; experiment 2 Kruskal-Wallis, p = 1.7e-08). Physicochemical
tests revealed that New Afton New Gold mine subsoil’s high pH and salinity posed a
hindrance to overcome for vegetation growth. Additionally, throughout both experiments, the
contents of total nitrogen, mineralizable nitrogen, and ammonium decreased, while the nitrate
content increased. That in turn might indicate that in the early stage of reclamation, when
biosolids are applied as a source of nitrogen, the nitrification step of the nitrogen cycle takes
prevalence. Seven native plant species were used in this research and all demonstrated that
they can cope with harsh growing conditions created by two mines’ subsoils, however, they
need to be provided with initial nutrient sources. This research provided valuable information
about subsoil, soil amendments, and native vegetation (including nitrogen fixers) to consider
when undertaking mine lands reclamation.
Keywords: mine reclamation, subsoil, soil amendments, native plant species, nitrogen cycling,
nitrogen fixers

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Acknowledgements
I would like to thank the following people for their contribution to my project:
My supervisor, Dr. Lauchlan Fraser, for imparting his broad knowledge and for
consistent guidance throughout the entire course of my studies at Thomson Rivers University.
Dr. John Karakatsoulis for sharing his experience, personal engagement in plant material
acquisition, and hours spent to help transplant shrubby seedlings. Dr. Eric Bottos and
Dr. Jonathan Van Hamme for being always ready to help whenever I came across any
problem related to microbiology.
Students often struggle with statistics. I was not an exception. I would like to thank
Dr. Shane Rollans and Dr. Jay Prakash Singh who have helped find my path through
the thicket of numbers and data.
One person, Dr. Bogumiła Kwiatkowska, encouraged me to continue my education on
the master program. Thank you for this inspiration, support, and guidance.
This project would never have come to its realization if it were not for my partner,
Dorota Wójcik, and my entire family multifaceted support, understanding, and steadfast faith
in me.
Everyone at the Fraser Lab for sharing their thoughts in number of extraordinary
discussions, helping me learn lab procedures and instrumentation use, supporting in
greenhouse hard work with plants and soil, and for entire friendship they have showed me.
I would like to give great thanks to Metro Vancouver, as well as to New Afton New
Gold and Teck Highland Valley Copper mines for provision of research materials and
equipment, and for designation of very helpful and obliging employees.

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Table of Contents
Abstract ........................................................................................................................................... i
Acknowledgements ........................................................................................................................iii
List of Figures ................................................................................................................................ vi
List of Tables................................................................................................................................. xv
CHAPTER 1: GENERAL INTRODUCTION........................................................................................... 1
RECLAMATION ................................................................................................................................ 1
Mine Reclamation ......................................................................................................................... 1
Applied Reclamation Tools........................................................................................................... 4
NITROGEN......................................................................................................................................... 6
Nitrogen as an Element and its Cycling in the Environment .................................................... 7
Nitrogen Biogeochemical Cycle .................................................................................................... 7
How Nitrogen Transformation Influence on Other Soil Properties ....................................... 13
Importance of Nitrogen in Mine Reclamation .......................................................................... 13
AREA OF RESEARCH .................................................................................................................... 15
Mines’ Characteristics ................................................................................................................ 15
RESEARCH OBJECTIVES.............................................................................................................. 17
REFERENCES .................................................................................................................................. 18
CHAPTER 2: HOW AN ADDITION OF BIOSOLIDS, BIOCHAR, AND WOODCHIPS INFLUENCE
SUBSOIL PHYSICOCHEMICAL PROPERTIES, GRASSES AND LEGUME PERFORMANCE, AND
NITROGEN CYCLING IN GREENHOUSE CONDITIONS. ................................................................ 22
INTRODUCTION ............................................................................................................................. 22
METHODS........................................................................................................................................ 25
Study Site...................................................................................................................................... 25
Experiment Design ...................................................................................................................... 25
Soil Sampling ............................................................................................................................... 27
Tests and Instrumentation Used ................................................................................................ 27
Statistical Analyses ...................................................................................................................... 29
RESULTS.......................................................................................................................................... 30
Mortality, Productivity, Overall Subsoils’ Difference ............................................................. 30
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended .............. 39
Quantitative and Qualitative Nitrogen Content Analyses ....................................................... 43
DISCUSSION ................................................................................................................................... 51
Mortality, Productivity, Overall Subsoils’ Difference ............................................................. 51
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended .............. 54
Quantitative and Qualitative Nitrogen Content Change ......................................................... 63

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CONCLUSION ................................................................................................................................. 67
REFERENCES .................................................................................................................................. 70
CHAPTER 3: CHANGED SOIL AMENDMENTS PROPORTIONS AND ITS INFLUENCE ON
SUBSOIL PHYSICOCHEMICAL PROPERTIES, SHRUBBY PLANTS PERFORMANCE, AND
NITROGEN CYCLING IN OPEN-AIR CONDITIONS. ........................................................................ 76
INTRODUCTION ............................................................................................................................. 76
METHODS........................................................................................................................................ 78
Study Site...................................................................................................................................... 78
Experiment Design ...................................................................................................................... 78
Soil Sampling ............................................................................................................................... 81
Tests and Instrumentation Used ................................................................................................ 81
Statistical Analyses ...................................................................................................................... 81
RESULTS.......................................................................................................................................... 82
Mortality, Productivity, Overall Subsoils’ Difference ............................................................. 82
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended .............. 88
Quantitative and Qualitative Nitrogen Content Analyses ....................................................... 92
DISCUSSION ................................................................................................................................. 100
Mortality, Productivity, Overall Subsoils’ Difference ........................................................... 100
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended ............ 102
Quantitative and Qualitative Nitrogen Content Change ....................................................... 105
CONCLUSION ............................................................................................................................... 109
REFERENCES ................................................................................................................................ 111
CHAPTER 4: RESEARCH CONCLUSIONS....................................................................................... 114
RESEARCH SYNTHESIS.............................................................................................................. 114
MANAGEMENT IMPLICATIONS & FUTURE RESEARCH..................................................... 115
REFERENCES ................................................................................................................................ 117
Appendix A – First Study Remaining Elements Results.......................................................... A1
Appendix B – Second Study Remaining Elements Results ...................................................... B1

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List of Figures
Figure 1.1 Schematic diagram of biological nitrogen cycle. Yellow arrows stand for oxidation
reactions, green arrows stand for reduction. Anammox – anaerobic ammonium oxidation; DRNA –
dissimilative nitrate reduction to ammonia. ............................................................................................ 8
Figure 1.2 Nodulation on roots of field locoweed (Oxytropis campestris) on the left and soopolallie
(Shepherdia canadensis) on the right. Photos credit Piotr Dzumek...................................................... 10
Figure 1.3 New Afton New Gold and Teck Highland Valley Copper mines location. Retrieved from
Google Earth.......................................................................................................................................... 17
Figure 2.1 Mortality comparison between two mines’ subsoils: HVC – Teck Highland Valley Copper,
NA – New Afton New Gold. Boxplots describe the number of dead plants per pot. Horizontal line
indicates the median of plants per pot which did not survive to the end of the experiment. KruskalWallis test was used to compare the medians of plant mortalities. n = 175 .......................................... 30
Figure 2.2 Mortality comparison between the New Afton New Gold (New Afton) subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Boxplots
describe the number of dead plants per pot. Horizontal line indicates the median of plants per pot
which did not survive to the end of the experiment. Kruskal-Wallis test was used to compare the
medians of plant mortalities. Wilcoxon test was used for pair-wise comparison: * - p < 0.05, ** - p <
0.01, **** - p < 0.0001, ns – difference not significant. n = 35........................................................... 31
Figure 2.3 Mortality comparison between the Teck Highland Valley Copper (HVC) subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Boxplots
describe the number of dead plants per pot. Horizontal line indicates the median of plants per pot
which did not survive to the end of the experiment. Kruskal-Wallis test was used to the medians of
plant mortalities. Wilcoxon test was used for pair-wise comparison: ** - p < 0.01, *** - p < 0.001,
**** - p < 0.0001, ns – difference not significant. n = 35..................................................................... 32
Figure 2.4 Plant species mortality comparison between two mines’ subsoils: HVC – Teck Highland
Valley Copper, NA – New Afton New Gold. Horizontal line indicates the median mortality rate.
Kruskal-Wallis test was used to compare the medians of plant species mortalities. n = 35 ................. 33
Figure 2.5 Plant species mortality comparison on two mines’ subsoils: HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median mortality rate. KruskalWallis test was used to compare the medians of plant species mortalities. n = 35 ............................... 34
Figure 2.6 Dry above-ground biomass productivity comparison between two mines’ subsoils: HVC –
Teck Highland Valley Copper, NA – New Afton New Gold. Boxplots describe dry biomass weight.
Horizontal line indicates the median of weights of individual plants at the end of the experiment.
Kruskal-Wallis test was used to compare the medians of plant dry above-ground biomass productivity.
n = 700................................................................................................................................................... 35
Figure 2.7 Mean dry above-ground biomass productivity comparison between two mines’ subsoils:
HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Error bars are standard error of
the mean. n = 700 .................................................................................................................................. 35
Figure 2.8 Dry above-ground biomass production comparison between the New Afton New Gold
(New Afton) subsoil treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended
by 25% of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone,
95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture
of amendments. Boxplots describe the weights of individual plants’ dry above-ground biomass.
Horizontal line indicates the median of plants’ above-ground biomass. Kruskal-Wallis test was used to
compare the medians of plant productivity on particular subsoil treatments. Wilcoxon test was used for

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pairwise comparison * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns – difference not
significant. n = 140 ................................................................................................................................ 36
Figure 2.9 Dry above-ground biomass production comparison between the Teck Highland Valley
Copper (HVC) subsoil treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil
amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips
alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture - subsoil amended by a
mixture of amendments. Boxplots describe the weights of individual plants’ dry above-ground
biomass. Horizontal line indicates the median of plants’ above-ground biomass. Kruskal-Wallis test
was used to compare the medians of plant productivity on particular subsoil treatments. Wilcoxon test
was used for pairwise comparison * - p < 0.05, *** - p < 0.001, **** - p < 0.0001, ns – difference not
significant. n = 140 ................................................................................................................................ 37
Figure 2.10 Principal Component Analysis of two mines’ subsoils in terms of 21 predictors: pH, EC,
SOM, total N and C content, mineralizable N content, NH4+ and NO3- contents, as well as the content
of 13 basic elements. hvc – Teck Highland Valley Copper, new afton – New Afton New Gold. ........ 38
Figure 2.11 Principal Component Analysis of two mines’ subsoil treatments: 100%sub – unamended
subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - Subsoil
amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone,
mixture - subsoil amended by a mixture of amendments. hvc – Teck Highland Valley Copper, new
afton – New Afton New Gold. .............................................................................................................. 38
Figure 2.12 Two mines’ subsoil pH comparison in the initial and final stages of the experiment (left),
comparison of the subsoil pH in the initial and final stages of the experiment within each mine (right).
HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the
median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25 ..................... 39
Figure 2.13 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
pH comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub –
unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 39
Figure 2.14 Two mines’ subsoil electroconductivities comparison in the initial and final stages of the
experiment (left), comparison of the electroconductivities in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 25 .......................................................................................................................... 40
Figure 2.15 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
electroconductivities comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 40
Figure 2.16 Two mines’ subsoil soil organic matter content comparison in the initial and final stages
of the experiment (left), comparison of the soil organic matter content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
Initial n= 5, final n= 25.......................................................................................................................... 41
Figure 2.17 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
soil organic matter contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids

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alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n=
5 ............................................................................................................................................................. 41
Figure 2.18 Two mines’ subsoil sodium content comparison in the initial and final stages of the
experiment (left), comparison of the sodium content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
5, final n= 25 ......................................................................................................................................... 42
Figure 2.19 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
sodium contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 42
Figure 2.20 Two mines’ subsoil total nitrogen content comparison in the initial and final stages of the
experiment. HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 25, final n= 75
............................................................................................................................................................... 43
Figure 2.21 Comparison of the total nitrogen content in the initial and final stages of the experiment
within each mine. HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 25, final n= 75
............................................................................................................................................................... 43
Figure 2.22 New Afton New Gold subsoil treatments total nitrogen contents comparison in the initial
and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
*** - p < 0.001, **** - p < 0.0001, ns – difference not significant. initial n= 5, final n= 15 ............... 44
Figure 2.23 Teck Highland Valley Copper subsoil treatments total nitrogen contents comparison in
the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
**** - p < 0.0001, NS. – difference not significant. initial n= 5, final n= 15 ...................................... 45
Figure 2.24 New Afton New Gold subsoil treatments total nitrogen contents comparison in the initial
and final stages of the experiment to the reference of undisturbed topsoil. Subsoil treatments: 100%sub
– unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison * - p < 0.05, ** - p < 0.01, *** - p < 0.001, ns – difference not
significant. initial n= 5, final n= 15, undisturbed n=5 .......................................................................... 46
Figure 2.25 Teck Highland Valley Copper subsoil treatments total nitrogen contents comparison in
the initial and final stages of the experiment to the reference of undisturbed topsoil. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil

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amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison ** - p < 0.01, *** - p < 0.001, ns, NS. – difference not significant.
initial n= 5, final n= 15, undisturbed n=5 .............................................................................................. 46
Figure 2.26 Two mines’ subsoil mineralizable nitrogen content comparison in the initial and final
stages of the experiment (left), comparison of the mineralizable nitrogen content in the initial and final
stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New
Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 25........................................................................................................... 47
Figure 2.27 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
mineralizable nitrogen contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 47
Figure 2.28 Two mines’ subsoil NH4+ content comparison in the initial and final stages of the
experiment (left), comparison of the NH4+content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
5, final n= 25 ......................................................................................................................................... 48
Figure 2.29 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
NH4+contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 48
Figure 2.30 Two mines’ subsoil NO3- content comparison in the initial and final stages of the
experiment (left), comparison of the NO3- content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
5, final n= 25 ......................................................................................................................................... 49
Figure 2.31 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
NO3-contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5
............................................................................................................................................................... 49
Figure 2.32 Comparison of the sum of NH4+ and NO3- in the initial and final stages of the experiment.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
4, final n= 20 ......................................................................................................................................... 50
Figure 2.33 Comparison of the ratio of NH4+ to NO3- in the initial and final stages of the experiment.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
4, final n= 20 ......................................................................................................................................... 50
Figure 2.34 The greenhouse trial with graminoids and legume arranged in 7 blocks.
Photo credit Piotr Dzumek…………………………………………………………………….……….69
Figure 3.1 Second study mortality comparison between two mines’ subsoils: HVC – Teck Highland
Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of: 0 – plants which

x
did not survive to the end of experiment; 1 - plants which survived to the end of experiment. KruskalWallis test was used to compare the plant mortalities. n = 150............................................................. 82
Figure 3.2 Second study plant species mortality comparison between two mines’ subsoils: HVC –
Teck Highland Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of: 0
– plants which did not survive to the end of experiment; 1 - plants which survived to the end of
experiment. Kruskal-Wallis test was used to compare the plant mortalities. n = 50............................. 83
Figure 3.3 Second study plant species mortality comparison on two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of: 0 –
plants which did not survive to the end of experiment; 1 - plants which survived to the end of
experiment. Kruskal-Wallis test was used to compare the plant mortalities. n = 50............................. 83
Figure 3.4 Stem and twigs elongation comparison between two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Boxplots describe stem and twigs elongation of
all individual plants per mine subsoil at the end of the experiment. Horizontal line indicates the
median of stem and twigs elongation of all individual plants. Kruskal-Wallis test was used to compare
the medians. n = 150.............................................................................................................................. 84
Figure 3.5 Mean stem and twigs elongation comparison between two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Error bars are standard error of the mean. n =
150 ......................................................................................................................................................... 84
Figure 3.6 Stem and twigs elongation comparison between the New Afton New Gold (New Afton)
subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Boxplots describe stem and twigs elongation of all individual plants per subsoil treatment at the end of
the experiment. Horizontal line indicates the median of plants’ stem and twigs elongation. KruskalWallis test was used to compare the medians of plant stem and twigs elongation on particular subsoil
treatments. Wilcoxon test was used for the pairwise comparison. * - p < 0.05,, *** - p < 0.001, ns –
difference not significant. n = 30........................................................................................................... 85
Figure 3.7 Stem and twigs elongation comparison between the Teck Highland Valley Copper (HVC)
subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Boxplots describe stem and twigs elongation of all individual plants per subsoil treatment at the end of
the experiment. Horizontal line indicates the median of plants’ stem and twigs elongation. KruskalWallis test was used to compare the medians of plant stem and twigs elongation on particular subsoil
treatments. Wilcoxon test was used for the pairwise comparison. ** - p < 0.01, *** - p < 0.001, **** p < 0.0001, ns – difference not significant. n = 30 ................................................................................ 86
Figure 3.8 Second study Principal Component Analysis of two mines’ subsoils in terms of 21
predictors: pH, EC, SOM, total N and C contents, mineralizable N content, NH4+ and NO3- contents, as
well as the contents of 13 basic elements. hvc – Teck Highland Valley Copper, new afton – New
Afton New Gold. ................................................................................................................................... 87
Figure 3.9 Second study Principal Component Analysis of two mines’ subsoil treatments: 100%sub –
unamended subsoil, 95%sub5%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip
- subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. hvc – Teck Highland Valley Copper,
new afton – New Afton New Gold. ....................................................................................................... 87
Figure 3.10 Second study two mines’ subsoil pH comparison in the initial and final stages of the
experiment (left) Comparison of the subsoil pH in the initial and final stages of the experiment within
each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 1588

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Figure 3.11 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments pH comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3
............................................................................................................................................................... 88
Figure 3.12 Second study two mines’ subsoil electroconductivities comparison in the initial and final
stages of the experiment (left) Comparison of the subsoil electroconductivities in the initial and final
stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New
Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 15........................................................................................................... 89
Figure 3.13 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments electroconductivities comparison in the initial and final stages of the experiment.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
1, final n= 3 ........................................................................................................................................... 89
Figure 3.14 Second study two mines’ subsoil organic matter contents comparison in the initial and
final stages of the experiment (left) Comparison of the subsoil organic matter contents in the initial
and final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA
– New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to
compare the medians. initial n= 5, final n= 15 ...................................................................................... 90
Figure 3.15 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments soil organic matter contents comparison in the initial and final stages of the
experiment. Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by
5% of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone,
95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture
of amendments. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 1, final n= 3............................................................................................................. 90
Figure 3.16 Second study two mines’ subsoil sodium content comparison in the initial and final stages
of the experiment (left), comparison of the sodium content in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15 .......................................................................................................................... 91
Figure 3.17 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments sodium contents comparison in the initial and final stages of the experiment.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
1, final n= 3 ........................................................................................................................................... 91
Figure 3.18 Second study two mines’ subsoil total nitrogen content comparison in the initial and final
stages of the experiment. HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
25, final n= 75 ....................................................................................................................................... 92
Figure 3.19 Second study comparison of the total nitrogen content in the initial and final stages of the
experiment within each mine. HVC – Teck Highland Valley Copper, NA – New Afton New Gold.

xii
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
25, final n= 75 ....................................................................................................................................... 92
Figure 3.20 Second study New Afton New Gold subsoil treatments total nitrogen contents comparison
in the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip - subsoil amended by
25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
**** - p < 0.0001, ns, NS. – difference not significant. initial n= 5, final n= 15.................................. 93
Figure 3.21 Second study Teck Highland Valley Copper subsoil treatments total nitrogen contents
comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub –
unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. Wilcoxon test was used for pairwise
comparison ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not significant.
initial n= 5, final n= 15 .......................................................................................................................... 94
Figure 3.22 Second study New Afton New Gold subsoil treatments total nitrogen contents comparison
in the initial and final stages of the experiment to the reference of undisturbed topsoil. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison ** - p < 0.01, *** - p < 0.001, ns – difference not significant. initial n=
5, final n= 15, undisturbed n=5 ............................................................................................................. 95
Figure 3.23 Second study Teck Highland Valley Copper subsoil treatments total nitrogen contents
comparison in the initial and final stages of the experiment to the reference of undisturbed topsoil.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon
test was used for pairwise comparison ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns, NS. –
difference not significant. initial n= 5, final n= 15, undisturbed n=5.................................................... 95
Figure 3.24 Second study two mines’ subsoil mineralizable nitrogen contents comparison in the initial
and final stages of the experiment (left), comparison of the mineralizable nitrogen contents in the
initial and final stages of the experiment within each mine (right). HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was
used to compare the medians. initial n= 5, final n= 15.......................................................................... 96
Figure 3.25 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments mineralizable nitrogen contents comparison in the initial and final stages of the
experiment. Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by
5% of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone,
95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture
of amendments. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 1, final n= 3............................................................................................................ 96
Figure 3.26 Second study two mines’ subsoil NH4+contents comparison in the initial and final stages
of the experiment (left), comparison of the NH4+ contents in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15 .......................................................................................................................... 97

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Figure 3.27 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments NH4+contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3
............................................................................................................................................................... 97
Figure 3.28 Second study two mines’ subsoil NO3-contents comparison in the initial and final stages
of the experiment (left), comparison of the NO3- contents in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15 .......................................................................................................................... 98
Figure 3.29 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right)
subsoil treatments NO3-contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3
............................................................................................................................................................... 98
Figure 3.30 Second study comparison of the sum of NH4+ and NO3- in the initial and final stages of
the experiment. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 4, final n= 12........................................................................................................... 99
Figure 3.31 Second study comparison of the ratio of NH4+ to NO3- in the initial and final stages of the
experiment. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 4, final n= 12........................................................................................................... 99
Figure 3.32 Common rabbitbrush individual covered by a film of white salt crystals debilitating
plant’s resistance to wind. Plant is torn apart and killed. Photo credit Piotr Dzumek....................... 100
Figure 3.33 The second study open-air trial with three shrubby species: big sagebrush (Artemisia
tridentata), common rabbitbrush (Ericameria nauseosa), and soopolallie (Shepherdia canadensis).
Plants are randomized in 10 blocks. Photo credit Piotr Dzumek ...................................................... 110
Figure A.1 Two mines’ subsoil total carbon content comparison in the initial and final stages of the
experiment (left), comparison of the total carbon content in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 25, final n= 75 ........................................................................................................................ A1
Figure A.2 New Afton New Gold subsoil treatments total carbon contents comparison in the initial
and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison * - p < 0.05,
** - p < 0.01, **** - p < 0.0001, ns – difference not significant. initial n= 5, final n= 15 ................... A1
Figure A.3 Teck Highland Valley Copper subsoil treatments total carbon contents comparison in the
initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
*** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not significant. initial n= 5, final n= 15 ....... A2

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Figure A.4 Two mines’ subsoil aluminum content comparison in the initial and final stages of the
experiment (left), comparison of the aluminum content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n=
5, final n= 25 ......................................................................................................................................... A2
Figure A.5 Two mines’ subsoil boron, calcium, copper contents comparison in the initial and final
stages of the experiment (left), comparison of the boron, calcium, copper contents in the initial and
final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA –
New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare
the medians. initial n= 5, final n= 25..................................................................................................... A3
Figure A.6 Two mines’ subsoil iron, potassium, magnesium contents comparison in the initial and
final stages of the experiment (left), comparison of the iron, potassium, magnesium contents in the
initial and final stages of the experiment within each mine (right). HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was
used to compare the medians. initial n= 5, final n= 25.......................................................................... A4
Figure A.7 Two mines’ subsoil manganese, molybdenum, phosphorus contents comparison in the
initial and final stages of the experiment (left), comparison of the manganese, molybdenum,
phosphorus contents in the initial and final stages of the experiment within each mine (right). HVC –
Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25................................... A5
Figure A.8 Two mines’ subsoil sulfur, zinc contents comparison in the initial and final stages of the
experiment (left), comparison of the sulfur, zinc contents in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 25 .......................................................................................................................... A6
Figure B.1 Second study two mines’ subsoil total carbon content comparison in the initial and final
stages of the experiment (left), comparison of the total carbon content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 25, final n= 75 ........................................................................................................................ B1
Figure B.2 Second study New Afton New Gold subsoil treatments total carbon contents comparison
in the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip - subsoil amended by
25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison * - p < 0.05,
** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns – difference not significant. initial n= 5, final n=
15 ........................................................................................................................................................... B1
Figure B.3 Second study Teck Highland Valley Copper subsoil treatments total carbon contents
comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub –
unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. Wilcoxon test was used for pairwise
comparison * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not
significant. initial n= 5, final n= 15 ...................................................................................................... B2
Figure B.4 Second study two mines’ subsoil aluminum content comparison in the initial and final
stages of the experiment (left), comparison of the aluminum content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New

xv
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15 .......................................................................................................................... B2
Figure B.5 Second study two mines’ subsoil boron, calcium, copper contents comparison in the initial
and final stages of the experiment (left), comparison of the boron, calcium, copper contents in the
initial and final stages of the experiment within each mine (right). HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was
used to compare the medians. initial n= 5, final n= 15.......................................................................... B3
Figure B.6 Second study two mines’ subsoil iron, potassium, magnesium contents comparison in the
initial and final stages of the experiment (left), comparison of the iron, potassium, magnesium contents
in the initial and final stages of the experiment within each mine (right). HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was
used to compare the medians. initial n= 5, final n= 15.......................................................................... B4
Figure B.7 Second study two mines’ subsoil manganese, molybdenum, phosphorus contents
comparison in the initial and final stages of the experiment (left), comparison of the manganese,
molybdenum, phosphorus contents in the initial and final stages of the experiment within each mine
(right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates
the median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 15 ............... B5
Figure B.8 Second study two mines’ subsoil sulfur, zinc contents comparison in the initial and final
stages of the experiment (left), comparison of the sulfur, zinc contents in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15 .......................................................................................................................... B6

List of Tables
Table 2.1 First study experiment soil medium composition percentage breakdown. ........................... 26
Table 3.1 Table presents plant material provenience and the age when transplanted. .......................... 78
Table 3.2 Second study experiment soil medium composition percentage breakdown. ....................... 80

1

CHAPTER 1: GENERAL INTRODUCTION
RECLAMATION
Reclamation is a complex process aimed at rebuilding the functionality of areas
previously disturbed by either human activity or an occurrence of a forceful natural disaster.
Reclamation is a deliberate human intervention to restore an ecosystem to a functioning, selfsustaining, and desirable state (Brown and Amacher, 1999, Garris et al., 2016). Reclamation
of disturbed areas usually consists of the reconstruction of the physicochemical and biological
features of the soil layer in the first phase, re-vegetated soon after. Therefore the reclamation
of soil is generally the first step in the process. Soil is a living space of organisms that form
the beginning of most land food chains. When soil structure, fertility, and productivity are
restored along with the diversity of microorganisms, it facilitates the colonization of other
organisms (Rigby et al., 2016). Environmental disturbance can disrupt soil biophysical
processes, such as elemental cycling of nitrogen. Vegetation plays a pivotal role in nitrogen
cycle. If, as a result of disturbance, the vegetation cover is destroyed, the cycle becomes
strongly interrupted and the pool of nitrogen in the soil quickly decreases (Wanic and Pająk,
2012). In reclamation, plants that enrich the soil with organic matter and biogenic elements
are particularly beneficial. In this context, plants from the Leguminosae family, such as
lupine, pea, alfalfa, and vetch are particularly valued because they fix atmospheric nitrogen
(Jefferies et al., 1981 (1); Jefferies et al., 1981 (2); Aschenbach et al., 2012).
Mine Reclamation
British Columbia, Canada, mining regulations require that land disturbed as a result of
human mining activity must be reclaimed (MAC, 2018; BC Mines Act, 2021; Ministry of
Energy and Mines, 2017). A modern approach to mine reclamation does not begin at the
moment of operation termination but much earlier, even before the operation commences. It
takes place by planning and ensuring the availability of funds, technical solutions, and
specialist knowledge (BC Mines Act, 2021; Ministry of Energy and Mines, 2017;
Malaschenko et al., 2017).
Post-mining Soils
Mine soils which are dedicated for reclamation are typically low in fertility
(Bradshaw, 1997). Moreover, often such soils are also contaminated with heavy metals such
as copper, molybdenum, or zinc (Qiu and Sego, 2001; Wijesekara et al., 2016). In such
oligotrophic soils, only a few species of specialized microorganisms can survive and thrive

2

(Madigan et al., 2012). Nitrogen-fixing bacteria can often cope with such conditions and
enrich the soil with essential nitrogen, but their growth is strongly inhibited by the
concentration of heavy metals (Wanic and Pająk, 2012).
High pH constitutes a twofold effect: firstly it negatively affects soil microorganisms,
and secondly, it influences the solubility of some soil minerals and in this way reduces the
availability of some necessary elements for enzymes construction such as iron (Campbell et
al., 2008; Freeman et al., 2011; Brown and Chaney, 2016). Excessively high pH cannot be
tolerated by many bacteria that play important roles, for example in the decomposition of
dead matter. In post-mining soils, adverse pH seems to be one of the most important factors
suppressing the composition and function of the microbial community (Bru et al., 2011).
Low soil organic matter is typical for mine subsoil. The use of organic amendments
can help remediate this problem (Brown and Chaney, 2016). Organic amendments change pH
to more preferable, neutral or slightly acidic levels, provide organic substances which bond
with metallic cations, provide nutrients that could be utilized at least at the beginning of the
succession process.
Legumes and other symbiotic plants with nitrogen-fixing microorganisms may be
excellent tools for post-mining soil restoration (Elias and Chadwick, 1979; Jefferies et al.,
1981 (1); Wanic and Pajak, 2012; Santi et al., 2013; Perry et al., 2014 (1); Perry et al., 2014
(2)). For the period when the co-operation with symbiotic microorganisms is being
established, legumes use fertilizer as a source of nitrogen. Then, after developing the nodules
and N-fixing bacteria activation, they switch to nitrogen provided by hosted diazotrophs
(Strzelczyk, 2000). Legumes, though, are rarely good competitors; therefore, after
transforming the soil to a more preferable state for other plants, they can be displaced by
stronger competitors (Smyth, 1997). That naturally increases biodiversity and drives
succession. Moreover, legumes are often good forage for wildlife and livestock, which
stimulates the introduction of organic matter and nutrient cycling through feces (Elias and
Chadwick, 1979; Jefferies et al., 1981 (2)).
Soil restoration should actively use a range of tools such as soil agricultural
preparation, phytomelioration, fertilization, and soil amendments application.

3

Ecological Background for Mine Reclamation
Organisms sharing the same ecological niche constantly interact. They compete for the
same resources and living space, or conversely, form networks of symbionts to co-operate for
more efficient resource exploitation (Ratzke et al.; 2020). Therefore, mine soil reclamation
should initiate and steer processes targeting the rebuilding of these complex interactions.
Microorganisms play a pivotal ecological role. They conduct decomposition of dead matter
releasing nutrients for other organisms, they are plant and animal pathogens, but also
symbionts e.g. Frankia (Pokojska-Burdziej and Strzelczyk, 2000). Therefore, the condition of
successful reclamation is to rebuild the complexity of microbial communities and their
interactions because the course of most of the processes that take place in the soil depends on
microbes (Zornoza et al., 2016).
It is a challenge to reclaim mine soils as an environment for living organisms. Every
organism to thrive must find the necessary conditions and resources. These, according to
ecological Shelford’s law, must be embedded within brackets defining their minimum and
maximum of the tolerance. Shelford (1931) presented that each of resources and conditions
cannot go beyond either required minimum or maximum of a certain organism. If that
happens, that organism would not be able to remain in a given environment. Mine soils are
often difficult to colonize for most organisms because many conditions and/or resources go
beyond either minimum or maximum level of tolerance. For instance, often the nutrient
content is below the minimum, while simultaneously the concentration of heavy metals
exceeds the maximum of tolerance. Due to low nitrogen concentrations, only microorganisms
able to fix this element from the atmosphere may be able to primarily colonize such soils, but
the microbial activity is strongly inhibited by high heavy metal concentration and salinity
(Campbell et al., 2008; Freeman et al., 2011; Madigan et al., 2012). A deliberate provision of
nitrogen at relatively low amounts at the initial stage of reclamation could act as a succession
starting factor and heavy metals/salinity deactivation. The addition of selected soil
amendments and/or organic fertilizers target at both aims at once (Larney and Angers, 2012;
Zornoza et al., 2016).
Some organisms have special adaptations and abilities to harvest such resources that
are unavailable to other organisms. Nitrogen-fixing microorganisms are a good example here.
Their unique ability to fix atmospheric nitrogen opens a range of possibilities as they become
demanded partners for a symbiotic co-operation with a range of plants. Plants cannot fix
nitrogen on their own. Plants may only uptake nitrogen in the mineral forms from soil water

4

solution with an exceptions of some small organic molecules (Carson and Phillips, 2021). The
alternative to that is to be provided with nitrogen by microorganisms. Thus, many species of
plants and microorganisms established mutual co-operation. Plants that have the ability to coexist with nitrogen-fixing organisms might play a special ecological role in reclamation
because they quickly enrich the soil with organic matter as well as release available nitrogen
while decaying (Brożek and Wanic, 2002; Wanic and Pająk, 2012).
In the soil, at any given moment, despite the complex composition of the soil
microbiome, most bacteria are present in the inactive forms such as spores or cysts. Many
microorganisms can survive the presence of adverse conditions as spores that can persist for
many decades (Madigan et al., 2012; Bottos et al., 2014). Factors that inhibit bacterial
development are of two natures. The first is anthropogenic, e.g., depleted fertility and
productivity, ruined soil structure and heavy metals contamination caused by industrial
processes (Wijesekara et al. 2016). The second is natural and seminatural factors such as
bacterial phages, adverse climate or weather, lack or excess of oxygen, nutrient-poor soilforming rock, secreted antibiotics, and many others (Zornoza et al., 2016; Kumar et al., 2010).
Therefore, debilitating the negative influence of the above-mentioned factors by reclamation
techniques should enhance microbial life and thus increase chances for successful mine soils
reclamation.
Applied Reclamation Tools
The upper layer of stripped soil, so-called topsoil, is collected separately from the
material excavated from deeper ‘sub-soil’ layers. Topsoil is the preferred material in the soil
restoration process because it is rich in plant nutrients and has a developed microbial
community, but it is almost always scarce in mine reclamation projects. There is an interest
whether subsoil, if not heavily contaminated, might play a role of the basic soil substrate in
mine reclamation. This master thesis proposes experiments in which two mine subsoils,
various soil amendments, and a range of plant species with nitrogen fixers among them will
be used as the tools for effective mine reclamation with the proper nitrogen cycling
restoration.
Subsoil
The proposed experiments will be based on the use of subsoil from the two mines:
New Afton New Gold [NA] and Teck Highland Valley Copper [HVC]. Subsoils from those
two mines have a list of common features, however, they also differ largely.

5

New Afton New Gold Subsoil
Initial observations were that subsoil collected from New Afton New Gold was light
brown, rather dense but with rocks. Rocks tended to break easily and fall apart when hit.
Physicochemical tests show such features of the New Afton New Gold subsoil as pH
above 9, very high electroconductivity (EC), rather low organic matter content which
translates to low organic carbon and low nitrogen content. Additionally, phosphorus and
potassium content was rather low, calcium and boron elevated, but sodium concentration was
very high. Additionally, New Afton New Gold subsoil heavy metals content is elevated in
terms of copper and molybdenum (See Chapters 2 and 3, Appendices A and B).
Teck Highland Valley Copper Subsoil
Subsoil collected from Teck Highland Valley Copper was grayish in color, rather
sandy in texture but with a large amount of rocks. Rocks were solid and did not tend to break
apart. Some rocks had intensive green color resulting from a content of oxidized copper.
Physicochemical tests show such features of HVC subsoil as pH above 8, EC low,
very low organic matter content that translates to very low organic carbon. HVC subsoil
characterizes by extremally low nitrogen content, very low phosphorus content, potassium
content low, calcium, sodium not elevated and boron only slightly elevated. Additionally,
HVC subsoil heavy metals content is strongly elevated in terms of copper and molybdenum
(See Chapters 2 and 3, Appendices A and B).
Soil Amendments
Over the last decades, many ecological restoration experiments have been carried out
in which various soil amendments were tested (Pichtel et al., 1994; Gould Gizikoff, 2002;
Wanic and Pająk, 2012; Brown et al., 2014; Kelly et al., 2014). One of the important features
of soil amendments is its ready availability, in terms of proximity to site and economic
feasibility. Often they are produced as waste in many industrial or municipal processes. Thus,
reclamation practitioners may easily acquire valuable products useful to them.
Each soil amendment has different properties, therefore, it must be used properly and
in a manner well thought through. The selection of soil amendments depends on the
objectives. They are often used to enrich the soil with organic matter (Larney and Angers,
2012), provide nutrients, increase water holding capacity, introduce strains of beneficial
microorganisms (Zornoza et al., 2016), improve soil buffering, reduce the negative impact of

6

toxic substances such as heavy metals, change pH, and increase cation exchange capacity
(Curtis and Claassen, 2009; Brown et al., 2014; Kelly et al., 2014; Aschenbach and Poling,
2015; Hunt et al., 2015; Brown and Chaney, 2016).
The proposed experiments will use three different soil amendments: biosolids, biochar,
and wood chips, as well as the mixture thereof. Each of the above soil amendments targets to
ameliorate specific subsoil problems.
Vegetation
Vegetation is a crucial element of most reclamation projects. Once successfully
established, plants transform reclaimed post-mining soils on their own. When reclamation is
intended on soil substrate deeply transformed to unfavorable for vegetation, the process of
spontaneous colonization may be unsatisfactorily slow. It might even collapse if additional
factor such as harsh climate or herbivores pressure is exerted on plants. On deeply
transformed soil substrates, as mines’ subsoils are, it is recommended to support revegetation
(Smyth, 1997).
With constantly growing interest and shifting cultural values, more attention is being
paid to increase our understanding and appreciation of native plants. Scientists and
reclamation practitioners highlighted a selection of native plant species that may be
successfully used in reclamation (Elias and Chadwick, 1979).
NITROGEN
Nitrogen is an intrinsic part of the most important molecules building organisms’ cells
(Galloway et al., 2004; Salt, 2004). This element constitutes parts of such biological
molecules as DNA, RNA, ATP, amino acids, chlorophyll, and many others. Therefore, it is
indispensable in such processes as photosynthesis, respiration, heredity, tissue building, or
regulation (Imsande and Touraine, 1994; Salt, 2004; Campbell, 2008; Freeman et al., 2011).
However, the uptake of nitrogen for living organisms is often difficult. Nitrogen is
present in the lithosphere in abundance but usually absent in the rock bed on which the soil is
formed. Therefore, the only primary source of nitrogen for organisms is the air possessing
only 6.2% of Earth’s nitrogen (Rosswall, 1981). Moreover, this source of nitrogen is still
unavailable for most organisms. Only some microorganisms have the ability to fix
atmospheric nitrogen. That element, on one hand, is necessary for all living organisms, but on
the other hand, it is difficult to obtain. That is why the low nitrogen content in the topsoil is

7

often the growth limiting factor. Plants deprived of nitrogen grow slowly and are unhealthy
(Troeh and Thompson, 2005; Bernhard, 2010).
Nitrogen as an Element and its Cycling in the Environment
Nitrogen may exist in oxidation states from -3 to +5, which allows this element to
form a broad array of compounds, both organic or inorganic. The biological uptake of this
element is extremely specific. Earth’s atmosphere contains 38x108 Tg of nitrogen, while the
entire biosphere maintains only 0.009 x108 Tg (Rosswall, 1981). Even though N is present in
abundance in the atmosphere, constituting as much as 78% of it, in the atmospheric form (N2)
is unavailable to the vast majority of organisms due to its inertia. Strong triple bonds hold two
nitrogen atoms together (Bernhard, 2010). This, in turn, means that a lot of energy and
specific enzymes are required to take up this element from the atmosphere and transform it
into mineral forms before it can be utilized (Campbell, 2008; Freeman et al., 2011; Madigan
et al., 2012). Although every organism needs nitrogen only certain prokaryotes can fix it from
atmosphere. Those microorganisms are responsible for the primary provision of nitrogen to
the entire animate world (Galloway et al., 2004; Bernhard, 2010; Santi et al., 2013).
To restore nitrogen cycling in reclaimed mine soil substrate it is necessary to know
what nitrogen transformations happen at every step of its cycling, and what influences those
steps. Knowing this, the mine reclamation practitioners may exert efforts supporting those
organisms which are involved in each step of the cycle.
Nitrogen Biogeochemical Cycle
In terms of nitrogen acquisition, all living organisms may be divided into two
fundamental groups: nitrogen fixers and non-nitrogen fixers. The first group acquires nitrogen
through atmospheric nitrogen fixation. That is called diazotrophy. Non-nitrogen fixers, in
turn, divide into two groups again: organisms intaking nitrogen in mineral forms e.g. NH4+ or
NO3- from the soil water solution or organisms feeding on living or dead organic matter
containing nitrogen. Organisms that take up nitrogen through the consumption and digestion
of organic matter deplete at first the environmental pool of available nitrogen. However, while
they carry out their metabolism, they excrete end products of digestion and metabolism which
are still rich in organic nitrogen (undigested food residues) or mineral nitrogen (e.g. urea),
returning it to the environment. At the end of their life, the whole bodies, which consist of
about 15% nitrogen, return it to the environment in which organisms lived (Rosswall, 1981).
That is how nitrogen once introduced into the system moves alternately in between living

8

biomass and dead matter, forming a small sub-cycle until it ultimately comes back to the large
cycle and gets returned to the atmosphere (Fig. 1.1).
Many organisms that are not able to self-provide the needed nitrogen tackle this
problem by entering into symbiosis with organisms that do have the ability to provide them
with available nitrogen forms. Such co-symbionts divide into two categories:
 The first category is formed by organisms able to extract intensively the nitrogen
compounds from the soil water solution, as in the case of fungi.
 The second category of symbiosis is formed with organisms that have the ability for
direct nitrogen fixation from the atmosphere.
The final stage of this multi-step process is usually the conversion of mineral nitrogen
back to atmospheric nitrogen. All these processes, starting from the fixation of atmospheric
nitrogen, through its transformation, up to the return back to the atmosphere, make up the
nitrogen cycling in the environment. In terrestrial ecosystems, most of these processes are
carried out by soil microbes (Rosswall, 1981; Campbell, 2008; Zornoza et al., 2016).

Figure 1.1 Schematic diagram of biological nitrogen cycle. Yellow arrows stand for oxidation
reactions, green arrows stand for reduction. Anammox – anaerobic ammonium oxidation;
DRNA – dissimilative nitrate reduction to ammonia.

9

Nitrogen Fixation
Introducing nitrogen from the atmosphere into the biosphere by nitrogen fixation is a
first step of the cycle (Figure 1.1; Bernhard, 2010). It occurs according to the following
reaction: N2 + 8H+ + 8e− + 16ATP → 2NH3+ 16ADP + H2 + 16Pi
The nif genes encode the enzyme nitrogenase which is responsible for N-fixation.
Many varieties of this enzyme are known. N2 is fixed and transformed into NH3 or glutamine.
NH3 is either taken up by plants straight away or after the reaction with water in which it is
converted into an ammonium ion (NH4+) (Imsande and Touraine, 1994).
In the soil, nitrogen-fixing microorganisms live freely or enter into symbiotic
relationships with plants or fungi. Many forms of such co-operation have developed during
early evolution (Pokojska-Burdziej and Strzelczyk, 2000). The strength of these relationships
varies. Bacteria from the families of Arthrobacter, Azotobacter, Azospirillum and some others
live freely in the soil, but often their accumulations occur in the rhizosphere of the plant roots
(rhizospheric bacteria) as plants create preferential living conditions for them (Zornoza et al.,
2016). In turn, plants use nitrogen-rich compounds produced by these microorganisms. Other
bacteria colonize outer spaces in root tissues. These are still rhizospheric bacteria but their cooperation with plant hosts is stronger. The strongest form of symbiotic relations happens
when bacteria penetrate plant root tissues. When some bacteria only colonize the apoplastic
root space, the co-evolution of other bacteria along with the host plant led to the penetration
and colonization of the interior of the host root cells. The host plant often produces special
tissues – nodules (See Figure 1.2). In nodule’s cells symbiotic bacteria find a favorable living
space. Plants that can co-exist with nitrogen-fixing bacteria often gain a competitive
advantage and are first colonists in areas of succession (Santi et al., 2013).
In terrestrial ecosystems, in terms of amounts of fixed atmospheric nitrogen, bacteria
have varying efficiency. Bacteria that live freely in the soil are less productive. It is estimated
that soil bacteria are able to fix 5 - 50 kg N/ha/year. The intensity of nitrogen fixation depends
on many abiotic and biotic factors such as temperature, pH, the presence of competitors,
predators, antibiotics, etc. Bacteria that form advanced symbioses with plants can fix up to ten
times more nitrogen than free-living ones (Król and Zielewicz-Dukowska, 2005). The
efficiency of such endosymbionts in terms of the production of mineral forms of nitrogen is
strongly dependent. The performance depends on the species, and even strain of bacterium,
plant species, the season, plant physiological state, the presence of other nutrients, climate,

10

etc. It is estimated that some plant species of the Fabaceae family fix up to 500 kg N/ha/year
(Troeh and Thompson, 2005).
Bacteria that form a symbiosis with vascular plants can be divided into two groups.
The first is built by bacteria from the Rhizobiaceae family that form a symbiosis with
legumes. Those are genera: Rhizobium, Bradyrhizobium, Azorhizobium and others (Madigan
et al., 2012). The second group is formed by bacteria that form a symbiosis with nonleguminous plants. Representatives of eight plant families form the symbiosis with
Actinobacteria from the genus Frankia. Such a relationship with Actinobacteria is called
actinorrhizae (Huguet et al., 2004; Strzelczyk, 2000; Pokojska-Burdziej and Strzelczyk, 2000;
Santi et al., 2013).
In the mine reclamation process supporting nitrogen fixation may play an important
role (Jefferies et al., 1981) as this will take over the duty of nitrogen provision to the system
when the pool of this element provided initially by fertilization gets eventually depleted.

Figure 1.2 Nodulation on roots of field locoweed (Oxytropis campestris) on the left and soopolallie
(Shepherdia canadensis) on the right.
Photos credit Piotr Dzumek

Decomposition and Decay
From the mine reclamation point of view decomposition and decay are very important
processes that must be supported because they enrich mine soil substrate with strongly needed
organic matter and release the entire list of nutritional elements with nitrogen among them.
Nitrogen-containing compounds excreted by one organism become available for the uptake
and metabolism to other organisms. Substances excreted by many organisms are further

11

decomposed by a range of microorganisms and fungi equipped with the appropriate enzymes
(Imsande and Touraine, 1994; Bernhard, 2010; Figure 1.1). Dead animal and plant tissues
pass atrophy and end up in the soil, where they undergo rotting and decomposition. Dead
organic matter plus excrements build the pool of soil detritus (Biology Online, 2020). There is
a broad range of organisms which are soil detritivores, they feed on detritus, decomposing it,
to obtain nutrients and energy. The production of mineral forms of nitrogen as a result of
decay and decomposition is called nitrogen mineralization. This way nitrogen is mobilized
and becomes again available for uptake (Rigby et al., 2016).
As a result of decomposition of organic matter an amine group -NH2 becomes
detached from an organic compound and becomes transformed to ammonia (NH3). This
process is named ammonification (Figure 1.1). In the soil, the ammonia, readily reacts with
water forming ammonium ion: NH3 + H2O → NH4++ OH−. Plants intake nitrogen from the
soil mostly in the mineralized forms. These forms, however, account only for around 1 - 2%
of total nitrogen in the soil (Carson and Phillips, 2021). Organic nitrogen is gradually
mineralized and slowly released. Mineralization might provide 20 to 200 kg N/ha (Curtin and
Campbell, 2008). Overall nitrogen uptake may exceed 100 kg N/ha/year (Imsande and
Touraine, 1994; Troeh and Thompson, 2005).
NH4+ produced primarily by nitrogen fixers and secondarily in a process of
decomposition and decay is taken up by organisms e.g. plants through roots for their
metabolism or is utilized by nitrification microorganisms to produce energy.
Nitrification
The next group of organisms in the nitrogen cycle are bacteria and archaea carrying
out the nitrification process (Figure 1.1; Bernhard, 2010). Nitrification is the oxidation of
ammonium ion to nitrogen oxides carried out by obligatory aerobic nitrifiers (Madigan et al.,
2012). Therefore, the nitrification process takes place in aerated soils. Nitrification does not
occur in soil horizons that are permanently flooded. The nitrification has two major stages.
The first stage involves the transformation of ammonium ion and leads to the formation of
nitric acid, the second stage involves the conversion of nitric acid and leads to the formation
of nitrous acid. Typically, each step is carried out only by one type of nitrifying bacteria.
Nitrosomonas conduct oxidation of NH4+ to NO2−. Nitrobacter conduct oxidation of NO2−
to NO3−.

12

The deeper in the ground, the less oxygen. Here the denitrification bacteria begin to
manifest their activity (Madigan et al., 2012).
Denitrification
Denitrification consists of the gradual conversion of nitrogen oxides back to nitrogen
gas (Figure 1.1; Bernhard, 2010). However, for this process to be energy-efficient, the
denitrification stages require different conditions than the nitrification ones. Here, nitrogen
oxides are back reduced while the organic compounds are oxidized. Therefore, denitrifying
bacteria are usually anaerobic.
Complete denitrification from NO3- to N2 encompasses a series of steps. Passing every
step entails certain enzymes which are produced only by certain types of bacteria. Therefore,
the entire sequence of various bacteria is needed to complete the entire denitrification. The
denitrification takes place in the following stages: 1) NO3-→ NO2-, 2) NO2- → NO,
3) NO → N2O, 4) N2O → N2 (Madigan et al., 2012).
Denitrification leads to nitrogen loss, therefore is not required at the initial steps of
mine reclamation. However, it is an intrinsic step closing entire nitrogen cycling.
Dissimilative Nitrate Reduction to Ammonia (DNRA)
DNRA is a process similar to denitrification, but the final product is not atmospheric
nitrogen but NH4+ (Madigan et al., 2012). DNRA takes place in two stages: 1) NO3- → NO2-,
2) NO2- → NH4+. Not only prokaryotic organisms conduct DNRA but also some eukaryotes
(Putz et al., 2018). As DNRA does not lead to nitrogen loss, it is more preferred than
denitrification in the attempt of preserving nitrogen in the reclaimed mine soil substrate.
Anammox
In some ecosystems anammox conducting bacteria are responsible for most of the
entire N2 return to the atmosphere. Those bacteria inhabit anoxic environments. Based on
metagenomic studies, it is believed that there may be thousands of species carrying out
anammox in specific environments (Long et al., 2013; Solanki et al., 2017). Anammox is the
oxidation of NH4+ by nitrogen oxides with molecular nitrogen as the final product. Reaction
conducted is: NH4+ + NO2- → N2 + 2H2O (Madigan et al., 2012). Anammox is not demanded
in the initial phases of mine reclamation as this process leads to even faster nitrogen loss.

13

How Nitrogen Transformation Influence on Other Soil Properties
Nitrogen cycling influences a wide spectrum of other soil processes, and vice versa.
All soil processes remain in a sort of balance as well as all elements’ cycles interact with each
other (Rosswall, 1981). Having at least a bit of nitrogen to start, pioneering organisms can
begin to influence reclaimed land, exerting further transformation (Ratzke et al., 2020).
Nitrogen available in the environment has usually a positive effect on the production of plant
biomass. This, in turn, increases the content of organic matter in the soil and the carbon
sequestration (Brożek and Wanic, 2002). Thus nitrogen influences the carbon cycle.
Phosphorus has a strong influence on nitrogen cycling because P together with N are
important elements of many molecules in organisms’ cells. Deficiency of phosphorus limits
especially N fixation as well as nitrification (Rosswall, 1981).
The individual stages of the nitrogen cycle often have opposing vectors of bio and
physicochemical change in the soil (Ratzke et al., 2020). For instance, nitrogen fixation and
denitrification increase pH by uptake of H+. Protein decay also increases the pH by releasing
of OH- anions. In turn, the nitrification results in acidification due to H+ release. If nitrogen
cycling is balanced, the pH of the soil does not change much, but if any of those processes
overweight, the soil pH changes. This is an important hint for mine reclamation practitioners
indicating that all steps of the nitrogen cycle have to be rebuilt to maintain the proper pH. Soil
pH influences the rate of N-fixation. Nitrogenase has atoms of Fe and Mo in the structure. So
Fe and Mo are necessary in the N-fixation (Campbell et al., 2008; Freeman et al., 2011). In
soils of high pH, Fe and Mo are not soluble. Thus, high pH may inhibit nitrogenase synthesis.
Apart from plants, some microbes use products of nitrifiers’ metabolism: anammox
and DNRA bacteria, as well as denitrifiers. Their role in the reclaimed soil should not be
forgotten. They prevent leaching of nitrogen oxides to the groundwater, take part in organic
matter mineralization, take part in soil microbial food chain, and increase pH (Campbell et al.,
2008; Freeman et al., 2011; Madigan et al., 2012).
Importance of Nitrogen in Mine Reclamation
Mine reclamation will not be successful if nitrogen cycling is not rebuilt, often
requiring human intervention to increase the speed of recovery. There are two major targets.
Firstly, it is to provide an initial nitrogen portion ensuring starting it to cycle, and secondly, it
is to prevent nitrogen loss at least at the initial stage of reclamation. Nitrogen may be provided

14

to the reclaimed mine soils in two ways: artificially by fertilization or naturally by nitrogen
fixation. Artificial fertilization in the case of deeply transformed mine soils is often necessary.
That initial introduction of nitrogen along with other nutrients intends to increase the level of
lacking elements above the minimum of tolerance for organisms to colonize. Often microbes
are present in the soil substrate in an abundance, however, they try to wait out the adverse
conditions in a form of spores (Madigan et al., 2012). Fertilizers may activate them (Suhag,
2016). This may be done with the use of mineral fertilizers, organic ones, or even with the use
of nutrient-rich soil amendments. Commercial organic fertilizers or soil amendments seem to
be better solution than mineral for initial complex nutrients provision in reclaimed mine soils
(Larney et al., 2009, Gardner et al., 2010). Some soil amendments may remediate the problem
of lacking soil nutrients.
When initial portions of nutrients are provided, it is a time to introduce organisms that
will facilitate further nitrogen provision as these nutrients may be quickly depleted or lost.
Plants that co-exist with N-fixing bacteria can fulfill this role. They are used by people in
agriculture, in the phytomelioration process, as well as in the bioremediation or the
reclamation (Elias and Chadwick, 1979; Jefferies et al., 1981; Santi et al., 2013, Wanic and
Pająk, 2012). Legumes such as Lupinus, Trifolium, Medicago are most commonly used here.
BC flora has many species forming co-existence with N-fixing microbes (Brown and
Amacher, 1999; Antos et al., 1996).
In mine reclamation nitrogen cycling may be supported and its loss limited. In the case
of mine soils, in which microbiological life is negatively affected (Baker et al., 2011; Larney
and Angers, 2012), it is unlikely that the plant will find its symbiotic partner. To increase the
chances, bacteria can be inoculated by commercial inoculants. To support plants forming
actinorrhizae with Frankia, it may be most effective to add a portion of natural soil
(Strzelczyk, 2002). Nitrifying bacteria are obligately aerobic. In clay and loamy soils, the
ground tends to long water holding. This results in unfavorable aerobic conditions. Therefore,
an intervention, such as tillage, aiming in better aeration of reclaimed soils, supports nitrifiers
and other topsoil aerobic organisms. Tillage, by oxygen provision, limits denitrifiers, and
anammox bacteria in the topsoil (Long et al., 2013). Therefore, if their activity is limited,
tillage contributes to available nitrogen lost prevention. Supporting DNRA bacteria also
prevents nitrogen loss. Providing of organic matter and sulfide (S2-) also supports DNRA
bacteria while inhibiting denitrifiers (Putz et al., 2018).

15

AREA OF RESEARCH
New Afton New Gold is located just 15 km away from the city of Kamloops, BC.
Teck Highland Valley Copper is located around 50 km away. Both mines lie within
Thompson-Nicola Regional District of British Columbia. Although not very far from each
other both these mines differ strongly (New Gold Inc., 2021; Teck Resources Limited, 2019;
“Highland Valley Copper”, 2021). Differences encompass such aspects as elevation above the
see level, climate, soils, vegetation, bedrock, and others.
New Afton New Gold lies in the area of Bunchgrass Biogeoclimatic Zone which is
characterized by high biodiversity and its uniqueness. Bunchgrass covers merely about 1% of
the BC area (Province of British Columbia, 1999) but only within this zone 30% of
endangered species of BC have their populations.
HVC, on the other hand, is characterized by its large size and, consequently, a
significant impact on the environment (Teck Resources Limited, 2019; “Highland Valley
Copper”, 2021; Malaschenko et al., 2017).
Reclamation undertakings withing both mines are challenging due to several facts:
climatic severity, large scale, a vulnerability to species invasiveness, areas’ local water
retention importance, elevated fire threat and many more.
Mines Characteristics
Both mines extract and pre-treat ores and possess open pits. NA pit is inactive, while
HVC has several active and inactive pits. As a result of an ore production within the mines,
there is a large scale constant hauling and heavy machinery movement in soil and rock
transportation. Earthy waste material is gathered on large stockpiles that transforms the
surface creating vast areas for future reclamation. Additionally, both mines possess tailings
storage facilities (TSF).
New Afton New Gold Mine
New Afton site is one of New Gold Inc. assets. All New Gold’s claims within the New
Afton Group span the area of 124.5 km2. Chiefly it comprises an open pit, underground
mining operation, support facilities, a concentrator, and tailings facilities. An underground
operation began in 2012, located underneath an open pit which had been exploited under the
operation of the historic Afton Mine (New Gold Inc., 2021). The New Afton reserves are
estimated for 1.0 million ounces of gold, 2.8 million ounces of silver, and 802 million pounds

16

of copper. The mine produces annually over 75 thousand ounces of gold and over 85 million
pounds of copper (New Gold Inc., 2021).
New Afton is located within the Bunchgrass biogeoclimatic zone (Province of British
Columbia, 1999). The Bunchgrass zone’s principal environmental factor determining its
distinctiveness is mean annual precipitation below 300 mm. Most of the precipitation falls
within the coldest period of the year. This zone is classified as semi-arid characterized by cool
winters and hot, dry summers. Plants native to these growing conditions have developed
mechanisms for the effective use of water from the thaw, and to endure long periods of
drought. Here grasses are dominating, e.g. bluebunch wheatgrass Pseudoroegneria spicata or
prairie junegrass Koeleria macrantha. Other plants well adapted to these conditions are shrubs
with the dominants big sagebrush Artemisia tridentata and common rabbitbrush Ericameria
nauseosa. Their adaptive strategy is intensive root growth. The soils of this zone, mainly due
to the abundance of grasses, are more fertile and have a favorable pH. Besides, they are
sufficiently airy and are not excessively dense. (Antos et al., 1996; Province of British
Columbia, 1999).
Teck Highland Valley Copper Mine
Highland Valley Copper mine is one of many Teck assets located in Canada. It is one
of the largest open-pit mines in the world. This site exploits mainly copper and molybdenum.
The entire mine area comprises the open pits, the transportation network, rocks grinding and
ores concentration facilities, tailings storage facilities and large areas of terrains already
subjected to reclamation. HVC produces over 150,000 tonnes of copper annually (Teck
Resources Limited, 2019). The Teck Highland Valley Copper mine is one of British
Columbia’s high-value assets but, being one of the world’s largest open-pit production
highlights the need for reclamation research. A large number of reclamation projects are
conducted presently either on an operational level as well as on experimental (Teck Resources
Limited, 2019; “Highland Valley Copper”, 2021; Malaschenko et al., 2017).
HVC is located around 1,300 m above sea level, while NA’s elevation is only around
700 m. Therefore, in the place of HVC location, there are different climatic conditions. HVC
biogeoclimatic zones are Interior Douglas-Fir and Montane Spruce. These zones, in particular
Montane Spruce, are characterized by a cool climate, shorter vegetation season, but receiving
a significantly larger mean precipitation. These zones are covered with woody vegetation,
where douglas fir Pseudotsuga menziesii, white spruce Picea glauca, and even subalpine fir

17

Abies lasiocarpa dominate alternately. The nitrogen fixer soopolallie Shepherdia canadensis
is also typical here. In these zones, the soils are usually characterized as nutrient-poor, stony
with an acidic pH. Nitrogen fixers can play an extremely important role here as the suppliers
of nitrogen (Imsande and Touraine, 1994; Antos et al., 1996; Province of British Columbia,
1999).
New Afton

HVC
Figure 1.3 New Afton New Gold and Teck Highland Valley Copper mines location.
Retrieved from Google Earth.

RESEARCH OBJECTIVES
Two proposed experiments focused on:
I.

Testing whether the mine subsoils from New Afton New Gold and Teck Highland
Valley Copper have a potential to be applied as a mine reclamation starting
medium, without the addition of an amendment, on selected native grasses, forb
and shrubs.

II.

Testing whether applied soil amendments influence plant productivity and
mortality of selected native grasses, forb and shrubs on the New Afton New Gold
and Teck Highland Valley Copper subsoil substrates;

III.

The qualitative and quantitative change of total nitrogen, mineralizable nitrogen,
NH4+, NO3- in the soil following soil amendments and plant species application.

First study experiment 1 was conducted in optimal plant-growth conditions of the
greenhouse. In contrast, second study experiment was conducted in semi-natural conditions in
open-air. Eventually, results of both experiments were compared.

18

REFERENCES
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22

CHAPTER 2: HOW AN ADDITION OF BIOSOLIDS, BIOCHAR, AND WOODCHIPS
INFLUENCE SUBSOIL PHYSICOCHEMICAL PROPERTIES, GRASSES AND LEGUME
PERFORMANCE, AND NITROGEN CYCLING IN GREENHOUSE CONDITIONS.
INTRODUCTION
As awareness of environmental values increase, an ecosystem-oriented reclamation
constantly gains importance (Brown and Amacher, 1999; MAC, 2018). This branch of human
activity is complex and interdisciplinary, combining knowledge from many fields of science.
Reclamation achieves the best results when it emulates natural processes (Jefferies et al.,
1981; Bradshaw, 1997; Bradshaw, 2000). To rebuild an ecosystem, the original ecosystem
must be well known. The ecosystem is a very complex network of mutual interdependencies
between its biotic and abiotic components. Moreover, organisms occupying a certain niche
organize in food webs (McCann, 2007; Egerton, 2007). Energy and matter flow through every
level of trophic interactions, allowing for the cycling of elements (Madigan et al., 2012).
Restoration of such relations between living organisms and their ecological niche should be
the aim of the reclamation process to make it effective (Fraser et al., 2015). Reclaiming areas
disturbed by mining is challenging. Mines alter the landscape, transform and remove soils,
influence hydrology, increase dust emission, and can leach contaminants into water bodies
and soils (Wijesekara et al., 2016). Additionally, mine degraded soils are usually infertile.
These issues, and other factors like high salinity or extreme pH, result in the low productivity
of these soils (FAO et al, 2020).
An efficient way to reclaim mine barren land is to cap with a layer of fresh topsoil.
The problem is that topsoil is scarce or is deeply transformed after many years of stockpiling
(Strohmayer, 1999). In such a case attention is turned toward subsoil, which is abundant on
mine sites, also stored as stockpiles. There is interest in whether subsoil can be used as a
reclamation starting soil medium. In subsoil characterization, the potential obstacles to
ecosystem restoration might be identified, and then these obstacles can be addressed in a
planned manner.
Another question is whether soil amendments could help transform subsoil.
Knowledge about the properties of soil amendments is increasing, particularly as applied to
mine reclamation (Brown et al., 2014; Brown and Chaney, 2016; Gunarathne et al., 2020),
nonetheless, still requires deepening, in particular about the proper selection, doses, and
methods of application. Little is known about the influence of the soil amendments on the

23

properties of mine subsoils. Three amendments have received particular attention in this
study: class A biosolids, biochar, and woodchips, as well as the mixture thereof. Biosolids
result from wastewater treatment (Sullivan et al., 2015; Ippolito et al., 2021). Annacis
Biosolids used in this study were provided by Metro Vancouver. This class A biosolids,
through anaerobic digestion and dewatering, pass very strict requirements for pollutants,
pathogens and vector attraction reduction along with others. Biosolids is an amendment very
rich in readily available nutrients, as well as with organic matter (Sullivan et al., 2015).
Biosolids provide a large portion of available and mineralizable nitrogen. Available nitrogen
is predominantly in a form of NH4+. Apart from nitrogen, biosolids enrich the soil substrate
with phosphorus and zinc, and also increase soil buffering, cation exchange capacity, reduce
substrate salinity, as well as the adverse impact of heavy metals (Brown and Chaney, 2016).
The biosolids application rate strongly depends on substrate N and P deficiency. It might also
vary regarding to the biosolids class, composting time etc. (Sullivan et al., 2015). Cogger and
Stahnke (2013) suggest an application of 15 to 20% by volume to land being prepared for new
lawn but also inform that the rate for new gardens should be larger. Larney and Angers (2012)
state that reclaimed mine soils are usually subjected to the largest organic amendment dosing
due to its deep degradation. Woodchips is an amendment rather poor in readily available plant
nutrients (Cheng, 2008). Moreover, this material decomposes very slowly, mainly due to
lignin content (Datta et al., 2017). Woodchips get produced as a waste in the sawmills
processes. This material is rough, contains different sized fractions of woody tissues, mainly
coniferous tree cortex. The addition of woodchips lowers the pH of overly alkaline soil
substrates, helps in increasing recalcitrant carbon, increases water penetration and aeration,
and constitutes a food source for a range of organisms (Yuan et al., 2020). In his work on
potting media Bugbee (2008) combined 10, 20 and 30% (v/v) of hardwood sawdust
(woodchips) with municipal biosolids compost to observe an influence of this amendment on
Coreopsis grandiflora and Rudbeckia hirta individuals performance, as well as on mineral
nitrogen leaching. He found that an application of woodchips in such rates did not affect NH4+
leaching from biosolids and did not affect plants performance. Cheng (2008) determined that
an application of 30% sawdust applied with NPK fertilizer gave the highest yield. Biochar is a
charcoal. It is a slow-release fertilizer rich in phosphorus and potassium and acting
beneficially on soil physicochemical properties (Taylor, 2010; Canadian AgriChar, 2020).
This soil amendment has a strongly alkaline pH – around 10. It enriches soil substrate with
organic matter. Biochar may increase soil buffering, cation exchange capacity, reduce salinity,
and an adverse impact of heavy metals (Kelly et al., 2014). AgriChar, which is a manufacturer

24

of biochar product used in this research, suggests using 5% of this product by volume
(Canadian AgriChar, 2020). Gunarathne et al. (2020) in their experiment with salt-affected
acidic soil used 1.0% - 5.0% detecting that eventually an addition of 5.0% of biochar brought
the best results.
Vegetation is one of the key factors in mine reclamation. Intentionally introduced
plants species are generally selected according to plant traits such as rate of growth,
ecological requirements and function, the potential in competition with other plant species,
the ability to enrich the soil substrate with essential nutrients, for example nitrogen (Elias and
Chadwick, 1979). The ecosystem will likely not be rebuilt stably if the normal cycle of
elements is not restored. Nitrogen-fixing plants can play a special role in reclamation of mine
soils that are particularly nutrient-poor. Lupine, clover, or alfalfa are more commonly used
nitrogen-fixers for phytomelioration and green fertilization worldwide. However, many of
these plants do not belong to the local flora. Four native plant species were selected: three
grass species: bluebunch wheatgrass Pseudoroegneria spicata, rough fescue Festuca
campestris, prairie junegrass Koeleria macrantha, and one legume field locoweed Oxytropis
campestris. Three selected graminoids are turf-forming perennial bunchgrasses. They may be
found in many ecosystems but are typical and commonly present in grasslands. Those plants
are well adapted to semi-arid conditions with hot and dry summers (Antos et al., 1996). That
legume may be found in many ecosystems but thrives the best on rocky, gravelly soils with
plenty of insolation. It stands well with harsh climate pressure but is not a good competitor
(Douglas at al., 1998), which potentially, coupled with an ability for nitrogen fixation, makes
this species a good candidate for post-mining reclamation tool.
The study objectives were:
I.

To determine similarities and differences between New Afton New Gold and Teck
Highland Valley Copper subsoils with or without amendments by observing plant
mortality, above-ground biomass productivity, and subsoil physicochemical
properties;

II.

To test the relative effects of various proportions of the three soil amendments
(biosolids, woodchips, biochar) on subsoil physicochemical properties;

III.

To measure qualitative and quantitative change of nitrogen compounds related to
soil amendments.

25

METHODS
Study Site
Subsoil substrate was collected from stockpiles at two mine sites: Teck Highland
Valley Copper (August 17, 2018) and New Afton New Gold (September 9, 2018). The
experiment was located in the Research Greenhouse at Thompson Rivers University. The
experiment began on December 22, 2018, and ended on April 13, 2019. It lasted 16 weeks
counting from when the seeds were sown to the day of the above-ground biomass harvesting.
Experiment Design
Pots and Seeding
1.2 L square-shaped pots were filled with 10 combinations of subsoils with soil
amendments (See Table 2.1). To increase chances for successful germination, seeds were
sown in batches instead of sowing just one seed. One batch of seeds (20 seeds in each) was
sown in each of the pot corners. This way four one-species monoculture treatments and one
four-species mix treatment were created. Grass seeds were sown on the surface of the
substrate and legume seeds were covered by a thin layer of soil substrate. After germination,
excess seedlings were eliminated such that one individual plant at each pot corner remained.
Pots were marked and numbered.
To boost the presence of N-fixing microbes, the legume seeds were inoculated with
use of commercial preparation McKENZIE SEEDS GARDEN INOCULANT containing
bacteria Rhizobium leguminosarum and Bradyrhizobium sp. Before seeding, legume seeds
were subjected to scarification and 24 h water imbibition (Baskin and Baskin, 1998).
Greenhouse Settings
The greenhouse was on a 14h/10h day/night lighting schedule with three 1,000W
lamps operating. 24-hour temperature variation was kept within 15 - 30°C interval.
The substrate was kept moist but not wet. As this experiment did not account for the drought
effect, the watering need was visually assessed only. To maintain proper air humidity, the
automatic mister was switched on and activated whenever the humidity sensors detected the
air humidity below 40%. Apart from that, the mister was activated daily at 7.30 AM for 15minutes-continuous misting regardless of the sensor readings. The pod was ventilated by the
built-in ventilator or by the automatic side and rooftop whenever the temperature approached
30°C or the air humidity exceeded 80%.

26

Pest Control
The principal adopted was to not influence the physicochemical properties of the soil
substrate. Applied: 1. insecticide DOCTOR DOOM GO GREEN TOTAL RELEASE
FOGGER containing the active ingredients Pyrethrins 0.4% and Piperonyl Butoxide 2.0%;
2. yellow color sticky traps to fight flying pest; 3. plant protection product NEMASYS, which
contains nematodes Steinernema feltiae, was applied to the subsoil treatments to fight fungus
gnats as this nematode species is a parasite of fungus gnat larvae living in the soil.
Applied Subsoil Substrate Combinations
Subsoils collected from both mines were treated the same way with the addition of soil
amendments. Amendments were applied in the volumetric proportions shown in Table 2.1.
Table 2.1 First study experiment soil medium composition percentage breakdown.
SUBSOIL

BIOSOLIDS

WOODCHIPS

BIOCHAR

CONTROL

100%

0%

0%

0%

BIOSOLIDS

75%

25%

0%

0%

WOOD CHIPS

75%

0%

25%

0%

BIOCHAR

95%

0%

0%

5%

75%

10%

10%

5%

MIXTURE OF ALL
AMENDMENTS

Regarding low subsoil organic matter content it was decided to apply high volumetric
dosing of soil amendments (Larney and Angers, 2012). Biochar was an exception because this
material has very high pH (Canadian AgriChar, 2020) and an addition of biochar in larger
quantity could potentially even elevate the subsoil pH which was already high (above 8).
An addition of 5% biochar followed the AgriChar suggestions of application.
Experiment 1 Overall Treatments Combination
The experiment was a 2 x 5 x 5 factorial design, with 50 treatment combinations:
 2 subsoil types: 1. New Afton New Gold subsoil, 2. Teck Highland Valley Copper subsoil;
 5 soil amendment treatments: 1. control [no soil amendments], 2. biosolids alone, 3.
woodchips alone, 4. biochar alone, 5. mixture of all three soil amendments (see Table 2.1);
 5 plant treatments: 1. bluebunch wheatgrass alone, 2. rough fescue alone, 3. prairie
junegrass alone, 4. field locoweed alone, 5. mix of all four species.

27

Replicated 7 times for a total of 350 individual pots. A randomized block design was arranged
in the greenhouse.
Soil Sampling
Before seeds were sown, but after subsoil substrate mixing with amendments, and
following 7 days for the mix to settle, ten soil samples were collected (2 mine subsoils x 5
subsoil treatments). Additionally, two reference sites were selected near the vicinity of the
two mine sites, which served as a source of undisturbed topsoil samples from the natural
ecosystem. All samples were collected in labeled 1L plastic bags and frozen at -20°C
immediately after sampling. Each time, prior to testing, a portion of the frozen sample was
removed from the bag and the rest was deposited back into the freezer to avoid unwanted
defrosting. At the experiment termination, the soil substrate from 7 pot-replicates was mixed
thoroughly together, and then one sample was collected in a 1L labeled plastic bag. The same
procedure was repeated for each of the 50 treatments. Then, the samples were frozen at -20°C
immediately after collection. This way all together a pool of 62 1L-samples were obtained: 10
samples of the initial subsoil substrates combinations, 50 treatment-resulting samples at the
end of the experiment, and 2 undisturbed topsoil samples for reference. All samples were
collected and stored in the same manner until the test was performed.
Tests and Instrumentation Used
Plant Productivity
Plant productivity was measured by harvesting above-ground biomass of each
individual plant from each corner of each pot for a total of 1400 specimens. All plants ovendried in Constant Temperature Oven DKN 812 for 48h at 45°C right after collection. Dried
plants were weighed on an analytical scale Fisher Scientific accuSeries accu-225D.
Mortality
Mortality was assessed at the moment of the above-ground biomass clipping and
collection.
Soil Physicochemical Tests
Part of the tests was conducted in Dr. Lauchlan Fraser's laboratory at TRU. These
were: the soil pH, soil electroconductivity, soil organic matter (SOM) content, as well as total
nitrogen and total carbon contents. In addition, soil samples were sent to the Ministry of

28

Environment and Climate Change Strategy - Analytical Lab in Victoria to analyze them for
the content of mineralizable nitrogen and available nitrogen forms: NH4+, NO3-, as well as
basic elements content: Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, S, Zn.
Before testing, according to the procedures, all soil samples were subjected to
desiccation in Constant Temperature Oven DKN 812 at 65°C for 24h, then manually crushed
in a mortar and sieved through a 0.5 mm laboratory soil sieve. Samples for pH and
electroconductivity were excepted from this procedure, as fresh soils are used for these tests.
Also, SOM samples were exempted from sieving. In the case of SOM, the desiccation was
conducted for 24h at 105°C.
pH and Electroconductivity
Measurements were taken by PALINTEST WATERPROOF 800 pH
/CONDUCTIVITY/TDS METER from fresh substrate samples sunk in the distilled water.
Prior to measurements taking, the instrument was calibrated.
SOM
Immediately after drying, samples were weighed on analytical scale Fisher Scientific
accuSeries accu-225D and results were noted. Samples’ weights were between 1.00 – 1.50 g.
Then, samples were placed in a muffle oven Furnace 62700 and subjected to 500°C for 5h to
volatize all organic matter. After burning samples were weighed again and then the SOM got
calculated from the difference between pre-burning and after-burning results.
Total C, Total N
Prior to testing 10 to 15 µg samples got prepared in small tin containers, weighed on
analytical scale Sartorius CP 2P, then tightly sealed and weighed again for the weight
confirmation. Each of the 62 1L-sample-bags were tested three to five times using
FLASHSMART Elemental Analyzer / Flash IRMS EA IsoLink operating with EagerSmart
software. Proper levels of gases: N, O, He were checked every time before testing. Technical
gases were provided by Praxair. Additionally, proper functioning of the instrumentation was
verified every time before and after testing by observing of reactor’s temperature and other
specific measurements accordingly to the manual. Eventually, the final results were subjected
to a comparison with standards provided by the manufacturer.

29

Mineralizable Nitrogen, Available Nitrogen Forms: NH4+, NO3Mineralizable nitrogen and available nitrogen forms tests were conducted by the
Ministry of Environment and Climate Change Strategy - Analytical Lab in Victoria. Tests
performed by the lab were KCl Extraction with UV Analysis for mineralizable nitrogen and
KCl Extraction with Autoanalyzer Analysis for available nitrogen forms.
Basic Elements
Basic elements test was conducted by the Ministry of Environment and Climate
Change Strategy - Analytical Lab in Victoria. Test performed by the lab was Metals via Acid,
Microwave Digestion with ICP-OES Analysis.
Statistical Analyses
The software applied was R Studio 1.3.1093 and Excel Microsoft 365. Statistical
analyses were divided into 3 sections which align with the study objectives.
Section 1 - mortality and productivity were analyzed to answer the first study question
on similarities and differences between two mine subsoils. As a first step, the plant
productivity and mortality data sets were tested for following the parametric assumptions
(Greenacre and Primicerio, 2013; Lander, 2014). Shapiro-Wilk test followed by residuals plot
and Normal Q-Q plot revealed that the data sets violate the normality and homoscedasticity
assumptions, therefore, non-parametric models had to be applied. Mortality and productivity y
data were subjected to Kruskal-Wallis one-way analysis of variance. To compare subsoil
treatments, when the Kruskal-Wallis test was showing a significant difference, populations
were subjected to Wilcoxon pairwise comparison. To confirm the revealed overall difference
between New Afton New Gold and Teck Highland Valley Copper subsoils the entire sets of
both subsoil physicochemical parameters were compared using PERMANOVA test based on
Euclidean distance and then visualized by Principal Component Analysis.
Section 2 - After detection that NA and HVC subsoils differ strongly 17 predictors
were analyzed one by one to determine which stand behind the detected difference. Tests
between two mine subsoils were followed by comparison between subsoil treatments within
each mine separately. Analyzed predictors were: pH, electroconductivity, soil organic matter
(SOM), total carbon, as well as all the basic elements. All mentioned predictors were analyzed
the same way. After revision of results, charts of the most influential predictors were prepared
and presented in the main body of the thesis, while all others are presented in the Appendix A.

30

Section 3 - The remaining 4 predictors related to nitrogen: total nitrogen content,
mineralizable nitrogen content, available NH4+ and NO3- contents were analyzed separately in
section 3. Additionally, the last set of analyses takes on qualitative and quantitative change in
NH4+ and NO3- contents throughout the experiment. Firstly the sum of initial NH4+ and NO3contents were compared with the final sum and then the initial and final ratios of those two
available N-forms were compared as second. This analysis worked only with biosolidscontaining subsoil treatments, these were: “75%sub25%bios” and the “mixture”. All other
subsoil treatments poor in available nitrogen were disregarded for these tests. Boxplots
combine together datapoints from both New Afton New Gold and the Teck Highland Valley
Copper. These tests intended to resolve which nitrogen cycling step is prevailing at the initial
stage of the mine reclamation with the uncured biosolids application.
RESULTS
Mortality, Productivity, Overall Subsoils’ Difference
Section 1: Mortality
Kruskal-Wallis test showed that mortality of plants growing on New Afton New Gold
subsoil combinations was significantly larger than mortality of plants growing on Teck
Highland Valley Copper subsoil combinations (Figure 2.1).

Figure 2.1 Mortality comparison between two mines’ subsoils: HVC – Teck Highland Valley Copper,
NA – New Afton New Gold. Boxplots describe the number of dead plants per pot. Horizontal line
indicates the median of plants per pot which did not survive to the end of the experiment. KruskalWallis test was used to compare the medians of plant mortalities. n = 175

31
New Afton New Gold Plant Mortality Analysis

Kruskal-Wallis test showed that the mortalities of New Afton New Gold subsoil
treatments were not equal (Figure 2.2).
Wilcoxon pairwise comparison test showed that: the treatment with an addition of
25% of biosolids alone resulted in significantly larger mortality than unamended subsoil, than
treatment with 25% of woodchips, than treatment with 5% of biochar, as well as than
treatment with a mixture of soil amendments. The treatment with a mixture of soil
amendments resulted in significantly larger mortality than unamended subsoil, than treatment
with 25% of woodchips, than treatment with 5% biochar, as well as significantly lower
mortality than treatment with 25% of biosolids. The unamended subsoil treatment resulted in
significantly larger mortality than the treatment with 25% of woodchips. There were no
significant differences in terms of mortality between unamended subsoil and the treatment
with 5% of biochar as well as between the treatment with 5% of biochar and the treatment
with 25% of woodchips (Figure 2.2).

Figure 2.2 Mortality comparison between the New Afton New Gold (New Afton) subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Boxplots describe the
number of dead plants per pot. Horizontal line indicates the median of plants per pot which did not
survive to the end of the experiment. Kruskal-Wallis test was used to compare the medians of plant
mortalities. Wilcoxon test was used for pair-wise comparison: * - p < 0.05, ** - p < 0.01,
**** - p < 0.0001, ns – difference not significant. n = 35

32

Teck Highland Valley Copper Plant Mortality Analysis
Kruskal-Wallis test showed that the mortalities of Teck Highland Valley Copper
subsoil treatments were not equal (Figure 2.3).
Wilcoxon pairwise comparison test showed that: the treatment with an addition of
25% of biosolids alone resulted in significantly larger mortality than unamended subsoil, than
treatment with 25% of woodchips, than treatment with 5% of biochar, as well as than
treatment with a mixture of soil amendments. The treatment with a mixture of soil
amendments resulted in significantly larger mortality than unamended subsoil, than treatment
with 25% of woodchips, than treatment with 5% biochar, as well as significantly lower
mortality than treatment with 25% of biosolids. There were no significant differences in terms
of mortality between unamended subsoil, the treatment with 5% of biochar, and the treatment
with 25% of woodchips (Figure 2.3).

Figure 2.3 Mortality comparison between the Teck Highland Valley Copper (HVC) subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Boxplots describe the
number of dead plants per pot. Horizontal line indicates the median of plants per pot which did not
survive to the end of the experiment. Kruskal-Wallis test was used to the medians of plant mortalities.
Wilcoxon test was used for pair-wise comparison: ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns
– difference not significant. n = 35

33

Plant Species Mortality Analysis
Kruskal-Wallis tests showed that individuals of bluebunch wheatgrass, field locoweed,
and rough fescue growing on New Afton New Gold subsoil combinations experienced larger
mortality than individuals of these species growing on Teck Highland Valley Copper subsoil
combinations. Mortalities of prairie junegrass on two mines’ subsoils did not differ
significantly (Figure 2.4).

Figure 2.4 Plant species mortality comparison between two mines’ subsoils: HVC – Teck Highland
Valley Copper, NA – New Afton New Gold. Horizontal line indicates the median mortality rate.
Kruskal-Wallis test was used to compare the medians of plant species mortalities. n = 35

34

Kruskal-Wallis tests showed that on New Afton New Gold subsoil treatments
mortalities of plant species were not equal. Wilcoxon pairwise comparison tests showed that
prairie junegrass individuals performed significantly better than bluebuch wheatgrass, field
locoweed, and rough fescue. There was no significant difference between all other species in
terms of survivorship/mortality on New Afton New Gold subsoil treatments (Figure 2.5 right).
Kruskal-Wallis tests showed that on Teck Highland Valley Copper subsoil treatments
all species performed equally in terms of survivorship (Figure 2.5 left).

Figure 2.5 Plant species mortality comparison on two mines’ subsoils: HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median mortality rate. KruskalWallis test was used to compare the medians of plant species mortalities. n = 35

35

Section 1: Productivity
Kruskal-Wallis test showed that dry above-ground biomass productivity of plants
growing on New Afton New Gold subsoil combinations was significantly lower than dry
above-ground biomass productivity of plants growing on Teck Highland Valley Copper
subsoil combinations (Figure 2.6).

Figure 2.6 Dry above-ground biomass productivity comparison between two mines’ subsoils: HVC
– Teck Highland Valley Copper, NA – New Afton New Gold. Boxplots describe dry biomass weight.
Horizontal line indicates the median of weights of individual plants at the end of the experiment.
Kruskal-Wallis test was used to compare the medians of plant dry above-ground biomass
productivity. n = 700

Average dry above-ground biomass productivity of plants growing on New Afton
New Gold subsoil combinations was more than three times lower than average dry aboveground biomass productivity of plants growing on Teck Highland Valley Copper (Figure 2.7).

55.69

HVC

17.20

NA

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

Dry above-ground biomass (mg)
Figure 2.7 Mean dry above-ground biomass productivity comparison between two mines’
subsoils: HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Error bars are
standard error of the mean. n = 700

36

New Afton New Gold Subsoil Treatments Productivity Analysis
Kruskal-Wallis test showed that the dry above-ground biomass productivities of New
Afton New Gold subsoil treatments were not equal (Figure 2.8).
Wilcoxon pairwise comparison test showed that the treatment with a mixture of soil
amendments resulted in a significantly larger dry above-ground biomass productivity than
unamended subsoil, than treatment with 25% of woodchips, than treatment with 5% of
biochar, as well as than the treatment with 25% of biosolids. The treatment amended by 25%
of biosolids resulted in a significantly larger dry above-ground biomass productivity than
unamended subsoil, than treatment with 25% of woodchips, than treatment with 5% of
biochar. The treatment with unamended subsoil resulted in a significantly larger dry aboveground biomass productivity than the treatment with 25% of woodchips only. There was no
significant difference in terms of dry above-ground biomass productivity between the
treatment with 5% of biochar and the treatment with 25% of woodchips (Figure 2.8).

Figure 2.8 Dry above-ground biomass production comparison between the New Afton New Gold (New
Afton) subsoil treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25%
of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar
– subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Boxplots describe the weights of individual plants’ dry above-ground biomass. Horizontal line
indicates the median of plants’ above-ground biomass. Kruskal-Wallis test was used to compare the
medians of plant productivity on particular subsoil treatments. Wilcoxon test was used for pairwise
comparison * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns – difference not
significant. n = 140

37

Teck Highland Valley Copper Subsoil Treatments Productivity Analysis
Kruskal-Wallis test showed that the dry above-ground biomass productivities of Teck
Highland Valley Copper subsoil treatments were not equal (Figure 2.9).
Wilcoxon pairwise comparison test showed that the treatment with a mixture of soil
amendments resulted in a significantly larger dry above-ground biomass productivity than
unamended subsoil, than treatment with 25% of woodchips, than treatment with 5% of
biochar, as well as than the treatment with 25% of biosolids. The treatment with 25% of
biosolids resulted in a significantly larger dry above-ground biomass productivity than
unamended subsoil, than treatment with 25% of woodchips, and than the treatment with 5%
of biochar. The treatment with unamended subsoil resulted in a significantly larger dry aboveground biomass productivity than the treatment with 25% of woodchips only. There was no
significant difference in terms of dry above-ground biomass productivity between the
treatment with 5% of biochar and the treatment with 25% of woodchips (Figure 2.9).

Figure 2.9 Dry above-ground biomass production comparison between the Teck Highland Valley
Copper (HVC) subsoil treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil
amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips
alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture - subsoil amended by a
mixture of amendments. Boxplots describe the weights of individual plants’ dry above-ground
biomass. Horizontal line indicates the median of plants’ above-ground biomass. Kruskal-Wallis test
was used to compare the medians of plant productivity on particular subsoil treatments. Wilcoxon test
was used for pairwise comparison * - p < 0.05, *** - p < 0.001, **** - p < 0.0001, ns – difference not
significant. n = 140

38

Section 1: Both Mines’ Subsoil Overall Difference; Principal Component Analysis
The two mines’ data sets of predictors characterizing physicochemical properties were
compared and the analysis presented strong evidence that the two subsoils were overall
significantly different: Pseudo F = 322.34; p < 0.001 (Figure 2.10). Datapoints representing
pots containing subsoil with biosolids as the only amendment clustered together separately
from all other treatments showing that they distinguished from other treatments. The same
with pots containing subsoil with a mixture of amendments. Datapoints representing three
other treatments clustered altogether showing that there was similarity between them (Figure
2.11).

Figure 2.10 Principal Component Analysis of two mines’ subsoils in terms of 21 predictors: pH,
EC, SOM, total N and C content, mineralizable N content, NH4+ and NO3- contents, as well as the
content of 13 basic elements. hvc – Teck Highland Valley Copper, new afton – New Afton New Gold.

Figure 2.11 Principal Component Analysis of two mines’ subsoil treatments: 100%sub – unamended
subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - Subsoil
amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone,
mixture - subsoil amended by a mixture of amendments. hvc – Teck Highland Valley Copper, new
afton – New Afton New Gold.

39

Physicochemical Properties of Mines’ Subsoils When Unamended and Amended
Section 2: pH
New Afton New Gold subsoil pH was significantly higher than Teck Highland Valley
Copper both in the initial and the final stages (Figure 2.12 left). Within each mine separately
there was no significant difference between the initial and the final pH either in the case of
New Afton New Gold or in the case of Teck Highland Valley Copper (Figure 2.12 right).

Figure 2.12 Two mines’ subsoil pH comparison in the initial and final stages of the experiment (left),
comparison of the subsoil pH in the initial and final stages of the experiment within each mine (right).
HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the
median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25

In the case of both mines the final pHs were statistically not equal (Figure 2.13). In
both cases, the biosolids-containing treatments had the lowest pH. In both cases as well, pHs
seemed to drop throughout the experiment (Figure 2.13).

Figure 2.13 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
pH comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub –
unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip
- subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

40

Section 2: Electroconductivity (EC)
New Afton New Gold subsoil electroconductivity was significantly higher than Teck
Highland Valley Copper both in the initial and the final stages (Figure 2.14 left). Within each
mine separately there was no significant difference between the initial and final
electroconductivities either in the case of NA or in the case of HVC (Figure 2.14 right).

Figure 2.14 Two mines’ subsoil electroconductivities comparison in the initial and final stages of the
experiment (left), comparison of the electroconductivities in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 25

In the case of both mines the final pHs were statistically not equal (Figure 2.15). In the
case of Teck Highland Valley Copper subsoil treatments, the addition of biosolids or mixture
seemed to increase the EC (Figure 2.15 right). Such a pattern was not visible though in the
case of New Afton New Gold subsoil treatments (Figure 2.15 left).

Figure 2.15 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
electroconductivities comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

41

Section 2: Soil Organic Matter (SOM) Content
New Afton New Gold subsoil SOM content was significantly larger than Teck
Highland Valley Copper but in the case of the final stage only (Figure 2.16 left). Within each
mine separately there was no significant difference between the initial and the final SOM
content either in the case of NA or in the case of HVC (Figure 2.16 right).

Figure 2.16 Two mines’ subsoil soil organic matter content comparison in the initial and final stages
of the experiment (left), comparison of the soil organic matter content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
Initial n= 5, final n= 25

In the case of both mines the final SOM contents were statistically not equal. In both
cases, the biosolids-containing treatments had the largest SOM contents (Figure 2.17).

Figure 2.17 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
soil organic matter contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 1, final n= 5

42

Section 2: Sodium (Na) Content
New Afton New Gold subsoil Na content was significantly larger than Teck Highland
Valley Copper both in the initial and final stages (Figure 2.18 left). Within each mine
separately there was a significant difference between the initial and final Na contents both in
the case of NA and in the case of HVC (Figure 2.18 right).

Figure 2.18 Two mines’ subsoil sodium content comparison in the initial and final stages of the
experiment (left), comparison of the sodium content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial
n= 5, final n= 25

Final Na contents were statistically not equal in the case of NA subsoil treatments only
(Figure 2.19 left). In the case of both mines, nearly each subsoil treatment contained more
sodium in the initial than in the final stage of the experiment (Figure 2.19).

Figure 2.19 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
sodium contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

43

Quantitative and Qualitative Nitrogen Content Analyses
Section 3: Total Nitrogen (N) Content
New Afton New Gold subsoil total nitrogen content was significantly larger than Teck
Highland Valley Copper one both in the initial and final stages (Figure 2.20). Within each
mine separately there was no significant difference between the initial and the final total
nitrogen content either in the case of New Afton New Gold or in the case of Teck Highland
Valley Copper (Figure 2.21).

Figure 2.20 Two mines’ subsoil total nitrogen content comparison in the initial and final stages of the
experiment. HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 25, final n= 75

Figure 2.21 Comparison of the total nitrogen content in the initial and final stages of the experiment
within each mine. HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 25, final n= 75

44

In the case of New Afton New Gold and the case of Teck Highland Valley Copper
subsoil treatments, the initial and final total N contents were statistically not equal (Figures
2.22, 2.23). Both, in the case of New Afton New Gold and the case of Teck Highland Valley
Copper subsoil treatments initially there was a statistical difference between the
“95%sub5%bios” treatment and the “mixture” treatment. The difference persisted until the
end of the experiment. Also, these two treatments differed statistically from all other
treatments. This had place in the case of both mines’ subsoil treatments. Treatments
“75%sub25%wchip”, “95%sub5%bchar” and “100%sub” in the case of both mines did not
differ statistically either in the initial or final stage (Figures 2.22, 2.23).

Figure 2.22 New Afton New Gold subsoil treatments total nitrogen contents comparison in the initial
and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
*** - p < 0.001, **** - p < 0.0001, ns – difference not significant. initial n= 5, final n= 15

45

Figure 2.23 Teck Highland Valley Copper subsoil treatments total nitrogen contents comparison in
the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
**** - p < 0.0001, NS. – difference not significant. initial n= 5, final n= 15

In the case of New Afton New Gold and the case of Teck Highland Valley Copper
subsoil treatments only the “mixture” treatment was not significantly different at the end of
the experiment from the reference of the undisturbed topsoil in terms of the total nitrogen
content (Figures 2.24, 2.25). All other treatments differed significantly from the reference. In
the case of “75%sub25%wchip”, “95%sub5%bchar”, and “100%sub” treatments the total
nitrogen contents were significantly lower than the reference’s total nitrogen content both in
the initial and the final stages of the experiment. “75%sub25%bios” treatment’s total nitrogen
content was significantly larger than the reference’s one both in the initial and the final stages
of the experiment (Figures 2.24, 2.25).

46

Figure 2.24 New Afton New Gold subsoil treatments total nitrogen contents comparison in the initial
and final stages of the experiment to the reference of undisturbed topsoil. Subsoil treatments:
100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison * - p < 0.05, ** - p < 0.01, *** - p < 0.001, ns – difference not
significant. initial n= 5, final n= 15, undisturbed n=5

Figure 2.25 Teck Highland Valley Copper subsoil treatments total nitrogen contents comparison in
the initial and final stages of the experiment to the reference of undisturbed topsoil. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison ** - p < 0.01, *** - p < 0.001, ns, NS. – difference not significant.
initial n= 5, final n= 15, undisturbed n=5

47

Section 3: Mineralizable Nitrogen Content
NA subsoil mineralizable nitrogen content did not differ statistically from HVC
subsoil mineralizable nitrogen content in the initial stage of experiment. At the end of
experiment the difference became significant (Figure 2.26 left). Within each mine separately
there was no significant difference between the initial and final mineralizable N contents
either in the case of NA or in the case of HVC (Figure 2.26 right).

Figure 2.26 Two mines’ subsoil mineralizable nitrogen content comparison in the initial and final
stages of the experiment (left), comparison of the mineralizable nitrogen content in the initial and final
stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New
Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 25

In the case of NA and the case of HVC subsoil treatments the final mineralizable N
contents were statistically not equal (Figures 2.27). In both cases, biosolids-containing
treatments had the largest mineralizable N contents. Mineralizable N contents seemed to drop
during the experiment (Figures 2.27).

Figure 2.27 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
mineralizable nitrogen contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

48

Section 3: Available Nitrogen Content – Ammonium Cation (NH4+)
New Afton New Gold subsoil NH4+ content differed statistically from Teck Highland
Valley Copper subsoil but only at the final stage (Figure 2.28 left). Within each mine
separately there was no significant difference between the initial and final NH4+ contents
either in the case of NA or in the case of HVC (Figure 2.28 right).

Figure 2.28 Two mines’ subsoil NH4+ content comparison in the initial and final stages of the experiment
(left), comparison of the NH4+content in the initial and final stages of the experiment within each mine
(right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the
median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25

In the case of NA and the case of HVC subsoil treatments the final NH4+ contents
were statistically not equal (Figures 2.28). In both cases, biosolids-containing treatments had
the largest NH4+ contents. NH4+ contents seemed to drop throughout the experiment (Figures
2.29).

Figure 2.29 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
NH4+contents comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub
– unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

49

Section 3: Available Nitrogen Content – Nitrate Anion (NO3-)
NA subsoil NO3- content did not differ significantly from HVC NO3- content either in
the initial or in the final stages (Figure 2.30 left). Within each mine separately there was no
significant difference between the initial and final NO3- contents either in the case of NA or in
the case of HVC (Figure 2.30 right).

Figure 2.30 Two mines’ subsoil NO3- content comparison in the initial and final stages of the
experiment (left), comparison of the NO3- content in the initial and final stages of the experiment within
each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25

In the case of NA and the case of HVC subsoil treatments the final NH4+ contents
were statistically not equal (Figures 2.31). In both cases, biosolids-containing treatments had
the largest NO3- contents. NO3- contents seemed to increase throughout the experiment
(Figures 2.31).

Figure 2.31 New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil treatments
NO3-contents comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub
– unamended subsoil, 75%sub25%bios – subsoil amended by 25% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 5

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Section 3: Available Nitrogen Content – NH4+, NO3-; Sum and Ratio
There was no significant difference between the sums of NH4+ and NO3- in the initial and
final stages of the experiment at α= 0.05. However, at α= 0.1 the difference was significant
(Figure 2.32).

Figure 2.32 Comparison of the sum of NH4+ and NO3- in the initial and final stages of the
experiment. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 4, final n= 20

There was a significant difference between the ratios of NH4+ to NO3- in the initial and
final stages of the experiment at α= 0.05 (Figure 2.33).

Figure 2.33 Comparison of the ratio of NH4+ to NO3- in the initial and final stages of the experiment.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 4,
final n= 20

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DISCUSSION
Results demonstrated that New Afton New Gold and Teck Highland Valley Copper
subsoils differed strongly in terms of many physicochemical properties. Differences in
physicochemical properties resulted in a broad variability in plant responses such as mortality
and above-ground biomass productivity on these two mines’ subsoil combinations. The
influence of individual soil amendments on subsoil properties also differed strongly.
The nitrogen analyses confirmed that this element's presence and transformation have great
importance on the reclaimed subsoil metamorphosis and vegetation response.
Mortality, Productivity, Overall Subsoils’ Difference
Mortality
Results showed that the more biosolids added, the higher mortality. Biosolids-free
subsoil combinations resulted in significantly lower mortality rates (See Figures 2.2, 2.3).
Biosolids contain large amounts of organic but also inorganic, readily available nitrogen
(Sullivan et al., 2015; Brown and Chaney, 2016). Nitrogen is essential to plant development,
however, overfertilization might be harmful to vegetation by increasing tissue concentration
to toxicity levels (Elhanafi et al., 2019). Additionally, overfertilization enhances negative
interactions between soil microbial species leading to decrease on biodiversity and
elimination of some microbial functions (Ratzke et al., 2020). Thorne et al. (1998) found that
overdosing of nitrogen-rich organic amendments can be harmful to arbuscular mycorrhizal
fungi. Subsoil, due to low organic matter content, has low potential to bond nutrients such as
nitrogen compounds causing vegetation being exposed to high concentration of inorganic
nitrogen when large amounts of organic amendments are applied. Castillejo and Castello
(2010) pointed out the risk of over-fertilization as an important issue in applying organic
amendments to degraded quarry soils. Fenn et al. (1998) reported that observed forests
experienced increased mortality and decline in productivity as a result of excessive nitrogen
input.
The mortality of the plants growing on New Afton New Gold subsoil treatments was
significantly higher than plants growing on Teck Highland Valley Copper subsoil treatments
(See Figure 2.1). In terms of mortality rate all applied species except prairie junegrass
performed significantly worse on New Afton New Gold than on Teck Highland Valley
Copper subsoil treatments (See Figure 2.4). This difference might be associated to the large

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difference in physicochemical properties of these two mine subsoils. New Afton New Gold
subsoil might be named saline and sodic due to high concentrations of salts and sodium, while
Teck Highland Valley Copper subsoil is not burdened with this problem. Hanay et al. (2004)
demonstrated that vegetation on degraded soils might suffer from high soluble salts and
exchangeable sodium content. They proposed gypsum application coupled with organic soil
amendments as possible remedy.
Plant mortality in experiment 1 was increased by the infestation of the plant pest
fungus gnat. The larvae of this insect feed on dead matter, fungi thallus, but also on tiny living
plant roots (Cransaw and Cloyd, 2021). Indeed, the insect attacked in particular these plants
which were growing on subsoils amended by biosolids solely or by a mixture, finding in these
treatments the best food base. It was noticeable that the more biosolids, the greater the
infestation. Additionally, the insects selected those pots in which field locoweed seedlings
germinated. Attacked young plants of this species got destroyed quickly after germination
causing larger mortality of this species seedlings (See Figure 2.5). Graminoids seemed to be
not selected by insects that much. Especially prairie junegrass was avoided by fungus gnats
what resulted in significantly larger survivorship of this species individuals growing on New
Afton New Gold subsoil treatments (See Figure 2.5). Combinations containing biosolids (lots
of SOM and nutrient), field locoweed (preferred plant), and New Afton New Gold subsoil
were visibly attacked more than the same combinations but containing Teck Highland Valley
Copper subsoil. This is another indicator that subsoils from those two mines differ. The insect
control measures gave an unsatisfactory and short-lived effect.
Productivity
The dry above-ground biomass productivity achieved by all plants growing on Teck
Highland Valley Copper subsoil combinations was over three times larger than the one
achieved by all plants growing on New Afton New Gold subsoil combinations (See Figure
2.7). That demonstrated that physicochemical properties of New Afton New Gold subsoil
posed a hindrance for vegetation striving to develop. Plants growing on Teck Highland Valley
Copper subsoil combinations did not have to overcome such obstacles to perform. That
explains why the above-ground biomass productivity of plants growing on Teck Highland
Valley Copper subsoil combinations was significantly larger (See Figure 2.6).
It is striking that, despite the highest mortality rates, biosolids-containing subsoil
treatments from both mines were more productive than biosolid-free subsoil treatments.

53

Biosolids contain large amounts of nutrients in forms ready to take up (Sullivan et al., 2015).
Thus, plants growing on subsoils with addition of biosolids reacted immediately (Larney and
Angers, 2012). Applied biosolids were provided straight after production, not composted prior
to delivery and application. This amendment at the moment of application had an uncured
form. Despite the fact that the most of total nitrogen is still in organic form, biosolids in uncomposted form contain and release also available nitrogen giving an outburst of fertility
(Senesi and Loffredo, 1999; Paschke et al. 2005). Results showed that within a short term of
the experiment plants response to organic soil amendments were significant only to this
treatments which obtained biosolids. Larney et al. (2000) demonstrated that an addition of
organic amendment (manure) rich with available nitrogen and phosphorus to reclaimed soil
increased the yield much more than the additions of topsoil or fertilizer. The single addition of
organic amendment kept bringing better results over topsoil and fertilizer in terms of the
productivity even 16 years after first application (Larney et al. 2009). Shrestha et al. (2009)
demonstrated that after 5 years from application nutrient-rich cow manure resulted with
significantly larger above-ground biomass productivity on reclaimed coal mine sites than
nutrient-poor oat straw. This study presented similar results as an addition of the nutrient-rich
biosolids resulted with significantly larger above-ground biomass productivity than nutrientpoor woodchips and biochar (See Figures 2.8, 2.9).
In the case of both Teck Highland Valley Copper and New Afton New Gold subsoils
the treatment amended by a mixture of soil amendments resulted in significantly larger aboveground biomass productivity than all other treatments (See Figures 2.8, 2.9). The “mixture”
treatment contained biosolids but in lower concentration (10% v/v) than the
“75%sub25%bios” treatment (25% v/v). Albornoz (2016) states that high nitrogen
fertilization rates are detrimental for crop yield. It seems that the addition of biosolids in a
dose of 10% v/v, as in the “mixture” treatment, did not cause the overfertilization problem
unlike 25% v/v of “75%sub25%bios” treatment. Additionally, other organic amendments
acted in protective way. The addition of woodchips and biochar blended into the mixture
bettered the soil physicochemical properties facilitating the plant development. Both
woodchips and biochar have potential for exchangeable binding with mineral nutrients
slowing down its availability and eventual release. The slower release of nutrients such as
nitrogen is important in establishing viable and resilient plant communities on reclaimed
disturbed sites (Claassen and Carey, 2007). de Varennes and al. (2010) states that
combinations of amendments may work better than each applied singly. In a blend more

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reluctant to decomposition amendments may provide nutrients and carbon slowly but long
lasting, while readily decomposable amendments can act intensively but for shorter time
initiating the nutrients cycling (Larney and Angers, 2012).
Overall Difference of Both Mines’ Subsoil Physicochemical Properties
Principal Component Analysis confirmed clearly that in terms of physicochemical
properties the subsoils from Teck Highland Valley Copper and New Afton New Gold mines
differ strongly (See Figure 2.10).
Datapoints representing “75%sub25%bios” as well as the “mixture” treatments tended
to cluster together and separately from other treatments which mean that they possessed their
specific features different from other treatments. All biosolid-free treatments tended to cluster
together which means that an addition of 5% biochar or 25% woodchips did not change
enough to cause a distinct difference in terms of physicochemical properties (See Figure
2.11).
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended
pH
Both mine subsoils are alkaline. New Afton New Gold unamended subsoil pH (9.3)
was much higher than Teck Highland Valley Copper’s one (8.50). The difference between
both mines’ subsoil treatments in terms of pH was statistically significant (See Figure 2.12).
Majority of plants perform the best in neutral or slightly acidic pH. That results from nutrients
solubility which is optimal in such conditions (Rodriguez, 2020). Elevated pH, especially in
the case of New Afton New Gold subsoil, could be a reason of lower above-ground biomass
productivity and elevated plant mortality rate. Brown and Chaney (2016) point out the
substrate pH as critical variable for metal uptake, availability, and toxicity. While most of
heavy metal solubility is limited by high pH, molybdenum acts conversely. Solubility of this
element increases along with pH increase (Kaiser et al. 2005; Brown and Chaney, 2016). The
Teck Highland Valley Copper subsoil molybdenum content exceeds the CCME guidelines for
agricultural, residential and parkland use (CCME, 2021). However, the Teck Highland Valley
Copper subsoil’s pH was closer to neutral what might result in lesser mobility of
molybdenum.

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Throughout the experiment pH seemed to drop overall. However, the change was not
statistically significant either in the case of New Afton New Gold or Teck Highland Valley
Copper subsoils. It seems that 16 weeks of the experiment duration may be a too short period
to allow for significant pH change (See Figure 2.12).
Soil amendment addition might influence the substrate pH. The extent of pH change
mainly depends on the applied amendment features (Larney and Angers, 2012). That applies
to organic amendments as well. Lower pH was adopted by these mine subsoil treatments
which contained biosolids. That is because the innate pH of this material is around 8.0 - 8.5.
Biosolids contain lots of NH4+. The acidifying reaction occurring is NH4+ + H2O ⇌ NH3 +
H3O+. The pH of biosolids-containing treatments also dropped the strongest over the time of
the experiment (See Figure 2.13). That is attributed to the oxidation of organic N and S
compounds (Sullivan et al. 2015). At the same time, the pH of all other subsoil treatments
which did not contain biosolids remained high or dropped only slightly.
It is worth adding that the pH of New Afton New Gold undisturbed topsoil was just
slightly above 7.0. That indicates that local ecosystems naturally tend to neutral pH. In the
case of Teck Highland Valley Copper undisturbed topsoil, the pH was 5.5. That indicates that
local flora naturally tends to decrease the pH to acidic levels.
Electroconductivity (EC)
Soil electroconductivity strongly depends on the soluble salts concentration in the soil
water solution. In the soil water solution soluble salts are present in a form of inorganic ions
(McLachlan et al., 2004). Miller at al. (2017) specify that soluble cations and anions Na, K,
Ca, Mg, SO4-S, Cl are responsible for EC readings. Soils suffering from salinity demonstrate
high electroconductivity readings.
While New Afton New Gold and Teck Highland Valley Copper undisturbed topsoils
seemed not to differ much in terms of electroconductivity, the ECs of those two mines’
subsoils differed hugely (See Figure 2.14). New Afton New Gold subsoil treatments EC
median was within the interval 2.5 – 3.0 mS/m, while Teck Highland Valley Copper one’s
placed within the interval 0.3 – 0.6 mS/m. That is a very large difference indicating that New
Afton New Gold subsoil was burdened with a salinity problem. Electroconductivity indicates
the level of ions concentration. The higher EC reading the more ions in the soil water
solution. This indicator though does not specify which ions are present in the solution. Some

56

ions are desirable being nutrients for plants and microorganisms, while other ions are toxic or
act somehow negatively. New Afton New Gold subsoil must contain a significant amount of
ions which increased strongly the EC, concurrently suppressing the plants' development.
The highest EC readings of Teck Highland Valley Copper subsoil treatments were
lower than the lowest readings of New Afton New Gold subsoil treatments (See Figure 2.15).
That strongly suggests that New Afton New Gold subsoil treatments contained lots of
adversely-acting ions, while Teck Highland Valley Copper subsoil either did not possess
those ions at all or they were present but at the inconspicuous levels. Soil organic
amendments may alleviate the salinity problem. Zeynep (2020) successfully used
vermicompost to reclaim sodium affected soil. Miller (2017) proposed woodchips as an
amendment lowering excessive EC together with associated with it high pH.
It may be noticed as well that throughout the experiment, the electroconductivity
dropped (See Figure 2.14). However, the drop was not large enough to cause the difference
between the initial and the final electroconductivity readings to be statistically significant.
Finally, all that means that the change of reclaimed subsoil in terms of EC is relatively a slow
process.
Soil Organic Matter (SOM)
Soil organic matter plays a pivotal role in soil (Brown and Chaney, 2016). Content of
this soil fraction decides about such soil features as productivity, nutrient preservation and
cycling, water holding capacity, soil microbial activity and many others (Larney and Angers,
2012). Because SOM plays such a great role, mine reclamation efforts need to concentrate on
rebuilding this fraction in the top horizon. Organic amendments once again seem to be best
tool to achieve this goal. Larney and Angers (2012) state that on degraded substrates an oneoff large applications of organic amendments can boost reclamation and lead to sustaining net
primary productivity. Gardner et al. (2010) demonstrate that at reclaimed copper mine tailings
sites in British Columbia an application of biosolids was more efficient in terms of restoring
soils with its functions and productivity than the use of traditional fertilizers. Reid and Naeth
(2005) report that an application of organic amendments such as biosolids or composted paper
mill sludge gave better results than fertilizers when attending to establish vegetation cover on
tundra kimberlite mine tailings in Northwest Territories, Canada. Winter Sydnor and Redente
(2002) reported that soil amelioration with organic matter resulted in significant increase of
above-ground biomass in reclamation of a high elevation gold mine in Colorado.

57

Fierro et al. (1999) applied paper sludge supplemented with mineral nitrogen and
phosphorus. In their reclamation attempt on an abandoned sandpit they adopted surrounding
non-degraded land as a reference. They reported that in the second season such measures as
soil carbon and nitrogen neared the natural system levels. In the case of both New Afton New
Gold and Teck Highland Valley Copper, the undisturbed topsoil organic matter content
measured for this study was near to 4%. Unamended New Afton New Gold subsoil SOM was
3%, constituted though only 3/4 of its topsoil’s, while in the case of the Teck Highland Valley
Copper unamended subsoil SOM was 1%, constituted therefore not more than 1/4 of its
topsoil reference. After amendments addition to mine subsoils the amount of SOM increased,
whereof the “mixture” treatment SOM departed the least from its reference of undisturbed
topsoil in the case of both mines.
Organic matter provided by organic amendments may have many various forms.
Biosolids provide predigested organic matter, well shredded, easy in terms of cation
exchange, and prone to further decomposition (Senesi and Loffredo, 1999; Sullivan et al.,
2015), while woodchips are built from coarse, not predigested, and difficult for decomposition
pieces (Senesi and Loffredo, 1999). Biochar, in turn, is formed of organic matter deeply
transformed by high temperature and pressure. Such processes transform organic matter into a
material that is reluctant for decomposition (Taylor P, 2010; Canadian AgriChar, 2020).
75%sub25%bios” and “mixture” treatments were the richest in terms of soil organic
matter content in the case of both mines’ subsoils (Figures 2.17). Concurrently, the same two
treatments showed up as the most productive. The treatment “75%sub25%wchip” contained
fair amount of organic matter as well. However, this treatment did not result with significant
above-ground biomass productivity. The explanation of this discrepancy is that woodchips are
mainly built from cellulose and lignin. Both are hard for decomposition, especially lignin
(Senesi and Loffredo, 1999; Datta et al., 2017). The “mixture” treatment contains both easy
for decomposition and recalcitrant organic matter. This treatment brought the best results in
the case of both mines’ subsoils. Many researchers suggest blending organic amendments in
order to achieve better and longer lasting results (Claassen and Carey, 2007; de Varennes et
al. 2010; Larney and Angers, 2012). Cheng (2008) concludes that saw dust gives good results,
but when incorporated with suitable amounts of clay, ammonium nitrate and organic
amendments.

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Basic Elements
Aluminum is one of the most prevailing elements in the Earth's lithosphere (Weil and
Brady, 2017). However, it is not important in either plant or animal nutrition. Moreover, large
concentrations may cause negative consequences. Its activity increases though only in a low
pH environment (Haynes and Mokolobate, 2001). In a higher pH environment aluminum is
harmless. New Afton New Gold subsoil, as well as topsoil, contained significantly more
aluminum than Teck Highland Valley Copper (See Figure A.4). Nonetheless, regarding the
high pH, such concentrations of aluminum should not have any influence on biomass
productivity or plant mortality.
In the case of both mines’ subsoils, boron content strongly exceeded the CCME
guidelines for agricultural land use (CCME, 2021). Boron is a micronutrient for plants. That
means that low concentrations of this element are necessary for proper plant development.
Biosolids is a good source of boron (Sullivan et al., 2015). However, in larger concentration
boron may act negatively on plant development. New Afton New Gold subsoil had
significantly larger boron content (See Figure A.5) which may be one of the causes of lower
New Afton New Gold subsoil biomass productivity. The boron concentration in NA subsoil
and its influence requires more studies.
Calcium is one of the most important nutrients and its roles for plants and animal life
is countless (Weil and Brady, 2017). Deficiency of this element may cause improper plant
tissue development, cellular membranes deterioration, necroses. Calcium is also responsible
for proper soil structure formation. Additionally, Ca has the pH elevation ability (Troeh and
Thompson, 2005). The larger concentration of calcium in the New Afton New Gold subsoil
(See Figure A.5), may constitute one of the explanations of higher pH and EC. However, both
mines’ subsoil calcium concentrations seemed to be rather in a norm, and not requiring any
additional supplementation.
In the case of both mines’ subsoils, copper content exceeded the CCME guidelines for
all types of land use (CCME, 2021). Additionally, biosolids provides an additional portion of
Cu (Sullivan et al., 2015). Teck Highland Valley Copper subsoil excess of copper content was
outstanding. Copper is a heavy metal but also a microelement necessary in plant metabolism.
Cu is a cofactor in many enzymes, however in high concentrations might be toxic especially
to microorganisms (Trevors and Cotter, 1990). Although the HVC subsoil copper content was
significantly larger than the NA subsoil’s content (See Figure A.5), and exceeded remarkably

59

the CCME guidelines, it all seemed to not have any negative influence on plants growth and
health. Alkalinity of the substrate poses a hindrance for plant Cu uptake (Rodriguez, 2020).
High NA subsoil pH could lead to Cu deficiency despite the fact that the NA subsoil had
larger Cu content than needed. The copper concentration in NA as well as in HVC subsoils
and its influence requires more studies.
Iron plays many important roles in plant physiology (Imsande, 2002). It is worth
emphasizing that iron is an intrinsic part of the nitrogenase enzyme. The concentration of iron
seemed to be sufficient in the case of both mines’ subsoils and having no significant influence
on biomass productivity. However, alkalinity of the substrate poses a hindrance for plant Fe
uptake (Rodriguez, 2020). High NA subsoil pH could lead to Fe deficiency despite the fact
that the NA subsoil had large Fe content. The iron uptake on the NA subsoil requires more
studies.
Potassium is one of the most important plant macronutrients (Weil and Brady, 2017).
Canadian AgriChar (2020) declares that their biochar is a good source of K. The
concentration of potassium in the case of the New Afton New Gold unamended subsoil was
low in comparison to New Afton New Gold undisturbed topsoil. Also, there was a strongly
significant difference between the NA subsoil and HVC subsoil in terms of potassium content
(See Figure A.6). NA subsoil seemed to be lacking with potassium more than the HVC
subsoil, which in turn could be one of the reasons why the NA subsoil is less productive.
Annacis biosolids were also a good provider with K. This amendment contains around 1,100
mg/kg of an available K in the dried weight.
Magnesium is another important plant nutrient (Shaul, 2002). It plays a crucial role in
many processes, for example, photosynthesis by co-building chlorophyll (Madigan et al.,
2012). Magnesium concentration was significantly larger in the case of the New Afton New
Gold subsoil (See Figure A.6). However, a lower concentration of magnesium in the case of
Teck Highland Valley Copper subsoil seemed to not act adversely on plant development.
Manganese is one of the micronutrients. It plays a pivotal role in the reactive centers
of many enzymes and chlorophyll (Rodriguez, 2020). Plants may suffer either from
deficiency or excess on Mn (Campbell and Nable, 1988). New Afton New Gold subsoil
contained significantly larger amounts of manganese (See Figure A.7). However, alkalinity of
the substrate poses a hindrance for plant Mn uptake (Rodriguez, 2020). High NA subsoil pH

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could lead to Mn deficiency despite the fact that the NA subsoil had sufficient Mn content.
The influence of Mn in NA subsoil requires more research.
Molybdenum is a heavy metal. Its excess may cause negative consequences on plant
development as well as on their consumers. At the same time, molybdenum is also a
micronutrient playing a role in many enzymes including nitrogenase (Kaiser et al. 2005).
Sullivan et al. (2015) states that biosolids is a good source of this element. Teck Highland
Valley Copper subsoil contained significantly more molybdenum than New Afton New Gold
subsoil (See Figure A.7). Moreover, the HVC subsoil molybdenum concentration exceeded
CCME guidelines for agricultural and parkland land use, but did not exceed for industrial and
commercial uses (CCME, 2021). The above indicates that HVC subsoil molybdenum
concentration was high but bearable for vegetation.
Sodium is sometimes accounted as a micronutrient. It plays a role in osmotic
regulation in tissues because sodium dissociates in water to cations which have a positive
charge. Accordingly, when placed on one side of a cellular membrane, this creates a charge
potential (Subbarao et al. 2003). Besides its certain roles in living organisms and its nontoxicity, sodium may act adversely on plants and soil-inhabiting organisms. When the
concentration of sodium is elevated it leads to the sodic soil problem (Hanay et al., 2004,
Clancy, n.d; FAO et al., 2020). First of all, sodium is an antagonistic cation to calcium cation.
Calcium plays an important role in the formation of proper soil structure. While sodium
overcomes calcium, the soil becomes compacted, difficult for water, and oxygen penetration
which eventually acts negatively on plants’ root systems (Hanay et al., 2004; Clancy, n.d;
FAO et al., 2020). New Afton New Gold subsoil contained several times more sodium than
Teck Highland Valley Copper subsoil (See Figures 2.18, 2.19), and indeed during the
experiment it was observed that New Afton New Gold subsoil combinations in pots did
compacted and did not drain water properly. This element’s cations are also highly soluble in
water and it is expected that those cations were standing behind the high electroconductivity
readings of all NA subsoil combinations. Sodium cations tend to increase pH and this may be
also one of the explanations for the NA subsoil elevated pH. When comparing two mines’
subsoil departures from its references in terms of sodium content it may be noticed that NA
undisturbed topsoil had a pretty low content of sodium in comparison to NA subsoil, while
HVC topsoil sodium content was similar to the subsoil’s one. Sodium content is one of the
largest differences between NA and HVC subsoils.

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Phosphorus is one of six biogenic elements. On the molecular level, it consists part of
many crucial molecules including DNA or ATP (Madigan et al., 2012). Often soils require
supplementation with this element. Sullivan et al. (2015) states that biosolids is a good source
of this plant and microorganism essential nutrient. Annacis biosolids contain around 1,900
mg/kg of an available P in the dried weight. New Afton New Gold unamended subsoil
contained around three times more phosphorus than the unamended Teck Highland Valley
Copper subsoil (See Figure A.7). Limited availability of phosphorus, alike in the nitrogen
case, may suppress the vegetation development. However, an addition of biosolids applied
with rates adjusted to the nitrogen levels usually cover the phosphorus needs as well (Sullivan
et al., 2015). Similar to nitrogen, treatments amended by 25% of biosolids could obtain even
too large portion of this element at once which might act rather adversely than positively.
Sulfur is acknowledged as the last biogenic element. This element constitutes part of
many vitamins or proteins, therefore, it co-builds enzymes as well (Madigan et al., 2012).
Together with iron and molybdenum, sulfur co-builds molecular structures thanks to which
nitrogenase can actively fix atmospheric nitrogen (Tanifuji K, Ohki Y. 2020). Even though
there may be plenty of sulfur in the soil profile, its elemental form is hydrophobic and
unavailable for plants. Soil microorganisms must transform this form before being taken up
by plants (Fuentes-Lara et al., 2019). One of the sulfur significations is the fact that this
element takes part in the regulation of nitrogen uptake (Salvagiotti et al., 2009). New Afton
New Gold unamended subsoil contained a significantly larger amount of sulfur than Teck
Highland Valley Copper unamended subsoil (See Figure A.8). Again, an addition of biosolids
applied with rates adjusted to the nitrogen levels usually cover the sulfur needs as well
(Sullivan et al., 2015). It seems though that plant demand for sulfur is covered by both mine
subsoils sufficiently. However, sulfur provided by subsoil may be unavailable or even toxic
for plant roots. In terms of sulfur provision, it is important to restore proper soil microflora.
By that time elevated sulfur content may act rather adversely than positively.
The last of the analyzed elements - zinc is one of the very important micronutrients.
Hundreds of enzymes contain Zn (Weil and Brady, 2017). The deficiency of Zn in plants
causes chlorosis and leaf stunting. Nonetheless, zinc is heavy metal as well, therefore, an
excess of this element may cause poisoning (Broadley, 2007). When comparing both mines’
undisturbed topsoils it is noticeable that both contained similar amounts of zinc. Unamended
subsoils contained more zinc than their relevant topsoil, however, the content did not exceed
CCME guidelines (CCME, 2021). An elevated amount of zinc, not exceeding the CCME

62

guidelines, came with an addition of biosolids which is a good source of this element
(Sullivan et al. 2015). Nonetheless, unamended subsoils contained amounts of zinc similar to
the undisturbed topsoil, therefore, it seemed that subsoil alone constitutes sufficient provision
of this element without additional supplementation. However, alkalinity of the substrate poses
a hindrance for plant Zn uptake (Rodriguez, 2020). High NA subsoil pH could lead to Zn
deficiency despite the fact that the NA subsoil had sufficient Zn content. The influence of Zn
in NA subsoil requires more research.
Total Carbon
Restoration of carbon cycling is essential in mine reclamation from the perspective of
successful revegetation (Larney and Angers, 2012). Soil organic matter contains most of soil
carbon. Many researchers proposes organic amendments as the effective tools to rebuild soil
organic carbon pool. Shrestha et al. (2009) when conducting experiment on coal mine sites
found that the treatment with cow manure was the best in terms of bettering many of substrate
properties including the pool of organic carbon. Tian et al. (2009) demonstrated that a longterm application of biosolids to calcareous strip-mined land brough by far better results in
terms of soil organic carbon buildup than traditional fertilization.
If comparing two mines ’unamended subsoils in terms of total carbon content it may
be noticed that the New Afton New Gold subsoil contained slightly more than 1%, while Teck
Highland Valley Copper subsoil contained less than 0.5% (See Figures A.2, A.3). Both
readings were low. The pool of organic carbon increased after soil amendments addition.
Even the proper provision of carbon but only carbon is still not sufficient to ensure
sustainable vegetation. Such soil amendments as woodchips and biochar provided carbon in
decent amounts whereas the above-ground biomass production on such treatments remained
still poor. Additionally, the form of provided carbon (easy or reluctant to biological
processing) plays a pivotal role. The “mixture” treatment provided both readily degradable
and recalcitrant carbon. The complex provision of carbon by the “mixture” treatment might be
one of the reasons why this treatment achieved the best results in terms of the above-ground
biomass production. Recalcitrant carbon contained in woody amendments will exert longlasting effect on reclaimed substrate (Senesi and Loffredo, 1999), but for better results should
not be applied solo.

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Quantitative and Qualitative Nitrogen Content Change
Total Nitrogen
Nitrogen fulfils countless biochemical and physiological functions and is one of the
most essential resources for plants (Geng and He, 2020). For its proper development plants
require large amounts of N to be supplied (Leghari et al., 2016). Deficiency of nitrogen cause
number of malfunctions such as impaired growth, leaves decolouration and chloroses,
reduction in flowering and others (Silva and Uchida, 2000). Same as deficiency, the excess of
nitrogen can act adversely on plant growth (Fenn et al., 1998, Elhanafi et al., 2019). That
refers to reclaimed lands as well (Castillejo and Castello, 2010).
If comparing two mines ’unamended subsoils in terms of total nitrogen content it may
be noticed that the New Afton New Gold subsoil contained slightly more than 0.02% (See
Figure 2.22), while the Teck Highland Valley Copper subsoil content was below the detection
level (See Figure 2.23). Both readings were extremely low. In the case of both mines'
referencing topsoils, the total nitrogen content levels were pretty similar to each other and
placed around 0.15%. Regarding the above, both unamended subsoils contained by far less
total nitrogen than their references (See Figures 2.24, 2.25). On such substrates the vegetation
suffers sever nitrogen deficiency. To remediate that problem reclamation researchers propose
fertilization, but rather by nitrogen-rich organic amendments than by traditional fertilizers
(Bradshaw, 1997; Chambers et al., 2002; Reid and Naeth, 2005; Gardner et al., 2010). Larney
et al. (2000) states that the greater the degradation, the better plant response to organic
amendments. Various organic amendments were proposed as a source of nitrogen. Some
proposed cattle manure (Larney and Janzen 1997; Shrestha et al., 2009), but more recently,
researchers attention is turned toward biosolids (Gardner et al., 2010; Sullivan et al., 2015).
The biosolids application rates depend on substrate nitrogen supplementation need (Sullivan
et al., 2015). Annacis biosolids contain around 5.5% of total nitrogen in the dried weight.
Both initially and finally New Afton New Gold subsoil treatments’ total nitrogen
content was significantly larger than Teck Highland Valley Copper one (See Figure 2.20).
That was because the NA unamended subsoil contained more N overall, however, even NA
unamended subsoil total nitrogen content was low. If comparing the initial and the final total
N content it may be noticed that they did not differ statistically either in the case of NA
subsoil or in the case of HVC. However, in both cases, the final total nitrogen content was
lower than the initial one (See Figure 2.21). That suggests that nitrogen gets depleted or lost

64

over time, but the 16 weeks of the experiment duration is too little time to capture that process
statistically. That is good information meaning the loss of nitrogen occurs relatively slowly.
That gives time to plants and soil microorganisms to acquire more nitrogen and import it to
the cycling. Larney et al. (2009) reported the positive effect of nitrogen-rich cattle manure
being present 16 years after one-off application.
It is noticeable that the largest provision of total nitrogen was present in subsoils
treated by 25% of biosolids. Treatments amended by mixtures came as second. Those two
treatments provide a statistically significant amount of total nitrogen when comparing to
biosolids-free treatments (See Figures 2.22, 2.23). Other treatments did not provide much
nitrogen at all. The “mixture” treatments resulted as the most productive. Its total nitrogen
contents were in the interval 0.1% - 0.2%. The “75%sub25%bios” treatment’s total N median
reached initially nearly 0.6% in the case of HVC and even exceeded 0.6% in the case of the
NA. These results indicated that total nitrogen content above 0.2% might start acting
adversely. The top level of total nitrogen when it is still harmless to vegetation depends on
other factors such as quantity and quality of soil organic matter (Elhanafi et al., 2019).
It is notable that only in the case of both mines’ subsoil “mixture” treatments the
differences from the references (topsoils) in terms of total N content were not statistically
significant (See Figures 2.24, 2.25). That indicates that only these treatments simulated the
natural conditions in terms of total nitrogen content.
Mineralizable Nitrogen
Mineralizable nitrogen indicates how much of nitrogen tied up in complex organic
residues could be potentially mineralized by soil microbial community to the plant available
form of ammonium. Mineralizable nitrogen constitutes a pool of this intrinsic element which
is gradually released throughout decomposition. This process depends on an abundance and
activity of certain soil microorganisms. Thus, plants and soil microorganisms get provided
with required inorganic nitrogen for a prolonged time. Mineralizable nitrogen is a significant
fraction of the total nitrogen (van Es et al., 2017). Nitrogen-rich organic amendments, such as
biosolids (Sulivan et al., 2015) are good source of mineralizable N. Chambers et al. (2002)
reported that the use of biosolids and other organic materials when attempting to reclaim a
landfill site in the United Kingdom resulted in eventual increase of readily mineralizable
organic nitrogen content.

65

In this study the amount of mineralizable N dropped throughout the experiment in the
case of both mine subsoils (See Figures 2.26, 2.27). Lost mineralizable nitrogen either got
mineralized or washed away during watering. In the both mine cases the initial mineralizable
nitrogen content did not differ statistically from the final mineralizable nitrogen at α= 0.05
(See Figure 2.26). That means that mineralization/loss occurred relatively slowly. The
mineralization rate depends among others on the climatic conditions, the microbial
community composition, type of substrate and type of soil amendment applied. Cordovil et al.
(2007) after conduction of potting experiment with municipal solid waste compost, secondary
pulp mill sludge, horn meal, poultry manure, solid phase from pig slurry and composted pig
manure reported that poultry manure was the fastest mineralized organic amendment.
However, too fast mineralization is unwanted. Organic nitrogen mineralization supposed to be
relatively slow and steady (Claassen and Carey, 2007) to help plant and microorganisms
benefit the most from released available nitrogen and to limit nitrogen loss by leaching
(Brown and Chaney, 2016).
Those subsoil treatments that contain more readily degradable organic matter hold at
the same time more mineralizable nitrogen. Regarding the above, here again, those subsoils
that were amended by biosolids alone or in a mixture contained noticeably more mineralizable
nitrogen than subsoil treatments unamended or amended by biosolids-free amendments (See
Figure 2.27).
Initially, the treatments containing 25% of biosolids stood out with an amount of
mineralizable nitrogen. In such cases, readings indicated the concentration of mineralizable
nitrogen exceeding 1,000 mg/kg. Treatments amended by a mixture of amendments (biochar
5%, biosolids 10%, woodchips 10%) contained much less of mineralizable nitrogen, which
was around 450 mg/kg (See Figure 2.27).
Available Nitrogen Forms: NH4+ and NO3Inorganic N includes soluble forms (NO2- and NO3-), exchangeable NH4+, and clayfixed nonexchangeable NH4+ (Rutherford et al., 2008). Therefore, readily available inorganic
nitrogen comprises soluble nitrate, nitrite, and exchangeable ammonium. However, the fixed
nonexchangeable NH4+ starts being slowly released when the pool of available ammonium
becomes depleted (Drury and Beauchamp,1991). Available NO3-and NH4+ are among the
most essential plant nutrients (Geng and He, 2020). Available NH4+ mainly results from
organic matter first decomposition and then mineralization (Soon and Liang, 2006).

66

NO3- though in the soil results from the nitrification process conducted by Nitrobacter,
Nitrospira, and Comammox bacteria (Madigan et al., 2012). Therefore, in natural soils, the
available nitrogen is a fraction of mineralizable nitrogen being released to the soil gradually.
However, in the case of an amending with biosolids, the provision with available nitrogen is
instant as this amendment contains large amounts of available NH4+ (Sulivan et al., 2015).
Annacis biosolids contain around 9,500 mg/kg of NH4+ and 12.2 mg/kg of NO3- in the dried
weight. Results of this study are consistent with above as initially measured pool of available
nitrogen provided by biosolids was nearly entirely comprised of ammonium (See Figures
2.28, 2.30).
In the case of both mines, the unamended subsoils were poor with available nitrogen
(See Figures 2.29, 2.31). It is not surprising because those substrates did not contain enough
organic matter for decomposition and mineralization (See Figure 2.17). Among all three
amendments applied in this study a significant amount of available nitrogen was provided
only by biosolids, and that was mainly in a form of NH4+. Initially the quantity of NH4+ was
several hundred times larger than NO3- (See Figure 2.33). Geng and He (2020) collected 240
field soil samples and measured NH4+ + NO3- and NH4+ : NO3-. Maximum sum they found
was 80 mg/kg, while the ratio was ranging from 100:1 to 1:22.16. For their further
experimental proceeding they adopted 36 mg/kg as a high amount of available nitrogen. In
this study the maximum sum was near to 800 mg/kg in the case of NA “75%sub25%bios”
treatment, while the ratios were ranging from 800:1 to 100:1. In the case of both mine
subsoils amended by 25% of biosolids v/v the amount of available nitrogen (mainly NH4+) at
initial stage of the experiment highly exceeded values which Geng and He (2020) adopted as
high. Throughout the experiment though the sum NH4+ + NO3- dropped (See Figure 2.32), and
the ratio NH4+ : NO3- changed drastically (See Figure 2.33). The ratio converted to nitrateprevailing over ammonium. This is a result of nitrification. It might indicate that nitrifying
bacteria in the reclaimed substrate have become strongly active. On the other side, the drop in
sum was not statistically significant at α=0.05. That means that the loss of available N forms
is relatively slow and vegetation together with microorganisms can profit from available NH4+
and NO3- for prolonged time.
High levels of available N in the case of both “75%sub25%bios” and “mixture”
suggest overfertilization when compared with figures quoted by Geng and He (2020). While it
is likely true in the case of the “75%sub25%bios” treatment, the “mixture” treatments’ NH4+
+ NO3- levels were below 300 mg/kg. That is still much more than the level which Geng and

67

He (2020) adopted as high (36 mg/kg), however, they based that value on field soil samples
(not reclaimed substrate as in this study). And in the nature the soil inorganic N is among the
most limiting nutrients (Geng and He, 2020).
Field Locoweed Performance
Although field locoweed individuals seemed to develop well on some subsoil
treatments, at the end of experiment 1 the nodulation was not observed neither in the case of
treatments well provided with readily available nitrogen nor on subsoil treatments deprived of
nitrogen. That might result from several reasons (Epp, 2015; Midwest Laboratories, 2021):
1) The symbiosis partner was absent in the substrate;
2) The symbiosis partner was inactive due to conditions such as salinity or lack of
necessary nutrient;
3) The experiment timing was not proper for the symbiosis formation;
4) The experiment was too short and plants did not manage to form nodules yet.
CONCLUSION
The results identified series of findings and answers to the study objectives. First and
foremost, two analyzed mines’ subsoils differed strongly in terms of their innate
physicochemical properties. That, in turn, affected vegetation mortality and productivity.
What they had in common is that in unamended form both subsoils were highly unfertile, and
due to that unproductive. Although they possessed satisfactory, or in some cases even
excessive, quantity of some micronutrients, they lacked in NPK, which are essential, biogenic
elements. Additionally, New Afton New Gold was encumbered with saline and sodic
problems additionally hindering vegetation and microorganismal development (Miller et al.,
2017). These two mines’ unamended subsoils would rather not be a self-sufficient substrate
for mine land reclamation when applied alone. The situation could be changed by an addition
of organic amendments, however, not all of them. Results showed that an addition of biochar
and woodchips alone did not change much. That stemmed from the same reason - both
amendments lacked macronutrients, especially nitrogen, and readily degradable organic
matter. This study results showed that the simple addition of organic matter (for example by
addition of woodchips or biochar alone) is not enough. Organic matter must meet certain
conditions such as appropriate degradability and satisfactory nutrient content. Woodchips and
biochar can be added mixed with other amendments, but rather not on their own.

68

From applied organic amendments only biosolids significantly increased aboveground biomass productivity. That resulted from biosolids’ large nutrient content such as N,
P, and Zn, and predigested organic matter, which is more labile and easier for decomposition
and carbon release.
Additionally, as an answer to the first study objective, tests appointed many more
differences between the New Afton New Gold and Teck Highland Valley Copper subsoils in
terms of physicochemical properties. New Afton New Gold subsoil contained significantly
more Ca, but first of all Na cations. Those two elements increased the pH of New Afton New
Gold subsoil to highly alkaline which caused organisms to deal with this important hurdle.
Moreover, sodium can acts adversely on soil structure and soil solution osmotic features.
However, sodium also tends to be washed out. Na levels declined throughout the experiment,
which confirmed the washout. It can be expected that over time of reclamation, rainwater will
wash away the excess of sodium, and then one of the factors limiting the vegetation growth
and development would debilitate. New Afton New Gold subsoil was deprived of K.
Potassium is one of the essential elements for vegetation development. Lack of K may be one
of reasons for significantly lower New Afton New Gold subsoil productivity. Both subsoils
contained an excess of boron, however, New Afton New Gold subsoil went beyond CCME
guidance much more than the Teck Highland Valley Copper subsoil. Perhaps, the provision of
sulfur was too large too, especially in the case of New Afton New Gold subsoil treatments.
S in the soil can form toxic compounds. It requires additional research whether this is the case
in New Afton New Gold reclaimed subsoil. Teck Highland Valley Copper subsoil contained
increased amounts of Cu and Mo. While this did not affect plant health, it might step up to
higher levels in the food chain and accumulate there. This requires additional research.
The best results in terms of plant above-ground biomass production were achieved
when mines’ subsoils were amended by a mixture of all three soil amendments. This result
partially answered the second study objective. An addition of biosolids alone boosted the
vegetation strongly, however, an addition of 25% biosolids volumetrically largely increased
mortality and brought other signs of overfertilization. In the “mixture” treatment biosolids
content was only 10%. This much seemed to cover plant demand for nutrient concurrently not
increasing the mortality significantly. It also seems that an application of less stable organic
amendments, especially biosolids, is less preferable than the amendments pre-composted
(Fierro et al., 1999). Paschke et al. (2005) demonstrated that an addition of a uncured
biosolids, by a rapid release of nitrogen, can increase a risk from the unwanted, annual weeds.

69

The third study objective focuses on an aspect of nitrogen cycling in reclaimed mine
subsoil. Results showed clearly that whenever there was nitrogen present in the soil substrate,
the biomass productivity was significantly larger than in the treatments in which N was
scarce. Significantly larger productivity was also associated with larger provision of carbon.
Whenever both N and C were provided in proper quantities, the biomass production was
significantly greater. That statement was supported by observations done on treatments
amended by biochar and woodchips. Both provided mostly recalcitrant carbon concurrently
being poor in nitrogen content and both did not result in satisfactory biomass production.
Even within a short time of study 1 experiment lots of nitrogen transformations were
observed. Total N, mineralizable N, NH4+ content, and NH4+ + NO3- visibly dropped
throughout the experiment. However, the rate of depletion was slow enough for plants to
benefit from the nitrogen provision. On the other hand at the end of the experiment, there was
significantly more NO3- than at the beginning. That means that in the early stage of
reclamation with the application of biosolids the nitrification process prevails.
Legumes are not good competitors to grasses especially on phosphorus-rich soils
(Smith, 1992). In this study as well the legume field locoweed Oxytropis campestris did not
do well in terms of competing to other grassy species on subsoils amended with biosolids,
nonetheless, its performance on unamended subsoil was better than all other species. The
above plus the ability to fix nitrogen makes this species potentially a good candidate to be
used in mine reclamation.

Figure 2.34 The greenhouse trial with graminoids and legume arranged in 7 blocks.
Photo credit Piotr Dzumek

70

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76

CHAPTER 3: CHANGED SOIL AMENDMENTS PROPORTIONS AND ITS INFLUENCE
ON SUBSOIL PHYSICOCHEMICAL PROPERTIES, SHRUBBY PLANTS PERFORMANCE,
AND NITROGEN CYCLING IN OPEN-AIR CONDITIONS.
INTRODUCTION
In greenhouse conditions plants usually perform better (Vernon, 2019). That is
because all aspects of controlled conditions in the greenhouse trials are set for constant
optima, while in nature optimal conditions for plant development do not happen often. That
explains why the greenhouse experiments possess limited value when extrapolating to field
and operational conditions. However, if the results of experiment 2, conducted open-air way
in semi-natural conditions, turn out similar to experiment 1, that would strengthen overall
research results and the conclusions credibility.
The fertilization has to be done in proper doses adjusted to the nature of fertilizer,
vegetation requirements, climatic conditions, substrate properties, etc. The same rule applies
when soil amendments are applied. The second study used the same subsoils, the same soil
amendments, but in different proportions. This intends to find an optimal dose according to
Shelford’s law of tolerance (Shelford, 1931).
Reclamation efforts are usually laborious and costly (Bradshaw, 2000; Prach and
Hobbs, 2008). That is why mine reclamation practitioners when making decisions on
choosing certain reclamation technics need to be supported by solid research results. When
laboratory results are backed by experiments conducted in conditions similar to natural, such
research gives a stronger basis for practical decisions. The first study has shown that the
addition of biosolids in the dose of 25% volumetrically might be too much. It seemed that
such a quantity of biosolids increased plant mortality and decreases productivity. A mixture of
soil amendments in the study 1 experiment gave much better results than all other treatments.
This way a new question emerged: whether that was caused by a lower, but still significant
amount of biosolids that provided necessary nutrients in more preferred amounts, or because
of the beneficial influence of other soil amendments that were applied in a mixture. To answer
this question, in the second study less biosolids was applied - 5% in the case of both the
mixture treatment and the treatment with biosolids applied alone. This aimed also at testing
whether the lower provision of nitrogen would affect its cycling.
In local environments, shrubby plants play an extremely important ecological role,
especially in the lower and middle Bunchgrass zone (Province of British Columbia, 1999).

77

Three shrub species were selected to the second study: common rabbitbrush Ericameria
nauseosa, soopolallie Shepherdia canadensis, and big sagebrush Artemisia tridentata.
Common rabbitbrush and big sagebrush are typical and commonly present in lower grasslands
(Antos et al., 1996, Province of British Columbia, 1999), where New Afton New Gold mine is
located. Soopolallie, in turn, is present in higher elevations (Walkup, 1991; Douglas et al.,
1998), being common in Teck Highland Valley Copper mine vicinity. All three species are
well adapted to interior BC biogeoclimatic conditions, hence, they may have the potential to
be an efficient mine reclamation tool. Soopolallie is a nitrogen fixer forming actinorrhizae
with bacteria from various strains of genus Frankia (Huguet et al., 2004). This shrub is not a
part of typical Bunchgrass zone vegetation, but is commonly present in Interior Douglas-fir
and Montane Spruce zones. Soopolallie is often present near water reservoirs, but it also
tolerates dry areas with nutritionally poor, mineral soils (Walkup, 1991). It is a species that
copes well with various forest disturbances such as fire or stand clear cut because it has the
perfect ability to reproduce through root clippings or suckers (Walkup, 1991). Soopolallie
produces fruits often consumed by birds and rodents. Besides, this shrub also provides a food
base for various ungulates. Seeds of soopolallie experience deep dormancy what is an
ecological adaptation to harsh climatic conditions (Baskin and Baskin, 1998). Soopolallie’s
applicability in mine reclamation might increase due to its nitrogen fixation ability. However,
the potential of soopolallie remains still largely unknown, as it is a non-leguminous plant
(Diagne et al., 2013). So far, the attention of most reclamation researchers was oriented
toward legumes often omitting non-leguminous nitrogen fixers.
The second study objectives were:
I.

To confirm similarities and differences between New Afton New Gold and

Teck Highland Valley Copper subsoils with or without amendments by observing the
physicochemical properties of subsoils subjected to treatments in semi-natural
conditions, as well as by observing differences in three shrubby species mortality rates
and productivity as responses to the treatments,
II.

To test the relative effects of altered proportions of the three soil amendments

(biosolids, woodchips, biochar) on two subsoils’ physicochemical properties;
III.

To compare qualitative and quantitative changes of nitrogen compounds

observed in the open-air study with the greenhouse study results.

78

METHODS
Study Site
The second study was located in a garden within the Aberdeen residential area in
Kamloops, BC. Elevation was 866 meters above see level which locates it between New
Afton New Gold and Teck Highland Valley Copper elevations. It began on May 23, 2019,
and ended on November 7, 2019. It lasted 24 weeks counting from the moment of all pots
placing in the experimental site. Subsoil substrate remained the same.
Experiment Design
Pots
2.4 L, round-shaped, perforated at the bottom pots were filled with 10 combinations of
subsoils and soil amendments (See Table 3.2). Pots were marked and numbered. Because of
10 replicates of every combination, pots got assigned evenly to 10 blocks. The pot’s bottom
was lined with weed-blocking fine-perforated textile which was to hold the subsoil material in
the pot concurrently allowing water to drain.
Plants and Seeds Sourcing
Table 3.1 Table presents plant material provenience and the age when transplanted.
PLANT SPECIES
BIG SAGEBRUSH

COMMON
RABBITBRASH

SOOPOLALLIE

PROVENIENCE OF

Splitrock Native Plants

TRU Research

Splitrock Native Plants

THE PLANT

Nursery in Lillooet,

Greenhouse in

Nursery in Lillooet,

MATERIAL

BC

Kamloops, BC

BC

6 months

5 months

24 months

AGE OF THE
PLANT MATERIAL

Common rabbitbrush seeds were sourced from Splitrock Native Plants Nursery. Those
seeds were sown 5 months before the experiment commencement to the substrate containing
peat and sand in ratio 1:1. Seeds were sown to the substrate similar to the one being used in
Splitrock Native Plants Nursery to maintain consistency. The entire process of independent
common rabbitbrush growing in the TRU greenhouse emulated the process adopted in
professional nursery from Lillooet. That included the same type and size of the styrofoam
blocks used (one plaque 140 mL), similar fertilization and the greenhouse settings.
Prior to sowing, seeds were undergone cold-wet stratification (Baskin and Baskin, 1998).

79

Transplantation of Seedlings
Transplantation of seedlings to the pots containing the subsoil combinations took place
on May 9, 2019. To minimize the shock caused to young plants, after transplantation plants
were still kept in the greenhouse in controlled conditions for 14 days before transportation to
the experiment location. After the transplants’ stabilization and ensuring that all plants are in
equally good vigor, pots with transplants were moved to the open-air study site. All plants
were transplanted within one day.
To increase the transplantation survivorship, seedlings were planted with minimal root
system disturbance. That was achieved by transplantation with the entire root system together
with the already existing soil substrate in which roots were installed. However, the amount of
existing soil substrate was minimal (less than 140 mL) to force quick relying on the treated
subsoil.
Pest Control
During the common rabbitbrush germination, to keep greenhouse pests controlled the
same measures were taken as in the first study experiment (See Chapter 2; Methods;
Experiment Design; Pest Control).
To emulate natural conditions, during the open-air phase of the second study
experiment invertebrate pests were not controlled at all. Birds and rodents were not controlled
either, however, the study site was fenced to prevent ungulates to encroach.
Settings and Watering
During the common rabbitbrush germination, the greenhouse settings were the same as
in the first study experiment (See Chapter 2; Methods; Experiment Design; Greenhouse
Settings).
Shrubby plants were watered as required, therefore, in that aspect, the experiment did
not emulate the natural conditions. Plants were not left to rely on natural precipitation because
once grown in a greenhouse and then radically shifted to the open-air conditions might not be
prepared to survive the extended drought periods. Also, plants did not germinate on the mine
subsoil which would let them adjust to this substrate. The third reason was that plants were
planted in pots that contained a relatively low amount of soil substrate and that in turn could
cause increased desiccation.

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Applied Subsoil Substrate Combinations
Subsoils collected from both mine stockpiles received the same amounts of soil
amendments. Soil amendments were applied in the following proportions as shown in Table
3.2.
Table 3.2 Second study experiment soil medium composition percentage breakdown.
SUBSOIL

BIOSOLIDS

WOODCHIPS

BIOCHAR

CONTROL

100%

0%

0%

0%

BIOSOLIDS

95%

5%

0%

0%

WOOD CHIPS

75%

0%

25%

0%

BIOCHAR

95%

0%

0%

5%

75%

5%

15%

5%

MIXTURE OF ALL
AMENDMENTS

Regarding study 1 experiment results (Chapter 2) in second study it was decided to go
with much lower biosolids application to avoid overfertilization. This time the biosolids
volumetric application rate was equalized to biochar which did not change and remained on
5%. The application of biosolids was 5 times lower in comparison to first study experiment
(Chapter 2) in order to observe whether lowered amount of biosolids would maintain
beneficial influence on plant productivity with parallel reduction of plant mortality. While the
total amount of organic soil amendments in a mixture remained the same, the amount of
biosolids was lowered to 5% and the amount of woodchips was increased to 15%. This was to
better use the woodchips feature of limiting an excess of NH4+ brought in by biosolids and to
slow down its release.
Experiment 2 Overall Treatments Combination
The experiment was a 2 x 5 x 3 factorial design, with 30 treatment combinations:
 2 subsoil types: 1. New Afton New Gold subsoil, 2. Teck Highland Valley Copper subsoil;
 5 soil amendment treatments: 1. control [no soil amendments], 2. biosolids alone, 3.
woodchips alone, 4. biochar alone, 5. mixture of all three soil amendments (see Table 3.2);
 3 plant treatments: 1. big sagebrush, 2. common rabbitbrush, 3. soopolallie.
Replicated 10 times for a total of 300 individual pots. A randomized block design was
arranged in the open-air experimental site.

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Soil Sampling
Soil samples collection was identical to the one adopted in the study 1 experiment (See
Chapter 2; Methods; Soil Sampling). At the experiment termination, the soil substrate from 10
pot-replicates was mixed thoroughly together, and then one sample was collected in a 1L
labeled plastic bag and immediately placed in the refrigerator. The same procedure was
repeated for each of the 30 subsoil treatments. This way all together a pool of 42 1-L samples
was obtained: 30 samples of the final subsoil treatments stage, 10 samples of the initial stage,
and two undisturbed topsoil samples for reference.
Tests and Instrumentation Used
In the case of study 2 experiment, the same tests and instrumentation were used as in
the study 1 experiment (See Chapter 2; Methods; Tests and Instrumentation Used). The only
difference was in the productivity measuring.
Plant Productivity
Because young shrubs were not equal at the beginning of the experiment, the
productivity was assessed indirectly by measurement of growth. The lengths of each plant’s
stem and all twigs were measured and summarized altogether at the beginning of the
experiment. The procedure was repeated at the end of the experiment. The total length
increment was obtained by subtraction of the initial result from the final one. Lengths of stem
and all twigs were measured by a ruler and noted.
Statistical Analyses
In the second study statistical analyses did not differ from those used in the first study.
The only difference is in the case of mortality rate presentation as, unlike to experiment 1, in
the second study this aspect data has a binary nature. The mortality presenting chart has a
“violin” form to depict the 0/1 did-not-survived/survived frequencies.
Once again, statistical analyses got divided into 3 sections (See Chapter 2; Methods;
Statistical Analyses).

82

RESULTS
Mortality, Productivity, Overall Subsoils’ Difference
Section 1: Mortality
Kruskal-Wallis test showed that mortality of plants growing on New Afton New Gold
subsoil combinations was significantly larger than mortality of plants growing on Teck
Highland Valley Copper subsoil combinations (Figure 3.1).

Figure 3.1 Second study mortality comparison between two mines’ subsoils: HVC – Teck Highland
Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of: 0 – plants which
did not survive to the end of experiment; 1 - plants which survived to the end of experiment.
Kruskal-Wallis test was used to compare the plant mortalities. n = 150

Kruskal-Wallis tests showed that neither the mortalities on New Afton New Gold
subsoil treatments nor on Teck Highland Valley Copper subsoil treatments differed
significantly.

Plant Species Mortality Analysis
Kruskal-Wallis tests showed that individuals of common rabbitbrush and soopolallie
growing on New Afton New Gold subsoil combinations experienced larger mortality than
individuals of these species growing on Teck Highland Valley Copper subsoil combinations.
Mortalities of big sagebrush on two mines’ subsoils did not differ significantly. On Teck
Highland Valley Copper subsoil combinations soopolallie’s survivorship was 100% (Figure
3.2).

83

Figure 3.2 Second study plant species mortality comparison between two mines’ subsoils: HVC –
Teck Highland Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of:
0 – plants which did not survive to the end of experiment; 1 - plants which survived to the end of
experiment. Kruskal-Wallis test was used to compare the plant mortalities. n = 50

Kruskal-Wallis tests showed that on Teck Highland Valley Copper subsoil treatments
mortalities of plant species were not equal. Wilcoxon pairwise comparison tests showed that
soopolallie individuals performed significantly better than big sagebrush (Figure 3.3 left).
Kruskal-Wallis tests showed that on New Afton New Gold subsoil treatments
mortalities of all plant species were statistically equal (Figure 3.3 right).

Figure 3.3 Second study plant species mortality comparison on two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Violin plots describe the frequency of:
0 – plants which did not survive to the end of experiment; 1 - plants which survived to the end of
experiment. Kruskal-Wallis test was used to compare the plant mortalities. n = 50

84

Section 1: Productivity
Kruskal-Wallis test showed that total stem and twigs elongation of plants growing on
New Afton New Gold subsoil combinations was significantly lower than total stem and twigs
elongation of plants growing on Teck Highland Valley Copper subsoil combinations (Figure
3.4).

Figure 3.4 Stem and twigs elongation comparison between two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Boxplots describe stem and twigs elongation of
all individual plants per mine subsoil at the end of the experiment. Horizontal line indicates the
median of stem and twigs elongation of all individual plants. Kruskal-Wallis test was used to compare
the medians. n = 150

Average stem and twigs elongation of plants growing on New Afton New Gold
subsoil combinations was more than two times lower than average stem and twigs elongation
of plants growing on Teck Highland Valley Copper (Figure 3.5).

HVC

40.96

NA

20.08

0.00

10.00

20.00

30.00

40.00

50.00

60.00

Stem and twigs elongation (cm)
Figure 3.5 Mean stem and twigs elongation comparison between two mines’ subsoils: HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Error bars are standard error of the mean.
n = 150

85

New Afton New Gold Subsoil Treatments Productivity Analysis
Kruskal-Wallis test showed that the stem and twigs elongations of plants growing on
New Afton New Gold subsoil treatments were not equal (Figure 3.6).
Wilcoxon pairwise comparison test showed that: the treatment with a mixture of soil
amendments resulted in a significantly larger stem and twigs elongation than treatment with
25% of woodchips only. The treatment with 5% of biosolids resulted in a significantly larger
stem and twigs elongation than the treatment with 25% of woodchips and the treatment with
5% of biochar. The treatment with unamended subsoil resulted in a significantly larger stem
and twigs elongation than the treatment with 25% of woodchips only (Figure 3.6).

Figure 3.6 Stem and twigs elongation comparison between the New Afton New Gold (New Afton)
subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Boxplots describe stem and twigs elongation of all individual plants per subsoil treatment at the end of
the experiment. Horizontal line indicates the median of plants’ stem and twigs elongation. KruskalWallis test was used to compare the medians of plant stem and twigs elongation on particular subsoil
treatments. Wilcoxon test was used for the pairwise comparison. * - p < 0.05,, *** - p < 0.001,
ns – difference not significant. n = 30

86

Teck Highland Valley Copper Subsoil Treatments Productivity Analysis
Kruskal-Wallis test showed that the stem and twigs elongations of plants growing on
Teck Highland Valley Copper subsoil treatments were not equal (Figure 3.7).
Wilcoxon pairwise comparison test showed that: the treatment with a mixture of soil
amendments resulted in a significantly larger stem and twigs elongation than unamended
subsoil, than treatment with 25% of woodchips, and than the treatment with 5% of biochar.
The treatment with 5% of biosolids resulted in a significantly larger stem and twigs
elongation than unamended subsoil, than treatment with 25% of woodchips, and than the
treatment with 5% of biochar. The treatment with unamended subsoil resulted in a
significantly larger stem and twigs elongation only than the treatment with 25% of
woodchips. The treatment with 5% of biochar resulted in a significantly larger stem and twigs
elongation only than the treatment with 25% of woodchips (Figure 3.7).

Figure 3.7 Stem and twigs elongation comparison between the Teck Highland Valley Copper (HVC)
subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Boxplots describe stem and twigs elongation of all individual plants per subsoil treatment at the end of
the experiment. Horizontal line indicates the median of plants’ stem and twigs elongation. KruskalWallis test was used to compare the medians of plant stem and twigs elongation on particular subsoil
treatments. Wilcoxon test was used for the pairwise comparison. ** - p < 0.01, *** - p < 0.001,
**** - p < 0.0001, ns – difference not significant. n = 30

87

Section 1: Both Mines’ Subsoil Overall Difference; Principal Component Analysis
Once again the Principal Component Analysis with 21 predictors presented strong
evidence that the two mines’ subsoils were overall significantly different Pseudo F= 106.323,
p < 0.001 (Figure 3.8). Datapoints representing pots containing subsoil with biosolids alone or
mixture were distinct from all other (cluster separately). Other treatments cluster together
revealing that there were no large differences between them (Figure 3.9)

Figure 3.8 Second study Principal Component Analysis of two mines’ subsoils in terms of 21
predictors: pH, EC, SOM, total N and C contents, mineralizable N content, NH4+ and NO3- contents,
as well as the contents of 13 basic elements. hvc – Teck Highland Valley Copper, new afton – New
Afton New Gold.

Figure 3.9 Second study Principal Component Analysis of two mines’ subsoil treatments: 100%sub –
unamended subsoil, 95%sub5%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip
- subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. hvc – Teck Highland Valley Copper,
new afton – New Afton New Gold.

88

Physicochemical Properties of Mines’ Subsoils When Unamended and Amended
Section 2: pH
New Afton New Gold subsoil pH was significantly higher than Teck Highland Valley
Copper both in the initial and the final stages (Figure 3.10 left). Within each mine separately
there was no significant difference between the initial and final pH either in the case of New
Afton New Gold or in the case of Teck Highland Valle y Copper (Figure 3.10 right).

Figure 3.10 Second study two mines’ subsoil pH comparison in the initial and final stages of the
experiment (left) Comparison of the subsoil pH in the initial and final stages of the experiment within
each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15

In the case of both mines the final pHs were statistically not equal (Figures 3.11). In
both cases, the biosolids-containing treatments had the lowest pH. In both cases as well, pHs
seemed to drop throughout the experiment (Figures 3.11).

Figure 3.11 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments pH comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

89

Section 2: Electroconductivity (EC)
New Afton New Gold subsoil electroconductivity was significantly higher than Teck
Highland Valley Copper both in the initial and final stages (Figure 3.12 left). Within each
mine separately there was a significant difference between the initial and final
electroconductivities both in the case of NA and in the case of HVC (Figure 3.12 right).

Figure 3.12 Second study two mines’ subsoil electroconductivities comparison in the initial and final
stages of the experiment (left) Comparison of the subsoil electroconductivities in the initial and final
stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New
Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 15

In the case of NA subsoil treatments, there was no evidence that the final
electroconductivities were statistically not equal (Figure 3.13 left). In the case of HVC subsoil
treatments the final electroconductivities were statistically not equal (Figure 3.13 right).
Throughout the experiment, the EC dropped.

Figure 3.13 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments electroconductivities comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

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Section 2: Soil Organic Matter (SOM) Content
New Afton New Gold subsoil SOM content was significantly higher than Teck
Highland Valley Copper SOM content in both the initial and final stages (Figure 3.14 left).
Within each mine separately there was a significant difference between the initial and final
SOM contents in the case of New Afton New Gold subsoil only (Figure 3.14 right).

Figure 3.14 Second study two mines’ subsoil organic matter contents comparison in the initial and
final stages of the experiment (left) Comparison of the subsoil organic matter contents in the initial
and final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA
– New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to
compare the medians. initial n= 5, final n= 15

In the case of both mines the final soil organic matter contents were statistically not
equal (Figures 3.15). In both cases, the subsoil treatment containing woodchips as an only
amendment had finally the second-largest SOM content and the subsoil amended by a mixture
had the first largest SOM content (Figures 3.15).

Figure 3.15 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments soil organic matter contents comparison in the initial and final stages of the experiment.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

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Section 2: Sodium (Na) Content
New Afton New Gold subsoil Na content was significantly larger than Teck Highland
Valley Copper Na content both in the initial and final stages (Figure 3.16 left). Within each
mine separately there was a significant difference between the initial and final Na content
only in the case of the New Afton New Gold subsoil (Figure 3.16 right)

Figure 3.16 Second study two mines’ subsoil sodium content comparison in the initial and final
stages of the experiment (left), comparison of the sodium content in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15

Only in the case of NA subsoil treatments, the final Na contents were statistically not
equal (Figure 3.17 left). In the case of New Afton New Gold, every subsoil treatment
contained more sodium in the initial than in the final stage of the experiment (Figure 3.17
left).

Figure 3.17 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments sodium contents comparison in the initial and final stages of the experiment. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

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Quantitative and Qualitative Nitrogen Content Analyses
Section 3: Total Nitrogen (N) Content
New Afton New Gold subsoil total N content was significantly larger than Teck
Highland Valley Copper total N content both in the initial and final stages (Figure 3.18).
Within each mine separately there was no significant difference between the initial and final
total N contents either in the case of New Afton New Gold or in the case of Teck Highland
Valley Copper (Figure 3.19).

Figure 3.18 Second study two mines’ subsoil total nitrogen content comparison in the initial and
final stages of the experiment. HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial
n= 25, final n= 75

Figure 3.19 Second study comparison of the total nitrogen content in the initial and final stages of
the experiment within each mine. HVC – Teck Highland Valley Copper, NA – New Afton New Gold.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. initial
n= 25, final n= 75

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In the case of New Afton New Gold and the case of Teck Highland Valley Copper
subsoil treatments, the initial and final total N contents were statistically not equal (Figures
3.20, 3.21). Both, in the case of New Afton New Gold and the case of Teck Highland Valley
Copper subsoil treatments initially there was no statistical difference between the
“95%sub5%bios” and the “mixture” treatments, while those two treatments differed
significantly from all other treatments. The situation changed slightly at the end of the
experiment. The lack of statistical difference between the “95%sub5%bios” and the “mixture”
treatments remained in the case of New Afton New Gold only (Figure 3.20). In the case of
Teck Highland Valley Copper, the difference between the “95%sub5%bios” and the
“mixture” treatments became significant (Figure 3.21).

Figure 3.20 Second study New Afton New Gold subsoil treatments total nitrogen contents comparison
in the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip - subsoil amended by
25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
**** - p < 0.0001, ns, NS. – difference not significant. initial n= 5, final n= 15

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Figure 3.21 Second study Teck Highland Valley Copper subsoil treatments total nitrogen contents
comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub –
unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar
alone, mixture - subsoil amended by a mixture of amendments. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. Wilcoxon test was used for pairwise
comparison ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not significant.
initial n= 5, final n= 15

Both, in the cases of New Afton New Gold and Teck Highland Valley Copper subsoil
treatments only the “mixture” and the “95%sub5%bios” at the end of the experiment were not
significantly different from the reference of undisturbed topsoil in terms of total N content.
All other treatments differed significantly from the reference (Figures 3.22, 3.23). In these
cases, the total N contents were significantly lower than the reference’s one. In the case of
both the “mixture” and the “95%sub5%bios” treatments, the initial total N contents were
significantly larger than the reference, but finally the total N content dropped and the
differences became not significant (Figures 3.22, 3.23).

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Figure 3.22 Second study New Afton New Gold subsoil treatments total nitrogen contents comparison
in the initial and final stages of the experiment to the reference of undisturbed topsoil. Subsoil
treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids
alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil
amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon test was
used for pairwise comparison ** - p < 0.01, *** - p < 0.001, ns – difference not significant.
initial n= 5, final n= 15, undisturbed n=5

Figure 3.23 Second study Teck Highland Valley Copper subsoil treatments total nitrogen contents
comparison in the initial and final stages of the experiment to the reference of undisturbed topsoil.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians. Wilcoxon
test was used for pairwise comparison ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001,
ns, NS. – difference not significant. initial n= 5, final n= 15, undisturbed n=5

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Section 3: Mineralizable Nitrogen Content
There was no significant difference in terms of mineralizable N content between NA
and HVC subsoils either in the initial or final stage (Figure 3.24 left). Within each mine
separately there was no significant difference between the initial and final mineralizable N
contents either in the case of NA or in the case of HVC (Figure 3.24 right).

Figure 3.24 Second study two mines’ subsoil mineralizable nitrogen contents comparison in the initial
and final stages of the experiment (left), comparison of the mineralizable nitrogen contents in the initial
and final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA –
New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 15

In the case of NA and the case of HVC subsoil treatments the final mineralizable N
contents were statistically not equal (Figures 3.25). In both cases, biosolids-containing
treatments had the largest mineralizable N contents. Mineralizable N contents seemed to drop
strongly throughout the experiment (Figures 3.25).

Figure 3.25 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments mineralizable nitrogen contents comparison in the initial and final stages of the experiment.
Subsoil treatments: 100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of
biosolids alone, 75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar –
subsoil amended by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments.
Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 1, final n= 3

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Section 3: Available Nitrogen Content – Ammonium Cation (NH4+)
There was no significant difference in terms of NH4+ content between NA and HVC
subsoils either in the initial or final stage (Figure 3.26 left). Within each mine separately there
was no significant difference between the initial and final NH4+ contents either in the case of
NA or HVC (Figure 3.26 right).

Figure 3.26 Second study two mines’ subsoil NH4+contents comparison in the initial and final stages of
the experiment (left), comparison of the NH4+ contents in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 15

In the case of NA and the case of HVC subsoil treatments the final NH4+ contents
were statistically not equal (Figure 3.27). In both cases, biosolids-containing treatments had
the largest NH4+ contents. NH4+ contents seemed to drop strongly throughout the experiment
(Figure 3.27).

Figure 3.27 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments NH4+contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

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Section 3: Available Nitrogen Content – Nitrate Anion (NO3-)
New Afton New Gold subsoil NO3- content was significantly larger than Teck
Highland Valley Copper subsoil NO3- content only in the final stage of the experiment (Figure
3.28 left). Within each mine separately there was a significant difference between the initial
and the final NO3- content in the case of HVC subsoil only (Figure 3.28 right).

Figure 3.28 Second study two mines’ subsoil NO3-contents comparison in the initial and final stages of
the experiment (left), comparison of the NO3- contents in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15

Only in the case of NA subsoil treatments the final NO3- contents were statistically not
equal (Figures 3.29). In the New Afton New Gold case, the subsoil treatment containing
biosolids as an only amendment had the largest final NO3- content, and the subsoil amended
by a mixture had the second-largest NO3- content (Figure 3.29 left).

Figure 3.29 Second study New Afton New Gold (left) and Teck Highland Valley Copper (right) subsoil
treatments NO3-contents comparison in the initial and final stages of the experiment. Subsoil treatments:
100%sub – unamended subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone,
75%sub25%wchip - subsoil amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended
by 5% of biochar alone, mixture - subsoil amended by a mixture of amendments. Horizontal line
indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 1, final n= 3

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Section 3: Available Nitrogen Content – NH4+, NO3-; Sum and Ratio
There was a significant difference between the sums of NH4+ and NO3- in the initial
and the final stages of the experiment at α=0.05 (Figure 3.30).

Figure 3.30 Second study comparison of the sum of NH4+ and NO3- in the initial and final stages of
the experiment. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 4, final n= 12

There was a significant difference between the ratios of NH4+ to NO3- in the initial and
final stages of the experiment at α=0.05 (Figure 3.31).

Figure 3.31 Second study comparison of the ratio of NH4+ to NO3- in the initial and final stages of the
experiment. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 4, final n= 12

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DISCUSSION
Second study experiment confirmed most of the observations on mine subsoils which
were revealed in the first study experiment (Chapter 2). Lowering the quantity of biosolids
also resulted positively. Open-air conditions did not drive changes in subsoil to other
directions than greenhouse conditions of the experiment 1, despite the fact that applied
vegetation species were very different in the study 2 experiment. Applied shrubs
demonstrated that they were good indicators of reclamation effectiveness as well as they
themselves might be a promising tool for mine reclamation.
Mortality, Productivity, Overall Subsoils’ Difference
Mortality
In natural conditions, in ecosystems where the woody-stem plants play a dominating
role, at the early stage of regeneration young seedlings germinate in abundance but then
experience high mortality (Swaine and Hall, 1983). Seedlings are often eliminated by such
factors as competition with neighboring vegetation, browsing, extreme climatic events, insect
or disease infestation (Collet and Le Moguedec, 2007). On reclaimed mine sites Rizza et al.
(2007) demonstrated that overwintering stress kills a large percentage of seedlings. In this
study experiment, although insects infestations were observed they did not seem to increase
mortality. Adverse soil substrate conditions were rather these which played a major role in
causing shrubby seedlings mortality. A large part of deaths resulted from the formation of a
film of crystals on New Afton New Gold subsoil (See Figure 3.31). Crystals were debilitating
young plants. When windy weather, all plants have been jerked frequently in all directions.
After hundreds of wind jerks some plants, debilitated by salt crystals, got torn apart and
killed. Salts formation on NA subsoil, with its multifaceted adverse influence on young
seedlings, is most probably the explanation of significantly larger mortality among plants
growing on NA subsoil versus plants growing on HVC subsoil (See Figure 3.1).

Figure 3.32 Common rabbitbrush individual covered by a film of white salt crystals debilitating plant’s
resistance to wind. Plant is torn apart and killed. Photo credit Piotr Dzumek.

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However, on both mines’ subsoil treatments, the mortality rates were visibly lower
than in experiment 1 (Chapter 2). Most probably in can be explained by the lowering of
biosolids content to 5% vol. Statistical tests did not detect any significantly larger mortality
among seedlings growing on biosolids-containing subsoil combinations.
Productivity
In study 1experiment (Chapter 2) an addition of biosolids increased significantly the
grasses and leguminous forb biomass productivity. However, it remained an open question
whether the same effect biosolids would have on woody-stem plants. This is an important
question as in several reclamation sites in British Columbia shrubs and trees are intended to
be re-established. Teck implemented revegetation programs in Highland Valley Copper and in
Pinchi Lake Mine in which shrubs were intended for installation on reclaimed sites amended
by biosolids (Teck 2012 Sustainability Report; Teck, 2013). Hunt (2017) reports that on
reclaimed rock disposal sites in Mount Polley Mine coniferous seedlings grew better on sites
where soil substrate was amended with biosolids in comparison to unamended control sites.
Additionally, on unamended sites the young trees showed chloroses which indicated nutrient
deficiency.
Study 2 experiment results demonstrated that an application of 5% biosolids increased
the stem and twigs elongation. However, the differences between the unamended subsoil and
the “75%sub25%bios”, as well as between the unamended subsoil and the “mixture”
treatment were significant only in the case of Teck Highland Valley Copper mine subsoil (See
Figures 3.6, 3.7). Again, similarly to study 1 (Chapter 2), the productivity on Teck Highland
Valley Copper subsoil combinations was much larger than on New Afton New Gold subsoil
combinations (See Figure 3.4). This result confirmed that when amended with nutrient-rich
amendment Teck highland Valley Copper subsoil becomes well productive. It is striking that
here again the most productive on both mines’ subsoils were treatments amended by the
mixture of soil amendments. However, in both cases, New Afton New Gold and Teck
Highland Valley Copper, the differences between “mixture” treatments and treatments
amended by 5% biosolids alone were not statistically significant. That means that an addition
of woodchips and biochar in a mixture did not help increase productivity. In turn, similarly to
study 1 experiment (Chapter 2), the lowest productivities in the case of both mines were
achieved by plants growing on subsoils amended by 25% of woodchips (See Figures 3.6, 3.7).

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Overall Difference of Both Mines’ Subsoil Physicochemical Properties
Study 2 Principal Component Analysis confirmed the first study (Chapter 2) finding
that physicochemical properties of two analyzed mines subsoils differ strongly (See Figure
3.8).
In terms of particular subsoil treatments, the situation in study 2 experiment was
similar to the one of the first study (Chapter 2), but not identical. While in experiment 2, both
the “95%sub5%bios” and the “mixture” treatments contained the same amounts of biosolids,
they did not tend to differ from each other that much as in experiment 1 (See Figure 3.9). That
indicates that neither an addition of biochar nor woodchips to the mixture changed much in
terms of physicochemical properties.
Physicochemical Properties of Mines’ Subsoils When Unamended and Amended
pH
Most plants prefer pH which is around neutral or slightly acidic (Boeckmann, 2019).
Also soil bacteria conducting such processes as organic matter decomposition or nitrogen
fixation prefer around-neutral soil pH (Griep, 2020).
In the second study experiment the New Afton New Gold subsoil pH is significantly
higher than Teck Highland Valley Copper subsoil pH (See Figure 3.10). Both mines’
unamended subsoils were alkaline. The further pH departs either way from the value of 7, the
larger is the hindrance for plant and microorganisms to develop. New Afton New Gold
subsoil pH departed toward alkaline values more than Teck Highland Valley Copper subsoil.
Similar to experiment 1 (Chapter 2), pH values of New Afton New Gold subsoil treatments
seemed to approach to the top limits of the vegetation tolerance on the strongly alkaline side
(Shelford, 1931), while Teck Highland Valley Copper subsoil treatments seemed to adopt pH
values closer to preferred by most plant species. Once again, the lowest pH was adopted by
biosolids-containing treatments (See Figure 3.11). NH4+, provided by biosolids, reacts with
molecules of water and forms H3O+ which acidify the substrate. This reaction was strongly
present especially in study 1 experiment (Chapter 2), while in study 2 experiment this reaction
had minor importance as the amounts of biosolids used were several times lower. While in
study 2 the acidifying process presented above was limited due to lower amounts of biosolids,
the alkalinization remained strong.

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Electroconductivity (EC)
In terms of electroconductivity, study 2 experiment confirmed observations from study
1 experiment (Chapter 2) as results from both trials are very similar. From this it might be
concluded that even though experiment 2 has been conducted in different conditions,
processes deciding about the EC change have carried on similarly. The only difference was
that in experiment 2 the change of electroconductivity throughout the experiment was deeper
(See Figure 3.12), causing the difference between the initial EC and the final EC to become
statistically significant. It could be expected that if the experiment had been conducted for a
longer time, the tendency of EC decrease would have been maintained until it approaches the
EC level of undisturbed topsoils (references). Electroconductivity indicates the level of ions
concentration (Miller at al., 2017). The drop in EC throughout the experiment indicated the
decrease in the amount of ions. The deeper drop of ions concentration observed in study 2
experiment may indicate that the loss of certain ions depends on climatic conditions (profuse
precipitation happening occasionally, larger temperature amplitudes) as well as on the
experiment duration (experiment 2 was 8 weeks longer). That all suggests that not only the
use of an organic amendments can help alleviate the substrate salinity problem, but also water
wash out can be helpful. Brown and Chaney (2016) suggest that in some cases an irrigation
may be required to remove excessive salinity.
Soil Organic Matter (SOM)
Most of soil organic matter concentrates in topsoil. During surface mining entire
topsoil is removed. Topsoil removal immediately decreases the soil organic matter budget
(Larney and Angers, 2012). Recovery of soil organic matter and nutrient cycling is critical to
the success of rehabilitation of deeply disturbed post-mining sites (Banning et al., 2008). In
study 2 experiment results, the readings of soil organic matter content in unamended subsoils
did not differ much from the readings obtained from study 1 experiment (Chapter 2). It
confirms, therefore, the correctness of the test. Again, both New Afton New Gold and Teck
Highland Valley Copper unamended subsoils contained less SOM than the reference of
undisturbed topsoils, respectively 2 and 4 times. Especially the subsoil from Teck Highland
Valley Copper mine contained low amounts of SOM. New Afton New Gold subsoil SOM
content was significantly larger than Teck Highland Valley Copper one (Figure 3.14). Poor
Teck Highland Valley Copper subsoil SOM content might be one of the explanations why this
mine unamended subsoil was not productive. Bauer and Black (1994) reported that in their 4-

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year experiment the highest total aerial dry matter and grain yields were associated to these of
their study plots which possessed the highest SOM contents. Moreover, the loss of
productivity was associated with a depletion of SOM. Bauer and Black (1994) explained that
the deplation of SOM was associated with a loss of soil fertility which at the end resulted in
decreased productivity. In this study the largest amount of SOM in the case of both mines was
noted on subsoils amended by a mixture of soil amendments (See Figure 3.15). It is not
surprising as this subsoil treatment contained volumetrically 25% of combined organic soil
amendments. Plants growing on the “mixture” treatment produced extensive root system. That
increased the final SOM results as well.
Basic Elements
In terms of basic elements, many analyses’ results from study 2 experiment were
identical or very similar to study 1 experiment results (Chapter 2). Comparison in terms of
basic elements contents serves to find more similarities and dissimilarities existing between
New Afton New Gold and Teck Highland Valley Copper subsoils. Eventually, the differences
found may explain large overall discrepancy in terms of two mines subsoils’ vegetation
mortality and productivity. Results from both studies identified that New Afton New Gold
subsoil contained significantly more: B, Ca, Fe, Mg, Mn, Na, S, Zn. On the other hand, Teck
Highland Valley Copper subsoil contained significantly more: Cu, K, Mo (See Chapter 2 and
Chapter 3 Results for Na, Appendices A and B for the rest of elements). Some of elements
analyzed in both studies were listed by Marschner (1983) as essential mineral plant nutrients,
meaning that in plant nutrition these elements cannot be replaced by any other element. These
were Fe, Mn, B, Zn, Cu, Mo. From these list none seemed to scarce in analyzed unamended
subsoils. Moreover, B in the case of New Afton New Gold subsoil, and Cu, as well as Mo in
the case of Teck Highland Valley Copper were exceeding the CCME (2021) guidelines.
However, the availability and uptake of nutritional elements is chiefly governed by soil
substrate properties (Pandey, 2015), including soil microbial communities presence, activity,
and facilitation (Jacoby et al., 2017). Therefore, a simple abundance of certain element in the
soil substrate does not guaranty yet a proper elemental nutrition.
On the other hand, such elements as C, N, P, and Zn differentiated subsoil treatments
in both experiments. The concentrations of these elements were always higher in subsoil
combinations amended by biosolids alone or a mixture (containing biosolids too) (See
Chapter 2 and Chapter 3 Results for C and N; Appendices A and B for P and Zn). Since those

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two subsoil treatments were the most productive as well it may be speculated that the
concentration of one or more of these elements influenced positively the productivity.
Analyzing all elements separately, and knowing their potential influence on
vegetation, it seemed that a higher concentration of Na dragged the New Afton New Gold
subsoil’s productivity down, while a higher concentration of K pulled the Teck Highland
Valley Copper subsoil’s productivity up.
Total Carbon
Soil organic matter is a sink for carbon (Akala and Lal, 2000) and its role in CO2
sequestration cannot be overestimated. Carbon is stored mainly in a form of organic matter in
both living soil organisms and dead organic residues (Insam and Domsch, 1988) Ganjegunte
et al. (2009) demonstrate that over years following the mine land reclamation the total carbon
content in the soil substrate was shaped by the type of vegetation, substrate texture, as well as
by lignine content. Antonelli et al. (2018) revealed that a single biosolids application to
reclaimed mine tailings has facilitated plant biomass production and has enhenced the carbon
accumulation for more than a decade.
The results of study 2 experiment showed a large difference between both mines’
unamended subsoils in terms of total carbon content. While New Afton New Gold unamended
subsoil’s total carbon content seemed to be low placing around 1.5%, the Teck Highland
Valley Copper unamended subsoil’s total carbon was even around 6 times lower. The lack of
total carbon certainly translates to low microbial carbon as well as low dead organic matter.
That might be a reason for little Teck Highland Valley Copper's unamended subsoil
productivity. Extreme lack of soil carbon may pose a large hindrance in terms of spontaneous
recolonization by soil living organisms. Regarding this, a use of organic soil amendments,
which would increase the substrate’s pool of carbon, appears as necessary for a successful
ecosystem restoration on Teck Highland Valley Copper's subsoil.
Quantitative and Qualitative Nitrogen Content Change
Total Nitrogen
In the second study the amounts of added biosolids were lowered strongly. Biosolids
though was the only soil amendment among applied which provided noticeable amounts of
total nitrogen. It was revealed in study 1 experiment (Chapter 2) that applied there plants grew
the best on substrate possessing 0.10% to 0.20% of total nitrogen. In study 2 experiment all

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the most productive treatments at the end of the experiment possessed the concentration of
total nitrogen which fell into that interval (See Figures 3.20, 3.21). Wang et al. (2017)
conducted a large scale mapping of total nitrogen content in Liaoning, a Chinese province.
Their results showed that the largest concentration of total nitrogen noted in the 0-5 cm soil
layer was 0.16%. Marx et al. (1999) provided an information that a typical agricultural soil in
the Willamette Valley, which they analyzed, contained 0.10 to 0.15 % of total N. Therefore,
an addition of 5% of biosolids may be close to ideal in terms of total nitrogen provision. What
is worth noticing that with an addition of 5% biosolids volumetrically to the subsoil the wellgrowing plants did not show any negative signs of overfertilization.
New Afton New Gold subsoil total nitrogen content was significantly larger than Teck
Highland Valley Copper total nitrogen content (See Figure 3.18). Such results were aligning
to study 1 experiment (See Chapter 2 Results). Again the HVC unamended subsoil’s
concentration of total nitrogen was extremely poor. Insufficiency of this intrinsic element,
besides the lack of carbon, may pose an additional and potent hindrance in terms of
spontaneous recolonization of reclaimed subsoil by soil living organisms as well as by plants.
An availability of N strongly influences soil microorganisms abundance and activity. N deficit
causes reduced efficiency of microbial biosynthesis (Blagodatsky and Richter, 1998).
Additionally, microorganisms have to compete for nitrogen with plants (Kaye and Hart,1997).
It is noticeable that in the experiment these plants that were provided with a decent amount of
total C (in a form of biochar and woodchips), but deprived of N remained reluctant to grow.
It confirms that nitrogen is crucial for ensuring plant growth. Alike to study 1 (Chapter 2), in
study 2 in terms of total nitrogen, the closest treatment to the reference of undisturbed topsoil
(“mixture” and “95%sub5%bios”) turned out as the most productive (See Figures 3.22, 3.23).
What differed study 2 results from the study 1 (Chapter 2) was that the treatments amended
by biosolids alone joined to the group of treatments which did not differ significantly from the
undisturbed topsoil. That took place thanks to lowering the biosolids concentration to 5%
only.
For a second time, in the cases of both mines the final total N contents were lower than
the initial ones (See Figure 3.19). However, the initial and final total N contents did not differ
statistically either in the case of NA subsoil or HVC. That confirms that nitrogen gets
depleted or lost but the process is relatively slow, which is a good news as vegetation and soil
microorganisms may profit from the total N pool for a prolonged time.

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Mineralizable Nitrogen
Curtin and Campbell (2008) state that the rate of nitrogen mineralization depends on
the quantity of mineralizable organic N in the soil and on environmental conditions. They also
inform that large amounts of mineralizable N can accumulate under grassland. Only 1 to 4%
of total N becomes plant-available during a growing season through mineralization (Marx et
al.,1999). Thus, the mineralizable nitrogen plays a pivotal role in maintaining the entire N
cycle by the release of mineralized nitrogen on the one hand, and by reception of dead organic
matter for mineralization on the other one.
The mineralizable nitrogen concentration in the case of study 2 experiment was
remarkably lower in comparison to study 1 experiment (Chapter 2) which corresponds with
the lower amount of biosolids applied. Initially, the biosolids-containing treatments stood out
with an amount of mineralizable nitrogen among all other treatments, but similarly to total N,
the final amount of mineralizable N was lower than the initial one. However, here again in the
case of both mines, the differences between the initial and final mineralizable nitrogen
contents were not statistically significant (See Figure 3.24).
Initially, the treatments amended with 5% of biosolids alone contained around 140160 mg of mineralizable N/kg, while the treatments amended by a mixture of soil
amendments placed a bit higher, around 160-170 mg/kg (See Figure 3.25). In comparison to
the undisturbed topsoil, subsoils amended by biosolids alone or the mixture contained still
much more mineralizable N, but only in the initial phase. At the end of the experiment though
the amount of mineralizable nitrogen decreased strongly and approached to the reference of
undisturbed topsoil. It indicates that the amounts on mineralizable N tend to stabilize over
time.
Available Nitrogen Forms: NH4+ and NO3Alike to study 1 (Chapter 2), also in study 2 experiment the levels of available
nitrogen strongly exceeded the level which Geng and He (2020) in their study adopted as
high. Marx et al. (1999) stated that normal levels of NH4+ are ranging from 2 to 10 mg/kg and
levels of NO3- over 30 mg/kg would be excessive. However, above values refer to natural or
agricultural soils, but not necessarily to reclaimed soils. In natural soils the levels of available
nitrogen forms stabilize on rather low levels as this forms are quickly taken up and utilized by
soil organisms or plants. It is worth to notice that the amounts of available nitrogen forms in

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undisturbed topsoils collected from vicinities of both mines were also low. Additionally, in
these reference soils the NO3- prevailed. It testifies that in natural conditions nitrogen cycles
fast and living organisms do not let mineralized forms of nitrogen stay unutilized in the soil
for long. However, degraded soils are different. Mine reclaimed soils due to its deep
degradation supposed receive large organic amendments dozing (Larney and Angers, 2012)
providing more of available nutrient.
After a closer look upon the mineralized forms of nitrogen provided by biosolids, it
might be noticed that NH4+ strongly prevailed over NO3- (See Figure 3.31). Other two applied
amendments did not provide much of either NH4+ or NO3- (See Figures 3.27, 3.29). Initially,
the treatments amended by 5% of biosolids alone contained around 110-120 mg/kg of
mineralized nitrogen forms, whereof NO3- was only around 5%. Treatments amended by a
mixture of soil amendments contained a bit more of mineralized nitrogen, which was around
120-160 mg/kg, but again, initially NO3- constituted a lesser fraction. At the end of study 2
experiment though, unlike to the first study, the level of available mineral nitrogen forms
dropped significantly (See Figure 3.30). Additionally, the proportion NH4+: NO3- changed, but
not as drastically as in the study 1 experiment (See Figures 2.33, 3.31). However, NO3- started
to play a more important role. This again confirms that in the open-air conditions, same as in
the greenhouse, nitrification process prevails at the initial stage of reclamation after biosolids
application to the reclaimed subsoil.
Soopolallie Performance
On both subsoils amended by woodchips alone, soopolallie experienced the largest
average growth rate among all species. The same situation took place on Teck Highland
Valley Copper subsoil amended by biochar. That may suggest that soopolallie can cope with
harsh conditions of reclaimed mine unamended soils. Surprisingly, soopolallie didn’t take on
on New Afton New Gold subsoil combinations that contained biosolids but did better on
biosolids-free New Afton New Gold subsoils combinations. Many of soopolallie individuals
produced nodulation which indicates that the process of nitrogen fixation was at least
initiated. All above testifies that Shepherdia canadensis can be considered as a good
candidate to become an efficient tool in deeply degraded mine soils. The ability for nitrogen
fixation adds value to this species. Hendrickson and Burgess (1989) provided that in a
regenerating lodgepole pine Pinus contorta stand in southern British Columbia Shepherdia
canadensis fixed 0.78 kg/ha/year. It might seem not too much, however, once installed,

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soopolallie can have a long lasting positive effect on reclaimed land by its constant nitrogen
provision to the ecosystem. Rhoades et al. (2008) presented results showing that about half
the 600 g total N/m2 accumulated across the newly formed river terrace chronosequence
occurred during the 120 years when Shepherdia canadensis was dominant. Moreover, they
informed that over years Shepherdia canadensis has enriched soil nitrogen pool several times
more than willow Salix sp.
CONCLUSION
The second study experiment confirmed most of the observations from the first
experiment which strengthen the credibility of both studies findings. The first study objective
focused on differences and similarities between New Afton New Gold and Teck Highland
Valley Copper subsoils. When experiment 2 confirmed that these two subsoils
physicochemical properties differ strongly, one aspect is worth emphasizing –
electroconductivity value of New Afton New Gold subsoil at the end of the experiment 2
dropped much more than in the study 1 (Chapter 2) case. This means that in natural conditions
the elution of sodium excess happened quickly. Observing the sodium leaching rate, it could
be speculated that thanks to the wash out it rather would not take longer than two vegetation
seasons that the Na concentration approaches to the undisturbed topsoil level.
The second study objective focuses on soil amendments’ influence on mines’ subsoils.
In experiment 2, unlike to experiment 1 (Chapter 2), the same amounts of biosolids (5% vol.)
were used in two subsoil treatments ( 1. - “95%sub5%bios”, 2. - “mixture”). This aimed at:
- verification whether smaller amounts of biosolids support plant growth to a similar extent
as larger amounts applied in experiment 1 (Chapter 2),
- verification whether the greater productivity of the “mixture” treatment over the
“95%sub5%bios” treatment in experiment 1 (Chapter 2) was because other soil
amendments (biochar, woodchips) were applied in the mixture and they supported the
growth, or because an application of 25% of biosolids alone in “75%sub25%bios”
treatment was simply a too large dose eventually reducing the productivity and increasing
the plant mortality.
Results from study 2 experiment have answered to both these questions. Firstly, the addition
of only 5% of biosolids in both “95%sub5%bios” and “mixture” treatments resulted in much
larger plant growth in comparison to all biosolids-free subsoil treatments, and secondly, the
fact that there was no statistical difference in productivity and mortality between

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“95%sub5%bios” and “mixture” treatments showed that the other two soil amendments in a
mixture did not contribute to elevated productivity at least at the early stage of the
reclamation.
The third study objective focused on nitrogen cycling aspect. In study 2 experiment on
one hand the dynamics of nitrogen compounds changes over time of the experiment were
intensified by the open-air weather conditions such as high summer temperatures or occurring
heavy downpours, and limited on the other hand by the simple fact of much lower biosolids
application. The most important finding from both studies is that nitrogen cycling phases
contributing to nitrogen loss pace relatively slowly allowing vegetation to profit from elevated
nitrogen compounds concentration for longer time.
In the second study three shrubby plant species were used, including a non-legume
nitrogen fixer. Additionally, all three species are native plants of great ecological function
fulfilled by blooming in late summer and early fall, being grazed by wildlife, providing shede
for seedlings etc. It was observed that all three species did well on mine subsoils. That is why
all three species seem to be good candidates to become valuable tools for the usage in mine
reclamation.

Figure 3.33 The second study open-air trial with three shrubby species: big sagebrush (Artemisia
tridentata), common rabbitbrush (Ericameria nauseosa), and soopolallie (Shepherdia canadensis).
Plants are randomized in 10 blocks.
Photo credit Piotr Dzumek

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Landscape and Urban Planning, Vol. 51, (89-100). DOI: 10.1016/S0169-2046(00)00099-2
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CHAPTER 4: RESEARCH CONCLUSIONS
RESEARCH SYNTHESIS
Fraser et al. (2015) state that mine reclamation should rebuild the entire ecosystem
with its biodiversity rather than just restoring vegetation cover. Only restoration of a complete
and fully functional ecosystem may bring sustainable and meaningful results. Simple
revegetation by seeding agronomics, often non-native, might achieve temporary results only
but lead to eventual collapse of the reclamation endeavor. It is clear that to restore a functional
ecosystem all its components including ecological interactions and elements cycling need to
be rebuilt. Usually, degraded mine substrates are not able to facilitate the sustainable and
resistant ecosystem (Gardner et al., 2010). What makes the situation worse are the improper
physicochemical properties of post-mining soils (Sheoran et al., 2010) that often contain high
concentrations of heavy metals (Brown and Chaney, 2016). Both study experiments from
chapters 2 and 3 demonstrated that the subsoils from New Afton New Gold mine and Teck
Highland Valley Copper mine differed strongly. However, what they had in common was a
deep lack of organic matter as well as plant and microorganisms nutrients. As both are
essential for vegetation and microbiota, bare mine subsoils from two analyzed mines are
rather not suitable to fulfill the role of topsoil restoration substrate in mine reclamation
projects. This answers the first research objective. Nonetheless, both experiments
demonstrated as well that the unfavorable features of analyzed mines’ subsoils could be
bettered by soil organic amendments application.
Three organic soil amendments were applied in both research studies. From biochar,
woodchips, and biosolids only the latter have brought positive, statistically significant results
in both short-term experiments. Biochar and woodchips applied alone, as low in nutritional
value and containing mostly the recalcitrant carbon, did not show any positive effect on either
plant survivorship or productivity. Only biosolids demonstrated a positive effect on plant
productivity, but when applied in excess demonstrated a negative influence on plant
survivorship especially on New Afton New Gold subsoil. This may be attributed to the fact
that biosolids is an organic material containing large concentrations of essential plant and
microorganisms nutrients such as nitrogen, phosphorus, zinc, and sulfur. Additionally, these
elements are present in chemical forms easy for biological uptake (Sullivan et al., 2015).
However, when the dose is too large, an excess of the above elements can act adversely on
plant tissues (Elhanafi et al., 2019). When woodchips and biochar applied alone did not act
beneficially in short term due to their low elemental nutrients content and recalcitrant form of

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carbon, they seemed to play important roles as overfertilization prevention and favorable
substrate structure supporters when applied together with biosolids in the soil amendments
mixtures. Results of this research confirm that neither biochar (Canadian AgriChar, 2020) nor
woodchips (Cheng, 2008) should be applied on their own only but they act supportively to
other organic or inorganic fertilizers. The above findings brought answers to the second
objective of this research.
The third research objective focused on the qualitative and quantitative change of
nitrogen in the mine subsoil subdued to reclamation treatments. Nitrogen is essential for all
living organisms (Campbell et al., 2008). Therefore, the constant provision of this element
and restoration of its cycling is necessary for mine reclamation to be successful. The main
observation from both studies was that unamended subsoils from both mines were strongly
deprived of nitrogen. None of the applied organic amendments but biosolids only was a good
source of this intrinsic element. Plants take up nitrogen mainly in the mineral forms of NH4+
and NO3- (Geng and He, 2020). However, biosolids provide large amounts of ammonium only
but hardly any nitrate. Some plats exhibit a preference for either mineral nitrogen forms
(Tylova-Munzarova et al., 2005; Tang et al., 2020). This suggests that nitrate-oriented plant
species would not perform well on either of mine subsoils even when amended by biosolids.
Chalk and Smith (2020) demonstrate though that plants can be flexible in respect to the use of
mineral nitrogen forms. Both studies' results of this research showed that throughout the
length of experiments the total nitrogen content, along with mineralizable nitrogen, and NH4+
content dropped, while NO3- content increased. That in turn showed that nitrate-preferring
plants can be applied in mine reclamation projects using subsoils amended by biosolids. Such
plats would obtain their more suitable mineral nitrogen which would eventually result from
the process of nitrogen transformation.
MANAGEMENT IMPLICATIONS & FUTURE RESEARCH
As topsoil is scarce, there is an interest in whether the subsoil could be an effective
substitute for topsoil in mine reclamation. However, as demonstrated in this research mine
subsoils can differ strongly. Subsoils from one location may possess strongly negative
physicochemical features that would eliminate such material from usage in mine reclamation.
In other instances, a simple amelioration could alter subsoil features to the extent that such
material might become a very valuable mine reclamation material. Concluding from this,
subsoils vary strongly from place to place and because of this need to be well analyzed prior

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to application in mine reclamation. Results from one mine might be not representative for
another mine.
Organic amendments often turn out to be better than commercial fertilizers. Moreover,
they can be combined bringing various beneficial features into the system. A combination of
biochar, woodchips, and biosolids resulted in the largest plant productivity. Moreover, organic
soil amendments result often as waste from local industry e.g. woodchips or communities
habitation, and day-to-day functioning e.g. gardens’ waste compost or biosolids. That makes
these potential soil amendments cheap and available locally which in turn minimizes the
transportation needs lowering the cost (Larney and Angers, 2012; Piorkowski et al., 2015) and
CO2 emission. However, organic soil amendments have also their downsides. For example,
biosolids come as a by-product from municipal wastewater treatment (Sullivan et al., 2015).
As such, this product might contain various unwanted substances like antibiotics, traces of
household chemicals, and metabolites of medicines just to mention a few. There is no study
which has demonstrated negative effects of biosolids to human health, nonetheless, abovementioned substances may possess ecotoxic features. Waterhouse et al. (2014) demonstrate in
their study that despite plant productivity's strong increase an application of biosolids caused
elevated mortality among native earthworms. Earthworm mortality in biosolids-amended soils
was 100%, compared with 42% and 25% in stockpiled and unmodified soils respectively.
This demonstrates a need for more research on soil amendments' ecotoxicity,
bioaccumulation, the influence of composting times on organic amendments’ adverse
features, proper proportions and dosing, as well as on potential substances leaching problem.
Until recently, in British Columbia, agronomics were domint plants used in
revegetation. This resulted from several facts: such plants had adaptability to a wide range of
conditions, grew fast, prevented soil erosion quickly after being sown, and often presented
high forage value, their propagules were easily available and significantly cheaper than native
plants. Non-native graminoids or forbs such as legumes (e.g. alfalfa or clover) have been
widely used for example as cover crops (Elias and Chadwick, 1979; Jefferies et al., 1981;
Tribouillois et al., 2014). On the other hand, the importance and potential use of native plants
in the field of revegetation was not well recognized and, therefore, few economic entities have
dealt with the propagation of these plants. However, non-native plants have their
disadvantages too. Often, these plants did not give way to native plants during the succession
process (Davis et al., 2005). Besides, some of the introduced species could change their
character to invaders. Another consequence of the presence of non-native plants is the

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possible resistance to locally present population limiting factors such as diseases, or
herbivory, which builds their competitive advantage over native plants. Yet, the non-native
organisms are accused of bringing new, previously unknown diseases or other harmful
organisms, such as non-native herbivorous insects, to these areas. With scientific investigation
and shifting cultural values, more attention is being paid to increase our understanding and
appreciation of native plants. Regarding the above further research on native plant species
applicability in mine reclamation, especially species involved in nitrogen fixation such as
field locoweed or soopolallie, would be a continuation of a good trend of transition from
introduced agronomics to species native in British Columbia.

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1

Appendix A – First Study Remaining Elements Results
Carbon

Figure A.1 Two mines’ subsoil total carbon content comparison in the initial and final stages of the
experiment (left), comparison of the total carbon content in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 25, final n= 75

Figure A.2 New Afton New Gold subsoil treatments total carbon contents comparison in the initial
and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison * - p < 0.05,
** - p < 0.01, **** - p < 0.0001, ns – difference not significant. initial n= 5, final n= 15

A2
2

Figure A.3 Teck Highland Valley Copper subsoil treatments total carbon contents comparison in the
initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
75%sub25%bios – subsoil amended by 25% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison ** - p < 0.01,
*** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not significant. initial n= 5, final n= 15

Aluminum

Figure A.4 Two mines’ subsoil aluminum content comparison in the initial and final stages of the
experiment (left), comparison of the aluminum content in the initial and final stages of the experiment
within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal
line indicates the median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25

A3
3

Boron

Calcium

Copper

Figure A.5 Two mines’ subsoil boron, calcium, copper contents comparison in the initial and final
stages of the experiment (left), comparison of the boron, calcium, copper contents in the initial and
final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA –
New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare
the medians. initial n= 5, final n= 25

A4
4

Iron

Potassium

Magnesium

Figure A.6 Two mines’ subsoil iron, potassium, magnesium contents comparison in the initial and
final stages of the experiment (left), comparison of the iron, potassium, magnesium contents in the
initial and final stages of the experiment within each mine (right). HVC – Teck Highland Valley
Copper, NA – New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was
used to compare the medians. initial n= 5, final n= 25

5
A5

Manganese

Molybdenum

Phosphorus

Figure A.7 Two mines’ subsoil manganese, molybdenum, phosphorus contents comparison in the
initial and final stages of the experiment (left), comparison of the manganese, molybdenum,
phosphorus contents in the initial and final stages of the experiment within each mine (right). HVC –
Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the median.
Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 25

A6
6

Sulfur

Zinc

Figure A.8 Two mines’ subsoil sulfur, zinc contents comparison in the initial and final stages of the
experiment (left), comparison of the sulfur, zinc contents in the initial and final stages of the
experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 25

B1

Appendix B – Second Study Remaining Elements Results
Carbon

Figure B.1 Second study two mines’ subsoil total carbon content comparison in the initial and final
stages of the experiment (left), comparison of the total carbon content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 25, final n= 75

Figure B.2 Second study New Afton New Gold subsoil treatments total carbon contents comparison
in the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended subsoil,
95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip - subsoil amended
by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone, mixture subsoil amended by a mixture of amendments. Horizontal line indicates the median. Kruskal-Wallis
test was used to compare the medians. Wilcoxon test was used for pairwise comparison * - p < 0.05,
** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns – difference not significant.
initial n= 5, final n= 15

B2
2

Figure B.3 Second study Teck Highland Valley Copper subsoil treatments total carbon contents
comparison in the initial and final stages of the experiment. Subsoil treatments: 100%sub – unamended
subsoil, 95%sub5%bios – subsoil amended by 5% of biosolids alone, 75%sub25%wchip - subsoil
amended by 25% of woodchips alone, 95%sub5%bchar – subsoil amended by 5% of biochar alone,
mixture - subsoil amended by a mixture of amendments. Horizontal line indicates the median. KruskalWallis test was used to compare the medians. Wilcoxon test was used for pairwise comparison
* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, ns, NS. – difference not significant.
initial n= 5, final n= 15

Aluminum

Figure B.4 Second study two mines’ subsoil aluminum content comparison in the initial and final
stages of the experiment (left), comparison of the aluminum content in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15

B3
3

Boron

Calcium

Copper

Figure B.5 Second study two mines’ subsoil boron, calcium, copper contents comparison in the initial
and final stages of the experiment (left), comparison of the boron, calcium, copper contents in the initial
and final stages of the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA –
New Afton New Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the
medians. initial n= 5, final n= 15

B4
4

Iron

Potassium

Magnesium

Figure B.6 Second study two mines’ subsoil iron, potassium, magnesium contents comparison in the
initial and final stages of the experiment (left), comparison of the iron, potassium, magnesium
contents in the initial and final stages of the experiment within each mine (right). HVC – Teck
Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the median. KruskalWallis test was used to compare the medians. initial n= 5, final n= 15

5
B5

Manganese

Molybdenum

Phosphorus

Figure B.7 Second study two mines’ subsoil manganese, molybdenum, phosphorus contents
comparison in the initial and final stages of the experiment (left), comparison of the manganese,
molybdenum, phosphorus contents in the initial and final stages of the experiment within each mine
(right). HVC – Teck Highland Valley Copper, NA – New Afton New Gold. Horizontal line indicates the
median. Kruskal-Wallis test was used to compare the medians. initial n= 5, final n= 15

B6
6

Sulfur

Zinc

Figure B.8 Second study two mines’ subsoil sulfur, zinc contents comparison in the initial and final
stages of the experiment (left), comparison of the sulfur, zinc contents in the initial and final stages of
the experiment within each mine (right). HVC – Teck Highland Valley Copper, NA – New Afton New
Gold. Horizontal line indicates the median. Kruskal-Wallis test was used to compare the medians.
initial n= 5, final n= 15