Bachelor of Science Thesis, Environmental Science Programme, 2020
Jesper Ekström Hoonk & Elin Johansson
Phytoremediation using Lupinus
mexicanus and biochar in arsenic
contaminated soil an experimental
study.
Rapporttyp Report category Licentiatavhandling Examensarbete AB-uppsats C-uppsats D-uppsats Övrig rapport Språk Language Svenska/Swedish Engelska/English Titel
Fytoremediering med Lupinus mexicanus och biokol i arsenikkontaminerad jord en experimentell studie.
Title
Phytoremediation using Lupinus mexicanus and biochar in arsenic contaminated soil an experimental study.
Författare
Author
Jesper Ekström Hoonk & Elin Johansson
ISBN _____________________________________________________ ISRN LIU-TEMA/MV-C—20/10--SE _________________________________________________________________ ISSN _________________________________________________________________
Serietitel och serienummer
Title of series, numbering
Handledare
Tutor Joyanto Routh
Date 2020-06-02
URL för elektronisk version http://www.ep.liu.se/index.sv.html
Sammanfattning
Markföroreningar och föroreningar har visat sig vara ett av de största hoten som påverkar mark- och ekosystemtjänster på global nivå. Arbetet med förebyggande av föroreningar i marken och reningsarbeten, såsom marksanering har en stark koppling till nästan alla mål för hållbar utveckling. Med fytoremediering som metod och biokol som medel använde vi växter för att extrahera arsenikförorening från jord. I ett försök att minska kunskapsklyftan kring hur biokol påverkar fytoremidering av föroreningar kombinerades dessa två. Kombinationen av biokol tillverkad av vass och L.mexicanus testades i denna studie för dess lämplighet för fytoremediering av As i jord. Frön från L.mexicanus odlades i 18 olika krukor spetsade med As (80 mg kg-1 torrsubstans) och i en variation av andelen biokol ändring (0,1,2,3,4,5%). Två ytterligare krukor planterades med frön men helt utan arsenik eller biokol. Dessa krukor fungerade som kontroll för att säkerställa grobarheten hos fröerna eller de förhållanden som växterna odlades i. Efter 5 veckor skördades växterna och den högsta koncentrationen av arsenik i rötter var på 3094 mg kg-1 och fanns i krukorna som hade 2% biokol. Den högsta koncentrationen av arsenik i plantdelar ovanjord var på 168 mg kg-1 och var i krukorna med 5% biokol. L. mexicanus visade sig kunna bioackumulera, och resultatet visade att bioconcentrations faktorn uppmätte 40,6 (2%) och 29,4 (5%), varav koncentrationen i löv och skälk var 1-2 gånger högre än vad som återfanns i jorden. Resultaten visade även att biokol påverkade translokeringsförhållandet och upptaget av arsenik i L. mexicanus, tillsättningen av biokol i jord gav inga stora förändringar i pH-värdet med tillsatt koncentration (5% av biokol eller mindre). Studien var begränsad i att förklara de mekanismer som är ansvarig för ökningen av As upptag men visar en lovande applikationspotential i arbetet med sanering av jordar från As.
Abstract
Soil contamination is one of the main threats affecting food and water safety and ecosystem services on a global scale. Contamination of toxic inorganic and organic pollutants in soils and its remediation is closely related to the implementation of Sustainable Development Goals. With phytoremediation as a method and biochar as a tool, we used a common plant type Lupin Mexicanus to remediate soil from arsenic contamination further, we wanted to study how biochar affects the phytoremediation of contaminants. The combination of biochar made from reeds and L.
mexicanus was in this study tested for its suitability for phytoremediation of arsenic (As) in soil. Seeds from L. mexicanus was grown in 18 different
pots spiked with As (80 mg kg-1, dry weight) and different percentage of biochar amendment (0, 1, 2, 3, 4,5%). Two additional pots were planted with seeds but completely without any arsenic or biochar. The plants were grown for 5 weeks. The highest concentration in roots was 3094 mg kg-1 found in the pots with 2% biochar. The highest concentration of arsenic in the aerial parts was at a level of kg-168 mg kg-kg-1 and found in the pots with 5 % biochar. L.mexicanus showed a potential of bioaccumulating arsenic from soil with a bioconcentration factor of 40,6 (2%) and 29,4 (5%), the concentration in leaves was 1-2 times higher compared to that in the soil. The results showed that biochar affected the translocation ratio and uptake of As in L. mexicanus, However, the biochar amended soil did not show anything major to the pH-value at these proportions (5% of biochar or less). This study was limited in explaining the mechanisms responsible for the increase in As uptake but shows a promising application for potential remediation of soils contaminated with high As levels.
Nyckelord
Arsenik, Jord, Lupinus mexicanus, Biokol, pH, Translokeringsfaktor, Fytoremediering
Keywords
Arsenic, Soil, Lupinus mexicanus, Biochar, pH, Translocation factor, Phytoremediation
Department, Division Tema Miljöförändring, Miljövetarprogrammet
Department of Thematic Studies – Environmental change Environmental Science Programme
Preface
This thesis was written under extraordinary circumstances in the midst of the corana pandemic forcing us to make many compromises. We would therefore like to thank our supervisor Joyanto Routh for his expertise, engagement and guidance in writing the thesis. We would like to specially thank Mårten Dario and Chen Luo for helping us with the laboratory analysis which was restricted for us during the past several weeks.
Elin Johansson & Jesper Ekström Hoonk 2020-05-10
1
Sammanfattning:
Markföroreningar och föroreningar har visat sig vara ett av de största hoten som påverkar mark- och ekosystemtjänster på global nivå. Arbetet med förebyggande av föroreningar i marken och reningsarbeten, såsom marksanering har en stark koppling till nästan alla mål för hållbar utveckling. Med fytoremediering som metod och biokol som medel använde vi växter för att extrahera arsenikförorening från jord. I ett försök att minska kunskapsklyftan kring hur biokol påverkar fytoremidering av föroreningar kombinerades dessa två. Kombinationen av biokol tillverkad av vass och L.mexicanus testades i denna studie för dess lämplighet för fytoremediering av As i jord. Frön från L.mexicanus odlades i 18 olika krukor spetsade med As (80 mg kg-1 torrsubstans) och i en variation av andelen biokol ändring (0,1,2,3,4,5%). Två ytterligare krukor planterades med frön men helt utan arsenik eller biokol. Dessa krukor fungerade som kontroll för att säkerställa grobarheten hos fröerna eller de förhållanden som växterna odlades i. Efter 5 veckor skördades växterna och den högsta koncentrationen av arsenik i rötter var på 3094 mg kg-1 och fanns i krukorna som hade 2% biokol. Den högsta koncentrationen av arsenik i plantdelar ovanjord var på 168 mg kg-1 och var i krukorna med 5% biokol. L. mexicanus visade sig kunna bioackumulera, och resultatet visade att bioconcentrations faktorn uppmätte 40.6 (2%) och 29.4 (5%), varav koncentrationen i löv och skälk var 1-2 gånger högre än vad som återfanns i jorden. Resultaten visade även att biokol påverkade translokeringsförhållandet och upptaget av arsenik i L. mexicanus, tillsättningen av biokol i jord gav inga stora förändringar i pH-värdet med tillsatt koncentration (5% av biokol eller mindre). Studien var begränsad i att förklara de mekanismer som är ansvarig för ökningen av As upptag men visar en lovande applikationspotential i arbetet med sanering av jordar från As.
Abstract:
Soil contamination is one of the main threats affecting food and water safety and ecosystem services on a global scale. Contamination of toxic inorganic and organic pollutants in soils and its remediation is closely related to the implementation of Sustainable Development Goals. With phytoremediation as a method and biochar as a tool, we used a common plant type Lupin Mexicanus to remediate soil from arsenic contamination further, we wanted to study how biochar affects the phytoremediation of contaminants. The combination of biochar made from reeds and L. mexicanus was in this study tested for its suitability for phytoremediation of arsenic (As) in soil. Seeds from L. mexicanus was grown in 18 different pots spiked with As (80 mg kg-1, dry weight) and different percentage of biochar amendment (0, 1, 2, 3, 4,5%). Two additional pots were planted with seeds but completely without any arsenic or biochar. The plants were grown for 5 weeks. The highest concentration in roots was 3094 mg kg-1 found in the pots with 2% biochar. The highest concentration of arsenic in the aerial parts was at a level of 168 mg kg-1 and found in the pots with 5 % biochar. L.mexicanus showed a potential of bioaccumulating arsenic from soil with a bioconcentration factor of 40.6 (2%) and 29.4 (5%), the concentration in leaves was 1-2 times higher compared to that in the soil. The results showed that biochar affected the translocation ratio and uptake of As in L. mexicanus, However, the biochar amended soil did not show anything major to the pH-value at these proportions (5% of biochar or less). This study was limited in explaining the mechanisms responsible for the increase in As uptake but shows a promising application for potential remediation of soils contaminated with high As levels.
Body text counting’s:
8695 words
2
Table of content
1. Introduction ... 3
2. Aim and research questions... 4
3. Background ... 5
3.1 Biochar ... 5
3.1.1 Soil stability ... 6
3.1.2 Interactions of Biochar and Arsenic ... 6
3.2 Phytoremediation ... 7
3.3 Lupinus ... 13
3.3.1 L. mexicanus ... 13
3.4 Arsenic ... 14
4. Materials & Methods ... 16
4.1 Biochar ... 16
4.2 Preparation of soil ... 16
4.2.1 Spiking of soil ... 16
4.2.2 Dry weight ... 17
4.3 Preparing the soil for the pots ... 17
4.3.1 Biochar homogenization ... 18
4.4 Growing of plants ... 18
5. Analysis - ICP-MS ... 19
5.1 Preparation of Lupin samples. ... 19
5.2 ICP-MS ... 20
6. Results ... 20
6.1 Germination and harvesting of plants ... 20
6.2 Arsenic uptake by plants ... 21
7. Discussion ... 24
7.1 L. mexicanus for As-contaminated soil ... 24
7.2 Phytoremediation ... 25 7.3 pH and adsorption ... 25 7.4 Biochar ... 26 7.5 Research questions ... 27 7.6 Sources of error ... 28 7.7 Further studies ... 28 8. Conclusion ... 28 9. References ... 30
3
1. Introduction
The value that soils have in ecosystems around the world should not be underrated (Pepper et al., 2009). Soils play a primary role in food production, food quality, regulating the climate and provides raw materials. The World Soil Resources Report (FAO and ITPS, 2015), shows that soil contamination and pollution is one of the main threats affecting global soils and ecosystems services. Soil pollution has major consequences on human health through several pathways, both from physical exposure from inhalation of soil particles or through intake of food or water. Methods of expensive physical remediation such as chemical inactivation or simply to move the contaminated soils to landfills are being replaced with biological-based methods such as phytoremediation or enhanced microbial degradation of pollutants (Rodríguez-Eugenio, McLaughlin and Pennock, 2018). The remediation and the prevention of soil pollution is a major contributor to increased food safety, soil degradation, adaptation and mitigation for climate change (Food and Agriculture Organization of the United Nations, 2018). Soil remediation and the work associated with preventing the pollution in soils is strongly connected to almost all the Sustainable Development Goals (SDGs), and its importance cannot be understated (ibid).
Up until year 2007, when the Swedish industries stopped using arsenic completely, arsenic was introduced into the environment primarily as a component of wood preservation and from the manufacturing of glass (Swedish Chemicals Agency, 2020). Other anthropogenic sources of arsenic would be burning of fossil fuels and through the process of smelting of arsenic-bearing minerals (Smedley & Kinniburgh, 2002). Because As doesn’t decompose and is therefore still in the soil or water (Bisone et al. 2016) this causes problems when cities and different housing projects grow into contaminated areas and people encounter the contaminated soil. As-contaminated sites also needs to be remediated to make the land resource available to agricultural production without it being a potential way of harm through plant uptake of As. In Sweden, there are approximately 85000 contaminated sites and 26000 of these sites are classified according to the potential risks posed by them. Category 1 “Very high risk to health and the environment” and category 2 “High risk”. As of now, 1 100 sites are classified as category 1 and 7 700 sites are classified as category 2 (The Swedish Environmental Protection Agency, 2019). Natural concentration of arsenic is 1-40 mg kg-1 in bedrock and generally around 6 mg kg-1 in Swedish soil (Aastrup et al., 2005). Elevated levels of As in Sweden are mostly found in hydrothermally altered volcanic rocks, metamorphic schist and in coal. Schist with a high amount of organic matter have been observed with arsenic levels of 15mg kg-1 in Sweden (Selinus, 2010).
4 The importance of soil remediation is a worldwide issue focusing on innovative and cheap
techniques (Rodríguez-Eugenio, McLaughlin and Pennock, 2018). Several methods currently exist in dealing with these contaminated soils, but a general lack of knowledge regarding the risks associated with contaminated sites and how they should be handled is a major setback (Törneman et al, 2009). Phytoremediation is a method that has been extensively studied (Sun, Zhang & Su, 2018) and has a growing scientific, economic and environmental relevance. The relatively low potential cost of phytoremediation opens the possibility to treat many sites that cannot be addressed with current ex-situ methods such as excavation and treatment because of high costs. Additionally,
phytoremediation preserves the topsoil and reduces hazardous material generated during cleanup. Phytoremediation uses plants to extract heavy metals from soil into the plant biomass and then harvested to remove specific pollutants, leaving only small levels in the soil or water (Ensley, 2000). But the method is slow in comparison to ex-situ methods and not always suitable to use because of the timeframe in remediation-projects today.
Many studies have shown that phytoremediation in combination with biochar, which is a porous, carbon-rich material made from biomass, have been effective at immobilizing heavy metal(loid)s in contaminated soils and affecting the bioavailability of various heavy metal(loid)s (Paz-Ferreiroet al., 2014). Immobilizing is to alter the soil metals to more geochemically stable phases and lessen its solubility. There is a general knowledge gap in understanding how different types of biochar function during phytoremediation methods for sustainable and safe use and application. The understanding of biochar and its characteristics, together with the interactions of biochar with plants is necessary to assess the impacts of biochar on phytoremediation of heavy metal contaminated soil (Sun, Zhang & Su, 2018).
2. Aim and research questions
The overall aim of this study is to investigate the uptake of As by Lupinus mexicanus and how biochar as an amendment to the soil, effects the plants ability to bioaccumulate As. We will measure the amount of arsenic in the biomass by examining the plant's uptake of arsenic from garden soil spiked with As. Our specific aim is to establish how biochar made from reeds is affecting the
phytoremediation capacity (both uptake and immobilization) in L. mexicanus.
L. Mexicanus is not a top contender in phytoremediating As from soils, but with biochar as
amendment in soil, it may show promising results. Combination of the two (Lupin and biochar) may overrun the lack of hyperaccumulating characteristics in Lupins, so that a reasonable level of arsenic
5 can be extracted without being phytotoxic to the plant. We hope to answer two questions and will be discussing our results compared to these questions.
• What effects do the combination of biochar and phytoremediation have on the potential of phytoremediation in arsenic-contaminated soils?
• How do different proportions of biochar amendment affect potential uptake of arsenic (As III) in L. mexicanus?
3. Background
3.1 Biochar
Biochar is a porous, carbon-rich material made from organic material that is derived from a pyrolysis process (figure 1). Biochar can be made from a range of organic materials such as wood, sewage sludge, grass, hay, fruits, nuts, seeds, and other food and agricultural products commonly known as feedstock in the pyrolysis process. The properties of the biochar are as diverse as the feedstocks themselves (Paz-Ferreiro et al., 2014). Biochar has several benefits for soils. For example, biochar increases biological activity in soil (Lehmann et al., 2011; Paz-Ferreiro et al., 2014), soil greenhouse gas emissions are reduced from agricultural sources and resulting in increasing the amount of carbon stored in the soil helping greenhouse gas mitigation (Gascó et al., 2012). Biochar is similar to charcoal but is often used for environmental management of soils and not as an energy source as in the case of charcoal. A defining feature of biochar is the formation of organic carbon fused ring structures. The ring structure in biochar affects its chemical properties associated with
mineralization and adsorption. The ring structures are formed during pyrolysis process (figure 1). During the process of making biochar, the carbon structures from the original material are fundamentally changed and the biochar is depleted of hydrogen (H) and oxygen (O). The
characteristic look of the material made into biochar does not change much, except it turns black, however, the microstructures in the material changes. There are many variables that come into play in determining the structure of the material after pyrolysis, like the type of material used, cooling speed, max temperature, time of pyrolysis, chemical properties of the material, and other factors. Different biochar has, therefore, different traits and characteristics. All biochar material generally has some uniform characteristics, the material is very porous, has a high cation exchange capacity and high alkalinity to name a few (Lehmann and Joseph, 2015).
6
3.1.1 Soil stability
In soils when biochar is added, it will exist as isolated particulate matter that attaches to mineral surfaces, gets stuck in soil pores, or absorb aggregates. Biochar with high carbon content will persist longer in the soil due to its longer half-life, compared to other forms of organic carbon in the soil and presents an interesting pathway for enhancing carbon sequestration in the soil (Sun, Zhang & Su, 2018). One study, for example, indicated that the mineralization of biochar was low with a half-life between 102 and 107 years (Zimmerman, 2010). This is both an advantage, as well as a
disadvantage. While the addition of biochar to soils, presents long-term management strategies, the possible negative effects in the future are not well understood, and once applied to the soil, biochar is not easily removed.
3.1.2 Interactions of Biochar and Arsenic
Metal(loid)s in the environment are influenced by biochemical reactions that play a central role in determining the fate, transportation, and transformation of these metal(loid)s. These metal(loid) ions may exist in different forms, as cations and anions, and depending on the charge of the ions, they interact differently with the positive or negatively charged surface of the biochar. When mixed with topsoil, biochar with a negative charge to its surface adsorb positively charged metal(loid)s, while other types of biochar with negatively charged surfaces adsorb positively charged metal(loid)s. The characteristics of the biochar can be attributed to several factors such as the pyrolysis temperature and feedstock. This makes itimportant to explore different types of biochar for the management of different types of metal(loid) polluted soils (Vithanage et.al, 2017; Sun, Zhang & Su, 2018).
Metal(loid)s such as Arsenic exists in the environment in anionic forms such as arsenite (H2AsO3-) or
arsenate (H2AsO34-). Because arsenic commonly occur as negatively charged ions, it is important to
7 surface on the biochar to be able to immobilize the As ions (Sun, Zhang & Su, 2018). There is
evidence suggesting that adding biochar to polluted soils can increase the mobility of As in the soil (Beesley et.al, 2010; Hartley et.al, 2009). The proposed mechanisms for the increased mobilization of the As could be the electrostatic repulsion between the biochar surface with negative charge and the anionic arsenic (Sun, Zhang & Su, 2018). The importance cannot be understated to make
distinctions between different types of biochar and collecting information regarding its effect on different types of metal(loid)s in order to establish safe soil management activities and not exacerbating the problem.
3.2 Phytoremediation
Methods for dealing with contaminated soils can be divided into two groups. Group one, ex situ, is when the soil or water is removed from the contaminated site to be “cleaned” elsewhere. These intrusive methods require dredging as well as transportation of the material from the sites and result in high costs. The other group of methods involved in cleaning soils on site and referred to as in situ treatment. In situ methods either clean soil or water through chemical or biological processes by breaking down or removing harmful substances from the material (Willey, 2007). These methods using biological pathways could generally be called bioremediation techniques and is of growing interest. One such method involves phytoremediation with amended biochar has shown increasingly promising results and recognized as a viable method for dealing with various types of contaminants in soils (Sun, Zhang & Su, 2018).
There are different methods of phytoremediation, but the main categories for remediating soil with plants is phytovolatilization, phytostabilization, rhizofiltration, and phytoextraction (Ghosh and Singh, 2005; Wang et. al., 2007) (see Table 1). Out of these, phytoextraction and phytostabilization are the most predominant ones when dealing with heavy metal contamination.
Table 1: The different mechanisms of phytoremediation (Ghosh and Singh, 2005; Wang et. al., 2007).
Phytoremediation types
Mechanism Contaminant Scope of application
Phytovolatilization Volatilization by leaves
Organic/Inorganic Volatile contaminants
Phytostabilization Complexation Inorganic Mining contamination
Rhizofiltration Rhizosphere accumulation
Organic/Inorganic Wastewater
Phytoextraction Hyperaccumulation Inorganic
8 In mine sites, phytostabilization is the more suitable remediation type because of the high levels of contamination (Bundschuh, Holländer & Ma, 2015) that can either kill the phytoplant or encounter wildlife and have a negative effect on the ecosystem.
Phytoextraction refers to the removal of heavy metals or organic pollutants from soil or water through plant tissue. The contaminants are removed by harvesting the plant's biomass. The mechanism is dependent on the uptake of contaminants by plant roots and
translocation within the plants (Raskin & Ensley, 2000). The translocation within plants is expressed through the translocation factor (TF). Translocation factor is the ratio of the contaminant concentration in the aerial parts of the plant to the concentration in roots.
Phytostabilization involves reducing the mobility of heavy metals in soils. This includes several mechanisms to immobilize the heavy metals, by having plants covering the soil to reduce wind erosion and in the process reduce the solubility or bioavailability of the toxic metal in the food chain (Raskin & Ensley, 2000). Phytoremediation is primarily a solar-powered process where contamination-resistant plants
Figure 2: Simplified version of the phytoextraction process. The removal of contamination from soil or water is trough root uptake and translocation within the plant to its above-ground parts. The leaves and shoots with accumulated contaminant are then removed by harvest.
Figure 3: Phytostabilization is the method of using plants and plant-associated microbes to reduce mobility of contaminants in the soil by preventing dispersal by wind and water.
9 are used to extract, contain, degrade, or immobilize contaminants in soil, air, and water. The process often goes under other names, for example under the name of phytotechnology. But the process is the same, i.e., you use plants to remediate soil and reduce pollutant levels. The technique of using plants to remediate soil from contaminants have been used long before the name of
phytoremediation was first used by Raskin in 1991 (McCutcheon & Schnoor, 2003).
The use of phytoremediation is most appropriate when dealing with medium to low levels of contamination that is spread over a large area that is not under any acute timespan to be remediated (Ghosh and Singh, 2005). Acute situations are not suitable for this process due to its time-consuming manner. The lower span of contaminants is because they are the areas that are not cost-effective to remediate through conventional methods. Phytoremediation is appropriate in situations when there is a slow increase in contamination levels in the soil or in voluntary soil cleaning efforts from private property owners. There is also proper to use phytoremediation for greenfield projects in urban development or in cities waste-management, as a buffer for leaching. Lastly it has potential to control contaminant levels in areas with non-point sources of pollution (McCutcheon & Schnoor, 2003).
Indication about which plant to use in the process of cleaning soil from contaminants has been expressed from various experiments. Plants used in phytoremediation according to these studies should be easily propagated and fast-growing. Other favorable traits for phytoremediation such as high biomass, large root system, and capability of accumulating high levels of the contaminant, were also expressed (Moreno et. al., 2005; Pedron et. al., 2013; Pedron et. al., 2014).
From the beginning, phytoremediation was based on the assumption, that the use of metal hyperaccumulator plants, which are characterized by the ability to accumulate high amounts of heavy metals or metalloids (Brooks, 1998), was the best way to influence contaminations levels. In As contaminated soils, phytoremediation methods that have been traditionally used and its success rate is represented in table 2. The table show that phytoremediation is still in its early days as a method to remediate soil from As and is still in the experimental stage. 11 greenhouse or laboratory pot trials with different plants, soils and As speciation are represented below, but equally many studies have advanced to field trials which is represented as field trials or fieldstudys. The difference between the two are that field trials plant specific species and then harvest and field studies harvest plants that already grow at the site. The longest ongoing field trial have lasted since 1999
(Dominguez et al, 2008). The As hyperaccumulating plant P. vittata is the preferred plant to use when phytoextracting As from soil. Salix and Populus have shown good results in phytostabilizing arsenic in soil. Lupinus Albus showed results for decreasing soluble As in soil (Table 2). The results
10 from using hyperaccumulators for remediation is lessened by the hyperaccumulators reduced production of biomass and by the ability to accumulate only one specific element. This makes the choice of hyperaccumulators impractical in some cases (e.g. multi-contaminated soils)(Pedron et. al., 2015).
Table 2: Phytoremediation studies of As contaminated soil and their results (Voiant Tangahu et al, 2011; Bundschuh, Holländer & Ma, 2015; Singh et al, 2015).
Contaminant Uptake mechanism
Scale and Duration
Plant Results References
Total As Phytoextraction field plot experiments. 5 months
Chinese Brake Ferns (Pteris vittata)
Co-planting of Pteris vittata and Ricinus communis is a promising approach. Uptake of As and Pb by P. vittata was enhanced by co-planting.
Yang, Yang & Huang (2017)
Multicontamination Phytostabilization Field experiment Epilobium dodonaei Vill
Epilobium dodonaei has phytostabilization potential at hot spot mine site Bor. E. dodonaei accumulated 3.98 mg kg-1 in root and 4.69 mg kg-1 in shoots. Randelović et al. (2016) Aqueous solution containing 0.1041 g of sodium arsenate heptahydrate (Na2HAsO4· 7H2O), the mixture which contained 50 mg kg-1 of As (wet weight)
Phytoextraction Greenhouse pot experiment, 16 weeks
rice-cut grass (Leersia oryzoides)
The data show that 120, 130, and 130 g/ha(-1) of arsenic were absorbed by the shoots at 6, 10, and 16 weeks. Periodic mowing of Leersia oryzoides on contaminated land could maintain the high arsenic uptake at 6 weeks.
Ampiah-Bonney, Tyson, & Lanza (2007)
Sodium (meta-) arsenite (50 uM, 150 uM and 300 uM)
Phytoremediation treatments (Agropeat and half strength Hoagland solution)
Laboratory—pot experiment (12 days)
Brassica juncea var. Varuna and Pusa Bold
Increase/decrease of antioxidant enzymes activities showed not much changes at the given concentrations.
Gupta et al., (2009)
Total As (up to 200 mg kg-1)
Phytoextraction Laboratory (April 1996, 2 months)
Hybrid willow (Salix sp.) and hybrid poplar (Populus sp.)
The willows were able to remove 1% of the total arsenic from the contaminated soil. The less mature poplars removed 0.1% of the total arsenic from the same soil. In the sand experiment, the willows took up about 30 to 40% of the administered arsenic.
Hinchman, Negri, & Gatliff (1995)
As, Cd, Mo, Pb, Zn 18 different phytoremedation treatments.
Laboratory Grasses: (Sorghum bicolor L.) and (Sorghum sudanense)
The concept of the integrated phytoremediation was successfully applied to vegetate Gyöngyösoroszi spoil. The biomass production was different, depending on the technology variant. Murányi & Ködöböcz (2008)
11
Arsenic (As) average 82mg kg-1
Phytoextraction Field try, 3 months Chinese Brake Ferns (Pteris vittata)
Fern shoot arsenic concentrations were (average 1640mg kg-1 DW) 20 times higher than the arsenic concentrations in the soil for this field try. Indication of rising soil pH improves arsenic removal. Salido et al. (2003) Multicontaminated As 886 mg kg-1 Phytoextraction and phytostabilization (soil) Pot experiment (2004-2005) and field trial (2005)
Three poplar species (Populus alba, Populus nigra, Populus tremula) and Salix alba
Roots provided a significant sink for pollutants. The highest accumulations in roots were measured in P. nigra and S. alba. Salix alba foliage contained highest
concentrations of As in stem. The overall removal of trace elements was only significantly higher in P. alba than in S. alba.
Vamerali et al (2009)a Multicontaminated As 886 mg kg-1 Phytoextraction and phytostabilization (soil)
Pot experiment and field trial (2 years (2004-2005) for pot experiment and field trial on May– September 2005) P. alba L. (white poplar) P. nigra L.(black poplar) P. tremula L. (European aspen), Salix alba L. (white willow)
The result shown that the elevated concentrations of As and other elements could be leached from the remediated wastes and these fluxes could also be influenced by soil amendments, changes in the rhizosphere or both. Immobilisation of trace elements in both coarse and fine roots may reduce leaching in As. Vamerali et al (2009)b Multicontaminated with As (as Na2HAsO4) (As 100 mg kg-1 )
Phytoextraction Pot experiment 5.5 months
Salix spp. Although As uptake was slightly increased in Suchdol-Zn soil compared to Suchdol-Pb soil, the contaminant removal from soil was significantly higher in Suchdol-Pb soil. due to a significant reduction of aboveground biomass yield in Suchdol-Zn soil. The yield reduction decreased the uptake of plant-available elements by biomass; thus higher plant-available portions of As were found in Suchdol-Zn soil. Vysloužilová et al (2003)
Total As Phytoextraction Field survey. 3months (September to November)
Samples of 24 fern species were collected from 12 different mine sites.
Pteris multifida and P. oshimensis can (hyper-) accumulate As in their fronds with high concentrations. Total As concentrations in soils associated with P. multifida and P. oshimensis varied Wang et al (2006)
12 from 1262 to 47.235 mg kg-1 . P. oshimensis is more suitable for remediating As-contamination in soil despite lower concentration in fronds than P. multifida, due to its high above ground biomass Cd, Cr, Pb, As, and Hg Field experiment (155
days (May– November))
Rice (Oryza sativa L.) The results showed the rice grain contained significantly lower amounts of five metals than straw and root in all sampling sites. Rice root accumulated As from the paddy soil.
Liu et al (2007)
Available As 40microM
Phytostabilization 3 week pot study; and 1-5months field study
White Lupin (Lupinus Albus) Decreased soluble As in planted soil Vazquez et al (2006) As 60-70 mg kg-1 Phytostabilization with 20 % Biochar amendment and 30% GreenWasteCompost( GWC) Greenhouse pot experiment, 8 months Miscanthus x giganteus Biomass yield increased by 2-4 times. Both TF and BCF <1
Hartley et al (2009)
Multi As 49-339
phytostabilization Site study, Since 1999 Holm oak (Quercus ilex)
Olive tree (Olea europaea)
White poplar (Populus alba L.)
Narrow-leafed mock privet (Phillyrea angustifolia L.) Mastic shrub (Pistacia lentiscus L.) Rosemary
(Rosmarinus officinalis L.)
Yellow retama brusch (Retama
sphaerocarpa L.) and tamarisk (Tamarix Africana Poir.) It showed limited transfer of the contaminating elements to the aboveground parts, except for white poplar. BCF <0.03 for As Dominguez et al (2008) Multi, As 4.9-5266mg kg-1
phytostabilization Field trials, 3 years Larix × eurolepis, Betula pendula, Alnus incana L., 2 different Populus: Populus deltoides × nigra Populus trichocarpa 5 different Salix: Salix caprea × cinerea × viminalis, Salix viminalis, Salix caprea × viminalis, Salix burjatica, Salix viminalis × schwerinni
Positive results for phytostabilization with low mobility and planttransfer with BFC <1
French et al (2006)
Arsenate 190 mg kg-1 Phytoextraction 2 years field study Chinese Brake Fern (P.vittata) The uptake of As in fronds (DW) was 3186-4575 mg kg-1 Kertulis-Tartar et al (2006)
As 393-1903 mg kg-1 Phytoextraction Field study, 5 months Chinese Brake Fern (P.vittata) The uptake of As in fronds (DW) was 775-2569 mg kg-1 Niazi et al (2009) Multi 8.8-3580 mg kg-1
Phytoextraction Pot trial, 9 months Chinese Brake Fern (P.vittata) The uptake of As in fronds (DW) was 1.2-229 mg kg-1 Shelmerdine et al (2009)
13
Multi 23-640 mg kg-1 Phytoextraction Greenhouse Pot trial, 16 months
Chinese Brake Fern (P.vittata)
The uptake of As in fronds (DW) was 110-6151 mg kg-1
Gonzaga et al (2008) As 98 mg kg-1 Phytoextraction Greenhouse pot trial,
5 months
Chinese Brake Fern (P.vittata)
The uptake of As in fronds (DW) was 6000 mg kg-1
Tu et al (2002)
Dw= dry weight, BCF = Bioconcentration Factor, the amount of target element in plant compared to that in the soil where 1 is the same concentration in plant as in the soil.
3.3 Lupinus
Lupinus comes in many forms and colors and is commonly known as lupin or lupine. This group of species includes both annual and perennial herbaceous species. Shrubby and tree types of lupin also fit within this diverse group. There are around 280 species of lupin that are generally accepted, but not all of them have been included into the Integrated Taxonomic Information System (ITIS). Lupinus is generally treated as a polymorphic species and its origin, taxonomical positioning and species names have been debated for a long time (Ainouche & Bayer, 1999). Lupinus is classified
taxonomically within the order of Fabales. The family name for lupins in taxonomy is Fabaceae, the tribe is Genisteae and the genus is Lupinus L. (Clements et al, 2005a).
Lupin's root morphology varies widely between species and in different soil types. Generally, lupins have a taproot system. A taproot is when the plant has one dominant large root that the other roots grow from laterally and resembles a carrot. The variations range from a dominant taproot to cluster root systems, (Clements et al, 1993) which is also called fibrous roots. The roots can adapt in soils to increase nutrition uptake, which means that a taproot system can form a cluster root system when the conditions in the soil are deficient in e.g. phosphorus and iron. The root system varies between the depth of 1-2 meters (Office of the Gene Technology Regulator, 2013; Kurlovich, 2002). With the herbaceous species of lupin, the stem is fascicular, meaning that the nutrients are transported through vascular bundles. But within the group of annual lupin species, the stem differs in size and shape of the cross-section (Kurlovich, 2002). If the plant has more and larger of one of the organic or inorganic way of transport it would affect the uptake of the more toxic inorganic As species. It could also affect the transport-time of nutrients and therfore enable the plant to have more biomass because of increased nutrient uptake. Lupins have a characteristic palmate shape of the leaf and the surface of the leaflets often have silver three-celled hair on them (Kurlovich, 2002).
3.3.1 L. mexicanus
Lupinus Mexicanus was the first taxon in the genus lupin named for Mexico. This particular lupin is hard to distinguish from other lupins because later work with the taxonomy of lupins in Mexico the name was ignored. This has resulted in some confusion. In 1832, it was stated that Lupinus
14 mexicanus was lost to science (Dunn, 1972). It is important to state that L. mexicanus is not the same as L. neomexicanus because the latter is a perennial.
The taxonomy of Lupins is still in chaos and there is multiplicity in the names assigned to many species including L. mexicanus. L. mexicanus is close relative to Lupinus hartwegii, L. bilineatus and L. persistens (Ainouche & Bayer, 1999). Nowadays, L. hartwegii and L. bilineatus are often synonymous with L. mexicanus (Impecta, n.d).
Some of the distinctive traits that separate L. mexicanus from other lupins is the shaggy-pilose hairs. The upper surface of the leaflets is smooth, and the bottom is shaggy-pilose, and the petals is also glabrous (smooth and non-hairy). L. mexicanus have been confused with L. aschenbornii. This should be avoided because L. aschenbornii is alpine and a perennial, and is significantly different from L. mexicanus (Dunn, 1972).
The common name for L. mexicanus (L. hartwegii) is Hartweg´s Lupin or Sunrise Lupin. The plant can grow in full sun or half shade and can grow up to 70 cm in height. It takes 10-40 days to reach full bloom and the flowers have mixed colors. It is an annual plant.
The major sink for As when using Lupins to phytoremediate is the roots. That is because of the low root-shoot ratio of heavy metal(loids)s (Vazquez et al, 2006). Casado et al. (2007) showed that the average uptake of As from L. hispanicus measured 0.94 mg kg-1 (Casado et al,2007). The
translocation factor (TF) e.g. root-shoot ratio for Lupins was (0.14-0.15)(Vetterlein, Jahn & Mattusch, 2009). This could be compared to corn (0.33)(ibid), or hyperaccumulators (>1) or Chinese brake fern (Pteris vittata) (24) (Bundschuh, Holländer & Ma, 2015).
3.4 Arsenic
Arsenic is a metalloid that can be found almost everywhere in the environment. In the periodic table arsenic (As) is organized under Group 5 along with Phosphorus (P) and Nitrogen (N). The
biogeochemical traits of As are similar to that of phosphorus (Aastrup et. al., 2005). They use the same transporters for uptake in plants and are chemical analogs which means they can substitute each other (Ullrich-Erebius et al., 1989; Meharg and Macnair, 1992). This poses a challenge because plants cannot distinguish between As and P when the roots draw water and nutrients from soil. Sources of arsenic found in nature (inorganic or organic) can be either natural or anthropogenic. The predominant natural sources of arsenic are volcanism or weathering of bedrock. The anthropogenic sources are wood preservation, manufacturing of glass, use of pesticides, burning of fossil fuels and smelting of some minerals (Bisone et al. 2016; Bissen and Frimmel 2003). If these sources lead to high concentrations of arsenic at different locations, it can cause both environmental- and health
15 problems. The problem with arsenic is that it is phytotoxic and hazardous to essentially all living organisms (plants, humans, and animals). Arsenic can neither be decomposed biologically nor chemically (Bisone et al. 2016) and if As was to leach from highly contaminated soils it would be a major risk for groundwater quality (Sadiq 1995; Violante and Pigna 2002).
Depending on the conditions in soil, As occurs in different oxidation states. The most common form in the natural environment is arsenite and arsenate. The most important factors that affect As speciation is the redox potential (reduction/oxidation potential, Eh) and pH. The different oxidation states of As that can occur in natural soils and water are elemental arsenic (0), arsenite (+III),
arsenate (+V), and arsine (-III) (Bissen & Frimmel 2003). Oxygen has a high affinity for As in soil and it is mainly found as oxyanions. e.g., arsenite as AsO33- and arsenate as AsO43- (Sadiq 1995; Bissen &
Frimmel 2003). The abiotic redox transformation for As is quite slow, and therefore, As3 and As5 can
be found in the same environment (Smedley & Kinnigburgh, 2002).
The speciation of arsenic gives important information about its potential for leaching and toxicity. Typically, organic arsenic compounds are less toxic than inorganic ones (Smedley & Kinnigburgh, 2002; Bissen & Frimmel 2003). As(III) is more mobile and toxic compared to As(V). However, it is not only speciation that affects the mobility and risk of leakage of As into soils. A number of other factors can also have a potential effect. Factors stated above such as pH value, type of adsorbing components in soils, redox conditions and residence time are important to take into consideration when testing uptake of As by plants.
The soil that best represents the natural variations in concentrations of As in the bedrock in Sweden, is moraine. Some of the variations may be due to differences in soil properties (grain size and amount of organic matter). Larger grainsize and a low cation exchange capacity (CEC) have a positive effect on As uptake by plants (Aastrup et al., 2005).
Generally, the major part of arsenic in soil is bound to metal hydroxides, clay minerals and organic matter (Aastrup et al., 2005). This means if there is high total concentration of As in soil it does not imply that all of this As fraction is phytoavailable. In fact, studies show that the amount of As in soil often correlates with the amount found in the bedrock and As concentrations in plants correlate with the concentration in the bioavailable state in solutions (Smedley & Kinniburgh, 2002). Arsenic uptake by plants from As-contaminated soil therefore depends mainly on the access to water-soluble arsenic (Pigna et al, 2015). In addition, the transformation of As(V) to As(III) affects the phytoavailability of As. Because As(III) has higher solubility (Smedley & Kinniburgh, 2002) it has a higher availability for plants.
16
4. Materials & Methods
In this experimental pot study, 40 Lupin seeds was planted in 20 different pots. 19 of these pots had arsenic spiked soil and one was left clean to ensure the growing conditions and the quality of the seeds. The spiked soil had a concentration of 80 mg kg-1 As which is double the concentration set for the protection of terrestrial environment by the Swedish environmental protection agency (2016). All pots with different percentage of biochar amendments was in three replicates. 6 plants grew in 3 pots with 0 % biochar, 6 plants grew in 3 pots with 1% biochar, 6 plants grew in 3 pots with 2% biochar and so on, all the way up to the highest percentage of biochar amendment of 5%. The study was limited to maximum of 5% biochar added because it is not practical to add more than 5% biochar to the soil when dealing with larger areas when applying in field conditions. The plants were watered with deionized water to ensure to not add any more of the contaminant to the soil. The Lupin-plants was left to grow for 5 weeks before harvest. The above-ground parts were separated from the roots when analyzed in ICP-MS to see were the As was accumulated in the plants. The plants growned in different percent of biochar was compared to each other to detect any differences in phytotoxicity from As, As-uptake, As- translocation and As-accumulation.
4.1 Biochar
The biochar used in this study was derived from dried, dead common reeds (Phragmites australis). The feedstock was chosen from biomass that we had access to at the start of the experiment. 10 liters of reeds were collected in a bag from a typical beach in Östergötland. The Reeds were put in a retort kiln, design shown in figure 1. The reeds were burned for 4 hours in a temperature of about 450-600°C, the temperature wasn’t measured, but other studies put the double barrel retort kiln within this temperature range (Stensson, 2018). The reeds were then removed from the kiln and allowed to cool. After cooling the biochar was pulverized and passed through a 2mm sieve.
4.2 Preparation of soil
4.2.1 Spiking of soil
Regular plant store soil (see table 3) was dried for 24h at 55 °C and then sieved with a 2 mm sieve to make homogenization with the solution easier by making the capillary property and grainsize homogenic. For most standard analysis of soil (chemical, physical, and mineralogical), the soil
samples is air-dried, crushed, and sieved to < 2 mm beforehand (Soil Survey Staff, 2014). To spike the soil to 80 mg kg-1 (recommended Swedish level for soils in 40 mg kg-1 ), a solution of NaAsO2 was
17 the target value of 80 mg kg-1 in the soil. The spiked soil was mixed thoroughly under a fume hood. The soil was weighed before (1435g) and after (2850g) adding deionized water and As-solution. Once mixed, the spiked soil was tested for its pH value using method 9040C (United States Environmental Protection Agency, n.d.), and then given time to settle for 48 h before adding the biochar to homogenize further.
4.2.2 Dry weight
Around 3500 g of the moist soil sample was dried at 55 °C to remove the moisture without baking the soil, for about 24 h. The soil was 1435 g (DW) after drying. The dry weight was calculated according to the following equation:
4.3 Preparing the soil for the pots
Materials used in this test was a stainless steel tray, 2 mm sieve, kitchen scale, measuring cups (stainless steel), plastic pots (with a diameter of 8 cm), plastic container with lid, spoon, soil,
deionized water, mask, gloves and lab coats. The soil was tested of its particle composition (see table 3) before spiking it with As and adding the amendment (see table 4). The soil that was used in the study was composed by mostly sand (93.8%) and small part of silt (6.1%) and a fraction of clay (0.1%). The grain size analysis was done on a Sedigraph. There is no std method for this analysis. The grain size is calculated based on the settling velocity by laser. The samples were first crashed and sieved through 2 mm, followed by sieving through 63 μm. Samples (after sieving through 63 μm) were then dispersed with 0.05 % sodium hexametaphosphate and ultrasounded, before analyzed on Sedigraph Ⅲ (Micrometrics, US).
Table 3: Soil particle composition of soil used in the pot experiment. Samples from the analysis which are larger than 63 µm is represented as sand, silt represent particles that are between 2-63 µm in size, and everything smaller than 2µm is clay.
Clay Silt Sand
0.1% 6.1% 93.8%
The pots with spiked soil and biochar amendment (0-5%) was done in three replicates for each amount (see table 4). To make sure that it wasn´t anything wrong with the planted seeds or the conditions that the experiment was held in, two pots were added to the ones that was spiked with As. These two extra pots (A0 and A1) contained only regular garden soil and the Lupin seeds completely without any amendments.
Table 4: Illustration of replicates where two seeds was planted in each pot. Every percent of biochar had three replicates. With the exeption for the Soil only-column, wich only had two pots with two seeds each because it was the controlsample. All was spiked with the same concentration of As except for A0 that was not spiked.
Soil only 0% biochar 1% biochar 2% biochar 3% biochar 4% biochar 5% biochar
18
A0 B0 C0 D0 E0 E0 G0
A1 B1 C1 D1 E1 F1 G1
B2 C2 D2 E2 F2 G2
4.3.1 Biochar homogenization
1. Each sample container had 250 cc of soil, for a total of 750 cc for 3 replicates. Volume is used instead of weight to accurately apply the biochar, due to biochar's porous structure the weight can vary due to moisture content and other factors (Brewer CE, Levine J, 2015). Soil and biochar were added to the tray so that biochar represented 1-5 % of total soil volume. For example, in group C with 1% biochar, 742.5 cc soil was added to the tray and 7.5 cc of biochar for a total volume of 750, with biochar representing 1% by volume. The total volume was spread out on a stainless-steel tray for homogenization.
2. The soil was placed on the tray, and biochar added to represent 1 - 5 % of soil volume, the soil and biochar was divided into four quarters, and each quarter was thoroughly mixed separately with a spoon.
3. The four quarters were mixed in the center of the tray.
4. The soil amended with biochar was then mixed into four quarters again; each quarter mixed separately; combined in the center of the tray again as step 2 and 3.
5. The procedure was repeated until complete homogenization, evidenced by the physical appearance of the soil.
6. Once mixing was complete, the soil in the tray was divided into two halves. 7. Each sample container was filled by alternating the soil from each half.
8. Each container was filled with 250 cc of soil mixed with biochar and the container was labeled. After 250 cc was added to each container, we added 20 ml of water to increase moisture for growing and then each sample was weighed to establish a consistent watering plan in the future and to ensure each plant gets an equal amount of water.
4.4 Growing of plants
The plants were kept in room temperature (19 °C) with access to daylight from window and under a LED light to extend the growth time until harvest. The exposure to light for each plant was 16 h a day. Each pot was planted with two seeds of Lupin mexicanus to increase the chance of germination. Throughout the experiment, all the samples were watered with deionized water. To ensure that each pot had the same amount of moisture, the pots were weighed in the beginning and then once a
19 day. Deionized water was added to compensate the uptake by plants and adjust it to the original weight of individual pots. Each part of the plant was harvested separately, first the leaves where pinched of and rinsed with deionized water. Then the shoots were harvested and washed, and lastly the roots where rinsed and gently brushed with a brush to remove the soil particles from the roots. The separate parts were then weighed, homogenized and kept in separate bags in a freezer until further analysis.
5. Analysis - ICP-MS
The method that was used to perform the analysis of As uptake was the Swedish standard SS28150. This was modified to be more suited for organic matter. The modification includes addition of hydrogen peroxide and the use of a microwave at 180 degrees Celsius.
5.1 Preparation of Lupin samples.
The plant samples were homogenized by using a mortar and pestle. Then 0.3 g of each sample was then dissolved with 8 ml nitric acid and 2 ml hydrogen peroxide in teflon tubes for the microwave digestor. This was performed under a fume hood due to nitric acid and hydrogen peroxide being oxidizing agents, corrosive and an irritant when it is in contact (Pubchem a & b, n.d). Additional safety procedures were taken; the use of lab coats, gloves and safety googles. The samples were then processed in a microwave (model) at 180 °C
After the digestion, the samples were transferred into 50 ml Sarstedt tubes and diluted up to 50 ml. When the particulate matter had sunk to the bottom, 1250 µl was extracted by using an autopipette and added into 15 ml Sarstedt tubes, so that the nitric acid in the new solution was 2%. 100 µl of the internal standard (Rh) was added as well and the extract diluted with deionized water to 10 ml. Rhodium was chosen as internal standard because it has a low contamination risk and can be converted into a chloride free nitrate solution which is compatible and stable (Gaines, n.d.). In order to calibrate the ICP-MS for the target metalloid, standard multielement solutions were prepared. These standards include the same concentration of HNO3 and internal standard as the
analyte, however each of the 4 standards were diluted to 0, 10, 50, 100 µg/l respectively with the multielement standard solution.
A NIMT test was used to calculate precision, the precision is the actual NIMT value ± the measured value by the ICP-MS, see table 5. Arsenic was analyzed in the standard - an Ombrotrophic peat (NIMT/UOE/FM001) to ensure analytical precision and reproducibility. While the certified value for
20 As in NIMT should be close to 2.22 mg kg-1, the value is 3.2 mg kg-1 (see table 5) which is within an acceptable range (Yafa et al, 2004).
Table 5: Arsenic concentration in the standard (NIMT), biochar and garden soil (without arsenic) used for the experiment.
Substance mg kg-1 NIMT 3.2 Soil replica 1 0.7 Soil replica 2 0.8 Biochar 1.2
5.2 ICP-MS
The ICP-MS utilizes an inductively coupled plasma atomizer (ICP) with a mass spectrometer (MS) to ionize the sample atoms and subsequently separate them based on their mass to charge ratio. After the sample atoms have been separated, the MS creates a current based on the intensity of one “band” of separated ions. This is repeated for every ion with a distinct mass to charge ratio (Wolf, 2005). The ICP-MS was chosen as a method for this analysis because the arsenic analysis on the instrument is stable, it has relatively good precision and accuracy, a low limit of detection, and quantitative multielement capabilities (Crouch, 2009).
6. Results
6.1 Germination and harvesting of plants
The plants were grown for about 5 weeks before harvesting. The plants did not have time to reach flowering stages. The different biomass weights after freeze drying is shown in table 6.
Plants from group C (1% biochar) and E (3% biochar) were not harvested, either because of no germination or as in the case of group E (3% biochar), the plants were too small to be harvested and digested for analysis. The other groups were harvested by separating them into leaves, shoots, and roots from each pot. Individual pots could not be analyzed due to the small amount of biomass. The leaves and shoots had to be combined within groups to reach the weight acquired for analysis. The combining of biomass from all 3 pots was done in all except the control whick only had 2 pots, the leaves and shoots were combined, shown in table 6. We needed as close to 0.3 g of dry biomass as possible from the samples to be digested. The shoots and leaves from each group were combined because the total biomass after freeze-drying the samples was less. Likewise, roots from each group
21 were also combined. Therefor a mean-value was not possible to be calculated from the result and similar assessment of ingroup variation.
Table 6: Biomass from lupins in mg after harvest and freeze drying. All replicates had to be combined to reach the 0.3 g of biomass (DW) required for digestion and later on analysis in ICP-MS.
Weight (mg) DW of plant biomass Percentage of biochar
No seeds germinated Root Total above-ground
(leaves+shoots) Total biomass 91.4 464 (312.2+152.1) 555.4 0% (control) 4/4 1.9 155.4 197.3 0% 4/6 8.9 42.7 51.6 2% 3/6 7.8 28.5 36.3 4% 4/6 13.4 57.3 70.7 5% 5/6
6.2 Arsenic uptake by plants
The pH value was measured on the pots after harvesting. The pH value for all the samples was in the range of being acidic to neutral (see figure 4). The control sample and the sample without biochar amendment (B 0%) had the lowest pH-value of 5.4. This value increased to 5.8 for all samples except in G 5%. The sample with 5% biochar showed a pH-value of 6.
Figure 4: Measured pH-value from the different pots after harvest. Biochar represents the sample with 100% biochar made from common reeds and the following is pH-values from the spiked soil with 0-5% added biochar.
All result of arsenic uptake will be presented in mg kg-1 As dry weight (DW). The control sample had 5.4 mg kg-1 As in total as uptake in its biomass. Plants grown in this laboratory experiment, and hereafter, referred to as the controls indicated 4.7mg kg-1 As in the roots, 0.2 mg kg-1 As in shoots and 0.5 mg kg-1 As in leaves. The soil indicated a low level of bio-accessible As. The control indicated
22 that As primarily accumulated in the roots. Arsenic levels were a little more than 6 times higher in the root than the areal parts in the control sample. The pH-value shown is the same for the control sample and the spiked soil with 0% biochar (see figure 4).
Arsenic uptake in the spiked soil in plant roots ranged from 2169-3094 mg kg-1 As. The highest uptake was in plants grown in the soil with 2% biochar. The lowest level of As was found in plants from the soil with 0% biochar. This can be compared to the results from the control sample which also had 0% biochar but was not spiked. The bioconcentration factor of As in roots is 27(0%) and 38.7 (2%).
Figure 5: Arsenic concentration in mg kg -1 (dry weight) of combined roots from all samples within the harvested groups. The control sample without As and biochar added to the soil showed a concentration of 4.7 mg kg -1.
Arsenic concentration in the areal parts of the plants ranged from 79.6mg kg-1 to 168 mg kg-1. The highest concentration was in samples grown in soil with 5% biochar and the lowest was found in samples from the soil with 0% biochar amendment (Figure 7). Arsenic concentration in above-ground parts increased by 78.2 mg kg-1 As from samples in 0% compared to the ones in 2% biochar. The value decreased between 2% and 4% biochar amendment by 15.5mg kg-1 As. In contrast, As levels increased by 25.7 mg kg-1 between 4% and 5% biochar amendment in soil. The
bioconcentration factor of As in leaves and shoots is the same level as in the soil, while the rest of the biochar percentages showed around 2 times As concentration in leaves and shoots compared to soil concentration.
23
Figure 7: Arsenic concentration in mg kg-1 (dry weight) of combined shoots and leaves from all samples within the harvested groups. The control sample without As and biochar added to the soil, had a biomass large enough to be separated in roots, shoots and leaves for analysis. The control sample showed a concentration of 0.5 mg kg-1 in leaves and 0.2 mg kg-1 in shoots.
The translocation ratio for the measured samples is low for low levels of added biochar but increased in the experiment with higher amendment (Table 7). Arsenic accumulation in roots compared to aerial parts showed a similar trend between the amendments (Table 7). The
translocation ratio of As in roots compared to the aerial parts is also indicated. The accumulation of As in aerial parts compared to roots decreased in the spiked soil from 27 times in roots (0%
amendment) to 12 times higher (5% amendment). Translocation ratio (TR) is calculated: TR= concentration in foliage / concentration in roots.
Table 7: The bioconcentration factor and the translocation ratio of As for Lupins between the different percentage of biochar in soil.
Biochar percentage Bioconc. factor whole plant(BCF) Translocation ratio Control 0.07 0.15 0% 28.1 0.04 1% - - 2% 40.6 0.05 3% - - 4% 35.9 0.05 5% 29.4 0.08
24
7. Discussion
When using an experimental design instead of doing a field study we were able to control factors that was not relevant for the study. By controlling the soil particle size, amount of As, water, time in light etc. we could focus on observing how biochar affected the As uptake and translocation. But by doing the trial this way is at the expense of the study’s external validity.
An experiment can say that biochar affect in a certain way in an isolated situation not how it is going to behave in contact with other factors e.g. contaminants, nutrients, other plants, flooding, wind etc. We can therefor not generalize our conclusions or say anything about real conditions.
If we would have done a field study it wouldn´t have showed us that it was the biochar that had any effect on the As-uptake in plants. When doing a field study, it is externally valid but not for all soils or conditions and not for testing hypothesis or to find specific and significant relationships between variables.
7.1 L. mexicanus for As-contaminated soil
The results indicate that the claim of As tolerance in Lupin to phytotoxicity holds; the plant is able to grow despite the high levels of As in the soil. The plant accumulates As in the roots primarily and does not accumulate so much in the leaves or shoots. The translocation or root to shoot ratio does not favor L. mexicanus as a serious contender in phytoextracting As from contaminated soils. A previous study using P. vittata in soil with 82 mg kg-1 As, showed that P. vittata had 20 times more As in its above-ground foliage than the surrounding soil (Salido et al, 2003). Compared to P. vittata our experiments with L. mexicanus in this study showed 1-2 times As concentration in the foliage compared to the soil.
The highest concentration of As was found in the roots which is a negative trait in the context of phytoextraction, where harvesting above-ground biomass is the key. The low root shoot ratio however still makes L. mexicanus valuable in phytostabilization, which is by increasing adsorption of As and decreasing the availability of As in the soil to either leach or move through other pathways and have a negative effect on the ecological balance.
The assumption that uptake in plants increased in a linear way, with a higher percentage of biochar amendment, is not represented in the result. Initially, the added biochar seems to have increased As concentration with the 2 % and 4% amendment but then comes down to the same level at 5% as with 0 % amendment. This is surprising and shows that there is something else that affects the uptake except pH and adsorption. However, the non-linear pattern observed coincides with the general knowledge about natural variation, which is for the most time non-linear.
25
7.2 Phytoremediation
The combination of biochar in the phytoremediation method for extracting As from the soil showed promising results that are worth further investigation. The translocation factor changed with higher percentages of biochar which in the context of phytoextraction is promising. With increased levels of As in the foliage, it would reduce the time factor for soil remediation, and thereby, support
phytoremediation efforts. Quick turnover and time are important factors in consideration when assessing the phytoremediation potential (Raskin & Ensley, 2000). In this context, the addition of biochar could be a solution in reducing the time aspect by doubling the uptake, which could perhaps reduce the time to half.
It is, however, important to point out this study was not done under field conditions. Moreover, the soil might not be representative of polluted soil and to assess the effects of biochar with other types of soil composition are beyond the scope of this study. Other plant species might not respond the same way with the addition of biochar, and this also requires further investigation. We did not measure the leachability of As which some studies mentioned (Sun, Zhang & Su, 2018) could be a result of adding biochar due to electrostatic repulsion. This is an important aspect to consider in pilot trials and also when remediating other metals. The increased leachability of As on adding biochar could cause a problem in its containment when remediating contaminated sites, and counterintuitively it may harm ecosystems.
7.3 pH and adsorption
Adsorption describes the potential of a dissolved chemical substance to adhere to a solid surface. Negatively charged ions can, for instance, adhere to iron- and aluminum oxides which have
positively charged surfaces, and clay particles have some ability to attract negative ions as well. pH is a deciding factor in determining the charge. The largest adsorption of negatively charged ions occurs under low pH range, below pH 6. One effect of adsorption is the decreased movement of metals through the soil because they adhere to the soil particles. However, changes in the soil environment can re-dissolve these metals and thus increase its leachability to groundwater (Bissen & Frimmel 2003).
This is important in the context of this study, the pH-value of the biochar (Figure 4), is 10.1. Since biochar has a high cation exchange capacity (CEC), the effects of high pH on negatively charged metal ions, such as As in this study, could increase the leachability effect. The increase in pH (Figure 4), is most likely due to the addition of biochar. However, the pH difference across the samples is not variable and not significant enough to explain the decrease in As concentration as seen in samples with 4% biochar and 5% biochar added. The addition of biochar in higher percentages could have
26 decreased adsorption to such an extent that the As in the top part of the soil in the pots, moved with the water down into the pots, decreasing the phytotoxicity in the top part of the pots allowing roots to grow.
The results could indicate that with the increase of biochar percentages and decreased uptake of As in the roots is caused by adsorption and pH, but there is also a shift in where the As is located within the plants suggesting that there could be other processes which affect the uptake . One study found that increasing pH, above pH7, resulted in higher uptake of As, this study had close to the same concentration (82 mg kg-1 ) as in this study (Salido et al, 2003). The study used P. vittata and different conditions in the experiment and cannot be applicable to this study. Nonetheless, the relation between pH and increased uptake of As by the plants can be observed to some extent in this study as well, with 0% amendment having 5.4 pH with foliage concentration of 79.6 mg kg-1 As. In comparison, the 2% amendment had pH of 5.8 and 158mg /kg in the foliage. The pH variation is small compared to the As-concentration in the results. The variation in As concentration between the biochar amendments is therefore hard to explain with respect to pH alone. Our results show a decrease in concentration even with stable or slightly increasing pH towards higher percentages of biochar, which would suggest that the effect of pH in this study is not an important variable in As uptake.
Electrostatic repulsion is another mechanism that could explain the low germination and growth seen in some of the pots with biochar amendment (Sun, Zhang & Su, 2018). The authors proposed increased mobilization of As could be due to the electrostatic repulsion between the negative surface of the biochar and the anionic arsenic. This would result in decrease of adsorption, increase As in the water table, and resulting in higher phytotoxicity (apparent from stunted growth in plants).
7.4 Biochar
The effects of biochar are hard to determine in this study. We can clearly see increased uptake by plants grown in biochar amended soil compared to the plants grown in group B with no biochar. Biochar in low concentrations (1 % and 3%) indicated very little or no growth, suggesting
phytotoxicity occurred and the plants could not survive the high As levels. But, the plants in the pots with 2% added biochar did grow. Perhaps the seeds may not have been viable and therefore did not germinate. The experiment was replicated with exactly the same condition in 3 separate pots with 1% and 3% amendment (i.e. 6 pots). Therefore, the explanation about viable seeds is probably flawed. The proposed explanation about electrostatic repulsion between the biochar and As sounds reasonable. However, this too is not reflected in the results. With this explanation, we would expect to see increasing toxicity with higher amounts of As in groups with higher percentages of biochar. In
