Erato polymnioides – A novel Hg hyperaccumulator plant in ecuadorian rainforest acid soils with potential of microbe-associated phytoremediation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Erato polymnioidesa novel Hg hyperaccumulator plant in 1 ecuadorian rainforest acid soils with potential of microbe-2 associated phytoremediation 3 4 Irene Chamba 5 Departamento de Química 6 Universidad Técnica Particular de Loja 7 San Cayetano Alto s/n, 8 110104 Loja, Ecuador. 9 Phone: (+593) 7 370 1444 ext 3024 10 [email protected] 11 12 Daniel Rosado* 13 Departamento de Química y Ciencias Exactas 14 Universidad Técnica Particular de Loja 15 San Cayetano Alto s/n, 16 110104 Loja, Ecuador. 17 Phone: (+593) 7 370 1444 ext 3041 18 [email protected] 19 20 Departamento de Ingeniería Química y Ambiental 21 Universidad de Sevilla 22 Camino de los descubrimientos s/n, 23 41092 Sevilla, Spain. 24 Phone: (+34) 954 487 274 25 [email protected] 26 27 Carolina Kalinhoff 28 Departamento de Ciencias Naturales 29 Universidad Técnica Particular de Loja 30 San Cayetano Alto s/n, 31 110104 Loja, Ecuador. 32 Phone: (+593) 7 370 1444 ext 3024 33 [email protected] 34 35 Thangaswamy Selvaraj 36 Departamento de Ciencias Naturales 37 Universidad Técnica Particular de Loja 38 San Cayetano Alto s/n, 39 110104 Loja, Ecuador. 40 Phone: (+593) 7 370 1444 ext 3024 41 [email protected] 42 43 Aminael Sánchez-Rodríguez 44 Departamento de Ciencias Naturales 45 Universidad Técnica Particular de Loja 46 San Cayetano Alto s/n, 47 *Revised manuscript with no changes marked Click here to view linked References HIGHLIGHTS:  Hg accumulation capacity was studied in three plant species.  Hg in acid soil studied is higher than the Ecuadorian threshold (0.1 mg kg-1).  Concentrations of Hg in roots and leaves were higher than in stems.  All plants showed arbuscular mycorrhizal fungi colonization in their roots.  Erato polymnioides showed high potential as an Hg hyperaccumulator. *Highlights (3 to 5 bullet points (maximum 85 characters including spaces per bullet point) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 110104 Loja, Ecuador. 48 Phone: (+593) 7 370 1444 ext 3024 49 [email protected] 50 51 Manuel Jesús Gazquez 52 Departamento de Física Aplicada 53 Escuela Superior de Ingeniería 54 Universidad de Cádiz 55 Campus de Puerto Real avenida República Saharahui s/n 56 11510, Puerto Real, Cádiz, España 57 Phone: (+34) 956 01 60 78 58 manuel.gazquez.[email protected]m 59 60 CORRESPONDING AUTHOR: 61 * Departamento de Química y Ciencias Exactas, Universidad Técnica Particular de Loja, San Cayetano Alto 62 s/n, 11 01 608 Loja, Ecuador. Tel.: (+593) 7 370 1444 ext 3041; E-mail address: djrosad[email protected] 63 (Daniel Jesús Rosado Alcarria). 64 65 66 Abstract 67 Mercury (Hg) accumulation capacity was assessed in three plant species (Axonopus 68 compressus, Erato polymnioides, and Miconia zamorensis) that grow on soils polluted by 69 artisanal small-scale gold mines in the Ecuadorian rainforest. Individuals of three species 70 were collected at two sampling zones: i) an intensive zone (IZ, 4.8 mg Hg kg-1 of soil) 71 where gold extraction continues to occur, and ii) a natural zone (NZ, 0.19 mg Hg kg-1 of 72 soil). In addition, the percentage of arbuscular mycorrhizal fungi (AMF) colonization was 73 determined in plant roots and seven fungal morphotypes isolated from rhizospheric soil. 74 Results suggest a facilitation role of native and pollution adapted AMF on Hg 75 phytoaccumulation. E.g., E. polymnioides increased Hg accumulation when growing with 76 greater AMF colonization. We concluded that E. polymnioides is a good candidate for the 77 design of microbe-assisted strategies for Hg remediation at gold mining areas. The 78 consortia between E. polymnioides and the AMF isolated in this study could be 79 instrumental to get a deeper understanding of the AMF role in Hg phytoaccumulation. 80 81 Keywords: Heavy metals; bioremediation; pollution; artisanal scale gold mining; 82 arbuscular mycorrhizal fungi; southern Ecuador 83 84 1. Introduction 85 Gold (Au) price has tripled in the last 10 years and artisanal small-scale gold mining 86 (ASGM) has increased substantially in rural areas all over the world (García et al., 2015; 87 Veiga et al., 2014). Around 16 million people are directly involved in this activity 88 nowadays, producing 380-450 tons of gold annually. In many cases, the process used in 89 ASGM for Au extraction is mercury (Hg) amalgamation, that consists in grinding the 90 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 mineral (raw material) and mixing it with Hg (Seccatore et al., 2014). During 91 amalgamation, an Au-Hg alloy is produced and heated in open vessels to separate both 92 metals from undesired matter. Pure Au and volatilized Hg are then obtained. 93 94 Hg is one of the most toxic heavy metals and a global pollutant that biomagnifies through 95 the food chain, posing a threat to human and animal health (Navarro et al., 2009; Pirrone et 96 al., 2009). ASGM using amalgamation is estimated to release into the environment 37% of 97 global air emissions of Hg and 1400 tons year-1 of Hg directly to the soil and water sheds 98 (Gibb and O’Leary, 2014). It is also responsible for Hg emissions that lead to massive 99 aerial Hg contamination and pollution of terrestrial ecosystems through rainfall deposition 100 (727 tons year-1) not only at ASMG sites but also at other areas (Seccatore et al., 2014; 101 United Nations Environment Programme, 2013). Anomalously high concentrations of Hg 102 (over 4 mg Hg kg-1 soil) are reported by several studies wherever the Hg amalgamation 103 process is practiced (García et al., 2015; Terán-Mita et al., 2013). However, volatilization 104 of Hg from soil and water occurs and Hg returns to the atmosphere generating a cycle, with 105 volatilization from soil being more relevant than from the oceans (Bjerregaard and 106 Andersen, 2007). Plants contribute to volatilization of Hg by uptaking Hg from soil and 107 releasing it into the atmosphere through stomata in a process called phytovolatilization (Ali 108 et al., 2013). 109 110 Communities living nearby and downstream ASGM are exposed to Hg vapor, and regularly 111 consume food heavily contaminated with methyl mercury (MeHg) (Gibb and O’Leary, 112 2014). Elemental Hg and MeHg are toxic to the nervous, digestive, and immune system. 113 They can cause mental retardation, seizures, vision and hearing loss, delayed development, 114 language disorders, and memory loss, being fatal in some cases (World Health 115 Organization, 2006). Reducing the impacts of ASGM on human health and the 116 environment has become a major concern for society and many governments worldwide. 117 Thus, reducing the use of Hg in ASGM was included in the Minamata Convention on Hg, 118 signed in 2013 in Minamanta, Japan (United Nations Environment Programme, 2013). 119 120 Several techniques to encapsulate and stabilize Hg are being researched to reduce the 121 impact of Hg emissions at ASGM sites, such as bioremediation and, especially, 122 phytoremediation (Mani and Kumar, 2014). Hg phytoremediation with so called metal 123 hyperaccumulator plants has attracted interest as an inexpensive, low-impact and visually 124 benign technique compared to traditional physical approaches (Lorestani et al., 2012). 125 126 Historically, the term "hyperaccumulator" has undergone significant changes since it was 127 defined by Brooks et al. (1977). The first definition was "plants with Ni concentrations 128 higher than 1000 mg kg-1 dry weigh”. Later, Baker and Brooks (1989) extended the concept 129 to more metals and defined hyperaccumulators as plant species which accumulate greater 130 than 100 mg kg-1 dry weight Cd, or greater than 1000 mg kg-1 dry weight Ni, Cu and Pb or 131 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 greater than 10000 mg kg-1 dry weight Zn and Mn in their shoots. However, other authors 132 suggest that criteria are unnecessarily conservative (van der Ent et al., 2013). Nowadays, 133 hyperaccumulators are defined as plants that achieve 100-fold higher shoot metal 134 concentration, compared to crop plants or common non-accumulator plants (Barceló and 135 Poschenrieder, 2003; Chamba et al., 2016; Redondo-Gómez, 2013). Thus, they have 136 tremendous potential for remediation of metals in the environment. 137 138 Many plant species have been cataloged as Hg hyperaccumulators (Angle et al., 2001; 139 Lorestani et al., 2012), e.g. Jatropha curcas and Piper marginathum from tropical 140 rainforests and Rumex induratus and Marrubium vulgare from the Iberian Peninsula. P. 141 marginathum reached Hg tissue concentrations in the range of 0.53-6 mg kg-1 (Marrugo-142 Negrete et al., 2016) and those from the Iberian Peninsula showed 8.3-67.2 mg kg-1 of Hg 143 in their roots (Moreno-Jiménez et al., 2006). Prasad and De Oliveira (2003) found 144 Asteraceae family members as one of the best hyperaccumulator plant species and a good 145 candidate for phytoremediation compared to several organism, including mycorrhizal and 146 non-mycorrhizal fungi, about 400 hyperaccumulating agricultural, vegetable crops and 147 ornamentals plants (Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, 148 Cunouniaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, Violaceae, and 149 Euphobiaceae). 150 151 Finding native hyperaccumulator plants to the ASGM areas has gained attention during 152 recent years. Native metal hyperaccumulator plants are harmless to native biodiversity, 153 require less handling and acclimatization and are also, in most cases, highly tolerant to the 154 contaminants that need to be removed (Sarma, 2011). However, native hyperaccumulator 155 plants are difficult to cultivate sometimes, and may have low growth rates and biomass, 156 lowering the metal uptake (Goltapeh et al., 2013). In this sense, mycorrhizal fungi live 157 associated to most of the higher plants in different forms, with arbuscular mycorrhizal fungi 158 (AMF) associations with the roots of terrestrial plants being the most widespread. AMF 159 modify the bioavailability and mobility of toxic metals in the soil and facilitate metal 160 uptake by the plants as well as increase the host plants biomass production in polluted 161 ecosystems (Leung et al., 2013; Teixeira et al., 2014). AMF constitute a bridge for nutrient 162 and heavy metal transport from soils to plant roots, such as N, P, K, Ca, S, Zn, Co, Ni and 163 Cu through extensive hyphal network. They also bind metals in the hyphae outside the root, 164 preventing metals to move to the aerial parts of the plant and, in the case of Hg, preventing 165 volatilization too (Sarwar et al., 2017). Then, native AMF have arisen as promising 166 alternative and as an innovative tool to replace or supplement present treatment processes 167 in order to assist phytoremediation. 168 169 This research has two main objectives. Firstly, to assess the potential of three native plants 170 as Hg hyperaccumulators in Chinapintza (Zamora-Chinchipe province, southeast Ecuador), 171 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 a site with a long ASGM tradition. Secondly, to evaluate AMF colonization in the plant 172 species and the spores of the most prominent AMF isolated. 173 174 2. Materials and Methods 175 2.1. Study area 176 As shown in Figure 1, the study area is located in Chinapintza (Zamora-Chinchipe 177 Province, southeast Ecuador), beside the Condor mountain range, near to the Peruvian 178 border (at 1854 masl; 4°02'16.6"S 78°34'14.9"W). Zamora-Chinchipe Province is well 179 known by its ASGM activity: around 23% (282.998 ha) of the total surface is dedicated to 180 ASGM (Sacher and Báez, 2011). The study area was divided into two zones: (1) a natural 181 zone (NZ) free of influence of ASGM and (2) an intensive zone (IZ) highly deteriorated by 182 ASGM that continues to occur. 183 184 Figure 1. Map of the sampling points. 185 186 2.2. Plant and soil sampling and processing 187 Three abundant plants present in NZ and IZ were selected as potential Hg 188 hyperaccumulators: Axonopus compressus (SW.) P. Beauv (Poaceae), Erato polymnioides 189 DC. (Asteraceae), and Miconia zamorensis Gleason (Melastomataceae). Seven individuals 190 of each species and their rhizosphere soil were taken as samples in each of the two zones, 191 i.e. a total of 42 individuals and its surrounding soil. 192 193 According to the recommendations of Tack and Verloo (1996), sampling took place far 194 from active roads and the surface of the fresh plant material was checked to be free of dust. 195 In the laboratory, plant samples were washed with ultra-pure water (Merck-Millipore Milli-196 Q), placed into paper bags and dried in an oven at 60°C for one week. Soil samples (0.5-1.0 197 kg each) were dried at 60°C until constant weight. Dried samples (plant tissues and soil) 198 were weighed and mechanically grounded using a stainless steel grinder (particle diameter 199 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 100 μm) for digestion. In the case of A. compressus, experiments were carried out only in 200 roots and leaves, since it lacks of a real stem. A. compressus shows a pseudo stem formed 201 by tightly packed overlapping leaf sheaths, like in other members of Poaceae family. 202 203 2.3. Soil physicochemical parameters 204 Soil pH was determined on a soil:water mixture (1:2.5) by a potentiometric method 205 (Jaworska et al., 2016). Soil organic matter (SOM) was determined by the Walkley & 206 Black method (Rial et al., 2016; Walkley and Black, 1934). Available phosphorus (P), 207 inorganic nitrogen (NH4) and potassium (K), were extracted in an Olsen modified extract 208 pH 8.5 (Olsen and Sommers, 1982), and quantified by photocolorimetry-blue 209 phosphomolybdate (P), photocolorimetry-blue indofenol (NH4), and atomic absorption 210 spectroscopy (AAS). Cationic exchange capacity (CEC) was measured in ammonium 211 acetate buffered at pH 7 with barium chloride, and finally soil texture was determined by 212 the method of Bouyucos (Day, 1982). All measurements were performed on seven 213 replicates. 214 215 2.4. Hg quantification in soil and plant samples 216 Dried samples were weighed, 0.2 g of each plant tissue (roots, stem and leaves) per 217 individual and 1 g of rhizosphere soil, and were left to soak in aqua regia, i.e. a mixture of 218 HCl and HNO3 in a 3:1 ratio (v/v), for a week. Next, they were digested on an open heat 219 block (environmental express 54 Hot block SC154) for 2 h. After cooling, the samples were 220 diluted to 100 ml with HCl 0.1 M and stored until metal determination. Total Hg 221 concentration was determined following the hydride-generation technique in an atomic 222 absorption spectrophotometer (Perkin-Elmer, AANALYST-400). A Hg standard calibration 223 curve (100, 200, and 300 μgl-1) was prepared in 10 ml of an acid mixture containing 1.5% 224 HNO3 by triplicates. Two blank samples were also run simultaneously to estimate 225 background metal contamination from the digestion procedure. For each sample, 10 ml of 226 acid mixture containing 1.5% HNO3 were added to 5 mL of the digestion mixture (prepared 227 by triplicates). Hg was determined using an aqueous solution of 3% (w/v) NaBH4 in a 1% 228 (w/v) NaOH solution freshly prepared and filtered as reducing agent. An electrodeless 229 discharge lamp was used (Olmedo et al., 2013). Analytical grade chemical reagents and 230 highly purified deionized water were always used. 231 232 2.5. Hg phytoextraction capacity calculation 233 To evaluate the Hg phytoextraction capacity of selected plant species, the bioaccumulation 234 factor (BF) was calculated as the ratio between the Hg concentrations in 3 plant tissues 235 (root, stem, leaves) and those in its corresponding rhizosphere soil sample: BF=Ctissue/Csoil 236 (González and González-Chávez, 2006). Ctissue and Csoil are Hg concentrations (mg Hg kg-1 237 of dry weight). To evaluate the plants ability to transfer Hg from soil to their aerial parts, 238 the translocation factor (TF) was calculated as the ratio between Hg concentrations in aerial 239 plant parts (leaves and stem) and those in the plant root: TF= Caerial/Croots (Chopin et al., 240 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 2008; Conesa et al., 2006). Caerial and Croots are Hg concentrations (mg Hg kg-1 of dry 241 weight). 242 243 2.6. Quantification of AMF colonization and spores morphotypes 244 Root samples from the three species were collected at the NZ and the IZ to determine the 245 presence of AMF colonization (%AMFcol). Roots were washed thoroughly with running 246 water to remove organic debris and soil particles and cut into small segments of 1 cm in 247 length. AMF colonization was assessed in each root segment following the method of 248 (Giovannetti and Mosse, 1980). Briefly, root segments were stained with 0.05% tryphan 249 blue in lactophenol, and then washed with clear lactophenol to remove the excess of the 250 colorant. A total of 25 stained root segments were randomly selected for each plant species 251 and mounted on microscopic slides (five segments per slide). AMF colonization was then 252 assessed microscopically based on the presence of a blue stain. The percentage of root 253 colonization was calculated per plant species using the ratio of effectively stained segments 254 (presence of colonization) to the total of microscopically analyzed root segments for the 255 particular species per 100. Measurements were performed for three replicates of each plant 256 species in two sampling zones. 257 258 AMF morphotypes associated with the roots of E. polymnioides and M. zamorensis were 259 isolated through the establishment of trap cultures. Five rizospheric soil samples per plant 260 species were placed in 2 l pots planted with a highly colonizable plant host (Avena sativa 261 L.) during five months. To fill each pot, fresh rizospheric soil samples were 1:1 mixed with 262 sterilized quarzitic sand (steam-sterilizered sand at 100°C forth periods of two hours each). 263 After planting, A. sativa plants were subjected to a regime of irrigation suspension for two 264 weeks to further promote AMF sporulation (Morton et al., 1995). AMF spores that 265 occurred in trap pots after five months were isolated by sieving 50 g of soil followed by 266 centrifugation in sucrose, as recommended by Sieverding et al. (1991). Pools of spores 267 sharing the same morphology were created under a stereomicroscope (40x) and further 268 separated into morphotypes by color and size. Photographs of each morphotype were 269 obtained from permanent microscope slides prepared using polyvinyl alcohol-lactic acid-270 glycerin (PVLG) (Koske and Tessier, 1983) as mounting media under a compound 271 microscope. Morphotypes were morphologically described based on the number, size and 272 ornamentation of spore walls, reaction to Melzer reagent (iodine-potassium iodide-chloral 273 hydrate), and other criteria such as type of hyphal attachments and cicatrix if present. 274 Taxonomic identification down to genera level and to species level when possible was 275 performed according to an on-line AMF species catalogue and descriptions available at the 276 International Culture Collection of Mycorrhizal Fungi web site 277 (http://invam.caf.wvu.edu/index.html) following the classification given by some authors 278 (Redecker et al., 2013). 279 280 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 2.7. Statistical analysis 281 SPSS version 20 software was used to carry out t-tests and ANOVA tests. The t test was 282 used to determine whether there were significant differences between two means 283 corresponding to two groups. For example, t test was used to elucidate if there were 284 significant differences between both zones in soil physicochemical parameters. The 285 ANOVA test was used to establish significant differences between more than two means, 286 corresponding to several groups. It allows drawing conclusions about Hg concentrations in 287 plant tissues, bioaccumulation and translocation factors and AMF colonization and 288 richness. 289 290 3. Results 291 3.1. Soil physicochemical parameters and Hg concentrations 292 Acidic pH, high Fe and low N, P and K concentrations were recorded in all soil samples 293 from both the NZ and the IZ (Table 1). There were small differences between soils from the 294 sampling zones on most of the assayed parameters with the exception of N, and as 295 expected, Hg concentrations (p<0.05). N and Hg were found at higher concentrations in 296 soil samples from the IZ. 297 298 Table 1. Soil parameters from the sampling zones. NZ: natural mining zone; IZ: intensive 299 mining zone. Values are expressed as mean of seven independent measurements ± standard 300 deviation 301 Soil parameter Sampling zone NZ IZ pH (H2O) 4.3 ± 0.5 4.1 ± 0.5 SOM (%) 2.8 ± 0.9 2.4 ± 0.5 N (mg kg-1) 44.8 ± 6.5 85.3 ± 10.5 P (mg kg-1) 6.9 ± 0.9 8.6 ± 2.3 K (cmol kg-1) 0.14 ± 0.03 0.12 ± 0.07 CEC (cmol kg-1) 6.3 ± 1.4 6.9 ± 1.2 Fe (mg kg-1) 505 ± 154 664 ± 207 Hg (mg kg-1) 0.19 ± 0.09 4.8± 1.2 S (mg kg-1) 33.2 ± 3.9 123.1 ± 41.5 302 As it can be seen in Table 1, sulfur is an important component of the acidic soils we 303 studied. Sulfur concentration in the IZ is almost four times that of the NZ. The substantial 304 increase of sulfur in the IZ compared to the NZ reflects the gold mining practices in the IZ. 305 In addition, it is important to discuss the potential implications of high sulfur contents for 306 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 0.5-1.0 mg Hg kg-1 in plant tissues can produce growth depression (Kabata-Pendias, 2011), 462 E. polymnioides showed an elevated tolerance to this heavy metal. In addition, E. 463 polymnioides displayed the least root-to-leaves Hg translocation, in contrast to A. 464 compressus, which has the ability to translocate an important fraction of the accumulated 465 Hg from the roots to its leaves, especially at the IZ (2.16). Based on these findings, E. 466 polymnioides could be seen mainly as a Hg stabilizer species while A. compressus capable 467 of phythoextracting Hg (incorporation and redistribution to the aerial parts of the plant). 468 469 Regardless the final goal of a remediation strategy, heavy metal phytoextraction is desirable 470 in stages where appropriately cutting and final disposal of the aerial plant biomass has been 471 previously covered. In contrast, phytostabilization could be more favorable in less 472 accessible locations, where plant roots and rhizospheric microbial interactions can 473 inmobilize Hg, reducing its incorporation into ground and/or water bodies and food chain 474 (Sarma, 2011). In a geographical setting like that of Chinapintza, with limited accessibility, 475 highly dense tropical forests and many ASGM sites around a water stream that goes 476 directly into downstream communities, Hg phytostabilization is preferred over 477 phytoextraction. A highly efficient accumulator and tolerant plant such as E. polymnioides 478 could represent an attractive strategy for the environmental remediation of Chinapintza 479 soils affected by ASGM. Even more, considering it is native to the area, an increase in its 480 population size for remediation purposes should not represent an ecological treat 481 482 On the other hand, volatilization of Hg0 by transpiration is a possible route for Hg 483 detoxification in plants (Moreno et al., 2008). Future studies are essential to evaluate the 484 phytovolatilization of Hg and the possible contribution to air pollution of this native plant. 485 486 487 5. Conclusions 488 E. polymnioides (Asteraceae family) showed high potential as an Hg hyperaccumulator 489 plant with higher affinity of mycorrhizal association helpful to survive against metal 490 toxicity. Thus, it is a suitable candidate to be considered as an Hg hyperaccumulator when 491 designing environmental friendly and sustainable strategies for Hg remediation at ASGM 492 areas. A deeper understanding of AMF role in Hg phytoextraction should be paid a special 493 attention in future studies. The availability of E. polymnioides, together with the associated 494 AMF diversity isolated in the present study, could be instrumental for this task. 495 496 Acknowledgements 497 This work had the financial support of: (1) National Secretary of Higher Education, 498 Science, Technology and Innovation of the Republic of Ecuador (SENESCYT from its 499 acronym in Spanish) in the frame of the Prometeo Project; (2) UTPL SmartLand initiative, 500 research program PROY_CCNN_1138. 501 502 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 6. 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