Efficacy of Lime, Biosolids, and Mycorrhiza for the Phytostabilization of Sulfidic Copper Tailings in Chile: a Greenhouse Experiment

International Journal of Phytoremediation

ISSN: 1522-6514 (Print) 1549-7879 (Online) Journal homepage: http://www.tandfonline.com/loi/bijp20

Efficacy of Lime, Biosolids, and Mycorrhiza for the Phytostabilization of Sulfidic Copper Tailings in Chile: A Greenhouse Experiment

César Verdugo , Pablo Sánchez , Claudia Santibáñez , Paola Urrestarazu , Elena Bustamante , Yasna Silva , Denis Gourdon & Rosanna Ginocchio

To cite this article: César Verdugo , Pablo Sánchez , Claudia Santibáñez , Paola Urrestarazu , Elena Bustamante , Yasna Silva , Denis Gourdon & Rosanna Ginocchio (2010) Efficacy of Lime, Biosolids, and Mycorrhiza for the Phytostabilization of Sulfidic Copper Tailings in Chile: A Greenhouse Experiment, International Journal of Phytoremediation, 13:2, 107-125, DOI: 10.1080/15226510903535056

To link to this article: http://dx.doi.org/10.1080/15226510903535056

Published online: 13 Aug 2010.

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Download by: [Pontificia Universidad Catolica de Chile] Date: 11 January 2017, At: 08:14 International Journal of Phytoremediation, 13:107–125, 2011 Copyright C Taylor & Francis Group, LLC ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226510903535056

EFFICACY OF LIME, BIOSOLIDS, AND MYCORRHIZA FOR THE PHYTOSTABILIZATION OF SULFIDIC COPPER TAILINGS IN CHILE: A GREENHOUSE EXPERIMENT

Cesar´ Verdugo,1 Pablo Sanchez,´ 1 Claudia Santiba´nez,˜ 1 Paola Urrestarazu,1 Elena Bustamante,1 Yasna Silva,1 Denis Gourdon,1 and Rosanna Ginocchio1,2 1Centro de Investigacion´ Minera y Metalurgica,´ CIMM, Vitacura, Santiago, Chile 2Facultad de Agronom´ıa e Ingenier´ıa Forestal, Pontiﬁcia Universidad Catolica´ de Chile, Santiago, Chile

Inadequate abandonment of copper mine tailings under semiarid Mediterranean climate type conditions has posed important environmental risks in Chile due to wind and rain erosion. There are cost-effective technologies for tailings stabilization such as phytostabilization. However, this technology has not been used in Chile yet. This study evaluated in a greenhouse assay the efﬁcacy of biosolids, lime, and a commercial mycorrhiza to improve adverse conditions of oxidized Cu mine tailings for adequate establishment and grow of Lolium perenne L. var nui. Chemical characterization of experimental substrates and pore water samples were performed; plant density, biomass production, chlorophyll content, and

metal content in shoots was evaluated in rye grass plants after an eight-week growth period. Results showed that neutralization of tailings and superﬁcial application of biosolids increased both aerial biomass production and chlorophyll content of rye grass. Increased Cu solubilization and translocation to shoots occurred after biosolids application (mixed), particularly on unlimed tailings, due to formation of soluble organometallic complexes with dissolved organic carbon (DOC) which can be readily absorbed by plant roots. Positive effects of mycorrhizal inoculation on rye grass growth were restricted to treatments with superﬁcial application of biosolids, probably due to Cu toxicity effects on commercial mycorrhiza used (Glomulus intraradices).

KEY WORDS: phytotoxicity, copper, rye grass, bioavailability, biosolids

INTRODUCTION Large-scale extraction and concentration of porphyry copper deposits has been one of the main economic activities in Chile since the XIX Century (Toledo and Zapater, 1989). This long history of Cu mine exploitation, besides the sustained reduction of metal concentration in ores, has resulted in accumulation of huge amounts of solid wastes (Lagos, 1994; CONAMA, 2000; Lagos and And´ıa, 2000). In the case of Cu sulfide ores, the economically interesting Cu sulfides are extracted by flotation process whereas pyrite and

Address correspondence to Rosanna Ginocchio, Centro de Investigacion´ Minera y Metalurgica,´ CIMM, Av. Parque Antonio Rabat 6500, Vitacura, Santiago, Chile. E-mail: [email protected]

107 108 C. VERDUGO ET AL. other gangue minerals are depressed from ﬂotation and discarded (Dold and Fontbote,´ 2001). This waste (tailings) represents 80–90% of total discarded solids throughout the pyrometallurgic process. Copper tailings are a water solution of 30–35% solids (<2 mm in diameter) having elevated concentrations of metals (i.e. Cu, Zn, Pb, Fe, and Mo) and metalloids (i.e., As Benkhe, 1973; Marshall, 1982; Bradshaw, 1983; Gutierrez´ and Hoffmann, 1991) that are generally characterized as toxic, radioactive and/or hazardous (Petrisor et al., 2004), particularly after inadequate disposition and management (Bradshaw, 1983; Lagos, 1994; McCall et al., 1995; Badilla-Ohlbaum et al., 2001; Ginocchio et al., 2006). In the past, Cu tailings were directly washed out into rivers and ravines, therefore reducing soil and surface water quality and affecting intertidal environments of north-central Chile (Castilla and Nealler, 1978; Correa et al., 1999; Ram´ırez et al., 2005). After severe environmental damages, mining regulations established by the Chilean Ministry of Mines in the late 1970s required deposition on tailings storage facilities (TSF), but no closure management was requested by law after abandonment. Abandonment of TSF’s in north-central Chile under Mediterranean semi-arid climate conditions leads to high water evaporation and tailings desiccation in few years (Dold and Fontbote,´ 2001). Without proper closure management, this ﬁne, homogenous and non-cohesive metal-rich particulate matter material has been left exposed to physical and chemical environmental forces (Bradshaw, 1983; Ginocchio, 2000). Erosion by wind and heavy rains due to El Nino˜ phenomenon, beside dam-wall failure after strong earthquakes, has released tailings into surrounding areas, posing risks to human health, agricultural activities and wildlife (SERNAGEOMIN, 1989; Lagos, 1994; CONAMA, 2000; Lagos and

And´ıa, 2000; Badilla-Ohlbaum et al., 2001). Furthermore, depending on their mineralogy (i.e. content of metal sulfides such as pyrite, FeS2) and geographic location (i.e. amount of precipitation), acid drainage and lixiviation of metals may also occur from sulfidic tailings (Dold and Fontbote,´ 2001; Evangelou, 2001) with some consequent environmental impacts on surface and ground waters, such as salinization, metal/metalloid enrichment and acidification (Gray, 1998; Dinelli et al., 2001). It is generally accepted that metal sulfide oxidation, and in particular of pyrite, is the main reason for the formation of sulfuric acid and solubilization of metals, a phenomenon known as acid mine drainage (AMD; Evangelou, 2001). Closure regulations for TSF were finally established by the Chilean government in 2002 and therefore a need for adequate and cost-effective stabilization techniques is increasing in the country. Several technologies for surface stabilization of mine tailings are internationally available, such as cementation, vitrification, and coverage of tailings with water or geomembranes, which have been effectively used in developed countries (Salt et al., 1995; Masscheleyn et al., 1996; Vangronsveld and Cunningham, 1998; Glass, 1999; Vigneault et al., 2007). However, these methods are expensive and sometimes inappropriate due to their ongoing maintenance requirements or climatic conditions (Van der Lelie et al., 2001). Therefore, cheaper and environmentally friendly methods have been developed, such as phytostabilization (Vangronsveld and Cunningham, 1998; Flynn et al., 2002; Petrisor et al., 2004). Phytostabilization has been reported to be an effective in situ, inexpensive, aesthetically pleasing, environmentally friendly, and socially accepted green technology to decrease land erosion and spreading of metals from mining areas (Salt et al., 1995). Copper tailings are usually a compacted substrate with bad drainage, low content in macronutrients and organic matter, high-metal/metalloid contents and scarce microbial communities (Vangronsveld et al., 1995; Zhu et al., 1999, Petrisor et al., 2004, De la Iglesia PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 109 et al., 2006; Ginocchio et al., 2006). Therefore, establishment of an adequate plant cover over dry TSF through phytostabilization requires the improvement of physical, microbio- logical, and chemical characteristics of tailings. In developed countries, biosolids and other organic wastes and by-products generated by cellulose producing plants or agricultural activities have been effectively used as amendments of metal polluted soils and hard-rock mine wastes (i.e., Sabey et al., 1975; Sopper, 1993; Haering et al., 2000; Brown et al., 2003). However, chemical characteristics of organic materials, such as biosolids, may greatly vary in different regions of the world, particularly in its nitrogen and metal contents; furthermore, chemical evolution of biosolids on tailings under different climate and management conditions may also differ. Therefore, experiences gained in other countries (i.e. temperate) may not be directly applied to semiarid Mediterranean areas of north-central Chile. Besides of this, use of biosolids on copper sulfidic tailings have not been deeply studied at least in terms of its affects on substrate metal speciation and on metal uptake and toxicity to plants. This study evaluates the efficacy of biosolids produced in north-central Chile to improve physical and chemical limiting conditions of copper sulfidic tailings for phytostabilization. With this option, management problems related to biosolids disposal could have also a potential solution in north-central Chile as they are currently being landfilled with scarce recycling options. Secondarily, application of a commercial mycorrhiza on the establishment, growth, and metal uptake of Lolium perenne L. var nui is also evaluated as a means to improve the development of plant covers on Cu tailings and reduce transportation and application costs by reducing the amount of organic amendment required. Mycor- rhizas are known for improving nutrient absorption by plants, particularly P in low nutrient substrates, by increasing the explored soil volume, increasing solubilization of minerals,

including metal-containing rock phosphates, and improving water absorption (Brundrett et al., 1996; Estaun´ et al., 1997; Leyval et al., 1997; Enkhtuya et al., 2002; Estaun´ et al., 2002; Bi et al., 2003; Barea et al., 2005).

MATERIAL AND METHODS Bulk Materials Cauquenes TSF is one of three abandoned TSF of the El Teniente copper mine, the world’s largest underground copper mine. Cauquenes TSF, with a surface of 640 ha, is located 12 km southeast of the town of Rancagua, north-central Chile; it operated until 1975. Cauquenes tailings resulted from an unusual acid flotation circuit (pH 4.5) due to the high content of kaolinite and montmorillonite of the ore and they were released with this pH to the TSF (Dold and Fontbote,´ 2001). Tailings were collected from the upper oxidation zone (approx. 1 m depth), where secondary mineralogical processes has occurred after TSF abandonment leading to a strong enrichment of bivalent cations (i.e., Cu, Zn, Ca, K, Mo, Mn) in the form of water-soluble phases, mainly as sulfates due to the upwards mobilization during the summer season via capillary forces (Dold and Fontbote,´ 2001). The presence of metals in water-soluble form at the top of Cauquenes tailings could lead to strong limiting conditions for the establishment of a plant cover through phytostabilization. A composite sample (60 kg, 0–20 cm depth) of sulfidic copper tailings was collected from the central area (approx. 2 ha) of the Cauquenes TSF. As particle size of tailings is less than 50 µm they were used as collected from the field; sample was homogenized by hand and dried in a forced-air drying cabinet at 35◦C; general chemical characteristics of sampled tailings are given in Table 1 and in Ginocchio et al. (2006). 110 C. VERDUGO ET AL.

Table 1 General chemical characteristic of tailings and biosolids used in the study

Total Total Total Total Total Total T.O.C. Nitrogen Phosphorous Potassium Copper Zinc Arsenic C.E.C. E.C. Substrate (%) (%) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (meq/100 g) pH (dS/m)

Tailings 0.02 0.02 0.16 0.31 3,996 107 68 14.23.77.2 Biosolids 54.50 3.90 21,080 3,150 524 1,385 20 n.d. 7.57.8

T.O.C. = total organic carbon; C.E.C. = cation exchange capacity; E.C. = electric conductivity; and n.d. = not determined.

A large sample (45 kg) of stabilized biosolids (water content of 35%) was obtained from El Trebal municipal water treatment plant owned and operated by Aguas Andinas Ltd., located nearby Santiago city, the capital of Chile, and approx. 100 km northwest from the town of Rancagua. After the final stage of centrifugation in the plant, biosolids are dumped into a monofill were they air-dried during the summer period before their transportation to a landfill for final disposal. General physicochemical characteristics of biosolids are given in Table 1. Agglomerates of biosolids were broken by hand using a wooden mallet before manual homogenization in order to get particle size <5 mm.

Plant Cultivation Studies A short-term greenhouse experiment was performed following a completely random- ized design. Seven substrate treatments were deﬁned for the study as indicated in Table 2. A unique dose of biosolids was added (200 ton ha−1, dry weight) which lead to either a 2% organic matter (OM) in the mixture of tailings with biosolids or a 30% OM when added on tailings surface. Due to the high acidity of Cauquenes tailings (pH 3.7, Table 1), half of the tailings’ treatments were neutralized with quick lime (calcium oxide, CaO, from Qu´ımica Universal). Experimental mixtures of tailings with biosolids and/or lime were carefully homogenized by hand. Experimental substrates were placed in six replicated 1-L plastic containers. All containers were sown with 0.6 g of seeds of Lolium perenne L. var nui (perennial ryegrass). This plant species has been frequently used in laboratory experiments of phytostabilization studies of metal polluted soils (i.e., Lepp et al., 1997; Antoniadis and Alloway, 2002; Arienzo et al., 2004) and was considered as an adequate laboratory species for this study.

Table 2 Experimental substrates used in the plant cultivation study

Treatment code Description of treatment

T Sulﬁdic tailings alone TL Limed tailings (7 kg lime per ton of tailings, 7 kg ha−1) TLB-S Limed tailings (7 kg ha−1) with biosolids (200 ton ha−1, d.w.) applied on surface TLB-M Limed tailings (7 kg ha−1) with mixed biosolids (200 ton ha−1, d.w.) TB-S Tailings with biosolids (200 ton ha−1, d.w.) applied on surface TB-M Tailings with mixed biosolids (200 ton ha−1, d.w.) B Biosolids alone

T = Sulﬁdic tailings; L = lime; B = biosolids; S = surface application; M = mixed application; and d.w. = dry weight. PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 111

Half of experimental containers (N = 3) were inoculated with Mycosym TritonR at a dose of 0.72 g per experimental pot (600 kg ha−1) as suggested by manufacturer (Mycosym Intl AG, Basel, Switzerland). Mycosym TritonR is a commercial product that contains spores and hyphae of the arbuscular mycorrhizal fungus Glomulus intraradices [(synonymous of G. fasciculatum according to Biermann and Linderman (1983)], as well as inoculated root fragments. Expanded clay, a light and porous granular material is used as inert carrier. It contains at least 200 infective units per mL, of which more than 50 are spores. The expanded clay (0.72 g pot−1) was mixed with ryegrass seeds (0.6 g−1) and evenly distributed into experimental substrates. Seeds and Mycosym TritonR were covered (5 mm depth) with the same substrate of every experimental treatment and pots were irrigated with deionized water to saturation. Experimental containers were randomly placed in a plant growth room under controlled environmental conditions (temperature of 22 ± 2 ◦C, 260 µmol s−1 m−2 of light intensity, photoperiod of 12:12, and relative humidity of 44%). Containers were relocated once a week and irrigated with deionized water to 60% ﬁeld capacity every 2–3 days or as required (no lixiviation allowed).

Pore Water Characterization Pore water samples were taken at the beginning of the seventh week from all experimental containers to assess pH, concentrations of total dissolved copper (Cu), zinc (Zn), and arsenic (As), and total dissolved organic carbon (DOC). Samplers were perpendicularly

fitted into the first 3–4 cm of substrates in all experimental containers (rhizospheric zone). An aliquot of 5 ml of filtered pore water (0.1 µm pore size) was taken from each container with a 5-cm-length RhizonR Soil Pore Water Sampler (Rhizosphere Research Product, Wageningen, Holland), following the method described in Vulkan et al. (2000). RhizonR samplers were acid-washed before their use with 30 mL high purity (>18 M cm−1) deionized water, 30 mL 1 N HNO3 (Suprapur, Merck), and 30 mL high purity deionized water. Pore water samples were kept in acid-washed polyethylene plastic vials (15 mL) for pH determination. Method 1638 of the U.S. Environmental Protection Agency (USEPA, 1996) was used for the acid washing of all polyethylene plastic vials (24 h with 0.5% Extran MA O2 neutral (Merck), 24 h with 1 N HNO3 (Suprapur, Merck), 24 h with 1 N HCl (Suprapur, Merck), and four washings with high purity deionized water). All pH determinations were done up to 10 min after sample collection with a combination pH electrode (Sensorex 120C, Garden Grove, CA, USA). Samples were then acidified with HNO3 (Suprapur, Merck) and analyzed for total Cu, Zn, and As using an inductively coupled plasma-mass spectrometer (ICP-MS; ELAN6100 with autosampler AS90; Perkin Elmer). The standard calibration solution was ICP-MS multielements, 99.99% purity, high purity, and reference material ION-20 (National Water Research Institute, Burlington, ON, Canada). Sample calibration standard and reference material were in 0.2% HNO3. DOC was determined by combustion with the U.S. EPA method 415.1 (Keith, 1996) in a carbon analyzer (Tekmar Dohrmann Carbon Analyzer, model Apollo 9000, Cincinnati, OH, USA). The calibration standard solution used was 1 mg L−1 as carbon of dipotassium phtalate for organic carbon. As reference material ION-20 was used (National Waters Research Institute). 112 C. VERDUGO ET AL.

Plant Performance After eight weeks, density of individuals was determined in all containers. The total number of individuals developed per container was directly counted and then the number of individuals per square centimeter was estimated from the total area of experimental containers (113.1 cm2). Plants were then harvested, washed with deionized water, and separated into shoots and roots. A sample of leaves per container (270 mg, fresh weight) was used for determination of total chlorophyll (a and b chlorophylls) according to Harborne (1975). Shoots and roots were dried to constant weight in an air-forced drying cabinet at 45◦C for determination of shoot and root biomass production per container (dry weight basis) and root to shoot biomass ratio. Shoots were then ﬁnely crushed in the RETSCH S100 agate ball mill and stored in polyethylene sample bottles. Plant tissue samples were digested according to the USEPA protocol (1996) for copper and zinc determination. Every digestion batch included one blank sample, one standard reference material sample known as SRM 1573a tomato leaves or SRM 1570a spinach leaves (National Institute of Standards and Technology, USA), one duplicate sample and one quality control sample for the Quality Assurance and Control, QA/QC, criteria. Copper was analyzed by ICP-MS (ELAN6100 with auto sampler AS90, PERKIN ELMER, Uberlinger, Germany).

Statistical Analysis The non-parametric multiple comparison Kruskal-Wallis test (Siegel and Castellan, 1988) was used for testing response variables among treatments. The Mann-Whitney test was used as a posteriori test (Siegel and Castellan, 1988). Spearman non-parametric simple correlations (Siegel and Castellan, 1988) were used to evaluate statistical relationships among chemical characteristics of pore water samples among experimental substrates. All statistical analyses were performed using the Statistica statistical package Version 6.0 (StatSoft Inc., 2001).

RESULTS Chemical Characteristics of Pore Water Significant differences in pore water pH were found among treatments (H = 39.93, P < 0.01, N = 42). Addition of lime into tailings (TL, TLB-S, TLB-M) significantly increased pH from 3.5 to neutrality, particularly on non-inoculated (m-) treatments (Table 3), while addition of biosolids alone, either on surface (TB-S) or mixed with tailings (TB- M), resulted in a significant but smaller increase in pore water pH when compared to lime (1–2 pH units versus 4–5 units, respectively; Table 3). In general, mycorrhizal inoculation (m+) of experimental substrates significantly reduced pore water pH, with the exception of both pure materials (tailings, T, and biosolids, B) where no effect was detected. In terms of metal/metalloid concentrations in pore water, there were significant differences in total dissolved Cu (H = 35.28, P < 0.01, N = 42), Zn (H = 38.58, P < 0.01, N = 42), and As (H = 37.3, P < 0.01, N = 42) among treatments as shown in Table 3. Neutralization of tailings with lime (TL, TLB-S, TLB-M) resulted in significant reductions of soluble metals (Cu and Zn) but increased solubilization of arsenic in pore water (Table 3). However, there was no clear effect of mycorrhizal inoculation (m+)on metal/metalloid levels in pore water (Table 3). Specifically, significant negative correlations

Table 3 Chemical characteristics of pore water of experimental mixtures. Mean and standard deviation of treatments without (m−) and with (m+) inoculation of mycorrhiza are given

D.O.C. Copper Total dissolved metal pH (mg/L) (mg/L) Zinc (mg/L) Arsenic (µg/L)

Substrates m− m+ m− m+ m− m+ m− m+ m− m+

T3.5 ± 0.04a 3.5 ± 0.02a n.d. 84 ± 47a 1966 ± 1189a 1665 ± 1119a 11.2 ± 6.7a 8.7 ± 1.8a 75 ± 0.1ac 41 ± 6.3a TL 7.3 ± 0.12b 6.6 ± 0.35e n.d. 28 ± 20a 0.08 ± 0.02d 0.2 ± 0.06d 0.2 ± 0.01b 0.2 ± 0.03b 49 ± 14ac 12 ± 1.5b TLB-S 7.2 ± 0.05b 5.7 ± 0.76c n.d. 186 ± 70b 2.7 ± 1.1b 1.9 ± 1.6b 0.8 ± 0.1c 0.5 ± 0.05c 105 ± 22c 122 ± 43c TLB-M 7.5 ± 0.04b 6.6 ± 0.25c n.d. 710 ± 175b 2.3 ± 0.2b 3.6 ± 2.0b 0.1 ± 0.08b 1.8 ± 0.5c 171 ± 9.7c 78 ± 15ac TB-S 5.7 ± 0.13c 4.6 ± 0.01d n.d. 493 ± 180b 655 ± 260a 6.8 ± 1.7b 18.3 ± 3.0a 2.6 ± 0.4c 39 ± 4.9a 75 ± 26ac TB-M 4.1 ± 0.19d 5.6 ± 0.06c n.d. 482 ± 53b 6.0 ± 1.5b 317 ± 361a 2.4 ± 0.7c 23 ± 5.8a 53 ± 26a 60 ± 10a B7.5 ± 0.08b 7.6 ± 0.12b n.d. 6931 ± 1601c 1.4 ± 0.2c 3.1 ± 3.6c 2.1 ± 0.3c 2.1 ± 0.3c 454 ± 67d 352 ± 48d Kruskal Wallis ANOVA by ranks d.f. 13 6 13 13 13 H 39.93 18.63 35.28 38.58 37.30 P <0.01 <0.01 <0.01 <0.01 <0.01 N4221424242

Different letters indicate signiﬁcant differences among treatments per parameter. D.O.C. = dissolved organic carbon; T = tailings; L = lime; B = biosolids; -S = surface application of biosolids on tailings; -M = biosolids mixed with tailings; n.d. = not determined; and d.f. = degrees of freedom. 113 114 C. VERDUGO ET AL.

1,E+07 (A) m-, Rs = -0.82, P< 0.01, N=14 1,E+06 m+, Rs = -0.74, P< 0.01, N=21

1,E+05

1,E+04

1,E+03

1,E+02 Cu in Pore Water (ug/L) Water Pore in Cu 1,E+01

1,E+00 0123456789 pH in Pore Water

1,E+03 (B)

1,E+02

1,E+01 Cu in Pore Water (um/L) Water Cu Pore in

m+, Rs = 0.70, P< 0.01, N=15

1,E+00 1,E+00 1,E+01 1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 DOC (mg/L)

Figure 1 Relationship among total dissolved copper in pore water and both (A) pore water pH and (B) dissolved organic carbon or DOC. In the case of graph B, treatments T and B were excluded. Open circles and solid rhombus represent the treatments without (m−) and with (m+) inoculation of mycorrhiza, respectively. Rs = Spearman correlation coefficients; P = probability value; and N = number of samples are given for treatments (m−) without and (m+) with inoculation of mycorrhiza. were found among Cu in pore water and pH in both non-inoculated (m-) and inoculated (m+) substrates (Figure 1A). Application of biosolids into tailings significantly increased DOC concentration in pore water (H = 18.63, P < 0.01, N = 21; Table 3), irrespective of application form (mixed or on top of tailings). In general, incorporation of biosolids on limed tailings increased total dissolved metal and metalloid levels in pore water (Table 3), thus posing a confounding effect. A significant and positive relationship was found among DOC and total dissolved Cu (Figure 1B) for all treatments excluding T and B; as the level of DOC increases in biosolids amended tailings, total dissolved Cu in pore water increases. The effect of mycorrhizal PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 115

b m-

) b m+

2 4

b 3 b

2 aa a a aa a

1 Plant Density (inds/cm (inds/cm Density Plant c

0 T TL TLB-S TLB-M TB-S TB-M B

Figure 2 Density of Lolium perenne var. nui plants on experimental treatments. White and black bars represent the treatments (m−) without and (m+) with inoculation of mycorrhiza, respectively. Mean and standard deviation are given. T = tailings; L = lime; B = biosolids; −S = surface application of biosolids on tailings; and −M = biosolids mixed with tailings. Different letters indicates signiﬁcant differences. inoculation on this parameter could not be evaluated as DOC levels were only determined in pore waters extracted from inoculated (m+) treatments.

Plant Performance Germination of L. perenne was completely inhibited on tailings (T), but neutralization of tailings with lime and incorporation of biosolids allowed germination and seedling development (Figure 2). After an 8-week growth period, significant differences were found for plant density among treatments (H = 39.09, P < 0.01, N = 42). It reached mean values of 1.0–1.5 individuals per square centimeter among non-inoculated substrates, irrespective of the treatment. Potted substrates inoculated with mycorrhiza had a 2–3 times higher plant density than non-inoculated substrates (Figure 2), except treatment TB-M where no inoculation effect was observed and treatment B where inoculation significantly decreased plant density (Figure 2). Shoot and root biomass markedly varied among treatments (Figures 3A and B). In relation to shoot biomass (H = 40.33, P < 0.01, N = 42), significant differences were found across treatments without inoculation (Figure 3A). Shoot biomass was highest on tailings with surface application of biosolids, with or without liming, and lowest in treatments with mixed incorporation of biosolids, with or without liming (Figure 3A). Inoculation with mycorrhiza resulted in a significant and positive effect on shoot biomass in treatments TLB-S and TB-S but a marked reduction (TB-M and B) or no effect (TLB-M) in this parameter was detected in the other treatments (Figure 3A). Root biomass significantly varied (H = 38.71, P < 0.01; N = 42) across inoculated and non-inoculated treatments (Figure 3B). Highest root biomass for non-inoculated substrates occurred on treatments TL, TLB-S and TB-S while treatments TB-M and B showed the lowest values (Figure 3B). Inoculation with mycorrhiza had either no effect (TLB-M 116 C. VERDUGO ET AL.

4500 (A) d m- 4000 m+ 3500 d

3000 b

2500 b

2000

1500 a aa

c c 1000 c

500 Shoot Biomass (mg/pot, d.w.) e e 0 T TL TLB-S TLB-M TB-S TB-M B

700 a (B) m- 600 m+ aa

500 a a

400 a

300

200 b b c c 100 c Root biomass (mg/pot, d.w.) d 0 T TL TLB-S TLB-M TB-S TB-M B

Figure 3 Shoot (A) and root (B) biomass (dry weight, d.w.) of Lolium perenne var. nui plants on experimental treatments. White and black bars represent the treatments (m−) without and with (m+) inoculation of mycorrhiza, respectively. Mean and standard deviation are given. T = tailings; L = lime; B = biosolids; −S = surface application of biosolids on tailings; and −M = biosolids mixed with tailings. Different letters indicates significant differences. and TB-M) or a significant reduction (B) in root biomass (Figure 3B). Furthermore, root morphology varied across treatments; roots were thin and spread throughout containers in treatments TL, TLB-M and TB-M but they were thick, short and restricted to the upper organic matter layer on treatments TLB-S and TB-S. When root to shoot biomass ratios were estimated across treatments (Figure 4) significant differences were found (H = 37.71, P< 0.01, N = 42). Among non-inoculated substrates, plants significantly allocated more resources to root biomass when grown on limed tailings than on tailings amended with biosolids, irrespective of lime application PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 117

1,0 m- m+ 0,8 a

0,6 aa

a 0,4 b

b 0,2 bb b b b

Root biomass biomass / Shoot (d.w.) 0,0 T TL TLB-S TLB-M TB-S TB-M B

Figure 4 Root to shoot biomass (d.w. = dry weight) ratio of Lolium perenne var. nui plants on experimental treatments. White and black bars represent the treatments (m−) without and (m+) with inoculation of mycorrhiza, respectively. Mean and standard deviation are given. T = tailings; L = lime; B = biosolids; −S = surface application of biosolids on tailings; and −M = biosolids mixed with tailings. Different letters indicates signiﬁcant differences.

(Figure 4), while the opposite trend was found for resource allocation to shoot biomass. The same pattern was found across inoculated substrates with the exception of treatments TB-M and B where plants allocated more resources to root biomass (Figure 4). Chlorophyll content in leaves significantly varied among treatments (H = 36.65, P < 0.01, N = 42; Figure 5). Among non-inoculated substrates, chlorophyll content was highest on tailings with surface application of biosolids, with or without liming, and lowest in treatments with mixed incorporation of biosolids, with or without liming (Figure 5). In these last treatments chlorotic leaves were observed. Inoculation with mycorrhiza significantly increased chlorophyll content in almost all treatments, except TB-M (Figure 5). In treatment B, leaf production was inadequate for chlorophyll determination (Figure 5). Shoot Cu concentration significantly varied among treatments (H = 38.94, P < 0.01; N = 42) and so was shoot Zn concentration (H = 38.57, P < 0.01, N = 42; Figure 6). Cu concentration was highest on unlimed tailings (TB-S and TB-M) with values up to 180 to 220 mg kg−1 and lowest on limed tailings (Figure 6A) where shoot Cu concentrations varied within 29.6 mg kg−1 and 99.7 mg kg−1. Treatments inoculated with mycorrhiza showed the same tendency, but Cu concentrations were duplicated after inoculation on treatments TB-S and TB-M (Figure 6A). In the case of shoot Zn concentration, no significant differences were detected among non-inoculated and inoculated substrates, but accumulation pattern across treatments differed from the Cu pattern (Figure 6A and B). Shoot Zn concentrations where significantly higher (2 to 4 times) in plants grown on tailings amended with biosolids, irrespective of lime application, than on limed tailings (TL; Figure 6B), but it also varied across the biosolids amended tailings; it was higher on TB-S (203.3–240.7 mg kg−1), intermediate on TLB-S, TB-M and B (133.4–156.9 mg kg−1) and lower on TLB-M (73.2–94.3 mg kg−1; Figure 6B). 118 C. VERDUGO ET AL.

4 m- m+ c 3 bc

b b 2 b a

a a

1 a a a Total chlorophyll (mg/mg of leaf) of (mg/mg chlorophyll Total

0 T TL TLB-S TLB-M TB-S TB-M B

Figure 5 Total chlorophyll (a + b) content in leaves of Lolium perenne var. nui plants on experimental treatments. White and black bars represent the treatments (m−) without and (m+) with inoculation of mycorrhiza, respectively. Mean and standard deviation are given. T = tailings; L = lime; B = biosolids; −S = surface application of biosolids on tailings; and −M = biosolids mixed with tailings. Different letters indicates signiﬁcant differences.

DISCUSSION

Neutralization of oxidized sulfidic tailings has an important effect on the establishment and growth of L. perenne (rye grass) as acidity by itself can negatively affect plant establishment and performance (Marschner, 1986). Neutralization with liming agents (i.e. CaCO3, Ca(OH)2, CaO) is a traditional practice in acidic agricultural soils to allow crop production but also in revegetation practices of acidic mine spoils (Williamson et al., 1982; Simon, 2005). In the last case, substrate neutralization has a double purpose; to reduce both acidity and elevated concentration of soluble metals and, therefore, the bioavailability of these elements to plants. In acidic metal-rich substrates, free ionic metal species are dominant (Sauve´ et al., 2000; Impellitteri et al., 2001); however, solubility of most metals (i.e., Cu, Zn) decreases as pH increases (Bourg, 1995). In this study, a significant negative correlation was found among total dissolved Cu and pH in pore water of experimental substrates, as a small increase in pH can decrease Cu solubility by several orders of magnitude, as reported for metal-polluted soils (Sauve´ et al., 2000; Vulkan et al., 2000; Ginocchio et al., 2002; Peijnenburg and Jager, 2003). Neutralization resulted, however, in increased solubilization of As into pore water, as this metalloid has an opposite behavior when compared to metals (Adriano, 2001). This response does not pose any risk to rye grass as As is not as toxic to plants as metals (Adriano, 2001); however, care should be taken when restoring both As and metal-rich mine wastes under field conditions as As may be disseminated through food webs posing risks to herbivores and humans. Although neutralization of tailings significantly increased rye grass germination and development, addition of biosolids improved plant performance, particularly when added on top of tailings. Specifically, shoot biomass and total chlorophyll content in rye grass were higher on tailings with a surface layer of biosolids than on only limed tailings. This result may be explained by the high-nitrogen and other macronutrient contents of PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 119

700 c (A) m- 600 m+

500 c

400

300 c c 200 b b Copper in shoot (mg/Kg) 100 aa aa a a

0 T TL TLB-S TLB-M TB-S TB-M B

300 (B) d m- m+ 250 d

200 b bb b bb 150

cc 100

Zinc in shoot (mg/Kg) aa 50

0 T TL TLB-S TLB-M TB-S TB-M B

Figure 6 Copper and zinc contents in shoots of Lolium perenne var. nui plants on experimental treatments. White and black bars represent the treatments without (m−) and with (m+) inoculation of mycorrhiza, respectively. Mean and standard deviation are given. T = tailings; L = lime; B = biosolids; −S = surface application of biosolids on tailings; and −M = biosolids mixed with tailings. Different letters indicates signiﬁcant differences. biosolids, essential elements for making proteins and chlorophyll in plants (Larcher, 1995; Blackmer, 1997; Garnier et al., 1999; Chiu et al., 2006). Better nutritional conditions of biosolids amended tailings (treatments TLB-S and TB-S) may be also inferred from relative allocation to root and shoot tissues and according to the “root-foraging” responses of plants. In low-nutritional substrates (i.e., limed tailings, TL) plants showed long and thin roots as resources were mainly allocated to root growth due to the need to “forage” for nutrients. This response pattern was also detected in Anaphalis maragaritacea, a plant that 120 C. VERDUGO ET AL. grows naturally on tailings (Kramer et al., 2000). On the other hand, short and thick roots, restricted to the upper layer of nutrient-rich biosolids and the interface with the tailings, where present on plants growing on treatments TLB-S and TB-S (nutrient-rich substrates), as resources were mainly allocated to shoot growth; under suitable level of nutrients in the substrate, plants may allocate more resources to the development of aerial structures (i.e., Noble and Marshall, 1983; Ginocchio, 1994). Differences on rye grass performance according to biosolids application, either on surface or mixed with tailings, may be explained by both excess of salinity and metal toxicity avoidance strategies than by nutritional status of substrates. Avoidance to metal exposure may be occurring on TB-S and TLB-S treatments as plant roots were mainly restricted to the upper biosolids layer. Biosolids has a neutral pH (7.5) and are rich in organic matter (55%), a material described as a good metal-chelating agent on metal polluted soils (Kramer et al., 2000; Brown et al., 2003; Ginocchio et al., 2006). Organic matter amendments, such as composted yard waste, spent mushroom compost, peat, sludges, and cattle manure, can form stable organic-metal complexes when incorporated into metal-rich substrates, such as tailings, as they have high cation exchange capacities due to its negatively charged surface (Hetrick et al., 1994; Sauve´ et al., 2000; Simon, 2005). However, the high content of low molecular weight organic acids in biosolids at dressing time, such as fulvic acids, measured as dissolved organic carbon (DOC), may counteract this effect, particularly on biosolids mixed tailings, as DOC showed to increase metal solubility and bioavailability in the substrate solution (i.e. Santiba´nez˜ et al., 2007; Navarro and Mart´ınez, 2008). Metal cations, such as Cu2+, are complexed by DOC and these soluble organo-metallic complexes can be readily absorbed by plant roots (Lamy et al., 1993; McBride et al., 1997; Antoniadis and

Alloway 2001; Coninck and Karam, 2008), as found in this study, and/or leached into deep substrate layers (Klitzke and Lang, 2007). Indeed, higher copper contents were detected in shoots of plants growing on biosolids mixed tailings than on surface applied biosolids. A decrease in the concentration of soluble metals would be expected on the longer term, once the mineralization of labile organic matter in biosolids leads to stabilized organic matter, as it has been shown by Al-Wabel et al. (2002). In acidic tailings (no lime added), free ionic copper species and dissolved organic- bound copper forms may be dominant species as it has been described in other acidic substrates (Sauve´ et al., 2000; Impellitteri et al., 2001), thus explaining the significantly higher copper levels in shoots detected on TB-S and TB-M treatments when compared to treatments TLB-S and TLB-M. It is well known that as the substrate pH decreases the Cu solubility increases (Arienzo et al., 2004) and so is the bioavailability of this element to plants. Biosolids have high electric conductivity values (7.8 dS m−1) which may limit L. perenne growth as this plant species is a non salt tolerant grass species (Ashraf et al., 1986). Indeed, very limited rye grass development was produced on treatment B (only biosolids). Contrary to expected, negative effects of biosolids were not detected when added on top of tailings (treatments TLB-S and TB-S) but when mixed with tailings (treatments TLB-M and TB-M). This result may be explained by the effect of irrigation of experimental pots with demineralized water, which could have washed out excess salts from biosolids to tailings below, away from the root development zone, thus reducing electrical conductivity of biosolids. As no water lixiviation was allowed from experimental pots, the wash out effect of salts did not occur on treatments B, TLB-M, and TB-M. Inoculation of rye grass with commercial mycorrhiza improved plant establishment in almost all treatments (TL, TLB-S, TLB-M, and TB-S) as higher plant densities developed PHYTOSTABILIZATION OF CU-TAILINGS WITH LIME, BIOSOLIDS, AND MYCORRHIZA 121 in all inoculated treatments than on not inoculated ones. This finding is in agreement with other studies that have shown that mycorrhiza improve plant establishment in degraded substrates, despite the substrate toxicity (Jeffries and Barea, 1994). However, positive effects of mycorrhizal inoculation on rye grass growth, measured as shoot biomass production, were very restricted to treatments TLB-S and TB-S. These results do not agree with results of other studies that show positive effects of mycorrhizal inoculation on plant growth in mine tailings and metal-polluted soils (i.e., Hetrick et al., 1994). This difference may be explained by metal tolerance capability of inoculated mycorrhiza, as Shetty et al. (1995) demonstrated a strong relationship between the adaptation of mycorrhizal fungi to elevated soil metal concentrations and the resulting increase in plant growth from mycorrhizal symbiosis. In the present study, a common commercial mycorrhiza was used which may be not necessarily adapted to metal-rich environments. Secondary substrate acidification of experimental substrates resulting from mycorrhizal inoculation may further explain this finding, as copper solubility increases with acidification of substrate. Although grasses infected by fungal endophytes often exhibit increased growth, particularly in nutrient poor substrates (Brundrett et al., 1996; Estaun´ et al., 1997; Enkhtuya et al., 2002; Estaun´ et al., 2002; Bi et al., 2003; Barea et al., 2005), results of infection may be manifested as decreased growth in plants growing under stressful conditions (Cheplick et al., 1989), such as copper and/or salinity toxicities detected in this study. The extent of mycorrhizal colonization of plant roots depends also on the nutrient status of plants; high N and P contents can inhibit mycorrhizal infection (Hartwig et al., 2002), which may have been the case in treatment B where inoculation decreased both plant density and biomass production. Even though symbiosis among rye grass and G. intraradices has

been demonstrated under natural (Gollote et al., 2004) and artiﬁcial (Hartwig et al., 2002) conditions, we did not check root colonization or degree of infected plants by mycorrhiza on experimental treatments. This important aspect should be evaluated in future studies.

CONCLUSION This study shows the importance of adequate management of oxidized copper mine tailings for allowing adequate plant establishment and growth. Neutralization of tailings is a key aspect for reducing stressful parameters of tailings to plants, as this reduces acidity and metal toxicity. Although, there is a potential for reusing biosolids on phytostabilization programs on abandoned mine tailings in north-central Chile, care must be taken when defin- ing both the dose and application form (surface layer or mixed with tailings) of biosolids to avoid (i) salinization of substrate, (ii) metal/metalloid solubilization and thus toxicity to plants and/or lixiviation to groundwater, and (iii) trace element dissemination through food webs in natural ecosystems. Finally, potential benefits of mycorrhizal inoculation should be further evaluated, particularly using metal tolerant mycorrhizas and more adequate plant species for field applications.

ACKNOWLEDGMENTS The authors wish to thanks the International Copper Association, ICA, Luis Par- raguez´ of Division El Teniente—CODELCO Chile, and Paola Arata of Aguas Andinas Ltd. Research funded by grant Innova Chile CORFO 04CR9IXD-01 to R. Ginocchio. 122 C. VERDUGO ET AL.

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